Proteomics 2007, 7, 4555–4564 DOI 10.1002/pmic.

200700503 4555

RESEARCH ARTICLE

Proteomic identification of tyrosine nitration targets in

kidney of spontaneously hypertensive rats

Raymond Tyther1, Ahmad Ahmeda2, Edward Johns2 and David Sheehan1

1
Proteomics Research Group, Department of Biochemistry, University College Cork, Ireland
2
Department of Physiology, University College Cork, Ireland

Nitrosative and oxidative stress are implicated in the development of hypertension. Events in the Received: May 26, 2007
renal medulla may play a key role in the development and progression of hypertension. This may Revised: August 17, 2007
arise through disruption of nitric oxide signalling in the medulla and be accompanied by Accepted: September 3, 2007
enhanced nitrosative and oxidative stress as indicated by the presence of proteins containing 3-
nitrotyrosine. Here we demonstrate enhanced protein nitration in the medulla of spontaneously
hypertensive rats. We have identified several nitrated proteins with both varied subcellular loca-
tion and functional roles. These proteins are involved in nitric oxide signalling, antioxidant
defense and energy metabolism. Moreover, increased nitration was observed in conjunction with
enhanced oxidative damage as evidenced by the presence of protein carbonyl oxidative stress
biomarkers. Our results suggest that kidney medulla is subject to enhanced nitrosative and oxi-
dative stress, and that resulting protein modifications may contribute to the progression of
hypertension.

Keywords:
Carbonylation / Hypertension / Kidney / 3-Nitrotyrosine / Rat

1 Introduction variety of sources including NAD(P)H-oxidase [7], leakage

Renal dysfunction is crucial to onset of hypertension [1]. The
spontaneously hypertensive rat (SHR) [2] and other models
have revealed ROS as contributors to kidney pathophysiology
[3, 4]. Nitric oxide (NO) signalling, essential for vascular and
renal function [5], appears to be disrupted by ROS in some
examples of kidney dysfunction [5, 6]. ROS such as super-
oxide (O22) and hydrogen peroxide (H2O2), arise from a
Correspondence: Dr. David Sheehan, Proteomics Research
Group, Department of Biochemistry, University College Cork,
Lee Maltings, Prospect Row, Mardyke, Cork, Ireland
E-mail: d.sheehan@ucc.ie
Fax: 1353-21-4274034
Abbreviations: ADMA, asymmetric dimethylarginine; AKR, aldo-
keto-reductases; CA II, carbonic anhydrase II; DDAH1, dimethylar-
ginine dimethylaminohydrolase 1; DNPH, 2,4-dinitrophenylhy-
drazine; HAOX, hydroxyacid oxidase; NOS, nitric oxide
synthase; 3NT, 3-nitrotyrosine; OAT, ornithine aminotransferase;
RNS, reactive nitrogen species; SHR, spontaneously hyperten-
sive rat
from mitochondrial electron transport [8] and nitric oxide
synthase (NOS) activity [9].
Oxidative stress can generate nitrosative stress by reac-
tion of ROS with NO producing reactive nitrogen species
(RNS): (ONOO2); nitrosoperoxycarbonate (ONO2CO22);
nitrogen dioxide radical (NO2); N2O3. Mechanisms of RNS
formation are diverse and are influenced by local factors
such as the presence of CO2 and heme proteins [10]. The
proportion of nitration due to ONOO2 and its protonated
form, peroxynitrous acid (ONOOH), is not yet known be-
cause nitration also occurs through oxidation of nitrite to
nitrogen dioxide by myeloperoxidase and other enzymes [11–
13] and nonenzymatic acidification of nitrite [14]. Modifica-
tion of tyrosine to 3-nitrotyrosine (3NT) is a key nitration
biomarker associated with Alzheimer’s disease and diabetes
[15–17]. Some nitrated proteins show increased turnover
[18], but others accumulate [19]. Interestingly, some anti-
oxidant enzymes including manganese-superoxide dis-
mutase (Mn-SOD) [20] and catalase [21] are themselves
nitration targets, perhaps further compromising ROS/RNS
protection.

