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Conference Abstracts

60th Annual Fall Conference and Scientific Sessions of


the Council for High Blood Pressure Research in Association with
the Council on the Kidney in Cardiovascular Disease
October 47, 2006 San Antonio Marriott Rivercenter San Antonio, Texas
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ADVANCING RESEARCH.
APPLYING KNOWLEDGE.
IMPROVING THE QUALITY
OF HEALTHCARE.
e26 Hypertension Vol 48, No 4 October 2006

Oral Presentations
1 dimensions and function, we could examine the role of ACE2 in BP regulation without the
Nuclear Hormone Receptor LXR Induces Murine Mesenchymal Stem Cells potential confounding effects of impaired heart physiology. Baseline BPs were measured in the
into Renin Expressing Cells: Possible Origin of Juxtaglomerular Cells ACE2 KO lines with radiotelemetry. Mean BPs were not significantly different between
B6-Ace2/y and -/y mice, and 129-Ace2/y and -/y mice (1282 vs. 1323 mmHg, n6 all
Kenichi Matsushita, Fulvio Morello, Richard E Pratt, Victor J Dzau; Duke Univ Med Cntr, groups). As in vitro studies suggest that ang II may be a substrate for ACE2, we examined the
Durham, NC effects of ACE2 deficiency on susceptibility to ang II-dependent hypertension, using the inbred
lines. Ace2/y (MWT) and -/y (MKO) mice were infused subcutaneously with angiotensin II (1000
Mesenchymal stem cell can differentiate into skeletal muscle, smooth muscle, cartilage, fat ng/kg/min) with an osmotic pump, while BP was monitored by radiotelemetry. With ang II
and bone. It may play an important role in developmental biology contributing to the genesis infusion, BP increased significantly in both MWT (1332 to 1696 mm Hg; p0.0005) and
of various tissues. Indeed, it has been reported that juxtaglomerular cells may originate from MKO mice (1312 to 1956 mm Hg, p0.0002), but the magnitude of this increase was
the metanephric mesenchyme. In this study we examined for direct evidence that mesenchy- almost 2-fold greater in the MKO animals (64 mm Hg) compared to MWT (36 mm Hg). After
mal stem cells (MSCs) can be induced to become renin expressing cells. Previously we have 2 weeks of ang II infusion, BPs were significantly higher in the MKO mice than WT (p0.01).
reported that the nuclear hormone receptor liver X receptor (LXR) is a major transcriptional To examine the mechanism for the more marked BP response to ang II in the ACE2-deficient
regulator of renin gene expression. Accordingly, we studied the effect of LXR activation of MSC mice, we measured kidney ang II levels after ang II infusion using MALDI-TOF mass
renin gene expression. Methods: Murine MSCs were subjected to serum starvation for 16 spectrometry. Renal ang II content was nearly six-fold higher in MKO mice compared to MWT
hours. We then added the LXR synthetic ligand T0901317 (10-6 M) and investigated mRNA (0.280.05 vs. 0.050.002 relative intensity; p0.0008). In summary, we find no effect of
expression of renin, ATP-binding cassette transporter A1 (ABCA1) which is well known ACE2-deficiency on basal BP regulation or CV function. However, ACE2 protects against ang
upregulated by LXR activation, LXR-alpha and LXR-beta after 6 hours of pharmacological II-dependent hypertension by regulating ang II levels in the kidney.
treatment by using real-time RT-PCR. We further examined the renin mRNA expression by
adding LXR natural ligand 22-hydroxycholesterol (10-7 M) or cyclic AMP (10-3 M). Results:
Murine MSCs treated acutely (6 hours) with T0901317 exhibited an increase in expression of 4
renin (1.9 0.4 fold change for MSCs without treatment, P 0.05). There was an increase Exaggerated Blood Pressure Variability Aggravates Hypertensive Cardiac
in ABCA1 expression (3.7 0.5 fold change for MSCs without treatment, P 0.05) and no Remodeling through the Angiotensin II-Mediated Inflammation
significant change in LXR-alpha or LXR-beta expression, suggesting that T0901317 effectively
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acted only at the post-transcriptional level. Furthermore, 6 hours of 22-hydroxycholesterol or Hiroshi Kudo, Hisashi Kai, Narimasa Takayama, Takahiro Mori, Yusuke Sugi, Daisuke Fukui,
cyclic AMP treatment also upregulated renin mRNA expression (2.0 0.4 fold and 6.0 1.8 Kiyoko Takemiya, Ayami Ikeda, Masayoshi Futamata, Hideo Yasukawa, Nobuhiro Tahara,
fold changes for MSCs without treatment, respectively. P 0.05). We have also overexpressed Mitsuhisa Koga, Yumiko Kawai, The Cardiovascular Rsch Institute, Kurume, Japan;
LXR-alpha in MSC using gene transfection. Upon prolonged stimulation with 22- Yoshitaka Hirooka, The Dept of Cardiovascular Medicine, Fukuoka, Japan; Tsutomu
hydroxycholesterol for 6 weeks or cyclic AMP for 2 weeks, our preliminary results show that Imaizumi; The Cardiovascular Rsch Institute, Kurume, Japan
the phenotype of the MSC changed to become cells containing renin granules. Conclusion: Our
results suggest that the activation of LXR induces renin mRNA expression and renin granule It was demonstrated that hypertensive patients with large blood pressure (BP) variability had
formation in mesenchymal stem cells and suggest that these cells may be the origin of greater risk of cardiovascular events and exaggerated end-organ damages. However, the
juxtaglomerular cells during embryonic development. pathogenesis is currently unknown. The aims of this study were to create a new rat model of
chronic hypertension with exaggerated BP variability and to investigate the effects of
exaggerated BP variability on hypertensive cardiac remodeling and the underlying mechanism.
2 For this purpose, we performed bilateral sino-aortic denervation (SAD) in spontaneous
c-Src Inhibition Attenuates Development of Hypertension and Associated hypertensive rats (SHRs). Seven weeks after SAD or sham operation (week 7), 24-hour BP was
Cardiovascular and Renal Damage in Ang II-Infused Mice through monitored telemetrically in SADSHRs and shamSHRs. Although mean BP was similar in the
Redox-Sensitive Mechanisms two groups, SADSHRs showed greater BP variability parameters, such as standard deviation
and covariance of mean BP, compared with shamSHRs. At week 7, both groups showed
Glaucia E Callera, Ying He, Augusto C Montezano, Univ of Ottawa, Ottawa, Canada; Alvaro concentric LV hypertrophy, and SAD enhanced it by 1.3-fold. SADSHRs enhanced myocyte
Yogi, Rita C Tostes, Univ of Sao Paulo, Sao Paulo, Brazil; Ernesto L Schiffrin, McGill Univ, hypertrophy and myocardial fibrosis by 1.4-fold and 4.7-fold, respectively, relative to
Montreal, Canada; Rhian M Touyz; Univ of Ottawa, Ottawa, Canada shamSHRs. Perivascular macrophage infiltration was evident in SADSHRs, but not in
shamSHRs. SAD remarkably upregulated expressions of myocardial angiotensinogen and
c-Src plays a critical role in Angiotensin II (Ang II)-mediated signaling. Whether this tyrosine monocyte chemoattractant protein-1 (MCP-1). To determine the role of angiotensin II in the BP
kinase influences development of hypertension and associated target organ damage induced variability-induced cardiac remodeling, non-depressor dose of angiotensin II receptor blocker,
by Ang II remains unclear. Here we investigated effects of Ang II on blood pressure (BP) and candesartan, was orally given to SHRs everyday from 1 week after SAD operation
renal and cardiac fibrosis in mice treated with the c-Src inhibitor, CGP 077675 and in mice (SADCandSHRs). At week 7, although the small dose of candesartan did not affect BP
deficient in c-Src (c-Src -/- and c-Src /- ). The systolic blood pressure (SBP) increase induced variability, the SAD-enhanced LV and myocyte hypertrophy was significantly reduced and the
by Ang II infusion (400 ng/kg/min, 2-weeks) in c-Src/ mice was significantly reduced by CGP SAD-induced myocardial fibrosis was almost abolished. Moreover, in SADCandSHRs, the
077675 (25 mg/Kg/day). Ang II-induced hypertension was blunted in c-Src-/- and c-Src/- mice SAD-induced MCP-1 upregulation and macrophage accumulation were almost reversed to the
(1678 vs 1378 and 1057 mmHg). Ang II-induced increase in plasma TBARS (control, levels of shamSHRs. In conclusion, exaggerated BP variability aggravates hypertensive
14.40.6 vs Ang II, 19.60.7 nmol/mL) and NAD(P)H oxidase-mediated vascular generation cardiac hypertrophy and fibrosis through chronic activation of inflammatory process. And,
of .O2- (3,5-fold) were reduced by CGP 077675 (p0.05). c-Src inhibition also inhibited the angiotensin II would play a key role in the activation of the inflammatory process, independently
increased phosphorylation of ERK1/2 (Ang II, 868 vs Ang II CGP, 613, arbitrary units) and of the presser effect.
JNK (Ang II, 12619 vs Ang II CGP, 431, arbitrary units) in aorta and resistance
(mesenteric) arteries. Ang II infusion failed to increase MAPK phosphorylation and .O2-
generation in c-Src/-, c-Src-/- mice (p0.05). The greater expression of proliferating cell 5
nuclear antigen (PCNA), a marker of cell growth, in mesenteric arteries from Ang II-infused Kidney-Specific Enhancement of Angiotensin II Initiates Renal Injury in
c-Src/ mice was inhibited with CGP 077675 treatment (Ang II, 1309 vs Ang II CGP, Gene-Targeted Mice
1044, arbitrary units). PCNA protein expression was similar in c-Src/- mice infused with Ang
II and vehicle. Cardiac and renal collagen content induced by Ang II was significantly lower in Hiroyuki Kobori, Yuri Ozawa, Yuki Suzaki, Tulane Univ Health Sciences Cntr, New Orleans,
in c-Src-/- and c-Src/- mice and in CGP 077675-treated mice. In conclusion, inhibition of c-Src LA; Curt D Sigmund, Univ of Iowa, Iowa City, IA; L. G Navar; Tulane Univ Health Sciences
activity by CGP077675 attenuates Ang II signaling and ameliorates oxidative stress, BP Cntr, New Orleans, LA
elevation and cardio-renal fibrosis by Ang II. Studies in c-Src- deficient mice support these
results. Our findings identify c-Src tyrosine kinase as a novel target to blunt Ang II -dependent We recently reported that concomitant increases in proximal tubular angiotensinogen (AGT)
hypertension and associated cardiovascular and renal damage. mRNA and protein participate in increased intrarenal angiotensin (Ang) II leading to progressive
renal injury in AngII-infused rats. However, it has not been established if selective increases in
intrarenal AngII can be responsible for renal injury. Using a transgenic mouse model in which
3 human AGT (hAGT) is expressed only in the kidney, experiments were performed to determine
Enhanced Susceptibility to Ang II-Induced Hypertension and Impaired Ang if selective renal overproduction of AngII elicited by stimulating hAGT present only in the kidney
II Metabolism in ACE2-Deficient Mice in the presence of human renin (hR) will cause renal injury. We used 3 groups of mice: 1) single
transgenic (A, N14) expressing hAGT only in the kidney regulated by kidney-specific
Susan B Gurley, Thu H Le, Robert Griffiths, Nisha Phillip, Timothy A Haystead, Thomas M androgen regulated protein promoter, 2) double transgenic (D, N13) expressing hR
Coffman; Duke Univ, Durham, NC systemically in addition to hAGT only in the kidney, and 3) wild type mice (W, N12).
Exogenous hAGT protein is inactive in A mice because endogenous mouse renin cannot cleave
In order to clarify the physiological roles of ACE2, lines of mice with targeted disruption of the hAGT to AngI due to a high species-specificity. All mice were monitored from 12 to 18 wks of
Ace2 gene have been generated. However, phenotypic features of the different ACE2-deficient age with free access to a regular diet and water. Systolic BP was progressively increased from
(KO) mouse lines have been variable, especially with regard to blood pressures (BP) and 116/-5 mmHg (12 wks) to 140/-7 (18 wks) in D mice during this period. This increase was
cardiovascular (CV) functions. One potential source of this variability might be heterogeneity of not observed in A or W mice. Intrarenal hAGT mRNA and protein were similar in A and D mice;
genetic background. Thus, we generated inbred ACE2 KO mice by sequential back-crossing of however, hAGT mRNA or protein was not detectable in kidneys of W mice. While plasma AngII
the Ace2 null mutation onto C57BL/6 (B6) or 129/SvEv (129) background for more than 6 concentrations were similar among the 3 groups, kidney AngII levels were increased in D
generations. Both lines of inbred ACE2 KO mice are viable, have normal reproduction, and lack (216/-43 fmol/g) compared with A (117/-16) and W (118/-17) mice. Interstitial
any gross anatomical or structural abnormalities. Because of these mice have normal cardiac collagen-positive area, stained by PicroSirius Red, was significantly increased in D (0.52/-
CHBPR ConferenceOral Presentations e27
0.06%) compared with A (0.36/-0.05) and W (0.34/-0.03) mice. Interstitial macrophage 8
infiltration, evaluated by CD68-positive cell number, was significantly increased in D (46/-5 Association between Variants of the Human GSTM Gene Family and
cells/mm2) compared with A (20/-3) and W (19/-2) mice. Afferent arteriolar wall thickness, Hypertension
stained by alpha actin, was significantly increased in D (3.31/-0.41 m) compared with A
(2.21/-0.12) and W (2.16/-0.11) mice. These data indicate that the selective renal Christian Delles, Univ of Glasgow, Glasgow, United Kingdom; Ana C Braga-Marcano, Patricia
overproduction of AngII initiates renal injury in the gene-targeted mice even before the B Munroe, Barts & The London Med & Dental Sch, London, United Kingdom; Sandosh
development of marked hypertension. Padmanabhan, John D McClure, Nick J Brain, Univ of Glasgow, Glasgow, United Kingdom;
Morris J Brown, Univ of Cambridge, Cambridge, United Kingdom; Nilesh J Samani, Univ of
Leicester, Leicester, United Kingdom; David Clayton, Univ of Cambridge, Cambridge, United
Kingdom; Martin Farrall, Univ of Oxford, Oxford, United Kingdom; John Webster, Aberdeen
Royal Infirmary, Aberdeen, United Kingdom; John M Connell, Univ of Glasgow, Glasgow,
United Kingdom; Mark J Caulfield, Barts & The London Med & Dental Sch, London, United
Kingdom; Anna F Dominiczak; Univ of Glasgow, Glasgow, United Kingdom
6
A Novel Regulatory Effect of AT1 Receptor-Interacting Molecule on Background: Glutathione S-transferases (GSTMs) are involved in cellular defences against
Vascular Smooth Muscle Cells oxidative stress. The human GSTM gene cluster consists of 5 genes, GSTM4, 2, 1, 5 and 3. We
have shown that the rat orthologue, Gstm1, is a positional and functional candidate gene for
Koichi Azuma, Kouichi Tamura, Masashi Sakai, Yuko Tsurumi, Toyoichiro Shigenaga, Yutaka rodent hypertension. The aim of the current study was to test the hypothesis that this discovery
Tanaka, Motoko Ozawa, Miyuki Matsuda, Tomoaki Ishigami, Yokohama City Univ, can be translated to man. Methods: First analysis was performed in 138 white subjects in
Yokohama, Japan; Marco Lopez-Ilasaca, Harvard Univ, Boston, MA; Masatsugu Horiuchi, whom exons, flanking introns, 3 and 5 regions of GSTM4, 2, 5 and 3 were sequenced to
Ehime Univ, Shigenobu, Japan; Satoshi Umemura; Yokohama City Univ, Yokohama, Japan detect single nucleotide polymorphisms (SNPs) within the GSTM gene family and to examine
linkage disequilibrium (LD) between these SNPs. We have then chosen 10 SNPs across the
Activation of tissue AT1 receptor (AT1R) signaling plays an important role in cardiovascular GSTM gene cluster and genotyped 1151 families (3453 individuals) from the MRC BRIGHT
hypertrophy and remodeling and the C-terminal domain of AT1R is involved in the receptor Study for these SNPs and for the common GSTM1 deletion genotype. Analysis was performed
internalization and its downstream pathway. We previously cloned a novel molecule interacting by Transmission Disequilibrium Test (TDT) using FBAT Software. Results: We detected 18, 5,
with the C-terminal domain of AT1R, ATRAP (for AT1R-associated protein), using the yeast 6, and 5 SNPs in GSTM4, 2, 5 and 3, respectively, with minor allele frequencies of 5% or
two-hybrid strategy. In this study, we tested the hypothesis that vascular smooth muscle cells greater. There were two distinct LD blocks, one between GSTM4 and 2 and one between
(VSMC) express ATRAP and that ATRAP modulates Ang II-induced responses in VSMC. We GSTM5 and 3. In the BRIGHT TDT Study we detected a highly significant association between
identified that the ATRAP mRNA and protein were endogenously expressed in VSMC, and found a SNP in the 3 region of GSTM5 (rs11807) and hypertension (z2.698, P0.007) and
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a substantial co-localization of ATRAP and AT1R in intracellular compartments in Ang significant associations for two more SNPs within the GSTM5/3 LD block (rs11101992:
II-stimulated VSMC. Overexpression of ATRAP by adenoviral gene transfer significantly inhibited z2.443, P0.015; rs3814309: z2.215, P0.027). The GSTM1 deletion was also over-
Ang II-mediated increases in TGF- mRNA expression (p0.05, n6) and TGF- production transmitted to affected offspring (z2.339, P0.019). Conclusions: The GSTM1 deletion
into the medium (p0.05, n6). Furthermore, this phenomenon was accompanied by genotype and three SNPs in the GSTM5/3 LD block are associated with hypertension as a
inhibition of Ang II-induced activation of BrdU incorporation (p0.05, n6). These results qualitative trait. This is an example for successful translation of findings from a rodent model
indicate that ATRAP significantly promotes internalization of the AT1R and attenuates Ang to human cardiovascular disease. GSTMs, and in particular GSTM1, 5 and 3, are robust
II-mediated proliferative response and synthesis of extracellular matrix in VSMC, and may candidate genes for human essential hypertension. Genetic variants in GSTMs may lead to
suggest a novel strategy to inhibit vascular remodeling. reduced defences against oxidative stress and thereby to endothelial dysfunction and relentless
progression of cardiovascular disease.

9
Effect of Age and Angiotensinogen 235 Genotype on Renal Plasma Flow
Responsiveness to Angiotensin II
7 Tejas V Patel, Gordon H Williams, Naomi D Fisher; Brigham and Womens Hosp, Boston, MA
Human G Protein-Coupled Receptor Kinase Type 4 (hGRK4) Wild-Type
Prevents Salt Sensitivity While its Variant, hGRK4 486V, Promotes Salt Objective: In essential hypertensives(HT), blunted renal plasma flow (RPF) response to Ang II
Sensitivity in Transgenic Mice: Role of Genetic Background is associated with the Angiotensinogen AGT235TT genotype, suggesting a pathologic increase
in intrarenal Ang II. RPF and its responsiveness to Ang II fall with age. We sought to determine
Zheng Wang, Laureano D Asico, Crisanto S Escano, Georgetown Univ Sch of Medicine, the interaction of age and genotype on RPF responsiveness in both HT and normotensives (NT).
Washington, DC; Robin A Felder, Univ of Virginia Health Sciences Cntr, Charlottesville, VA; Method: A total of 315 subjects in high sodium balance had RPF response to Ang II (3ng kg-1
Pedro A Jose; Georgetown Univ Sch of Medicine, Washington, DC min-1) measured by PAH clearance. Subjects were divided into 45 yrs (N83 HT, 65 NT) and
45 yrs (N145 HT, 22 NT) for age analysis, as 45 was the median age. Result: Age and
The hGRK4 486V variant, either by itself or in conjunction with other hGRK4 variants (65L AGT235 genotype independently predicted RPF response to Ang II. For both HT and NT, the Ang
and 142V), is associated with salt-sensitive hypertension (Clin Chem.2006;52:352), as II induced fall in RPF was significantly lower in older than in younger subjects (p0.03 and
evidenced by its development in hGRK4 486V transgenic mice (but not in hGRK4 wild-type 0.004, respectively). Among HT, this fall with age was seen in subjects carrying both the
transgenes) generated in our laboratory. The influence of genetic background on the expression AGT235MM and MT genotypes (p0.005, Fig. A). However, AGT235TT homozygotes did not
of salt sensitivity was studied by introducing the transgenes into mice with varying gene ratios demonstrate a fall in RPF responses with age (p0.72, young vs. old, Fig. B), as younger
of salt-resistant SJL and salt-sensitive C57BL/6J (B6/J) backgrounds. The mice (6 8 mo old) subjects already showed blunted responses. Among NT, all AGT235 genotypes had a significant
were maintained on normal (0.9%) or high (6%) NaCl diet for 3 weeks. With normal NaCl intake, fall in RPF response to Ang II. This association between age, RPF response to Ang II and
aortic BPs (measured via the femoral artery under pentobarbital anesthesia) were similar in genotype was not seen with 3 other genes in RAAS: ACE I/D, ATR1 and aldosterone synthase.
transgenic and non-transgenic mice. However, with high NaCl intake, BPs differed among the Conclusion: In HT but not NT, the AGT235TT variant is associated with a blunted RPF response
groups, and influenced by genetic background (Table). BPs of non-transgenic littermates (NT) to Ang II that does not fall with age. This suggests that young HT with the AGT235TT genotype
with at least 12% SJL background were not affected by high NaCl. In contrast, high NaCl warrant a more aggressive management to preserve renal function. This is the first report of
increased BPs of 486V mice, despite having 25% SJL background (P0.001). The ability of age and genotype interaction, which may have important implications in managing essential
high NaCl to elevate BPs of 486V mice decreased as the SJL background increased to 56%. HT. #
High NaCl increased BPs of NTs with more than 94% B6/J background. However, hGRK4
wild-type prevented the salt-sensitive hypertension of B6/J mice (P0.001). We conclude that
hGRK4 prevents, while the 486V variant promotes, salt-sensitive hypertension in transgenic
mice. The extent of salt-resistant or salt-sensitive genetic background is crucial in uncovering
the roles of hGRK4 wild-type and 486V variant in salt sensitivity. Inconsistencies in the
association of candidate genes with hypertension may be explained by differences in genetic
background.
% Background Normal NaCl (0.9%) High NaCl (6%)

SJL B6/J
(salt- (salt- SBP SBP
Mice esistant) ensitive) (mm Hg) N (mm Hg) N P#
10
SJL 100 0 970.6 4 972.2 5 0.87 Disruption of Natriuretic Peptide Receptor A Gene Increases Adrenal
B/6J 0 100 1031.2 4 1231.8* 5 <0.001
Angiotensin II and Aldosterone Levels
NT 25 75 1000.9 4 1003.7 6 0.97
NT 12 88 1052.9 4 1027.7 5 0.76
Di Zhao, Naveen K Somanna, Elangovan Vellaichamy, Kailash N Pandey; Dept of Physiology
NT 6 >94 1021.8 8 1221.2 11 <0.001
and Hypertension and Renal Cntr of Excellence, Tulane Univ Health Sciences Cntr & Sch of
GRK4 6 >94 1012.3 7 1032.3** 7 0.5
Medicine, New Orleans, LA
486V 25 75 1000.5 2 1254.6*** 9 0.03
486V 56 44 963.1 6 1103.6**** 5 0.01
The disruption of guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) gene (Npr1)
#P 0.9% vs 6% NaCl, ANOVA, Newman-Keuls (6% NaCl): *P0.01, SJL vs B/6J; **P0.001, NT(94% leads to elevated arterial blood pressure, cardiac hypertrophy, congestive heart failure, and
B6/J) vs GRK4; ***P0.001, NT(25% SJL) vs 486V; ****P0.05, 486V (25% SJL) vs 486V (56% SJL). sudden death in mice lacking NPRA. ANP-NPRA signaling is known to counteract the
SBPsystolic blood pressure, Data are MSE. renin-angiotensin-aldosterone system (RAAS). We studied whether Npr1 gene copy number
e28 Hypertension Vol 48, No 4 October 2006

affects adrenal angiotensin II (Ang II) and aldosterone (ALDO) levels in a gene-dose dependent 13
manner in Npr1 gene-targeted mice. Adrenal Ang II and ALDO levels increased in 1-copy Sexual Dimorphism in the Regulation of NOS 3 and Oxidative Stress in the
(gene-disrupted heterozygous allele, 0.2-fold, p0.05, 0.4-fold, p0.05) mice as compared Renal Cortex of Spontaneously Hypertensive Rats (SHR)
with 2-copy (wide type) mice, but decreased in 3-copy (gene-duplicated heterozygous allele,
0.2-fold, p0.05, 0.1-fold, p0.05), and 4-copy (gene-duplicated homozygous allele, 0.3-fold, Jennifer C Sullivan, David M Pollock, Jennifer S Pollock; Med College of Georgia, Augusta,
p0.01, 0.4-fold, p0.001) mice. Interestingly, renal Ang II levels decreased in 1-copy GA
(0.3-fold, p0.05), 3-copy (0.4-fold, p0.01), and 4-copy (0.4-fold, p0.01) mice as
compared with 2-copy mice. Low salt diet increased adrenal Ang II and ALDO levels in 1-copy Female SHR have a slower progression of renal injury compared to male SHR, although the
(0.2-fold, p0.05, 24-fold, p0.001), 2-copy (0.2-fold, p0.05, 23-fold, p0.001), 3-copy mechanisms protecting females are unknown. NO is an important regulator of kidney function,
(0.2-fold, p0.05, 4-fold, p0.001), and 4-copy (0.3-fold, p0.05, 5-fold, p0.001) mice. and NO levels have been reported to be greater in females. The aim of this study was to test
On the other hand, high salt diet decreased adrenal Ang II levels in 1-copy (0.5-fold, p0.001), the hypothesis that (1) NOS3 is differentially regulated in the renal cortex of male and female
2-copy (0.4-fold, p0.01), 3-copy (0.3-fold, p0.01) mice, and decreased adrenal ALDO SHR, and (2) oxidative stress is less in female SHR such that there is greater NO bioavailability
levels in 1-copy (0.3-fold, p0.05), 2-copy (0.2-fold, p0.05) mice. Low salt diet increased in females. Experiments used 14 16 week-old male and female SHR (n6 7). Microalbumin
renal Ang II levels in 1-copy (0.5-fold, p0.05), 2-copy (0.4-fold, p0.05), 3-copy (0.6-fold, (ualb) excretion and macrophage-specific ED-1 staining in the renal cortex were used to assess
p0.05), and 4-copy (0.5-fold, p0.05) mice, whereas high salt diet decreased renal Ang II injury. Blood pressure was measured by telemetry. At 14 weeks of age, blood pressures were
levels in 1-copy (0.3-fold, p0.05), and 2-copy (0.3-fold, p0.05) mice. The results suggest comparable in male and female SHR (1293 and 1311 mmHg respectively). Male SHR had
that ANP-NPRA signaling antagonizes adrenal Ang II and ALDO levels in a gene-dose dependent greater ualb excretion (4.40.3 mg/day/gram) compared to females (1.00.2 mg/day/gram,
manner. Our findings implicate that increased adrenal Ang II and ALDO levels play an important p0.05). In addition, male SHR had increased ED-1 staining (ED-1 cells/glomerulus: male,
role in elevated blood pressure in Npr1 gene-disrupted mice. 2.00.3, female, 1.30.3, n3) verifying a gender difference in renal injury. Total NOS 3
protein expression was comparable in the renal cortex from male and female SHR (male:
1.40.11 RDU, female: 1.50.12 RDU). Phosphorylation of serine 1177, serine 633 and
dephosphorylation of threonine 495 have been associated with increased NO production in
11 endothelial cells. Renal cortical expression of phosphoSer1177 NOS3 in female SHR was
The T8590C Polymorphism of CYP4A11 and 20-HETE in Essential increased compared to male SHR (male: 0.050.02 RDU, female: 0.110.04 RDU, p0.04).
Hypertension However, phosphoSer633 and phosphoThr495 were comparable from male and female SHR
(phosphoSer633NOS3: male: 0.210.01 RDU, female: 0.220.01 RDU; phosphoThr495NOS3:
Cheryl L Laffer, New York Med College, Valhalla, NY; James V Gainer, Nancy J Brown, male: 0.160.01 RDU, female: 0.160.01 RDU). Basal superoxide production (lucigenin
Michael R Waterman, Jorge H Capdevila, Vanderbilt Univ Med Cntr, Nashville, TN; Michal chemiluminescence in renal cortical homogenates) was higher in male SHR (male: 3.30.7
Laniado-Schwartzman, Alberto Nasjletti, Fernando Elijovich; New York Med College, Valhalla, cpm/g; female: 1.60.2, p0.04). Glomerular nitrotyrosine staining was also greater in male
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NY SHR compared to female SHR. Therefore, these data indicate that female SHR have a greater
capacity to maintain NO bioavailability in the renal cortex than male SHR through enhanced
CYP4A11 synthesizes 20-HETE in humans. The C allele of the T8590C polymorphism which phosphorylation of NOS3 as well as maintaining reduced levels of superoxide; this effect
codes for an enzyme with a 50% reduction in catalytic activity, is linked to essential appears to be unrelated to blood pressure.
hypertension (EH, population odds ratios of 1.232.31). We have previously shown that salt and
furosemide increase, whereas hyperinsulinemia reduces urine 20-HETE in EH. We now
investigated the effect of C on 20-HETE in 32 hypertensive subjects (18 black, 14 white) 14
genotyped for T8590C and phenotyped for insulin sensitivity, salt-sensitivity of blood pressure 17b-Estradiol (e2)-Mediated Protection from Renal Injury is Associated
(BP), and responses of renin, aldosterone, endothelin and catecholamines to salt-loading and with Angiotensin Converting Enzyme 2 (ace2) Up Regulation
salt-depletion. Genotypes were: 13 TT, 17 CT and 2 CC. Frequency of C was 33% and higher
in blacks (39%) than in whites (25%) as previously reported. C carriers had higher diastolic BP Hong Ji, Georgetown Univ Schl Of Med, Washington, DC; Carlo Pesce, Stefano Menini, Univ
(943 mmHg vs 862, p0.05) and non-significantly greater waist-to-hip ratios, lower of Genova, Genova, Italy; Xie Wu, Wei Zheng, Kathryn Sandberg; Georgetown Univ Schl Of
plasma renin, and blunted aldosterone responses to salt, compared to TT. The slope of the Med, Washington, DC
pressure-natriuresis curves (C1.530.02 radians, TT1.490.02), insulin sensitivity index
(HOMA model, C0.1540.020 ml*L*U-1*mmol-1, TT0.1460.025), glomerular filtration The newly discovered member of the renin angiotensin system, angiotensin converting enzyme
rate and salt-balance were similar between groups. However, fractional excretions of Na and 2 (ACE2), exhibits cardioprotective effects through its conversion of angiotensin II (Ang II) to
K in response to furosemide (1.90.2% vs 2.30.2, p0.06 and 11.30,6% vs 14.01.3, Ang-(17). Objective: To investigate whether ACE2 contributes to the clinical observation that
p0.05) were lower in C than in TT. Urine 20-HETE was similar between C (1.590.29 g/hr) chronic renal disease from several etiologies progresses at a slower rate in women compared
and TT (1.600.41). However, both insulin and the C-allele significantly and independently to men, we determined if E2 regulates the activity of renal ACE2 in the renal wrap model of
blunted the 20-HETE response to salt-loading (20-HETEsalt-load2.36 - 0.054xInsulin - hypertension (RW). Methods: Sprague Dawley rats (n6 8/group) were divided into 2
0.49xC-allele, F11.2, p0.001). From this multivariate model it can be calculated that the sham-operated control groups: male (Sham-M) and female (Sham-F) and 4 RW groups: RW-M,
C allele and a 9 U/ml increase in serum insulin are equipotent in determining a 0.5 g/hr RW-F, RW-OVX and RW-OVX-E2 (0.24 mg E2/60 day pellet). After 6 wk on a high sodium (4%)
lesser response of urine 20-HETE to salt. Our data suggest that C influences 20-HETE diet, the RW kidney was removed for determination of renal pathology by morphometry. In
responses to salt and 20-HETE-mediated, furosemide-induced natriuresis and kaliuresis. addition, renal cortical expression of ACE2 mRNA and protein were determined by real-time
However, allele-based analysis does not reveal relationships with salt-sensitivity of BP. This will PCR, expressed as fold increase over RW-F (Fold), and Western blot, expressed in arbitrary
likely require enough homozygous CCs for genotype-based analysis, as suggested by units normalized to -actin (AU); significant differences were defined as *p0.05. Results: In
population studies in which odds ratios for hypertension are higher in CC homozygous subjects the RW kidney, ovariectomy reduced the expression of ACE2 mRNA [Fold: RW-OVX, 0.26
than in C allele carriers. 0.03* vs RW-F, 1.00 0.17 or RW-OVXE2, 1.07 0.3] and protein [AU: RW-OVX, 35.9
4.2* vs RW-OVXE2, 82.8 17], while E2 replacement prevented these effects. Compared to
ovariectomized females (RW-OVX) and male (RW-M) rats, estrogen replete females (RW-F &
RW-OVXE2) also exhibited less renal injury including tubulointerstitial fibrosis, glomeruloscle-
12 rosis, and mean glomerular volume (MGV) [MGV (m3 x 106): RW-M, 2.25 0.16* vs RW-F,
Characterization of Blood Pressure and Protein Excretion in Chromosome 1.25 0.06; RW-OVX, 2.02 0.11* vs RW-OVXE2, 1.33 0.03 or RW-F]. Conclusions:
These results demonstrate that E2-mediated protection from renal damage induced by RW
18 Congenic Strains of Dahl S Rats hypertension is associated with E2-mediated up regulation of renal ACE2, suggesting that
females are protected from progressive renal disease by increased metabolism of Ang II to
Carol P Moreno Quinn, Roberta Rogge, Mary L Kaldunski, Bernardo Lopez, Jozef Lazar,
Ang-(17).
Allen W Cowley, Jr, Howard J Jacob, Howard J Jacob; Med College of Wisconsin,
Milwaukee, WI

The aim of the present study was to identify the region within Chr 18 involved in the 15
development of salt-induced hypertension in the Dahl salt-sensitive (SS) rat. Previous linkage Ovariectomy is Protective Against Salt-Induced Renal Injury in the Older
and chromosomal substitution studies suggest the presence of a locus for salt-sensitive Female mRen2.Lewis Strain
hypertension in chromosome 18. Consomic SS-18BN rats were backcrossed to the SS rat, and
the offspring intercrossed to generate a series of overlapping congenic strains covering the Liliya M Yamaleyeva, Brian M Westwood, Leanne Groban, Mark C Chappell; Wake Forest
entire length of chromosome 18. Blood pressure and protein excretion was measured by Univ Health Sciences, Winston-Salem, NC
telemetry in male rats during low salt and three weeks of high salt diet (8% NaCl) in parental
(SS and SS-18BN) and congenic strains. Mean arterial pressure (MAP) did not differ significantly Estrogen depletion (ovariectomy) at an early age increases blood pressure and exacerbates
between any of the strains studied during low salt diet (ranging from 110 to 115 mmHg). After salt-dependent hypertension and renal injury in the congenic mRen2.Lewis strain. The loss of
three weeks of high salt, SS rats developed hypertension (MAP 16710 mmHg), while in the estrogen in mRen2.Lewis rats is associated with impaired endothelial nitric oxide synthase
SS-18BN consomics the development of hypertension was significantly attenuated (MAP 1405 expression in the kidney, as well as enhanced expression of Ang II, aldosterone and ACE, which
mmHg). Protein excretion was also significantly higher in the SS than in the SS-18BN consomic is reversed by estrogen replacement. Although these and other studies clearly support the
rats. Initial characterization of the overlapping congenic strains suggests that there is a 17 Mbp protective effects of estrogen, recent clinical trials in older females do not substantiate this role.
region in chromosome 18 that provides significant protection from the effects of high salt diet Therefore, the current study assessed the outcome of estrogen depletion in older mRen2.Lewis
in the SS rats. We also phenotyped consomic BN-18SS rats, in which choromosome 18 from the rats subjected to a high salt diet. Heterozygous intact or ovariectomized (OVX, 15 weeks of age)
hypertensive SS rat was introgressed onto the genetic background of the normotensive Brown mRen2.Lewis were aged to 60 weeks and then placed on a high salt (HS, 4% sodium) diet for
Norway (BN) rat, and found that this reverse consomic strain had no increased blood pressure 4 weeks. The systolic blood pressures were similar between groups [intact: 182 7 versus
sensitivity to a high salt diet. These results suggest that there is a region in chromosome 18 OVX: 169 6 mmHg, p0.22] following the 4 week diet; however, the HS diet significantly
that confers protection to the development of salt-sensitive hypertension but is not enough by increased proteinuria in the intact versus OVX rats [intact: 31.3 7; OVX: 1.8 0.3 mg/mg
itself to increase susceptibility to hypertension, probably needing the additional contribution of creatinine/kg body weight, p0.01, n6]. Creatinine clearance (GFRc) tended to decrease in
other genes. intact rats maintained on the HS diet relative to the high salt OVX mRen2.Lewis rats. Both the
CHBPR ConferenceOral Presentations e29
mean kidney-to-body weight (MK/BW) and left ventricle to BW (LV/BW) ratios were significantly (MA), 15 month old middle-aged rats that were ovariectomized at 4 months of age (ovx), and
greater in the intact HS group [intact MK/BW: 3.7 0.2 versus OVX: 2.5 0.04 mg/gm, 15 month old middle-aged rats that were ovariectomized at 4 months of age and supplemented
p0.01, n6 and intact LV/BW: 2.8 0.1 versus OVX: 2.1 0.1 mg/gm, p0.01, n6]. In with estrogen (E; n6 each group). LV levels of extracellular matrix (ECM) proteins, matrix
summary, ovariectomy of adult mRen2.Lewis rats prevents proteinuria, renal and cardiac metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs) were evaluated
hypertrophy as well as maintains GFRc during the course of a HS diet. We conclude that in older by immunoblotting, with densitometry values normalized to actin. Results. LV mass increased
female hypertensive mRen2.Lewis rats with increased sodium intake, ovarian hormones may from 6976 mg in Y to 125731 mg in MA and 119925 mg in ovx (p0.05 Y vs MA and
have deleterious actions within the kidney. ovx). LV mass in the E group was not significantly different from Y. Fibronectin (Fn) levels
increased from 1.390.03 units in Y to 1.670.04 units in MA (p0.05). Collagen III and
collagen IV levels were also elevated in MA, indicating increased fibrosis. Multiple MMPs/TIMPs
16 increased in MA, including MMP-3, MMP-7, MMP-9, MMP-13, MMP-14, and each TIMP (-1, -2,
Gender Differences in Cortical and Medullary Function in Adults Rats with -3, and -4). Ovx showed similar increases in fibrosis and MMP/TIMP profiles. The increase in
a Reduction in Ang II during Nephrogenic Period collagen levels was attenuated in the E group. MMP-12 increased from 1.190.03 units in MA
to 1.360.1 units in ovx (p0.05) and the 150 kD Fn fragment increased from 1.200.02
Analia S Loria, Virginia Reverte, Francisco Salazar, Fara Saez, M T Llinas, F. J Salazar; Dept units in MA to 1.270.03 units in ovx (p0.05). Compared to MA levels, MMP-2, MMP-8, and
of Physiology, Sch of Medicine, Univ of Murcia, Murcia, Spain TIMP-4 increased in E, as did the 75 kD fragment of collagen III. Compared to ovx, MMP-8
increased 18025% and the collagen III fragment increased 1135% in the E group.
A recent study of our group has demonstrated that there are important gender differences in Conclusion. Hypertension superimposed on aging stimulated ECM turnover through increased
renal structural response to the reduction of angiotensin II (Ang II) during the nephrogenic MMP/TIMP production and ECM degradation. Estrogen replacement attenuated the hypertro-
period. It was found that renal cortex was more affected in males than in females treated with phic response and increased collagenolysis, which may serve as one mechanism for the
an AT1 Ang II receptor antagonist (ARAII) during the first two weeks of life and that only treated protective effects of estrogen.
males had an important papillary atrophy. This study was designed to test the hypothesis that
there are gender differences in the cortical and medullary function in adult rats in which Ang
II effects were reduced during the nephrogenic period. Newborn SD rats were treated with
vehicle or an ARAII (L-158.809, 7 mg/kg/day) during the first 14 postnatal days. Systolic arterial
pressure, renal clearances, proteinuria and the urinary concentrating ability in response to
dehydration was examined in rats at 12 months of age (n8 in each group). Arterial pressure
was greater (P0.05) in treated males (134 0.9 mmHg) and treated females (131 0.5 19
mmHg) than in control rats (cr) (115 0.3 mmHg in both sexes). Urinary protein excretion MicroRNAs in the Kidney: Regional Distribution and Physiological
(mg/ml/24hrs) increased (P0.05) in treated males and females, but the elevation was greater
(P0.05) in treated males (264 24 vs. 101 10 in cr) than in treated females (95 19
Implications
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017

vs. 40 3 in cr). As compared to the values found in the control group, basal urine osmolality
Mingyu Liang; Med College of Wisconsin, Milwaukee, WI
was lower in male than in female treated rats. In response to a prolonged dehydration period,
the increment in urinary osmolality was significantly greater (P0.05) in female (706 92
MicroRNAs are a class of small regulatory RNA, the discovery of which has been hailed as one
mOsm/kg) than in male treated rats (386 54 mOsm/kg). Changes in tubular solute free water
of the most significant breakthroughs in biology in recent years. Mature microRNAs are
reabsorption (TcH2O) (ml/24 hr/gr kidney w.) in response to dehydration were not affected in
approximately 20 nucleotides long, and can bind to the 3 untranslated region of their target
female treated rats with respect to female control rats. However, the decrease of TcH2O in
mRNAs and suppress protein translation. A mammalian genome has at least a few hundred
response to dehydration was lower (P0.05) in male treated rats (8.0 0.8 to 3.9 0.5) than
genes that specifically encode microRNAs. The known mammalian microRNAs, which are
in male control rats (12.8 1.8 to 7.0 0.9). In summary, the results of this study present
highly conserved among mammalian species, have been predicted to regulate the protein
new evidences showing that the reduction of Ang II during the nephrogenic period elicits the
development of hypertension in males and females. This reduction alters more significantly expression of thousands of genes since microRNAs only need to match their targets partially.
renal cortical and medullary function in adult males than in adult females. The relevance of specific microRNAs to complex mammalian physiology, however, is largely
unknown. To begin analyzing the physiological role of microRNAs in mammalian species, we
examined the expression profile of microRNAs in the renal cortex and the renal medulla of male
17 Sprague-Dawley rats (n6). We constructed a microRNA microarray containing probes for
nearly 400 microRNAs that have been identified in human, mouse, or rat. MicroRNAs were
Phytoestrogens Inhibit Human Aortic Smooth Muscle Cell Growth and
isolated from the renal cortex and the renal medulla using size-based fractionation. A 3
ERK1/2 Expression via PPAR-, but not Estrogen, Receptors extension method was used to label microRNAs, which were then hybridized to microRNA
microarrays. Approximately 100 microRNAs were detectable in each region of the kidney.
Raghvendra K Dubey, Univ Hosp Zurich, Zurich, Switzerland; Edwin K Jackson, Delbert G
Statistical analysis identified 11 and 9 microRNAs as preferentially expressed in the renal cortex
Gillespie, Univ of Pittsburgh Med Cntr, Pittsburgh, PA; Bruno Imthurn, Marinella Rosselli;
and the renal medulla, respectively, four of which were further validated using a modified
Univ Hosp Zurich, Zurich, Switzerland
real-time PCR method. Computationally predicted target genes for the differentially expressed
microRNAs include transporters, transcriptional regulators, and cell cycle regulators, several of
Plant-dervied estrogens (phytoestrogens), such as genistein (abundant in soybeans), may
which appeared to be consistent with known physiological characteristics of the renal cortex
induce cardioprotective effects by inhibiting smooth muscle cell (SMC) growth and neointima
and the renal medulla. The present study was the first to have systematically analyzed
formation. The prevailing view is that the biological effects of phytoestrogens are mediated by
microRNAs in mammalian kidneys. The results suggest that microRNAs might play an important
estrogen receptors (ERs). Our recent findings that the effects of estradiol are ER independent
role in controlling renal function through regulating the expression of their target genes.
and mediated via its metabolite (methoxyestradiol) suggest that the actions of phytoestrogens
may also be due to alternative mechanisms. Because PPAR- is a nuclear receptor that can
interact with steroids, we investigated the role of PPAR- in mediating the antimitogenic
actions of phytoestrogens in human aortic SMCs. In cultured human aortic SMCs, treatment
with 0.1100M genistein or estradiol inhibited fetal calf serum (2.5%)-induced DNA synthesis
(3H-thymidine incorporation), cell proliferation (change in cell number), collagen synthesis
(3H-proline incorporation), cell migration (modified Boydens chamber) and ERK 1/2 expession.
All of the inhibitory effects of genistein, but not estradiol, were completely blocked by GW9662 20
(2M, PPAR- antagonist). For example, genistein (50M) inhibited cell proliferation by Collecting Duct-Specific Knockout of PPAR Impairs Sodium Retaining
724%, and GW9662 reduced this to 71%. Treatment of SMCs with rosiglitazone (PPAR- Ability and Reduces Blood Pressure during Sodium Depletion
agonist) inhibited SMC growth, and this effect also was blocked by GW9662. Importantly,
genistein was more potent than rosiglitazone with regard to inhibiting SMC growth (50M Tianxin Yang, Aihua Zhang, Zhanjun Jia, Hui Zhang; Univ of Utah, Salt Lake City, UT
inhibited SMC growth by 72% and 30%, respectively). The ER-antagonist ICI182780 (10M)
did not attenuate the inhibitor effects of either estradiol or genistein. Moreover, inhibitors of We have recently created mice with collecting duct (CD)-specific disruption of PPAR (termed
CYP450 and COMT (inhibit metabolism of estradiol to methoxyestradiol) blocked the antimi- CD PPAR KO) by crossing PPARf/f mice with AQP2-Cre mice and the KO mice were resistant
togenic actions of estradiol, but not genistein. Similar to genistein, the inhibitory effects of other to thiazolidinedione-induced fluid retention, revealing a novel role of PPAR in regulation of
phytoestrogens equol and daidzein on SMC growth were abrogated by GW9662. In conclusion, distal tubular fluid reabsorption (Zhang et al. PNAS 104:9406 11, 2005). The present study
our findings suggest that some phytoestrogens exert inhibitory effects on SMCs via activation examined a potential physiological role of CD PPAR in the setting salt depletion. We used
of PPAR-. Regardless of whether this is a direct or indirect effect, activation of PPAR- by telemetry to monitor daily mean arterial blood pressure (MAP). During one-week low salt (LS)
phytoestrogens may confer cardiovascular protection. diet PPARf/f mice were able to maintain constant MAP while CD PPAR KO mice exhibited a
significant reduction of MAP (105.0 3.1 vs. 94.6 2.4 mmHg, on day 7, n7, p0.05).
After switching from low to normal salt diet, MAP in CD PPAR KO mice returned to normal.
18 Sodium balance studies showed that CD PPAR KO mice had impaired ability to effectively
Estrogen Effects on Left Ventricular Hypertrophy and Matrix reduce urinary sodium excretion during LS intake. This was associated with exaggerated
Metalloproteinase Profiles in Dahl Salt-Induced Hypertension plasma renin and plasma aldosterone responses to LS. In response to LS, PPARf/f mice
exhibited a 2.5-fold increase in the abundance of the subunit of the epithelial sodium channel
Qiuxia Dai, Teresa Craig, Carmen Hinojosa-Laborde, Merry L Lindsey; Univ of Texas Health (-ENaC) in the kidney as assessed by immunoblotting and a nearly complete trafficking of
Science Cntr, San Antonio, TX -ENaC to the apical membrane of the CD by immunohistochemistry, that were both abolished
in CD PPAR KO mice. LS treatment induced more than 10 fold increases in urinary excretion
Background. Female Dahl salt-sensitive (DS) rats fed a low salt diet develop hypertension with of the endogenous ligand of PPAR 15d-PGJ2 in both mouse strains. In primary cultures of wild
age. DS rats ovariectomized at 4 months of age show accelerated hypertension, which is type CD cells, 1 M rosiglitazone treatment for 24 hours induced a 2-fold increase in Sgk1
attenuated with estrogen replacement. While acute pressure overload is known to induce mRNA as assessed by real time RT-PCR that was significantly suppressed in the PPAR KO
structural and functional changes in the left ventricle (LV), the effects of chronic hypertension cells. In summary, this study has characterized 15d-PGJ2/PPAR/Sgk1/-ENaC pathway in the
on LV size and structure have not been examined in the DS rat model. Methods. Four groups distal nephron that appears to be important for maintenance of sodium balance and blood
of DS rats were examined: young intact control rats (Y), 15 month old middle-aged intact rats pressure during sodium depletion.
e30 Hypertension Vol 48, No 4 October 2006

21 and feature the metabolic syndrome. We studied these transgenic and non-transgenic control
Enhanced Renal Interstitial Fluid ATP and the Tubuloglomerular Feedback rats (SD) under standard diet for 22 weeks. Body weight (BW) gain was similar in both groups
Mechanism in Dahl Salt-Sensitive Hypertensive Rats until the age of 7 weeks, although energy expenditure (EE) and lipid oxidation (LOX) were
greater in TGR(hREN) vs. SD. Thereafter, BW gain was significantly greater in TGR(hREN) vs.
Akira Nishiyama, Matlubur Rahman, Youichi Abe, Masakazu Kohno, Kagawa Univ Med Sch, SD. At age 22 weeks, BW was 5709 g in TGR(hREN) and 49720 g in SD. Food intake was
Kagawa, Japan; L G Navar; Tulane Univ Health Sciences Cntr, New Orleans, LA significantly higher in TGR(hREN) than SD, but EE and LOX were similar in both groups.
MRI-determined body composition analysis showed a significantly higher body fat mass in the
Participation of renal interstitial fluid (RIF) ATP as a mediator of the tubuloglomerular feedback TGR(hREN) (886 g) vs. SD (553 g) rats. Additionally, TGR(hREN) had increased serum
(TGF) mechanism has been indicated in recent studies. Here, we investigated the possible triglyceride levels (1088 vs. 665 mg/dl) and impaired glucose tolerance tests, compared
contributions of RIF ATP and TGF mechanism to the development of salt-dependent with SD. Telemetric mean arterial blood pressure was not different in TGR(hREN) and SD
hypertension. Dahl salt-sensitive (DS) rats were maintained on a low (L: 0.3% NaCl) or high salt (1042 and 1032 mm Hg). Chronic ACE inhibitor treatment did not influence BW,
(H: 8% NaCl) diet for 4 weeks. Using an intravital tapered-tip lens-probe videomicroscopy triglycerides, or glucose tolerance impairment. When pair-fed, TGR(hREN) rats were restricted
system, the superficial afferent arteriolar diameter (AAD) was measured before and during to the caloric intake of SD rats, the developed a lower BW than SD. In vitro, we found that
enhanced TGF activity with acetazolamide (2 mg/kg, bolus 4 mg/kg/h, infusion, i.v.) in neither renin nor renin-inhibitor treatment had any influence on adipogenesis of human
anesthetized rats. RIF ATP levels were measured by a microdialysis method. P0.05 was preadipocytes. We conclude that renin initiates a process leading to increased appetite, obesity
considered statistically significant. Systolic blood pressure in DS/L (n28) and DS/H (n36) and metabolic alterations independent of blood pressure. Human renin does not seem to have
rats was 1342 and 2178 mmHg, respectively. Compared to DS/L rats (9.90.8 m), DS/H a direct effect on adipogenesis.
rats showed smaller basal AAD (7.70.9 m). In DS/L rats, acetazolamide significantly
decreased AAD (8.70.7 m) without changing the efferent arteriolar diameter. Inhibition of
TGF activity with furosemide (1 mg/kg, bolus 4 mg/kg/h, infusion, i.v.) reversed the
acetazolamide-induced afferent arteriolar vasoconstriction (9.60.8 m). In DS/H rats,
acetazolamide did not alter basal AAD (7.6 0.9 m), whereas the addition of furosemide
significantly increased AAD (8.51.0 m). Basal RIF ATP levels were much higher in DS/H rats
(4.30.5 nmol/L) than DS/L rats (1.20.4 nmol/L). Acetazolamide increased RIF ATP levels in 24
DS/L rats (2.20.7 nmol/L), whereas ATP levels were not altered by acetazolamide in DS/H Impaired Renal Vasodilation in SHR Fed a High Fat Diet Precedes Changes
rats (3.80.8 nmol/L). Treatment with suramin (20 mg/kg/day, i.p.), a non-selective P2 in Insulin Sensitivity
receptor antagonist, markedly attenuated the development of hypertension in DS/H rats
(1383 mmHg, n25). Suramin also significantly increased basal AAD (10.60.9 m) in Sarah Knight, Ahmed El-Marakby, John D Imig; Med College of Georgia, Augusta, GA
DS/H rats. However, AAD did not change during the administration of acetazolamide and
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017

furosemide in suramin-treated DS/H rats. These results suggest that augmented TGF activity Obesity, hypertension and insulin resistance have been identified as cardiovascular risk factors
associated with increases in RIF ATP levels contributes to the increased afferent arteriolar tone which are believed to contribute to the progression of end-stage renal disease. To examine the
and the development of salt-dependent hypertension. mechanism by which a HF diet and hypertension contribute to endothelial dysfunction, 8 week
old male spontaneously hypertensive rats (SHR) were fed either a high fat (HF, 36% fat) or a
normal fat (NF, 7% fat) diet for 6 weeks (n6). Systolic blood pressure increased in a similar
22 manner in NF (from 163 4 mmHg to 219 5 mmHg) and HF diet fed rats (from 166 3
Localization of the Renin Receptor in Renal Tubules and its Upregulated in mmHg to 217 4 mmHg). In the NF group body weight increased from 218 7 g to 288
Diabetes 6 g, whereas the HF groups displayed a 15% greater increase, from 217 5 g to 319 9 g
after 6 weeks. Non-fasting blood glucose levels were 13% higher in the HF group than the NF
Chun Xue, Jiqian Huang, John Gildea, Univ of Virginia, Charlottesville, VA; Genevieve group at 109 3 mg/dL and 95 2 mg/dL respectively (P0.05). Using the euglycemic
Nyugen, INSERM Tenon Hopital, Paris, France; Helmy M Siragy; Univ of Virginia, clamp method, glucose infusion rates of 40 2 mg/kg/min in the NF group and 39 3
Charlottesville, VA mg/kg/min in the HF group were required to maintain blood glucose levels of 125 mg/dL,
indicating comparable insulin sensitivity (n3 4). We measured renal afferent arteriole
Recent studies demonstrated presence of the renin receptor (RR) in the glomerular mesangium vascular reactivity to acetylcholine (ACh) (n6). Mean baseline diameter averaged 23 2 m
and the subendothelial layer of the renal arteries. In this study we hypothesized that in addition and 26 5 m in the NF and HF groups respectively. The relaxation of pre-constricted
to the presence of RR in glomerular mesangium, it is also present in renal tubules and is arterioles in response to doses of 1.0x10-8 to 1.0x10-5 ACh was reduced in the HF compared
upregulated in diabetes via angiotensin AT1 receptor (AT1R) and oxidative stress. Using real with the NF group. Maximum relaxation reached 84% of baseline diameter in the NF but only
time PCR, western blot analysis and immunostaining, we studied renal RR expression at 6 57% of baseline in the HF group (P0.05). In order to elucidate a possible mechanism for the
weeks after induction of diabetes in rats injected with streptozotocin (65 mg/kg ip) and in reduced renal vascular relaxation to ACh we measured the cortical expression of CYP2C23, an
response to 1 week treatment with the AT1R blocker valsartan (10mg/kg/d) or the NADPH enzyme responsible for the production of vasodilatory EETs, by western blot in rats fed a HF
oxidase inhibitor DPI (0.5mg/kg/d). Both RR mRNA and protein expressions were present in or NF diet. We found that cortical CYP2C23 protein expression was reduced in the HF group
renal glomeruli and tubules in normal rats. Diabetes significantly upregulated RR expression in with levels of 20 4 densitometric units (DU) compared to 29 1 DU in the NF group (n4).
the kidney (Figure A& B). Valsartan or DPI treatments reduced the RR expression in diabetic rats Our results indicate that a 6 week HF diet with hypertension impairs the renal vascular
(Figure A& B). To confirm the presence of the RR expression in renal glomerular mesangium response to ACh, in the absence of insulin resistance or a change in blood pressure and the
and tubules, we used Western blot analysis to detect RR protein in isolated renal mesangial and decrease in CYP2C23 metabolites could contribute to the impaired renal vasorelaxation.
proximal tubular cells (Figure C). Similarly, immunostaining localized the RR in glomeruli and
tubules. These results demonstrate that RR is present in renal glomeruli and tubules and
upregulated in diabetes. AT1R and oxidative stress upregulate RR expression.

25
Glucose Reduces Renal Medullary Circulation by Induction of Oxidative
Stress in Renal Medulla

Chun-hua Jin, Takefumi Mori, Susumu Ogawa, Satoshi Endo, Yoshimi Yoneki, Kazuhiro
Nako, Sadayoshi Ito; Div of Nephrology, Endocrinology and Vascular Medicine, Tohoku Univ
Graduate Sch of Medicine, Sendai, Japan

Although high blood pressure is often observed in diabetes and metabolic syndrome, little is
known whether renal medullary circulation is involved. Previous studies have shown that
glucose increase superoxide production in medullary thick ascending limb. Studies were
designed to determine the role of glucose and insulin in the regulation of renal medullary
circulation. 50% glucose solution was infused from femoral vein at a rate of 0.5ml/h in
anesthetized male Sprague-Dawley rats and blood pressure was monitored with the catheter
implanted from the femoral artery. Local renal blood flow was measured with implanted optical
fiber attached to the laser-Doppler flowmetry. Drugs were infused from catheter implanted to
the border of inner and outer medulla to determine the local renal medullary effect. Intravenous
23 infusion of glucose resulted in 215 mg/dl increase of blood glucose and 124% (n7)
Obesity in Human Renin Transgenic Rats - A Novel Role for Renin? reduction of medullary blood flow (MBF) without significant reduction on mean arterial
pressure. Interstitial infusion of 50% glucose solution at a rate of 0.5 ml/h also reduced MBF
Petra Gratze, Ralf Dechend, Michael Boschmann, Jeanette Malchow, Stefan Engeli, Jurgen by 165% (n6) without reduction of blood glucose. However, the level of MBF was remained
Janke, HELIOS, Franz-Volhard Clinic, Charite, Berlin, Germany; Jochen Springer, Dept of low when 20mU/kg/min. of insulin were additionally infused to medullary interstitial space even
Cardiology, Charite, Berlin, Germany; Ralph Plehm, Max-Delbruck Cntr, Berlin, Germany; with 358 mg/dl reduction of blood glucose. Renal medullary infusion of superoxide scavenger
Susanne Klaus, German Institute of Human Nutrition, Potsdam, Germany; Friedrich C Luft, Tiron (90g/kg/min., n7) and sodium-glucose transporter (SGLT) inhibitor phloridzin
HELIOS, Franz-Volhard Clinic, Charite, Berlin, Germany; Dominik N Muller; Max-Delbruck (6.9g/kg/min., n5) abolished the reduction of MBF by intravenous infusion of glucose. We
Cntr, Berlin, Germany conclude that glucose but not insulin reduces medullary blood flow with induction of oxidative
stress, and SGLT is involved in the glucose transport for reduction of MBF. We conclude that
Renin is known to function as a protease initiating angiotensin (Ang) II formation. However, we glucose induced reduction of MBF could involve in the mechanism of high blood pressure in
found that transgenic rats over-expressing the human renin gene (TGR(hREN)) develop obesity diabetes and metabolic syndrome.
CHBPR ConferenceOral Presentations e31
26 constriction in MA but not MV; the effects of yohimbine were impaired in tissues from
Dynamic Renal Autoregulation: Abnormal in Diabetes DOCA-salt rats. Cocaine (10 M) which blocks NE uptake increased the amplitude and duration
of NE oxidation currents and constrictions in MA but not MV; the effect of cocaine was larger
Gerald DiBona, Univ. Ia. College of Medicine, Iowa City, IA; Tracy D Bell, Michael W Brands; in MA from DOCA-salt rats. The latency to onset and duration of constriction in MA were shorter
Med College of Georgia, Augusta, GA than MV but the rate of constriction and rise time of NE release were faster in MV from control
rats; there were no differences in the rise time of constriction or NE oxidation currents in MA
Within 24 hours of induction of diabetes mellitus (DM, stretptozotocin) in conscious rats, arterial and MV from DOCA-salt rats. These data indicate that there are fundamental differences in the
pressure (AP) and renal blood flow (RBF) are increased, achieving levels of 10% and 22% dynamics of sympathetic neurotransmission to MA and MV. NE uptake and prejunctional 2
respectively above control at DM day 7 (submitted). In addition, dynamic RBF autoregulation (AP autoreceptors play a more prominent role in modulating NE disposition in MA compared to MV.
to RBF transfer function) was impaired on DM day 1 with positive gain values at both Furthermore, DOCA-salt hypertension is associated with an impairment of autoreceptor
tubuloglomerular feedback (TGF, 0.02 0.05 Hz) and myogenic (myo, 0.1 0.3 Hz) frequencies. function and upregulation of NE uptake in the neuroeffector junction of MA. These changes
To identify whether there was a change in the RBF frequencies and amplitudes used by TGF contribute to the hemodynamic disturbances occurring in DOCA-salt hypertension. #
and myo during evolution from Control to DM, the dominant frequency in both the TGF (Ftgf)
and myo (Fmyo) ranges was identified and the amplitude at that dominant frequency (Atgf, Atgf)
was extracted over 1000 seconds during both Control and DM periods using a time-frequency
representation approach (Wang et al, Ann Biomed Eng, 2006). *P0.01 DM vs Control, 29
P0.01 Atgf vs Amyo. The dominant frequencies chosen for the TGF (Ftgf) and myo (Fmyo)
components of dynamic RBF autoregulation were not different between Control and DM.
Renal Denervation Does not Abolish Sustained Baroreflex-Mediated
However, the RBF amplitude at both the dominant frequencies for TGF (Atgf) and myo (Amyo) Reductions in Arterial Pressure
was significantly increased in DM compared to Control. Atgf was greater than Amyo during both
Control and DM. As increased RBF amplitude at the TGF and myo frequencies indicates less Thomas E Lohmeier, Drew A Hildebrandt, Terry M Dwyer, Austin M Barrett, Univ. of
attenuation by the renal vasculature of the corresponding AP amplitude at the same Mississippi Med Cntr, Jackson, MS; Eric D Irwin, North Memorial Med Cntr, Robbinsdale,
frequencies, this is consistent with impairment of both the TGF and myo mechanisms for MN; Martin A Rossing, Robert S Kieval; CVRx, Inc., Maple Grove, MN
dynamic RBF autoregulation in DM. That these changes are apparent on DM day 1, prior to any
renal structural damage, suggests that this is a functional alteration related to the altered We previously demonstrated that prolonged bilateral electrical stimulation of the carotid sinus
metabolic state. activates the baroreflex producing sustained reductions in plasma norepinephrine concentra-
tion (NE), mean arterial pressure (MAP), and heart rate (HR) in normotensive and hypertensive
Control DM dogs. Prolonged baroreflex activation (PBA) also decreases renal sympathetic nerve activity and
increases renal excretory function, responses expected to lead to long-term reductions in MAP.
Ftgf, Hz 0.0330.001 0.0340.001
The goal of this study was to determine whether PBA mediated reductions in MAP are
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Fmyo, Hz 0.1740.001 0.1680.001


Atgf, amplitude 0.1010.006 0.3960.007* dependent upon renal innervation. Six dogs were chronically instrumented to allow PBA and
Amyo, amplitude 0.0320.001 0.0900.001* continuous recordings (24h/day) of MAP and HR. Sodium intake was maintained at 60
mmol/day. After control measurements, the animals underwent 7 days of PBA. On day 1,
sodium excretion was reduced by 20 mmol while MAP (control 953 mmHg) and HR
(control653 bpm) decreased to 813 mmHg and 552 bpm, respectively. On subsequent
27 days of PBA, sodium balance was achieved despite a sustained fall in MAP and HR (day 7
The Splanchnic Circulation as a Critical Target for Chronic Low Dose 794 mmHg and 552 bpm, respectively). Plasma NE concentration decreased 50%
Angiotensin II Hypertension during PBA (control 9613 pg/ml). After termination of PBA, there was a natriuresis on day
8, followed by sodium balance as MAP and HR returned to control levels during 7 days of
Andrew J King, Melissa W Li, Gregory D Fink; Michigan State Univ, East Lansing, MI recovery. Two weeks after bilateral denervation, the above experiment was repeated. MAP, HR,
and NE were 914 mmHg, 704 bpm, and 1008 pg/ml before PBA. During 7 days of PBA,
We have shown that chronic low dose angiotensin II (AngII), only when administered in reductions in MAP, HR, and NE were equivalent to those observed when the renal nerves were
combination with a high salt diet, engages the sympathetic nervous system (SNS) to increase intact. Tissue levels of NE were reduced 100-fold in denervated kidneys (control 98780
venomotor tone. We propose this contributes to AngII hypertension by increasing the driving ng/g), confirming denervation. Thus, the renal nerves are not essential for achieving long-term
force for venous return to the heart, resulting in a translocation of blood from the highly reductions in MAP during PBA. As denervated kidneys may be supersensitive to NE, decreased
compliant venous system to the less compliant arterial circulation. In particular the splanchnic NE during PBA may account for increased renal excretory function and attendant reductions in
venous bed, which is richly innervated by the SNS mostly via the celiac ganglion, and is the MAP in the absence of the renal nerves.
most important active capacitance bed in the body, may be an important target for AngII
mediated sympathoactivation. Therefore we hypothesized that celiac ganglionectomy (CGx) will
attenuate the hypertensive response to AngII only in rats fed a high salt diet. To test this, rats
were acclimatized to a 2% or 0.4% NaCl diet for 7 days and then instrumented with a
radiotelemetry blood pressure transmitter at the same time as surgical CGx or sham operation 30
(SHAM). Following 10 days of recovery and 4 days of control, an AngII or saline filled minipump ASIC2-/- Mice: A Genetic Model of Impaired Baroreceptor Activation and
was implanted subcutaneously to deliver AngII (150ng/kg/min) or vehicle for 14 days. During Oxidative Stress-Dependent Neurogenic Hypertension
control CGx reduced MAP in rats on 2% (SHAM 102 1.4, CGx 95 1.7 mmHg, p0.05) and
0.4% NaCl (SHAM 100 1.3, CGx 93 2.3 mmHg, p0.05). CGx markedly attenuated the Rasna Sabharwal, Harald M Stauss, Eric Lazartigues, Carol A Whiteis, Margaret P Price,
hypertensive response to AngII in rats on 2% NaCl (SHAM 145 6.3, CGx 114 7.9 mmHg, Michael J Welsh, Robin L Davisson, Francois M Abboud, Mark W Chapleau; Univ. of Iowa,
p0.05), but had little effect on rats fed 0.4% NaCl (SHAM 114 4.2, CGx 104 4.5 mmHg). Iowa City, IA
CGx was confirmed by measuring norepinephrine levels in splanchnic organs by HPLC. To
investigate the role of the renal nerves and the possibility that CGx was exerting its effects via Acid Sensing Ion Channel 2 (ASIC2) is highly expressed in sensory neurons and brain including
renal denervation (RDx), a separate group of rats fed 2 % NaCl were subjected to the same medulla. Our previous work suggests a role of ASIC2 in baroreceptor activation. Most recently
protocol but received selective bilateral RDx. RDx decreased MAP (SHAM 103 1.7, RDx 97 we reported that ASIC2-/- mice exhibit decreased baroreflex sensitivity (BRS), sympathoexci-
1.9 mmHg, p0.05) during control but had no protective effect (SHAM 128 9.9, RDx 127 tation, and mild hypertension (FASEB J. 20(5):2006). Neuronal oxidative stress contributes to
4.9 mmHg) on the hypertensive response to AngII and appeared to exacerbate the pressor sustained sympathoexcitation in a variety of pathological states. In this study, we tested the
response over the first 5 days. We conclude that CGx attenuates the hypertensive response to hypothesis that oxidative stress mediates the baroreflex/autonomic dysfunction in ASIC2-/-
chronic low dose AngII, only in rats fed a high salt diet, and this suggests the splanchnic mice. Blood pressure (BP) and heart rate (HR) were measured by telemetry in conscious control
circulation is a critical target for AngII induced sympathoactivation. (n7) and ASIC2/ (n8) male mice before and after administration of the antioxidant tempol
(drinking water, 1 mM) for one week. Results are shown in the table. BRS calculated from
spontaneous fluctuations in BP and HR using the sequence method was markedly decreased
28 in ASIC2-/- mice. Cardiac parasympathetic and sympathetic activities as determined from HR
Probing Functional Differences in Sympathetic Input to Mesenteric Arteries responses to methylatropine and propranolol were abolished and enhanced, respectively in
and Veins in Normotensive and DOCA-Salt Hypertensive Rats Using in vitro ASIC2-/- mice. HR and BP (24 hr. avgs.) were significantly elevated. Tempol reversed the
baroreflex/autonomic impairment and normalized HR and BP in ASIC2-/- mice, but had no
Continuous Amperometry and Video Imaging effect in control mice (see Table, *ASIC2-/- vs. control, P0.05; Tempol vs. baseline). We
conclude that oxidative stress causes baroreflex and autonomic dysfunction and mild
Greg M Swain, Jinwoo Park, James J Galligan, Gregory D Fink; Michigan State Univ, East
hypertension in ASIC2-/- mice. The role of baroreceptor sensory vs. central nervous system
Lansing, MI
defects in mediating the functional changes as well as the mechanism linking loss of ASIC2 and
oxidative stress remain to be determined. (NIH HL14388 and AHA#0525745Z)
Arteries and veins make different contributions to overall hemodynamics and there are likely
to be differences in sympathetic transmission to arterial and venous smooth muscle cells in Control Control Tempol
normal and hypertensive subjects. Therefore, we used amperometric methods with diamond or
carbon fiber microelectrodes and video imaging to study neurogenic constrictions and the 24 hr. avg. BP (mmHg) 1093 1083
24 hr. avg. HR (bpm) 54713 53316
kinetics of norepinephrine (NE) release and clearance in perivascular sympathetic neuroeffector BRS (ms/mmHg) 2.400.17 2.270.21
junctions in mesenteric arteries (MA) and veins (MV) in vitro. We used tissues from control and Atropine ( bpm) 15029 9220
DOCA-salt rats. Electrically-evoked constrictions and NE release in MA and MV were blocked Propranolol ( bpm) -8821 -7020
by tetrodotoxin (TTX, 0.3 M) indicating that these responses were neurogenic. NE release and ASIC2-/- ASIC2-/- Tempol
contractile responses were frequency dependent (130 Hz) with maximal responses at 20 Hz. 24 hr. avg. BP (mmHg) 1173* 1083
The frequency-response curves for constriction and NE release in MV were shifted to the left 24 hr. avg. HR (bpm) 5788* 5337
of those for MA. The half maximum stimulation frequency was 1.9 0.2 Hz in veins and this BRS (ms/mmHg) 0.940.07* 2.070.52
Atropine ( bpm) 1512* 1117
value was lower (P 0.05) than in MA (4.8 0.3 Hz). Peak responses were similar in MA and Propranolol ( bpm) -20233* -6411
MV. The 2-adrenergic receptor antagonist, yohimbine (1.0 M), increased NE release and
e32 Hypertension Vol 48, No 4 October 2006

31 unclear. We hypothesized that increased sensitivity of BP to N-arachidonoyldopamine (NADA),


Alpha-Glucosidase Inhibition, a Novel Treatment for Postprandial an endovanilloid metabolized from dopamine, occurs during HS intake. NADA (1, 4, 10 mg/kg,
Hypotension in Autonomic Failure iv) dose-dependently decreased mean arterial pressure (MAP) in conscious Wistar male rats fed
a HS diet for 10 days, and its depressor effect was greater in HS vs. normal salt (NS)-treated
Cyndya Shibao, Alfredo Gamboa, Andre Diedrich, Cynthia Dossett, Ginnie Farley, Italo rats (e.g. at 4mg/kg, 16 3 vs. 8 2 mmHg, p0.05, n6 7). NADA (4 mg/kg) induced
Biaggioni; Vanderbilt Univ, Nashville, TN depressor effect was abolished by capsazepine (CAPZ, 3 mg/kg), a selective TRPV1 antagonist,
or CGRP8 37 (1 mg/kg/min), a selective CGRP receptor antagonist (p0.05). Capsaicin (CAP, 10
Postprandial hypotension (PPH), is an important clinical condition associated with syncope, or 30 g/kg, iv), a selective TRPV1 receptor agonist, or CGRP (1 or 5 g/kg, iv),
falls, angina and cerebrovascular events. Those at most risk are elderly and patients with dose-dependently decreased MAP in HS and NS rats, with a greater effect in the former (CAP,
autonomic dysfunction. Because the enteric glucose availability has been proposed to 11 2 vs. 6 1; 21 3 vs. 12 2; CGRP, 22 3 vs. 12 2; 41 4 vs. 25 3, p0.05).
contribute to the pathophysiology of postprandial hypotension (PPH), we hypothesized that CGRP levels in plasma were higher in HS compared to NS rats (58.7 5.7 vs. 40.3 4.6
acarbose, an alpha-glucosidases inhibitor, that decrease the glucose absorption, and the pg/ml, p0.05), and that NADA (4 mg/kg) caused a greater increase in plasma CGRP levels in
postprandial peak of insulin, would improve PPH. The effect of 100 mg of acarbose was studied HS compared to NS rats (84.2 7.2 vs. 55.3 4.6 pg/ml, p0.05). TRPV1 receptor protein
in 7 patients with pure autonomic failure (5 females, 2 males, age 657 years, BMI 24.2 expression in the mesenteric arteries was increased in HS compared to NS-treated rats
4.9 Kg/m2) in a double blind, non-randomized, crossover study. All participants had severe PPH, (0.81 0.06 vs. 0.59 0.04, p0.05). Our data show that 1) HS upregulates mesenteric
defined as a decrease in systolic blood pressure of at least 20 mm Hg within 2 hours of meal TRPV1 expression; 2) HS increases sensitivity of BP to NADA; 3) HS increases basal and
ingestion. Baseline measurements were taken for 30 minutes every 5 minutes on the supine NADA-induced release of CGRP; and 4) the enhanced depressor effect induced by NADA during
position. Acarbose (or placebo) was administered 20 minutes before ingestion of a meal HS intake is blocked by antagonists of TRPV1 or CGRP receptors. These results indicate that
consisting of 423 Kcal. Blood pressure and heart rate were monitored for 90 minutes. NADA may serve as a novel endogenous TRPV1 agonist to prevent salt induced increases in BP
Neurohumoral parameters were measured at baseline, 15, 30, 45, 60 and 90 minutes of meal via enhancing CGRP release.
ingestion. During placebo, the fall in systolic blood pressure (SBP) was 429 mm Hg as
compared to 206 mm Hg with acarbose, P0.05 (Figure). Acarbose significantly reduced
plasma levels of insulin (193 at 60 min compare to 285 pg/ml with placebo, P0.028). We
conclude that acarbose 100 mg successfully improved postprandial hypotension in patients
with severe autonomic failure.

34
Cardiovascular and Sympathetic Responses to Direct Injection of Leptin
into the Arcuate Nucleus of the Hypothalamus
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017

Kamal Rahmouni, Donald A Morgan; Univ of Iowa, Iowa City, IA

Leptin is an adipocyte-derived hormone that plays an important role in the regulation of energy
homeostasis through its action in the central nervous system. The arcuate nucleus (ARC) is
considered as a major hypothalamic nucleus for leptin action on energy homeostasis. Here, we
tested whether leptin action in the ARC plays a dual role in the regulation of sympathetic nerve
activity (SNA) deserving thermogenic and cardiovascular tissues. The sympathetic and
cardiovascular responses to intra-ARC leptin were compared to those evoked by intracerebro-
ventricular (ICV) administration of leptin. Sprague-Dawley rats were equipped with an ICV or
intra-ARC cannula one week before the study. Arterial pressure and multifiber SNA to
thermogenic brown adipose tissue (BAT) and kidney were recorded simultaneously under
anesthesia (-chloralose), at baseline and for 6 h after treatment. As expected, ICV
administration of leptin (10 g in 2 L, n6) caused a significant increase in BAT (250
16%, P0.00001 vs. vehicle) and renal (73 20%, P0.003 vs. vehicle) SNA. ICV leptin also
increased mean arterial pressure (from 83 4 to 100 8 mmHg, P0.04). Intra-ARC
administration of vehicle (200 nL) caused no significant change in regional SNA or arterial
pressure. However, intra-ARC leptin mimicked the effect of ICV administration of this hormone.
Indeed, direct injection of leptin (500 ng, n7) into the ARC increased both BAT (244 51%,
32 P0.0036 vs. vehicle) and renal (95 29%, P0.0035 vs. vehicle) SNA. Intra-ARC
Antenatal Betamethasone Causes Angiotensin II-Mediated Impairment of administration of leptin also increased mean arterial pressure (from 82 3 to 100 7 mmHg,
Cardiovascular Function P0.02). These data demonstrate that leptin action in the ARC is important for the control of
thermogenic sympathetic nerve activity. These results also suggest that the arterial pressure
Hossam A Shaltout, Wake Forest Univ, Sch of Medicine, Hypertension and Vascular Disease effects of leptin might be evoked by the action of this hormone in the arcuate nucleus of the
Cntr, Winston Salem, NC; Jorge P Figueroa, James C Rose, Wake Forest Univ, Sch of hypothalamus.
Medicine, Dept of Obstetrics and Gynecology, Winston Salem, NC; Mark Chappell, Debra I
Diz, David B Averill; Wake Forest Univ, Sch of Medicine, Hypertension and Vascular Disease
Cntr, Winston Salem, NC

Exposure of fetuses that are candidates for premature delivery in the early part of the third
trimester to synthetic steroids is an accepted strategy to accelerate lung development and to
protect neonates from other early developmental problems. However, recent studies show that
exposure of the fetus at this point of gestation to synthetic steroids perturbs normal 35
nephrogenesis and may lead to elevated blood pressure as these individuals reach adoles- Cardiovascular and Renal Function in Dahl Salt-Sensitive Rats Vaccinated
cence. We hypothesize that exposure of the fetus to synthetic steroids leads to activation of the against Endogenous Inhibitors of Na/K ATPase
renin-angiotensin system as the individual enters early adulthood. To investigate this
hypothesis, fetal sheep were exposed to betamethasone (Beta) or vehicle (Veh) at the 80th day Christine G Schnackenberg, Melissa H Costell, Roberta E Bernard, Mark A Pullen, Robert W
of gestation and then delivered at full term. Sheep (average age 1.8 years) were instrumented Coatney, Karen F Kozarsky, Theodore M Danoff; GlaxoSmithKline, King of Prussia, PA
for conscious recording of arterial pressure and heart rate and infusion of drugs. Beta sheep
(n6) had significantly (p 0.01) higher baseline mean arterial pressure (MAP) (93 2 mm Endogenous inhibitors of Na/K ATPase, which are thought to be structurally related to ouabain,
Hg, n6) than Veh sheep (84 2 mm Hg, n5). Blockade of AT1 angiotensin (Ang) II are reportedly increased in hypertension. We tested the hypothesis that antibodies directed
receptors with candesartan (CV; 0.3 mg/kg, i.v.) reduced MAP in Beta sheep (82 3 mm Hg)
against these postulated inhibitors of Na/K ATPase attenuate the development and maintenance
whereas MAP was not changed in Veh sheep (82 3 mm Hg). Baroreceptor reflex control of
of salt-sensitive hypertension and cardiac hypertrophy. Dahl salt-sensitive (DahlS) rats with
cardiac interval was evaluated by sequential infusion of phenylephrine and sodium nitroprus-
side. Prior to AT1 blockade baroreflex sensitivity was significantly less in Beta sheep compared radiotelemetry were maintained on low salt diet and divided into three groups: control (n8),
to Veh sheep (11 13 vs. 26 3 msec2 mmHg-1, respectively; p0.0.01). Moreover, 45 vehicle vaccinated (n9), and ouabain vaccinated (n8). When the ouabain binding capacity
minutes after CV injection baroreflex sensitivity was increased in Beta (21 5 msec2 mmHg-1) of the plasma antibodies from ouabain vaccinated rats was high (ELISA), baseline measure-
and Veh (35 4 msec2 mmHg-1) sheep. Finally, the left ventricle to body weight ratio (LV/BW) ments were determined and high salt (8% NaCl) feeding began in all groups. There were no
of Beta sheep was significantly greater than Veh sheep (p0.01). These results demonstrate differences in basal MAP (125 3 mmHg), renal function, or cardiac function (ECHO) in DahlS
that in utero exposure of the fetus to synthetic steroid can have long-lasting programming rats on low salt diet. Four weeks of high salt intake significantly increased mean arterial
effects to alter the circulatory control. pressure (182 5, 167 6, 171 6 mmHg), proteinuria (186 4, 141 8, 169 27
mg/d/100g) and left ventricular mass/body weight ratio in all groups. Ouabain vaccinated DahlS
rats produced ouabain specific, nM affinity IgG that neutralized ouabain inhibitory activity of
33 Na/K ATPase in vitro. Despite the blockade of ouabain activity, ouabain vaccinated rats had
Enhanced Depressor Response by Activating TRPV1 During High Salt similar blood pressure, renal function, and cardiac function as vehicle vaccinated rats. Similar
Intake: Role of a Novel Endovanilloid N-Arachidonoyldopamine results were found in digoxin vaccinated DahlS rats. Finally, in a separate group of naive
hypertensive DahlS rats (n5), intravenous administration of antibodies to digoxin that also
Youping Wang, M, Donna H Wang; Michigan State Univ, East Lansing, MI bind ouabain (Digibind, 60 mg/kg) had no effect on mean arterial pressure. These studies
suggest that neutralizing antibodies to endogenous inhibitors of Na/K ATPase do not prevent the
Ttransient receptor potential vanilloid type 1 (TRPV1) channels play a role in preventing high salt development of salt-sensitive hypertension, renal dysfunction, and cardiac hypertrophy or
(HS) induced increases in blood pressure (BP), but mechanisms for TRPV1 activation by HS is attenuate the maintenance of high blood pressure in DahlS rats.
CHBPR ConferenceOral Presentations e33
36 Results: Within 2 days, NaCl loading induced similar increases in PV and CO in SS (MAP
High Plasma Norepinephrine Levels Determined By 2-Adrenoceptor 10mmHg, n9) and SR (MAP 0mmHg, n11). In both SS and SR, CO reached its
Polymorphisms Predict the Future Renal Injury in Nonobese, Normotensive maximum on day 3, 8% above its low NaCl value, and returned to baseline by day 7 of the NaCl
load. In SR but not in SS the NaCl-induced increase in CO was accompanied by significant initial
Subjects
decreases in BP (-6%) and SVR (-14%). In SR, SVR returned to low NaCl levels by day 7 of the
NaCl load, whereas in SS, SVR rose above low NaCl levels by day 4 and continued to rise to
Kazuko Masuo, Baker Heart Rsch Institute, Melbourne Vic, Australia; Tomohiro Katsuya,
a final value of 11% above low NaCl. NaCl load induced an increase in bodyweight (BW) of 2.1
Hiromi Rakugi, Hideki Kawaguchi, Toshio Ogihara, Osaka Univ Graduate Sch of Medicine,
kg in SS and 2.5 kg in SR, respectively, Pns. This increase in BW was accompanied by a
Suita City, Osaka, Japan; Michael L Tuck; Sepulveda VA Med Cntr and the UCLA Sch of
positive NaB of 390 mmol in SS and of 520 mmol in SR, P0.05. Changes in BP as early as
Medicine, Sepulveda, CA
9 hours after initiating NaCl loading were highly predictive of salt-sensitivity, i.e. changes in
MAP on days 5 and 6, r0.64, P0.003 for DBP. Changes in NaB, BW, PV and CO are not
Background: Renal injury is one of the common organ damage caused by hypertension. The
predictive of salt-sensitivity at any time point. Conclusions: We propose a vasogenic
2-adrenoceptor polymorphisms and heightened sympathetic nerve activity are associated
hypothesis of salt-sensitivity: In African Americans, the pressor effect of dietary NaCl depends
with hypertension. In the present study, we examined the relationships between alterations of
from its outset on an abnormal vascular response to NaCl loading.
renal function (blood urea nitrogen (BUN) and creatinine), sympathetic nerve activity (SNA) and
the 2-adrenoceptor polymorphisms in a longitudinal design over 5 years. Methods: In 219
nonobese, normotensive, normal renal function men at entry, we measured serum BUN,
creatinine, plasma norepinephrine (NE) levels, the homeostasis model assessment of insulin-
resistance (HOMA), BMI, total body fat-mass, blood pressure (BP), and the 2 (Arg16Gly, 39
Gln27Glu)-adrenoceptor polymorphisms annually over 5 years. The subjects were stable in
body weight over 5 years and non-diabetic. Results: Hyper-SNA was defined as a plasma NE
The Na-K-2Cl Cotransporter as a Mediator of the Na Retention in African
level of mean1SD of 1.37 pmol/mL in the participants at entry. There were 37 subjects who Americans
had entry hyper-SNA and 182 subjects who had entry normal-SNA (plasma NEmean1SD).
At entry, plasma NE was significantly higher in subjects with hyper-SNA compared to those Tae-Yon Chun, Indiana Univ Sch of Medicine, Indianapolis, IN; Lise Bankir, INSERM Unite
with normal-SNA, but BUN, creatinine, HOMA, BMI, total body fat-mass and BP levels were 652, Paris, France; George J Eckert, Chandan Saha, Mary Anne Wagner, Beth Deem,
similar. Changes in serum BUN, creatine, uric acid, plasma NE, HOMA, total body fat-mass, BMI Indiana Univ Sch of Medicine, Indianapolis, IN; Daniel G Bichet, Hopital du Sacre-Coeur de
and BP levels over a 5-year period were significantly greater in subjects with hyper-SNA. Montreal, Montreal, Canada; J. Howard Pratt; Indiana Univ Sch of Medicine and the VA Med
Subjects with hyper-SNA had significantly higher frequencies of the Gly16 and Glu27 alleles of Cntr, Indianapolis, IN
2-adrenoceptor polymorphisms. Subjects carrying the Gly16 or Glu27 alleles had higher levels
of plasma NE, HOMA and total body fat-mass at entry, and greater increases in BUN, creatinine, Identifying differences between ethnic groups in the renal handling of Na could provide insight
into mechanisms for hypertension. In the current study, we made measurements in African
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017

plasma NE levels and total body fat-mass over a 5-year period. Conclusions: The Gly16 and
Glu27 alleles are associated with high plasma NE at entry and elevations in BUN and creatinine Americans (AAs) and European Americans (EAs) that had potential relevance to the renal
over a 5-years period accompanied by greater increases in plasma NE. Although renal injury Na-K-2Cl cotransporter (NKCC2). Urine osmolality (Uosm) served as an indirect index of NKCC2
is known of hypertension-related end-organ damage, high plasma NE (heightened sympathetic function since it is highly dependent on the medullary Na concentration achieved by NKCC2.
nerve activity) and high HOMA (insulin-resistance) in part determined by 2-adrenoceptor Healthy subjects (99 AAs, 78 EAs; 18 35 yr) were admitted to the GCRC where they had free
polymorphisms might be related with the onset of renal injury. access to water overnight (12 hr) and during an early morning meal. This was followed by a
continuous infusion of 0.9% saline (60 ml/hr x 8 hr) (no additional food or water). After
equilibration and 2 hr basal period, 40 mg furosemide (F) was administered i.v. Analysis of
covariance adjusted for sex was used to compare the 2 groups. During the overnight period,
37 Uosm (mosm/kg H2O) was higher in AAs than EAs, 552 25 (SE) vs. 413 17 (p0.0001),
Blood Pressure Lowering Effect Of The Mitochondrial Inhibitor Rotenone In whereas during saline infusion baseline values were not significantly different: 537 19 and
Mice With DOCA-salt Hypertension 492 21 in AAs and EAs, respectively (p0.12). Thirty min after F administration, Uosm fell
in both groups close to plasma osmolality (300), indicating complete inhibition of NKCC2. In
Tianxin Yang, Aihua Zhang; Univ of Utah, Salt Lake City, UT the subsequent recovery period, Na excretion was less in AAs (p0.0013 in initial 2 hr)
consistent with greater Na reabsorption. Following the post-F nadir, Uosm increased but more
Oxidative stress plays a pivotal role in the pathogenesis of various forms of hypertension but slowly in AAs than EAs (p0.0001). This appeared explainable in part by reduced levels of
its source is debatable. Despite emphasis on NADPH oxidase, attention has been paid to the renin activity (PRA) (p0.0041) and aldosterone (PA) (p0.0008) in AAs than EAs. Recovery of
mitochondria as a potential source of oxidative stress in the development of hypertension. The Uosm correlated with both PRA (r0.17; p0.057) and PA (r0.24; p0.0027) leading us to
present study was undertaken to examine the effect of the mitochondrial complex I inhibitor speculate that in AAs the suppressed levels resulted in less vascular tone in vasa recta thereby
rotenone on blood pressure (Bp) in mice treated with deoxycorticosterone acetate and 1% salt enhancing washout of the medullary gradient. Plasma vasopressin measured in a subset of 60
(DOCA-salt). Wild type 129-C57/BL6 mice received DOCA-salt treatment for 4 days followed by subjects did not explain the ethnic differences. In summary, AAs had higher Uosm under free
an one-week treatment with vehicle or rotenone at 600 ppm in a gelled diet, and daily mean living conditions, less Na excretion in post-F recovery period, and slower Uosm recovery that
arterial pressure (MAP) was monitored by telemetry. The second day of DOCA-salt, MAP in the correlated with PRA/PA. Although the indirect assessments limit making specific conclusions,
vehicle treated mice increased from 110 1.33 to 138 2.04 mmHg that remained constant the findings suggest that increased Na retention in AAs could be representative of greater
throughout the entire experimental period. Rotenone treatment induced an immediate and NKCC2 activity.
significant reduction of MAP (136.0 2.9 vs. 114 3.5 mmHg, n6, p0.01). DOCA-salt
increased urinary excretion of 8-isoprostane from 275.47 44.65 to 1654.97 316.15
pg/24h that was reduced to 826.04 141.24 pg/24h after rotenone treatment. Conversely,
DOCA-salt decreased urinary excretion of nitrate/nitrite from 466.73 84.14 to 68.30 15.17
that was restored to 238.11 44.53 pmol/24h by rotenone treatment. Reactive oxygen 40
species (ROS) production in isolated kidney mitochondria as measured by enhanced luminal or Non-Modulating Hypertension is Associated with Insulin Resistance and
isoluminol chemiluminescence was 1.0 0.1, 2.48 0.4 (p0.01), and 1.26 0.28 the Lys528Arg Variant of Human Adipocyte-Derived Leucine
(p0.01) (fold changes over control) in vehicle, DOCA-salt, and DOCA-salt plus rotenone
Aminopeptidase
groups, respectively; levels of protein carbonyls in the three groups in the same order were
4.38 1.35, 11.10 5.56 (p0.01), and 5.91 2.28 (p0.05) nmol/mg protein.
Jonathan S Williams, Annaswamy Raji, Gordon H Williams, Paul R Conlin; Brigham and
Histological analysis of heart and kidneys did not reveal significant abnormalities after rotenone
Womens Hosp/Harvard Med Sch, Boston, MA
treatment. In contrast to the significant Bp lowering effect of rotenone, DOCA-salt hypertension
in mice with mutation of the p47phox gene did not appear to be affected. In conclusion, the
Intermediate phenotyping provides a powerful means to test susceptibility loci for human
present study provides new evidence favoring mitochondria as an important source of oxidative
hypertension. Two intermediate phenotypes, insulin resistance (IR) and adrenal responsiveness
stress in DOCA-salt hypertension.
to angiotensin II (Ang II) have demonstrated familial aggregation in hypertensive sib-pair
studies. Adipocyte-derived leucine aminopeptidase (ALAP) degrades Ang II in the kidney,
suggesting its possible role in BP regulation. This is supported by reports that the Lys528Arg
38 polymorphism, which reduces ALAP activity, is associated with human hypertension. Non-
Salt-Sensitivity in African Americans: Is it Initiated by an Abnormal modulating hypertension (NM) is characterized by blunted renal vascular and adrenal responses
Vascular Response? to Ang II, which are corrected by ACE-inhibition. NM patients are also more likely to have IR.
We investigated the heritability of NM and its association with IR and the Lys528Arg variant of
Olga Schmidlin, Alex Forman, Anthony Sebastian, R. C Morris, Jr; Univ. of California San ALAP. We analyzed 436 hypertensive individuals categorized by their adrenal response to Ang
Francisco, San Francisco, CA II (NM aldosterone response to Ang II 15 ng/dl, N98). Within this cohort, results from 138
sib-pairs were examined to test familial aggregation of the NM phenotype. IR was estimated
It is widely formulated that salt-sensitivity is: 1) initiated by an increased renal reclamation of using homeostasis model assessment (HOMA). Sib-pair analysis demonstrated familial
salt that induces a transient increase in cardiac output (CO) by expanding plasma volume (PV): aggregation of NM (OR 2.6, 95%CI, 1.1, 6.78). NM was also associated with IR (OR 1.8, 95%CI
and later, 2) sustained by a persisting increase in systemic vascular resistance (SVR). Although 1.1, 2.9). Allele frequencies for Lys528Arg were in Hardy-Weinberg equilibrium. NM was
many observations accord with this nephrogenic hypothesis, recent observations in animal associated with a higher odds of carrying the 528Arg variant of ALAP (OR 2.2, 95% 1.0, 4.9,
models suggest that an abnormal vascular response to salt may initiate salt-sensitivity. Neither P 0.047). There was a significant genotype-HOMA dose-response relationship for Lys528Arg
of these hypotheses has been rigorously tested in humans. In 20 mostly normotensive African with higher HOMA values associated with the 528Arg genotype (P-trend 0.03). Individuals
Americans we determined a) whether dietary NaCl loading induces greater increases in Na with both IR and NM were more likely to have the 528Arg polymorphism (OR 4.55, 95%CI, 1.4,
balance (NaB), PV and CO in those who are salt-sensitive (SS) compared to those who are 15.0, P 0.008). These results show that NM is associated with IR, aggregates in families,
salt-resistant (SR); and b) whether initial NaCl-induced hemodynamic events can predict and that both NM and IR are associated with the 528Arg variant of ALAP. Our results support
salt-sensitivity. Employing impedance cardiography, we measured CO daily at 4-hr intervals the involvement of ALAP in both blood pressure regulation and IR in NM. This suggests a
during the last 3 of 7 days of low NaCl intake, 35 mmol/d, and on all 7 days of NaCl loading, common underlying mechanism for both IR and hypertension in NM, which may be linked
250 mmol/d. We estimated changes in PV from changes in serum protein concentration. through abnormal ALAP activity.
e34 Hypertension Vol 48, No 4 October 2006

41 and HO-dependent CO generation and to assess the significance of HO products on the internal
Reactive Oxygen Species (ROS)-Dependent Hypertension in D2 Receptor diameter of renal interlobular arteries from treated and untreated rats. DETC increased vascular
Deficient Mice superoxide production (475 vs. 746 cpm/ug protein; p0.05) and HO-1 protein, without
altering HO-2 protein levels. Yet, total CO generation was reduced (112696 vs. 682147
Ines Armando, Xiaoyan Wang, Van A Villar, John E Jones, Laureano Asico, Pedro A Jose; pmol/mg/hr), reflecting a near abolition of HO-dependent CO generation (52963 vs. 2920
Georgetown Univ Med Cntr, Washington, DC pmol/mg/hr) while HO-independent generation was marginally increased. Pretreatment with
tempol significantly reduced HO-independent CO and minimized DETC-induced inhibition of
Alterations in dopamine D2-like receptor function have been reported in essential hypertension. HO-dependent CO production (2920 vs. 28075 pmol/mg/hr; p0.05). Assessment of the
A polymorphism in exon 6 of the D2 receptor (D2R) gene is associated with elevated blood functional response to HO inhibition in pressurized renal interlobular arteries revealed that CrMP
pressure and a Taq1 polymorphism is associated with human essential hypertension. We have reduced internal diameter in vehicle-treated rats while having no effect in DETC-treated
previously shown that disruption of D2R in mice (D2-/-) results in high blood pressure that is animals (-28.30.8 vs. -1.00.6 um; p0.05). These data suggest an inhibitory role of
associated with increased aldosterone production and defective aldosterone suppression by O2- or one of its derivatives on HO activity that prevents the vascular HO system from supporting
high salt diet. Aldosterone has been shown to increase the expression of NADPH oxidase vasodilatory mechanisms. Hence, vascular dysfunction during acute oxidative stress may be
subunits as well as oxidative stress. We hypothesized that the increased blood pressure in D2-/- related, in part, to deficient generation of HO-derived products.
is related to increased NADPH oxidase activity and ROS. We determined the mRNA expression
of the NADPH oxidase subunits p22phox, p40phox, p47phox, p67phox, Rac1, Rac2, Nox1, Nox2 and
Nox4 by RT-PCR in kidneys of D2-/- and their wild type littermates, D2/.The mRNA
44
expression of Nox1, Nox2 and Nox4 was increased 303, 141 and 292%, respectively in Chronotropic Actions of Angiotensin II Are Mediated by a PKC-Dependent
D2-/- (p0.05 vs D2/, n5 per group).The mRNA expression of the other subunits tested Activation of NADPH Oxidase in Brain Neurons
was similar in both groups. The protein expression of Nox1, Nox2 and Nox4, determined by
western blot, was increased in kidneys of D2-/- mice by 458, 8910 and 555%, Chengwen Sun, Colin Sumners, Mohan K Raizada; Dept of Physiology and Functional
respectively (p0.05 vs D2/). The urinary excretion of 8-isoprostane (7715 vs 358 Genomics, College of Medicine, Univ of Florida, Gainesville, FL
ng/mg creatinine [D2/]; p0.04, n9), a marker of oxidative stress, and renal NADPH
oxidase activity (121,30015,310 vs 81,2009,870 [D2/] LU/mg prot; p0.05; lucigenin We have previously established that via stimulation of AT1 receptor (AT1R), Angiotensin II (Ang
assay) were also increased in D2-/- mice. Treatment with hemin, an inducer of heme II) increases neuronal firing, which induces cascades of signaling events leading to cardiovas-
oxigenase-1 (50 mol, IP, for 24 h), normalized blood pressure in anesthetized (Avertin) D2-/- cular effects of this hormone in the brain. Although we have identified NADPH oxidase-derived
mice (systolic: vehicle: 1235; hemin: 896 mmHg; p0.05; n5) but had no effect in ROS as a key signaling intermediate in the chronotropic actions of Ang II, the precise
D2/ mice (systolic: vehicle: 1032; hemin: 1001 mmHg; n5). Treatment with mechanism underlying this effect remains elusive. We hypothesized that NADPH oxidase
apocynin, a NADPH oxidase inhibitor (3 mg/k/day, via osmotic mini-pumps, 10 days), also activation involves protein kinase C (PKC) since inhibition of this enzyme prevents Ang
normalized blood pressure in D2-/- mice (systolic: D2-/-, 962; D2/, 961 mmHg; n5 II-induced neuronal activity. Thus, our objective in the present study was to examine the role
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per group). Our results show that the D2R is involved in the regulation of ROS production and of PKC-mediated NADPH oxidase activation in the chronotropic actions of Ang II in the neurons
suggest that by direct and indirect mechanisms altered D2R function may result in co-cultured from hypothalamus and brainstem. Incubation of neurons with Ang II (100 nM)
ROS-dependent hypertension. causes 2 fold increases in NADPH oxdase activity. This action of Ang II, mediated by
neuronal AT1R, is abolished by a PKC inhibitor GF109203X (GF, 1 M) and mimicked by a PKC
activator, Phorbol 12-myristate 13-acetate (PMA, 1 M). In addition, Ang II-induced increase
in neuronal firing rate (from 0.99 0.18 Hz to 1.81 0.29 Hz) is blocked by GF (1 M).
42 Treatment of neurons with PMA (1 M) causes a 2-fold increase in neuronal firing rate that is
Endothelin-1 Induced Oxidative Stress Reduces Endothelial Progenitor Cells attenuated by a ROS scavenger, TEMPOL (100 M) or a NADPH oxidase inhibitor, gp91ds (5
via an ETA-NADPH Oxidase Pathway in Salt-Sensitive Hypertension M). Finally, treatment of neurons with Ang II (100 nM, 5 min) increases membranous p47phox
and decreased cytoplasmic p47phox protein as measured by Western blotting. This Ang
Dandan Chen, Michigan State Univ, East Lansing, MI; YuGang Dong, The First Affiliated II-induced translocation of p47phox is abolished by pretreatment of neurons with GF (1 M).
Hosp of Sun Yat-Sen Univ, Guangzhou, China; Eric J Marrotte, Jame J Galligan, Gregory D These observations indicate that Ang II-induced stimulation of neuronal firing is mediated by
Fink, Alex F Chen; Michigan State Univ, East Lansing, MI activation of NADPH oxidase in a PKC-dependent manner and that PKC is proximal to NADPH
oxidase activation in ROS-mediated actions of Ang II in the regulation of neuronal activity.
Background: The circulating endothelial progenitor cells (EPCs) derived from the bone marrow
home to the ischemic site to form mature EC and new blood vessels. Circulating EPCs are
reduced in patients of cardiovascular diseases including hypertension, and correlates inversely 45
with their mortality. However, the mechanisms underlying reduced availability of EPCs in Reduction of Oxidative Stress-Induced Vasoconstriction by Tempol is
hypertension are poorly understood. DOCA-salt hypertension exhibits increased levels of ET-1 Mediated by Hydrogen Peroxide
and oxidative stress. We tested the hypothesis that ET-1-induced oxidative stress contributes
to reduced EPCs in DOCA-salt rats. Methods: Rat EPCs were isolated from peripheral blood and Adam Pearlman, Christopher Wilcox, Yifan Chen; Georgetown Univ, Washington, DC
cultured in EC growth medium-2 for 5 days. EPCs that were DiI-acLDL and lectin
double-positively stained were counted. The level of intracellular reactive oxygen species (ROS) Reduction of oxidative stress in hypertension by the superoxide dismutase (SOD)-mimetic
in EPCs was assessed by dichlorofluorescein (DCF) fluorescence microscopy. Results: The tempol improves vascular dysfunction. Tempol reduces the concentration of superoxide anion,
number of EPCs was significantly reduced in DOCA-salt compared to Sham rats (3.940.30 (O2-), but increases the concentration of hydrogen peroxide (H2O2). We tested the hypothesis
vs. 10.320.25cells/hpf, n3 6, p0.01). Treatment with ROS generator LY83583 (10 M, that the vasodilatory effect of acute exposure to tempol is primarily due to generation of H2O2.
18 hrs) profoundly elevated the ROS level in EPCs of DOCA-salt rats (212.48.3% vs. Sham, We applied tempol to mouse cremaster arterioles in vivo (n7) and to pressurized rat
n3, p0.05), but only slightly in EPCs of normal rats (110.01.0%, n3, p0.05). ET-1 mesenteric arteries in vitro (n9). Vessels were exposed to angiotensin II (cremaster) or
treatment (10 nM) significantly reduced the number of EPCs in a time-dependent manner U46619, a thromboxane receptor agonist, (mesenteric) for 1530 minutes. Acute application of
(24 96 hrs) in normal rats. ET-1 (48 hrs) induced decrease of EPCs (6.910.11 vs. tempol caused significant vasodilation in vivo (18935%) and in vitro (212%) with preserved
10.320.25 cells/hpf, n3 6, p0.01) was rescued by the selective ETA receptor antagonist vasodilation in endothelium denuded vessels. The vasodilatory effect of tempol was signifi-
ABT-627, NADPH oxidase inhibitor apocynin, or adenoviral-mediated gene transfer of cantly reduced in vivo (397%) or in vitro (102%) in the presence of catalase. Similarly,
dominant-negative Rac1 that inhibits endogenous NADPH oxidase subunit Rac1 (9.850.28, addition of 30mM KCl to the mesenteric vessel bath abrogated the effect of tempol. Tempol
10.480.38, and 9.130.38 cells/hpf, respectively, n3 6, p0.01), but not by ETB receptor was not effective following prolonged treatment with alternative preconstrictors not associated
antagonist BQ788 (7.620.41 cells/hpf, n3, p0.05). ET-1 treatment also significantly with substantial production of O2-: phenylephrine in vivo (154%) or norepinephrine in vitro
augmented the ROS level in EPCs of normotensive rats (212.48.3%, n3, p0.05), which (0%). In contrast to tempol, nitro blue tetrazonium, (NBT), a O2- scavenger of equal potency to
was blunted by ABT-627 and apocynin (103.32.0% and 114.52.5%, respectively, n3, tempol that does not result in increased H2O2, was an ineffective vasodilator when applied in
p0.05). Conclusion: These results demonstrated, for the first time, that EPCs of DOCA-salt vivo (105%) or in vitro (03%). Direct applications of H2O2 to the vessels caused a
hypertensive rats exhibit both reduced number and antioxidant capacity, mediated at least in dose-dependent dilation in vitro (0.01100M) that was independent of intact endothelium. We
part, via an ETA-NADPH oxidase pathway. conclude that prolonged stimulation of angiotensin II or thromboxane receptors produce
vascular oxidative stress. Subsequent vasodilation by tempol is largely due to generation of
H2O2 which likely produces a hyperpolarizing effect. In a variety of pathophysiological
43 conditions associated with oxidative stress, an adequate amount of H2O2 may be an important
Acute Oxidative Stress Inhibits HO-Dependent Generation of Vascular CO in counterbalance to the vasoconstrictive effect of O2-.
Renal Arteries

Brian D Lamon, Frank F Zhang, Huan Deng, Michael S Wolin, Alberto Nasjletti; New York 46
Med College, Valhalla, NY Angiotensin II Causes Elevated Superoxide Levels in the Paraventricular
Nucleus during Hypertension
Carbon monoxide (CO) is produced endogenously via HO-dependent and HO-independent
pathways. CO is liberated during the metabolism of heme by HO-1 and -2 along with biliverdin Carrie A Northcott, Andrew J King, Gregory D Fink, Joseph R Haywood; Michigan State
(BV) and iron. HO-independent CO is generated by peroxidizing lipids under conditions of Univ, East Lansing, MI
elevated oxidative stress. The source of endogenous CO may have functional implications, as
CO alone causes vasoconstriction while the combination of CO and BV causes vasodilation. Circulating angiotensin II (Ang II) acts on the brain to stimulate neural pathways to the
These studies were designed to determine the effects of oxidative stress on CO generation by paraventricular nucleus (PVN) that use Ang II as a neurotransmitter. In renal wrap hypertension,
renal interlobar arteries (RIA). We studied rats treated with vehicle, with the superoxide an Ang II dependent model, superoxide (O2-) levels in the PVN are elevated. Thus, we
dismutase (SOD) inhibitor diethyldithio-carbamate trihydrate (DETC; 1g/kg, i.p.) or with DETC hypothesized that Ang II was the stimulus for increased O2- in the PVN during hypertension. We
tempol (an SOD mimetic). RIA were obtained 3 hours after treatment and used to measure examined this hypothesis in several ways; ex vivo addition of Ang II (100 nmol Ang II for 15,
superoxide generation, HO protein levels, and endogenous CO release. An inhibitor of HO, 30 and 60 minutes) to PVN brain punches to determine if Ang II could directly stimulate O2-
chromium mesoporphyrin (CrMP; 30uM), was used to discriminate between HO-independent production in the PVN, measurements of O2- levels in PVN punches from rats treated acutely
CHBPR ConferenceOral Presentations e35
with Ang II (30 ng/kg/min, 15ul/min intravenously for 1 hour) and rats treated chronically with 49
Ang II (150 ng/kg/min subcutaneously for 14 days). Because high blood pressure itself has Agonistic Autoantibodies to the AT1 Receptor in Rat Models of
been shown to increase O2- levels in some tissues, we examined O2- levels in the PVN of chronic Preeclampsia: Induced by Chronic Reductions in Uterine Perfusion Pressure
DOCA-salt hypertensive rats, a model lacking elevated circulating Ang II. Blood pressures were
(RUPP) and Low Dose TNFalpha Infusion
measured in the conscious state. The brains were removed, quick frozen on dry ice and
immediately cryostat sectioned (600 um) and PVN punched for chemiluminescent O2-
Ralf Dechend, Helios-Klinikum, Franz-Volhard-Klinik, Charite, Berlin, Germany; Mytae Llinas,
measurements. After a 15 minute ex vivo incubation of the PVN with Ang II there was a
Univ of Mississippi Med Cntr, Jackson, MS; Silvia Caluwaerts, Universitair Ziekenhuis
significant increase in O2- (p0.05), demonstrating direct Ang II-induced O2- production. Acute
Gasthuisberg, Leuven, Belgium; Florian Herse, Helios-Klinikum, Franz-Volhard-Klinik, Charite,
Ang II infusion resulted in a significant increase in blood pressure (p0.05), and O2- in the PVN
Berlin, Germany; Babbette Lamarca, Univ of Mississippi Med Cntr, Jackson, MS; Dominik N
(Saline: 0.22 0.02 nmol/min*mg, Ang II: 0.36 0.02 nmol/min*mg, p0.05). Chronic Ang
Muller, Max-Delbrueck Cntr, Berlin, Germany; Robert Pijnenborg, Universitair Ziekenhuis
II infusion also resulted in a significant increase in blood pressure (Mean Arterial Pressure: 104
Gasthuisberg, Leuven, Belgium; Gerd Wallukat, Max-Delbrueck Cntr, Berlin, Germany; Joey
3 mm Hg vs. 134 8 mm Hg, p0.05) and increased O2- in the PVN (Vehicle: 0.23 0.02
P Granger; Univ of Mississippi Med Cntr, Jackson, MS
nmol/min*mg, Ang II: 0.44 0.09 nmol/min*mg, p0.05). In contrast, in DOCA-salt rats
[Systolic Blood Pressure (SBP) 178 3 mm Hg] there was no significant increase in O2- in
The IgG fraction from preeclamptic women contains an angiotensin II receptor autoantibody
the PVN compared to sham normotensive rats (SBP136 5 mm Hg). These data
(AT1R-AA) that stimulates the angiotensin II type I (AT1) receptor. AT1R-AA could contribute to
demonstrate that circulating Ang II acts on neural pathways in the brain to increase PVN O2-.
the pathogenesis of preeclampsia. We investigated the uterine perfusion pressure (RUPP) and
Further, the O2- changes in the PVN are not due to increased blood pressure per se. In
low-dose TNF infusion models to see if these models develop AT1R-AA. We detected
conclusion, Ang II-mediated changes in O2- may contribute to increased excitatory neurotrans-
AT1R-AA by the chronotropic responses to AT1 receptor-mediated stimulation of cultured
mitter activity in the PVN and ultimately result in hypertension.
neonatal rat cardiomyocytes coupled with receptor-specific antagonists. Mean arterial pressure
(MAP) was significantly higher in RUPP rats (1371 mm Hg) than in normal pregnant (NP) rats
(1071 mm Hg). The hypertension in the RUPP rats was associated with significant elevations
in agonistic antibodies capable of activating the angiotensin AT1 receptor (RUPP 15.31.6 vs.
NP 0.60.3 beats/min). Pregnant rats with low dose TNF infusion (5 days at a rate of 50
ng/d during days 14 to 19 of gestation in pregnant rats) had higher blood pressures (125
47
1 mm Hg) than NP rats (1012 mm Hg). They also developed AT1R-AA (9.22.3 vs 1.0 0.8
Heterozygous eNOS-Deficiency Produces Oxidative Stress and Endothelial beats/min). Low dose TNF in virgin rats did not increase MAP nor lead to AT1R-AA. TNF
Dysfunction in a Model of Diet-Induced Obesity and Type II Diabetes expression in the placentas of RUPP rats was upregulated (p 0.05) Serum TNF levels were
nearly 3-fold higher in the RUPP rats compared with NP (p 0.05). These findings suggest that
Sean P Didion; Univ of Iowa Carver College of Medicine, Iowa City, IA the generation of AT1R-AA is a secondary event to reduction in placental perfusion leading to
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017