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4556 R. Tyther et al.
Renal medulla and cortex fulfil distinct roles. In the cor-
tex, which is well perfused and oxygenated, fluids/electro-
lytes are filtered from proteins at the glomerulus, glucose,
water and electrolytes are reabsorbed along the nephron and
regulatory hormones are produced. The principal function of
the medulla is to concentrate urine. In view of the dynamics
of 3NT formation, the kidney presents an interesting model
system because of the dichotomy between a well-perfused
and oxygenated cortex, and a medulla on the threshold of
anoxia [22]. Medulla cells are adapted to function in this
hypoxic milieu, but O22 may perturb this situation causing
dysfunction [23]. These contrasts extend to NO signalling/
metabolism: NO and nitrite/nitrate levels of medulla exceed
those of cortex [24] as does NOS expression [25]. Kidney
nitrosative stress is also associated with hypertension and
induction of hypertension via angiotensin II is accompanied
by an increase in 3NT [26]. The cortex and medulla pro-
teomes in normotensive rats are quite similar [27], but activ-
ities/abundance of key antioxidant enzymes like SOD, cata-
lase and glutathione peroxidase differ between the two kid-
ney components in normotensive rats [28] and SHR [29].
Oxidative/nitrosative stress may be differentially regulated in
the medulla and cortex [30]. Development of hypertension in
SHR is accompanied by proteomic changes in myocardial
tissue [31], but it is not known if kidney protein expression
profiles are similarly altered.
In this study, protein nitration in kidneys from hyper-
tensive SHR and age-matched normotensive Wistars was
studied. The relative amount of protein nitration in cortex
and medulla was also investigated and we have identified
several protein targets of tyrosine nitration; potential bio-
markers for nitrosative stress. Characterisation of the ‘nitro-
proteome’ of SHR kidney may provide insights into the
pathogenesis and progression of hypertension.
2 Materials and methods
2.1 Animals and tissue preparation
Rats were obtained from Harlan (UK) and maintained in
the Biological Services Unit (University College Cork) for at
least 1 wk prior to use. Animals received regular laboratory
diet and tap water ad libitum. All procedures were per-
formed in accordance with National guidelines and the
European Community Directive 86/609/EC and approved
by the Animal Experimentation Ethical Committee of Uni-
versity College Cork. Male Wistar and SHR weighing 250–
300 g were anaesthetised with 0.75–1.0 mL of a chloralose/
urethane mixture (16.5/250 mg/mL, respectively). Kidneys
were exposed via retroperitoneal incision, quickly removed
and placed on ice. Cortex was dissected from medulla, and
tissues were weighed, diluted to 25% with homogenisation
buffer (250 mM HEPES, pH 7.7/1 mM EDTA/0.1 mM
neocuproine), and homogenised with a Polytron PCU2
Tissue Homogeniser (Kinematica, Switzerland). The
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics 2007, 7, 4555–4564
homogenate was centrifuged at 20 0006g at 47C for
20 min, and the supernatant collected (cytosolic fraction).
The pellet was washed three times in PBS, resuspended in
homogenisation buffer containing 1% C7BzO (Sigma,
Germany) and incubated on ice for 20 min with frequent
vortexing. It was then centrifuged for 10 min at 14 0006g
and the supernatant was collected (membrane fraction).
Protein concentrations were determined using the BioRad
protein assay (BioRad, Germany) and fractions stored at
–707C until use.
2.2 Protein preparation and electrophoresis methods
Protein fractions (100 mg) were rehydrated in buffer con-
taining 5 M urea, 2 M thiourea, 2% CHAPS, 4% carrier
ampholyte (Pharmalyte 3–10, Amersham-Pharmacia Bio-
tech, UK), 1% DeStreak reagent (Amersham-Pharmacia
Biotech) and a trace amount of bromophenol blue. Final
volumes of 125 mL were loaded on 7 cm pH 3–10 nonlinear