chronic inflammation.
Although the incidence of obesity and type II diabetes is increasing, very little is known
regarding mechanisms that contribute to endothelial dysfunction in these conditions. The goal
of this study was to test the hypothesis that loss of a single gene for endothelial nitric oxide
synthase (eNOS) produces endothelial dysfunction in a mouse model of diet-induced obesity
50
and type II diabetes. Responses of carotid arteries from wild-type and heterozygous RGS2 Ameliorates Angiotensin II-Induced Hypertension in Mice
eNOS-deficient (eNOS) mice were examined in vitro following 32 weeks on either a control
(10% kcal from fat) or a high fat (45% kcal from fat) diet. Body weight, fat deposition (ie, Michael Obst, MDC, Berlin, Germany; Jens Tank, Med Faculty of the Charite, Franz Volhard
perirenal and reproductive fat mass) and blood glucose levels were greater (P0.05) in high-fat Clinic, HELIOS Klinikum-Berlin, Berlin, Germany; Ralph Plehm, MDC, Berlin, Germany; Jens
fed mice compared to mice fed a control diet and were not affected by genotype. Responses Jordan, Med Faculty of the Charite, Franz Volhard Clinic, HELIOS Klinikum-Berlin, Berlin,
to acetylcholine (ACh, an endothelium-dependent agonist) and nitroprusside (NP, an Germany; Friedrich C Luft, MDC and Med Faculty of the Charite, Franz Volhard Clinic,
endothelium-independent agonist) were similar in wild-type and eNOS mice fed a control diet HELIOS Klinikum-Berlin, Berlin, Germany; Volkmar Gross; MDC, Berlin, Germany
(eg, 10 mol/L ACh produced 926 and 853% relaxation in wild-type and eNOS mice,
respectively). In contrast, responses to ACh were markedly reduced (P0.05) in eNOS, but The regulator of Gq G protein signaling (RGS) 2 accelerates the rate of G protein deactivation
not wild-type, mice fed a high-fat diet (eg, 10 mol/L ACh produced 853 and 496% by stimulating GTP hydrolysis. Angiotensin II (Ang II) activates signaling pathways predomi-
relaxation in control and high-fat fed eNOS mice, respectively). This effect was selective nantly through the G-protein-coupled Ang II type 1 receptor (AT1AR). We hypothesized that RGS2
since NP produced similar (P0.05) relaxation in vessels from high-fat fed wild-type and deletion increases the response to Ang II and changes blood pressure regulation because
eNOS mice. Responses to ACh in high-fat fed eNOS mice were restored toward normal by signaling would be abnormally prolonged. To address this issue, we infused Ang II chronically
tempol (a superoxide scavenger, 1 mmol/L) suggesting a role for superoxide in this response. in conscious RGS2 deleted (RGS2 -/-) and wild-type (RGS2 /) mice. We combined
These findings provide the first genetic evidence that loss of a single eNOS gene produces telemetric arterial blood pressure recordings with fast Fourier analysis (FFT) of mean arterial
oxidative stress and endothelial dysfunction in a model of diet-induced obesity and type II blood pressure (MAP) and heart rate (HR) and pharmacological autonomic testing to elucidate
diabetes. These findings have important implications in terms of disease states and/or genetic Ang II effects in these mice. During Ang II infusion, BP increased to a greater degree in RGS2
polymorphisms associated with reductions in eNOS expression and/or activity. -/- animals ( MAP day (113 vs. 33 mmHg and ( MAP night 123 vs. 73 mmHg) while
HR was not affected and ranged between 56315 and 59515 beats/min. Baroreflex
sensitivity was not different between the groups. Therefore, baroreflex resetting was more
pronounced in RGS2 -/- compared with wild type animals. Together, these observations
suggest, that RGS2 attenuates Ang II mediated resetting of baroreflex heart rate regulation. The
profoundly decreased urinary norepinephrine excretion rates in Ang II-treated RGS2 -/- and
48 RGS2 / mice (17.25.0 vs. 24.16.9 ng/day), compared to untreated RGS2 -/- and RGS2
Sympathetic Neurons Isolated from Apolipoprotein E Deficient Mice Exhibit / mice (332.864.6 vs. 172.216.2 ng/day), suggest a reduced sympathetic activity.
Furthermor, the depressor response to prazosin was similar before and after Ang II infusion in
Increased Excitability: Role of Oxidative Stress RGS2 -/- animals. Thus, the increased pressor response in RGS2 -/- mice cannot be explained
by an excessive increase in alpha-adrenoreceptor mediated vasoconstriction. The results
Xiuying Ma, Zhi-Yong Tan, Carol A Whiteis, Francois M Abboud, Mark W Chapleau; Depts of
suggest that the interaction between RGS2 and Ang II-dependent vasoconstriction via the AT1A
Internal Medicine and Physiology & Biophysics, Univ of Iowa, and Veterans Affairs Med Cntr,
receptor is important for blood pressure homeostasis. Deletion of RGS2 sensitizes Ang
Iowa City, IA
II-dependent signaling in vivo. 1712
We recently demonstrated that O2- is increased in sympathetic ganglia of apolipoprotein E
deficient (apoE-/-) mice with atherosclerosis. Previous studies have shown that oxdative stress
in the central nervous system increases sympathetic nerve activity. In the present study, we 51
tested the hypothesis that oxidative stress in apoE-/- sympathetic ganglia increases membrane AAV Delivery of NADPH Oxidase gp91-shRNA Attenuates Cold-Induced
excitability of postganglionic neurons. Neurons were isolated from sympathetic ganglia of Hypertension and Vascular Hypertrophy
wild-type (WT, n6), apoE-/- (n6), and apoE-/- mice treated with the SOD mimetic tempol
(n4) for 2 weeks in the drinking water (30 mg/kg/day). Resting membrane potential (RMP) Xiuqing Wang, Lucille H Skelley, Zhongjie Sun; Univ Fl Coll Med, Gainesville, FL
and action potentials (APs) were recorded by patch-clamp at rest and in response to
depolarizing current injection. RMP was more negative in apoE-/- (n24) than in WT (n20) Cold exposure increases NADPH oxidase gp91 protein expression. The aim of the present study
neurons (-43.60.6 vs. -41.10.9 mV, P0.05) but membrane resistance did not differ. The was to determine the effect of RNAi inhibition of gp91 expression on cold-induced
majority neurons from WT mice (17 of 20, 85 %) were electrically quiescent at RMP with only cardiovascular dysfunction. We hypothesized that RNAi inhibition of gp91 expression attenuates
3 of 20 neurons (15 %) firing APs spontaneously. In contrast, a large percentage of apoE-/- cold-induced hypertension (CIH) and vascular hypertrophy. Recombinant AAV carrying short-
neurons fired APs spontaneously at RMP (10 of 24, 42 %, P0.05 vs. WT). In quiescent hairpin small interference (si)RNA for gp91 (AAV.gp91-shRNA) was constructed and tested for
neurons, the current threshold for triggering a single AP was significantly lower in apoE-/- than the ability to inhibit gp91 to control CIH. Three groups of rats received AAV.gp91-shRNA
in WT mice (7.51.9 vs. 14.52.4 pA), and the frequency of AP firing with equivalent current (1.25X1010 particles/rat, IV), AAV carrying scrambled shRNA (AAV.Control-shRNA., 1.25X1010
injection (100 pA for 1 s) was higher (15.01.7 vs. 9.52.1 spikes) (apoE-/- vs. WT, P0.05). particles/rat, IV), and phosphate buffer solution (PBS), respectively. Following gene delivery, all
Chronic treatment of apoE-/- mice with tempol reduced the proportion of neurons firing APs rats were exposed to moderate cold (44F) continuously. Blood pressure was monitored using
spontaneously (22 vs. 42 %, tempol treated vs. untreated apoE-/-, P0.05). Interestingly, a telemetry system. AAV delivery of gp91-shRNA significantly attenuated cold-induced
tempol-treatment did not affect either current threshold or AP firing during current injection in elevation of blood pressure (BP). BP was 1215 mmHg, 1486 mmHg, and 1545 mmHg
quiescent apoE-/- neurons. We conclude that membrane excitability is increased in sympa- for the AAV.gp91-shRNA-treated group, AAV.Control-shRNA-treated group, and PBS-treated
thetic neurons isolated from apoE-/- mice. The reduction in number of spontaneously firing group, respectively. One single injection of AAV.gp91-shRNA can attenuate hypertension for up
neurons isolated from tempol-treated apoE-/- mice suggest oxidative stress plays a role in the to 10 weeks (length of the study). Western blot analysis indicated that gp91 protein expression
increased neuronal excitability of apoE-/-sympathetic neurons. We speculate that increased was inhibited by AAV.gp91-shRNA. DHE staining showed that the in situ superoxide production
excitability of sympathetic neurons may contribute to excessive sympathetic nerve activity and was significantly decreased in aorta by AAV delivery of gp91-shRNA. AAV.gp91-shRNA
baroreflex impairment in atherosclerotic states. (VA and NIH HL14388) attenuated vascular hypertrophy. AAV delivery of gp91-shRNA significantly decreased TGF-
e36 Hypertension Vol 48, No 4 October 2006

expression. Conclusions: (1) AAV delivery of gp91-shRNA effectively silenced gp91 expression unknown. We studied effects of brief perinatal molsidomine (NO donor) treatment in
in vivo. (2) RNAi inhibition of gp91 may serve as a new approach for long-term control of Fawn-Hooded Hypertensive (FHH) rats, a genetic model of hypertension-associated renal injury
oxidative stress and related cardiovascular dysfunction. (Supported by NIH R01 HL077490). with defective regulation of preglomerular resistance. FHH dams received oral molsidomine
(120 mg/l) during the last 2 wk of pregnancy and all 4 wk of lactation. Systolic BP (SBP),
proteinuria and urinary excretion of NO metabolites (NOx) were measured in male and female
52 offspring. Mean arterial pressure (MAP) and renal vascular resistance (RVR) was measured
Total Body Na and Osmotically Inactive Na Content Are Dependent on under pentobarbital anesthesia at 36 wk in males and 42 wk in females and during NO
the Agt Genotype in Mice synthase inhibition with L-NNA in males. Perinatal molsidomine persistently reduced SBP from
4 to 42 wk in females: 1231 vs. 1373 and from 4 to 36 wk in males: 1434 vs. 1513
Jens Titze, Nada Cordasic, Claudia Handtrack, Bernd Klanke, Friedrich-Alexander-Univ mmHg (all P0.05). Urinary NOx excretion was significantly increased at 8 wk, i.e. 4 wk after
Erlangen-Nurnberg, Erlangen, Germany; Peter Dietsch, Charite, Berlin, Germany; Hubertus stopping molsidomine treatment in males and females (P0.05). At the end of the experiment
Wagner, Federal Cntr for Nutrition and Food, Kulmbach, Germany; Karl F Hilgers; MAP was lower, significantly in females (P0.05). Despite lower MAP, renal vascular
Friedrich-Alexander-Univ Erlangen-Nurnberg, Erlangen, Germany resistance (RVR) increased, significantly in females (P0.05). During acute NOS inhibition at 36
wk in male FHH the increase in RVR was significantly higher (P0.05) in perinatally treated
Water-free Na retention, either by osmotically inactive Na storage, or by osmotically neutral FHH. There were no significant effects on proteinuria or glomerular counts, but perinatal
Na/K-exchange, may play an important role for volume and blood pressure homeostasis. molsidomine caused about 50% decrease in glomerulosclerosis (GS) in males and females
However, the molecular mechanisms of water-free Na retention are unclear. We hypothesized (p0.01). Perinatal NO supplementation had clear programming effects on BP, NOx excretion
that increasing the gene dose of angiotensinogen (Agt) gene would increase the total body Na and development of GS in female and male FHH. In contrast to perinatally treated SHR, delta
content and alter the Na-to-water ratio in mice. From mice carrying either a deletion or a MAP and delta RVR during NOS inhibition in FHH were higher in perinatally treated males
duplication of the Agt gene, we bred fifty-eight animals with one, two, three or four copies compared to controls, supporting that perinatal administration of a NO donor permanently
(Agt-1: n20; Agt-2: n 7; Agt-3: n13; Agt-4: n18) which were fed a normal (0.6%) salt increases NO dependency of BP and RVR in FHH. Despite lower BP, RVR tended to increase,
diet. At the end of the experiment, the carcasses were ashed and the total body Na, K, and possibly indicating restoration of preglomerular resistance, either by structural adaptation or
water content was measured. Increasing the Agt gene dose increased the total body Na increased activity of a vasoconstrictor.
(0.1340.028 versus 0.1440.029, versus 0.1660.017, versus 0.1740.021 mmol/g dry
weight in Agt-1, Agt-2, Agt-3 and Agt-4 mice, respectively; p0.05), while the total body water
content was only moderately increased (0.6060.046 versus 0.6310.059, versus 55
0.6450.013, versus 0.6520.033 ml/g wet weight; p0.05). As increases in total body Na Effect of Cardiomyocyte Overexpression of Angiotensin II Type 2 Receptor
were not balanced by a corresponding K loss in the mice, the presence of increasing AGT on Cardiac Remodeling and Dysfunction in Mice with Myocardial Infarction
gene copies increased the total body (NaK)-to-water ratio (0.1940.015 versus
0.2000.010, versus 0.2080.013, versus 0.2110.007 mmol/ml; p0.05), indicating
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017

Ying Sun, Oscar A . Carretero, Jiang Xu, Chunxia Lin, Edward G. Shesely, Xiao-Ping Yang;
osmotically inactive Na storage. In separate animals (n218), we confirmed that there was Henry Ford Hosp, Detroit, MI
a small but significant gene-dose effect on systolic blood pressure which increased by 3.51.1
mmHg for each Agt gene copy (p0.05). We conclude that increasing the Agt gene dose leads Angiotensin II (Ang II) acts mainly on two receptors: AT1 and AT2. AT1 is known to mediate most
to total body Na surplus and pronounced osmotically inactive Na storage in the mice. of the biological actions of Ang II, whereas the role of AT2 remains controversial. We previously
Whether pronounced osmotically inactive Na storage in Agt-4 mice is secondary to total body found that lack of AT2 diminished the beneficial cardiac effect of AT1 receptor blockers (ARB),
Na surplus, or directly regulated by the renin-angiotensin-system, remains to be investigated. indicating a cardioprotective role of the AT2 receptor. Using transgenic mice overexpressing AT2
We speculate that osmotically inactive Na storage may contribute to buffer the effect of Na in ventricular myocytes (9 copies of the AT2 transgene, Tg-AT2), we tested the hypothesis that
accumulation on volume retention and blood pressure in this model. cardiac remodeling and dysfunction post-myocardial infarction (MI) would be less severe and
the response to ARB would be greater in Tg-AT2 mice. Both Tg-AT2 and their wild-type
littermates (WT) were divided into 1) sham MI; 2) MI vehicle; and 3) MI ARB (losartan, 30
53 mg/kg/day, via drinking water). Treatment were started 4 weeks after MI and continued for 8
P38 Map Kinase Inhibition Ameliorates Cardiac Damage without Affecting weeks. Systolic blood pressure (SBP) was measured weekly by tail cuff. Left ventricular ejection
Albuminuria in Angiotensin (Ang) II-Induced End-Organ Damage fraction (EF) and systolic (LVDs) and diastolic dimension (LVDd) were evaluated by echocar-
diography. In sham-MI, these parameters did not differ between strains. MI increased LVW and
Joon-Keun Park, Dept of Nephrology, Hannover Med Sch, Hannover, Germany; Ralf chamber dimensions and decreased EF to a similar extent in both strains. Losartan decreased
Dechend, Robert Fischer, Erdenechimeg Shagdarsuren, Andrej Gapeljuk, Maren Wellner, LV weight (LVW), LVDd and LVDs and increased EF in WT and these effects were diminished
HELIOS, Franz-Volhard Clinic, Charite, Berlin, Germany; Silke Meiners, Dept of Cardiology, in Tg-AT2 mice (Table). Our data suggest that overexpression of the AT2 receptor only in
Charite, Berlin, Germany; Petra Gratze, Nidal Al-Saadi, Anette Fiebeler, HELIOS, cardiomyocytes does not provide cardiac protection, nor aggravate cardiac dysfunction and
Franz-Volhard Clinic, Charite, Berlin, Germany; Jeffery B Madwed, Boehringer Ingelheim remodeling post-MI; however, it diminishes the cardioprotective effect of AT1 blockade. The
Pharmaceuticals, Ridgefield, CT; Alexander Schirdewan, HELIOS, Franz-Volhard Clinic, precise mechanism needs to be studied further.
Charite, Berlin, Germany; Hermann Haller, Dept of Nephrology, Hannover Med Sch,
Hannover, Germany; Friedrich C Luft, HELIOS, Franz-Volhard Clinic, Charite, Berlin, Sham MI MIvehicle MI losartan
Germany; Dominik N Muller; Max-Delbruck Cntr, Berlin, Germany WT TG WT TG WT TG

Ang II induces cardiac and renal damage. We investigated whether or not p38 inhibition LVW (mg/10g) 321 331 431* 442* 391# 412
ameliorates end-organ damage in a rat model with high Ang II levels. We studied cardiac and EF (%) 782 781 373* 353* 484# 362
renal damage in double transgenic rats harboring both human renin and angiotensinogen genes LVDd (mm) 2.70.1 2.50.1 4.10.2* 4.20.2* 3.50.2# 4.30.1
(dTGR) in protocol I from week 4 to 7 and protocol II from week 4 to 8, with or without p38 LVDs (mm) 1.20.1 1.20.1 3.00.2* 3.20.2* 2.40.3# 3.40.1
inhibitor (p38i; 30 mg/kg/d in the diet) treatment. Non-transgenic Sprague-Dawley (SD) rats *p0.01 vs sham MI within strains; #p0.05 vs MIvehicle within strains;
were controls. Systolic blood pressure (BP) was moderately increased at week 5 (1613 mm
Hg) and reached 2044 mm Hg at week 7 in untreated dTGR. P38i treatment partially reduced p0.05 vs WT-MI losartan
BP (1667 mm Hg), while SD were normotensive. Albuminuria was not different between
untreated and p38i treated dTGR (335 vs. 305 mg/d), but increased compared to SD
(0.30.09 mg/d). While cardiac hypertrophy index (heart weight/tibia) was unchanged in 56
untreated and p38i-treated dTGR (3106 vs. 3076 mg/cm), the beta-MHC expression of Genetic Variation at the CYP11B Locus Accounts for Heritabilities of
p38i-treated hearts was significantly lower compared to dTGR, indicating a delayed isoform Aldosterone Metabolite (THAldo) Excretion and 11Beta-Hydroxylase Activity
switch to the fetal phenotype. Fibrosis, macrophage infiltration, TNF- and IL-6 immunostain-
ing were significantly reduced in the heart by p38i, but only slightly reduced (IL-6, TNF- and Marie Freel, Univ of Glasgow, Glasgow, United Kingdom; Bernard Keavney, Peter Avery,
macrophages) or not (fibrosis) in the kidney. Vascular damage seemed to be improved in the Univ of Newcastle, Newcastle, United Kingdom; Bongani Mayosi, Univ of Cape Town, Cape
heart and the kidney. In protocol II, we focused on mortality and arrhythmogenic electrical Town, South Africa; Nicole Gaukrodger, Helen Imrie, Michelle Baker, Univ of Newcastle,
remodeling. Mortality of untreated dTGR was 100% (10/10) at week 8, whereas only one of 10 Newcastle, United Kingdom; Robert Fraser, Mary Ingram, Univ of Glasgow, Glasgow, United
died in the p38i group (10%). Cardiac Magnetic Field Mapping showed prolongation of Kingdom; Hugh Watkins, Martin Farrall, Univ of Oxford, Oxford, United Kingdom; Eleanor
depolarization and repolarization in untreated dTGR compared to SD (QRS 24.30.4 ms vs. Davies, John Connell; Univ of Glasgow, Glasgow, United Kingdom
18.70.8 ms, QT 118.614.3 ms vs. 821.7 ms), which was partially reduced by treatment
with p38i (QRS 22.60.3 ms, QT 99.33 ms). Our findings indicate that p38i improved Aldosterone is a key cardiovascular hormone: 15 % of hypertensives have altered aldosterone
survival, vascular, and cardiac damage/arrhythmogenic electrical remodeling, with a slight regulation, defined by a raised ratio of aldosterone to renin. However, the causes of aldosterone
effect on BP. In contrast, renal damage was not ameliorated to the same extent. Therefore, p38 excess are not understood. Polymorphic variation in the gene encoding aldosterone synthase
inhibition appears to benefit the heart more than the kidney in this model. (CYP11B2) is associated with hypertension, but the best characterised intermediate phenotype
is a relative reduction in efficiency of 11-hydroxylation (conversion of deoxycortisol to
cortisol), which reflects function of the enzyme,11-hydroxylase, encoded by the adjacent
54 gene (CYP11B1). This is expressed in zona fasciculata, and is not involved in aldosterone
Long-Term Effects of Perinatal Nitric Oxide Supplementation on Blood synthesis. To characterise better the genetic regulation of aldosterone synthesis we have used
Pressure and Glomerulosclerosis in Fawn-Hooded Hypertensive Rats a family study to define heritability of steroid phenotypes and identify key genetic determinants.
We genotyped 6 polymorphisms in CYP11B2 and 3 in CYP11B1 in 248 nuclear families and
Maarten P Koeners, Branko Braam, Hein A Koomans, Roel Goldschmeding, Jaap A Joles; measured urinary excretion rates of the major metabolites of aldosterone (THAldo), deoxycor-
UMC, Utrecht, The Netherlands tisol (THS) total cortisol (F) and androgens in 573 subjects from 105 families. The efficiency of
11-hydroxylation, previously noted to be associated with CYP11B2, was assessed by the
Perinatal treatments, that supposedly increase NO bioavailability, persistently reduced adult THS/F ratio. THAldo and THS/F were highly heritable (p0.0001). THAldo excretion and THS/F
blood pressure (BP) in SHR (Racasan ea 2004). Whether such perinatal treatment can also associated most strongly with polymorphisms in CYP11B1 (exon 1 and intron 3) (0.001).
prevent hypertension and associated renal injury in rats with a different genetic background is THAldo excretion was closely correlated with F, androgens and THS/F (all p0.001). We have
CHBPR ConferenceOral Presentations e37
shown, for the first time, that aldosterone production is heritable, reflecting genetic regulation,
and confirmed the heritability of THS/F, the index of 11-hydroxylation. The same polymor-
phisms in CYP11B1 account for variability in THAldo excretion and reduced efficiency of
11-hydroxylation, consistent with the hypothesis that a genetically determined change in
11hydroxylase efficiency leads to an increase in adrenal ACTH drive that, over years, amplifies
aldosterone production. This proposal is strongly supported by the correlations demonstrated
between THAldo and ACTH-dependent steroids (cortisol and androgens) and between THAldo
and the index of 11hydroxylase efficiency. These data provide novel insights into the possible
origins of aldosterone-associated hypertension.

57
Adrenal Steroids and the Metabolic Syndrome in African Americans

Srividya Kidambi, Jane M Kotchen, Clarence E Grim, Shanthi Krishanswami, Hershel Raff,
Theodore A Kotchen; Med College of Wisconsin, Milwaukee, WI

The Metabolic Syndrome describes the cluster of hypertension with metabolic CVD risk factors.
African Americans have a high prevalence of hypertension-related CVD and adrenal cortical
adenomas/hyperplasia. We evaluated the hypothesis that adrenal steroids are associated with
metabolic syndrome risk factors in African Americans. Ambulatory blood pressures, anthropo- 59
metric measurements, and standardized measurements of PRA, aldosterone, fasting lipids,
glucose, and insulin were obtained in 261 African Americans (48% hypertensive, 47% female),
Rapid Aldosterone-Mediated Effects in Vascular Smooth Muscle Cells
studied on an inpatient CRC after discontinuing antihypertensive and lipid lowering medications
Robert Gros, Qingming Ding, Souzan Armstrong, Caroline ONeil, J. Geoffrey Pickering, Ross
for 1 and 4 weeks, respectively. Hypertension was defined as an average ambulatory blood
D Feldman; Robarts Rsch Institute, London, Canada
pressure 130/85 mmHg. Insulin resistance was estimated with the Homeostasis Model
Assessment (HOMA). Late night and early morning salivary cortisol, and 24-hour urine free
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It has been increasingly appreciated that aldosterone (ALDO) elicits acute vascular effects
cortisol excretion were measured in a sub sample of 68 subjects (41% hypertensive). Results
through non-genomic signalling pathways. Our previous studies demonstrated that ALDO
are described in the table below.
attenuated phenylephrine-mediated constriction in intact vessels (via PI3 kinase-dependent
METABOLIC/HORMONAL TRAITS IN STUDY SUBJECTS NOS activation) but enhanced vasoconstrictor responses in endothelium-denuded arteries. To
determine the mechanism of this contractile response, we assessed the effect of ALDO on
Normotensives Hypertensives p-value
myosin light chain (MLC) phosphorylation and contraction in clonal adult human vascular
Age (years) 42 1 SE 45 1 0.01 smooth muscle cells (HITC6). Acute incubations of HITC6 cells with increasing doses of ALDO
BP (mmHg) 115 1/69 1 147 1/89 1 (0.1100 nmol/L) mediated a dose-dependent increase in MLC phosphorylation. ALDO (100
Waist circumference (cm) 87 1 94 1 0.0001 nmol/L)-mediated MLC phosphorylation was inhibited by pretreatment with either spironolac-
Plasma glucose (mg/dl) 88 1 90 1 ns
tone (10 mol/L) or the PI3 kinase inhibitor, LY294002 (50 mol/L). These rapid effects of
Plasma insulin (mIu/ml) 11.2 0.5 12.7 0.5 0.01
Total cholesterol (mg/dl) 175 3 185 4 0.05 ALDO on MLC phosphorylation were mimicked by 17-estradiol (100 nmol/L) and hydrocor-
LDL (mg/dl) 108 3 117 3 0.04 tisone (100 nmol/L). Paralleling the effects on MLC phosphorylation, ALDO-mediated a
HDL (mg./dl) 50 1 47 1 0.01 dose-dependent contraction response in HITC6 cells (EC50 of 19910 pmol/L, Emax 596%
Triglycerides (mg/dl) 93 4 105 5 0.09 of ionomycin-mediated contraction), that was inhibited by pretreatment with spironolactone as
HOMA index 1.4 0.1 1.6 0.1 0.01 well as by LY294002. In addition, comparable contractile responses were seen with both
Plasma Aldosterone (ng/dl) 6.3 0.4 8.4 0.4 0.0001 17-estradiol (EC50 of 9.20.1 nmol/L, Emax 778% of ionomycin-mediated contraction) and
PRA (ng/ml/hr) 1.7 0.2 1.1 0.1 0.01
hydrocortisone (EC50 of 1.70.1 nmol/L, Emax 599% % of ionomycin-mediated contrac-
Late night salivary cortisol 1.5 0.2 2.3 0.3 0.02
(nmol/L) tion). In total, these data are consistent with a mechanism of acute ALDO-mediated contraction
acting via PI3 kinase-dependent activation. Further, this effect is common to both glucocor-
ticoids and estrogen. Steroid-mediated vasoconstriction may represent an important patho-
Hypertensives had greater waist circumference, less favorable lipid profiles, were more insulin
biological mechanism of vascular disease- especially in the setting of pre-existing endothelial
resistant, had lower PRA and higher plasma aldosterone and late night salivary cortisol
dysfunction.
concentrations. Early morning salivary cortisol and 24-hour urine free cortisol did not differ.
Based on Adult Treatment Panel III criteria, 45% of hypertensive and 4% of normotensive
subjects had the Metabolic Syndrome (p 0.0001). Overall, ambulatory systolic blood pressure
was correlated with plasma aldosterone (r 0.31, p 0.0001) and late night salivary cortisol
(r0.31, p 0.01). The relationship of blood pressure with both aldosterone and cortisol raises
the possibility that adrenal mineralocorticoids and glucocorticoids contribute to hypertension
and associated metabolic CVD risk factors in African Americans. 60
Aldosterone and Salt-Induced Cardiac Fibrosis is Exaggerated in
Angiotensin II Type 1a Receptor Knockout Mice

Shuntarou Kagiyama, Kiyoshi Matsumura, Masayo Fukuhara, Kanae Higuchi, Koji Fujii,
Mitsuo Iida; Kyushu Univ, Fukuoka, Japan
58
Aldosterone Deficiency Attenuates Angiotensin II-Stimulated Plasminogen Background: Aldosterone infusion with high salt treatment induces interstitial and perivascular
Activator Inhibitor-1 Gene Expression fibrosis in the heart of rats. Several studies reported that aldosterone enhanced angiotensin II
(AngII) induced proliferation and increased the expression of AngII receptor mRNA and AngII
James M Luther, Zuofei Wang, Ji Ma, Vanderbilt Univ, Nashville, TN; Natalia Makhanova, binding in vascular smooth muscle cells. To investigate the role of AngII type 1a receptor
Hyung-Suk Kim, Univ of North Carolina, Chapel Hill, NC; Nancy J Brown; Vanderbilt Univ, (AT1aR) in aldosterone and salt (Ald-NaCl)-induced cardiac fibrosis, we used AT1aR knockout
Nashville, TN mice (AT1aR-KO). Methods and Results: Aldosterone (0.15 g/hr) was infused subcutane-
ously with 1% NaCl (Ald-NaCl) in drinking water on AT1aR-KO or wild type mice (Wt). To
To test the hypothesis that Ang II increases plasminogen activator inhibitor-1 (PAI-1) expression examine the role of NaCl on cardiac fibrosis, we fed some of the AT1aR-KO receiving
mediated by aldosterone, we administered Ang II (600ng/kg/min IV x 4 hrs) or vehicle to male aldosterone with tap water (Ald-H2O). After Ald-NaCl treatment, systolic BP in both strains was
wild type (WT) and aldosterone synthase deficient (KO) mice, both on a C57BL/6 background significantly higher than that in mice not receiving aldosterone, but systolic BP in Ald-NaCl-
(n5/group). Two additional KO groups were given aldosterone (500 ng/d via minipump) for 7 treated AT1aR-KO was still significantly (p0.05) lower than that in Wt. Left ventricular
days prior to study. Carotid arterial and jugular venous catheters were implanted 35 days prior weights/body weights in Ald-NaCl-treated AT1aR-KO and Wt were significantly higher than in
to study. Baseline systolic blood pressure in KO mice was lower than in WT (1253 vs 1113 the mice receiving only NaCl but did not differ between the strains.Severe cardiac fibrosis was
mmHg, P0.006) and K (5.50.2 vs. 4.70.2 mmol/L, P0.02) was higher. Urinary K seen only in Ald-NaCl-treated AT1aR-KO and not in Ald-NaCl-treated Wt. Ald-H2O-treated
(38040 mol/24hr) and Na (300130 mol/24hr) excretion was similar between groups. AT1aR-KO did not show any cardiac fibrosis. We performed RT-PCR to investigate the effect of
Plasma BUN (263 vs. 171 mg/dL, P0.01) was higher, while Na (142.10.6 vs. Ald-NaCl treatment on the expressions of mRNA of sodium-handling related genes in cardiac
146.81.8 mmol/L, P0.004) and Cl- (1120.7 vs. 115.71.6 mmol/L, P0.03) were lower tissues. Epithelial Na channel and subunit were not detected, and subunit was detected
in KO mice. Aldosterone replacement normalized SBP (1206 mmHg), K (4.30.2, P0.001 but did not differ in strain or treatment. Aldosterone increased the expression of Na-K
vs. KO), Na, and BUN. Ang II infusion increased SBP in WT (19.67 mmHg) and KO (16.49), ATPase 1 subunit in Wt but not in AT1aR-KO. The expression of 2 subunit was decreased
and increased SBP to a greater extent in KO mice after aldosterone replacement (34.84.4 in AT1aR-KO, but 1 subunit was unchanged. Na/Ca2 exchanger was decreased in
mmHg, P0.05 for time x group). Body weight and relative heart weight (5.40.4 vs. 5.70.2 AT1aR-KO and was further decreased by aldosterone treatment.Conclusions: These results
mg/g) were similar between groups while relative kidney weight was lower in KO (6.10.3 vs. suggest that the Ald-NaCl-induced cardiac fibrosis required both aldosterone and NaCl
7.70.4 mg/g, P0.003). Ang II increased cardiac PAI-1 gene expression in WT but not KO administration. As cardiac fibrosis was exaggerated in AT1aR-KO mice, AT1aR may not be
(Figure 1). Aldosterone replacement normalized Ang II-stimulated cardiac PAI-1 gene expres- essential for this phenomenon. Although precise mechanisms remain unresolved, alterations in
sion. Aldosterone enhances Ang II-stimulated PAI-1 gene expression in vivo. These data have sodium handling genes transcription were present and might be implicated in the present
implication for the development of pharmacological inhibitors of aldosterone synthesis. findings.
e38 Hypertension Vol 48, No 4 October 2006

61 experiments were performed in the presence of catalase (100 U/ml). We found that O2-
Comparative Effects of Mineralocorticoid Receptor Antagonism and treatment selectively increased PKC alpha activity by 177% (from 4.7 1.2 to 13.2 1.7 A.U;
Aldosterone Synthase Inhibition on Angiotensin II-Induced End Organ p0.02; n5) and PKC delta activity by 37% (from 12.6 1.0 to 17.3 1.4 A.U.; p0.05;
n5). O2- did not activate the other isoforms tested (beta, gamma, epsilon, theta, iota, lambda
Damage
and zeta). In isolated perfused tubules, O2- treatment increased JCl by 42 13% (p0.05;
n5). In the presence of the PKC alpha/beta-selective inhibitor Go6976, O2- did not significantly
William B Lea, Eun Soo Kwak, Agnes B Fogo, Nancy J Brown; Vanderbilt Univ, Nashville, TN
increase JCl (from 132.2 24.5 to 136.7 28.2 pmol/min/mm; n5). Pre-treatment of thick
ascending limb tubules with rottlerin induced cell detachment from the basal membrane, cell
Mineralocorticoid receptor antagonism or the aldosterone synthase inhibition reduces Ang
swelling and necrosis at all concentrations tested. We conclude that: 1) O2- stimulates specific
II-induced end organ damage. However, the effect of the two pharmacologic strategies has
isoforms of PKC in the thick ascending limb; 2) activation of PKC alpha is required for O2- to
never been compared. We compared effects of spironolactone (5.8mg/kg/d) and FAD286
stimulate NaCl absorption in the thick ascending limb; and 3) the role of PKC delta in O2-
(4mg/kg/d) in uninephrectomized Sprague-Dawley rats treated with high sodium intake (1%
-stimulated NaCl absorption in the thick ascending limb could not be tested because it appears
saline) and either saline or Ang II (1/h) via osmotic minipump for 8 weeks. Spironolactone or
to be necessary for cell survival.
FAD286 prevented the hypertensive response to uninephrectomy alone but did not attenuate
the SBP response to Ang IIuninephrectomy. FAD286, but not spironolactone, significantly
decreased plasma aldosterone during Ang II infusion. Serum potassium, sodium and urine 64
potassium and sodium excretion were comparable in spironolactone and FAD286 treatment
groups. Ang II induced cardiac hypertrophy and either spironolactone or FAD286 prevented this
cGMP Decreases Surface NKCC2 Levels in Thick Ascending Limbs
effect. Unilateral nephrectomy caused hypertrophy of the remaining kidney, which was
Gustavo R Ares, Pablo A Ortiz; Henry Ford Hosp, Detroit, MI
increased by Ang II. Spironolactone and FAD286 prevented Ang II induced renal hypertrophy,
albuminuria and azotemia. Mineralocorticoid receptor antagonism and aldosterone synthase
NaCl absorption in the TAL is mediated by the apical Na/K/2Cl cotransporter, NKCC2. We
antagonism similarly protect against Ang II induced cardiac hypertrophy and renal injury
showed that NKCC2 levels in the apical membrane determine NaCl absorption by the TAL. Nitric
through pressure-independent mechanisms. Aldosterone synthase inhibition also attenuated
oxide and atrial natriuretic peptide decrease NaCl absorption in the thick ascending limb (TAL)
Ang II-induced aortic medial thickening.
via cGMP. However, the mechanism by which cGMP decreases NaCl absorption in TALs is not
Uninephrectomy Uninephrectomy known. We hypothesized that cGMP decreases NKCC2 levels in the apical membrane of TALs.
Uninephrectomy Uninephrectomy Uninephrectomy Ang II Ang II We used surface biotinylation and Western blot to measure NKCC2 levels in the apical
Sham Uninephrectomy Spironolactone FAD286 Ang II Spironolactone FAD286 membrane of rat TAL suspensions. Under basal conditions, surface NKCC2 levels were 3.4
SBP 157.72.7 168.02.4* 159.12.2 162.92.5 172.22.5* 171.72.5* 167.42.4*
0.7% (n9) of total NKCC2, similar to what we previously reported. We first tested whether the
(mmHg) membrane-permeant cGMP analog, dibutyryl cGMP (db-cGMP) could decrease surface NKCC2
levels. Adding db-cGMP (100 M) to the bath for 20 min decreased surface NKCC2 levels by
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Aldosterone (pg/ml) 20875 16840 14218 13136 472159* 374160 13910


HW/BW (mg/g) 3.50.2 3.80.3 3.80.3 3.80.1 4.90.2* 3.80.1 4.10.3 26 7% (n 4, p0.02) whereas db-cGMP at 500 M decreased surface NKCC2 by 46
Aortic media (m) 139.010.1 149.26.8 171.215.8 141.55.4 181.315.2* 181.812.6* 151.816.6 10% (n 5, p0.01). A higher concentration of db-cGMP (1 mM) did not decrease surface
KW/BW (mg/g) 3.80.3 5.60.2* 5.50.3* 5.80.4* 6.80.6*|| 5.30.2* 5.80.3*
NKCC2 levels further (n5). Parallel control experiments showed no biotinylation of intracel-
BUN (mg/dL) 22.52.1 24.92.0 26.61.1 26.21.9 46.78.0* 23.80.9 30.80.6
Albumin/Cr (g/mg) 460226 1027407 28272514 1062488 35831606* 26211074 25371694
lular proteins, indicating that only surface proteins were being biotinylated. To test whether
cGMP regulates NKCC2 trafficking rather than total NKCC2 expression, we measured the effect
*P0.01 versus sham, P0.05 versus uninephrectomy, uninephrectomyspironolactone, or uninephrectomyFAD286, P0.05 versus
uninephrectomyAng IIFAD286, P0.05 versus uninephrectomyAng IIspironolactone, || P0.05 versus uninephrectomyAng IIspironolactone,
of db-cGMP (500 M) on total NKCC2. We found no change in total NKCC2 expression
P0.05 versus uninephrectomyFAD286 compared to basal (basal: 100%, db-cGMP: 114 14 %, n 4, n.s.). We concluded that
cGMP decreases NKCC2 levels in the apical membrane of TALs by regulating NKCC2 trafficking.
This may be the primary mechanism by which NO and other natriuretic hormones that act via
cGMP inhibit NaCl absorption by the TAL.
62
Synergetic Interaction between Aldosterone and Angiotensin II Induces
Activation of c-Src and NADPH Oxidase but not of MLC in Vascular Smooth 65
Muscle Cells Extracellular Renal Cyclic Gmp is Required for Nitric Oxide-Induced
Natriuresis in the Rat
Augusto C Montezano, Glaucia E Callera, Ottawa Health Rsch Institute, Ottawa, Canada;
Ernesto L Schiffrin, Lady Davis Institute for Med Rsch, Montreal, Canada; Rhian M Touyz; Farah Ahmed, Nancy L Howell, Brandon A Kemp, Helmy M Siragy, Robert M Carey; Univ
Ottawa Health Rsch Institute, Ottawa, Canada Virginia Sch Med, Charlottesville, VA
Aldosterone (Aldo) exerts a synergistic mitogenic effect with angiotensin II (Ang II) through Previous studies from our laboratory have demonstrated that extracellular guanosine cyclic 3,
ERK1/2 activation in vascular smooth muscle cells (VSMC). Whether similar interactions 5-monophosphate (cGMP) inhibits Na transport directly in proximal tubule cells and that renal
influence contractile and redox signaling pathways remain unclear. Here we investigated the interstitial (RI) cGMP induces natriuresis and modulates pressure-natriuresis in vivo in the rat.
cross-talk of c-Src-sensitive NADPH oxidase-generated O2- signaling between Aldo and Ang II The present study addresses the hypothesis that nitric oxide (NO)-induced natriuresis is
and assessed whether this interaction influences pro-contractile events. Cultured rat mesen- abolished when cGMP is prevented from being transported outside its synthesizing cells in vivo.
teric vascular smooth muscle cells were studied. Activation of c-Src was determined by Sprague-Dawley rats (N13) were anesthetized and uninephrectomized and the remaining
immunoblotting using a phospho-specific antibody. NADPH-dependent generation of O2- was kidney was implanted with a renal cortical interstitial micro-infusion catheter and a separate
measured by enhanced lucigenin (5mol) chemiluminescence. Phosphorylation of myosin light microdialysis probe for measuring RI cGMP. Rats were studied during a 1 h control period
chain (MLC) was assessed as a molecular signaling marker of contraction. Whereas high (vehicle infusion) following which they received a RI infusion of NO donor S-nitroso-N-
concentrations (10-8 mol/L) of Aldo and Ang II significantly increased activation of c-Src acetylpenicillamine (SNAP; 0.12 nmol/kg/min; N7) or SNAP combined with organic anion
(2-fold) and NADPH oxidase (2-fold), low concentration (10-10 mol/L) of Aldo and Ang II transporter inhibitor probenecid (PB; 10 g/kg/min; N6) for two consecutive 1 h experimental
were without significant effect. High dose Ang II and Aldo induced phosphorylation of MLC. periods, followed by a 1 h post-control period when vehicle was infused. All RI infusions were
Co-stimulation of VSMCs with combined low dose Aldo and Ang II significantly increased c-Src at 2.5 l/min and blood pressure (BP), urinary Na excretion (UNaV) and RI cGMP were
phosphorylation (0.5-fold above basal, p0.05) and NAD(P)H-dependent generation of O2- quantified for each control and experimental period. In response to intrarenal infusion of SNAP
(1-fold, p0.05). MLC phosphorylation was not affected by low dose Aldo/Ang stimulation. alone, UNaV increased from a control of 0.05 0.02 to 0.12 0.06 mol/min (PNS) at 1 h
c-Src phosphorylation was inhibited by a selective AT1 receptor antagonist, Ibersartan (10-6 and to 0.14 0.03 mol/min (P0.01) at 2 h, followed by a reduction to 0.07 0.03
mol/L), or a mineralocorticoid receptor antagonist, eplerenone (10-6 mol/L). Our results suggest mol/min (PNS) during the post-control period. In contrast, SNAP failed to increase UNaV
that Aldo exerts a synergistic c-Src/redox signaling effect with Ang II. However, signaling events when co-infused with PB during either experimental period. Indeed, PB decreased UNaV from
associated with activation of MLC, typically involved in VSMC contraction, are independent of 0.07 0.01 to 0.03 0.01 mol/min during the second SNAP infusion period (P0.01 from
synergistic actions between Aldo and Ang II. Whereas synergism may be important in mitogenic SNAP alone). SNAP alone increased RI cGMP from 2.2 0.6 to 3.6 1.1 pmol/ml (PNS) in
responses in VSMCs, pathways involved in contraction may not be. These findings indicate the first experimental period and to 4.5 1.3 pmol/ml (P0.01) during the second
differential regulation of Ang II-mediated signaling events by Aldo. experimental period. In contrast, PB abolished the increase in RI cGMP in response to SNAP
alone in both experimental periods (P0.01 from SNAP alone in the second period). The data
demonstrate that export of cGMP from its synthesizing cells into the RI compartment is required
63 for the natriuretic action of NO donor SNAP. Extracellular RI cGMP mediates NO-induced
PKC Alpha Activation is Required In O2- -Stimulated NaCl Absorption in natriuretic responses.
Thick Ascending Limb
Guillermo B Silva, Pablo A Ortiz, Jeffrey L Garvin; Henry Ford Hosp, Detroit, MI 66
Increased Renal Na Transporters and Altered AT1R and Dopamine
Superoxide (O2-) is an important regulator of kidney function. The thick ascending limb Receptors Are Associated with Hypertension in D3 Dopamine Receptor
reabsorbs 20 30% of the filtered load of NaCl. Abnormal sodium retention in this segment has Deficient Mice
been shown to be involved in hypertension. O2- stimulates NaCl absorption by the thick
ascending limb by enhancing Na/K/2Cl- co-transport activity. Protein kinase C (PKC) Xiaoyan Wang, Ines Armando, Laureano Asico, John E Jones, Zheng Wang, Van Anthony M
stimulates thick ascending limb transport. Thus we hypothesized that O2- stimulates NaCl Villar, Crisanto S Escano, Pedro A Jose; Georgetown Univ Med Cntr, Washington, DC
absorption by selective activation of specific PKC isoforms. To test this, we measured the effect
of O2- on activation of classical, novel and atypical PKC isoforms by membrane fractionation and D3 dopamine receptor (D3R) deficient mice (D3-/-) have impaired ability to excrete a sodium
Western blot. In isolated perfused tubules we measured the effect of the PKC alpha/beta- load and renin-dependent hypertension. Lack of one dopamine receptor gene may alter the
selective inhibitor Go6976 and the PKC delta-selective inhibitor rottlerin on O2--induced Cl- expression of other G protein-coupled receptors (GPCR) and renal Na transporters which may
absorption (JCl). Medullary thick ascending limbs were exposed to O2- by adding xanthine contribute to hypertension. We studied the expression of dopamine receptors and renal Na
oxidase (1 mU/mL) and hypoxanthine (0.5 mmol/L) to the bath. To avoid formation of H2O2, transporters in D3-/- and their wild type (D3/) littermates. On a normal NaCl (0.8%) diet,
CHBPR ConferenceOral Presentations e39
systolic blood pressure (SBP) (telemetry), was higher in D3-/- than in D3/ (night time: vasculature may lead to vascular dysfunction and hypertension. To test this hypothesis, we
1323 vs. 1223 mm Hg; n 7; p 0.001). Renal D1R and D2R proteins were the same; generated transgenic mouse models targeting dominant negative mutants of hPPAR (either
D4R was decreased (599%) while D5R was increased in D3-/- (16923%) relative to P467L or V290M) to the vascular E and SM using the vascular E-specific Ve-cadherin promoter
D3/ (10010%; p0.05). Infusion of SCH 23390, a D1/D5R antagonist (120 ng/k/min x or the SM-specific SM myosin heavy chain promoter, respectively. The dominant negatives
1h) into anesthetized mice increased BP in D3-/- (from 1131 to 1283, n3; p0.05) but should produce cell-specific knock-downs of PPAR. Expression of each transgene was
not in D3/, suggesting that D5Rs partially compensated for the lack of D3Rs. AT1R confirmed and differentiated from the endogenous PPAR via RNase protection assay. No
expression (autoradiography) in renal proximal tubules was greater in D3-/- (955 fmol/mg expression of the transgene was observed in non-transgenic (NT) littermates. Physiologic
prot) than in D3/ (76 5; p 0.05); AT1 receptor expression in other areas was similar. characterization of the E-V290M model revealed significant impairment of E-dependent
On a normal salt diet, NHE3 (major proximal tubule Na transporter) and NCC (distal convoluted relaxation of aorta to acetylcholine (Ach, 454% vs 544% in NT at 10M, n6), whereas
tubule Na transporter) proteins were increased in D3-/- (28427% and 59641%, respec- the response to nitroprusside was normal. In the basilar artery, there was also a trend toward
tively). On a low NaCl diet (0.04%), D3-/- had increased expression of alpha ENaC (39455%) impaired relaxation to Ach (656% vs 767% in NT at 100 M, n7) and the calcium
and gamma ENaC (14418%, 75 kDa, active form). On high NaCl diet (1.6%), NCC ionophore A23187 (585% vs 676% in NT at 0.1 M, n7). Interestingly, preliminary
(20924%) and alpha ENaC (25839%) proteins were increased in D3-/- (all data relative to studies suggest that the impairment in endothelial function was greater in females than males.
D3/). Our results suggest that by direct or indirect mechanisms the D3R regulates the renal We also examined contraction of the aorta in response to several vasoconstrictors. Contraction
expression of other GPCR (e.g., D4R, D5R, and AT1R) and Na transporters. The high BP in to PGF2 was greater in E-V290M (1.70.1g, n7) than NT littermates (1.40.1g, n6).
D3-/- is mitigated by upregulation of D5R. Eventual BP is the result of the interaction of several Contraction of the aorta in females in response to endothelin-1, a PPAR target gene, was
genes (e.g., dopamine receptors and AT1R), consistent with the polygenic regulation of BP. greater in E-V290M (585 mg, n5) than in NT (328 mg, n3). Our data suggest that
PPAR plays and important role in the regulation of vascular tone. Studies identifying the
specific molecular and signaling pathways regulating vascular function, and physiological
67 analysis of SM-P467L and SM-V290M mice are currently in progress.
Cholesterol Facilitates Hypertension by Stimulating the Renal Epithelial
Sodium Channel
70
He-Ping Ma, Jing Wang, You-You Liang, David G Warnock; Univ of Alabama at Birmingham, Ac-SDKP Attenuates Angiotensin II-Induced Collagen Deposition and
Birmingham, AL Macrophage Infiltration in the Aortic Media

Hypertension can be eventually developed due to the well-known effect of cholesterol on Chunxia Lin, Xiaoping Yang, Tangdong Liao, Martin D Ambrosio, Oscar A Carretero; Henry
vascular walls. However, a significant portion of the patients with hypercholesterolemia Ford Hosp, Detroit, MI
appears to have hypertension before any morphological changes in vascular walls. This clinical
observation indicates that cholesterol may also cause hypertension by affecting an organ rather We previously reported that N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) inhibits interstitial
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017

than cardiovascular system. Since excessive sodium reabsorption by the kidney due to and perivascular fibrosis in the heart and kidney and this effect may be partly due to inhibition
enhanced activity of the renal epithelial sodium channel (ENaC) can result in hypertension, as of macrophage infiltration and collagen deposition. In the present study, we hypothesized that
seen in Liddles syndrome, we hypothesized that cholesterol might facilitate hypertension by in angiotensin (Ang II) - induced hypertension, Ac-SDKP prevents vascular inflammation and
stimulating the renal ENaC. To test this hypothesis, patch-clamp experiments and transepi- fibrosis by decreasing macrophage infiltration and collagen deposition. SD rats were divided
thelial current measurement were performed by using A6 distal nephron cells cultured on into 4 groups and treated for 1 week with: 1) vehicle; 2) Ac-SDKP (800 g/kg/D, via osmotic
permeable support. The data demonstrated that application of cholesterol (final concentra- minipump); 3) Ang II (750 g/kg/D, via osmotic minipump); or 4) Ang IIAc-SDKP. We found:
tion 50 g/ml) to the basolateral bath significantly increased ENaC open probability from 1) Ang II significantly increased systolic blood pressure (SBP), aortic medial area (hematoxylin
0.28 0.12 to 0.63 0.17 in five cell-attached patches (p 0.01) and also elevated and eosin staining), collagen I and III deposition (picrosirius red staining) and macrophage
amiloride-sensitive current across A6 cell monolayer (n 24; P 0.001). However, number (immunohistochemical staining) in the aortic media; 2) Ac-SDKP had no effect on Ang
application of cholesterol to the apical bath did not affect ENaC open probability (0.31 0.09 II-induced hypertension and medial hypertrophy; however, 3) Ac-SDKP greatly reduced Ang
versus 0.29 0.10) in four cell-attached patches and did not affect amiloride-sensitive current II-induced collagen I and III deposition and macrophage infiltration in the aortic media (see
across A6 cell monolayer (n 18). To monitor cholesterol transport across the cell membrane, table). These data suggest that in Ang II-induced hypertension, Ac-SDKP inhibits macrophage
confocal microscopy experiments were performed by using a fluorescence-labeled cholesterol infiltration and collagen deposition in the aortic media via a hypertension-independent
(NBD-cholesterol). The data demonstrated that there was an acute cholesterol uptake across mechanism.
the basolateral membrane, but not the apical membrane. These data together suggest that
basolateral cholesterol uptake, somehow, significantly stimulates ENaC in A6 distal nephron vehicle Ac-SDKP Ang II Ang IIAc-SDKP
cells. This finding may uncover a novel pathway for cholesterol to induce hypertension. SBP (6th day,mm Hg) 1191 1214 1897** 19113
Aortic medial area 59.166.00 66.302.11 102.301.66** 87.876.62
(10000, m2)
68 Collagen I (% of medial area) 10.751.66 11.640.40 19.082.03* 9.291.94#
Collagen III (% of medial area) 2.590.20 2.620.36 4.850.62* 1.550.37##
NOS Uncoupling in the Kidney of Dahl S Rats - Role of Dihydrobiopterin Macrophage number 2.370.85 1.870.94 19.380.35** 9.761.72##
(per mm2 of medial area)
Norman E Taylor, Med College of Wisconsin, Milwaukee, WI; Kristopher G Maier, SUNY Values are mean SE, *p0.05, p0.01, Ang II vs vehicle; # p0.05, p0.01, Ang II vs Ang
Upstate Med Univ, Syracuse, NY; Richard J Roman, Allen W Cowley, Jr.; Med College of IIAc-SDKP
Wisconsin, Milwaukee, WI

Nitric oxide synthase (NOS) can paradoxically contribute to the production of reactive oxygen 71
species (ROS) when L-arginine or the cofactor R-tetrahydrobiopterin (BH4) become limited. The Decreased Physiological Levels of Ac-sdkp Increase Perivascular Fibrosis
present study examined whether NOS contributes to superoxide (O2-) production in kidneys of
hypertensive Dahl salt-sensitive rats (SS) compared to an inbred consomic control strain Maria A Cavasin, Tang-Dong Liao, Xiao-Ping Yang, Oscar A Carretero; Henry Ford Health
(SS-13BN) and tested the hypothesis that elevated dihydrobiopterin (BH2) levels are importantly System, Detroit, MI
involved in this process. This was assessed by determining the effects of L-nitroarginine methyl
ester (L-NAME) inhibition of NOS on O2- production and by comparing tissue concentrations of We previously showed that prolyl oligopeptidase (POP) is the enzyme responsible for release of
BH4 and BH2. A RP-HPLC method was applied for direct measurements of BH4 and BH2 using N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) from its precursor thymosin-4, and oral
(S)-tetrahydrobiopterin as an internal standard. O2- concentrations were measured in-vivo treatment with a specific POP inhibitor (POPi) significantly decreased plasma and tissue
from medullary microdialysis fluid using dihydroethidine and in-vitro using lucigenin. The concentrations of Ac-SDKP. Chronic infusion with Ac-SDKP prevents and/or regresses fibrosis
results indicate: 1) O2- levels were doubled in the outer medulla of SS rats fed a 4% salt diet in hypertension and post-myocardial infarction; however, it is not known whether normal levels
and could be inhibited by L-NAME, from 17.72.9 to 11.421.9 RLU/min/mg dry tissue. In of this peptide have a physiological role in preventing collagen deposition. Therefore, we
contrast, L-NAME raised O2- levels in consomic SS-13BN rats from 7.32.1 to 13.12.2 hypothesized that decreasing circulating levels of Ac-SDKP using an oral POPi induces heart
RLU/min/mg dry tissue. 2) SS rats showed a reduced ratio of BH4/BH2 (1.40.2 vs. 5.90.6) and kidney fibrosis. SD rats were divided into two groups: a) control, and b) POPi (S17092) and
in the outer medulla that was driven by increased concentrations of BH2 (14315 vs. 558 killed after 60 days of treatment. Myocardial and renal hydroxyproline were measured, and
pg/mg tissue). 3) A 28% decrease in superoxide dismutase activity together with a 42% tissue slices were stained with picrosirius red to determine collagen deposition. We found that
decrease in catalase activity contributed to elevated ROS in SS samples. Based on the shift of rats treated with POPi had significantly lower cardiac and renal levels of Ac-SDKP compared
BH4 to BH2 and the observation of L-NAME inhibitable O2- production, we conclude that NOS to the controls. POPi significantly increased total collagen content in the heart and kidney as
uncoupling occurs in the renal medulla of hypertensive SS rats fed a high salt diet and well as perivascular fibrosis (PVF). To confirm that the increase in collagen was due to the
contributes to the observed medullary renal injury. decrease in Ac-SDKP and not to an increase in any other POP substrates with potential fibrotic
effects, we measured the neuropeptides arg-vasopressin and substance P in tissue. We found
no changes in concentration in the heart and kidney, although both neuropeptides were
69 significantly increased in the brain. We conclude that Ac-SDKP may participate in the regulation
Vascular Dysfunction in Transgenic Mice Expressing a Dominant Negative of collagen deposition, since decreasing its normal concentration in tissue promotes fibrosis.
Mutant of PPAR
Groups Control POPi (40 mg/kg/day).
Andreas M Beyer, Carmen M Halabi, Henry L Keen, Mary L Modrick, Frank M Faraci, Curt D HEART Hydroxyproline (g/mg tissue) 3.270.3 4.270.3*
Sigmund; Univ of Iowa, Iowa City, IA Cardiac PVF (collagen / vessel area) 0.570.06 0.700.04*
KIDNEY hydroxyproline (g/mg dry tissue) 3.400.15 4.430.24*
Patients carrying dominant negative mutations in the PPAR gene exhibit an attenuation of Renal PVF (coll / vessel area) 0.630.02 0.790.06*
PPAR transcriptional activity and present with insulin resistance and hypertension. Gene HEART Ac-SDKP (pg / mg tissue) 34.33.3 5.71*
targeted mice carrying equivalent dominant negative mutations also exhibit hypertension with KIDNEY Ac-SDKP (pg / mg tissue) 11324 37.25.8*
HEART Arg-vasopresin (fg/mg tissue 173 205
varying degrees of metabolic abnormalities. As PPAR is expressed in both endothelial (E) and KIDNEY Arg-vasopresin (fg/mg tissue 8722 7316
smooth muscle (SM) cells, we hypothesized that diminished activity of PPAR in the
e40 Hypertension Vol 48, No 4 October 2006

Groups Control POPi (40 mg/kg/day).


74
Interferon-Gamma-Mediated Pathway -New Target for Prevention of
HEART Substance P (pg / mg tissue) 0.330.12 0.250.03 Vascular Remodeling after Vascular Injury-
KIDNEY Substance P (pg / mg tissue) 0.100.01 0.100.01

* p 0.05 vs control. Ken Kusaba, Hisashi Kai, Mitsuhisa Koga, Narimasa Takayama, Takahiro Mori, Yusuke Sugi,
Kiyoko Takemiya, Hiroshi Kudo, Daisuke Fukui, Ayami Ikeda, Masayoshi Futamata, Yumiko
Kawai, Nobuhiro Tahara, Hideo Yasukawa, Tsutomu Imaizumi; Kurume Univ, Kurume, Japan

Interferon-gamma is a multi-functional cytokine with both pro- and anti-atherogenic properties.


Thus, it is still controversial whether the net effect of interferon-gamma promotes or attenuates
72 vascular remodeling after vascular injury. In vascular smooth muscle cells (SMCs), interferon-
Inhibition of Progression and Stabilization of Plaques by Therapeutic regulating factor-1 (IRF-1) is induced by interferon-gamma, and in turn upregulates expression
Interferon- Function Blocking in Apoe-/- Mice of inducible nitric oxide synthase (iNOS), which results in SMC proliferation. Recently, we have
shown that blocking of interferon-gamma by overexpressing a soluble mutant of the
Koga Mitsuhisa, Cardiovascular Rsch Institute, Kurume, Japan; Hisashi Kai, Kurume Univ, interferon-gamma receptor (sIFNgR), an interferon-gamma inhibiting protein, regresses and
Kurume, Japan; Mamiko Kai, Fukuoka Univ, Fukuoka, Japan; Nobuhiro Tahara, Kiyoko stabilizes advanced atherosclerotic plaque in apoE-deficient mice. Thus, we investigated the
Takemiya, Hiroshi Kudo, Yusuke Sugi, Takahiro Mori, Narimasa Takayama, Daisuke Fukui, role of interferon-gamma on neointima formation after vascular injury by inhibiting the
Ayami Ikeda, Masayoshi Futamata, Kurume Univ, Kurume, Japan; Yumiko Kawai, Fukuoka interferon-gamma-mediated pathway in vivo. For this purpose, naked DNA plasmid encoding
Univ, Fukuoka, Japan; Hideo Yasukawa, Yasufumi Kataoka, Tsutomu Imaizumi; Kurume the sIFNgR or the empty plasmid (mock) was injected into the thigh muscle of male Wistar rats
Univ, Kurume, Japan 2 days before balloon injury (day -2)(n6/group). sIFNgR gene transfer significantly elevated
serum levels of sIFNgR protein at days 2 to 14. In the mock-treated rats, balloon injury induced
A role of interferon- (IFN) is suggested in early development of atherosclerosis. However, it a transient proliferation of neointimal SMCs with a peak at day 7, assessed by PCNA labeling,
remains unknown whether inhibition of IFN function will prevent progression of advanced and the maximum neointimal thickening was observed at day 14. At day 7, balloon injury
atherosclerotic plaques and stabilize them. Atherosclerotic plaques were induced in ApoE-/- markedly upregulated IRF-1 and iNOS mRNAs in the mock-treated rats. sIFNgR gene transfer
mice by feeding with high-fat diet from 8-week old. IFN function was postnatally inhibited by almost inhibited the balloon injury-induced inductions of IRF-1 and iNOS at day 7. And, the
repeated gene transfers of a soluble mutant of IFN receptors (sIFN-R), an IFN inhibiting number of proliferating neointimal SMCs was reduced by 50% in the sIFNgR-treated rats
protein, into the thigh muscle every 2 weeks. Prophylactic protocol: sIFN-R or the mock relative to the mock-treated rats. Moreover, at day 14, sIFNgR gene transfer prevented
plasmid treatment was initiated at 8-week old (pre-atherosclerotic stage). Mock-treated neointima formation, resulting in 45% reduction of the intima/media area ratio compared with
ApoE-/- mice showed atherosclerotic plaques in the ascending aorta, extending to the aortic mock-treated rats. In conclusion, the role of interferon-gamma was suggested in the neointima
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017

arch, at 12 week old. sIFN-R prevented the early plaque formation, resulting in 60% reduction formation through IRF-1 regulation of iNOS expression and subsequently SMC proliferation in
of the en-face plaque area relative to the mock treatment. Therapeutic protocol: sIFN-R or the balloon-injured artery. The present study provided an insight into a new strategy for prevention
mock treatment was started at 12-week old (atherosclerotic stage). Mock-treated ApoE-/- mice of vascular remodeling after vascular injury by targeting interferon-gamma.
showed marked progression of atherosclerotic plaques, extending to the abdominal aorta, at 16
week old. The plaque of the mock-treated mice consisted of massive lipid core and bulky
macrophage accumulation, but had very thin fibrous cap with little smooth muscle cells, 75
suggesting low plaque stability. sIFN-R prevented further plaque progression, and the en-face Sleep Disordered Breathing in Children with Hypertension
plaque area at 16-week old was similar to the pre-treatment level at 12 week old. Also,
sIFN-R stabilized advanced plaques: sIFN-R decreased the lipid and macrophage accumu- Alisa A Acosta, Kathy Franco, Monesha Gupta-Malhotra, Richard J Castriotta, Ronald J
lations and increased fibrotic area with more smooth muscle cells. Moreover, sIFN-R Portman; Univ of Texas - Houston, Med Sch, Houston, TX
downregulated expressions of pro-inflammatory cytokines (IL-1 and IL-6), chemokines
(MCP-1 and macrophage inflammatory protein-1), vascular cell adhesion molecule-1, and Obstructive sleep apnea (OSA) is known to be a risk factor for hypertension (HTN) in adults, and
matrix metalloproteinases-9 and -13, but upregulated type I pro-collagen. sIFN-R did not few studies have shown a similar association in children. Recently, we have included in our
affect serum cholesterol levels. Accordingly, the role of IFN was suggested in not only the Pediatric Hypertension Clinic protocol a routine screen of risk factors for sleep disordered
early plaque development but also further progression and destabilization of advanced plaques. breathing (SDB). In the past year, 26 out of 350 (7.4%) of the hypertensive patients were
Blocking of IFN function would be a new strategy to inhibition plaque progression and to identified for nocturnal polysomnography based on the following risk factors: snoring, enlarged
stabilize advanced plaques through the anti-inflammatory effects. tonsils, BMI 85th percentile, or nocturnal HTN by ambulatory blood pressure monitoring.
Twenty patients completed nocturnal polysomnography. The mean age at the time of
polysomnography was 12.6 3.97 years (range 4 18 years), 15 were male (75%), all 20 had
a diagnosis of HTN, 11 (55%) also had nocturnal HTN, 17 (85%) had adenotonsillar
hypertrophy, and all 20 patients had a history of snoring. At the time of the initial visit to
73 Pediatric Hypertension Clinic, 17 (85%) had a BMI the 95th percentile, and one was at the
90th percentile. Of the 20 patients who completed the nocturnal polysomnography, 12 (60%)
Differential Effects of Hypertension and Hypercholesterolemia on Carotid had SDB: 7 (35%) had obstructive sleep apnea ( 1 obstructive apnea/hour), 4 (20%) had
Vasa Vasorum Density and Endothelial Function obstructive hypoventilation (maximum pCO2 53 torr during sleep and/or 50 torr for 10%
of sleep), and 1 (5%) had mild SDB (1.5 apneas hypopneas/ hour). Of the remaining 8
Daniele Versari, Mario Gossl, Dallit Mannheim, Offer Galili, Lilach O Lerman, Amir Lerman; subjects studied, 6 (30%) had primary snoring disorder (PSD), and 2 had normal polysom-
Mayo Clinic Rochester, Rochester, MN nography without snoring. Furthermore, SDB was found in 10 (59%) pts with adenotonsillar
hypertrophy and HTN [OR2.16, CI 1.18 - 3.95, p0.007 vs adenotonsillar hypertrophy alone]
Objective: Hypertension and hypercholesterolemia are two of the most important risk factors and 11 (65%) pts with obesity and HTN [OR2.18, CI 1.19 4.01, p0.007 vs obesity alone].
for carotid atherosclerosis and cerebrovascular ischemic events. However, little data are This compares to an overall prevalence of 2% in all children, 40% in children with
available on the different effects of these risk factors on carotid function and structure. The aim adenotonsillar hypertrophy, and 46% in obese children reported in the literature. These data
of the present study was to compare the effects of hypertension and hypercholesterolemia on suggests HTN in children constitutes an additional risk factor for SDB, and further studies are
carotid endothelial function, structure and vasa vasorum (VV). Methods: Sixteen pigs were needed to explore the relationship between SDB and HTN.
randomized to 12-week normal diet without (N; n5) or with renovascular hypertension (HT;
n4), or high-cholesterol diet (HC; n6). For each pig one carotid artery was injected with a
lead chromate-doped silicon polymer and scanned by microCT for evaluation of VV structure.
Tissue from the other carotid was obtained for staining (trichrome and Sirius red for fibrosis), 76
immunoblotting (VEGF, bFGF), and assessment of endothelial function by organ chambers Difference in Aldosterone Levels in Male and Female Subjects with
(vasodilation (VD) to acetylcholine, ACH, and sodium nitroprusside, SNP). Results: HT and HC Resistant Hypertension
showed increased carotid artery fibrosis as compared to N, but in HT Sirius red revealed a
higher content of organized collagen fibers than in HC; also intima-media thickness was Krishna K Gaddam, Monique N Pratt-Ubunama, Mari K Nishizaka, David A Calhoun; Univ of
significantly (p0.05) increased in HT (0.780.06 mm) compared to N (0.540.04 mm) and Birmingham at Alabama, Birmingham, AL
HC (0.590.02 mm). Immunoblotting densitometry, microCT and endothelial function results
are shown in the Table. Conclusions: Both HT and HC induce endothelial dysfunction in pig Background: Several centers worldwide including ours have recently reported a high incidence
carotid artery. However, HT is also associated with greater fibrosis and vascular wall of primary aldosteronism (PA) among subjects with resistant hypertension. The reason for the
thickening, which might impair endothelium-independent VD, as well as VV growth. On the high prevalence of hyperaldosteronism in this high risk group is unknown. The current study
other hand, HC is associated with increased VEGF expression and VV vascularization of the was designed to identify potential stimuli of aldosterone secretion in subjects with resistant
arterial wall, suggesting that carotid atherosclerosis can evolve in several different pathways. hypertension. Methods: Consecutive subjects referred for resistant hypertension (blood
These findings might have significant implications for our understanding of the pathophysiology pressure 140/90 on 3 antihypertensive agents) were prospectively evaluated with plasma
and complications of carotid atherosclerosis. aldosterone concentration (PAC), plasma rennin activity, a 24-hr urine collection for aldoste-
rone, sodium and potassium during a normal diet. All subjects were on a stable antihyper-
Mean VV diameter (um) 48.013.65 0.340.02* 18.3722.21* tensive regimen without use of potassium sparing diuretics. Results: A total of 271 subjects
N HC HT were evaluated, including 132 males and 139 females. Age (5411 vs. 5511), BMI (335.3
VV density (n/mm2) 0.080.01 0.340.02* 0.090.01 vs. 338.2), clinic blood pressure (14522/8717 vs. 14421/8316) were similar in men
Mean VV diameter (um) 64.51.3 63.02.1 66.91.1 and women. PAC was higher in men compared to women (14.89.54 vs. 11.89.28ng/dl,
VEGF/actin 0.10.1 1.920.17* 0.490.63 p0.0091). In a subset of subjects (n96) 24-hr urinary cortisol, aldosterone, sodium,
FGF/actin 0.460.33 0.440.25 0.470.21 potassium and protein were measured. In this subset, PAC (11.437.6 vs. 10.46.2ng/dl) and
Max %VD ACH 48.013.65 23.821.6* 18.3722.21* urinary aldosterone (13.27.2 vs. 9.55.3g/24-hr, p0.0053) were higher in men
Max %VD SNP 85.09.7 82.68. 61.617.61*
compared to women. Urinary cortisol (l0842.1 vs. 65.630g/24-hr, p0.0001), sodium
* p0.05 vs N; p0.05 vs HC (233.8111.8 vs. 155.984.2meq/24-hr, p0.0002) and potassium (79.633.6 vs.
CHBPR ConferenceOral Presentations e41
50.523.9meq/24-hr, p0.0001) were also higher in male subjects. Urinary aldosterone 79
excretion was significantly correlated with cortisol excretion in males (p0.0003, r 0.51) Higher Ambulatory Blood Pressure and Less White-Coat Effect in Subjects
with a similar trend in females (p0.062, r0.27). Conclusion: This data is the first to with Hyperaldosteronism and Resistant Hypertension
demonstrate a gender difference in plasma aldosterone and urinary aldosterone excretion
levels in subjects with resistant hypertension. Based on our findings, we propose that potential Eduardo Pimenta, Sr., Monique N Pratt-Ubunama, Krishna K Gaddam, Mari K Nishizaka,
stimuli of aldosterone underlying this gender difference include greater dietary ingestion of David A Calhoun; Univ of Alabama at Birmingham, Birmingham, AL, USA, Birmingham, AL
potassium by the male subjects; and/or ACTH stimulation as suggested by the same gender
difference in aldosterone and cortisol excretion. Background: Ambulatory blood pressure (BP) better predicts cardiovascular outcomes than
office BP. The effects of hyperaldosteronism on ambulatory BP levels and circadian BP variation
are not well established. Objective: The purpose of this study was to compare the 24-hr
ambulatory blood pressure monitoring (ABPM) profile and the degree of white-coat effect (WCE
- daytime ambulatory BP at least 10 mmHg office BP) in resistant hypertensive subjects with
77 or without hyperaldosteronism. Methods: One hundred thirty-eight subjects with resistant
Plasma Aldosterone is Related to Severity of Obstructive Sleep Apnea in hypertension were prospectively evaluated with an early-morning plasma aldosterone and
Subjects with Resistant Hypertension but not Subjects with Normal Blood plasma renin activity (PRA), 24-hour urinary aldosterone and sodium, and 24-hr ABPM.
Pressure Daytime, nighttime and 24-hr blood pressure as well as nocturnal BP decline and the morning
BP surge were determined. Hyperaldosteronism (H-Aldo) was defined as suppressed PRA
Monique N Pratt-Ubunama, Mari K Nishizaka, UAB Hypertension Program, Birmingham, AL; (1.0 ng/mL/hr) and elevated 24-hr urinary aldosterone excretion (12 g/24h) in sodium
Krisha K Gaddam, UAB Hypertension Prgm, Birmingham, AL; Robyn L Boedefeld, UAB Sleep replete subjects (200 mEq/24-hr). Results: Overall, the mean office BP was 160.325.9/
Disorders Cntr, Birmingham, AL; Stacey S Cofield, UAB Dept of Biostatistics, Birmingham, 89.816.3 mmHg on an average of 4 medications. There was no difference in mean office BP
AL; Susan M Harding, UAB Sleep Disorders Cntr, Birmingham, AL; David A Calhoun; UAB values between the 45 subjects with H-Aldo and the 93 subjects with normal aldosterone levels
Hypertension Program and Sleep Disorders Cntr, Birmingham, AL (N-Aldo). However, systolic daytime (149.215.2 vs 138.617.4mmHg, p0.0007), diastolic
daytime (89.48.8 vs 80.513.7mmHg, p0.0001), systolic nighttime (143.116.4 vs
Objective: Untreated obstructive sleep apnea (OSA) may contribute to the development of 130.917.8mmHg, p0.0002), diastolic nighttime (82.810.1 vs 73.514.2mmHg,
resistant hypertension by stimulating aldosterone release. This study compares plasma p0.0001), 24-hr systolic (146.814.6 vs 135.717.0mmHg, p0,0003) and 24-hr diastolic
aldosterone, renin levels and OSA severity in subjects with resistant hypertension and in control (86.618.8 vs 77.913.8mmHg, p0.0002) BP were all higher in H-Aldo than N-Aldo
subjects without resistant hypertension but with equally severe obstructive sleep apnea. subjects. A WCE was present in 46% of the H-Aldo subjects but only 13% of N-Aldo. Nocturnal
Methods: Seventy-one consecutive subjects (41 males, 31 females) referred to University of dipping was not different in the 2 aldosterone groups. The systolic morning surge was
Alabama at Birmingham for resistant hypertension (blood pressure uncontrolled with 3 17.411.3 mm Hg in the H-Aldo subjects and 19.213.7 mm Hg in the N-Aldo subjects
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medications) and 29 consecutive subjects (15 males, 14 females) referred to the UAB Sleep (pNS). Conclusions: In spite of similar office BP, daytime and night ambulatory BP levels are
Disorders Center for suspicion of OSA and without resistant hypertension (normotensive or higher and there is less WCE in resistant hypertensive subjects with high aldosterone levels.
blood pressure controlled on 2 or less antihypertensive medications) were evaluated. All These results suggest that high aldosterone levels impart increased cardiovascular risk not
subjects were prospectively evaluated by an early morning, paired plasma aldosterone reflected by office blood pressure measurements.
concentration (PAC) and renin level and overnight, attended polysomnography. The number of
apnea and hypopnea events/hr (apnea-hypopnea index or AHI) and the percent of sleep time
spent with oxygen saturation 90% (hypoxic index or HI) was determined in all subjects. 80
Results: The prevalence of OSA (AHI 5 events/hr) was 85% in subjects with resistant
hypertension (median AHI 15.3 events/hr) and 83% in control subjects (median AHI 14.3
Blood Pressure and Mortality in Patients with Cardiovascular Disease
events/hr). Median PAC was significantly higher (11.0 vs. 5.5 ng/dL, p0.002) and renin levels
Jing Chen, Tulane Univ, New Orleans, LA; Donfeng Gu, Chinese Acadewmy of Med
were significantly lower (8.0 vs. 19.0 Units/mL, p0.0005) in subjects with resistant
Sciences and Peking Union Med College, Beijing, China; P. Whelton, Chung-Shiuan Chen,
hypertension compared to controls subjects. PAC correlated with AHI (rho0.44, p0.0002)
Tulane Univ, New Orleans, LA; Xigui Wu, Chinese Academy of Med Sciences and Peking
and HI (rho0.38, p0.001); there was no correlation between PDR and AHI or HI in subjects
Union Med College, Beijing, China; Xingfeng Duan, Chinese Academy Med Sciences and
with resistant hypertension. Median plasma aldosterone levels were not related to AHI
Peking Union Med College, Beijing, China; Kristi Reynolds, L Lee Hamm, Paul K Whelton,
(rho0.12, p0.52) or HI (rho0.002, p0.99) in control subjects. Conclusions: We
Jiang He; Tulane Univ, New Orleans, LA
demonstrate that PAC correlates with OSA severity in subjects with resistant hypertension. No
relationship between PAC and OSA severity was observed in control subjects with equally
Background: Blood pressure (BP) has been associated with an increased risk of cardiovascular
severe OSA but without resistant hypertension. These data suggests that OSA contributes to the
disease (CVD) mortality in the general population. However, direct and J-shaped associations
development of resistant hypertension by stimulating aldosterone release.
between BP and mortality have been reported among patients with CVD. Methods: We
examined the association between BP and CVD mortality among 2,253 male and 1,942 female
patients with CVD. Data on BP and co-variables were obtained at a baseline examination in
1991 using a standard protocol. Follow-up evaluation was conducted in 1999 2000 with a
78 response rate of 93.4%. Results: After adjustment for age, gender, cigarette smoking, alcohol
consumption, body mass index, physical inactivity, education, diabetes, geographic region
Sleep Apnea and Hypertension Prevalence in Chronic Kidney Disease (north vs. south) and urbanization (urban vs. rural), a direct association between BP and CVD
mortality was observed (p0.001). Compared to those with a systolic BP 120 mm Hg,
John J Sim, Stephen F Derose, Scott A Rasgon; Kaiser Permanente Los Angeles Med Cntr,
relative risks (95% CI) for patients with a systolic BP of 120 129, 130 139, 140 159,
Los Angeles, CA
160 179 and 180 mm Hg were 1.21 (0.79,1.84), 1.69 (1.15, 2.48), 2.09 (1.47, 2.96), 2.83
(1.98, 4.03), 3.83 (2.66, 5.52) for stroke, 1.29 (0.94, 1.77), 1.63 (1.22, 2.19), 1.96 (1.50, 2.56),
Introduction: Hypertension (HTN) is a known complication and risk factor for chronic kidney
2.37 (1.80, 3.12), 2.99 (2.25, 3.99) for CVD, and 1.05 (0.82, 1.33), 1.23 (0.98, 1.53), 1.34
disease (CKD). The rate of HTN in CKD correlates with declining renal function exceeding 80%
(1.09, 1.64), 1.62 (1.32, 2.00), 2.00 (1.61, 2.50) for all-cause mortality, respectively. The
in those with end stage renal disease. Sleep apnea (SA) also has a strong association with
associations between BP and risk of CVD and all-cause mortality were similar in males and
systemic HTN. Both CKD and SA have been associated with cardiovascular and cerebrovascular
females. Conclusion: These data indicate that there is no J-shaped association between BP
disease where HTN may be an intermediary variable. We evaluated the prevalence of HTN in
and mortality among patients with CVD. Furthermore, our findings support a lower BP treatment
patients who have CKD and SA compared to the CKD population overall. CKD was defined by
goal among patients with CVD in order to reduce mortality.
depressed glomerular filtration rate (GFR) and/or the presence of proteinuria and classified into
National Kidney Foundation Kidney/Disease Outcomes Quality Initiative (K/DOQI) stages. We
compared the prevalence of HTN in patients in each stage of CKD against those with CKD and
SA. Methods: The population of subjects age 18 years and enrolled in an integrated health 81
plan during 2002 - 2004 was studied. CKD was determined by GFR and proteinuria. GFR was Effect of Cardiac Overexpression of Bradykinin B2 Receptors in a Rat
estimated by the modified MDRD equation. Two GFRs taken 90 days apart were required to Model of Pressure Overload
establish stable GFR according to predefined algorithms. HTN was determined using a
combination of ICD-9 codes, HTN medications, and comorbid conditions. SA was defined by Marcos E Barbosa, Max-Delbruck-Cntr, Berlin, Germany; Heloisa A Baptista, Escola Paulista
ICD-9 codes for obstructive or central SA, or dispensation of CPAP or BIPAP. Rates of HTN and de Medicina, Sao Paulo, Brazil; Robert Fischer, Max-Delbruck-Cntr, Berlin, Germany; Joao B
SA were compared by chi-squared tests. Results: See table. HTN is significantly more Pesquero, Escola Paulista de Medicina, Sao Paulo, Brazil; Michael Bader;
prevalent in patients (1) with SA than without SA among CKD patients with GFR 1590 Max-Delbruck-Cntr, Berlin, Germany
mL/min/1.73m2 (p 0.001 for CKD Stage 2 & 3, p 0.05 for CKD Stage 4); (2) with SA than
without SA among patients with GFR 90 (p 0.001 for no CKD and CKD Stage 1); and (3) Hypertension resulting in left ventricular hypertrophy and fibrosis can lead to cardiac
with proteinuria than without proteinuria among patients with GFR 90 (p 0.001 for no SA dysfunction. Using a myosin light chain 2 promoter, a transgenic rat model overexpressing
and SA). Conclusion: HTN is very common in SA with or without CKD. Rates of HTN increase bradykinin B2 receptors (BKB2) specifically in the heart (TGR(MLC2B2)) was developed to
when SA coexists with CKD and in patients with normal GFR who have proteinuria. Rates of investigate the cardiac function of the kallikrein-kinin system (KKS). Overexpression of BKB2
HTN increase as GFR declines. receptors was verified by a ribonuclease protection assay, by radioligand binding studies as
HTN AND SLEEP APNEA BY CKD STAGE well as by the response to bradykinin in isolated hearts perfused in a modified Langendorff
preparation. Isolated hearts from TGR(MLC2B2) rats presented higher left ventricular pressure
CKD
Stage Renal Function Total Patients HTN (%) SA (%) HTN & SA (%)
(68.66.6mmHg) when compared with Sprague Dawley (SD) rats (49.51.3 mmHg,
p0.001). Accordingly, blood pressure measured by tail-cuff plethysmography was increased
0 GFR 90 No Proteinuria 395,634 109,144 (28%) 8,151 (2%) 4,182 (51%) in TGR(MLC2B2) (122.71.9 mmHg) compared to SD rats (107.81.9 mmHg, p0.001).
1 GFR90 and Proteinuria 4,917 3,626 (74%) 261 (5%) 228 (87%) Telemetric measurements of systolic blood pressure confirmed the difference between SD
2 GFR 6089 258,238 141,947 (55%) 8,486 (3%) 5,573 (66%) (120.92.8 mmHg) and TGR(MLCB2) rats (130.62.7 mmHg, p0.05). Treatment with the
3 GFR 3059 59,300 48,217 (81%) 1,901 (3%) 1,642 (86%)
4 GFR 1529 2,789 2,614 (94%) 91 (3%) 91 (100%)
BKB2 antagonist, icatibant, annulled this difference (121.52.5 mmHg). To study the function
of the KKS in the pathogenesis of cardiac hypertrophy, SD and TGR(MLC2B2) rats were
e42 Hypertension Vol 48, No 4 October 2006

submitted to abdominal aortic constriction. After 8 weeks, hemodynamic parameters were The increase in intracellular Ang II (in cell lysates) was even greater (64 25 and 70 12
measured by a Millar tip catheter and the pressure overload after aortic banding in TGR(MLCB2) %, in CMs and FBs, respectively) in the presence of candesartan, suggesting the increase was
(sham 113.23.9 to banded 166.39.1 mmHg) was 76% higher than the observed in SD rats due to intracellular synthesis, not to uptake. Similarly, high glucose treatment resulted in a
(sham 106.3 2.7 to banded 136.37.4 mmHg, p0.05). Cardiac Magnetic Field Mapping significant increase in total Ang II synthesis. However, in CMs the increase in the concentration
showed a slight prolongation of the early part of repolarization at baseline in TGR(MLCB2) of Ang II was mainly observed in the cell lysates (704 46 %), with a minimal increase in the
compared to SD rats (QTPeak 302.5 and 25.70.6, p0.053). Alterations in repolarization culture medium (14 1 %), compared to control. In the presence of candesartan, the Ang II
were more pronounced in TGR(MLCB2) submitted to aortic constriction compared to SD rats. concentration increased 556 78 % intracellularly and 34 4 % in the medium. This latter
Rotation in magnetic field orientation during early repolarization was higher in TGR(MLCB2) observation suggested that under hyperglycemic conditions, intracellularly synthesized Ang II
(137.08.0 to 101.06.7 degrees) compared to SD rats (134.06.0 to 122.09.0 degrees, in CMs is retained inside the cell. In contrast, Ang II synthesized by FBs is primarily secreted
p0.05). In conclusion, overexpression of the BKB2 receptor in cardiomyocytes of transgenic extracellularly. This study demonstrates intracellular synthesis of Ang II by isolated CMs and
rats results in increased cardiac performance at baseline and after pressure overload. FBs and suggests a possible role for intracrine (intracellular) Ang II in CMs, under high glucose
conditions.

82
The Nonpeptide Angiotensin-(17) Receptor Mas Agonist AVE 0991 85
Attenuates Heart Failure Induced by Myocardial Infarction Myocardial Performance Index in Childhood Onset Essential Hypertension
Anderson J Ferreira, Bruno J Jacoby, Ccero A Araujo, Filipe A Macedo, Gerluza A Silva, Monesha Gupta-Malhotra, Rabih K Hamzeh, Tim Poffenbarger, Karen McNiece, Ronald J
Alvair P Almeida, Robson A Santos; Federal Univ of Minas Gerais, Belo Horizonte, Brazil Portman; Univ of Texas, Houston, TX
The nonpeptide AVE 0991 has been reported as a selective ligand of the Ang-(17) receptor Objectives. As a measure of global ventricular systolic and diastolic function, the myocardial
Mas, which actions are similar to those attributed to the cardioprotective fragment of the performance index (MPI) may be an early indicator of left ventricular dysfunction in
renin-angiotensin system, Ang-(17). In this study we evaluated the cardiac effects of AVE hypertensive children. Thus, we performed the following study to determine the MPI in children
0991 (0.58 mol/kg, 7 days) in normal and infarted male Wistar rats. Myocardial infarction was with essential hypertension (HTN). Methods. Study subjects were untreated newly diagnosed
induced by left coronary artery ligation. At the end of the treatment, the Langendorff technique children with HTN and controls. HTN was defined as BP 95th percentile based on the Fourth
was used to analyze cardiac function. Left ventricles serial sections were stained with Gomori Working Group criteria and confirmed by 24-hour ambulatory blood pressure monitoring
trichrome stain to quantify the infarted area. AVE 0991 produced a significantly decrease in (ABPM). BP is reported as the patients BP factored by their age, gender, height-specific 95th
perfusion pressure and an increase in systolic tension, dT/dt, and heart rate. These effects %ile (BP index). Left ventricular mass index (LVMI) was calculated as LVM (g)/height (m) 2.7, and
were completely blocked by the perfusion of the hearts with a solution containing the selective left ventricular hypertrophy (LVH) was defined as LVMI 95th percentile. Control subjects had
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Ang-(17) antagonist, A-779 (2 nM). L-NAME treatment (10 mg/kg) abolished the AVE normal casual BPs and 24-hour ABPM. 2-D and M-mode echocardiography with pulse Doppler
0991-induced vasodilation in isolated hearts. AVE 0991 significantly attenuated the decrease were performed on all pts prior to therapy. Endocardial shortening fraction (SF), midwall left
in systolic tension (sham-operated: 13.00 1.02 g; infarction: 7.18 0.66 g; AVE-treated: ventricular shortening (MWS) and circumferential end systolic stress (cESS) were calculated.
9.23 1.05 g, n5), dT/dt (sham operated: 209.9 17.9 g/s; infarction: 104.5 12.5 MPI was calculated from simultaneous mitral valve inflow and left ventricular outflow velocities
g/s; AVE treated: 136.2 19.8 g/s, n5), - dT/dt and heart rate induced by myocardial using pulse Doppler. Results. The left ventricular diastolic parameters (peak E and A velocities,
infarction. Infartion-induced vasoconstriction was completely abolished by AVE 0991 treatment. E/A ratio, isovolemic relaxation time, acceleration and deceleration times) were similar between
Furthermore, AVE 0991 significantly decreased the infarted area. These data indicate that the the two groups. MPI was significantly higher among HTN children compared to non-
compound AVE 0991 produces beneficial effects in isolated perfused rat hearts involving the hypertensive controls. Conclusions. MPI is significantly higher in children with essential HTN
Ang-(17) receptor Mas and the release of nitric oxide. In addition, our results indicate that AVE compared to controls, albeit with preserved function as this early stage. These findings suggest
0991 attenuates post-ischemia heart failure. that MPI may be a sensitive parameter for detecting early cardiac dysfunction in hypertensive
children.

83 HTN Control P value


Genetic Ablation of Bach1, a Transcriptional Repressor of Heme Number 24 36
Oxygenase-1 Gene, Leads to Myocardial Protection against Pressure Age, years 13.5 1.8 13.5 1.7 NS
Overload in Mice BMI 27 9 26 3 NS
Casual systolic BP index 1.11 .06 1.05 .06 0.002
Casual diastolic BP index .95 .14 .93.12 NS
Shinji Mito, Ryoji Ozono, Yuichiro Watari, Yoshiyuki Yamamoto, Yoko Yano, Masao
Ambulatory systolic BP index 1.04 0.05 0.93 0.04 0.0001
Yoshizumi; Hiroshima Univ Graduate Sch of BioMed Sciences, Hiroshima, Japan Ambulatory diastolic BP index 0.93 0.1 0.87 0.07 0.006
Systolic BP load, % 65 17 21 13 0.0001
Bach1 is a stress-responsive transcriptional factor that is thought to control the expression Diastolic BP load, % 28 24 14 11 0.003
levels of cytoprotective factors including heme-oxygenase(HO)-1. In the present study, we LVMI, g/m2.7 36.7 9 35.8 8 NS
investigated the roles of Bach1 in the development of left ventricular hypertrophy (LVH) induced Relative wall thickness 0.37 0.05 0.35 0.04 NS
by transverse aortic constriction (TAC) in vivo using mice lacking the Bach1 gene (Bach1-/- SF, % 42 10 42 13 NS
MWS, % 20 0.4 15 0.4 NS
mice). Three weeks after TAC, the heart weight to body weight (HW/BW) ratio, myocardial
cESS, g/cm2 82 34 75 29 NS
cross-sectional area, and an index of interstitial fibrosis were significantly increased both in MPI 0.36 0.4 0.2 0.1 0.04
Bach1-/-mice and wild-type control (Bach1/) mice. However, the increases in all of these
parameters were significantly less in Bach1-/- than in Bach1/ mice (HW/BW: 5.741.17 vs.
7.250.53 mg/g, p0.05). Myocardial HO-1 protein expression was significantly greater in
sham-operated Bach1-/- than in Bach1/ mice. TAC increased myocardial expression levels of 86
HO-1 protein to a larger extent in Bach1-/- than in Bach1/ mice. Treatment of Bach1-/- mice Stimulation of Cytokines Gene Expression in Hypertrophied Hearts of Mice
with tin-protoporphyrin, an inhibitor of HO, completely abolished the hypertrophy-reducing Lacking Guanylyl Cylase/Natriuretic Peptide Receptor-A
effect of Bach1-knockout, indicating that the reduction of hypertrophy was dependent on the
HO activity.The results of present study suggest that Bach1 plays a significant role in repressing Elangovan Vellaichamy, Di Zhao, Naveen K Somanna, Kailash N Pandey; Dept of Physiology
the myocardial HO-1 expression both in basal and stressed conditions, thereby setting a and Hypertension and Renal Cntr of Excellence, Tulane Univ Health Sciences Cntr & Sch of
threshold of deployment of anti-hypertrophic factors. Inversely, an inhibition of Bach1 could be Medicine, New Orleans, LA
a novel approach to protect myocardium from pressure overload.
Cardiac hormones, atrial and brain natriuretic peptides (ANP and BNP), are released in the
circulation and elicit natriuretic, diuretic, vasorelaxant, and anti-proliferative responses, all
84 directed to the reduction of blood pressure and blood volume. Natriuretic peptide receptor-A/
Evidence of Intracellular Synthesis of Angiotensin II in Cardiac Myocytes guanylyl cyclase-A (NPRA/GC-A) is considered the principal receptor for both ANP and BNP, and
and Fibroblasts binding of these hormones to NPRA leads to the generation of the intracellular second
messenger cGMP. Mice carrying targeted-disruption of Npr1 gene (encoding for NPRA) exhibit
Vivek P Singh, Kenneth M Baker, Rajesh Kumar; Div of Molecular Cardiology, Cardiovascular hypertension, marked cardiac hypertrophy, and congestive heart failure with sudden death
Rsch Institute, The Texas A&M Univ System Health Science Cntr, College of Medicine; Scott after six months of age. In the present studies, we analyzed the cardiovascular phenotype in
&White; Central Texas Veterans Health Care System, Temple, TX Npr1 (genetic determinant of NPRA) gene-disrupted mutant mouse model. Homozygous null
mutant Npr1 (0-copy; 0/0) mice showed almost 35 40 mmHg higher systolic blood pressure
The current paradigm is that cardiac angiotensin II (Ang II) is synthesized in the interstitial space and 60 65% greater left ventricular weight as compared with Npr1 wild-type (2-copy; 1/1)
from components of the circulating or local renin angiotensin system. Local production of Ang mice. The expressions of cytokines and growth factors gene were significantly increased in
II has a significant role in cardiac homeostasis, through autocrine/paracrine actions. The recent ventricles of 0-copy Npr1 mice hearts as compared with 2-copy wild-type mice hearts. The
discovery of intracrine effects of Ang II in the heart, prompted us to determine if there was electrophoretic mobility shift assay (EMSA) showed a 4-to-5-fold increase in NF-B binding
intracellular Ang II synthesis. Cardiac myocytes (CMs) and fibroblasts (FBs) were isolated from activity in nuclear extracts of 0-copy mice hearts as compared with 2-copy mice hearts.
neonatal rat hearts and separated by double-differential plating. Before treatment, the cells Captopril or hydralazine treatments attenuated systolic blood pressure; however, only captopril
were incubated in serum-free medium for 24 h to allow for degradation of any exogenous treatment significantly decreased the left ventricular weight and fibrosis in 0-copy Npr1 mice
source of Ang II. Cells were treated with either isoproterenol (10 M) or high glucose (25 mM) hearts. Nevertheless, the captopril treatment also decreased cytokine expression in 0-copy
in the absence or presence of the AT1 antagonist, candesartan (1 M). The latter was included Npr1 mice hearts. cGMP levels were significantly reduced 5-fold in plasma and 10-fold in
to prevent receptor-mediated internalization of Ang II. After 24 h of treatment, Ang II was ventricular tissues of mutant mice hearts relative to wild-type controls. The present findings
measured in cell lysates and the culture medium using a competitive ELISA. Isoproterenol provide direct evidence that ablation of NPRA/cGMP signaling activates inflammatory process
treatment increased the Ang II concentration in cell lysates (38 18 and 24 12 %) and the leading to an enhanced cardiac hypertrophy and fibrosis in homozygous null mutant mice
medium (52 24 and 144 22 %), of both CMs and FBs, respectively, compared to control. lacking NPRA.
CHBPR ConferenceOral Presentations e43
87 HFD (96.6%3), but not in HT or FORKO. Angiotensin II-constrictor responses were not altered
A Novel Mechanism of Afferent Arteriole (Af-Art) Regulation: A Cross-Talk by HFD in all groups. Incubation of the vessels with antioxidant cocktail (SOD, catalase,
between the Connecting Tubule (CNT) and Af-Art TEMPOL) significantly increased the ACh response only in HFD-fed FORKO (59.6%5.9,
p0.005). Reactive oxygen species (ROS) generation was increased in FORKO (0.29.11
Yilin Ren, Jeffrey L Garvin, Ruisheng Liu, Oscar A Carretero; Henry Ford Hosp, Detroit, MI arbitrary unit) and further increased by HFD (10-fold). These findings suggest that endothelial
dysfunction in FORKO mice is mediated by ROS which is aggravated by HFD. Oil-red-O staining
In most nephrons the superficial connecting tubule (CNT) comes into close contact with the revealed deposition of fat in aortic and coronary sinuses that was associated with increased
afferent arteriole (Af-Art). However, the physiological significance of this contact is not expression of VCAM-1 in all HFD-fed groups. The percent change in lipid profile (total
clear. The NaCl concentration in the CNT varies over a wide range. We hypothesized that cholesterol, HDL, LDL and TRIG by FD) was not significantly different in FORKO compared to
there is cross-talk between the CNT and Af-Art that is initiated by changes in NaCl the same change by HFD in WT and HT. Our findings demonstrate that HFD aggravates vascular
concentration in the CNT lumen. Rabbit Af-Arts were perfused at 60 mm Hg while the NaCl function by increasing oxidant radicals in FORKO mice without worsening lipid profile.
concentration in the lumen of the adherent CNT was changed from 10 to 80 mM. We first
found that in nonconstricted Af-Art, increasing luminal NaCl in the CNT caused slight
dilatation but not constriction. Then we tested whether increasing luminal NaCl in the CNT
induced dilatation of Af-Arts preconstricted with norepinephrine (NE). With 10 mM NaCl in
the CNT lumen, adding NE to the bath decreased Af-Art diameter from 18.4 0.7 to
10.1 1.3 m. When the solution was changed to 80 mM NaCl, Af-Art diameter increased
to 17.3 1.6 m (n 6; p 0.05, high vs. low NaCl). We then tested whether entry 90
of Na, Cl- or some combination of these ions is required to induce Af-Art dilatation. During Differential Function and Trafficking of ETA and ETB Receptors in
the control period, high NaCl induced preconstricted Af-Art dilatation from 13.0 1.9 to Mesenteric Arterial and Venous Smooth Muscle and HEK 293 Cells
17.8 1.3 m (n 4; p 0.05, high vs. low). We then replaced Na in the CNT lumen
with choline chloride. Unlike NaCl, increasing choline chloride from 10 to 80 mM did not Xiaoling Dai, James J Galligan; Michigan State Univ, East lansing, MI
dilate the preconstricted Af-Art (from 11.9 1.6 to 12.4 1.8 m; n 3). We next
studied which Na transporter mediates these effects. When we blocked amiloride-sensitive ET-1 is more potent in contracting veins than arteries and this might be due to differential
Na channels by adding 10-6 M amiloride to the CNT lumen, the dilatation induced by 80 localization of ETA (ETAR) and ETB receptors (ETBR) on vascular smooth muscle cells
mM NaCl was eliminated (from 12.0 1.2 to 11.9 1.6; n 4). Finally, we added 10-3 (VSMCs). We investigated ETAR and ETBR trafficking in HEK 293 cells transfected with ETA
M thiazide to the CNT lumen to block NaCl cotransport. Thiazide did not block and/or ETB receptors and in rat mesenteric arteries (MA) and veins (MV). Using [Ca2]i
NaCl-induced dilatation of the preconstricted Af-Art. We concluded that: 1) there is imaging in HEK 293 cells transfected with ETAR and ETBR, we found that ETBR desensitized
cross-talk between the CNT and the Af-Art; 2) increases in CNT Na cause dilatation of faster (t1/2211s) than ETAR (t1/2481s) upon ET-1 (0.1 M) stimulation. The t1/2 for
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preconstricted Af-Arts; and 3) this phenomenon is mediated by amiloride-sensitive desensitization in cells co-transfected with ETAR and ETBR was 400s. In HEK 293 cells
epithelial Na channels. Cross-talk between the CNT and Af-Art may be a novel transfected with ETAR or ETBR alone, 83% of ETAR and 54% of ETBR, colocalized with the
mechanism of regulation of the renal microcirculation and may explain the dilatation plasma membrane marker pan cadherin. After ET-1 treatment for 1 hr 76% of ETAR, but
observed during high salt intake. only 11% of ETBR, colocalized with pan cadherin indicating a preferential internalization of
ETBR. When ETAR and ETBR were co-transfected, 93% of ETAR and 84% of ETBR
co-localized with pan-cadherin, and 79% of ETAR and 71% of ETBR colocalized with pan
88 cadherin after ET-1 treatment. We next studied ET-1-induced constriction of MA and MV
Angiotensin Type 2 Receptor Contributes to Vascular Responses in in vitro. The maximal ET-1 constriction was 50% for MV and 20% for MA. MA desensitized
Resistance Arteries of Type 2 Diabetic Hypertensive Patients Treated with by 50% during a 20-min ET-1 application, while MV maintained maximal constriction. In
Angiotensin Type 1 Receptor Blockers dissociated MA VSMCs, 26% of ETBR colocalized with ETAR in the membrane in contrast
to 43% in MV VSMCs. In MA VSMCs, 50% of ETAR and 15% of ETBR colocalized with pan
Carmine Savoia, Lady Davis Institute for Med Rsch, SMBD Jewish General Hosp, McGill cadherin. In MV VSMCs, 55% ETAR and 40% of ETBR colocalized with pan cadherin. After
Univ, Montreal, Canada; Rhian M Touyz, Kidney Rsch Cntr, Ontario Health Rsch Institute, 1 hr of ET-1 treatment, colocalization of ETAR and ETBR with pan cadherin increased to
Univ of Ottawa, Ottawa, Canada; Ernesto L Schiffrin; Lady Davis Institute for Med Rsch, 62% and 33% respectively; in MA VSMCs this increased to 60% and 53% for ETAR and
SMBD Jewish General Hosp, McGill Univ, Montreal, Canada ETBR, respectively. We conclude that co-expression of ETAR with ETBR in HEK 293 cells
increases ETBR membrane expression/stability and this is associated with decreased ETR
The role of angiotensin (Ang) AT2 receptors on blood pressure and vascular responses to desensitization. There is increased ETBR membrane expression in MV compared to MA and
Ang II in humans is unclear. We questioned whether AT2 is expressed and functionally MV are resistant to desensitization. ET-1 stimulation promotes increased membrane
active in peripheral resistance arteries of hypertensive diabetic patients treated for one localization of ETAR and ETBR and this effect is more prominent for ETBR in MV. These data
year with either the Ang receptor blocker (ARB) valsartan or the beta blocker (BB) atenolol. indicate ETBR receptors traffic to the plasma membrane more efficiently in MV than in MA
Twenty-six hypertensive type 2 diabetic patients (30 to 70 years of age) treated with oral and this difference may contribute to the enhanced contractile sensitivity of MV to ET-1.
hypoglycemic and antihypertensive agents (not receiving ARBs or BBs) were randomized
to one-year treatment with valsartan (80 to 160 mg) or atenolol (50 to 100 mg), added to
their previous therapy. Ten normal subjects were studied as a control group. Resistance
arteries dissected from gluteal subcutaneous tissue were assessed on a pressurized
myograph. Vasodilatory response curves to Ang II (10-9-10-6 mol/L) were performed in
norepinephrine pre-contracted vessels in presence of valsartan (10-5 mol/L) the AT2
inhibitor PD123319 (PD, 10-6 mol/L). AT2 expression in small resistance arteries was 91
evaluated by confocal microscopy. After one year of treatment, systolic and diastolic BP Glutathione-s-Transferase -1 is a Candidate Modifier Gene for Renal
were well controlled in both the valsartan and atenolol groups (1433/832mmHg vs Vascular Pathology
1222/732mmHg, p0.005; 1442/832mmHg vs 1283/752mmHg, p0.005,
respectively). Endothelium-dependent and -independent relaxation did not change in the Kelly K Parsons, Mitchel D Harris, Thomas M Coffman, Thu H Le; Duke Univ Med Cntr,
treated groups between the beginning and end of the study. Ang II evoked a significant Durham, NC
vasodilatory response only in norepinephrine-pre-contracted resistance arteries from
patients treated with valsartan. PD significantly blocked this effect. AT2 expression was On susceptible genetic backgrounds such as C57BL/6 (B6), mice with targeted disruption of the
increased (four-fold, p0.005) only in valsartan-treated patients. In conclusion, AT2 AT1A receptor gene locus (Agtr1a) develop renal vascular pathology characterized by medial
receptors are upregulated and contribute to Ang II-induced vasodilation in human thickening of interlobular arteries and arterioles, resembling lesions seen in patients with
resistance arteries of hypertensive diabetic patients treated with an ARB, and may mediate hypertensive nephrosclerosis. We identified a locus, msrvh1, from B6 conferring susceptibility
in part the beneficial actions of these drugs in the treatment of hypertension in high risk for renal vascular pathology. Msrvh1 spans 5 cM on chromosome 3 and contains at least
patients. 40 known genes. As one approach to identify candidate genes, we performed microarray
analysis with RNA from kidneys of Agtr1a-/- mice with susceptible (B6) and resistant (129/SvEv)
genetic backgrounds. We identified 2 differentially expressed genes located within msrvh1. Of
89 interest, both were members of the glutathione-s-transferase (Gst) super-family of enzymes
High Fat Diet Increases Oxidative Stress and Inflammatory Markers involved in the detoxification of reactive oxygen species (ROS). Expression of one of these, Gst
Expression without Worsening of Lipid Profile in Forko Mice -1 (Gstm1), was previously reported to be reduced in stroke-prone spontaneously hyperten-
sive rats, perhaps contributing to the increased oxidative stress seen in SHRSPs. In the arrays,
Danesh Javeshghani, Lady Davis Institute, Montreal, Canada; Malur R Sairam, IRCM, Gstm1 expression was reduced by 50% in kidneys from B6 compared to 129 Agtr1a-/- mice
Montreal, Canada; Ernesto L Schiffrin, Lay Davis Institute, Montreal, Canada; Rhian M (6.70.5 vs. 13.50.4; p0.004). To verify the differences in Gstm1 expression between the
Touyz; Kidney Rsch Cntr, Ottawa, Canada strains, we performed Northern analysis and found a similar, significant reduction in Gstm1
mRNA levels in kidneys from B6 compared to 129 Agtr1a-/- mice (270.2 vs. 392.7
The potential effect of ovarian hormone replacement therapy (HERS) on the development of %GAPDH; p 0.01). A corresponding difference in Gst1 protein levels was also seen by
atherosclerosis is unclear. To probe this question we determined the effect of 3 4 month high Western blotting. We next assessed Gst activity in kidney tissues of B6 and 129 Agtr1a-/- mice
fat diet (HFD) feeding on vascular function and changes in the expression of inflammatory using a spectrophotometric assay (Sigma). Total Gst activity tended to be lower in kidneys from
markers in 3 groups of adult mice : wild-type (WT), heterozygote (HT, lower estrogen) , and B6 compared to 129 Agtr1a-/- mice, p0.051. After treatment with Cibacron which inactivates
homozgygous (FORKO, Follitropin Receptor KnockOut, estrogen deficient). Bradykinin relaxation the Gst and classes, leaving Gst intact, we found a significant 30% reduction in Gst
response of resistance arteries was less in FORKO (33.7%13.7, p0.003) and HT activity in B6 kidney tissues compared to 129 (9.2 0.9, n6 vs 13.5 0.5 nmol/ml/min,
(44.4%5.4, p0.04) than WT (60.3%10.5). ACh relaxation response was also less in n6 p 0.002). In summary, we have identified strain-specific differences in expression of
FORKO than the WT (75.112.2 vs 96.6%3, p0.04). These responses were not reduced Gstm1 in the kidney. Genetic variants associated with reduced Gstm1 expression have a
further by HFD in HT or FORKO groups. In L-NAME-incubated vessels HFD-fed groups reduced capacity for ROS inactivation in tissue and this may confer susceptibility to renal
ACh-relaxation response was less in WT (28.2%12.4, p0.004) compared to WT without vascular damage.
e44 Hypertension Vol 48, No 4 October 2006

92 90mM KCl were also similar across groups (18758 and 13330 nM in AngHS and control
Chymase-Dependent Processing of Big ET-1 in Arteries but not Veins cells; respectively). Therefore, Ang II hypertension plus high salt markedly attenuates
preglomerular calcium signaling responses to P2 receptor activation, with particular impair-
Keshari Thakali, Catherine Rondelli, Gregory D Fink, Stephanie W Watts; Michigan State ment of P2X1 receptor-mediated responses. These data are consistent with the hypothesis that
Univ, East Lansing, MI P2X1 activation of calcium signaling pathways plays a central role in pressure-mediated
autoregulatory responses. Impairment of P2X1 receptor dependent responses may explain the
The endothelin (ET) family of peptides are vasoconstrictors more potent in contracting veins hypertension-induced impairment of renal microvascular autoregulatory capability.
than arteries. The precursor big endothelin-1 (big ET-1) can be processed by multiple vascular
enzymes to form contractile substances. We focused on metabolism of big ET-1 by endothelin
converting enzyme (ECE; to ET-1[121]), matrix metalloproteases (MMPs; to ET-1 [132]) and
chymase (to ET-1[131]). The overall hypothesis was that arteries and veins would be
differently dependent on these three systems. Immunohistochemical, western, zymographic
and isometric contractile assays in rat aorta and vena cava were used. Big ET-1 contracted 95
aorta [6017% maximal phenylephrine (PE) contraction] but was more efficacious in vena Role of CCR2 in the Development of Renal Damage in Angiotensin
cava [47861% maximal norepinephrine (NE) contraction]. Dependence on ECE in both artery II-Induced Hypertension
and vein was supported by immunohistochemical localization of ECE and its product ET-1
[121] to endothelial cells, and the reduction of big-ET-1 induced contraction by ECE inhibitors Tang-Dong Liao, Xiao-Ping Yang, Edward G. Shesely, Yun-He Liu, Jiang Xu, Mingzhu Tian,
phosphoramidon (10 uM; aorta: 33% control; vena cava: 37% control) and CGS-26393 (10 uM; Maria A. Cavasin, Oscar A. Carretero; Henry Ford Hosp, Detroit, MI
aorta: 26% control; vena cava: 80% control). By contrast, inhibition of MMPs with minocycline
(50 uM) and GM6001 (5 uM) did not reduce big ET-1 -induced contraction in aorta or vena cava, Angiotensin II-induced hypertension is associated with an inflammatory response that may
though zymographic analyses confirmed active MMPs in all tissues. The product of MMP2, contribute to the development of target organ damage. Monocyte chemoattractant protein-1
purified ET-1[132] (100 nM), contracted aorta (269% max PE) and vena cava (25288% (MCP-1) plays a role in the recruitment and activation of macrophages/ monocytes via CC
max NE). Chymase was detected in both aorta and vena cava via Westerns and immunohis- chemokine receptor-2 (CCR2). We tested the hypothesis that in Ang II-induced hypertension,
tochemistry; inhibition of chymase (chymostatin, 100 uM) reduced arterial (19% control) but not reduction of the inflammatory response will decrease target organ damage in CCR2 -deficient
venous constriction to big ET-1. Both vessel types contracted to the product of chymase, mice (CCR2 -/-). CCR2 -/- mice and age-matched C57BL/6J wild-type controls (CCR2 /)
ET-1[131] (100 nM; aorta 628% max PE; vena cava 16351 max NE). These were randomly divided into four groups: 1) CCR2 / controls, 2) CCR2 / infused with Ang
results suggest at least one significant difference, the role of chymase, in the impact made by II (5.2 ng/10g/min), 3) CCR2 -/- controls and 4) CCR2 -/- infused with Ang II. After 4 weeks of
enzymatic processing of big ET-1 in arteries and veins. These findings are relevant to Ang II infusion via osmotic mini-pump, systolic BP increased significantly compared to controls;
hypertension because chymase processes not only big ET-1, but also angiotensin peptides. however, there was no difference between the two strains. In CCR2 -/-, macrophage infiltration
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Because ET-1 and angiotensin II are mediators in the pathogenesis of hypertension, this work and cell proliferation in the kidney decreased significantly (table). 24-hour urinary albumin was
underscores the potentially different roles played by the arterial and venous circulations in lower in CCR2 -/-, while GFR was higher (table). Glomerular matrix and Renal collagen content
elevation of blood pressure. was significantly Lower in CCR2-/- than in CCR2/ with Ang II infusion. We concluded that
CCR2 is required in the development of renal damage in Ang II-induced hypertension.

93 CCR2 CCR2 CCR2 CCR2


Parameters /(controls) /(Ang II) -/-(controls) -/-(Ang II)
Reduction of NOX by siRNA Ameliorates Tubuloglomerular Feedback (TGF)
during Angiotensin II Infusion SBP (mm Hg) 1062.9 1563.7*** 952.4 1594.9***
Urinary Albumin (g/24 hr) 364.4 26287.6* 394.1 5910.8#
Renal Macrophages 333 7010** 395 352##
Pouneh Nouri, Pritmohinder Gill, Christopher S Wilcox, William J Welch; Georgetown Univ,
(No./mm2)
Washington, DC Renal proliferating Cells 375 9410** 665 597##
(No./mm2)
Angiotensin II infusion increases oxidative stress, renal vasoconstriction and tubuloglomerular GFR (ml/min/100gBW) 1.040.08 0.590.08* 1.080.10 0.980.08##
feedback (TGF), but the specific role and source of superoxide (O2-) in these responses in Glomerular Matrix 8.950.76 14.480.99** 8.820.60 8.210.76##
unclear. We showed that reduction of the p22phox subunit of NADPH oxidase (NOX), the major (% glomerular area)
source of superoxide (O2-) in the kidney, by small interfering RNA (siRNA) treatment reduced Renal collagen content 22.81.6 28.00.8* 20.71.6 22.01.1##
(g/mg dry weight)
the blood pressure response to a slow pressor infusion of Ang II (200 ng/kg/min). To determine
whether NOX mediates the effects of Ang II on TGF, we measured single nephron GFR from Values are Mean SE ***p 0.001, **p 0.01, *p 0.05 Ang II vs controls; ##p 0.01, #p 0.05,
samples collected in the proximal tubule (PT) and distal tubule (DT) in rats undergoing a slow CCR2 -/- Ang II vs CCR2 / Ang II; p 0.01 CCR2 -/- vs CCR2 /
pressor infusion of Ang II, 2 days after IV injection of siRNA directed to p22phox (Ang II
siRNA) or vehicle (Ang II). Proximal SNGFR (which does not reflect macula densa-TGF control)
was not different between groups (Ang II: 423 vs Ang II siRNA: 473 nl/min, ns). However,
distal SNGFR (which does reflect macula-densa TGF control) was higher in Ang II siRNA rats
(293 vs 403 nl/min, n79, p0.05). Distal SNGFR in turn reflected the differences in
whole kidney GFR (0.580.07 vs 0.790.07 ml/min/100 g BW, n79, p0.05). PT flow was 96
not different, but DT flow was higher in Ang II siRNA rats (DT: 5.60.7 vs 7.30.4 nl/min, Aquaporin-1-Dependent NO Transport is Essential for Full Expression of
p0.05), suggesting NOX-dependent O2- enhances reabsorption in the loop of Henle. The
regulation of SNGFR by TGF, determined by the difference between Prox-Dist SNGFR was
Endothelium-Dependent Relaxation
reduced by in Ang II siRNA (Ang II: 12.4 2.1 vs Ang II siRNA: 6.9 1.1 nl/min, n79,
Marcela Herrera, Jeffrey L Garvin; Henry Ford Hosp, Detroit, MI
p0.05). We conclude that O2- generated by NOX in the kidney contributes to the renal
vasoconstriction and fall in GFR during Ang II infusion via activation of the macula densa
Nitric oxide (NO) released by vascular endothelial cells relaxes adjacent smooth muscle cells.
mechanism that enhances the TGF response.
It has been generally assumed that NO freely diffuses out of the endothelial cells without need
of a specific transporter. However, we have recently shown that the water channel aquaporin-1
(AQP-1) transports NO across cell membranes. Vascular endothelial cells express AQP-1, but
94 the role these water channels play in the release of NO and endothelium-dependent relaxation
P2 Receptor Mediated Calcium Signaling Responses are Impaired in is unknown. We hypothesized that AQP-1 mediates the release of NO out of endothelial cells,
Preglomerular Smooth Muscle Cells (PVSMC) of Ang II Hypertensive Rats and thus endothelium-dependent relaxation is impaired in AQP-1 knockout (KO) mice. We
Fed a High Salt Diet measured a) NO release by endothelial cells from wild-type (WT) and AQP-1 KO mice, in
response to 1 M acetylcholine (Ach) using a NO-selective sensor and b) the ability of Ach to
Edward W Inscho, Andrea Clarke, Shali Zhang, Crista Royal, David Osmond; Med College of relax aortic rings from WT and AQP-1 KO mice, using a wire myograph. In confluent aortic
Georgia, Augusta, GA endothelial cells from WT mice, Ach increased NO release by 111.5 24.3 pmol NO/mg
protein after 1 min. In contrast, Ach increased NO release only by 32.9 12.2 pmol NO/mg
P2X1 receptors play a critical role in pressure-mediated afferent arteriolar autoregulatory protein in endothelial cells from AQP-1 KO mice, 76 % less (n 3, p 0.044). In intact aortic
responses. These autoregulatory responses are significantly impaired in kidneys from Ang II rings, the ability of Ach to cause vasorelaxation was significantly reduced in AQP-1 KO
hypertensive rats fed a high salt diet for 14 days (AngHS), and this impairment is associated compared to wild-type mice at almost all concentrations tested (table). We found no differences
with impaired afferent arteriolar responsiveness to P2X1 receptor activation. We tested the in the relaxation induced by the NO donor sodium nitroprusside in endothelium-denuded aortic
hypothesis that PVSMC from AngHS exhibit compromised calcium signaling responses to P2 rings among strains. We conclude that: 1) AQP-1 transports endogenously produced NO out of
receptor activation. Rats were infused with angiotensin II (65ng/min) for 14 days by osmotic endothelial cells; 2) this is essential for full expression of endothelium-dependent relaxation;
minipump while being fed an 8% salt diet. PVSMC were isolated and their calcium signaling and 3) the impaired relaxation observed in AQP-1 KO mice is not due to changes in the
responses were determined using a microscope-based fura 2 technique. Calcium signaling signaling cascade beyond NO. AQP-1-dependent NO transport could be an important regulatory
responses were determined for ATP, the P2X1 receptor agonist, , -methylene ATP, or the mechanism of vascular resistance. Inadequate transport of NO by AQPs may be an alternate
P2Y2 receptor agonist, UTP. Systolic blood pressure averaged 1165 mmHg in control rats explanation for diseases believed to be caused by reduced NO production or availability, such
(n8) compared to 2058 mmHg in AngHS rats after 14 days (P0.05; n9). The increase as hypertension.
in calcium induced by ATP (10M) in cells from AngHS rats was significantly reduced and
% Relaxation
averaged 19836 nM compared to 35780 in control cells (P0.05; n28 35). Consistent
with impaired P2X1-receptor mediated vasoconstriction in hypertensive rats, calcium signaling Ach [M] 0.003 0.01 0.03 0.1 0.3 1 3
responses were markedly attenuated in AngHS rats. , -methylene ATP (10M) increased WT 134 316 469 668 776 834 834
calcium by 699 nM in cells from AngHS rats compared to 33392 nM (P0.05; AQP-1 KO 00.5 51 204 477 577 576 577
n10 15). Responses to UTP (10M) were similar across the two groups and averaged p value 0.01 0.001 0.02 n.s 0.05 0.01 0.02
529125 and 737136 nM for the AngHS and control groups; respectively. Responses to
CHBPR ConferenceOral Presentations e45
97 hemodynamic response to Ang II in the renal medulla. It is suggested that HIF-1-
Resetting of Pressure Natriuresis by Decoy of Transcription Factor mediated gene activation importantly participates in the regulation of renal medullary
HIF-1alpha in the Renal Medulla function and pressure natriuresis. (Supported by NIH grants DK 054927 and HL 70726).