IPG strips (BioRad, CA, USA) and rehydrated overnight for
at least 15 h. IPG strips were focused on a Protean IEF Cell
(BioRad) with linear voltage increases: 250 V for 15 min;
4000 V for 2 h; then up to 20 000 V?h. Following IEF, strips
were equilibrated (20 min) in equilibration buffer (6 M urea,
0.375 M Tris, pH 8.8, 2% SDS, 20% glycerol) containing 2%
DTT, and then for 20 min in equilibration buffer containing
2.5% iodoacetamide. Equilibrated strips were electro-
phoresed on 12% SDS-PAGE gels at a constant voltage
(150 V) at 47C using an Atto AE-6450 mini PAGE system
(Atto, Japan). For analytical gels, loading was determined by
analyzing homogenate by 1-DE and staining using colloidal
CBB G250, and IEF and 2-DE were carried out as described
previously [32].
For 1-DE, protein fractions were diluted with sample
buffer, and electrophoresis was performed [33] using 12%
polyacrylamide gels on an Atto AE-6450 mini PAGE system
(Atto).
2.3 Detection of protein carbonyls
Protein carbonyls were detected using the 2,4-dinitro-
phenylhydrazine (DNPH) derivatisation method [34].
Briefly, 20 mg protein was derivatised with 10 mM DNPH in
10% TFA for 20 min with regular vortexing, and the reac-
tion was stopped by incubation with neutralisation buffer
(2 M Tris-base/30% glycerol) for 15 min. Samples were
combined with Laemmli buffer and 1-DE was performed as
described above.
Following 1-DE, proteins were transferred (100 mA per
blot, 55 min) to Protran NC (0.2 mM) membranes (Whatman
Germany) using an AE-6677 HorizBlot (Atto) and equivalent
protein loading was confirmed by staining with Ponceau S
(0.2%) in 5% acetic acid. Membranes were blocked for 1 h at
room temperature with 1% BSA in PBS containing 0.05%
Tween (PBST). Membranes were incubated overnight at 47C
with anti-DNP antibody (Dako Ref. V0401) at a 1/5000
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Proteomics 2007, 7, 4555–4564
dilution in PBST/1% BSA. Membranes were washed
2615 min with PBST before incubation for 1 h at room
temperature with goat antirabbit HRP antibody (Dako, Den-
mark) at 1/3000 dilution. Membranes were washed for
2615 min in PBST once more before carbonylated proteins
were detected on X-OMAT film (Sigma) using the Super-
Signal West Pico Chemiluminescence kit (Pierce). Blot im-
ages were acquired using an image scanner (GS-800 cali-
brated densitometer, BioRad) and protein carbonylation was
quantified by densitometric analysis using Quantity One
4.5.2 Analysis software (BioRad, CA, USA).
2.4 Detection of nitrated proteins
For 1-DE, 100 mg protein was loaded per well and electro-
phoresis and protein transfer performed as described above.
Following transfer, NC membranes were blocked for 1 h in
5% milk/TBS/0.05% Tween (TBST- 20 mM Tris, 150 mM
NaCl, 0.05% Tween 20, pH 7.5). Membranes were incubated
overnight at 47C with antinitrotyrosine, clone 1A6 antibody
(Upstate, Lake Placid, NY, USA) at a 1/1000 dilution in 5%
milk/TBST. Membranes were washed for 3615 min with
TBST, prior to incubation for 1 h at room temperature with
rabbit antimouse HRP-conjugated antibody (DAKO). Fol-
lowing 3615 min washes with TBST; immunodetection was
performed using the SuperSignal West Femto Chemilumi-
nescence kit (Pierce). Analyses involving 2-DE gel blots were
carried out as above, except equivalent protein loading was
confirmed using BLOT-FastStain™ (Geno Technology, MO,
USA). To specifically reduce 3-NT to aminotyrosine for con-
trol experiments [35], membranes were treated with sodium
dithionite (Na2S2O4) as previously described [20].
2.5 Preparation of nitrated BSA
To generate a positive control for Western blot analysis, BSA
(Sigma) was treated with 500 mM peroxynitrite (Cayman
Chemical, USA) at a final concentration of 1 mg/mL in
200 mM sodium bicarbonate (Sigma) at pH 7.8 [36]. Nitra-
tion of BSA was confirmed spectrophotometrically by detec-
tion of the yellow chromophore at 430 nm [37].
2.6 Image analysis and statistics