Ningjun Li, Li Chen, Pin-Lan Li; Med College of Virginia, Virginia Commonwealth Univ,
Richmond, VA 98
Preventing an Increase in RIHP Reduces Renal 20-Hete Levels and Inhibits
Hypoxia inducible factor-1 (HIF-1), a transcription factor, is abundantly expressed in the Pressure Natriuresis
renal medulla and regulates many oxygen-sensitive genes such as nitric oxide synthase (NOS),
cyclooxygenase-2 (COX-2) and heme oxygenase-1 (HO-1). Given that the expression of these Jan M Williams, Robert P Ryan, Averia K Flasch, Richard J Roman; Med College of
genes are importantly involved in the long term control of arterial blood pressure, the present Wisconsin, Milwaukee, WI
study is designed to test the hypothesis that HIF-1 serves as an antihypertensive factor to
regulate renal medullary function and sodium excretion by activating regional expression of Previous studies from our laboratory have demonstrated that inhibitors of the formation of
these oxygen-sensitive genes. A new gene transfection strategy with ultrasound- 20-hydroxyeicosatetraenoic acid (20-HETE) attenuate the pressure natriuretic response.
microbubble technique was used for local delivery of HIF-1 decoy oligodeoxynucleotide However, the signal that increases 20-HETE release following elevations in renal perfusion
(ODN) into the renal medulla in Sprague Dawley rats. This HIF-1 decoy ODN would block pressure (RPP) is unknown. This study examined the effects of changes in renal interstitial
the binding of HIF-1 and thereby inhibit the transcription of its target genes. Ten days hydrostatic pressure (RIHP) on renal 20-HETE levels and the pressure natriuretic response in
after ODN transfection, the HIF-1 binding activities were significantly inhibited by 45% male Sprague Dawley rats. In control animals, 20-HETE levels in renal cortical tissue rose from
and the mRNA levels of HO-1, one of the HIF-1 target genes, were decreased by 51% in 0.760.26 to 1.420.33 ng/g of tissue (n6) following elevations in RPP from 103 to 166
renal medullary tissues in decoy ODN transfected rats compared with the scrambled ODN mmHg. After opening the renal capsule to prevent the rise in RIHP, 20-HETE levels were not
transfected rats. The diuretic and natriuretic responses to the elevation of renal perfusion significantly altered and averaged 0.490.14 and 0.600.14 n/g of tissue (n6) at low and
pressure (RPP) (from 80 - 160 mmHg) were significantly blunted by 50% in HIF-1 decoy high pressure, respectively. Preventing a rise in RIHP by opening the renal capsule blunted the
OND infused rats, accompanied by a 37% inhibition in the increases of renal medullary pressure diuretic and natriuretic responses by 41% and 36%, respectively without altering
blood flow (MBF) in response to the elevations of RPP. Intravenous infusion of angiotensin glomerular filtration rate (GFR) or renal blood flow (RBF). Pre-treating the animals with
II (Ang II) at 0.2 nmol/Kg/min produced a much larger reduction of renal MBF in HIF-1 N-hydroxy-N-(4-butyl-2methylphenyl) formamidine (HET0016, 1 mg/kg/hr, i.v.), a selective
decoy ODN-treated rats than in scrambled ODN-treated rats (66 6.1% vs. 84 3.9 % inhibitor of the synthesis of 20-HETE, completely prevented the rise in 20-HETE levels in the
of the control MBF), while there was no difference in Ang II-induced decreases in cortical renal cortex following an elevation in RPP and blunted the diuretic and natriuretic responses by
blood flow in both rat groups. In summary, local delivery of transcription decoy ODN with 47% and 39%, respectively. HET0016 had no significant effect on GFR or RBF. These results
ultrasound-microbubble technique effectively blocked the activity of HIF-1 in the renal suggest that elevations in RPP increase 20-HETE levels in the kidney secondary to increasing
medulla, and that decoy of HIF-1 blunted pressure-natriuresis and enhanced the
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RIHP and that 20-HETE contributes to the pressure natriuretic response.


e46 Hypertension Vol 48, No 4 October 2006

Poster Presentations
P1 months of age. Female sheep were synchronized in their estrous cycles by withdrawal of a
Gender Differences in Baroreflex Sensitivity and Lifespan in progesterone-releasing implant. ACE and ACE2 activities were determined in serum by
Renin-Angiotensinogen Transgenic Hypertensive Mice assessing the metabolism of radiolabeled Ang I and Ang II to Ang II and Ang-(17), respectively.
Ang I was readily converted to Ang II, Ang-(17) and Ang-(15) in serum and Ang II production
Veronica A Peotta, Rasna Sabharwal, Harald M Stauss, Eric Lazartigues, Carol A Whiteis, was abolished with the ACE inhibitor lisinopril (LIS); however, Ang-(19) was not detected in
Francois M Abboud, Mark W Chapleau; Univ. of Iowa, Iowa City, IA control or LIS-treated samples. Both male and female sheep exhibited similar ACE activities
based on the rate of Ang I to Ang II conversion [male: 49 5 fmol/ml/min, n5; female: 45
Men and women differ in baroreflex sensitivity (BRS), susceptibility to cardiovascular disease, 7 fmol/ml/min, n3]. Ang II was primarily converted to Ang-(17) in serum and its formation
and lifespan. Decreased BRS predicts increased mortality post-myocardial infarction. The goal was abolished with the ACE2 inhibitor MLN4760. Serum ACE2 activity based on Ang II to
of this study was to characterize BRS and age-related mortality in male vs. female mice with Ang-(17) formation was higher in male versus female [male: 38 4 fmol/ml/min, n5;
and without hypertension (HT). We hypothesized that BRS is impaired at a young age in HT, female: 22 2 fmol/ml/min, n4; p0.05]. ACE2 inhibition increased the half-life of Ang II
most prominently in male mice; and that age-related mortality is greater in male vs. female HT 6 fold in male serum [101 24 min vs. 602 196 min, n3] and 2.9 fold [150 29 min
mice. Blood pressure (BP) was measured in conscious control and HT (renin-angiotensinogen vs. 439 150 min, n3] in female serum. Immunoblots using an N-terminally directed
transgenic, RA) mice by radiotelemetry. Spontaneous BRS was calculated using the antibody revealed a 120 kD band in both male and female serum samples consistent with the
sequence technique. BRS was markedly impaired in male RA mice compared with male activity measurements. ACE2 activity was readily detectable in sheep serum and constituted
control mice at a young age (20 wks) (see Table, *P0.05). BRS tended to be reduced in the primary pathway for Ang II metabolism, although males exhibited higher serum activity than
female control mice, yet was slightly but significantly higher in female RA compared with females. Serum ACE2 did not directly convert Ang I to Ang-(19), however, Ang I to Ang II
male RA mice (**P0.05). Consequently, the HT-induced BR impairment was not conversion lead to increased levels of Ang-(17) via ACE2. We conclude that a soluble form of
statistically significant in females. Age-related mortality defined by Kaplan-Meier plots ACE2 is evident in serum at activity levels comparable to ACE and that both enzymes may
(Controls, n90; RA, n36) was markedly increased at one year of age in male RA contribute to the circulating levels of Ang II and Ang-(17).
mice (43%), an effect not seen in female RA mice (8%). Mortality at one year was low in
male (7%) and female (3%) control mice. The differences in BRS and mortality could not be
attributed to BP which was comparably increased in male and female RA mice (1653 vs. P4
1564 mmHg, respectively). We conclude that male mice with angiotensin-dependent HT Role of Angiotensin II in Female Mouse Model of Systemic Lupus
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017

exhibit a profound decrease in BRS at a young age accompanied by very high premature Erythematosus
mortality while female RA mice are protected from HT-induced baroreflex dysfunction and
do not die prematurely. Decreased BRS along with other factors may contribute to increased Michael J Ryan, Gerald R McLemore, Jr.; Univ of Mississippi Med Cntr, Jackson, MS
mortality in males with angiotensin-dependent hypertension. (VA and HL14388)
Systemic Lupus Erythematosus (SLE) is an autoimmune inflammatory disorder that predomi-
Males-BRS (ms/mmHg) Females-BRS (ms/mm Hg) nantly affects women. Individuals with SLE have a high incidence of hypertension, renal, and
Control Mice 2.330.35 (n5) 1.610.40 (n8) vascular disease. While blockade of the renin angiotensin system (RAS) is commonly used to
RA Mice 0.720.10*(n8) 1.000.19**(n9) treat its cardiovascular complications, the contribution of RAS to the pathophysiology of
hypertension during SLE is unclear. We tested the hypothesis that activation of the RAS
contributes to hypertension and altered angiotensin (AngII) dependent renal blood flow (RBF)
P2 responses in a mouse model of SLE (NZBWF1). Anesthetized female SLE mice or controls
Gender Differences in the Renal Structural Changes in Response to the (NZW/LacJ) were instrumented with a RBF probe and carotid artery and jugular vein catheters.
Reduction of Ang II during Nephrogenic Period Mean arterial pressure (1083 vs. 873 mmHg, n8, p0.01) and renal vascular resistance
(212 vs. 152 RU, p0.03) were significantly increased in SLE mice. Acute infusion of the
Fara Saez, Dept of Physiology, Sch of Medicine, Univ of Murcia, Murcia, Spain; M T Castell, AngII receptor blocker losartan (10g, i.v. bolus) caused a similar increase in RBF in control and
Adelina Zuasti, Dept of Hystology, Sch of Medicine, Univ of Murcia, Murcia, Spain; Analia SLE mice (457% vs. 456%, n10); however, losartan caused a greater decrease in
Loria, Francisco Salazar, Virginia Reverte, F. J Salazar; Dept of Physiology, Sch of Medicine, mean pressure in SLE mice compared to controls (-142 vs. -82 mmHg, n10, p0.04).
Univ of Murcia, Murcia, Spain Compared to controls, the RBF response to infusion of exogenous AngII (1ng, i.v. bolus) in SLE
mice was blunted (284% vs. 123%, p0.01). After placing mice on a high salt diet (4%,
It is well documented that angiotensin II (Ang II) is involved in nephrogenic development, and 7 days) to suppress endogenous RAS, the pressor response to AngII infusion was unchanged
that Ang II inhibition during perinatal period induces an elevation in arterial pressure in adults. (4.71.1 vs. 4.41.0 mmHg) in control mice and increased in SLE mice (3.20.9 vs. 5.51.2
This study was designed to test the hypothesis that normal renal development is more Ang mmHg). Similarly, the RBF response to AngII was restored after high salt treatment in SLE mice
II-dependent in males than in females and consequently, there are gender differences in the (-224%, n8, p0.05) but unchanged in controls (-325%, n7). These data suggest an
renal response to Ang II inhibition during perinatal period. Newborn SD rats were treated with increased activity of RAS and support a potential role for RAS in SLE induced hypertension.
an AT1 Ang II receptor antagonist (ARAII) (L-158.809, 7 mg/kg/day) during the first 14 postnatal
days. Renal structural changes were examined at 3 and 9 10 months of age using appropriate
techniques in male and female rats treated with vehicle or ARAII. At three months of age, P5
systolic arterial pressure increased (P0.05) to the same extent in treated males and females Up-Regulation of Renal Ace2 is Associated with Gender Differences in a
(127 0.5 vs. 115 0.7 mmHg in control rats, cr). Nephron number/gram b.w. decreased Model of Fetal Programming of Hypertension Induced by Placental
similarly (P0.05) in treated males (54 5 vs. 98 3 in cr) and treated females (81 5 Insufficiency
vs. 133 10 in cr) with the consequent elevation in mean glomerular volume that was 21%
greater (P0.05) in males than in females. Glomerulosclerosis was greater (P0.05) in treated Daniela Grigore, Norma Ojeda, Elliott B Robertson, Barbara T Alexander; Univ of Mississippi
males (56 2%) than in treated females (31 3%). Tubulointerstitial damage was also Med Cntr, Jackson, MS
greater (P0.05) in treated males than in treated females in renal cortex (22 3% vs 10
2%), outer medulla (17 3 vs. 9 2), and inner medulla (36 13 vs. 8 2). The ratio Hypertension and cardiovascular disease are programmed by exposure to adverse conditions
arteriolar wall thickness/luminal diameter was elevated in adult rats treated with ARAII during in utero. Reduced uterine perfusion initiated at day 14 of gestation in the pregnant rat results
the nephrogenic period but the increment was greater (P0.05) in males (3.5 0.1) than in in intrauterine growth restriction (IUGR) and hypertension by 4 weeks of age. Gender
females (2.3 0.1). Another important gender difference in the response to the Ang II inhibition differences are present since only male IUGR offspring remain hypertensive after puberty. A role
during the nephrogenic period was that only treated males had a significant decrease in papilla for RAS involvement in IUGR hypertension is suggested as hypertension in adult male IUGR is
volume (50%, P0.05). At nine months of age, there was a further elevation of arterial abolished by RAS blockade. Thus, the purpose of this study was to determine if age and gender
pressure in treated males (134 0.9 mmHg) and treated females (131 0.5 mmHg), and the specific RAS alterations are associated with IUGR-induced hypertension. At 12 weeks of age
previously mentioned gender differences in renal structure were significantly accentuated. In MAP by telemetry was significantly elevated (P0.05) in male IUGR as compared to male
summary, the results of this study provide new evidences showing important gender control, female control, and female IUGR offspring (1351, 1211, 1171, 1262 mmHg,
differences in the response of renal structure to the reduction of Ang II effects during respectively). Real-time PCR was used to measure renal cortical mRNA expression of RAS
nephrogenic period. components in male and female IUGR and control offspring. At 6 weeks of age, no significant
alterations in renal RAS components were present in male or female IUGR offspring as
compared to their sex-specific control counterpart. However, by 12 weeks of age, or after
P3 establishment of hypertension, renal cortical mRNA expression of angiotensinogen (Aogen) was
Gender Differences in ACE2-Dependent Metabolism of Angiotensin II in the significantly increased 11-fold in male IUGR (n 6) compared to male control (n 6); renin
Serum of Sheep 24-fold and ACE 13-fold. Renal cortical mRNA expression of Aogen was also significantly
increased 1.6-fold in female IUGR (n 6) compared to female control (n 6) offspring as was
Brian M Westwood, Jorge Figueroa, James C Rose, Mark C Chappell; Wake Forest Univ Sch renin, 4.4-fold, and ACE, 5.5-fold. However, the difference in expression for each RAS
of Medicine, Winston-Salem, NC component, comparison of IUGR to control, was significantly less in female compared to male
offspring. At 12 weeks of age ACE2 which is known to counterbalance ACE activity was
Increasing evidence suggests that angiotensin converting enzyme 2 (ACE2) is an enzymatic significantly decreased 6-fold in male IUGR relative to male control offspring. ACE2 expression,
component of the renin-angiotensin system (RAS) that may modulate the conversion of Ang II however, was significantly increased 6-fold in female IUGR relative to female control offspring.
to Ang-(17). We have shown in heart and kidney that tissue-bound or membrane-associated Thus, these results suggest inappropriate activation of ACE pathway components may
ACE2 plays a key role in the metabolism of Ang II, but not Ang I. The issue of circulating ACE2 participate in the maintenance of hypertension in adult male IUGR offspring. However, an
and its contribution to angiotensin metabolism has not been investigated; therefore the current increase in ACE2 may be a compensatory mechanism that protects female IUGR offspring from
studies evaluated ACE2 activity in the serum from both male and female adult sheep at 18 sustained hypertension into adulthood.
CHBPR ConferencePoster Presentations e47
P6 AT1R and AT2R activation. Few studies have examined the effects of chronic AngII infusion in
Differential Vascular Tissue Distribution of Angiotensin II Receptor females, and even fewer measure blood pressure via the sensitive method of radiotelemetry.
Subtypes during Pregnancy This finding is potentially of great significance as it suggests that females have in place
mechanisms to counter the classic pressor effects of AngII to increase blood pressure, that are
Vera V Koledova, Yaroslavl State Med Academy, Yaroslavl, Russian Federation; Raouf A not present or less effective in males.
Khalil; Brigham and Womens Hosp, Boston, MA

Several forms of hypertension are associated with upregulation of the renin-angiotensin


P9
system. Although the renin-angiotensin system is upregulated during normal pregnancy, Angiotensin Ii Type I Receptor Blockade Prevents Interleukin (IL-6)-Induced
reduction in blood pressure and decreased vasopressor response to AngII are often observed; Hypertension in the Pregnant Rat
however, the vascular mechanisms involved are unclear. AngII activates not only angiotensin
type 1 receptor (AT1R) to induce vasoconstriction, but also AT2R to release vasodilator Babbette LaMarca, Josh Speed, Lillian Fournier, Kathy Cockrell, Joey P Granger; UMMC,
substances. We tested whether the pregnancy-associated reduction in the pressor response to Jackson, MS
AngII reflects differences in the vascular tissue distribution of AT1R and AT2R subtypes.
Systolic blood pressure was measured in pregnant and virgin Sprague-Dawley rats, the Increases in inflammatory cytokines such as tumor necrosis factor (TNF) alpha and
thoracic aorta was isolated and cryosections (6 micron) were prepared for histological interleukin-6 have been proposed to be an important link between placental ischemia,
morphometric analysis using hematoxylin and eosin staining, and immunohistochemical endothelial dysfunction, and hypertension in women with preeclampsia. We have recently
analysis using antibodies to AT1R and AT2R and VectaStain Ellite ABC Kit. Blood pressure was reported that blood pressure and IL-6 are significantly elevated in response to reductions in
lower in pregnant (1002 mmHg) than virgin rats (1043 mmHg). The vessel wall thickness uterine perfusion in pregnant rats. We have also reported that a 5 day infusion of IL-6, at a rate
was thinner in pregnant (158) compared with virgin rats (206 microns). The tunica intima to mimic serum levels in the rat placental ischemic model, significantly increases blood
thickness as % of total was smaller in pregnant (2) than virgin rats (4), while the % tunica pressure in normal pregnant rats. We also found an increase in plasma renin activity in
media thickness was greater in pregnant (68) than virgin rats (63), possibly related to increased pregnant rats with IL-6-induced hypertension, suggesting a role for activation of the
hemodynamic forces during pregnancy. Significant AT1R and AT2R staining could be identified renin-agiotension system. Therefore, the purpose of this study was to determine the role of
in tissue sections from pregnant and virgin rats. In virgin rats, AT1R staining was abundant and angiotensin II in mediating IL-6-induced hypertension in pregnant rats. To achieve this goal, we
evenly distributed in the tunica media and smooth muscle (49.5%) and smaller AT2R staining compared the effects of increasing serum IL-6 ( 5ng/day for 5 days, IV) in normal pregnant rats
was detected in the tunica intima and endothelium (26.6%). In pregnant rats, AT1R staining in and in pregnant rats pretreated with an angiotensin II type I receptor antagonist (Losartan, 40
the smooth muscle layer was significantly reduced (32.3%), while AT2R staining in the mg/kg/day). Arterial pressure increased in normal pregnant rats (n6) from 102 2 to 118
endothelium was enhanced (59.2%). AT1R and AT2R staining in the adventitia was not different 4 mm Hg when serum IL-6 levels were increased from 63 5 pg/ml to 230 54 pg/ml.
between virgin (18.3 and 22.9%) and pregnant rats (13.1 and 26.6%, respectively). These data In sharp contrast, blood pressure in losartan-pretreated pregnant rats infused with IL-6 was not
significantly different (854 vs 854 mmHg) than control pregnant rats pretreated with
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suggest differential recruitment of the vasodilator AT2R and vasoconstrictive AT1R subtypes
between the vascular endothelium and smooth muscle during pregnancy, and may in part losartan (n8). The results of this study indicate that activation of the angiotensin type 1
explain the reduction of blood pressure despite upregulation of the renin-angiotensin system receptors play an important role in mediating IL-6-induced hypertension in pregnant rats.
during pregnancy.
P10
P7 Ovariectomy Induces Hypertension in Adult Growth Restricted Female
Presence of Activating Autoantibodies against the AT1-Receptor in Women Offspring in a Model of Fetal Programming due to Placental Insufficiency
with a History of Preeclampsia Norma Ojeda, Daniela Grigore, Elliott B Robertson, Barbara T Alexander; Univ of Mississippi
Med Cntr, Jackson, MS
Ralf Dechend, Helios Klinikum, Franz-Volhard-Klinik, Charite, Berlin, Germany; Myles Wolf,
Renal Unit Massachusetts General Hosp, Boston, MA; Nina Markovic, Magee-Womens Rsch In a rat model of fetal programming induced by placental insufficiency, male intrauterine
Institute and Dept of Obstetrics and Gynecology, Pittsburgh, PA; Florian Herse, Helios growth restricted offspring (IUGR) are hypertensive in adulthood whereas female IUGR offspring
Klinikum, Franz-Volhard-Klinik, Charite, Berlin, Germany; Gerd Wallukat, Max-Delbrueck are normotensive. The renin angiotensin system (RAS) appears to play a role in IUGR-induced
Cntr, Berlin, Germany; Carl A Hubel; Magee-Womens Rsch Institute and Dept of Obstetrics hypertension as ACE inhibition abolishes hypertension in adult male IUGR offspring. The
and Gynecology, Pittsburgh, PA purpose of this study was to determine whether removal of estrogen would reveal an increase
in arterial pressure in female IUGR offspring through unopposed activation of the RAS. Female
Activating autoantibodies against G protein-coupled AT1-receptor (AT1R-AA) and the alpha- control and IUGR offspring underwent either ovariectomy (OVX) or sham surgery (intact) at 10
adrenergic receptor have been detected in the serum of preeclamptic patients. Whether or not weeks of age followed by insertion of a telemetry probe for 24 hour measure of mean arterial
these activating autoantibodies persist after delivery is not known. If they persist, they might pressure (MAP) from 12 to 16 weeks of age. ACE inhibition (Enalapril, 40 mg/kg/day) or vehicle
be implicated in the increased cardiovascular risk after preeclampsia. We included 29 was initiated in a subset of all groups at 14 weeks of age. At 16 weeks of age MAP was similar
normotensive women with a history of preeclampsia and 32 women with normal pregnancies upon comparison of intact female control (1178 mmHg) and intact female IUGR offspring
during their first completed (nulliparous) pregnancy at 18.0 9.7 months postpartum in the (1182 mmHg). Ovariectomy led to a significant increase in MAP in female IUGR offspring
study. The activating antibodies were detected by the chronotropic responses to AT1 (1422 mmHg, P0.05 vs. intact female IUGR), while it remained without effect in female
receptor-mediated stimulation of cultured neonatal rat cardiomyocytes coupled with receptor- control offspring (1242 mmHg). ACE inhibition abolished hypertension induced by ovariec-
specific antagonists (losartan and prazosin). 17% of former preeclamptic patients and 3% tomy in adult female IUGR offspring (961 mmHg, P0.05 vs. untreated OVX female IUGR).
former normal pregnant patients showed a positive bioassay for antibodies directed at the However the decrease in blood pressure in response to Enalapril, in IUGR offspring was greater
AT1-receptor (p0.05). Activating autoantibodies against the alpha-adrenergic receptor were in OVX group, treated vs. untreated, as compared to the intact group, treated vs. untreated
found in 10% of formerly normal pregnant and 14% of formerly preeclamptic patients. In an (difference of 462 mmHg vs. 245 mmHg, respectively, P0.001) indicating enhanced RAS
earlier study, we found that sFLT1, indicating altered angiogenesis, is upregulated in the activation in the absence of estrogen. However, circulating RAS components were not
formerly preeclamptic group (41.6 6.7 vs. 30.4 10.2; P 0.01). There was no correlation significantly altered following ovariectomy (plasma renin activity and plasma renin substrate)
between patients with AT1R-AA and sFLT-1. We conclude that AT1R-AA did not invariably suggesting activation of tissue RAS or increased sensitivity to angiotensin II may occur in the
disappear after delivery but persist in nearly 20% of formerly preeclamptic patients. Further absence of estrogen in female IUGR. Thus, these results suggest that development of
follow-up is necessary to determine if AT1R-AA represent a subsequent risk in these women. hypertension in ovariectomized female IUGR offspring may occur through unopposed activation
Lack of correlation between AT1R-AA and sFLT may indicate heterogeneous mechanisms of the RAS.
leading to preeclampsia.

P11
P8 Differential Expression of Sry and Tyrosine Hydroxylase in Adrenal Gland of
Low Dose Angiotensin II Paradoxically Lowers Blood Pressure in Female WKY and SHR/y Rats
Rats
Daniel L Ely, Amy Milsted, Gail Dunphy, Monte Turner; Univ of Akron, Akron, OH
Amanda Sampson, Rebecca L Flower, Robert E Widdop, Kate M Denton; Monash Univ,
Melbourne, Australia Our laboratory has shown that a locus on the SHR Y chromosome (Yc) increases blood pressure
(BP) in the SHR rat and in WKY rats that had the SHR Yc locus crossed into their genome (SHR/y
There are striking sex-related differences in the regulation of arterial pressure and the rat). We have shown a novel function for the locus of interest, Sry, a transcription factor on the
incidence of cardiovascular disease. The mechanisms are incompletely understood, but it is Yc which elevates tyrosine hydroxylase (TH) promoter activity and norepinephrine (NE)
suggested that sex-hormones contribute by differentially modulating the renin-angiotensin production. SHR/y rats have increased NE content and turnover in kidney and heart. Also we
system, one of the major regulators of blood pressure. The two main angiotensin receptors, have identified at least 6 different copies of the gene on the Yc. When Sry was transfected to
angiotensin type 1 receptor (AT1R) and angiotensin type 2 receptor (AT2R), exert opposing a normotensive WKY, BP increased. To determine the time course of both Sry and TH adrenal
effects on the cardiovascular system. All the classical excitatory effects evoked by AngII expression,the following study examined endogenous Sry and TH expression using real time
(vasoconstriction, sodium reabsorption) result from AT1R stimulation, while AT2R stimulation PCR in the adrenal glands in WKY and SHR/y strains, before and during the time when BP was
causes vasodilatation and natriuresis, although the latter effects may be masked by the increasing (215 weeks). The adrenal epinephrine content increased 60% between 215
overriding effects of AT1R stimulation. Indeed, it is now appreciated that there is a low level of weeks in both strains. Adrenal NE content showed a 70% increase between 2 6 weeks in the
AT2R expression in the vasculature, although these data are almost universally obtained from SHR/y strain, whereas the WKY strain showed only a 26% increase between 215 weeks. The
male animals. Thus there is a dearth of knowledge on the influence of gender on both AT1R adrenal NE content may be a trigger for the sharp rise in BP around 6 weeks in the SHR/y strain.
and AT2R expression and functional responsiveness in the cardiovascular system. We made the In order to determine if exogenous Sry would elevate adrenal TH, NE and BP, we delivered
remarkable observation that 2 weeks low dose AngII decreases blood pressure in female rats 10g of either the expression construct, Sry1/pcDNA 3.1 or control vector, into the adrenal
(-81 mmHg, P0.05) at levels (50 ng/kg/min s.c) that increase blood pressure in males medulla by electroporation. One week after exogenous Sry delivery there was a 98% increase
(51 mmHg, P0.05). This startling finding is consistent with opposing vascular effects of in TH compared to vector controls. After 3 weeks there were significant increases in resting
e48 Hypertension Vol 48, No 4 October 2006

plasma NE and adrenal TH content compared to vector controls. BP was 30mmHg higher in Sry II levels were higher in female SHR compared to males (male: 10.21.2 pg/mL; female:
injected animals (160mmHg) compared to vector controls (130mmHg) after 3 weeks. In 20.11.5, p0.001). Despite females having higher levels of plasma angiotensin II, male SHR
conclusion, Sry, a transcription factor on the Y chromosome, modulates adrenal medullary had greater AT1 protein expression in the renal cortex compared to female SHR (male:
function and blood pressure. 0.0450.003 RDU; female: 0.0190.002 RDU, p0.001). We then assessed the level of
activation of a known AT1 pathway target, JAK/STAT. Activated JAK1 and JAK2 levels in the
renal cortex from female SHR were significantly higher compared to male SHR (expressed as
P12 % phosphorylated/total protein, JAK1: male, 11.11.4 %; female, 44.78.3 %,p0.01; JAK
Decreased Autonomic Contribution to Obesity-Associated Hypertension in 2: male, 56.74.4 %; female, 72.59.9 %, p0.05). Male SHR had significantly higher levels
African Americans Women of STAT6 (male: 65.517.2 %; female: 32.44.3 %, p0.05) and STAT1 (male: 45.13.8 %;
female 31.26.2%, p0.05) activation in the renal cortex, while female SHR had significantly
Cyndya Shibao, Alfredo Gamboa, Andre Diedrich, Ginnie Farley, Italo Biaggioni; Vanderbilt higher levels of STAT5 (male: 4.03.2 %; female 25.65.5%, p0.05) activation. These data
Univ, Nashville, TN suggest that differential activation of the AT1/JAK/STAT pathways may be involved in promoting
renal injury in male SHR and the reno-protection afforded to female SHR.
Obesity affects nearly 30% of the US population and is a risk factor for hypertension. Those
most affected, with the higher prevalence of obesity and hypertension, are African-Americans,
particularly women. Increased sympathetic activation has been associated with obesity and it
may contribute to obese-related hypertension, however blood pressure is poorly controlled with
sympatholytics agents in hypertensive African-Americans. We tested the hypothesis that the
autonomic nervous system contributes less to blood pressure regulation in obese African- P14
Americans as compared to obese Caucasians. A total of 17 obese healthy females were studied Prolonged Enhancement of Vascular Inflammatory Process and Oxidative
(9 Caucasians, Age 374 years, BMI 351.2 Kg/m2, BP 1255/744 mm Hg, and 8 African Stress Aggravates Hypertensive Myocardial Fibrosis and Diastolic
Americans, Age 352 years, BMI 340.9 Kg/m2 ,BP 1267/744 mm Hg, P0.05).
Dysfunction in Ovariectomized Rats
Complete autonomic blockade with trimethaphan decreased mean arterial blood pressure
(MAP) more in Caucasians (-194 mm Hg as compared to African Americans (-82 mm Hg,
Takahiro Mori, Hisashi Kai, Hiroshi Kudo, Yusuke Sugi, Narimasa Takayama, Kiyoko
Figure). Intrinsic MAP, in the absence of autonomic influences, was higher in African Americans
Takemiya, Mitsuhisa Koga, Yumiko Kawai, Daisuke Fukui, Ayami Ikeda, Masayoshi
(836 vs 723 mm Hg in Caucasians, P0.124). The decreased in MAP was negatively
Futamata, Hideo Yasukawa, Tsutomu Imaizumi; Kurume Univ, Kurume, Japan
associated with the androgen to gynecoid ratio (A/G), an index of abdominal fat distribution
(R-0.6, P0.03). We conclude that the autonomic nervous system contributes less to blood
The prevalence of diastolic dysfunction is high in hypertensive women, particularly in the
pressure regulation in African Americans as compared to Caucasians obese women.
post-menopausal state. However, the underlying mechanism remains unknown. Recently, we
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have shown that in male Wistar rats with a supra-renal aortic constriction (AC), pressure
overload induces a transient perivascular inflammation, which leads to reactive myocardial
fibrosis and the resultant diastolic dysfunction. The aims of this study were to determine
whether the estrogen withdrawal would aggravate pressure overload-induced myocardial
fibrosis and the resultant diastolic dysfunction and if so to examine whether these changes
would be associated with inflammatory process. For these purposes, female Wistar rats were
randomized to the following 2 groups: ovariectomy (OVX)AC rats received bilateral OVX and
ShamAC rats received the sham-operation at 6 week-old. In both groups, rats underwent AC
at 10 week-old (Day 0). OVX had no effects on blood pressure elevation during the observation
period up to 28 days after AC (Day 28). There were no significant differences in LV weight/body
weight ratio and myocyte diameter between the two groups at Day 28. However, OVX enhanced
the AC-induced myocardial fibrosis and aggravated diastolic dysfunction assessed by Doppler
echocardiography. In ShamAC rats, myocardial MCP-1 induction and perivascular macro-
phage infiltration were transiently induced with a peak at Day 3, and they returned to the
baseline levels by Day 28. In OVXAC rats, the AC-induced MCP-1 induction and macrophage
infiltration at Day 3 were enhanced and remained high levels at Day 28. In ShamAC rats,
dihydroxyethidium (DHE) staining revealed a transient superoxide generation in the intramyo-
cardial arterioles, peaking at Day 3 and decreasing to the insignificant level by Day 7. In
contrast, the vascular DHE signals remained higher levels at Day 28 in OVXAC rats. In
conclusion, the estrogen withdrawal aggravated the pressure overload-induced myocardial
fibrosis and diastolic dysfunction through prolonged enhancement of vascular inflammatory
process and oxidative stress.

P15
Dysregulation of the Circulating and Local Renin-Angiotensin System in
Preeclampsia

Florian Herse, Ralf Dechend, Helios Klinikum, Franz-Volhard-Klinik, Charite, Berlin, Germany;
Nina Harsem, Dept of Obstetrics and Gynecology, Ulleval Univ Hosp, Oslo, Norway; Gerd
Wallukat, Max-Delbrueck Cntr, Berlin, Germany; Jurgen Janke, Henning Damm, Helios
Klinikum, Franz-Volhard-Klinik, Charite, Berlin, Germany; Dominik N Muller, Max-Delbrueck
Cntr, Berlin, Germany; Anne C Staff; Dept of Obstetrics and Gynecology, Ulleval Univ Hosp,
Oslo, Norway

The renin-angiotensin system (RAS) has been implicated in the pathogenesis of preeclampsia,
although conflicting results have been demonstrated, especially regarding locally tissue-related
RAS. Plasma renin activity (PRA) and local RAS gene expression was investigated in
P13 preeclamptic (n11) and uneventful pregnancies (n10) at cesarean delivery. Maternal
Molecular Mechanisms of Sexual Dimorphism in Renal Injury in serum, subcutaneous adipose tissue, placental biopsies and decidual vacuum suction material
Spontaneously Hypertensive Rats (SHR): Involvement of Angiotensin II and was collected. Autoantibodies against the AT1 Receptor (AT1R-AA) were measured by cardiac
the JAK/STAT Pathways contraction bioassay. We also analyzed mRNA expression of renin, renin receptor (ReRe),
angiotensinogen (Aogen), angiotensin-converting enzyme (ACE), AT1 Receptor and AT2
Amy Banes-Berceli, Mario B Marrero, Jennifer S Pollock, Jennifer C Sullivan; Med College Receptor in decidua, placenta and adipose tissue by quantitative real time RT-PCR. PRA was
of Georgia, Augusta, GA significantly reduced in the maternal circulation of preeclamptic patients. We detected AT1R-AA
in 78% of preeclamptic patients compared to 0% of control patients. AT1 receptor expression
Male SHR have a more rapid progression of renal injury compared to female SHR. The was 4-fold increased in the decidua of preeclamptic patients compared to controls. There was
molecular mechanism(s) responsible for the sexual dimorphism in renal injury, however, is a 1.5 fold increased AT1-expression in adipose tissue of preeclamptic patients. AT2 receptor
unknown. We hypothesized that altered angiotensin II receptor 1 (AT1) signaling to the Janus expression was nearly absent in preeclamptic placenta, whereas weak expression was
Kinase (JAK)/signal transducers and activators of transcription (STAT) pathway in the renal detectable in most of the controls (10% vs 60%). No difference between the two groups was
cortex from male and female SHR promotes renal injury and reno-protection in male and female observed for renin, ReRe, ACE or Aogen expression in either tissues investigated. However,
SHR respectively. While no studies have investigated differential regulation of the JAK/STAT renin expression was nearly 114-fold increased in decidua compared to placenta for both
pathways by gender, previous studies have demonstrated in male diabetic rats that angiotensin patient groups. The substrate Aogen and ACE were also much higher expressed in decidua as
II-induced activation of the JAK2/STAT1 pathway increases proteinuria and renal injury. compared to placenta biopsies. In conclusion,Tissue RAS genes and PRA are dysregulated in
Whereas, STAT5 activation has been shown to be anti-apoptotic in cultured endothelial cells. preeclampsia. The activating AT1 receptor autoantibody is present in most preeclamptic
Male and female SHR (14 16 week-old) were used in all studies, n5 8. A terminal plasma women. The AT1 receptor expression is upregulated in decidua and adipose tissue of
sample was collected and kidneys were isolated for Western blot analysis. Plasma angiotensin preeclamptic women. These findings suggest a role for the RAS in preeclampsia.
CHBPR ConferencePoster Presentations e49
P16 kins, may be important mediators of endothelial dysfunction and hypertension in preeclamptic
Estrogen Blunts Angiotensin II- Induced Activation of NAD(P)H Oxidase in women. Furthermore, the increased expression of the angiogenic cytokines from the ischemic
Female Mice: Role of Thioredoxin preeclamptic placenta, such as CNTF and angiogenin, may be evidence of an innate attempt
to improve placental blood flow via vascular remodeling.
Talin Ebrahimian, Ernesto L Schiffrin, Lady Davis Institute for Med Rsch, SMBD Jewish
General Hosp,McGill Univ, Montreal, Canada; Rhian M Touyz; Kidney Rsch Cntr, Ottawa
Health Rsch Institute, Ottawa, Canada
P19
Human Renal Mesangial Cells Can Produce Aldosterone in Response to
Thioredoxin (Trx) is a small ubiquitous protein, which could act directly as an antioxidant by Low-Density Lipoprotein(LDL)
modulating Reactive Oxygen Species (ROS). Estrogen has been shown to have protective
effects against oxidative stress. In addition there is gender dimorphism with respect to the Trx Tetsuo Nishikawa, Jun Saito, Sachiko Suematsu, Yuzuru Ito, Yoko Matsuzawa, Hiroko Ito,
system and differential regulation of the cardiac Trx system and NAD(P)H oxidase activity by Yokohama Rosai Hosp, Yokohama, Japan; Tomoshige Kino, George Chrousos; National
Angiotensin II (Ang II) in male and female mice. We questioned whether estrogen has a Institute of Child Health and Human Development, Bethesda, MD
protective effect in Ang II-induced hypertension and if the Trx system contributes to this
protective effect in female mice. C57bl/6 female intact (Cont) or ovariectomized (Ovx) mice Aldosterone is classically produced by the zona glomerulosa cells of the adrenal cortices.
were infused with Ang II (400 ng/kg/min) for 14 days. Systolic blood pressure (SBP) was Systemic aldosterone plays an important role in the development of the microvascular disease
measured by tail cuff. Oxidative stress was evaluated by dihydroethidine staining and and glomerular damage of the kidney. Here, we investigated the possibility of local production
chemiluminescence. The expression of Trx was measured by western- blot. The SBP, similar of aldosterone in the kidney, using human primary glomerular mesangial cells. These cells
in Cont and Ovx groups (P0.05, n6), increased by Ang II to similar levels in both groups produced both pregnenolone and aldosterone measured by specific radioimmunoassay and/or
(1253 vs. 897 mmHg in Cont and 1362 vs. 1126 mmHg in Ovx, P0.001 and GC/MS methods. The production of both steroids was significantly stimulated by treatment with
P0.0002 respectively). Aortic Trx basal expression similar in both groups, was decreased (0.4 LDL, while angiotensin II had a synergistic effect. ACTH and (Bu)2cAMP, on the other hand,
fold) by Ang II in Cont mice (P0.01, n6), and was unchanged in Ovx mice compared to the failed to stimulate aldosterone production by these cells, suggesting that the local production
control counterpart (P0.05, n4). Aortic *O2- level as an index of oxidative stress, was higher of this steroid by mesangial cells is regulated differently from that of adrenal zona glomerulosa
(1.5 fold) in Ovx group compared to Cont group, Ang II increased *O2- level (1.5 fold) only in Ovx cells. LDL-induced aldosterone production by mesangial cells was further enhanced when
mice compared to Cont mice (P0.01, n4). Moreover NAD(P)H oxidase activity was similar incubated with high concentration of glucose in the culture media. Mesangial cells expressed
under basal conditions between both groups, was decreased by Ang II in Cont group (P0.01, the mRNA of the LDL receptor and steroidogenic enzymes, such as P450scc, 3b-HSD,
n4 5), whereas it was significantly increased (1.3 folds) in Ovx group (P0.05, n4 5). 21-hydroxylase and CYP11B2. Expression of CYP11B2 mRNA was remarkably enhanced by
These results demonstrate that estrogen negatively modulates the Trx and AngII-induced LDL. Mesangial cells also expressed mRNA of the mineralocorticoid receptor (MR), and LDL
NAD(P)H oxidase activity in Ang II infused female mice independently of the increase in blood stimulated its abundance by 3 fold, while spironolactone completely abolished this LDL effect.
Since MR is a known mineralocorticoid-response gene as well as an intracellular receptor
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pressure.
molecule for this steroid, these results suggest that locally produced aldosterone is biologically
active, stimulating the transcription rates of the mineralocorticoid-responsive genes by
activating the MR in mesangial cells. Our data indicate that human mesangial cells are an
P17 aldosterone-producing tissue in which LDL plays a major regulatory role. Therefore, human
Differential Regulation by ER Alpha and ER Beta for ACE and ACE2 renal mesangial endocrine system may contribute to local aldosterone concentrations and
effects in the renal glomerulus independently of the systemic RAA system and may participate
K. Bridget Brosnihan, Liomar A Neves, Patricia Gallagher, Wake Forest Univ Sch of in the development and progression of glomerular damage in several pathologic conditions
Medicine, Winston-Salem, NC; Jeffrey B Hodgin, Oliver Smithies, Nobuyo Maeda; Univ of such as diabetic nephropathy.
North Carolina, Chapel Hill, NC

ACE and ACE2 are key enzymes in the regulation of Ang II and Ang-(17) formation. ACE is P20
known to be down-regulated by estradiol, but it is not known if estrogen (E2) regulates ACE2. Non-Genomic c-Src-Dependent Signaling by Aldosterone Occurs through
To investigate the role of estrogen receptor-alpha (ER) in estrogens regulation of ACE and
Caveolin-Enriched Membrane Microdomains
ACE2 mRNA in lung, we ovariectomized female mice lacking apolipoprotein E (AAee) or
apolipoprotein E and ER (ee) and treated them with estradiol (6 g/day) or placebo for 3
Glaucia E Callera, Augusto C Montezano, Univ of Ottawa, Ottawa, Canada; Alvaro Yogi, Rita
months. ACE and ACE2 mRNA were measured using RT-PCR. E2 treatment of ovariectomized
C Tostes, Univ of Sao Paulo, Sao Paulo, Brazil; Rhian M Touyz; Univ of Ottawa, Ottawa,
AAee mice downregulated ACE mRNA (1.00.05 vs 0.76 0.6 units, p0.005); a similar
Canada
reduction (1.10.1 vs 0.760.1 units) was observed in ee. E2 treatment had no effect on
ACE2 mRNA in AAee mice, but resulted in significant reduction in ACE2 mRNA in ee (0.96
We previously demonstrated that aldosterone (Aldo) rapidly increases activation of NAD(P)H
0.1 vs 0.640.6 units, p 0.01). These results suggest that an ER-independent receptor,
oxidase through c-Src-dependent pathways in vascular smooth muscle cell (VSMC). Aldo
most likely the ER, plays a role in ACE mRNA regulation. On the other hand, E2
induces c-Src trafficking into membrane cholesterol rich domains. Whether non-genomic
down-regulation of ACE2 mRNA is revealed only in the absence of ER. Under conditions of
c-Src-dependent signaling by Aldo involves the specialized caveolin-enriched membrane
estrogen treatment where E2 downregulates ACE mRNA and has no effect on ACE2 mRNA, the
microdomains remains unclear. In the present study we hypothesized that Aldo activates c-Src
formation of Ang-(17) would be favored.
through Caveolin-enriched/cholesterol rich domains. Detergent resistant fractions from rat
VSMC were obtained by sucrose gradient centrifugation and identified by flotillin-2 expression.
Methyl--cyclodextrin (10-2) was used to disrupt membrane cholesterol rich domains.
P18 Knockdown of Cav 1 protein by small interfering RNA was performed in VSMC. Aldo (10-7 mol/L)
Maternal Plasma and Placental Cytokine Profile in Hypertensive effects on c-Src expression and phosphorylation were assessed in total protein and in
Preeclamptic Women membrane fractions. NAD(P)H-dependent generation of .O2- were evaluated by lucigenin-
derived luminescence assay. The translocation of p47phox subunit was assessed in cholesterol
Babbette LaMarca, UMMC, Jackson, MS; Bartira E Pinheiro da Costa, Pos-graduacao em rich domains. Cav 1 knockdown abolished Aldo-induced c-Src phosphorylation (p 0.05). The
Medicina e Ciencias da Saude, FAMED/IPB/HSL, Pontifcia Universidade Catolica do RGS,, phosphorylation of cortactin, a c-Src downstream target, was inhibited by Cav 1 knockdown
Porto Alegre, Brazil; Lillian Fournier, UMMC, Jackson, MS; Kiele Hoffmann, Pos-graduacao (p 0.05). Aldo induced NAD(P)H-dependent generation of .O2- in VSMC (1-fold, p0.01) and
em Medicina e Ciencias da Saude, FAMED/IPB/HSL, Pontifcia Universidade Catolica do this effect was inhibited by cholesterol rich domains disruption (p 0.05). Aldo stimulated
RGS,, Porto Alegre, Brazil; Giovani Gadonski, Pos-graduacao em Medicina e Ciencias da p47phox translocation into cholesterol rich domains (2-fold, p0.05). Aldo-mediated c-Src
Saude, FAMED/IPB/HSL, Pontifcia Universidade Catolica do RGS, Porto Alegre, Brazil; Carlos translocation into cholesterol rich domains (1.5-fold) was decreased by PP2 (0.2-fold, p0.05),
E Poli de Figueiredo, Pos-graduacao em Medicina e Ciencias da Saude, FAMED/IPB/HSL, a c-Src activity inhibitor. Aldo actions were inhibited by eplerenone, a mineralocorticoid
Pontifcia Universidade Catolica do RGS,, Porto Alegre, Brazil; Joey P Granger; UMMC, receptor antagonist. Our results suggest that 1) c-Src activation by Aldo in VSMCs mediates
Jackson, MS NAD(P)H oxidase activation through cholesterol rich domains, 2) c-Src phosphorylation
precedes its translocation into cholesterol rich domains, 3) caveolin-enriched membrane
Preeclampsia is a hypertensive disorder of human pregnancy that causes high morbidity and microdomains contribute to non-genomic c-Src-dependent signaling by Aldo. Findings from
mortality. Women with preeclampsia present with increased blood pressure, systemic these studies further identify molecular mechanisms underlying Aldo-induced non-genomic
endothelial dysfunction, placental ischemia, and enhanced production of inflammatory actions, which are mediated through classical mineralocorticoid receptors.
cytokines. Cytokines alter many immune response pathways and other biological responses
like endothelial function and angiogenesis. The aim of the present study was to examine the
cytokine profile in maternal plasma and placenta in normal pregnant and preeclamptic women. P21
Recruitment of preeclamptic and healthy pregnant women was performed at So Lucas Hospital, Aldosterone Suppresses Insulin Signaling via the Downregulation of Insulin
Brazil. Patients with severe preeclampsia were diagnosed with blood pressure above 160/110 Receptor Substrate-1 in Rat Vascular Smooth Muscle Cells
mmHg after 20 weeks of gestation accompanied by proteinuria of at least 2 g/24 hour. Samples
of blood and placental bed biopsy were collected immediately before and after caesarian, Hirofumi Hitomi, Hideyasu Kiyomoto, Dept of Cardiorenal and Cerebrovascular Medicine,
respectively. Protein array analysis was used to characterize 120 cytokines in both plasma and Kagawa Univ, Kagawa, Japan; Akira Nishiyama, Dept of Pharmacology, Kagawa Univ,
placenta tissue. Relevant cytokines were classified as pro-angiogenic (n37), inflammatory Kagawa, Japan; Taiga Hara, Kumiko Moriwaki, Naoki Kondo, Kumiko Kaifu, Genei Ihara,
(n60), anti-angiogenic (n4). The Student t and Chi-square tests were used to determine Yoshiko Fujita, Masakazu Kohno; Dept of Cardiorenal and Cerebrovascular Medicine,
statistical significance, p0.05. Approximately 50% of all cytokines examined were signifi- Kagawa Univ, Kagawa, Japan
cantly up-regulated in preeclamptic patients with greater differences seen in protein isolated
from placenta. In plasma samples 2 pro-angiogenic, 14 inflammatory and 2 anti-angiogenic Reports indicate impaired glucose tolerance in the patients with primary aldosteronism;
were significantly altered in preeclamptic patients. In protein isolated from placenta, 16 however, the relationship between aldosterone and insulin signaling pathway has not been
pro-angiogenic, 19 inflammatory and 1 anti-angiogenic was significantly altered in preeclamp- clarified. In this study, we examined the effects of aldosterone treatment on insulin receptor
sia. In summary, we found that placental and plasma cytokine profile was significantly altered substrate-1 (IRS-1) expression and Akt phosphorylayion in rat vascular smooth muscle cells
in preeclamptic women. Proinflammatory cytokines, such as TNF alpha and various interleu- (VSMCs). IRS-1 protein expression and Akt phosphorylation were determined by Western blot
e50 Hypertension Vol 48, No 4 October 2006

analysis with anti-IRS-1 and phosphorylated-Akt antibodies, respectively. P0.05 was P24
considered significant. Aldosterone (1100 nmol/L) dose-dependently decreased IRS-1 protein Angiotensin II-Induced Protein Kinase D Activation is Mediated by Protein
expression with a peak at 18 h (n4 7 for each). Aldosterone-induced degradation of IRS-1 Kinase C in H295R Human Adrenocortical Cells
was markedly attenuated by treatment with a selective mineralocorticoid receptor antagonist,
eplerenone (10 mol/L, n4). Treatment with antioxidants, N-acetylcysteine (10 mmol/L) or Damian G Romero, Bronwyn L Welsh, Silvia Rilli, Licy L Yanes, Elise P Gomez-Sanchez,
ebselen (40 mol/L), also abrogated aldosterone-induced IRS-1 degradation (n4 7 for each). Celso E Gomez-Sanchez; Univ of Mississippi Med Cntr, Jackson, MS
Additionally, proteasome inhibitor, MG132 (1 mol/L) prevented IRS-1 degradation (n4).
Aldosterone treatment abolished Akt phosphorylation induced by insulin (100 nmol/L, 5 min, Angiotensin II (Ang II) is one of the most important physiological modulators of adrenal cell
n4). These data indicated that aldosterone decreases IRS-1 expression via reactive oxygen physiology where Ang II regulates aldosterone secretion and proliferation. Abnormalities in
species stimulation in VSMCs; thus aldosterone may be involved in the pathogenesis of aldosterone secretion have been associated with nearly 10 % of the cases of primary
vascular insulin resistance. hypertension. However, Ang II signaling in adrenal cell remains poorly understood. H295R
human adrenal cells are a widely used experimental model to study adrenal cell physiology.
Protein kinases are crucial mediators in intracellular signaling. We screened for protein kinase
expression levels using the KinetworksTM system in H295R cells after 3 h Ang II treatment.
Protein kinase D (PKD) was the protein kinase that exhibits the most dramatic changes. PKD
P22 is a member of a new class of serine/threonine protein kinases that is activated by
Spironolactone Reduces Ischemic Infarct Size in Male, but not Female phosphorylation. PKD has been implicated in the regulation of a variety of cellular functions,
Hypertensive Rats including signal transduction, membrane trafficking, protein transport, and cell survival,
migration, differentiation, and proliferation. We show that Ang II time- and dose-dependently
Christine S Rigsby, Anne M Dorrance; Med College of Georgia, Augusta, GA increase PKD phosphorylation in H295R cells. PKD phosphorylation occurs within 2 min of Ang
II treatment and at concentrations as low as 1 nM. PKD phosphorylation was dose-dependently
Data from the Framingham Heart Study suggests that women may be more sensitive to the increased by the protein kinase C (PKC) activator PMA. Ang II-mediated PKD phosphorylation
deleterious cardiovascular remodeling effects of aldosterone. Our laboratory has previously was blocked by several PKC inhibitors as Bisindolylmaleimide I, Go 6983, Ro-31 8220 and
shown that male spontaneously hypertensive stroke-prone rats (SHRSP) treated chronically Ro-32 0432. PKC inhibitor profiles and PKC isozyme expression profile in H295R cells suggest
with spironolactone, a mineralocorticoid receptor antagonist, have 50% smaller ischemic that PKC could mediate PKD phosphorylation. We used different experimental approaches to
infarcts after the induction of middle cerebral artery occlusion (MCAO), without a change in interfere with PKC function and study its role on PKD phosphorylation. PKC translocation
blood pressure. Therefore, we hypothesized that female SHRSP treated with spironolactone inhibitor peptide decreased Ang II-mediated PKD phosphorylation (-47 %, p 0.05). PKC
would also have less damage after MCAO. Female SHRSP were treated for six weeks from six expression downregulation by plasmid delivered RNA interference also decreased PKD
weeks of age with spironolactone (25 or 50 mg/kg/day) and compared to non-treated controls phosphorylation (-25 %, p 0.05) mediated by Ang II. This study reveals for the first time that
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017

and Wistar Kyoto (WKY) rats. At 12 weeks of age, cerebral ischemia was induced using the PKD is an intracellular signaling mediator of Ang II in human adrenal cells. These findings show
permanent MCAO technique. After 18 hours, the brain was removed, sliced (2 mm), and stained a novel intracellular signaling system in adrenal cells that could be involved in pathological
with 2% TTC to visualize the infarcted region; infarct volume was analyzed and the percent of conditions related to alterations in Ang II or aldosterone secretion.
the hemisphere infarcted (%HI) was determined using the Swanson equation. Surprisingly,
spironolactone-treated female SHRSP did not have decreased infarct sizes compared to control
SHRSP (table). Mean arterial pressure was not altered by spironolactone treatment (table), as P25
evidenced by the use of telemetry. We also examined the effect of acute administration of
spironolactone on ischemic infarct size in female SHRSP. Spironolactone (25 mg/kg, IP) was
The Effect of Aldosterone in Angiotensin Ii/High Salt Induced Heart
administered 24 hours before and again at the time of MCAO; infarct sizes were analyzed 24 Damage and the Relationship with Oxidative Stress
hours post-MCAO, as described above. Acute administration of spironolactone also had no
effect ischemic infarct size (65.0 1.2%HI control vs. 63.8 1.5%HI spironolactone-treated, Hong Wang, Tatsuo Shimosawa, Hiromitsu Matsui, Tomoyo Kaneko, Sayoko Ogura, Toshiro
p0.05). These interesting studies indicate an apparent sexual dimorphism in the actions of Fujita; the Univ of Tokyo, Tokyo, Japan
spironolactone.
TABLE. Angiotensin II can cause organ damage by increasing oxidative stress directly. Recently,
aldosterone, which is released by angiotensin II, has also been shown to induce cardiac
Experimental Group (n) Mean Arterial Pressure (mmHg) %HI damage partly by increasing oxidative stress. Most basic research revealed that aldosterone-
SHRSP - Control (9) 145.04.9 66.31.3
induced organ damage are closely related to salt intake. In order to clarify the relation between
SHRSP - Spir 25 mg/kg (8) 129.76.3 63.21.3 salt status and aldosterone in cardiac damage and the role of oxidative stress, we examined
SHRSP - Spir 50 mg/kg (7) - 61.81.3 the effect of circulating aldosterone by salt restriction, local aldosterone by angiotensin II and
WKY - Control (4) 98.21.6* 22.97.0* high salt loading and the aldosterone antagonist, eplerenone, in Sprague Dawly rat. Rats were
allocated to five groups as follows: 1. angiotensin II high salt diet; 2. angiotensin II low
All values are mean SEM. *p0.05 vs. SHRSP groups. Spir spironolactone
salt diet; 3. high salt diet; 4. low salt diet; and 5. angiotensin II high salt diet eplerenone.
The blood pressure was highest in the angiotensin II high salt group and eplerenone did not
decrease the blood pressure. Diastolic heart function was monitored by both deceleration time
(DcT; measured with Doppler echocardiography) and time constant (T; measured with Left
P23 ventricular pressure curve). Diastolic function was deteriorated by angiotensin II high salt
(DcT; 69.29 6.17 ms and T; 18.63 3.59 ms) which was reversed by aldosterone blockade
Pentoxifylline Decreases Cardiac Hypertrophy and Renal Damage in Rats
of eplerenone (DcT; 59.58 0.98 ms and T; 12.64 2.19 ms). Although aldosterone was
with Mineralocorticoid Dependent Hypertension stimulated by angiotensin II, angiotensin II low salt group did not show diastolic dysfunction.
The oxidative stress, which is known to induce heart damage, was higher in high salt group
Anita D Smith, Anne M Dorrance; Med College of Georgia, Augusta, GA and it was decreased by eplerenone. These results suggest salt status is essential in oxidative
stress and angiotensin-aldosterone axis to induce heart damage. Eplerenone can attenuate
Rats with deoxycorticosterone acetate (DOCA)-salt hypertension have elevated levels of heart dysfunction independent of blood pressure, but by blocking the aldosterone effect and
proinflammatory cytokines. Pentoxifylline (PTX) is a phosphodiesterase inhibitor that inhibits the decreasing NADPH oxidase-derived oxidative stress. Salt restriction is also effective in
release of tumor necrosis factor-, we have previously shown that treatment of DOCA-salt rats preventing cardiac dysfunction by the inhibition of oxidative stress.
with PTX reduces their blood pressure and increases the levels of the anti-inflammatory
cytokine interleukin-10. We hypothesized the PTX treatment would also reduce oxidative stress
and inflammation in DOCA-salt treated rats and that this would result in a reduction in cardiac
hypertrophy and urinary protein excretion. Male Sprague-Dawley rats were uninephrectomized P26
and treated with DOCA (200mg/kg) PTX (9.6mg/day), both groups of rats were given salt Changes in Mineralocorticoid Receptors (MR) and Epithelial Sodium
water to drink (1% NaCl and 0.2%KCl), control rats were uninephrectomized and were given Channels (ENaC) in Rat Brain by Central Sodium
regular tap water. After 28 days of treatment 24-hour urine samples and terminal blood
samples were obtained. PTX treatment significantly reduced the blood pressure in the Hong-Wei Wang, M.Shahrier Amin, Junhui Tan, Frans H Leenen; Univ of Ottawa Heart
DOCA-salt treated rats and reduced cardiac hypertrophy as evidenced by a reduction in the Institute, Ottawa, Canada
heart / body weight ratio. Plasma thiobarbitutic acid reactive substances (TBARS) were used as
a measure of oxidative stress; these were increased in the DOCA-salt rats and reduced by PTX Both MR and ENaC subunits (, , ) are expressed in rat brain on epithelia and neurons.
treatment. Plasma monocyte chemoattractant protein-1 (MCP-1) was used as a marker of Central blockade of MR or ENaC prevents sympathetic hyperactivity and hypertension induced
inflammation; this was also elevated in the DOCA-salt rats and reduced by PTX treatment. 24 by icv infusion of Na-rich aCSF. Mechanisms regulating ENaC in the brain are still unknown.
hour urinary albumin excretion was lower in the DOCA-salt rats treated with PTX compared to To assess transcriptional and post-transcriptional regulation, we evaluated MR and ENaC mRNA
the rats treated with DOCA-salt alone, suggesting that PTX treatment may reduce renal and protein levels in the brain in response to Na-rich aCSF by real-time qRT-PCR,
damage, interestingly, this occurred without a reduction in kidney hypertrophy (Table1). These immunohistochemistry and aldosterone receptor binding assay. mRNA levels of serum/
results suggest that PTX may be a valuable therapeutic agent for the treatment of hypertension glucocorticoid regulated kinase 1 (SGK1) , an important regulator of ENaC, were also examined.
related end-organ damage. Male Wistar rats were infused icv for 2 weeks with regular or Na-rich aCSF with or without
TABLE 1 spironolactone. Brain areas studied included ependyma of the anteroventral 3rd ventricle, SON,
PVN and SFO. In the ependyma, Na-rich aCSF increased protein levels (p0.05) for both
TBARS Urinary Heart/ Kidney/ and ENaC, which were prevented by spironolactone. In the SON, Na-rich aCSF did not affect
Blood Pressure MCP-1 (nmol protein body body
(mmHg) (pg/ml) MDA/ml) (mg/dl) weight weight the protein levels of the ENaC subunits but tended to increase (p0.05) and ENaC (p0.08)
mRNA levels. Na-rich aCSF also increased SGK1 transcripts (p0.005). Spironolactone
DOCA-salt 2202.91 71521185 2.490.28 13219 0.500.02 0.670.02 prevented the increased mRNA levels of ENaC subunits but not SGK1. Na-rich aCSF increased
DOCAPTX 1887.54* 2354526* 1.240.21* 4111* 0.440.01* 0.650.03 MR binding densities in the SON (p0.05), whereas the mRNA levels did not change. In the
SHAM 1533.41* 1741339* 1.450.11* 206* 0.340.01* 0.470.01
PVN, Na-rich aCSF increased ENaC protein levels (p0.05) and MR binding densities
Significantly different from DOCA-salt (p0.05), n6 10 in each group. (p0.06) but ENaC mRNA levels appeared to decrease (p0.08). Na-rich aCSF did not
CHBPR ConferencePoster Presentations e51

affect the transcription of , ENaC, MR and SGK1 in the PVN. In the SFO, Na -rich aCSF only if this paradigm increases PRA and leads to angiotensin II-dependent hypertension. We tested
significantly increased ENaC mRNA abundance and MR binding densities. Spironolactone did this hypothesis by measuring PRA and blood pressure in rats exposed during their sleep (7
not prevent the changes of ENaC expression in the PVN and SFO. These results suggest that hr/day) to 20 short periods every hour of hypoxia/hypercapnia (IH/HC, inhaled O2% 5%,
regulation of ENaC expression in response to Na-rich aCSF is quite region specific and may inhaled CO2% 5%) for14 days. Some rats from each group were instrumented with
reflect primary (ependyma, SON) and downstream (PVN, SFO) responses to CSF [Na]. The telemeters for recording arterial pressure and treated with the angiotensin converting enzyme
upregulation of the SGK1 and ENaC transcription by Na-rich aCSF in the SON suggests that (ACE) inhibitor, captopril in their drinking water (1.30.05 mg/kg/day) throughout the 14 days
SGK1 is involved in ENaC mRNA regulation. Spironolactone prevented the increases in ENaC of exposure to IH/HC. PRA measured with a radioimmunoassay kit was significantly elevated
subunits expression only in the ependyma and SON, implying that, in addition to aldosterone, in the IH/HC rats (19.7 2.6 ng/ml/hr, p 0.05) compared to sham rats (11.3 0.4
other regulators (e.g. vasopressin) play an important role. ng/ml/hr). In addition, mean arterial pressure (MAP) did not increase in IH/HC rats treated with
captopril in contrast to the increase in untreated IH/HC rats on day 14 (captopril 109 2.5
vs untreated 119 5 mmHg). These data suggest IH/HC exposure increases PRA to contribute
P27 to the development of angiotensin II-dependent hypertension. Therefore blocking the renin-
Aldosterone Augments the Vasoconstriction and Reduced Vascular angiotensin system may be a good first line treatment for sleep apnea patients with
Relaxation in AngII/L-NAME Treated Rats on High Salt Diet hypertension unless the ACE-inhibitor cough worsens the sleep apnea symptoms.

Jeffrey C Lin, Harvard Med Sch, Boston, MA; Amanda K Stennett, Gordon H Williams,
Brigham and Womens Hosp, Boston, MA; Raouf A Khalil; Harvard Med Sch, Boston, MA P30
Chemokine Receptor 2b Blockade Decreases Renal Nf-Kappa B Activity
Aldosterone (Aldo) is a major component of RAAS and renal control mechanisms of blood and Slows the Progression of Hypertension and Renal Damage in
pressure particularly during sodium overload; however, the role of Aldo in the vascular control Salt-Sensitive Hypertension
mechanisms is less clear. We tested whether Aldo enhances the vasocostrictive effects of AngII
particularly during high salt diet. Male Sprague-Dawley rats were placed on normal (NS, 0.4% Ahmed A Elmarakby, Jeffrey J Olearczyk, Aarthi Sridhar, Jeffrey E Quigley, David M Pollock,
NaCl) or high sodium diet (HS, 1% NaCl) for 14 days. To minimize endogenous Aldo, rats were John D Imig; Med College of Georgia, Augusta, GA
adrenalectomized (ADX) and supplemented with dexamethazone. Rats were treated with
L-NAME in drinking water (40 mg/kg/day for 14 days). During days 8 14, rats were treated Studies have demonstrated that inflammatory cytokines play a role in hypertension and its
with AngII (225 g/kg/day) and Aldo (80 g/kg/day) via subcutaneous osmotic minipumps associated renal damage. We have previously shown that urinary MCP-1 excretion increased
Aldo antagonist eplerenone (Epl, 100 mg/kg/day by oral gavage). Rats were sacrificed and in salt-sensitive hypertension. Thus, we hypothesize that MCP-1 and chemokine receptor
thoracic aortic segments were prepared for measurement of isometric contraction/relaxation activation contributes to the blood pressure elevation and renal injury in salt-sensitive
and NO production using DAF-FM fluorescence. Phenylephrine (10-5 M)-induced vascular angiotensin II (ANG) hypertension. Male Sprague Dawley rats were fed a high-salt diet (HS; 8%
contraction was enhanced in HS (0.66) compared with NS rats (0.47), but not in ADX HS rats
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017

NaCl) and osmotic minipumps were implanted to deliver ANG at a dose of 60 ng/min for 14
(0.61g/mg). AngIIL-NAME treatment did not enhance contraction in ADX rats, suggesting that days. Rats were divided into three groups: HS & ANG/HS & ANG/HS/ CCR2b selective
endogenous Aldo is needed to sensitize blood vessels to AngII. Aldo administration enhanced chemokine receptor antagonist, RS102895 (10 mg/kg/day). Mean arterial pressure (MAP)
vascular contraction in AngIIL-NAME treated rats. Acetylcholine (Ach, 10-5 M)-induced % increased from (1015 to 13815 mmHg) during the first week in the ANG/HS group.
vascular relaxation was smaller in HS (41) than NS rats (71) and further reduced in RS102895 slowed the progression of blood pressure elevation during the first week of ANG/HS
AngIIL-NAME treated HS rats (11%), suggesting significant NO production during HS diet. Epl treatment (984 to 11014 mm/Hg). At two weeks, MAP was elevated to 17415 mmHg in
partially restored Ach relaxation in AngIIL-NAME treated rats (25%). Aldo administration ANG/HS and 14418 mmHg in the RS102895 treated group. Albuminuria was significantly
abolished Ach relaxation in AngIIL-NAME treated rats, which was slightly restored by Epl elevated in ANG/HS group compared to HS rats (237 26 vs. 4 0.6 mg/day) and RS102895
(5%). Ach-induced vascular NO production was reduced in HS (0.103/mg) compared with NS treatment reduced albuminuria in ANG/HS hypertensive rats (111 12 mg/day). Renal cortical
(0.122/mg), but not in ADX HS rats (0.117/mg). The reduction in NO production in NF-kappa B activity increased in ANG/HS hypertensive rats compared to HS group (0.110.006
AngIIL-NAME treated rats (0.106/mg) was reversed by Epl (0.127/mg). Vascular relaxation to vs. 0.080.003 ng activated NF-kappa B/g cortical protein) and RS102895 treatment
exogenous NO donor SNP was also reduced in Aldo-treated rats. Thus Aldo enhances the lowered NF-kappa B activity (0.080.005 ng activated NF-kappa B/g cortical protein).
vasoconstrictive effects of AngII in part by reducing vascular NO production and by decreasing ICAM-1 and NF-kappa B mRNA expression also increased in renal cortex isolated from ANG/HS
vascular sensitivity to NO, which may compromise the vascular control mechanisms of blood hypertensive rats and these changes were attenuated with RS102895 treatment using real time
pressure particularly during high salt diet. PCR. These data suggest that CCR2b receptor contribute to blood pressure regulation, and renal
injury in salt-sensitive hypertension.

P28
Role of Rho-Kinase in Aldosterone-Induced Vascular Remodeling P31
Angiotensin II-Induced Hypertension and Subsequent Renal Pathology is
Kayoko Miyata, Yukiko Nagai, Shoji Kimura, Hideyasu Kiyomoto, Masanao Hosomi, Attenuated in Mice Lacking Soluble Epoxide Hydrolase
Masakazu Kohno, Akira Nishiyama; Kagawa Univ Med Sch, Kagawa, Japan
Steven M Weldon, Damian Matera, Jun Xu, Rhonda Chen, Richard Ingraham, Michael
Aldosterone plays a critical role in the pathogenesis of cardiovascular injury. We recently Thibodeau, Jeffrey B Madwed, Alisa K Kabcenell; Boehringer Ingelheim Pharmaceuticals
demonstrated that MR is abundantly expressed in cultured rat vascular smooth muscle cells Inc., Ridgefield, CT
(VSMCs). In this study, we investigated whether Rho-kinase, an effector of the small G protein
Rho, is involved in aldosterone-induced vascular remodeling. Phosphorylation of myosin Targeted disruption of the soluble epoxide hydrolase (sEH) gene has been shown to effect basal
phosphate target subunit-1 (MYPT1) was determined, as a marker of Rho-kinase activity, in blood pressure (BP) in mice (Sinal, 2000). The BP phenotype in these animals is presumed to
VSMCs by ELISA or Western blotting analysis with anti-phospho MYPT1 antibody. The presence be due to an increase in the vasoactive arachidonic acid metabolites, epoxyeicosatrienoic acids
of actin filament and stress fiber formation was visualized by direct staining with rhodamine- (EETs). We examined whether angiotensin II (ang II)-induced hypertension (HT) and associated
phalloidin. We also examined the effects of fasudil (10 mg/kg/day, s.c.), a selective Rho-kinase renal pathology would be attenuated in our independently generated sEH knock out (KO) strain
inhibitor, on MYPT1 phosphorylation and vascular remodeling in uninephrectomized rats treated compared to wild type (WT) controls. HT was induced in male (M) and female (F) mice by
with 1% NaCl (in a drinking solution) plus aldosterone (0.75 mg/H, S.C) for 5 weeks. P0.05 infusion of ang II (1 mg/kg/day) for 14 days. Mean BP (MBP) and heart rate (HR) were
was considered statistically significant. In cultured VSMCs, aldosterone (1 nmol/L) increased continuously recorded via telemetry. Kidney, heart and aortic tissue were collected for gene
MYPT1 phosphorylation with a peak at 90 min (by 1.70.1-fold). Aldosterone-induced MYPT1 expression profiling and renal histopathology at 14 or 26 days after the start of ang II infusion.
phosphorylation was markedly attenuated by pretreatment with eplerenone (10 mol/L), a Baseline (Day0, D0) MBP was similar across all groups: 1092; 1113; 1112 and 1095
selective MR antagonist, or Y27632 (10 mol/L), a specific Rho-kinase inhibitor (n4 6 for for F-WT, M-WT, F-KO and M-KO, respectively. AngII infusion resulted in a significant,
each). Aldosterone also significantly increased VSMC stress fiber formation (at 3 hours). The sustained increase in MBP in all groups. By D14 of angII, there was no difference in MBP
aldosterone-induced stress fiber formation was markedly attenuated by pretreatment with between F-WT and F-KO mice. However, there was a significant separation in MBP observed
eplerenone or Y27632 (n6 8 for each). Compare with rats treated with salt alone (n6), between M-WT and M-KO groups; beginning on D5 and sustained throughout the ang II
aldosterone/salt-treated rats showed hypertension (1263 vs. 1983 mmHg) and increased infusion, 1658 vs. 13410 (p0.012) for M-WT and M-KO, respectively (D14). HR was not
medial thickness of the aorta and MYPT1 phosphorylation in aortic tissues (by 1.60.2-fold, significantly different between any groups at D14. Although renal vascular fibrinoid necrosis
n7). Treatment with fasudil did not alter blood pressure (1933 mmHg), but prevented elicited by ang II was similar between M-WT and M-KO at D14, renal cortical infarcts observed
aldosterone-induced aortic MYPT1 phosphorylation in aldostreone/salt-treated rats (n7). in M-WT at D26 were not present in M-KO indicating renal protection. Female mice did not
Additionally, the aldosterone-induced increase in medial thickness of the aorta was ameliorated exhibit vascular fibrinoid necrosis. Interestingly, microarray data from sEH KO tissues exhibited
by treatment with fasudil. These data suggest that Rho-kinase is involved in the progression suppression of genes involved in calcium regulation. These data are the first to provide
of aldosterone-induced vascular remodeling, independent of blood pressure changes. evidence that sEH gene deletion attenuates hypertension and associated renal damage in mice,
possibly through calcium regulation. In addition, the effects exhibited in sEH KO mice may be
gender selective. These data provide additional evidence that sEH may have an intriguing role
P29 in blood pressure regulation and associated renal protection.
Captopril Prevents Hypertension in Rats Exposed to Intermittent
Hypoxia/Hypercapnia to Simulate Sleep Apnea
P32
Ana Quenia G Silva, Pooja Singh, Nancy L Kanagy; Univ of New Mexico Health Science Hypertension Caused by Prenatal Testosterone Excess in Female Sheep
Cntr, Albuquerque, NM
Andrew J King, N B Olivier, P.S. MohanKumar, Michigan State Univ, East Lansing, MI; J.S.
Sleep apnea has been associated with systemic hypertension and some studies suggest Lee, V Padmanabhan, Univ of Michigan, Ann Arbor, MI; Gregory D Fink; Michigan State
elevated plasma renin activity (PRA) contributes to the hypertension. However, other studies in Univ, East Lansing, MI
sleep apnea patients have not observed increased PRA and one clinical study observed that
enalapril actually exacerbated sleep apnea. Thus it is not clear what role PRA and angiotensin Polycystic ovary syndrome (PCOS), a leading cause of infertility, affects 10% of women of
II play in sleep apnea associated hypertension. We simulated sleep apnea in rats to determine reproductive age. The etiology and pathophysiology of PCOS are poorly understood. PCOS is
e52 Hypertension Vol 48, No 4 October 2006

multi-facetted and includes reproductive, metabolic and other aspects of the metabolic hypertensive renal injury, absent in injury-resistant hypertension. This program precedes the
syndrome, including insulin resistance, obesity and hypertension. Exposure to excess emergence of histological injury. This renal injury transcriptional program arises from
testosterone (T) during the prenatal period may predispose individuals to PCOS. The goal of this differential gene expression regulated by components of HNF1, a major renal TF that influences
study was to determine if hypertension occurs in a well-characterized model of PCOS produced expression of a large number of renal genes.
by prenatal treatment of sheep with T. Because abnormal responses to stress may occur in
PCOS, radiotelemetry was used to measure blood pressure over a 24-hour period in conscious,
undisturbed animals. Control (n4) and prenatal T-treated (100 mg T propionate im twice
weekly from days 30 to 90 of fetal life; n4) 2-year-old females were ovariectomized and
replaced with early follicular phase levels of estrogen using an implant. On the same day, the
catheter of a radiotelemetry pressure transmitter (DSI, Inc.) was implanted into the femoral
artery; the transmitter was placed subcutaneously at the inner thigh. Six days later, sheep were P35
housed in small rectangular pens (3 x 4 ft) with free access to food and water. Starting at noon,
a 24-hour recording period was begun. Some key measurements (24-hour averages, mean
Ac-SDKP Mediates Part of the Renal Antifibrotic Effect of ACEi in
SEM) are summarized in the Table. Aldosterone-Salt-Induced Hypertension

Systolic Diastolic Hongmei Peng, Oscar A Carretero, Tang-Dong Liao, Edward L Peterson, Nour-Eddine
Mean pressure pressure pressure Heart rate Rhaleb; Henry Ford Hosp, Detroit, MI
Group (mmHg) (mmHg) (mmHg) (beats/minute)

C-females 90.92.8 115.14.5 79.22.3 73.55.9 We reported that acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) reduced renal fibrosis and cell
T-females 100.52.7* 125.02.8 88.92.8* 82.57.4 proliferation in rats with aldosterone (ALDO)-salt-induced hypertension. Others reported that an
angiotensin-converting enzyme inhibitor (ACEi) prevented renal fibrosis in this model. Recently
* P 0.05 comparing C-females and T-females. Blood pressure differences were greater during the lights-on than during the
lights-off period. Body temperature and activity level did not differ between groups. In T sheep there was marked insulin resistance
we found that the antifibrotic effect of ACEi in the heart was partly mediated by Ac-SDKP in rats
and both total and LDL cholesterol tended to be higher. There was no difference in heart weight between groups. This first-ever use with angiotensin-induced hypertension. Thus we tested whether the antifibrotic effect of ACEi
of radiotelemetric blood pressure recordings in sheep demonstrates that mild hypertension, a risk factor reported in some women in the kidney is mediated by Ac-SDKP in ALDO-salt hypertension. An anti-Ac-SDKP monoclonal
with PCOS, is also a feature of the sheep model of PCOS produced by prenatal T treatment. antibody (mAb) was used to block the effect of Ac-SDKP. SD rats (BW: 310 g) were divided into:
1) sham, 2) ALDO-salt vehicle, 3) ALDO-salt captopril (100 mg/kg/d in drinking water),
4) ALDO-salt captopril mAb (400 g/kg, i.p. every other day), 5) ALDO-salt Ac-SDKP
P33 (800 g/kg/d), and 6) ALDO-salt Ac-SDKP mAb. ALDO (0.75 g/hr) or Ac-SDKP was given
Heart Rat and Blood Bressure Variability in L-NAME Treated NET-Deficient s.c. via osmotic minipump and salt (1% NaCl/0.2% KCl) in drinking water. Treatment lasted for
Mice 6 weeks. As expected, ALDO-salt induced renal fibrosis (hydroxyproline assay), glomerular
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017

matrix expansion (periodic acid-Schiff (PAS) staining) and increased phosphorylated p42/44
Volkmar Gross, Michael Obst, MDC, Berlin, Germany; Jens Tank, Med Faculty of the mitogen-activated protein kinase (MAPK) (P-p42/44) (Western blot). ACEi and Ac-SDKP
Charite, Franz Volhard Clinic, HELIOS Klinikum-Berlin, Berlin, Germany; Ralph Plehm, MDC, significantly reduced renal fibrosis and glomerular expansion (p 0.05) but not hypertension
Berlin, Germany; Jens Jordan, Med Faculty of the Charite, Franz Volhard Clinic, HELIOD or renal hypertrophy; while mAb reduced the antifibrotic effects of ACEi or Ac-SDKP on the
Klinikum-Berlin, Berlin, Germany; Friedrich C Luft; MDC and Med Faculty of the Charite, kidney. ACEi and Ac-SDKP also significantly reduced renal P-p42/44, and mAb blunted this
Franz Volhard Clinic, HELIOS Klinikum-Berlin, Berlin, Germany decrease. Phosphorylated p38 or JNK MAPK was not detectable. We concluded that the
antifibrotic effect of ACEi in the kidney is at least partly mediated by Ac-SDKP in ALDO-salt
To characterize the interaction between the blood pressure (BP) buffering effect of nitric oxide hypertension via inhibition of the p42/44 MAPK pathway.
(NO) and the norepinephrine transporter (NET), we blocked NO-generation with N-nitro-L- EFFECT OF MAB ON THE ANTIFIBROTIC EFFECT OF ACEI AND AC-SDKP ON THE KIDNEY
arginine methyl ester (5 mg L-NAME/10 ml tap water) in NET gene-deficient (-/-) mice. ALDO-salt ALDO-salt
Telemetric arterial blood pressure recordings were used for fast Fourier analysis (FFT) of BP ALDO-salt ACEi ALDO-salt Ac-SDKP
Parameters Sham ALDO-salt ACEi mAb Ac-SDKP mAb
and heart rate (HR). NET-/- had lower mean arterial blood pressure (MAP) than NET / mice
during the day (11008 vs. 1121 mmHg) and night (1151 vs. 1241 mmHg). HR was not SBP, mm Hg 1073 1977* 1767* 1844* 1938* 1847*
Renal collagen, 22.71.6 43.11.2* 22.92.6** 33.32.0 27.81.5** 34.22.0#
different between the groups, showed a clear-cut day/night rhythm, and ranged between g/mg dry K
5446 and 6093 beats/min. L-NAME increased MAP in both strains to the same extent ( glomerular 11.12.0 22.21.7* 10.91.3** 20.60.8 10.71.4** 18.22.3#
matrix,
MAP 10 mmHg). BP after acute L-NAME application and selective nNOS and eNOS blockade % of glomerular
were also not different between NET -/- and NET / mice. The absolute HF (8.02.6 vs. area
P-p42/44, fold 1.00.0 5.71.5* 1.50.4** 3.51.2 1.60.4** 4.00.8#
6.130.9 msec2), LF (21.66.0 vs. 16.63.0 msec2), and VLF (34.512.6 vs. 20.65.1 increase
msec2) heart rate variability power values were not different between NET -/- and NET / *: p 0.005 vs sham. **: p 0.05 vs ALDO-salt. : p 0.05 vs ALDO-salt ACEi. #: p 0.05 vs ALDO-salt
mice. In contrast to NET / mice, these parameters increased in NET-/- during L-NAME. BP
Ac-SDKP. Data: mean standard error.
variability in the low frequency range (SBP-LF), thought to reflect sympathetic activity to
resistance vessels, averaged 1.80.2 mmHg2 in NET -/- and 3.10.6 mmHg2 in NET /
mice and increased during L-NAME treatment more in NET / mice than in NET -/- mice
(6.90.9 vs. 3.40.7 mmHg2). Baroreflex sensitivity (BRS-LF) was higher in NET -/- mice than
in NET / mice. This difference between NET -/- mice and NET / mice reached
significance during L-NAME treatment (2.90.5 vs. 1.90.3 msec/mmHg, p0.05). These
results support the hypothesis that NO attenuates baroreflex mechanisms. However, only
L-NAME treatment unmasks the difference between strains. The findings suggest an interaction P36
between NO and NET. Acknowledgement: All animals used in this study were obtained from Cardiac-Specific EP4 Receptor Knock-Out Does not Affect Baseline Cardiac
Autonomic Dysfunction Service, Vanderbilt University, Nashville, TN, USA. Characters 1693 Function

Jian-Yong Qian, Ed Shesely, Yunhe Liu, Pamela Harding, Margot C LaPointe; Henry Ford
P34 Hosp and Health System, Detroit, MI
The Transcription Factor Hnf1 Contributes to Initiation of Hypertensive
Renal Injury via Coordinate Regulation of Redox Stress Prostaglandin E2 (PGE2) is a pro-inflammatory prostanoid that stimulates cardiomyocyte
hypertrophy in vitro through its EP4 receptor. The role of EP4 in normal heart function is
Renata I Dmitrieva, Cruz A Hinojos, Michael C Braun, Myriam Fornage, Eric Boerwinkle, unknown. Systemic EP4 gene knock out (KO) mice have been reported, but 95% of
Peter A Doris; Univ of Texas HSC Houston, Houston, TX homozygotes for the null allele have persistent ductus arteriosus after birth and soon die. Thus
understanding EP4 function in the heart requires cardiac-specific deletion. To generate mice
Redox stress (RS) has been implicated in the pathogenesis of hypertensive renal injury. We lacking EP4 only in cardiac myocytes (CM-EP4 KO), we bred 2 lines of transgenic mice: 1) an
used gene expression arrays to examine renal expression of 32 genes contributing to redox EP4 flox/flox mouse which has loxP sites flanking exon 2 of the EP4 gene; and 2) an -myosin
balance during the development and maintenance of hypertension in SHR lines varying in their heavy chain-Cre recombinase mouse (to localize expression of Cre to adult cardiac myocytes).
susceptibility to renal injury. Our array data, confirmed by QRTPCR, provides evidence that RS A breeding strategy was developed to generate roughly equal numbers of CM-EP4 KO mice and
involves down-regulation of multiple redox radical scavenging genes. By comparing renal- wild-type (WT) littermates. CM-EP4 KO and WT mice (male, 1314 week old) had similar
injury prone SHR-A3 with injury-resistant SHR-B2 and -C animals we observed that survival rates. CM-EP4 KO and WT mice were subjected to 2D M-mode echocardiography.
down-regulation was specific to the sub-strain experiencing renal injury and preceded There was no difference between CM-EP4 KO mice and WT regarding ejection fraction (KO
histopathological evidence of renal injury. A transcriptional mechanism was sought using a 78.83.2%, n3; WT 79.51.5%, n5), left ventricular diastolic dimension (KO
bioinformatics approach to assess the frequency of transcription factor (TF) binding sites in the 3.10.1mm; WT 3.00.1mm) and diastolic posterior wall thickness (KO 0.90.04mm;
regulatory regions of down-regulated radical scavenging genes. A single TF, HNF1, emerged as WT 0.90.03mm). There was also no difference in heart/body weight ratio (KO
a potential mediator of these changes. Expression of each of the three genes encoding subunits 40.31.9; WT 39.10.4 mg/10g BW), consistent with the sonographic posterior wall
of the HNF1 was lower in the renal injury-prone strain. Western blot analysis of the major HNF1 thickness data. We confirmed that the EP4 gene was deleted in heart, but not in other tissues.
subunit expressed in the kidney (Tcf2) indicated that loss of one of two alternatively spliced Organs from CM-EP4 KO and WT mice were divided into two sections from which genomic DNA
forms of Tcf2 exclusively in the injury-susceptible strain, SHR-A3. Isoform loss was shown to and RNA were extracted. PCR primers designed to detect a 346 bp floxed EP4 gene showed
be a post-transcriptional event. To support evidence that redox mechanisms of renal injury the presence of the gene in the heart, aorta, lung, liver, skeletal muscle and brain of WT
might be under control by HNF1, the capacity of HNF1 to bind to 5-regulatory regions of redox littermates, but a 93% decrease in the gene only in the CM-EP4 KO mouse hearts. Similarly,
scavenging genes was demonstrated by EMSA. Further evidence of HNF1 regulation of these an analysis of EP4 mRNA by RT-PCR of hearts showed that expression was significantly
genes was obtained by demonstrating conservation of HNF1 binding sites across species decreased in the heart of CM-EP4 KO mice vs. WT controls. In conclusion, CM-EP4 KO mice
(Consite, Karolinska Institute). Across 35 further genes known to be regulated by HNF1 and were successfully generated, showing EP4 deletion only in the heart. KO mice had no overt
expressed in the kidney, the mean expression level was 2.7 fold-lower in the injury-prone cardiac phenotypes, as expected. Additional experiments are underway to determine the role
strain. Our studies have revealed a transcriptional program for renal redox stress in of EP4 in models of hypertrophy and ischemia.
CHBPR ConferencePoster Presentations e53
P37 Systolic blood pressure (SBP) was determined by tail-cuff. Cardiac function was assessed by
The T-Type Calcium Channel Blocker (CCB), Mibefradil, Attenuates the echocardiography. Insulin resistance index, the homoeostasis model assessment (HOMA)
Acute Hindleg Edema Induced by the L-Type CCB, Nifedipine, in the score, was calculated from fasting glucose and insulin levels. SBP was significantly lower in
rats given LS (13214 mmHg), AC3174 (15517 mmHg), Cap (16516 mmHg) and
Spontaneously Hypertensive Rat (SHR) as Assessed via Magnetic AC3174Cap (16316 mmHg) than in HS rats (2097 mmHg). Left ventricular (LV) weight
Resonance Imaging (MRI) /body weight ratio was significantly lower in rats treated with LS, AC3174, Cap, AC3174Cap
than in rats treated with HS. Despite comparable antihypertensive efficacy, LV weight/body
Terry C Major, Shantanu Dhamija, Serguei Liachenko, Brandy Morenko; Pfizer Inc, Ann weight ratio in AC3174Cap treated rats was significantly lower than in rats given Cap alone.
Arbor, MI Compared with HS rats, LV wall stress was significantly reduced in LS (22%), AC3174 (30%),
AC3174Cap (28%) and Cap (20%) treated rats. Compared with LS treated rats, fasting insulin
Among the calcium channel blockers (CCB), particularly among the dihydropyridines (DHP) like levels and HOMA were significantly increased by 3.5 and 3.7 fold, respectively, in HS treated
nifedipine, a common adverse effect is vasodilatory edema. Newer CCBs such as the T-type rats. Treatment with AC3174, Cap and AC3174Cap normalized the insulin levels and HOMA
CCB, mibefradil, demonstrate antihypertensive efficacy similar to that of their predecessors but score. Treatment with AC3174, AC3174Cap and Cap significantly attenuated elevated serum
appear to have a reduced propensity to cause edema. Edema formation is currently determined creatinine levels and improved creatinine clearance rate compared with HS rats. These results
after long-term clinical trials by measuring patient-reported edema incidence, a fairly suggest that 4 week AC3174 infusion has anti-hypertensive, insulin sensitizing and renopro-
subjective parameter. In this study, we investigated the ability of MRI to measure static water tective effects.
accumulation in hindleg skeletal muscle of the SHR by the CCBs, mibefradil and nifedipine, both
alone or in combination. Isoflurane-anesthetized SHRs were cannulated in the jugular vein for
CCB delivery and the carotid artery for mean blood pressure (MBP) measurements. After a P40
1530 minute stabilization period, two baseline T2 relaxation scans in the lower leg muscles Measurement of Cellular Physiology in Individual Freshly Exfoliated Human
of the SHR were measured by a 7 Tesla Bruker MRI system utilizing multi-echo images and Renal Proximal Tubular Cells from Urine in Subjects Enrolled in a Genetic
calculating the integral increase in T2 relaxation (IIT2R;%# of voxels x ave change in Study of Salt Sensitivity
T2/voxels) as a quantitative marker for edema formation. Mibefradil (10 mg/kg IV) and
nifedipine (1 mg/kg IV) both lowered MBP equipotently -975 and -837 mmHg, respectively John J Gildea, Daniel C Huntley, Michael J Surace, Robert M Carey, Robin A Felder; Univ of
compared to vehicle (78 mmHg) . However, the edema formation or IIT2R was significantly Virginia, Charlottesville, VA
higher with nifedipine (2708 5%;p0.05) than with mibefradil (981171%) or vehicle
(9514%) measured 30 minutes after start of drug infusion. The combination of 1 mg/kg We developed a method to obtain living proximal tubular cells (PTC) from freshly voided urine
nifedipine3 mg/kg mibefradil (1N3M) and 1N0.3M lowered MBP -1016 and -644 specimens in order to study sodium transport phenotype and correlate it with genotype in
mmHg, respectively, compared to 1N0M (-723 mmHg) but the IIT2R was significantly subjects enrolled in a study of genetic salt sensitivity. PTC were concentrated from 150 mL of
reduced in the 1N3M (1139279%) and 1N0.3M SHRs (1725306), respectively, urine obtained as an aliquot of a 24 hour urine specimen by centrifugation at 150xG and then
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017

compared to the control SHR group, 1N0M (2539119%). These results show that IIT2R can covalently attached to a slide using the Cytospin method. PTC were selectively identified using
be used to quantify peripheral edema with high precision and reproducibility and that T-type conjugated Lotus tetragonolobus agglutinin conjugated to a fluorescent dye (Alexa 594-LTA),
CCBs at equipotent MBP lowering can produce significantly less edema compared to DHPs. In which is a specific PTC stain. PTC constituted 10%, n3, of the total cells in the urine of three
addition, T-type CCBs can dose-dependently attenuate the peripheral edema induced by DHPs. out of 10 processed specimens. Cells were found to be viable for 24 hours in culture media
This study indicates that T-type CCBs may equalize the pressure across the capillary, reduce at 37C in a 5% CO2 atmosphere. All cells were then loaded with corona green, a fluorescent
water leakage and reduce vasodilatory edema. dye that is sensitive to changes in cytoplasmic sodium concentration. Ouabain was used to
selectively block NaKATPase, and the change in intracellular sodium was followed over time
in both PTC and other living cells found in urine. We measured the differential change in
PTC/cuboidal cell corona green fluorescence as an indicator of PTC NaKATPase activity. A
P38
21% increase in fluorescence of PCT after 2 minutes of ouabain treatment indicated that we
Partial Normalization of Renal Transcriptome in Spontaneously were able to inhibit NaKATPase activity and increase intracellular sodium. We suggest that
Hypertensive Rats (SHR) Treated with Multiple Antihypertensive Agents exfoliated PTC may be used to directly measure cell physiology, in particular sodium transport.
This method may be a useful diagnostic tool for studying aberrant PTC sodium transport which
Sebastiaan Wesseling, Jaap A Joles, Farid Kantouh, Hein A Koomans, Branko Braam; Univ may be correlated with individual patient SNP genotype.
Med Cntr Utrecht, Utrecht, The Netherlands

SHR are a genetic model for essential hypertension (EH). Compared to WKY, SHR have P41
disturbed NO/O2- balance, dysfunctional Ca-regulation and are sensitive to Ang II signaling. In The Human Renin Kidney Enhancer Appears Dispensable for Tissue and
patients with EH the leukocyte transcriptome normalized upon antihypertensive therapy. We Cell-Specific Expression in Transgenic Mice
hypothesized that the renal transcriptome in SHR will be progressively normalized as more
hypertensive factors are inhibited. SHR (21 wks old) with losartan (Los), Los and tempol (LT), Xiyou Zhou, Curt D Sigmund; Univ of Iowa, Iowa City, IA
Los and amlodipine (LA) or Los, amlodipine and tempol (LAT) in food and/or drinking
water were studied vs. untreated SHR and WKY for 3 wks. Renal cortex was processed for rat Renin (REN) is the rate-limiting enzyme in the RAS and thus dictates the level of Ang-II.
7.5k oligonucleotide microarray (n5 6/group). All groups, also WKY, were compared to WKY Transcription of REN is expressed predominantly in renal JG cells where it is tightly regulated
with a dye swap. Differential gene expression was defined as absolute log2(GROUP/WKY)0.7. by physiological cues. We previously generated hREN transgenic mice with a 160 Kb
Systolic blood pressure for WKY, SHR, Los, LT, LA and LAT was 1312, 1935 P1-artificial chromosome (PAC160) containing REN, two upstream (GOLT1A and KISS1), and
(P0.001 vs. WKY), 1533, 1666, 1444 and 1285 mmHg, respectively (P0.01 treated one downstream (ETNK2) gene. hREN expression in these mice is tightly regulated, as is
SHR vs. SHR; N.S. LAT vs. WKY). SHR showed 183 modulated genes vs. WKY of which 56, expression of the linked genes. A kidney-specific enhancer of transcription (KE) is located 11
97, 125 and 125 genes were normalized by Los, LT, LA and LAT, respectively. kb upstream of the transcription start site which has been reported to markedly enhance
Hierarchical clustering analysis revealed: 1) Los normalized genes with reduced expression; 2) transcription in renin expressing cells. The importance of the KE in vivo remains unclear. We
few genes were oppositely regulated by treatments; 3) several genes were insensitive to any hypothesize that the KE mediates tissue- and cell-specific expression, and is responsible for
treatment. Gene Ontology classification was applied to the 183 genes to identify biological physiological regulation. To test this, we deleted the enhancer sequence from PAC160 using
processes. In most processes regulation was bi-directional, but in Na and K ion transport only homologous recombination in bacteria, and then developed several lines of transgenic mice
reversal of gene induction occurred which was normalized by LA and LAT. Drug (termed KE). Lines KE2 and KE4 have a single copy and multiple copies of hREN
combinations increasingly normalized regulated processes and no processes were totally integrated, respectively. We used the downstream gene- ETNK2 as an internal control. To our
insensitive to treatment. Only 14 modulated genes in SHR were insensitive to any treatment: surprise, deletion of the KE had no effect on tissue-specific expression of hREN. Expression of
among these were reduced Cd36 (insulin sensitive, important for cellular fatty acid uptake) and hREN protein remained localized to renal JG cells under baseline conditions in KE4, and after
induced cytosolic epoxide hydrolase 2 (Ephx2; degrades endogenous vasodilators such as EET). treatment with Captopril in KE2. Captopril (1000 mg/kg/day) significantly induced expression
Summarizing, renal cortex of SHR displayed differential gene expression sensitive to Ang II, of hREN in KE2 (10.6-fold) and KE4 (7.4-fold) to an extent similar to the unmodified hREN
Ca2 and, to a lesser extent, superoxide. Genes involved in Na and K transport were sensitive gene in PAC160 and to the endogenous mREN gene (5-fold). Ang-II administration (1000
to multiple treatments. Combining drugs not only normalized BP but also partially corrected the ng/kg/min) which raised BP by 40mmHg decreased hREN expression in KE4 mice (to 30%
transcriptome. of untreated) similar to wildtype PAC160 mice. We also identified another line (KE6)
containing a truncated PAC160 in which the KE as well as additional upstream sequences was
deleted. Whereas expression of hREN in this line was evident in all tissues tested, it was still
expressed renal JG cells. However, initial studies revealed a lack of repression by Ang-II. Our
P39 results suggest that sequences other than the KE, either further upstream or in the proximal
The Exenatide Analog Ac3174 Attenuates Hypertension, Renal Dysfunction promoter may be necessary for tissue-specific and regulated expression and for retaining
and Insulin Resistance in Dahl Salt-Sensitive Rats JG-cell specific expression of hREN.

Lisa M Adams, Rayne Fernandez, Alain Baron, David Parkes, Que Liu; Amylin
Pharmeceuticals, San Diego, CA P42
Rats with Ang II-Dependent Hypertension From Human Transgenes Develop
Glucagon-like peptide-1 (GLP-1) is a gut peptide incretin that enhances glucose-dependent Electrical Remodeling
insulin secretion. Accumulating evidence suggests that activation of GLP-1 receptors also
improves insulin sensitivity and induces vasodilatation and diuresis. Exenatide is a longer- Robert Fischer, Ralf Dechend, Andrej Gapelyuk, Erdenechimeg Shagdarsuren, Konstanze
acting incretin mimetic that exhibits antidiabetic properties in patients with type II diabetes. Gruner, Andreas Gruner, Petra Gratze, Maren Wellner, Anette Fiebeler, Helios Klinikum,
AC3174 is an analog of exenatide with similar pharmacologic properties. We hypothesized that Franz-Volhard-Klinik, Charite, Berlin, Germany; Dominik N Muller, Max-Delbrueck Cntr,
chronic treatment with AC3174 can attenuate salt-induced hypertension, insulin resistance and Berlin, Germany; Alexander Schirdewan; Helios Klinikum, Franz-Volhard-Klinik, Charite,
renal dysfunction in Dahl salt-sensitive (DSS) rats. DSS rats were fed low salt (LS, 0.3 % NaCl) Berlin, Germany
or high salt (HS, 8% NaCl) and were treated with vehicle or AC3174 (1.7 pmol/kg/min) via
subcutaneous infusion (Alzet pump) for 4 weeks. Other DSS rats were given captopril (Cap, Rats harboring the human renin and angiotensinogen genes (dTGR) develop hypertension and
2g/L drinking water) or AC3174 (1.7 pmol/kg/min) plus Cap (AC3174Cap) for 4 weeks. cardiac damage. We investigated electrical remodeling in this model. We used non-invasive
e54 Hypertension Vol 48, No 4 October 2006

cardiac magnetic field mapping (CMFM) to determine whether untreated dTGR or AT1 atherogenic index (the ratio between non-HDL cholesterol and HDL cholesterol) did not
receptor blocker (ARB)-treated dTGR exhibit electrical remodeling and arrhythmias. We enter the final model (adjusted R20.553). Circulating markers of oxidative stress were not
performed CMFM, ECG, and characterized cardiac damage in 5 and 7 week old dTGR, correlated with aortic compliance. Conclusions: This is the first report revealing a
losartan (Los)-treated dTGR (30 mg/kg/d), and Sprague Dawley (SD) controls. Heart significant relationship between oxidative stress and vascular stiffness. Detailed pheno-
weight-to-tibia ratio and systolic blood pressure (BP) increased in dTGR from week 5 to typing including MRI-based analysis of aortic compliance and measurement of vascular SO
week 7. Los treatment normalized heart weight-to-tibia ratio and BP. Already at early stage generation is required to uncover this important link.
of cardiac damage (week 5), untreated dTGR showed slightly increased perivascular
(collagen I) and interstitial fibrosis (fibronectin), connective tissue growth factor expression
(CTGF), and monocytes infiltration compared with SD. All differences were much more
prominent at week 7. Los reduced CTGF, fibrosis and inflammation to control levels.
Already at week 5, untreated dTGR showed prolonged de- and repolarization compared to P45
SD, which was even more pronounced at week 7 (QRS 22.90.5 ms vs. 18.70.8 ms, Withdrawn at Authors Request
QT 1133 ms vs. 821.7 ms, week 7; p 0.01). Parameters reflecting inhomogeneity
of depolarization (inhomogeneity index IHi 24.5 3.7 vs. 10 1.5, p 0.01) and
repolarization (TPeak-TEnd interval 68.6 2.3 ms vs. 54.7 2 ms, p 0.0001) also
increased with progression of cardiac damage. In contrast, Los treatment normalized all
electrophysiological parameters. LV mRNA expression of the potassium channel subunit P46
Kv4.3 and gap junction protein Cx43 was significantly reduced in dTGR at week 5 and 7 In Human Coronary Artery Smooth Muscle Cells (CASMCs) Adenosine
compared to Los and SD. We conclude that electrical remodeling is present early in Induces Antimitogenesis via A2B Receptors
angiotensin II/hypertension-induced cardiac damage. ARB treatment reduced fibrosis,
inflammation, and the pathological electrophysiological alterations. Non-invasive CMFM is Raghvendra K Dubey, Univ Hosp Zurich, Zurich, Switzerland; Nanette J Tomicek, Delbert G
a suitable tool to characterize the effects of cardiac electrical remodeling in rats. Gillespie, Edwin K Jackson; Univ of Pittsburgh Med Cntr, Pittsburgh, PA

Via A2B receptors, adenosine inhibits growth of human and rat arterial SMCs; however, a
recent study demonstrated that via A1 adenosine receptors adenosine induces growth in
porcine CASMCs. Because in contrast to porcine CASMCs, human CASMCs express A2B
P43 adenosine receptor, we investigated whether unlike porcine CASMCs, adenosine inhibits
In situ Direct Detection System of Reactive Oxygen Species (ros) and Nitric growth in human CASMCs. In human CASMCs, fetal calf serum (2.5% FCS) stimulated DNA
Oxide (no) in the Kidney synthesis (3H-thymidine incorporation), cellular proliferation (cell number), collagen
synthesis (3H-proline incorporation) and ERK1/2 expression. The adenosine receptor
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017

Naruya Tomita, Tamehachi Namikoshi, Yoshisuke Haruna, Souhachi Fujimoto, Masahito agonists adenosine and 2-chloroadenosine, but not N6-cyclopentyladenosine, CGS21680
Oozeki, Shinya Kobayashi, Norio Komai, Tamaki Sasaki, Naoki Kashihara; Kawasaki Med or IB-MECA, inhibited the growth effects of serum, an agonist profile consistent with an A2B
Sch, Kurashiki, Japan receptor-mediated effect. MRS1754 (selective A2B antagonist), but not 8-cyclopentyl-1,3-
dipropylxanthine (A1 antagonist), SCH58261 (A2A antagonist) or VUF5574 (A3 antagonist),
Increased production of reactive oxygen species (ROS) in hypertension and diabetes may blocked the growth-inhibitory effects of 2-chloroadenosine, an antagonist profile consis-
be one of the important pathways leading to the progression of renal damages induced by tent with an A2B receptor-mediated effect. Our findings strongly support the hypothesis that
hypertension and diabetes. Especially, an imbalance between ROS and nitric oxide (NO) is adenosine causes inhibition of human CASMC growth by activating A2B receptors coupled
occurring in the glomeruli from the early. Recently, we have developed the unique to inhibition of MAP kinase activity. Unlike the findings in porcine CASMCs, our findings
technique to determine the production of ROS and NO in glomeruli using confocal laser strongly suggest that pharmacological or molecular biological activation of A2B receptors
microscopy after perfusion with dichlorofluorescein diacetate (DCFH-DA) (a ROS marker) may prevent vascular remodeling associated with coronary artery disease, hypertension,
and diaminorhodamine-4M AM (DAR-4M) (an NO marker). In this study we investigated the atherosclerosis and restenosis following balloon angioplasty.
production of ROS and NO in hypertensive model and diabetic model rats. First, we used
Dahl salt sensitive (DS) rats given an 8%NaCl diet (DH-H) or 0.3%NaCl diet (DS-L) for 6
weeks. Systolic blood pressure was significantly elevated in DS-H group at 6 weeks (DS-H:
210 4 mmHg, DS-L: 132 8 mmHg, p0.01). In the DS-L rats little ROS production
and significant NO production were observed in glomeruli at 6 weeks. On the other hand,
accelerated ROS production and diminished bio-available NO were observed in glomeruli
of DS-H rats also at 6 weeks. Next, we used Spague-Dawley rats and induced diabetes by
intravenous injection of streptoztocin (STZ 65 mg/kg). Then, rats whose blood glucose on
day 3 was more than 300 mg/dl were used for further study. At 6 weeks after induction
of diabetes ROS was increased and No was decreased in glomeruli. On the other hand, in
control rats ROS was rarely observed, however, abundant NO production was noticed in
glomeruli. We have developed the method to detect ROS and NO directly in the kidney.
Although further detailed studies may be needed, this method may give a lot of information
concerning the relation between ROS and NO in the pathogenesis of hypertension and
diabetes.

P44 P47
Functional Relationship between Oxidative Stress and Vascular Stiffness in NAD(P)H Oxidase Activation Induced Reactive Oxygen Species
Humans Accumulation and Enhanced Vasoconstriction in Ren-2 Rat Aortas

Christian Delles, Lukas U Zimmerli, David J McGrane, Univ of Glasgow, Glasgow, United Yongzhong Wei, Craig S Stump, Zachary T Resch, Grace M Uptergrove, Shawna A Cooper,
Kingdom; Alan J McKay, Gartnavel General Hosp, Glasgow, United Kingdom; Vivek L Pathi, Suzanne E Clark, Univ of Missouri-Columbia Sch of Medicine, Columbia, MO; Carlos M
Western Infirmary, Glasgow, United Kingdom; Henry J Dargie, Carlene A Hamilton, Anna F Ferrario, Wake Forest Univ, Bowman Grey Sch of Medicine, Winston-Salem, NC; James R
Dominiczak; Univ of Glasgow, Glasgow, United Kingdom Sowers; Univ of Missouri-Columbia Sch of Medicine, Columbia, MO

Background: Oxidative stress is critically involved in the pathogenesis of cardiovascular Activation of the renin-angiotensin system (RAS) plays an important role in development of
disease by promoting endothelial dysfunction and reducing arterial compliance. However, cardiovascular diseases, possibly by increasing reactive oxygen species (ROS) generation and
a direct link between oxidative stress and vascular stiffness has not yet been demon- promoting insulin resistance. The TG(mREN-2)27 (Ren2) transgenic rat overexpresses the
strated. Methods: To cover the whole range of oxidative stress and vascular stiffness we mouse renin gene has elevated tissue RAS activity. We hypothesized that Ren-2 rats would
examined 73 patients (age: 6011 years). Of these, 49 patients had severe CAD and exhibit increased vascular ROS (aorta), and that ROS accumulation is mediated by increases in
underwent CABG surgery, and 24 underwent surgery for removal of varicose veins but NAD(P)H oxidase subunit expression and activity. When Ren2 rats (age 8 10 weeks) were
were otherwise healthy. Vascular superoxide (SO) production was examined by lucigenin- compared to age-matched Sprague-Dawley (SD) controls, Ren2 rats exhibited significantly
enhanced chemiluminescence in sections of saphenous vein. Reduced to oxidised increased aortic ROS as measured by a lucigenin assay (72.6%, P0.05), or by dihydro-
glutathione ratio (GSH/GSSG; whole blood) and thiobarbituric acid-reactive substances ethidium (DHE) and 4-hydroxy-2-nonenal (4-HNE) staining. Further, NADPH oxidase activity was
(TBARS; plasma) were measured as circulating markers of oxidative stress in subsets of significantly increased (82.2%, P0.05) in Ren-2 aorta when compared with SD. When Ren-2
patients (n58 and n14, respectively). Compliance of the ascending aorta was rats were treated with the AT1R blocker valartan (30 mg/kg) for three weeks, ROS level and
measured in 39 patients (22 CAD and 17 control patients) by MRI (1.5 T Siemens Sonata) NADPH oxidase activity were markedly reduced compared to untreated conditions. NADPH
using an ECG gated Fast Imaging with Steady-state Precession sequence from the change oxidase subunits (p67phox and Rac1) measured by Western blot were also increased in Ren-2
in volume of the aortic segment during the cardiac cycle divided by pulse pressure. aorta compared to SD, but valsartan treatment reduced levels in Ren-2. Arterial wall thickness
Results: Vascular SO generation (1.000.45 vs 0.760.43 nmol/mg/min; P0.034) and in Ren-2 rats was also significantly greater than that in SD rats. These findings were associated
GSH/GSSG (555352 vs 824384; P0.005) were significantly different between CAD with hypertension, increased vasoconstriction in ex vivo aorta preparations, and impaired
and control patients, whereas TBARS were similar between the groups (1.0670.242 vs insulin-induced Akt activation (ser 307 phosphorylation) in Ren-2 aortas. This study show that
1.1590.366 nmol/mL; P0.580). Neither GSH/GSSG (P0.496) nor TBARS (P0.213) increased NADPH oxidase activity leads to elevated ROS in Ren-2 aortas and that this is
were correlated with vascular SO generation. Stepwise multiple linear regression revealed associated with altered insulin signaling and ex vivo vasoconstriction. These observations
that age (P0.001) and vascular SO generation (P0.045) were independent determi- suggest that elevated RAS activity contributions to vascular disease via enhanced activation of
nants of aortic compliance, whereas sex, body mass index, systolic blood pressure and NADPH oxidase.
CHBPR ConferencePoster Presentations e55
P48 respective EC50 (0.380.19 and 1.850.44 mol/L ) and Rmax (57.97.9 and
Mechanisms of Oxidative Stress Induced Increase in Salt Sensitivity and 38.64.4% relaxation) values. Importantly, the inclusion of either the antioxidant tempol
Development of Hypertension in Sprague Dawley Rats (1 mmol/L) or ebselen (1 mol/L) into the bathing buffer restored all indices of
vasorelaxing responsiveness to Ach to values undistinguishable from those in aortic ring
ANEES AHMAD BANDAY, ABDUL BARI MUHAMMAD, MUSTAFA F LOKHANDWALA; Heart and of air-breating rats. Equally important, vasorelaxing responsiveness to a donor of nitric
Kidney Institute, College of Pharmacy, Univ of Houston, Houston, TX oxide, NONOate (10-8-10-4 mol/L), was not impaired in rats breathing 100 ppm CO. These
data suggest that supranormal levels of environmental CO brings about impairement of
High salt intake produces vascular changes which contribute to development of Ach-induced vasorelaxation, an indicator of endothelial dysfunction, via an oxidative-stress
hypertension in salt-sensitive individuals. The underlying causes for these alterations are dependent mechanism which presumably limits the bioavailability of nitric oxide.
not elucidated. There is evidence that oxidative stress plays a key role in the pathogenesis
of cardiovascular diseases. We wanted to investigate whether oxidative stress is a
contributing factor for salt sensitive hypertension. Male Sprague Dawley rats were divided
into five groups and received tap water (vehicle), 30mM L-buthionine sulfoximine (BS, an
oxidant), high salt (HS, 1% NaCl), BS plus HS, and BS plus HS with antioxidant tempol
(1mM) in drinking water for twelve days. Compared to vehicle, BS treatment caused
increase in renal tubular malondialdehyde and urinary excretion of 8-isoprostane and P51
reduced renal glutathione but had no effect on nitric oxide (NO) levels. Phenylephinephrine Increased ET-1-Mediated Vasoconstriction but not Impaired Vasorelaxation
(10M) pre-constricted thoracic aortic rings from BS rats showed decreased response to of Mesenteric Resistance Vessels in Type 2 Diabetic Gk Rats - Interaction
sodium nitroprusside (SNP) dependent vasorelaxation. Animals treated with HS had
significant decrease in NO levels despite normal oxidative state. Response to SNP was
of Endothelin Receptors
unaffected but acetylcholine (Ach) showed decreased vasorelaxation in these animals.
Kamakshi Sachidanandam, Alex K Harris, Jim R Hutchinson, Univ of Georgia, Augusta, GA;
Concomitant treatment of rats with BS and HS caused marked increases in oxidative
Adviye Ergul; Univ of Georgia, Med College of Georgia, Augusta, GA
stress, blood pressure and decreased NO levels. A rightward shift was observed to SNP
and Ach dose dependent vasorelaxation with increase in EC50. In these animals angiotensin
Diabetes-associated cardiovascular complications oftentimes result from vascular dys-
II (Ang II) caused increased and prolonged contraction with significant decrease in EC50. At
cellular level the incubation of vessels from BS rats with SNP showed decreased cGMP function, manisfested as hyperreactivity to vasoconstrictors or impaired relaxation to
accumulation while HS rats had decreased basal NO systhetase activity. Tempol decreased vasodilators. We previously demonstrated that endothelin-1 (ET-1) is elevated in the
the oxidative stress, normalized blood pressure and restored responses to SNP, Ach and Goto-Kakizaki (GK) model of Type 2 diabetes, causing hyperreactivity of resistance vessels.
Ang II in BS plus HS rats. In addition tempol also restored NO levels, signaling and NO However, the relative roles of ET receptors in this response remained unclear. This study
tested the hypothesis that ETA antagonism prevents increased vasoconstriction, whereas
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017

systhetase activity. We conclude that BS increases the oxidative stress and reduces NO
ETB blockade augments hyperreactivity to ET-1. Fourteen week-old GK rats were treated
signaling while HS reduces NO levels by decreasing the NO systhetase activity. These
for 4 weeks with antagonists to either the ETA (ABT-627, 5mg/kg/day) or the ETB receptors
phenomena collectively contribute to reduced activity of both endothelium dependent and
(A-192621, 15mg/kg/day or 30mg/kg/day). Vascular function of third order mesenteric
independent vasorelaxants and enhance vasoconstriction leading to development of
arteries, reported as sensitivity (EC50-nM) or magnitude (Rmax-% constriction or relaxation)
hypertension.
of responses to vasoactive agents, is outlined in the Table. Hyperreactivity to ET-1
(0.1100nM) was markedly reduced with ETA blockade, while lower dose ETB antagonism
did not worsen vascular function compared to untreated GK rats. However, blockade of ETB
P49 receptors at the higher dose blunted the vasoconstrictor effect of ET-1. Vasorelaxation to
Interaction of Apoptotic and Healthy Microvascular Endothelial Cells: acetylcholine (ACh, 1nM-10M) was similar between groups. Upon prior incubation with
Implications on Inflammatory Responses the NOS inhibitor L-NNA (100nM), relaxation to ACh was impaired in ETB antagonist-treated
and untreated GKs, whereas ETA antagonism completely restored vasorelaxation. Thus,
Torsten Kirsch, Alexander Woywodt, Joon-Keun Park, Hermann Haller, Marion Haubitz; Med ETA-mediated vasoconstriction is dominant in ET-1-hyperreactivity as well as decreased
Sch Hannover, Hannover, Germany relaxation in the presence of NOS inhibition in the GK model. The data suggests that ETB
antagonism does not exacerbate the constriction in resistance arteries. Indeed, at 30
Objective: Circulating endothelial cells have been detected in a variety of vascular disorders. mg/kg/day dose ETB blockade yields similar responses to ETA receptor antagonism by
However, the functional significance of these circulating endothelial cells and their putative ABT-627 suggesting an interaction between two receptor subtypes. (n3 8/group;
interaction with healthy endothelium remains unknown. The present study aims to evaluate the *p0.05 vs GK)
response of human microvascular endothelial cells to apoptotic or necrotic EC in an in vitro
model and to delineate molecular pathways of this interaction. Methods and Results: Human ET-1 ACh L-NNA/ACh
microvascular endothelial cells (HMEC-1) were exposed to their apoptotic or necrotic EC50 Rmax EC50 Rmax EC50 Rmax
counterparts and generation of chemokines and adhesion molecules was determined by ELISA
GK 0.50.1 15518 1614 902 5722 567
or quantitative real-time PCR. Adhesion of neutrophils was quantified using adhesion assays.
GKABT627 6020 9513* 108 9218 102 1082*
In addition, the fate of the apoptotic or necrotic EC in the co-culture was determined by
GKA192621 15mg/kg/day 0.30.1 14711 3616 953 329135* 5513
fluorescence microscopy. Exposure of HMEC-1 with apoptotic EC resulted in a temporary
GKA192621 30mg/kg/day 693195* 14424 45 1053 1415 6419
increase of monocyte chemotactic protein 1 (MCP1) and interleukin-8 (IL-8) expression
whereas exposure to lysed (necrotic) cells led to enhanced expression of IL-8 but no increase
in MCP-1 mRNA. Furthermore, adhesion of primary neutrophils to HMEC-1 that were
co-incubated with apoptotic EC was significantly increased compared to HMEC-1 exposed to
lysed cells or untreated controls. Moreover, we observed that both apoptotic and necrotic cells
were localized within the endothelial cells suggesting engulfment of damaged EC by HMEC-1.
Conclusions: Apoptotic EC induce an inflammatory response in microvascular HMEC-1. This
proinflammatory environment triggers increased chemokine production and enhances adhesion P52
and migration of primary neutrophils. These data suggest that in vivo circulating endothelial Microcirculatory and Conduit Artery Function Are Associated with Urinary
cells may induce vascular inflammation.
Albumin in Hypertensive Subjects: A Community-Based Study
Malik A Rauoof, Stephen T Turner, Iftikhar J Kullo; Mayo Clinic, Rochester, MN
P50
Environmental Carbon Monoxide is a Determinant of Oxidative Stress and Background. Increased urinary albumin is associated with adverse cardiovascular outcomes.
Defective Relaxing Responsiveness to Acetylcholine We investigated whether non-invasive measures of microcirculatory and conduit artery
endothelial function are associated with the urinary albumin/creatinine ratio (UACR), a
Frank F Zhang, Pawel M Kaminski, Brian Lamon, Michael S Wolin, Alberto Nasjletti; New commonly used index of albuminuria, in hypertensive adults without known coronary heart
York Med College, Valhalla, NY disease (CHD) or stroke. Methods. Non-Hispanic white hypertensive adults (n473) belonging
to hypertensive sibships were studied. UACR was measured in the first voided morning urine
Previous studies suggest that carbon monoxide (CO) produced in arterial vessels along with sample. Brachial artery ultrasonography was used to assess microcirculatory (reactive
the antioxidants biliverdin and bilirubin, during metabolism of heme by heme oxygenases, hyperemia, RH) and conduit artery (flow-mediated dilatation, FMD; nitroglycerin-mediated
promotes vasodilatation. However, exogenous CO fosters vascular oxidative stress and dilatation, NMD) function. Variables associated with UACR before and after adjustment for CHD
promotes vasoconstriction. This study was designed to test the hypothesis that exposure risk factors were identified using linear and ordinal (quartiles of UACR) regression models.
to increased levels of environmental CO causes oxidative stress-dependent attenuation of Results. Male sex, diabetes, higher serum creatinine, and lower high-density lipoprotein (HDL)
acetylcholine (Ach)-induced vasorelaxation. Rats were housed in chamber and allowed to cholesterol level were associated with higher UACR in univariable analyses. Body mass index,
breathe normal air or air enriched in CO (25100 ppm) for 1 hour. Immediately thereafter systolic BP (SBP), and statin use were positively associated with UACR in a borderline
the animals were anesthetized and the descending thoracic aorta was dissected out for significant manner (P0.10). In stepwise multiple linear and ordinal regression models that
immediate assessment of superoxide anion and relaxing responsiveness to Ach (10-9-10-4 included univariate predictors, diabetes and SBP were positively and HDL cholesterol inversely
mol/L) using vascular rings mounted on a wire-myograph and bathed with oxygenated associated with UACR. Among brachial reactivity variables, NMD was a significant independent
Krebs buffer containing 1 mol/L phenylephrine. The level of superoxide anion in the predictor of UACR in both multivariable linear (P0.0003) and ordinal (P0.001) regression
aorta of rats breathing normal air (115648 CPM/dry tissue ) was exceeded (P0.05) by analyses. RH (P0.022) and FMD (P0.042) were also associated inversely with higher UACR
that that in the aorta of rats breathing air containing 100 ppm CO (1901145 CPM/dry in ordinal regression analyses. Conclusion. Impaired microcirculatory and conduit artery
tissue). Ach elicited concentration-dependent reduction of isometric tension in aortic rings function are associated with increased urinary albumin in hypertensive adults. These findings
taken from air-breathing rats (EC50:0.290.04 mol/L; Rmax:99.00.7% relaxation). The indicate that increased urinary albumin may be a marker of vascular dysfunction in
concentration-response to Ach in aortic rings of animals breathing 50 and 100 ppm CO hypertension and suggest a potential mechanism underlying the association of this trait with
was shifted to the right , with attendant increase and decrease (P 0.05) in their increased CHD risk.
e56 Hypertension Vol 48, No 4 October 2006

P53 treatment of LGD1069 or 9-c-RA (P0.05).Both treatment significantly (P0.05) decreased


Endothelial Control of Vascular Remodeling in Hypertension LVW/BW ratio and LVMI, and reduced collagen deposition in the heart in SHR. Both treatments
also significantly augmented acetylcholine-induced relaxation in SHR (P0.01). Endothelium-
Aaron Baker, David Ettenson, Michael Jonas, Elazer Edelman; Massachusetts Institute of independent relaxation induced by sodium nitroprusside was not different between the groups.
Technology, Cambridge, MA Conclusion : Our results suggested that activation of RXR may attenuate the development of
hypertensive cardiac hypertrophy and improved endothelial function in SHR.
While many studies have focused on the response of isolated endothelial or vascular smooth
muscle cells (vSMCs) to mechanical forces, few studies have examined the dynamic interplay
between these two cell types in the context of vascular remodeling to hemodynamic forces. We P56
examined the affect of in-vitro mechanical strain and hypertension in-vivo on endothelial cell Increased Cerebrovascular Tortuosity Index is Associated with Increased
modulation of vSMC proliferation. Human umbilical vein endothelial cells grown to confluence Hemorrhagic Transformation in Acute Ischemic Stroke
on collagen coated silastic membranes were exposed to cyclic mechanical strain for 24 hrs
(maximal strain of 3% or 5%, 1 Hz). Strain increased the inhibitory properties of endothelial Adviye Ergul, Mary-Louise Middlemore, Kamakshi Sachidanandam, Vera Portik-Dobos, Anna
cells to vSMCs two-fold, endothelial production of soluble perlecan (2.2-fold) and cell- Kozak, Susan C Fagan; Univ of Georgia College of Pharmacy, Augusta, GA
associated syndecan-1 (3.2-fold) and conditioned media-associated heparan sulfate glycos-
aminoglycans (38%, by ion exchange and size exclusion chromatography analysis). Further, Occlusion or rupture of cerebral blood vessels results in acute ischemic stroke and further
mechanical strain causes endothelial cells to activate TGF-1 (163.09.1 pg/ml vs. not breakdown of the blood brain barrier (BBB) increases cerebral injury by the development of
detectable) and increase cellular uptake of FGF-2 (30.00.8 pg/ml vs. 19.40.8 pg/ml). Using vasogenic edema and secondary hemorrhage known as hemorrhagic transformation (HT). Type
inhibitors to p38 MAPK (SB293063) and ERK1/2 (U0126) we showed that activation of both of 2 diabetes not only is a predictor of poor outcome of stroke but also increases the risk of HT
these pathways was essential for load-induced HSPG production, TGF-1 activation and FGF-2 in stroke patients. Yet, the impact of chronic mild hyperglycemia as seen in majority of Type
uptake. Additionally, we exposed cells to strain in the presence of a neutralizing antibody to 2 diabetic patients on cerebral vessel structure and its mechanisms and functional conse-
TGF-1 and demonstrated that autocrine TGF-1 signaling was essential for load-induced quences especially in the acute ischemia/reperfusion injury setting remain unknown. The
HSPG production and sustained p38 MAPK and ERK1/2 activation. Further studies were carried current study tested the hypothesis that diabetes-induced changes in the cerebral vasculature
out comparing 20 week-old spontaneously hypertensive (SHR) rats to wild-type controls. increase the risk of hemorrhage and augment ischemic injury. Male Goto-Kakizaki rats, a model
Immunohistochemical staining on the aortae of these rats revealed increased endothelial HSPG of mild Type 2 diabetes, (260 290 g, n5) or control Wistar rats (260 290 g, n6) were
core proteins, TGF-1 and phosphorylated signaling intermediates, similar to our in-vitro used. Vascular structure was assessed by measuring cerebrovascular tortuosity index after
findings. Prior work has shown that mechanical strain and hypertension stimulate vSMC visualization of vascular tree by carbon black/latex infusion, which was significantly increased
proliferation and hypertrophy through several mechanisms. In this context, our findings suggest in the GK model (1.13 0.01 vs 1.34 0.06, P0.01) indicative of increased vascular density
a novel, integrated paradigm for understanding endothelial control of vascular remodeling to and remodeling. In additional experiments, GK (n5) or control (n6) rats underwent 3 hours
of middle cerebral artery occlusion and 24 h reperfusion. Neurologic function was assessed and
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017

hypertension.
then brain was harvested for infarct size and hemorrhage analyses. Infarct size as defined by
the % of the total hemisphere was significantly smaller in GK rats (8 4 vs 29 5, p0.01).
There was significant intracerebral bleeding and hematoma formation in the ischemic
P54 hemisphere in GK rats as opposed to controls (100 vs 20%, p0.0001). These findings provide
Synergistic Effect of Hypertension and Hypercholesterolemia on Myocardial evidence that there is cerebrovascular remodeling and potential neovascularization in diabetes.
Microvascular Permeability Response to Acute Coronary Artery Stenosis While, diabetes-induced neovascularization appears to prevent infarct expansion, development
of immature blood vessels increases the risk for hemorrhage and thereby exacerbates
Elena Daghini, Alejandro R Chade, Xiangyang Zhu, Daniele Versari, James D Krier, Amir neurovascular damage due to cerebral ischemia/reperfusion.
Lerman, Lilach O Lerman; Mayo Clinic College of Medicine, Rochester, MN

Background. The initial site of injury in early atherosclerosis is increasingly recognized to be P57
at the level of myocardial microcirculation. Exposure to hypertension (HT) or hypercholester- Endothelin Promotes Cerebrovascular Dysfunction in Type 2 Diabetes
olemia (HC) may aggravate cardiac response to ischemic insults, and their early co-existence Mainly Via ETA Receptor Activation
synergistically promotes cardiac events, conceivably as consequence of impaired microvas-
cular function. One of the early features of microvascular damage is represented by barrier Alex K Harris, Kamakshi Sachidanandam, Jim R Hutchinson, Univ of Georgia, Augusta, GA;
dysfunction, characterized by altered myocardial microvascular permeability. Therefore, this Adviye Ergul; Univ of Georgia, Med College of Georgia, Augusta, GA
study was designed to test the hypothesis that the combination of HT and HC would adversely
affect myocardial microvascular permeability in response to an acute coronary stenosis (CAS). Changes in both vascular function and structure may contribute to increased stroke risk as well
Materials. The effect of acute CAS on myocardial microvascular permeability (determined from as worsened stroke outcome in diabetes. Conduit arteries such as basilar and middle cerebral
dye time-attenuation curves obtained in the anterior cardiac wall) was explored in-vivo using artery (MCA) contribute significantly to microvascular pressure and we have previously shown
electron beam CT in pigs after 12 weeks of high cholesterol diet (HC; n4), renovascular HT that type 2 diabetes cause remodeling of MCAs. Since previous studies have demonstrated
without (HT; n5) or with concurrent HC (HTC; n4), and in a control group (N, n4). Each enhanced ET-1 induced vasoconstriction and impaired endothelium mediated relaxation of
animal was studied before and after a 30-minute 90% balloon-induced CAS achieved by basilar arteries in streptozotocin induced diabetes, the current study sought to elucidate the
inflating a balloon catheter in the LAD coronary artery. Results. Blood pressure was similarly potential vascular roles of the ET receptors in a non-obese, normotensive model of Type-2
elevated in HT and HTC, while cholesterol levels were similarly elevated in HC and HTC. Acute diabetes, the Goto-Kakizaki (GK) rat. Fourteen week old GK or control Wistar rats were treated
LAD CAS caused a significant increase in microvascular permeability in its perfusion territory with antagonists to either ETA (ABT-627, 5mg/kg/d) or ETB (A-192621, 15 mg/kg/d) receptors
in all groups (p0.05), which was particularly high in N (275137%), progressively reduced for four weeks. Basilar arteries were harvested and vascular function assessed using a wire
in HC (5321%) and HT (4418%), and further significantly blunted in HCHT (235%, myograph. Hypersensitivity of GK basilar arteries to ET-1 (0.1100 nM) was significantly
p0.01 vs. N). Conclusion. The current study suggests that an increase in microvascular reduced by ETA receptor antagonism while ETB blockade had no effect. Paradoxically, ETA
permeability may represent an adaptive myocardial response to acute ischemia. However, blockade increased ET-1 sensitivity in Wistars. Maximum constriction to ET-1 as well as
exposure to multiple risk factors may progressively attenuate this response, which may reflect acetylcholine (ACh, 1nM-10m) induced relaxation following ET-1 dose response was similar
inadequate microvascular response to ischemic insult, and may contribute to the observed among groups. Vasorelaxation to ACh following 70% pre-constriction with 5-hydroxytryptamine
increase in cardiac events. (5-HT) was impaired in diabetes. ETA receptor blockade restored relaxation to control values in
the GK animals with no significant effect in Wistars. ETB blockade had no effect in either group.
These findings demonstrate the presence of cerebrovascular dysfunction in diabetes and
suggest that the ETA receptor may exert a greater influence than the ETB in this process.
P55
*p0.05 vs Wis, **p0.05 vs GK
Effect of Retinoid X Receptor Agonist on Arterial Reactivity and Ventricular
Hypertrophy in Spontaneously Hypertensive Rats ET ET/Ach Ach

EC50 Rmax EC50 Rmax EC50 Rmax


Jun PU, Reji Hosp affiliated to Shanghai Jiaotong Univ Sch of Medicine, Shanghai, China,
Shanghai, China; Ben HE, Yan-Zhou ZHANG; Reji Hosp affiliated to Shanghai Jiaotong Univ W 132 10717 174.7 643 5023 537
Sch of Medicine, Shanghai, China W ABT 1.8.4* 13616 122 8515 4311 417
W A-192 10497 13929 2210 484* 167103 556
Background: It is well recognized that retinoids have potent anti-proliferative and anti- GK 5.42.8* 13927 9353 637 2815 224*
inflammatory effects. However, the effect of retinoid X receptor(RXR) agonist on endothelial GK ABT 4211** 1229 3816 747 2920 5514**
function and ventricular hypertrophy has not been investigated. The aim of this study was to GK A-192 74 17259 82 6011 7360 2710
assess the effects of long-term RXR agonist administration on high blood pressure, impaired
endothelial function in aorta, and damage observed in heart in spontaneously hypertensive rats
(SHR). Methods: Male SHR (14 weeks old) and age-matched normotensive Wistar Kyoto (WKY) P58
rats received either the selective RXR agonist LGD1069 (100 mg/kg of diet), 9-cis-retinoic acid Structural Alterations of Subcutaneous Small Resistance Arteries May
(9-c-RA, 100 mg/kg of diet), or placebo for 6 weeks. At the end of each treatment, arterial Predict Major Cardiovascular Events in Hypertensive Patients
pressure and heart rate were measured, the vascular reactivity of aortic rings was studied, the
heart weight-to-body weight ratio (HW/BW) and left ventricular mass index (LVMI) was Carolina De Ciuceis, Damiano Rizzoni, Enzo Porteri, Silvia Paiardi, Gianluca E Boari,
calculated, and collagen deposition in the heart was determined histochemically using Fracesca Zani, Marco Miclini, Maria Lorenza Muiesan, Dept. of Med and Surgical Sciences,
picrosirius red staining. Results: Our results showed a significantly (P0.01) higher HW/BW Univ of Brescia, Brescia, Italy; Francesco Donato, Chair of Hygiene, Univ of Brescia, Brescia,
ratio, LVMI and collagen deposition in vehicle-treated SHR compared to levels in corresponding Italy; Massimo Salvetti, Maurizio Castellano, Enrico Agabiti Rosei; Dept. of Med and Surgical
WKY. Systolic blood pressure was significantly higher in SHR (194 7mmHg, P0.01) than Sciences, Univ of Brescia, Brescia, Italy
in WKY (128 4 mmHg) at the end of the trial. Treatment with LGD1069 or with 9-c-RA
caused, from the third week on, a significant reduction of the systolic blood pressure of SHR Objective Structural alterations in the microcirculation may be considered an important
(P0.05). WKY did not present a significant change in blood pressure during the 6 weeks mechanism of organ damage. An increased media to lumen ratio of subcutaneous small
CHBPR ConferencePoster Presentations e57
resistance arteries (M/L) may predict the subsequent development of cardiovascular events in P61
a high risk population (Rizzoni et al, Circulation 2003). However, it is not presently known Endothelin B Receptor Antagonism Reduces Cerebrovascular Remodeling in
whether structural alterations of small arteries may predict also mortality and/or major Diabetes: Potential Interaction among ETA, ETB1 and ETB2 Subtypes
cardiovascular events. Design and Methods Three-hundred and four subjects were included
in the present study. They were 65 normotensive subjects, 112 patients with essential Alex K Harris, Kamakshi Sachidanandam, Jim R Hutchinson, Univ of Georgia, Augusta, GA;
hypertension, 29 patients with pheochromocytoma, 46 patients with primary aldosteronism, 25 Adviye Ergul; Univ of Georgia, Med College of Georgia, Augusta, GA
patients with renovascular hypertension, 9 with acromegaly and 18 normotensive patients with
non-insulin dependent diabetes mellitus. All subjects were submitted to a biopsy of Diabetes enhances vascular matrix accumulation, which can further contribute to the
subcutaneous fat. Small resistance arteries were dissected and mounted on an isometric development of vascular dysfunction. It has been demonstrated that endothelin-1 (ET-1) may
myograph, and the tunica media to internal lumen ratio was measured (M/L). The subjects were contribute to this process by stimulation of collagen synthesis and SMC migration. We have
re-evaluated after an average follow-up time of 6.9 years (0.6 13.5). Results Twelve subjects previously demonstrated that blockade of the ETA receptor attenuates dysregulation of matrix
died for fatal cardio-cerebrovascular event (FCE), 16 had a major, non fatal cardiovascular degrading enzymes (MMP) and blunts collagen deposition and matrix remodeling in Type-2
event (stroke or myocardial infarction) (SMI), 20 had a minor cardiovascular event (MCE) diabetes. A recent study demonstrated that inhibition of the ETB receptor by pharmacological
(chronic renal failure, cardiac pacemaker device implant, atrial fibrillation, angina pectoris or or genetic approaches promotes pathological vascular remodeling in a vascular injury model
heart failure, claudication intermittens, surgical intervention for aortic aneurism) (overall: 2.3 suggesting that ETB receptors are vasculoprotective. However, the relative role of the ETB
events % per year), while 256 of them had no cardiovascular event (NCE). A significant receptor in Type-2 diabetic vascular remodeling processes and its influence on the vascular
difference was observed in M/L between patients with FCESMIMCE and those with NCE matrix remained unknown. Accordingly, the current study tested the hypothesis that
(M/L: 0.1100.032 vs 0.0880.028, p0.00003) and between patients with FCESMI and pharmacological inhibition of the ETB receptor augments vascular remodeling of middle cerebral
those with NCE (M/L: 0.1050.037 vs 0.0880.028, p0.05). Restricting the analysis to arteries in Type-2 diabetes. Fourteen week old Goto-Kakizaki rats were treated for 4 weeks
patients with essential hypertension, a significant difference in M/L between those with with the ETB receptor antagonist A-192621 (15 or 30 mg/kg/day). High dose receptor blockade
FCESMIMCE and those with NCE (0.1140.022 vs 0.100.033, p0.05) was observed. significantly reduced MMP-2 protein (44 15* vs 201 13 pixels) and activity (12151
Conclusions Our results indicate, in a larger population than that previously investigated, that 1004* vs 18296 1180 pixels) in middle cerebral arteries while lower dose inhibition had no
structural alterations of small resistance arteries may predict all major cardiovascular events. significant effect. Morphometric analysis of cross sections demonstrated that similar to MMP-2
protein changes, low dose receptor blockade had no effect on vessel structure. However, high
dose blockade significantly reduced wall:lumen ratio (0.23 .03* vs 0.51 .04) in a manner
similar to that observed with ETA receptor blockade. These data indicate a potentially greater
involvement of the smooth muscle ETB2 receptor in Type-2 diabetes giving rise to ETA-like
effects in the vasculature as opposed to anticipated vasculoprotective effects via activation of
P59 ETB1 receptors on endothelial cells. These findings also point to a potential receptor interaction
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Crosstalk of AT1, AT2 Receptors and Aldosterone in Angiotensin among ETA, ETB1 and ETB2 subtypes and may be relevant for the future development and use
II-Rgulated Vascular Smooth Muscle Cell Senescence of ET receptor antagonists in diabetes. *p0.05 vs GK Untreated

Masaki Mogi, Li-Juan Min, Jian-Mei Li, Jun Iwanami, Kana Tsukuda, Masaru Iwai,
Masatsugu Horiuchi; Ehime Univ, Graduate Sch of Medicine, Tohon, Ehime, Japan P62
Preeclampsia Activates Neuropeptide-y (NPY) and its Receptors: Possible
Recent evidence has revealed a close association between vascular cell senescence and Role in Impaired Blood Flow and Ischemic Angiogenesis
vascular disorders, and the potential roles of angiotensin (Ang) II on vascular senescence have
been highlighted, although its detailed mechanism regulated by Ang II receptor subtypes is still Sara P Paiva, Lydia E Kuo, Jason U Tilan, Georgetown Univ, Washington, DC; Ann-Cathryn
unclear. To examine the roles of Ang II receptor subtypes in vascular senescence, we employed Rylander, AstraZeneca, Molndal, Sweden; Ullamari Pessonen, Univ of Turku, Turku, Finland;
vascular smooth muscle cells (VSMC) prepared from the thoracic aorta of AT1a receptor null Zofia Zukowska; Georgetown Univ, Washington, DC
mice (AT1KO) and AT2 receptor null mice (AT2KO). Daily administration of Ang II (10-7 M)
increased senescent cells evaluated with senescence-associated -gal (SA-gal) activity. In Hypertension in preeclampsia (PE) is associated with sympathetic hyperactivity and impaired
contrast, VSMC prepared from AT2KO showed an acceleration of both proliferation and placental angiogenesis. Early onset PE (34wks) may depend more importantly on defective
senescence compared to VSMC prepared from wild type mice. Moreover, we observed that Ang angiogenesis along with decreased availability of free VEGF. Late onset PE (34wks) may
II-induced senescence in VSMC prepared from AT1KO was markedly attenuated. Immunoblot depend less on defective placentation and more on maternal vasoconstriction. Both angiogen-
analysis showed 3.2-fold increase in Ki-ras2A expression 72 hours after Ang II stimulation in esis and vasoconstriction can be regulated by neuropeptide Y (NPY), a sympathetic co-
VSMC and knocking down of Ki-ras2A gene with small interfering of RNA strongly inhibited the transmitter, which acts via a family of receptors (Rs). NPY136 acts via Y1R as vasoconstrictor
increase in SA-gal stained cells, suggesting that crosstalk between AT1 and AT2 receptor and vascular mitogen but - when converted by endothelial dipeptidyl peptidase IV (DPPIV) to
could play some antagonistic roles in Ki-ras2A expression. We also observed that Ang II NPY336 - is angiogenic, in part via NO and VEGF, due to activation of Y2 and Y5Rs. We studied
treatment significantly increased cycline dependent kinase inhibitors, such as p16, p21, p27 expression of NPY, its Rs and DPPIV in placentas (Real Time RT-PCR), omental fat
and p53. Moreover, we demonstrated that treatment with non-effective doses of aldosterone (immunocytochemistry) and NPY levels in serum (RIA) from 20 women with early onset PE, 21
(10-12 M) and Ang II (10-10 M) synergistically increased Ki-ras2A activation and senescent VSMC. with late onset PE, and from 48 controls. NPY and Y2R mRNAs were highly expressed in
Treatment with a selective AT1 receptor blocker (ARB), valsartan, reduced SA-gal stained placentas from early PE but undetectable in the other 2 groups. Y1R mRNA expression was
cells, Ki-ras2A expression to a basal level and cancelled the effect by aldosterone. These highest in placentas from early PE and detectable but similar in the other 2 groups. NPY, Y1,
results suggest that AT1 receptor stimulation increased VSMC senescence via Ki-ras2A Y2, Y5, and DPPIV were elevated in omental fat from PE versus normal pregnancies, and serum
activation, whereas AT2 receptor activation antagonized these effects of AT1 receptor, and that NPY levels were 50% higher in late compared to early PE. We hypothesize that NPY contributes
AT1 receptor and aldosterone signaling synergistically enhanced VSMC senescence. to both early and late PE, by impairing blood flow and placentation. Activation of NPY-Y1R may
enhance maternal vasoconstriction and contribute to hypertension. The expression of NPY/Y2R
in early onset PE may reflect a compensatory mechanism which fails due to impaired
availability of free VEGF, its key angiogenic effector.

P60 P63
Veins Are not Arteries: A Story of Remodeling in Hypertension Targeted Deletion of Collectrin, a Novel Gene Upregulated after Subtotal
Nephrectomy in Mice, Results in a Urine Concentrating Defect Induced by
Catherine Rondelli, Mohammad H Pervaiz, Ralph E Watson, Keshari Thakali, Gregory D Fink, Amino Aciduria
Stephanie W Watts; Michigan State Univ, East Lansing, MI
Sandra Malakauskas, Duke Univ and Durham VA Med Cntrs, Durham, NC; Timothy Fields,
Arterial remodeling in hypertension is a process that is adaptive because it provides a stronger Duke Univ Med Cntr, Durham, NC; Hui Quan, CIIT Cntrs for Health Rsch, Rsch Triangle
artery that supports higher intra-arterial pressures, but also detrimental in that it contributes to Park, NC; Thu H Le; Duke Univ and Durham VA Med Cntrs, Durham, NC
organ damage. Remodeling occurs in response to mechanical and neurohumoral stimuli. We
hypothesized that veins, which are not exposed to higher pressures in hypertension, would Chronic kidney disease (CKD) represents a substantial health care burden, yet mechanisms
demonstrate less active remodeling than arteries. We assessed remodeling by measuring causing progression of CKD are poorly understood. Collectrin is a novel gene found to be
matrix metalloproteinase (MMP) expression and function, vessel morphometry and smooth upregulated after subtotal nephrectomy in mice, a model of CKD. In the adult mouse kidney,
muscle alpha-actin staining. Thoracic aorta and vena cava from sham normotensive (systolic collectrin is expressed in the proximal tubule and collecting duct. To understand its biological
blood pressure 1104 mm Hg) and deoxycorticosterone (DOCA)-salt hypertensive rats (1888 role, we generated a mouse line with targeted deletion of collectrin. We found that, at baseline,
mm Hg) were used in immunohistochemical, western and zymographic assays. Aorta and vena collectrin knockout (KO) mice excrete nearly twice the urine volume of WT, but urine
cava expressed the gelatinases MMP-2, MMP-9 and MT-MMP1; proteins localized to smooth osmolalities were similar. However, after water deprivation, KO mice have a modest urine
muscle in aorta, densely in the endothelium and single smooth muscle layer (subendothelial) concentrating defect, demonstrated by the inability to achieve maximal urinary concentration
of vena cava. Western and zymography analyses validated that MMP-2 was active in all vessels (3880 mosm/kg, n12 WT vs. 3189 mosm/kg, n13 KO; p0.00001). KO mice display a
but more active in aorta vs vena cava (150%). In hypertension, MMP-2 expression and greater corresponding increase in serum sodium (0.83 mEq/L, n8 WT vs. 6.72 mEq/L, n10
activity was increased in the aorta (127% control) but not vena cava of the DOCA-salt rat when KO; p0.0044). Similarly, after dDAVP administration, KO mice only partially concentrate their
compared to sham. Wall thickness was increased in DOCA-salt vs sham aorta (301/023 vs urine (3477 mosm/kg, n5 WT vs. 2498 mosm/kg, n5 KO; p0.0016), suggesting
21814 microns; p 0.05) but medial thickness was not different in vena cava. Moreover, the decreased renal responsiveness to vasopressin. To determine whether the enhanced urine
smooth muscle layer of the vena cava from DOCA-salt rats was not thicker when compared to volume is a result of increased solute or water clearance, we collected 24-hour metabolic data.
sham, differing from the hypertrophy observed in aorta. These findings suggest that large veins, KO mice display both an increase in osmolar clearance (7.1 cc/day, n11 WT vs. 11.2 cc/day,
at least using these measures, do not undergo vascular remodeling in systemic hypertension n12 KO; p0.0067) as well as TcH2O, a measure of free water reabsorption (5.9 cc/day,
and suggest that elevated pressures or other hemodynamic forces are a primary cause of n11 WT vs. 9.0 cc/day, n12 KO; p0.018). These data suggest that KO mice exhibit an
remodeling. osmotic diuresis. We further examined the solute composition of the inner medullary regions
e58 Hypertension Vol 48, No 4 October 2006

of the kidney. We found a trend toward decreased concentrations of solutes in KO mice, P66
suggesting a mild dilution of their inner medullary gradients. This is consistent with an osmotic Aminopeptidase N Down-Regulates Basolateral Na/K ATPase: Evidence of
diuresis, in which enhanced urine flow rates can result in a washout of the medullary Novel Signaling via Angiotensin IV and the Angiotensin IV Receptor
gradient. We next determined the causative osmolyte by examining the 24-hour excretions of
solutes known to be regulated or recovered by the kidney. We found that KO mice excrete twice Kumar Kotlo, Randal Skidgel, Robert S Danziger; Univ of Illinois at Chicago, Chicago, IL
the amount of amino acids (6.2 mol, n6 WT vs. 12.9 mol, n6 KO; p0.013). Taken
together, we propose that targeted deletion of collectrin results in decreased urine concen- Most experimental approaches to hypertension have sought to identify proteins and genes
trating ability due to an osmotic diuresis induced by amino aciduria. directly linked to salt-sensitivity and development of the hypertensive phenotype. Much less
attention has been paid to the identification of proteins linked to normal adaptation to high salt.
We have reported that aminopeptidase N/CD13 (Nepep), which cleaves an amino-terminal
P64 arginine from angiotensin III (Ang III) to yield the natriuretic hexapeptide angiotensin IV (Ang IV),
NFAT5 Mediates Osmotic Regulation of Sgk1 Gene Transcription exhibits greater renal tubular expression in the Dahl salt-resistant (SR) rat than its Dahl
salt-sensitive (SS) counterpart (Hypertension 43:2825, 2004). In this work, regulation of
Songcang Chen, Nada Nekrep, Chris Grigsby, Keith Olsen, David G Gardner; Diabetes Cntr, basolateral Na/K ATPase by Nepep was determined in LLC-PK1 cells. Transfection of cells with
San Francisco, CA siRNA to Nepep increased and Nepep over-expression plasmid decreased the surface
expression of the -subunit of Na/K ATPase, measured by biotinylation and Confocal
We recently reported that elevated extracellular tonicity increases the expression of serum and immunohistochemistry, and oubain-inhibitable activity ATPase activity. In transgenic mice
glucocorticoid-inducible kinase (sgk1) mRNA and protein (Chen S, et al Hypertension 2004; over-expressing Nepep in epithelial cells, there was decreased expression of basolateral Na/K
43:866). Sgk1 is thought to play an important role in aldosterone-dependent sodium handling ATPase, consistent with cell culture findings indicating that Nepep reduces Na/K ATPase. The
in distal tubular segments. In the present study we show that this increased expression derives role of Ang IV in the signaling pathway for Nepep mediated down-regulation of Na/K ATPase
from an increase in p38 MAPK-dependent sgk1 promoter activity. Increased extracellular was explored. Nepep over-expression increased (p 0.05) intracellular Ang IV abundance,
tonicity increased activity of a transfected -1400 sgk1-LUC reporter by 5 fold (p.01 vs measured by quantitative immunohistochemistry (QIHC) using a polyclonal Ab to Ang IV. siRNA
control) and this increase was suppressed by the p38 MAPK inhibitor SB203580 or by to the Ang IV receptor (AT4) inhibited the decrease in Na/K ATPase caused by Nepep
co-transfection of a dominant negative mutant of the p38 MAPK-activating kinase MKK6 over-expression. Together, these results indicate that Nepep down-regulates basolateral Na/K
(p.01 vs. NaCl-treated). Site-directed mutational analysis of the sgk1 promoter identified an ATPase and that the signal is mediated via Ang IV and the AT4 receptor. These results identify
osmoregulatory element (ORE) approximately 324 bp upstream from the transcription start site. a novel signaling pathway in nephrons that may play a mechanistic role in adaptation to high
Mutation of 3 bases within this site resulted in complete elimination of the osmotic induction salt and serve as a therapeutic target for hypertension.
while having no effect on basal promoter activity. Electrophoretic mobility shift analyses,
carried out with nuclear extracts from IMCD cells and oligonucleotides spanning this ORE,
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017

revealed a highly specific DNA protein interaction that was competed with wild type but not P67
mutant sequence. Immunoperturbation studies demonstrated that the complex was super- Role of VEGF in Renal Mechanisms of Hypertension: VEGF Receptor
shifted by antibody directed against the nuclear transcription factor NFAT5 but not by antibody Inhibition Enhances Dietary Salt-Induced Hypertension in Sprague-Dawley
directed against the p50 subunit of NF-B. Co-transfection of sgk1-LUC with an anti-sense (SD) Rats
construct or a dominant negative mutant directed against NFAT5, virtually eliminated the
osmotic induction of the promoter. Finally, chromatin immunoprecipitation analysis of IMCD Jian-Wei Gu, Megan Shparago, Wei Tan, Amelia P Bailey; Univ of Mississippi Med Cntr,
cells subjected to osmotic stress showed 8-fold increase in association of NFAT-5, as well Jackson, MS
as RNA polymerase II, with the sgk1 gene promoter in intact cells. Collectively, these data
identify sgk1 as the target of increased extracellular tonicity, and potentially other stress- Clinical evidence links the inhibition of vascular endothelial growth factor (VEGF) to hyperten-
related effectors, in IMCD cells and provide a clear picture of the mechanism underlying this sion. The present study examines: 1) if administration of VEGF receptor inhibitor (SU5416)
induction. enhances dietary salt-induced hypertension in SD rats; and 2) if VEGF or SU5416 directly
affects proliferation of cultured human renal proximal tubular epithelial cells (HRPTEC),
compared to human umbilical vein endothelial cells (HUVEC), and human aortic smooth muscle
P65 cells (HASMC). 3H-thymidine incorporation was determined in those cells following exposure to
VEGF (10 ng/ml), VEGF plus SU5416 (10 mol/L), SU5416 (1, 5, 10, & 20 mol/L), or vehicle
Regulation of the Prorenin and Renin Receptor in the Kidney by Salt intake
control for 18 hrs. Eight 10 wk old male SD rats received a high sodium diet (HS, 8%) and the
and Angiotensin II other 8 SD rats received a normal sodium diet (NS, 0.5 %) for 4 wks. After 2 wks of the dietary
program, 4 rats were administrated with SU5416 at 10 mg/kg/d, i.p. or DMSO (vehicle) for 14
Jiqian Huang, Chun Xue, John Gildea, Helmy M Siragy; Univ of Virginia, Charlottesville, VA days in HS and NS groups. In the end, mean arterial pressure (MAP) was determined in
conscious rats by continuous monitoring through a catheter placed in the carotid artery for 2
ATP6AP2 was cloned and found to encode the renin receptor (RR)which binds prorenin and days. There were no significant changes in MAP between SU5416 and DMSO-treated rats fed
renin. The deduced 350-amino acid protein has a calculated molecular mass of 39KD. with NS (126.4 4.1 vs. 115.1 6.3 mmHg; P0.213). In DMSO-treated rats, 4 weeks of HS
Bioinformatics computation deduced 4 transcripts and 2 significant proteins of 66 and 39KD did not significantly increase MAP, compared to NS rats (125.9 4.1 vs. 115.1 6.3 mmHg;
(proRR & RR). The physiologic and pathologic functions of proRR and RR are currently unknown. P0.233). However, MAP was significantly higher in rats treated with SU5416, as opposed to
In this study we investigated the physiologic factors that may regulate proRR and RR. We those treated with DMSO fed with HS for 4 wks (157.6 3.9 vs. 125.9 4.3 mmHg,
hypothesized that salt intake and angiotensin II(AngII) modulates proRR and RR expression. We P0.0016). SU5414 treatment further increased total urinary protein in SD rats fed with HS,
studied renal proRR and RR expression in response to 5 days of normal(NS), low(LS) or high(HS) compared to the vehicle HS group (53.72.1 vs. 42.5 4.6 mg/d; P0.05). There was a
salt intake, renal administration of AngII(100ng/kg/min/d) or AT1R antagonist valsartan (Val,10 significant decrease in proliferation of HRPTEC treated with VEGF plus SU5416, compared to
mg/kg/d). Western blot demonstrated the constitutive expression of RR at 66 and 39KD in rat the control (65%, P0.001). VEGF caused a 2-fold increase in proliferation of HUVEC,
renal cortex and medulla (Fig.B), rat mesangial cells (RMCs) (Fig.A & C) and rat proximal tubular compared to the control (P0.001), but its action was completely abolished by SU5416.
cells (RPTCs) (Fig.D). Using RR siRNA, proRR and RR were knocked down in RMCs (Fig.A). In Neither VEGF nor SU5416 exerted any effect on proliferation of cultured HASMC. Also, SU5416
the cortex, HS, AngIINS and ValLS enhanced the expression of proRR. In the medulla, AngII caused a dose-related inhibition on proliferation of HRPTEC. These results suggest that VEGF
and Val upregulated proRR expression. Similarly LS and HS upregulated, while valsartan inhibition enhances dietary salt-induced hypertension and kidney injury, and that VEGF exerts
downregulated RR at 39KD in the renal medulla (Fig.B). 48hrs treatment with NaCl(25mM), autocrine and paracrine functions on HRPTEC. (HL51971 & AA013821)
Glucose(30mM), AngII(100nM) and AngII plus Glucose upregulated proRR and RR protein in
RMCs (Fig.C) and in RPTCs(Fig.D). These results demonstrate that RR is ubiquitously and
constitutively expressed in the kidney, mainly in RMCs and RPTCs. ProRR(66KD) is the
precursor of RR(39KD). AngII via AT1R is involved in processing proRR to RR. In conclusion salt
P68
intake, glucose and Ang II modulate proRR and RR expression in the kidney. Antioxidant Treatment Reduces Renal Immune Infiltration, Renal TNF,
Renal MCP-1, Renal Damage and Dysfunction in Salt-Sensitive
Hypertension
Niu Tian, Sharkeshia Jordan, R. D Manning, Jr.; Univ of Mississippi Med Cntr, Jackson, MS

Renal free radical (O2.-) production increases and renal infiltration of immune cells occurs in
some forms of experimental hypertension. However, in salt-sensitive hypertension, it is not
clear whether elevations in renal oxidative stress increase renal immune infiltration and renal
cytokines and chemokines leading to renal damage, dysfunction and increased arterial
pressure. The importance of renal oxidative stress in initiating a renal immune response was
determined in Dahl salt-sensitive (S) rats subjected to a high sodium diet with and without
antioxidant treatment. S rats were equipped with arterial and venous catheters and subjected
to a 5-week diet of high Na (8% NaCl) or high NaVit C/E [vitamin C-(1g/L) in the drinking
water and vitamin E-(5000 IU/Kg) in the food]. Mean arterial pressure was measured
continuously throughout the last 10 days. The results at the end of week 5 are shown below
(values are meanSEM; P0.05 comparing Hi Na and Hi NaVit C/E groups). The data show
that antioxidant treatment of high sodium Dahl S rats with vitamin C/E effectively decreased
renal oxidative stress as evidenced by decreased renal O2.- release and increased renal
GSH/GSSG, the reduced-to-oxidized glutathione ratio. As a result, renal macrophage infiltration
(ED1 cells), renal TNF-alpha, and renal MCP-1 decreased. In addition, proteinuria and mean
arterial pressure decreased, and GFR and renal plasma flow increased markedly. In summary,
vitamin C/E treatment in Dahl S rats on high sodium intake decreased renal oxidative stress,
CHBPR ConferencePoster Presentations e59
renal macrophage infiltration, TNF-alpha and MCP-1, improved renal dysfunction, lessened P71
renal injury and decreased arterial pressure. In conclusion, in Dahl S rats on a high sodium diet, Female Protection from Renal Disease Progression is Associated with
renal oxidative stress plays an important role in mediating increased renal inflammation, which 17Beta-Estradiol (E2)-Mediated Attenuation of the Injury-Induced Increase
is associated with decreased renal hemodynamics, renal damage and increased arterial
in NAD(P)H Oxidase Activity
pressure.
GSH/GSSG ratio cortex 0.3 0.3 2.5 0.8 Wei Zheng, Hong Ji, Ana Paula Dantas, Kathryn Sandberg; Georgetown Univ, Washington,
DC
Renal macrophages 4.90.3 2.30.4
(count/mm2)
Chronic renal disease from several etiologies progresses at a faster rate in men compared to
Renal TNF-alpha (pg/mg) 1.20.06 0.90.05
Renal MCP-1 (OD/g) 1.20.1 0.740.05 women. We found that E2 protects against progressive renal disease induced by renal wrap
Proteinuria (mg/day) 1704 9612 hypertension (RW) and a high salt diet. Compared to male rats (RW-M) and ovariectomized
GFR (ml/min) 1.50.1 3.00.5 females (RW-OVX), estrogen replete females (RW-F & RW-OVXE2) were protected from
Renal plasma flow (ml/min) 3.80.4 7.40.5 indices of renal injury including proteinuria, mean glomerular volume, glomerulosclerosis, and
Mean arterial pressure (mmHg) 1915 1508 tubulointerstitial fibrosis. Progressive renal disease is associated with elevated levels of
reactive oxygen species in the kidney. Objective: To determine if female protection from renal
injury is associated with E2 attenuation in reactive oxygen species generation, we determined,
in RW hypertension, if E2 regulates the activity of renal NAD(P)H oxidase (ox), which is the major
enzyme that generates superoxide in the rat kidney. Methods: Sprague Dawley rats
(n6 8/group) were divided into 2 sham-operated control groups: male (Sham-M) and female
(Sham-F) and 4 RW groups: RW-M, RW-F, RW-OVX and RW-OVX-E2 (0.24 mg E2/60 day pellet).
P69 After 6 wk on a high sodium (4%) diet, NAD(P)H ox activity was measured in renal cortical
Suppression of Renal Immune Cell Infiltration Attenuates Hypertension and homogenates by the lucigenin method and expressed as relative light units/180 sec (RLU);
Kidney Disease in Dahl Salt-Sensitive (SS/Mcw) Rats *p0.05. Results: RW increased NAD(P)H ox activity in both males (1.4-fold*) and females
(2.3-fold*). Compared to females in the same group, both male control (2-fold*) and RW
David L Mattson, Hayley Lund; Med College of Wisconsin, Milwaukee, WI (1.3-fold*) rats had higher enzyme activity. In RW females, ovariectomy increased enzyme
activity by 1.3-fold* while E2 replacement prevented this effect. [RLU: Sham-M, 2840 110;
Previous studies from our laboratory demonstrated that systemic administration of the Sham-F, 1400 160; RW-M, 4080 240*; RW-F, 3200 260; RW-OVX, 4270 320*;
immunosuppressive agent mycophenolate mofetil attenuated the development of hypertension RW-OVXE2, 2730 680.] Conclusions: These results demonstrate that E2-mediated
and kidney disease in the Dahl SS/Mcw rat. The present experiments were performed to protection from RW-induced renal injury is associated with attenuation in RW-induced
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017

confirm that observation using a mechanistically different immunosuppressive agent and to increases in NAD(P)H ox activity, suggesting that E2 protects the kidney from progressive renal
determine the histological and immunohistochemical changes which occur in the kidneys of the disease in part, by attenuating the injury-induced increases in renal superoxide. Supported by
vehicle and immune-suppressed rats. Dahl SS/Mcw rats were fed a high salt (4.0% NaCl) diet NIH AG19291.
for three weeks. Rats were treated with a daily injection of vehicle (5% dextrose) or the
immunosuppressive agent tacrolimus (0.25 mg/kg/day, i.p.), a calcineurin inhibitor, during the
period of high NaCl intake (n5/group). High salt mean arterial blood pressure in the rats P73
administered tacrolimus (1523 mmHg) was significantly decreased compared to vehicle- High-Salt Diet Produces Severe Renal Dysfunction Independently of
treated rats (1718 mmHg). Furthermore, the rate of protein (6112 mg/day) and albumin Changes in Arterial Pressure in Spontaneously Hypertensive Rats (SHR)
excretion (209 mg/day) in the tacrolimus-treated rats was significantly lower than the protein
and albumin excretion rate in vehicle-treated rats (18129 and 7428 mg/day, respectively). Luis C Matavelli, Xiaoyan Zhou, Jasmina Varagic, Dinko Susic, Edward D Frohlich; Ochsner
Creatinine clearance and body weight were not different between the groups, averaging Clinical Foundation, New Orleans, LA
0.50.2 ml/min/gkwt and 34714 g, respectively, in the vehicle-treated group. A histological
examination demonstrated marked glomerular damage (fibrotic tissue and collapsed capillary We have previously shown that high dietary salt produced adverse cardiac effects in SHR
structure), blocked tubules in the outer medulla (protein deposition casts), and fibrotic vasa independent of increases in arterial pressure; however, the renal effects are unknown. In the
recta bundles in the kidneys of vehicle-treated rats. Furthermore, immunohistochemical present study we evaluated the role of step-wise increases in salt-loading on renal function,
localization of ED1, a marker of macrophage infiltration, demonstrated ED1-positive cells hemodynamics, and glomerular dynamics in SHR. At 8 weeks of age, male SHR were given 4%
throughout the kidneys of the vehicle-treated rats following the high NaCl intake with the (n11), 6% (n9), or 8% (n11) NaCl diet for the ensuing eight weeks; controls (n11)
greatest infiltration near damaged glomeruli and in the region of the protein casts in the outer received standard chow (0.6% NaCl). Each week, all rats had 24-h urinary albumin (Ualb)
medulla. These experiments indicate that the renal infiltration of immune cells plays an measured. After eight weeks, SHR had systemic and renal hemodynamics determined. The
important role in the development of hypertension and renal disease in Dahl SS/Mcw rats controls, 4%, and 6% salt-loaded SHR were studied further by renal micropuncture. After two
consuming a high NaCl diet. weeks salt-loading, Ualb increased in 6% and 8% salt-loaded SHR. At eight weeks, mean
arterial pressure (MAP) had increased and glomerular filtration rate (GFR) decreased in all
salt-loaded rats. The 6% and 8% salt-loaded SHR decreased renal plasma flow (ERPF) and
increased renal vascular resistance (RVR), left kidney mass index (control3.410.10,
4%3.720.04, 6%3.840.11*, 8%3.930.19* mg/g) and serum creatinine (con-
trol0.470.02, 4%0.560.04, 6%0.720.07*, 8%0.620.04* mg/dl). Although
P70 single nephron glomerular filtration rate was not different (control27.32.57,
4%24.91.1, 6%23.62.2 nl/min), the 4% and 6% salt-loaded rats decreased single
Enhanced Superoxide Activity and its Interaction with Nitric Oxide
nephron plasma flow (SNPF) and increased afferent (RA) and efferent arteriolar (RE)
Modulate Renal Function in Pre-Hypertensive Ren-2 Transgenic Rats resistances. Furthermore, 6% salt-loaded SHR increased glomerular hydrostatic pressure (PG).
In conclusion, salt-excess increased MAP minimally, but progressively deteriorated renal
Libor Kopkan, Tulane Univ Health Sciences Cntr, New Orleans, LA; Monika Thumova, function, hemodynamics, and glomerular dynamics. These findings demonstrate a causal
Zuzana Huskova, Zdenka Vanourkova, Ludsk Cervenka, Cntr for Experimental Medicine, relationship between salt excess and renal injury independent of a minimal increase in MAP.
Institute for Clinical and Experimental Medicine, Prague, Czech Republic; Dewan S Majid; All data are mean 1SEM; *p0.05 vs control.
Tulane Univ Health Sciences Cntr, New Orleans, LA
Control 4% NaCl 6% NaCl 8% NaCl
It has been demonstrated that pre-hypertensive heterozygous transgenic rats (TGR) harboring
Ualb (2nd wk; 0.480.04 0.490.05 0.720.06* 1.240.18*
the Ren-2 renin gene exhibit an excess of superoxide (O2-) production compared to
mg/24h)
aged-matched Hanover-Sprague Dawley rats (HanSD). This study was performed to examine MAP (mm Hg) 1862 1941* 1961* 1972*
the role of O2- and its interaction with nitric oxide (NO) in the regulation of renal hemodynamic GFR (ml/min/g) 1.090.13 0.800.09* 0.510.05* 0.510.07*
and excretory function in pre-hypertensive TGR that may be involved in the development of ERPF (ml/min/g) 3.160.41 2.580.18 1.600.18* 1.670.18*
hypertension in this model. Renal responses to acute infusion of O2- scavenger, tempol (150 RVR (U) 33.44.1 39.83.1 72.713.5* 77.615.5*
g/min/100g BW), and NO synthase inhibitor, nitro-L-arginine methylester (L-NAME, 5 SNPF (nl/min) 101.59.9 75.25.9* 70.35.7*
g/min/100g BW) directly into the renal artery were evaluated in anesthetized male TGR RA (U) 5.640.53 7.850.60* 8.800.86*
(n31) and HanSD rats (n29). Glomerular filtration rate (GFR) and renal plasma flow (RPF) RE (U) 1.600.15 2.200.19* 2.630.26*
PG (mm Hg) 49.20.9 50.70.8 53.61.0*
were estimated by inulin and para-aminohippurate clearances, respectively. Urinary concen-
ration of 8-isoprostane (index of endogenous O2- activity) was determined by enzyme
immunoassay. Although there were no differences in mean arterial pressure and basal renal
hemodynamics and excretory function between pre-hypertensive TGR and age-matched HanSD P74
rats, infusion of tempol alone caused significant increases in RPF and GFR (104 and 122%, Ac-SDKP Exerts Renal Protective Effects in Wild Type and eNOS-/- Mice
respectively; n9) in TGR but not in HanSD (n9). Compared to HanSD, 8-isoprostane with DOCA Salt Hypertension
excretion was significantly higher in TGR (41%) which was significantly attenuated during
tempol treatment. Intra-arterial L-NAME infusion in the kidney caused greater decreases in RPF Saraswati Pokharel, Nour-Eddine Rhaleb, Tang-Dong Liao, Umesh Sharma, Oscar Carretero;
and GFR in TGR (-344 and -224%, respectively; n10) than in HanSD rats (-193 and Henry Ford Hosp, Detroit, MI
-104%, respectively; n10). In TGR (n12), greater hemodynamic effects of L-NAME were
abolished by co-infusion of tempol. Moreover, co-infusion of tempol reversed the anti-diuretic Background: Hypertensive end organ damage is characterized by renal inflammation and
and anti-natriuretic effects of L-NAME. These findings indicate that there was greater proteinuria. In this study, we hypothesized that N-acetyl-Ser-Asp-Lys-Pro (Ac-SDKP) inhibits
enhancement of O2- activity during NO inhibition in TGR than HanSD rats. These data suggest hypertension induced nephropathy by decreasing inflammation and increasing nephrin.
that the enhanced O2- activity and its interaction with NO during the pre-hypertensive phase in Methods and Results: We treated 16-week old uninephrectomized wild type (WT) and
TGR modulates renal hemodynamics and excretory function and thus plays a role in the endothelail nitric oxide synthase (eNOS)-/- mice with 1) placebo, 2) DOCA and 3) DOCA
development of hypertension in this transgenic rat model. Ac-SDKP (800 g/Kg/day) for 12 weeks. We measured blood pressure, urine protein, renal
e60 Hypertension Vol 48, No 4 October 2006

macrophage infiltration and glomerular nephrin expression. DOCA salt increased blood pressure P77
in both strains, which was unaltered by AcSDKP. In vehicle-treated eNOS-/- mice, the urine Low Na Intake Suppresses the Expression of Cyp2c23 and the Arachidonic
albumin excretion was 65-fold higher than the WT controls. AcSDKP reduced albuminuria in Acid-Induced Inhibition of ENaC
both strains of mice treated with DOCA-salt (Table 1). Similarly, AcSDKP decreased the number
of the renal cortical macrophages in mice treated with DOCA salt in both strains, though to a Peng Sun, Dao-Hong Lin, Dept of Pharmacology,New York Med College,10595, Valhalla, NY;
lesser degree in eNOS-/- mice (Table 1). In search of the mechanism by which AcSDKP inhibits Tong Wang, Dept of Cellular & Molecular Physiology,Yale Univ of Sch of Medicine,06520,
proteinuria, we measured glomerular slit pore protein, nephrin, lack of which causes congenital New Haven, CT; Elisa Babilonia, Zhi-Jian Wang, Rowena Kemp, Alberto Nasjletti, Wen-Hui
nephritic syndrome in humans. Immunofluorescence showed that the nephrin expression in the Wang; Dept of Pharmacology,New York Med College,10595, Valhalla, NY
mice treated with DOCA salt was lower than the vehicle treated groups and AcSDKP
significantly increased the nephrin expression in both the strains. More importantly, nephrin We have previously demonstrated that arachidonic acid (AA) inhibits epithelial Na channels
expression was significantly lower in vehicle-treated eNOS-/- mice compared to WT mice (ENaC) through cytochrome P450(CYP) expoxygenase-dependent pathway. In the present study
(Table 1). This could be responsible for significantly higher amount of proteinuria in eNOS-/- we tested the hypothesis that low Na intake suppresses the expression of CYP2C23, which is
strain. Conclusion: The antiproteinuric effects of Ac-SDKP may, at least partly, be due to its mainly responsible for converting AA to epoxyeicosatrienoic acid (EET) in the kidney , and
ability to decrease renal inflammation and increase nephrin expression in hypertensive mice. attenuates the AA-induced inhibition of ENaC. Immonostaining showed that CYP2C23 is
Table 1: Effects of AcSDKP on urine albumin excretion, renal macrophage infiltration and expressed in the Tamm-Horsfall protein (THP)-positive and aquaporin 2 positive tubules. This
nephrin expression in WT and eNOS-/- mice suggests that CYP2C23 is expressed in the thick ascending limb (TAL) and collecting duct (CD).
mice Placebo DOCA DOCA AcSDKP Na restriction significantly suppressed the expression of CYP2C23 in the TAL and CD. Western
blot also demonstrated that the expression of CYP2C23 in renal cortex and outer medulla
Urine albumin (g/10 gm BW/24 hour) diminished in rats on Na deficient diet (Na-D) but increased in those on high Na diet (4%).
WT 10.81.7 41.55* 13.53.1 Moreover, the content of 11,12 EET decreased in the isolated CCD from rats on Na-D in
eNOS-/- 693271 1058357 297105
comparison to those on a normal Na diet (0.5%). Patch clamp study showed that application
Number of renal cortical macrophages/ high power field
WT 5.71.6 21.93.1* 6.40.7 of 510 M AA inhibited the activity of ENaC in the CCD in the control rats. But, the inhibitory
eNOS-/- 4.50.8 327.3* 209.8 effect of AA to ENaC was significantly diminished in rats on Na-D for 14 days. In contrast,
Nephrin expression (% fractional area/ glomerulus) 11,12-EET inhibits ENaC in the CD from rats on Na-D for 14 days with a similar potency as that
WT 240.7 123* 184.9 in control animals. We also used micropufusion technique to examine the effect of MS-PPOH,
eNOS-/- 92.9 5.81.4* 174.9 an inhibitor of CYP epoxygenase, on Na transport in the distal nephron. Application of MS-PPOH
*, P0.05 vs. placebo; , P0.05 vs. WT DOCA significantly increased Na absorption in the distal nephron in control rats but had no significant
effect on Na absorption in rats on Na-D for 14 days. We conclude that low Na intake
downregulates the activity and expression of CYP2C23 and attenuates the inhibitory effect of
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AA on Na transport.

P75
Increased GFR and Renal Excretory Function by Activation of TRPV1 in the P78
Isolated Perfused Kidney Angiotensin Converting Enzyme Inhibition Results in Hypoxia Selectively in
the Post-Clip Kidney in Two-Kidney, One Clip Hypertension
Jianping Li, Donna H Wang; Michigan State Univ, East Lansing, MI
Fredrik Palm, Margarida Mendonca, William J Welch, Christopher S Wilcox; Georgetown
To test the hypothesis that activation of the transient receptor potential vanilloid type 1 (TRPV1) Univ Med Cntr, Washington, DC
channels in isolated perfused kidney leads to natriuresis and diuresis via an increase in
glomerular filtration rate (GFR), recirculating Krebs-Henseleit buffer added with inulin and We have previously reported a decreased renal venous (RV) oxygen tension (pO2) in the
lithium was perfused at a constant flow, and perfusion pressure (PP) was pre-adjusted two-kidney, one clip hypertension (2K1C) model of renovscular hypertension after ACE
to185190 mmHg with phenylephrine. Capsaicin (Cap), a selective TRPV1 agonist, in the inhibition (ACEI). The present study evaluated the renal cortical pO2 and blood flow in both
presence or absence of capsazepine (Capz), a selective TRPV1 antagonist, CGRP8 37, a kidneys and the response to acute ACEI in 2K1C rats. Three weeks after left renal artery
selective calcitonin gene-related peptide (CGRP) receptor antagonist, or spantide II (Spa), a clipping, rats were inactin-anesthetized and both kidneys immobilized in plastic cups.
selective substance P (SP) receptor antagonist, were perfused. Cap (2, 10, 30 M, n5 6) Thereafter, cortical pO2 (Clark-type microelectrodes) and blood flow (laser-Doppler) were
dose-dependently decreased PP (-8 1, -33 3, -58 2 mmHg, P0.001) and increased measured before and after ACEI (enalaprilat; bolus 0.3 mg/kg bw, infusion 0.3 mg/kg bw/h).
urine flow rate (UFR, 43 4, 89 7, 164 11 l/min/g kidney weight, P0.01), Na RVpO2 was measured at the end of the experiments. The elevated baseline blood pressure was
excretion (UNaV, 2.9 0.2, 6.8 0.9, 11.7 1.0 mol/min/g kw, P0.05), GFR (51 5, significantly lowered by ACEI (1626 vs.1076 mmHg; n8; p0.001). The unclipped kidney
172 17, 301 21 l/min/g kw, P0.001), and the release of CGRP (1.4 0.1, 4.3 0.4, displayed hypertrophy compared to the post-clip kidney (1.50.1 vs. 1.10.1 g; p0.001).
8.0 1.4 pg/g kw, P0.05) and SP (0.31 0.03, 1.02 0.22, 1.48 0.23 pg/g kw, The renal cortical blood flow was similar in both kidneys before ACEI (37.63.1 vs. 31.23.0
P0.05) without changing fractional excretion of lithium (FELi ) and fractional distal sodium laser units; ns), but ACEI reduced blood flow significantly only in the post-clip kidney (17.53.4
excretion (FDSE). CGRP8 37 (0.1 M) or Spa (0.1 M) alone attenuated the effect of Cap on PP vs. unclipped 30.74.4 laser units). The cortical pO2 was lower during baseline in the post-clip
(Cap: -33 3, CGRP8 37 Cap: -20 3, Spa Cap: -22 1 mmHg, P0.05), UFR (89 kidney (37.21.7 vs. unclipped kidney 49.02.1 mmHg; p0.001). ACEI selectively lowered
7, 42 4, 58 6 l/min/g kw, P0.05), UNaV (6.8 0.9, 3.5 0.1, 3.7 0.5 mol/min/g the pO2 in the post-clip kidney (27.70.6 vs. unclipped 47.22.2 mmHg; p0.001)
kw,P0.01), and GFR (172 17, 101 7, 115 9 l/min/g kw, P0.05). However, Capz accompanied by a corresponding decrease in RVpO2 in the post-clip kidney (37.02.4 vs.
(30 M) alone or CGRP8 37 combined with Spa fully blocked the effect of Cap on PP, UFR, UNaV, unclipped 52.31.3 mmHg; p0.001). In conclusion, a functional renal artery restriction
GFR, and the release of CGRP and SP. Thus, activation of TRPV1 in the isolated kidney reduces baseline cortical pO2 in the post-clip kidney, which is decreased further by ACEI. This
decreases renal perfusion pressure and increases GFR and water/sodium excretion possibly via direct demonstration of a sustained reduction in the renal cortical pO2 could be attributed to
simultaneous activation of CGRP and SP receptors upon their enhanced release, suggesting reduced renal blood flow, which may be the limiting determinant of oxygen availability in the
TRPV1 plays a key role in modulating renal hemodynamics and excretory function. post-clip kidney.

P79
Role of Nitric Oxide in Renal Blood Flow Control during Diabetes as
P76 Revealed by Continuous Measurement
Diuresis and Natriuresis Induced by Hypertonic Saline Perfusion of the
Renal Pelvis: Role of TRPVl Tracy Bell, Michael Brands; Med College of Georgia, Augusta, GA

Yi Zhu, Donna H Wang; Michigan State Univ, East Lansing, MI Vasodilation of the afferent arteriole is thought to be the major cause for the increase in renal
blood flow (RBF) and glomerular filtration rate (GFR) during the early stages of diabetes mellitus.
To test the hypothesis that the transient receptor potential vanilloid type 1 (TRPVl) channels Previous studies from our lab have implicated an important role for the Nitric Oxide (NO) system
expressed in sensory nerves innervating the renal pelvis mediate diuresis and natriuresis in mediating this response, because giving NOS inhibitors prevented the increase in renal
induced by hypertonic saline (HS) perfusion, sodium chloride at 150, 300 and 600 mM was plasma flow and GFR during diabetes. However, a limitation of these studies is that single point
perfused into the left renal pelvis (LRP) of anesthetized rats at a rate that did not change renal measurements were taken and may not reflect the time-dependent role of NO. Therefore, in
perfusion pressure. Mean arterial pressure was not altered by LRP perfusion of HS at any of this study, we have developed a more precise method to measure the role of NO in the chronic
these concentrations. LRP perfusion of HS at 600 mM (equal to 1200 mOsm/L), but not at 150 control of RBF during diabetes. We measured RBF continuously, 18 hr/day using a Transonic
or 300 mM, induced 23 fold increases in urine flow rate (Uflow) and urinary sodium excretion flow probe in control (C, n2), diabetic (D, n4), L-NAME (L, n3) and diabetic/L-NAME (DL,
(UNa) in the contralateral kidney (n5 6, P0.05). The increases in contralateral Uflow and n6) treated rats. Mean arterial pressure averaged 880.8, 911.1, 1151.2 and 1220.7
UNa were blocked by ipsilateral renal denervation or by ipsilateral LRP perfusion of capsazepine mm Hg, in the C, D, L, DL groups, respectively, during the L-NAME vehicle control period. At
(CAPZ, 24 nM), a selective TRPVl antagonist, or RP67580 (12 M), a selective neurokinin-1 the onset of diabetes, MAP increased to an average of 1482.1 in the DL group. Renal blood
receptor (NK1) antagonist (p0.05). In contrast, LRP perfusion of mannitol at 1200 mOsm/L flow averaged 6.30.1, 4.70.1, 5.90.3 and 5.40.1 in the C, D, L, DL groups,
had no effect on contralateral Uflow and UNa. Incubation of the renal pelvis in vitro with respectively, during the L-NAME or vehicle control period and, increased immediately by
capsaicin (CAP, 2.4 nM), a selective TRPV1 agonist, or HS but not mannitol significantly 102.6% of control on day 1 of diabetes in the D group before increasing progressively to
increased substance P (SP) release (p0.05). Whereas HS-induced increases in SP release 524.7% of control by the end of diabetic week 2. Interestingly, RBF decreased to 813.2 and
was blocked by CAPZ or RP67580, CAP-induced increases in SP release was blocked by CAPZ 831.8% of control in the L and DL groups, respectively, by the end of diabetic week 2.
only (p0.05). Taken together, our data indicate that HS perfusion into the renal pelvis Glomerular filtration rate increased by 6033.3% of control in the D group, while decreasing
activates ipsilateral TRPV1 and/or NK1 receptors expressed in sensory nerves directly or via by 7924.5, and 8627.3% of control in the L and DL groups, respectively, by the end of
enhanced SP release, leading to contralateral diuresis and natriuresis. diabetic week 2. Our model provides a novel method to measure the chronic effects of the NO
CHBPR ConferencePoster Presentations e61
on RBF control during diabetes. Furthermore, by using this model we have demonstrated that vs Nicotine 51 9 RLU (n4, P 0.05). In response to protein kinase C (PKC) activation, MC
NO is required for the immediate increase in RBF. synthesize ROS via NADPH oxidase (Jaimes KI98). In the current studies we demonstrate that
calphostin C, a PKC inhibitor, as well as DPI and apocynin, two inhibitors of NADPH oxidase,
prevented the effects of nicotine upon MC proliferation and fibronectin production hence
P81 establishing ROS as second messengers of nicotines actions (table). These studies unveil
Activation of Gi Via Calcium Sensing Receptor (CaR) Regulates TNF in previously unrecognized mechanisms that indict nicotine, a component of CS, as an agent that
Medullary Thick Ascending Limb (mTAL) Cells may accelerate and promote the progression of kidney disease via alterations in the glomerular
mesangium.
Huda I Abdullah, Paulina L Pedraza, John C McGiff, Nicholas R Ferreri; NYMC, Valhalla, NY
Nicotine Nicotine Nicotine
Control Nicotine Calphostin C Apocynin DPI
We previously showed that TNF production via CaR activation is PI-PLC-mediated, NFAT-
dependent, and part of a mechanism that regulates ion transport in mTAL cells in a Thymidine Incorporation 100 1382* 857 708 605*
PGE2-dependent manner. Presently, we determined if activation of Gi in mTAL cells contributes (Percent)
to CaR-mediated: 1) TNF production in a calcineurin (CN)- and NFAT-dependent manner, and Fibronectin Expression 100 14110* 10230 9627 324*
(Percent)
2) regulation of ouabain-sensitive oxygen consumption, an in vitro correlate of Na transport.
CN activity in response to 1.2 mM Ca2 was inhibited in mTAL cells transiently transfected N 3; * P 0.05 vs Control
with dominant negative CaR, R796W, (control: 0.72 0.29; Ca2: 1.970.69; R796W:
0.990.45; Ca2 & R796W: 0.660.28 pmol/g protein; n4; p0.01), indicating that the
response to extracellular Ca2 is dependent on activation of the CaR. CaR-mediated TNF P84
production was abolished when cells were pretreated with pertussis toxin (PTX; 100 ng/ml; Targeted Ablation of the Chromogranin A Gene: Profound Alterations in
n3; p0.001). CN activity induced by CaR also was abolished by PTX (control: 0.720.29; Cardiovascular and Renal Physiology
Ca2: 1.970.69; PTX: 0.690.19; Ca2 & PTX: 0.690.40; n4; p0.01). The
contribution of Gi activation to NFAT-induced TNF production was assessed in cells Sushil K Mahata, Univ of California at San Diego & VA San Diego Healthcare System, La
co-transfected with either a NFAT cis-reporter construct (2 g/well) or pGV-B2-TNF promoter Jolla, CA; Nitish R Mahapatra, Univ of California at San Diego, La Jolla, CA; Sucheta M
construct (0.05 g/well) and a SV40 renilla luciferase promoter construct (0.025 g/well). PTX Vaingankar, Univ of California at San Diego System, La Jolla, CA; Manjula Mahata, Univ of
inhibited CaR-mediated increases in NFAT- and TNF promoter-luciferase activity (fold induction: California at San Diego, La Jolla, CA; Yusu Gu, Univ of California at San Diego & VA San
Ca2: 2.740.39; PTX: 1.010.26; Ca2 & PTX: 1.260.22; n5; p0.001); and (Ca2: Diego Healthcare system, La Jolla, CA; Michael G Ziegler, John Ross, Jr., Univ of California
3.530.49; PTX: 1.140.24; Ca2 & PTX: 1.520.06; n3; p0.01), respectively. Inhibition at San Diego, La Jolla, CA; Daniel T OConnor; Univ of California at San Diego & VA San
of Gi also attenuated COX-2-dependent PGE2 synthesis in cells challenged with Ca2 (fold Diego Healthcare System, La Jolla, CA
induction: Ca2: 10.222.88; PTX: 0.780.27; Ca2 & PTX: 2.861.81; n7; p0.001).
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017

Moreover, inhibition of ouabain-sensitive oxygen consumption induced by CaR activation was Chromogranin A (CHGA), a 48 kDa secretory pro-peptide, is co-stored and co-released with
reversed in the presence of PTX. Collectively, the data suggest that Gi-coupled signaling catecholamines from secretory vesicles in adrenal medulla and postganglionic sympathetic
contributes to NFAT-mediated TNF production by increasing CN activity, and is part of a axons. It gives rise to the catecholamine release-inhibitory peptide catestatin. CHGA is
mechanism by which CaR regulates mTAL function. Understanding CaR-mediated signaling over-expressed in essential hypertension, a complex trait with genetic predisposition.
pathways that regulate TNF production in the mTAL will be crucial to defining novel Conversely, the plasma concentration of the catestatin fragment of CHGA is diminished both in
mechanisms that regulate extracellular fluid volume and salt balance. established hypertension and in patients still-normotensive offspring at genetic risk of
developing the disease. These human findings suggest a mechanism whereby diminished
catestatin might increase the risk for hypertension. To gain a better insight into the functions
P82 of CHGA and catestatin in vivo we generated Chga null (Chga-/-) mice. Telemetric and tail-cuff
Inadequate Regulation of Renal B0AT1 Gene Expression in the Spontaneous studies on blood pressure (BP), transthoracic echocardiography, and hemodynamic studies
Hypertensive Rat reveal extreme cardiovascular and renal changes in Chga-/- mice: (i) Elevated systolic and
diastolic BP; (ii) Loss of diurnal BP variation; (iv) Increased left ventricular mass and cavity
Maria Joao Pinho, Patricio Soares-da-Silva; Fac. Medicine Porto, Porto, Portugal dimensions; (iii) higher heart rate, left ventricular pressure, ventricular contractility (max dP/dt)
and end diastolic pressure and lower R-R intervals; (iv) Increased plasma and urinary
We first reported that overexpression of Na-independent type 2 L-amino acid transport (LAT2) catecholamines; (v) increased plasma neuropeptide Y (Npy); (vi) low plasma creatinine and high
in the spontaneous hypertensive rat (SHR) kidney is organ specific and precedes the onset of GFR. Rescue of elevated BP to normalcy was achieved by either exogenous catestatin
hypertension (Hypertension, 42, 613 618, 2003). B0AT1 is a novel member of the Na- replacement, or humanization of Chga-/- mice with transgenic insertion of a human CHGA
dependent neurotransmitter transporter family (SLC6), which has a major role in the uptake of haplotype (Chga-/-:CHGA/). Pithing of the Chga null mice restored the BP to normalcy.
luminal neutral amino acids. The present study evaluated the expression of neutral amino acid Pithed Chga-/- mice displayed exaggerated BP response to electrical stimulation. Telemetered
transporter B0AT1 in the rat kidney and intestine. The effect of high salt intake was also Chga null mice also showed inflated BP response to immobilization stress. These findings
evaluated on the renal expression of B0AT1 in SHR and normotensive Wistar-Kyoto rat (WKY) suggest involvement of peripheral and central mechanisms in the development of BP. We
at 4 and 12 weeks of age. Animals were fed normal (NS) or high (HS - 1% saline as drinking conclude from these studies that the loss of the physiological brake catestatin in Chga-/- mice,
water) salt diet for 24 hours. The abundance of transcripts B0AT1 was evaluated by quantitative coupled with dysregulation of transmitter storage and release, may act in concert to alter
real-time PCR, using the specific rat primers (forward primer 5 AAC CAG AAT CAG ACA GGC autonomic control of the circulation in vivo, eventuating in hypertension.
TAT 3 and reverse primer 5 AGA ACA CTC CAG GCA CAT 3, position 466 and 606 bp in rat
B0AT1 sequence). Data was normalized to the expression of the constitutively expressed gene
GAPDH. At 4 and 12 weeks of age, the abundance of renal B0AT1 transcript in SHR was 49% P85
and 32% that in WKY, respectively. However, no significant differences were observed on the Genetic Architecture of the Salt Sensitivity of Blood Pressure and
expression of intestinal B0AT1 between SHR and WKY. High salt intake decreased by 30% the Correlation with Insulin Resistance in Hypertensive Hispanic Families
expression of B0AT1 in both 4- and 12-week old WKY and in 4-week old SHR. Surprisingly, HS
intake produced a significant increase (45% augment) in B0AT1 mRNA in 12-week old SHR. Anny H Xiang, Univ of Southern California, Los Angeles, CA; Leslie J Raffel, Cedars-Sinai
It is concluded that Na dependent B0AT1 amino acid transporter is under-expressed in the Med Cntr, Los Angeles, CA; Miwa Kawabubo, Univ of Southern California, Los Angeles, CA;
SHR and this is organ specific and precedes the onset of hypertension. Overxepression of the Xiuqing Guo, Mark O Goodarzi, Kent D Taylor, Cedars-Sinai Med Cntr, Los Angeles, CA;
Na-dependent B0AT1 at the kidney level during HS intake in adult SHR reveals their inability Manuel J Quinones, Willa A Hsueh, Univ of California Los Angeles, Los Angeles, CA;
for adequate sodium handling when hypertension is well established. Supported by grant Thomas A Buchanan, Univ of Southern California, Los Angeles, CA; Jerome I Rotter;
POCTI/SAU-OBS/57916/2004. Cedars-Sinai Med Cntr, Los Angeles, CA

Salt sensitivity (SS), the blood pressure change in response to changes in salt/water
P83 homeostasis, has been a variable of interest. Blood pressure (BP) and insulin resistance (IR) are
Nicotine: The Link between Cigarette Smoking and the Progression of under common genetic control. Whether SS is genetic and its relation with IR, however, have
Renal Injury? not been well studied. We assessed the genetic nature of SS and genetic relationship with IR
in 371 non-diabetic offspring from 116 Hispanic families, ascertained through a parent with
Edgar A Jaimes, Run-Xia Tian, Jessica Nigro, Leopoldo Raij; VA Med Cntr and Univ of hypertension. SS was measured by the Weinberger protocol with SBP sensitivity (SBP SS) and
Miami, Miami, FL DBP sensitivity (DBP SS) defined as the blood pressures between end of the salt loading
(day 1) and end of salt depletion (day 2). Three additional phenotypes were derived: blood
Cigarette smoke (CS) is the most important cause of preventable morbidity and mortality in the pressure between baseline and end of salt loading (SBP load, DBP load), blood pressure
U.S. Recent clinical studies have suggested that in addition of being a major cardiovascular risk between end of salt loading and baseline of salt depletion (afternoon overnight) (SBP night,
factor, CS promotes the progression of kidney disease. The mechanisms by which CS promotes DBP night), and blood pressure beween onset and end of salt depletion. Insulin sensitivity
the progression of chronic kidney disease have not been elucidated. The studies reported (SI) was measured by hyperinsulinemic euglycemic clamp. Heritabilities (h2) of these traits were
herein were designed to: a) determine whether mesangial cells poses nicotine receptors estimated and phenotypic, genetic, and environmental correlations between SS traits and SI
(nAChRs) and b) determine whether nicotine through activation of these receptors promotes were examined when h2 was significant. Variance component analysis was used, adjusting for
mesangial cell proliferation and extracellular matrix production via reactive oxygen species age, gender and BMI. Results: Several SS traits were significantly heritable (p0.05), including
(ROS). Here we demonstrate for the first time, that human mesangial cells (MC) are endowed SBP SS, SBP load, SBP night, and DBP night (h2 18%, 19%, 19% and 33%
with the nAChRs 4, 5, 7, 2, 3, 4 and 5 as assessed by western blot. Studies respectively), as was SI (h2 58%). Familial phenotypic correlations between the SS traits and
performed in other cell types have shown that these nAChRs are ionotropic receptors that SI were -0.06 (p 0.38) for SBP SS, -0.09 (p 0.21) for SBP load, -0.16 (p 0.005)
function as agonist-regulated Ca2 channels. At 10 -7 molar, a concentration found in plasma for SBP night, and -0.12 (p 0.048) for DBP night. When familial correlations were
of cigarette smokers, nicotine induced MC proliferation as assessed by 3H-thymidine partitioned, genetic correlations between SBP night, DBP night and SI were significant (rg
incorporation: Control 931 55 cpm vs nicotine 1318 82 cpm (N3, P 0.05) and -0.56, p0.03 for SBP night, rg -0.43, p0.04 for DBP night), while the environ-
increased synthesis of fibronectin, a critical matrix component involved in the progression of mental correlations were not. Conclusion: These results suggest overnight drop in BP following
chronic kidney disease, as assessed by western blot : Control 36 7 relative light units (RLU) salt loading is a better genetic phenotype than traditional salt sensitivity phenotypes. It also
e62 Hypertension Vol 48, No 4 October 2006

appears that this newly derived trait shares genetic determinants with insulin resistance. The 0.005 0.036), accounting for less than 1% of the total variance of systolic BP (SBP) at rest and
mechanism underlying this genetic variation and covariation warrant further study. during stress. On the contrary, the best multilocus models revealed multiple independent
genetic modification effects (gene-gene, gene-gender and gene-BMI, p value range: 0.003
0.009), accounting for 2.5% and 7.3% of the total variance for SBP levels at rest and during
P86 stress, respectively. As such, we speculate that the RAAS genetic modifications may contribute
Identical by Descent Mapping of Hypertension Genes in SHR more significantly to the dynamic BP regulation in response to behavioral stress compared to
the static BP value. Given the complexity of BP control, our data support the hypothesis that
Renata I Dmitrieva, Cruz A Hinojos, Eric Boerwinkle, Megan Grove, Myriam Fornage, Peter multiple RAAS genetic modifications underlie BP elevation. In addition, MARS could be a viable
Doris; Univ of Texas HSC Houston, Houston, TX model to test for the multiple genetic contributions to hypertension.

The SHR model of essential hypertension is inbred and was produced by selective breeding.
During inbreeding, it is common to create multiple lines in case fixation of deleterious alleles P89
causes loss of a line. In fact, SHR is a collection of several inbred lines that can be crossed Transcriptional Regulation of Angiotensinogen Haplotypes in Three
without segregation of the hypertensive trait, indicating extensive or complete sharing of Angiotensinogen-Expressing Human Cell Lines
hypertension alleles among SHR lines. Heritable traits are created by underlying genetic
variation that can affect both coding and regulatory sequence. The former can be localized only Matthew E Dickson, Curt D Sigmund; Univ of Iowa, Iowa City, IA
imprecisely via mapping studies. However, comprehensive gene expression arrays provide a
platform to identify genes that may harbor regulatory variation. We sought to identify Numerous association studies have correlated single nucleotide polymorphisms (SNPs) in the
hypertension alleles in SHR that act via altered expression. We have measured renal gene angiotensinogen (AGT) promoter with hypertension, and a few studies have reported differential
expression using Affymetrix arrays in SHR-A3, SHR-B2, SHR-C and WKY (Heid) animals. We transcription factor binding to individual SNPs. Most of these studies have been performed in
have identified and confirmed those genes and ESTs that are persistently differentially cultured hepatocyte cell lines, thus ignoring several other important sites of AGT synthesis. In
expressed between WKY and all SHR lines. We have screened these genes for polymorphism addition, AGT promoter polymorphisms are inherited in haplotype blocks (17 SNPs), of which
to identify allelic variation and have identified allelic variants that are inherited identical by there has been limited analysis. Analysis of naturally occurring haplotype blocks is an important
descent (IBD) by SHR-A3, -B2, -C and SHR/N, but not by WKY(Heid) and WKY/N. We then first step in determining which SNPs differentially regulate AGT expression. We cloned 1.3
generated an F2 cross between SHR-A3 and WKY (Heid) and determined which of these genes kb of the AGT promoter from 11 individuals of ethnically diverse background obtained from the
and ESTs exhibit allelic expression (AE) in F2 individuals. We found evidence of AE for Bhmt, Coriell Polymorphism Discovery Resource. An analysis of 21 chromosomal clones revealed 8
Ddt, Ela1, Gsto1, Gsst2, Ptprj, AA866432, AA892388 and AA893147. We measured the effect non-redundant haplotypes that appeared to accurately represent known haplotypes. Luciferase
of the SHR alleles of these genes by contrasting the blood pressure (radiotelemetry) differences assays were performed with these 8 haplotypes in 3 human cell lines of diverse origin, each
between F2 animals homozygous for WKY and SHR alleles of these strains and found a net expressing AGT endogenously (HepG2 derived from the liver, HK-2 derived from the renal
effect of homozygosity for the SHR alleles of 24.7mmHg, or 31% of the trait variance between proximal tubule, and CCF derived from astrocytes). Transcriptional assays were performed at
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017

the strains. This effect arose principally from the 5 genes underlined above. In contrast, the baseline ( the AGT enhancer) and following hormonal stimulation (100 nM dexamethasone,
cumulative effect on blood pressure of differentially expressed genes in which all SHR lines 50 nM testosterone, or 10 nM estradiol). Baseline reporter expression varied 23 fold between
share the same allele, but in which differential expression is not attributable to AE was minus haplotypes within each cell line and was very similar in HepG2 and HK-2 with the most
5.9mmHg. These 5 genes include plausible blood pressure genes that are IBD in SHR, act via significant differences resulting from the -20C/A SNP. The expression pattern in CCF was
allelic expression in all SHR lines and are associated with elevated blood pressure. dissimilar. Gel shift and supershift assays indicated preferential binding of the USF1/2
transcription factors to -20C, especially in the presence of -6A, in all three cell lines. The AGT
enhancer altered baseline expression in HK-2 cells but had minor effects in the other cells.
P87 Hormone treatments did not differentially regulate expression of haplotypes in any of the cell
lines, but treatment with dexamethasone or estradiol caused a haplotype-independent 1.9- or
Substitution of Chromosome 17 Influences Phenotypes Related to the
1.6-fold increase, respectively, in CCF cells. Our data suggest a major effect of the -20 SNP
Metabolic Syndrome in the Lyon Hypertensive Rat that may be modified by other SNPs, in particular -6. Some of the transcriptional effects may
also be cell-specific.
Anne E Kwitek, Med College of Wisconsin, Milwaukee, WI; Sophie Gilibert, Faculte de
Pharmacie - Universite Lyon, Lyon, France; Howard J Jacob, Med College of Wisconsin,
Milwaukee, WI; Jean Sassard, Alain Bataillard; Faculte de Pharmacie-Universite Lyon, Lyon, P90
France Association of the C-344T Aldosterone Synthase Gene Variant with
Essential Hypertension and Related Phenotypes: A Meta-Analysis
The Lyon Hypertensive (LH) rat strain is a genetically hypertensive strain associated with high
blood pressure (BP), exaggerated salt-sensitivity, and a metabolic syndrome made of Silvia C Sookoian, Tomas Fernandez Gianotti, Instituto de Investigaciones Medicas, A.
overweight together with increased plasma lipids. Genetic mapping in a cross between the LH Lanari. Universidad de Buenos Aires, Ciudad Autonoma de Buenos Aires, Argentina; Claudio
and the Lyon Normotensive (LN) strains previously showed the existenceof three clusters of Gonzalez, Departamento de Farmacologia, Facultad de Medicina, Universidad de Buenos
Quantitative Traits Loci (QTLs) related to the metabolic syndrome on rat chromosome 17. The Aires, Ciudad Autonoma de Buenos Aires, Argentina; Carlos J Pirola; Instituto de
first cluster was linked to morphological parameters, the second influenced blood pressure and Investigaciones Medicas, A. Lanari. Universidad de Buenos Aires, Ciudad Autonoma de
the third was linked to plasma lipid levels. In order to determine the functional role of these Buenos Aires, Argentina
QTLs we generated a consomic strain LH-17BN in which the LH chromosome 17 has been fully
substituted by a normotensive Brown Norway (BN) one. Male LH-17BN, LH, and BN rats were The renin-angiotensin-aldosterone system is an important regulator of blood pressure. Variants
phenotypically characterized. Measures included radio telemetric measurements of BP during in their component-encoding genes have been associated with essential hypertension and
normal and elevated salt intake (1% and then 2% in the drinking water) as well as the hypertrophic cardiomyopathy. Among them the CYP11B2 gene (CYP11B2) encoding aldoste-
determination of renal (creatinine clearance, proteinuria), metabolic (plasma triglycerides, rone synthase has been extensively studied but its role in hypertension and related phenotypes
cholesterol) and morphological parameters. Compared to parental LH rats, the LH-17BN rats remains controversial. Then, we performed a systematically review of the literature by means
exhibited significant decreases in both body weight and BP and its response to salt load. of a meta-analysis to evaluate the influence of the CYP11B2 C-344T polymorphism on the
Creatinine clearance was increased and proteinuria decreased. Finally plasma triglycerides and occurrence of arterial hypertension and related phenotypes. From 485 reports, we included 42
cholesterol were reduced in LH-17BN. Substitution of only BN chromosome 17 completely observational studies, case control and cohort at baseline. A fixed-effect model was used to
normalized triglycerides to the level of the BN parent. The present work confirms that the three pool data from individual studies. From 4741 essential hypertensive and 5444 control subjects,
clusters of QTLs found by a linkage analysis in the LH x LN strains can be recapitualted in the we found a significant association between hypertension and the C-344T variant showing a
consomic LH-17BN strain. Therefore, these chromosome 17 consomic rats will be useful to protective effect of the C allele (OR: 0.819, 95 % CI: 0.743 to 0.902, p 0.0001,n: 10185).
generate overlapping congenic rats in order to approach the genes possibly involved in the BP Besides, homozygous CC subjects had lower plasma renin activity (D: -0.109, 95 % CI: -0.213
and metabolic alterations shown by the Lyon model. Furthermore, the critical QTL region can to -0.006, p0.039, n: 1876) and posterior wall thickness (D: -0.151, 95 % CI: -0.265 to
be significantly narrowed by SNP haplotype analysis using all three strains. -0.037, p 0.01, n: 1235). No significant association was found for systolic arterial blood
pressure (D: -0.020, 95 % CI: -0.072 to 0.032, p0.44, n: 7432), diastolic arterial blood
pressure (D: -0.023, 95 % CI: -0.075 to 0.028, p0.37, n: 7432) and plasma aldosterone (D:
P88 0.016, 95 % CI: -0.085 to 0.117, p0.755, n: 1678). Although left ventricular mass (LVM) was
Comprehensive Analyses of RAAS Polymorphisms on Blood Pressure at not different in total combined studies (p 0.078, n: 1746), it was lower in Caucasians
Rest and during Behavioral Stress in Normotensive Youth homozygous CC (D: -0.106, 95 % CI: -0.207 to -0.006, p 0.04, n: 1591). In conclusion,
homozygous individuals for the -344C CYP11B2 variant are at 20% lower risk of developing
Haidong Zhu, Dongliang Ge, Ying Huang, Frank A Treiber, Gregory A Harshfield, Harold hypertension and may have decreased LVM.
Snieder, Yanbin Dong; Med College of Georgia, Augusta, GA

The renin-angiotensin-aldosterone system (RAAS) is a proteolytic cascade that regulates and P91
maintains blood pressure (BP). This study aimed to explore the interactive and integrative Various Inversions, Deletions, and Reinsertions on Chromosome 12p in
effects of multiple RAAS polymorphisms on BP at rest and during behavioral stress in a Autosomal-Dominant Hypertension with Brachydactyly - A Fish-Study in
normotensive population. A total of 920 young European- (EA) and African American (AA) twins Four Families and One Sporadic Case
(age: 12.0 30.0 yr, 45% AAs) were subjected to three 10-minute stress tasks (a social
competence interview, a virtual reality car driving simulation test and a video game). Thirteen Martin Kann, Sylvia Bahring, Max-Delbruck-Cntr for Molecular Medicine, Charite - Univ
potential functional polymorphisms from genes encoding angiotensinogen (AGT), angiotensin I Medicine Berlin, HELIOS-Klinikum Berlin-Buch, Berlin, Germany; Michaela Kirsch, Anita
converting enzyme (ACE), angiotensin II receptor type 1(AGTR1), and aldosterone synthase Rauch, Institute of Human Genetics, Friedrich-Alexander-Univ Erlangen-Nuremberg,
(CYP11B2) were genotyped. We performed multilocus prediction allowing for genetic modifi- Erlangen, Germany; Friedrich C Luft; Max-Delbruck-Cntr for Molecular Medicine, Charite -
cation effects [gene-gene, gene-gender, gene-ethnicity, and gene-body mass index (BMI)] Univ Medicine Berlin, HELIOS-Klinikum Berlin-Buch, Berlin, Germany
using multivariate adaptive regression splines (MARS) and generalized estimating equations
(GEE). Final multivariate models were selected by cross-validation. Single polymorphism We described a Turkish family with autosomal-dominant, salt-insensitive primary hypertension,
analyses showed modest effects of M235T (AGT) and A-239T (ACE) (p value range: and type E brachydactyly. Affected persons die of stroke 50 years of age. Genome-wide
CHBPR ConferencePoster Presentations e63
linkage analysis disclosed a 10 cM locus on chromosome 12p11.2 (LOD score, 9.29). We P94
sequenced all known genes within the candidate region, but found no mutations. However, Heart-Specific Inhibition of Protooncogene C-myc Attenuates Cold-Induced
interphase fluorescence-in-situ-hybridization (iFISH) studies, using a five-clone BAC array Cardiac Hypertrophy
spread across the locus, showed an inversion, deletion, and reinsertion mutation. We now
extended iFISH-analysis to three other families and one sporadic case (all non-Turkish), with Mahajoub Bello Roufai, Hua Li, Zhongjie Sun; Univ Fl Coll Med, Gainesville, FL
an array of 24 contiguous BAC clones. We confirmed rearrangements at chromosome 12p in
all studied patients. All affected persons had an inversion of the centromeric part of the locus. Background: The protooncogene c-myc is involved in the regulation of cell growth. Although
However, the inversions lacked a common breakpoint and differed greatly in size. In one increased c-Myc expression is found in hypertrophied hearts, the role of c-Myc in the
instance, the locus boundaries were extended. No disruptions of any known coding sequences development of cardiac hypertrophy has never been determined. The aim of this study was to
occurred. Finally, the sporadic case also had a partial deletion of the inverted sequence that test the effect of heart-specific inhibition of c-Myc expression on the development of
was reinserted in the telomeric part of the locus. The region affected by the rearrangements cold-induced cardiac hypertrophy (CICH), a natural form of cardiac hypertrophy. We hypothe-
does not contain any known genes but does include 5 ESTs that are differentially spliced. We sized that heart-specific inhibition of c-Myc expression attenuates CICH. Methods and
obtained full-length transcripts of these ESTs by RACE-PCR. These transcripts lack both open Results: We constructed c-Myc antisense (c-MycAS) plasmid and GFP plasmid driven by a
reading frames longer than 89 amino-acids and Kozak sequences. Expression analysis of the heart-specific promoter, -MHC. The cell culture study indicated that c-MycAS can selectively
transcripts in affected and unaffected individuals and different tissues, respectively, is inhibit c-Myc expression and that GFP can selectively express in the rat heart cells. Four groups
underway. In summary, these findings support a causative role of chromosome 12p of rats were used to test the effect of in vivo inhibition of cardiac c-Myc expression on the
rearrangements in the genesis of the hypertension-brachydactyly syndrome. Since no known development of CICH. Three groups received an intravenous injection of c-MycAS, GFP, and
genes are affected by the mutations, possible causes for the syndrome include as yet unknown buffer, respectively, before exposure to cold (44F), while the last group received buffer and
genes or non-coding RNA. Initial analysis of ESTs mapped to the rearrangements was was kept at room temperature (RT, 78F) to serve as a control. Blood pressure (BP) of the
inconclusive. A position effect must also be considered that could alter the interplay between cold-exposed groups receiving buffer or GFP increased significantly whereas BP of the
a regulatory element and its respective gene. We are currently investigating these possibilities. c-MycAS group did not increase until 28 days after exposure to cold. Thus, c-MycAS driven by
a heart-specific promoter delayed and attenuated cold-induced hypertension (CIH). The
antihypertensive effect of c-MycAS was probably due to the decreased cardiac output.
Magnetic resonance imaging showed that the in vivo left ventricle wall thickness was
decreased by c-MycAS at day 18 when cardiac c-Myc expression was inhibited by c-MycAS.
P92 Consistently, the cold-induced increase in heart weight was decreased by c-MycAS at day 18.
Reduced Capacity of Heme Oxygenese-1 Induction in Patients with The heart specificity of -MHC promoter was confirmed by the selective inhibition of c-Myc
Coronary Artery Disease expression in the heart and by the selective expression of both GFP mRNA and GFP protein in
the heart. Conclusion: Heart-specific inhibition of c-Myc expression attenuated the develop-
Andrei Brydun, Ryoji Ozono, Koichiro Okuhara, Fujiko Kohno, Tetsuya Oshima, Yuichiro
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017

ment of CIH and CICH. The increased c-Myc expression plays a key role in the pathogenesis
Watari, Kazuaki Chayama, Hiroshima Univ, Hiroshima, Japan; Kazuhiko Igarashi; Tohoku of CICH. Thus, heart-specific inhibition of c-Myc expression may provide a new and effective
Univ, Sendai, Japan approach for the control of cardiac hypertrophy.

Heme oxigenase-1 is a stress-inducible cytoprotective enzyme. There are some data


suggesting that the present of the long GT-repeat sequences (L-alleles) in the HO-1 promoter P95
region may be associated with susceptibility to the coronary artery disease (CAD). However the
results of the previous studies are controversial and HO-1 mRNA expression has never been
Genetic and Transcriptomic Analysis of Chromosome 1 Blood Pressure QTL
studied in humans. In the present study, we tested the hypothesis that reduced HO-1 in the Spontaneously Hypertensive Rat
expression may impair defense against oxidative stress, leading to progression of CAD. We
evaluated the basal and hemin-stimulated HO-1 expression and GT-repeat polymorphism in Fadi J Charchar, Jenny Clemitson, Richard J Dixon, Steve Haines, Laurence Hall, Univ of
peripheral blood mononuclear cells in the 60 patients with CAD and 30 control subjects, and Leicester, Leicester, United Kingdom; Andrew Bingham, Bhakti Patel, Univ of Liecester,
investigated the relationship between the HO-1 expression level and markers of oxidative stress Liecester, United Kingdom; Ming Lo, Jean Sassard, Departement de Physiologie et
and inflammation including plasma levels of 8-isoprostan, TNF-alpha, and IL-10. There was no Pharmacologie Clinique, Lyon, France; Nilesh J Samani; Univ of Leicester, Leicester, United
significant difference between the basal HO-1 expression levels of CAD and control groups, Kingdom
however stimulated HO-1 was significantly (p0.05) suppressed in CAD group compared with
control group. This difference was independent of age, sex, current smoking and the presence Introduction and Aims: Chromosome 1 in the rat contains a quantitative trait locus (QTL) with
of diabetes mellitus, hyperlipidemia, and hypertension in the multiple logistic regression model. a major effect on blood pressure (BP). We have previously confirmed the effect of this locus on
However, no significant correlation was found between HO-1 expression level, basal or BP by the production of reciprocal congenic strains (WKY.SHR-Sa) and (SHR.WKY-Sa) derived
stimulated, and the markers of oxidative stress and inflammation. Interestingly, measurement from a cross of the spontaneously hypertensive rat (SHR) with the Wistar-Kyoto rat (WKY).
of the stimulated HO-1 expression in the 16 patients before and after CABG showed no Furthermore, we constructed a congenic substrain (Sisa1) from the SHR.WKY-Sa strain with
significant difference, suggesting HO-1 upregulation capacity to be an intrinsic factor. LL only a 4.3 Mb introgressed region which also exhibited the BP effect. Our aims were to carry
genotype carriers showed lower HO-1 mRNA levels after the stimulation with hemin (p0.05). out further genetic dissection of this region and to identify strong positional candidate genes
However we did not observe prevalence of this genotype in the CAD group over the control through transcriptome analysis. Methods: We further fine mapped the QTL region by systemic
group. These results suggest that impaired ability to induce HO-1 may contribute to construction of two mutually exclusive congenic substrains. Genome-wide microarray expres-
development of CAD, although its roles in the control of oxidative stress and inflammation were sion profiling in whole kidney was undertaken to identify differentially expressed genes among
not detected. Measurement of the HO-1 mRNA upregulation capacity may better reflect its role the parental SHR, WKY, congenic strains (WKY.SHR-Sa), (SHR.WKY-Sa) and the congenic
in the pathogenesis of CAD than genotyping. substrain Sisa1, at both 6 and 24 weeks of age. Results and Conclusion: We have reduced the
congenic segment to 3Mb region by the construction of two strains (Sisa6000/Sisa9000) that
break down the Sisa1 region. Only Sisa6000 congenic exhibit reduced BP compared to the SHR
animals (166.1 2.5 mmHg, (n 17) vs 174.4 1.4 mmHg (n 20), P0.001). We have
identified 4 hypertension candidate genes (Spondin 1, Homer-2, Thumpd1 and XP 215009)
P93 that mapped within the defined boundaries of the initial BP QTLs on chromosome 1 and that
Regulation of Blood Pressure, Natriuresis, and Renal Thiazide/Amiloride exhibited significant differential expression between the WKY and SHR genotypes, at both 6 and
24 weeks of age. These differentially expressed genes were confirmed by performing
Sensitivity in PPAR-Null Mice quantitative RT-PCR. One of these genes, Spon1 maps within the boundaries of our new
minimal congenic region and has not been previously identified as a hypertension candidate
Patience Obih, Xavier Univ, New Orleans, LA; Adebayo Oyekan; Texas Southern Univ,
gene. The results of this study justify further investigation of these positional candidate genes
Houston, TX
and their potential involvement in BP control in hypertensive rat models and humans.
PPAR is a ligand-activated nuclear transcription factor that is expressed in the kidney and
many cardiovascular tissues and recent studies have linked defective fatty acid oxidation with
defective renal function and hypertension including salt-sensitive hypertension. However, salt P96
handling in these mice is not fully known. This study evaluated thiazide and amiloride Assessment of SNP-Based Genotypes in Two Independent Populations:
sensitivity and tested whether PPAR KO mice are hypertensive and if they are salt-sensitive. GRK4 A486V Association with Hypertension
PPAR KO and WT (129S1/Sv) mice were placed on a normal salt (NS, 0.3% NaCl) or high salt
(8% NaCl, HS) diet for 28 days and mean arterial pressure (MAP) and heart rate (HR) Theodore E Mifflin, Cynthia D Schoeffel, Daniel C Huntley, Univ of Virginia, Charlottesville,
determined (telemetry). Basal MAP and HR were similar in KO and WT mice (1166 mmHg, VA; Kelli K Ryckman, Scott M Williams, Vanderbilt Univ, Nashville, TN; Pedro A Jose,
58740 bpm, KO; 1164 mmHg, 55120 bpm, WT) placed on NS. HS diet increased MAP Georgetown Univ, Washington, DC; Christopher Rembold, Robert M Carey, Robin A Felder;
in WT mice (243 mmHg, p0.05) but not in KO mice. However, HS diet increased Univ of Virginia, Charlottesville, VA
proteinuria in KO mice much more than in WT mice (8- versus 2.5-fold, p0.05). We
hypothesize that ion transport differences might be important in determining rate of NaCl Background: We determined the contributions of individual gene(s) to the presence and
excretion (UNaV). In response to an acute NaCl-load test (2.5 ml/kg N/S i.p. for 5 hrs), PPAR development of essential hypertension (EH). We investigated 8 separate single nucleotide
KO mice excreted the sodium load faster than WT mice (4.311.11 vs 0.770.31 mol, polymorphisms (SNPs) and evaluated SNP genotypes (individual and combined) and haplotypes
p0.05). UNaV was compared in mice pretreated with amiloride (Amil; 200 g/kg) or for association with salt sensitivity (SS). A previous study searched for SNP genotype
hydrocholorothiazide (HCTZ; 3 mg/kg), in vivo measurements of sodium hydrogen exchanger correlations in hypertensive persons (HP). Our goal was to evaluate these combined populations
(NHE) or Na-Cl-cotransporter (NCC) activity, respectively. UNaV was blunted in HCTZ-treated KO (n122 252) for both genotype/haplotype results that could be related to EH. Methods: We
mice (335%, p0.05) mice but increased 15.5 fold in WT mice. In Amil-treated mice, sodium determined five SNP genotypes of genomic DNA from 122 HP study participants and eight SNP
load-induced increase in UNaV was blunted in KO compared with WT mice (3.3 versus genotypes from 252 SS study participants. We used standard linkage analysis software to
15.5-fold). These data suggest that PPAR is renoprotective and affects Na transport via NHE establish significance of genotypes (and inferred haplotypes) of the separate and combined
and NCC mechanisms. Thus, despite defective fatty acid oxidation and marked proteinuria, data sets. Results: In the HP study, GRK4 A486V was slightly associated with EH (p 0.044).
PPAR null mice are neither hypertensive nor develop salt-sensitive hypertension. For the Combined study, GRK4 A486V was significantly associated with EH (p0.0045).
e64 Hypertension Vol 48, No 4 October 2006

Multidimensional reduction analysis revealed no associations between the eight SNPs within were investigated in stained cardiac sections under microscopy. (i) Left ventricular fractional
the individual (SS or HP) studies or combined studies. For the HP study, the odds ratio (OR) for shortening (FS) was reduced in saline-treated MI rats (19% vs. pre-operative values of
EH and A486V, OR3.53 (CI 1.259.96, p0.0132). For the combined studies, odds ratio for 39 42%). This reduction was partially reversed by administration of 25 ng/kg/h OT (FS-33%)
EH and A486V, OR1.92 (CI 1.173.16, p0.0092). Conclusion: SNP genotype analysis of or 125 ng/kg/h (FS-31%). OT also improved ejection fraction and cardiac output. (ii)
374 persons indicates that GRK4 A486V is significantly associated with EH. A study of Immunohistochemical (IMM) and morpho-pathological analyses indicated OT-mediated in-
pseudo-haplotypes collectively created using the 8 SNP genotype results revealed none were crease in proliferating cells in the infarct zone (PCNA staining), augmented neovascularisation
associated with EH. (smooth muscle actin coloration) and reduced apoptosis (caspase-3 staining). (iii) OT treatment
resulted in reduction of neutrophils from 7% of total cell number in the infracted cardiac site
of saline-treated MI rats to 2% and 1.5% in MI rats treated at 25 and 125 ng/kg/h, respectively.
P97 OT treatment markedly decreased (40%) the number of macrophages in the infract zone
Angiotensinogen Gene Variants do not Affect Ambulatory Blood Pressure in compared to saline-treated MI (considered as 100%). (iv) IMM revealed increased staining of
the General Population ANP and eNOS in cells localized in the scar zone in response to OT treatment. Real-time PCR
showed OT-dependent reduction of a proinflammatory cytokine TNF mRNA elevated in left
Peter Braund, Martin Tobin, Gail Lavery, Claire Bodycote, Stuart Raleigh, Fadi Charchar, ventricular dysfunction. Therefore, OT treatment reduces inflammation and stimulates angio-
Paul Burton, Nilesh J Samani; Univ of Leicester, Leicester, United Kingdom genesis and improves function of the injured heart. In conclusion, OT improves the healing
process in experimentally-induced MI in rats. Supported by CIHR and CHSF
Background Over the last ten years several variants in the angiotensinogen (AGT) gene and its
promoter have been associated with risk of hypertension. However, the data are conflicting. An
important confounder could be the level of salt intake. We hypothesised that if AGT variants P100
influence risk of hypertension, they should have a discernible effect on blood pressure (BP), Peroxisome Proliferator-Activated Receptor Activator Attenuates Left
assessed as a quantitative trait, in the general population, especially if salt intake is also taken Ventricular Hypertrophy in Salt Sensitive Hypertension
into account. We analysed the effect of 8 AGT gene polymorphisms on 24 hour BP measured
by an ambulatory approach in 1533 subjects in whom 24 hour sodium intake was also Minori Nakamoto, Yusuke Ohya, Shuichi Takishita; Dept of Cardiovascular Medicine,
assessed. Methods and Results The 1533 subjects comprised parents (aged 40 59 years) Nephrology and Neurology, Univ of the Ryukyus, Okinawa, Japan
and adult offspring (18 y) from 386 families recruited from the general population as part of
the ongoing GRAPHIC (Genetic Regulation of Arterial Pressure of Humans in the Community) In salt-sensitive hypertension, importance of volume overload and local renin-angiotensin
study. Ambulatory BP was measured using a Spacelabs 90207 monitor. Sodium intake was system on the pathogenesis of left ventricular (LV) hypertrophy and heart failure has been
estimated from 24 hour urinary sodium excretion. The AGT single nucleotide polymorphisms demonstrated, however little is known about the role of peroxisome proliferator-activated
(SNPs) genotyped included 4 SNPs in the promoter region (rs5046, rs5049, rs5050, rs5051), receptor (PPAR) on these LV changes. We thus investigated whether pioglitazone, a PPAR
1 SNP in intron 1 (rs2004776), 1 SNP in exon 2 (rs4762), 1 SNP in intron 3 (rs11122575) and
Downloaded from http://hyper.ahajournals.org/ by guest on May 19, 2017

activator, has beneficial effects on LV hypertrophy in Dahl salt-sensitive (DS) rats, a model for
1 SNP in exon 5 (rs7079). The SNPs were chosen to provide a full coverage of the gene at a salt-sensitive hypertension, and compared the effects of pioglitazone to those of angiotensin II
D of 0.8 and to include all SNPs with functional effects or associated with hypertension. receptor blocker, candsartan. DS rats were fed either low salt (0.3 %: DSL) or high salt (8 %:
Genotyping was carried using Taqman assays. All analyses took account of familial DSH) diet from 6 weeks to 16 weeks. Pioglitazone (10 mg/kg per day) or candesartan (4 mg/kg
relationships and were adjusted for age, age2 and sex as covariates. The minor allele per day) was administrated with high salt. At 16 weeks of age, LV geometry, cardiac fibrosis,
frequencies for the SNPs ranged from 8.7% (rs11122575) to 40.2% (rs 5051). None of the gene expression of collagen, connective tissue growth factor (CTGF) and transforming growth
SNPs showed a significant association with either ambulatory SBP or DBP analysed either for factor-1 (TGF-1), and matrix metalloproteinase (MMP) activities were evaluated by
the full 24 hours or for day-time and night-time periods. For 24h SBP the 95% confidence echocardiography, histochemistory, real-time PCR, and zymography, respectively. Blood
intervals for the per allele BP effect excluded by the study ranged from -0.67 to 0.91 mmHg pressure (BP) was elevated, heart weight was increased, and LV wall thickness was increased
for rs5051 to -1.61 to 1.19 mmHg for rs11122575. No significant interactions between salt in DSH (2134 mmHg) compared to DSL (1451 mmHg). Candesartan and pioglitazone
intake and AGT SNPs were observed. Conclusion AGT gene variants do not significantly decreased BP in DSH (1813 mmHg and 1914 mmHg, respectively). The LV geometrical
influence ambulatory BP phenotypes in the general population. Our findings do not support the changes and interstitial and perivascular fibrosis, as well as myocardial hypertrophy in DSH
concept that variants in the AGT gene are a risk factor for hypertension. were recovered to the level of DSL by administration of candesartan or pioglitazone. Gene
expressions of TGF-1, collagen type I, collagen type III, and CTGF were significantly increased
in DSH. Candesartan decreased the expressions of all genes, but pioglitazone did those of
P98 TGF-1 and collagen type I. MMP activity was increased in DSH, but the correction of MMP
TRPV1 Gene Knockout Abolishes Preconditioning Protection Against activity by the two drugs was limited. These results suggest that pioglitazone reduced LV
Myocardial Injury in Isolated Perfused Hearts in Mice hypertrophy and fibrosis in salt-sensitive hypertension, as did candesartan. The mechanism for
the beneficial effects may be different between the two drugs, however decrease in BP and
Beihua Zhong, Donna H Wang; Michigan State Univ, East Lansing, MI decrease in collagen synthesis may be commonly involved.

We have shown that the transient receptor potential vanilloid type 1 (TRPV1) channels play an
important role in protecting the heart from ischemia /reperfusion (I/R) injury via an increase in P101
substance P (SP) release (Circulation 112(23):36173623, 2005). However, it is unknown Highly Increased Circulating Endothelial Cells in Women with Preeclampsia
whether TRPV1 is involved in preconditioning (PC)-induced protection of hearts. To test the
hypothesis that TRPV1 expressed in sensory nerves innervating the heart plays a role in Magdalena Grundmann, Alexander Woywodt, Bettina Hollwitz, Torsten Kirsch, Uta
PC-induced protection against myocardial injury and the lack of TRPV1 eliminates such Erdbruegger, Katrin Oehler, Hermann Haller, Marion Haubitz; Med Sch Hannover, Hannover,
protection, hearts of gene-targeted TRPV1-null mutant (TRPV1-/-) or wild-type (WT) mice were Germany
Langendorffly perfused in the presence or absence of capsazepine (CAPZ), a selective TRPV1
antagonist, CGRP8 37, a selective calcitonin gene-related peptide (CGRP) receptor antagonist, or Objective: Preeclampsia is primarily a disorder of the maternal endothelium resulting in
RP67580, a selective NK1 receptor antagonist when hearts were subjected to three 5-mins of endothelial dysfunction and injury. However, the degree of endothelial damage is difficult to
ischemia PC followed by 30 mins of global ischemia and 40 minutes of reperfusion. After I/R, quantify. As circulating endothelial cells (CEC) have been shown to be a marker of endothelial
increased left ventricular end-diastolic pressure (LVEDP) and decreased left ventricular injury in patients with different diseases involving the endothelial layer like systemic vasculitis
developed pressure (LVDP), coronary flow (CF), and LV peak positive dP/dt (dP/dt) occurred and thrombotic microangiopathy, we tested the hypothesis that CEC in patients with
in WT, TRPV1-/-, or TRPV1-/- PC compared to WTPC (n8, p0.05). CAPZ, CGRP8 37, or preeclampsia are elevated and reflect endothelial cell damage. Methods: Circulating endothelial
RP67580 abolished PC induced protection in WT but not TRPV1-/- hearts (p0.05). PC cells were measured in 8 patients with preeclampsia and 8 normotensive women before
decreased lactate dehydrogenase (LDH) release from WT but not TRPV1-/- hearts (p0.05). delivery, 1 day after delivery and 3 - 5 days later. Enumeration of CEC was performed with
Radioimmunoassay showed that the release of SP and CGRP after PC was higher in WT hearts anti-CD 146-driven immunomagnetic isolation and subsequent staining with Ulex Europaeus
than in KO hearts (p0.05), which was attenuated by CAPZ in WT but not KO hearts. Our data lectin 1. Results: Women with normal pregnancies had slightly elevated CEC (median 18
show that TRPV1 gene deletion abolishes PC induced protection of the heart, indicating TRPV1 cells/ml; p0.05) compared to healthy non-pregnant women (median 8 cells/ml). However,
plays a key role mediating the beneficial effects of preconditioning against I/R injury through women with preeclampsia showed highly elevated numbers of CEC (median 100 cells/ml;
release SP and CGRP. range 48 1200; p0.001). Cell numbers declined rapidly after delivery and after 35 days
before discharge both groups had comparable numbers of CEC (women with preeclampsia 28
cells/ml compared to 26 cells/ml). Conclusion: In women with preeclampsia CEC were highly
P99 elevated compared to healthy pregnant women and comparable to CEC in systemic vasculitis.
Oxytocin Improves Cardiac Healing in Experimentally-Induced Myocardial Our findings demonstrate the severity of endothelial injury in preeclampsia. Numbers seem to
Infarction in Rats correlate with the severity of the disease. After delivery CEC in women with preeclampsia
declined rapidly and 35 days later values were comparable to those of women without
Jolanta Gutkowska, Vickram Bissonauth, CHUM, Hotel-Dieu, Montreal, Canada; Rame Taha, preeclampsia, indicating a rapid repair of the endothelial damage in preeclampsia.
Gilbert Blaise, CHUM, Notre Dame, Montreal, Canada; Marek Jankowski; CHUM, Hotel-Dieu,
Montreal, Canada
P102
Oxytocin (OT), a reproductive hormone also plays a role in cardiovascular regulation, by Time Course of Circulating Endothelial Cells in Patients with a Stroke
mechanisms that include stimulation of the cardioprotective mediators, NO and atrial natriuretic
peptide (ANP). OT also stimulates stem cell differentiation into cardiomyocytes. Based on these Marion Haubitz, Stefan Gerdes, Alexander Woywodt, Torsten Kirsch, Uta Erdbruegger,
findings, we hypothesized that OT treatment could improve cardiac function in rats subjected Hermann Haller, Karin Weissenborn; Med Sch Hannover, Hannover, Germany
to experimentally-induced myocardial infarction (MI). An infarct in the left ventricle (LV) of
Sprague-Dawley male rats was generated by left coronary artery ligation. OT was then Objective: Circulating endothelial cells (CEC) have been shown to be a marker of endothelial
administered by ALZET pump at a concentration of 25 or 125 ng/kg/h during 3 or 7 days. damage. Elevated levels of CEC could be demonstrated in patients with vascular diseases such
Saline-treated MI and sham-operated rats served as controls. Cardiac function was analyzed as systemic vasculitis or thrombotic microangiopathy. We now tested the hypothesis that acute
by M-mode echocardiography. The infarct size, cell proliferation, apoptosis, and immune cells ischemic stroke is associated with endothelial injury and analyzed CEC at different time points
CHBPR ConferencePoster Presentations e65
after stroke. Methods: Circulating endothelial cells were measured in 38 patients with the P105
clincical diagnosis of stroke at admission and 1, 7, 14, 21 and 90 days after the event. 20 Duration of Breastfeeding and Aortic Pulse Wave Velocity in Young
patients had an ischemic stroke of the middle cerebral artery, 5 a lacunary infarct and 13 a Children: The Manchester Childrens Cardiovascular Health Study
thrombembolic event. Enumeration of CEC was performed with anti-CD 146-driven immuno-
magnet