Differentially nitrated protein spots were determined by
comparing 2-DE gel blots representing Wistar medulla cyto-
solic fraction versus SHR medulla cytosolic fractions. SHR
and Wistar samples were each represented by duplicate gels
originating from four individual animals. Each blot and
stained membrane was scanned (BioRad GS-700 densitom-
eter) and imported into BioRad PDQuest 2D analysis soft-
ware. Replicate gels were combined into groups, normalised
to the total density of detected spots, and 3NT immuno-
reactive spots were matched across the set of 16 blots.
Resulting spot intensities were an average across each repli-
cate group, and SDs were calculated based on spot intensities
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Animal Proteomics 4557
of individual gels. Blots were analysed for different spot
intensity (quantitative differences) and identification of spots
present in only one of the two sample groups (qualitative
differences).
Differences in spot intensity were determined using a
Mann-Whitney test within PDQuest with significance level
set to either 95% (p,0.05) or 99% (p,0.01). Resulting band
sets were visually inspected to verify band quality and the
integrity of the statistical significance. For 1-DE blot analy-
ses, the total intensity of the 3NT-positive bands in each lane
has been divided by the intensity of four prominent bands
(common to all lanes) from the corresponding lane in the
Ponceau S image. This normalised quantity was used for
statistical analysis so that the results would not be skewed by
any slight differences in protein loading. All data are
mean 6 SD, and significance (p,0.05) was investigated
using unpaired, two-sample unequal variance Student’s t-
test.
2.7 Spot excision, tryptic digestion and LC-MS/MS
Following 1-DE or 2-DE, proteins were visualised using col-
loidal CBB G-250 and spots of interest excised from the gel.
Proteins were in-gel digested with trypsin (sequencing
grade, modified; Promega, UK,) using an Investigator Pro-
Gest robotic workstation (Genomic Solutions, Huntingdon,
UK). Briefly, proteins were reduced with DTT (607C,
20 min), S-alkylated with iodoacetamide (257C, 10 min) then
digested with trypsin (377C, 8 h). The resulting tryptic pep-
tide extract was dried by rotary evaporation (SC110 Speedvac;
Savant Instruments, NY, USA) and dissolved in 0.1% formic
acid for LC-MS/MS analysis.
Peptide solutions were analysed using an HCTultra PTM
Discovery System (Bruker Daltonics, UK) coupled to an
UltiMate 3000 LC System (Dionex, UK). Peptides were sepa-
rated on a monolithic capillary column (200 mm id65 cm;
Dionex part no. 161409). Eluent A was 3% ACN in water
containing 0.05% formic acid, eluent B – 80% ACN in water
containing 0.04% formic acid with a gradient of 3–45% B in
12 min at a flow rate of 2.5 mL/min. Peptide fragment mass
spectra were acquired in data-dependent AutoMS (2) mode
with a scan range of 300–1500 m/z, three averages, and up to
three precursor ions selected from the MS scan 100–2200
m/z). Precursors were actively excluded within a 1.0 min
window, and all singly charged ions were excluded.
Peptide peaks were detected and deconvoluted auto-
matically using Data Analysis software (Bruker). Mass lists
in the form of MASCOT generic files were created auto-
matically and used as the input for MASCOT MS/MS ions
searches of the NCBI database using the Matrix Science web
server (www.matrixscience.com). Default search parameters
used were: enzyme = trypsin, max. Missed cleavages = 1;
fixed modifications = carbamidomethyl (C); variable mod-
ifications = oxidation (M); peptide tolerance 6 1.5 Da; MS/
MS tolerance 6 0.5 Da; peptide charge = 21 and 31;
instrument = ESI-TRAP.
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4558 R. Tyther et al.
3 Results
3.1 Protein nitration is enhanced in SHR compared to
normotensive kidney medulla
Cytosolic medulla fractions from SHR and Wistar cortex and
medulla were compared by Western blot with anti-3NT
(Fig. 1). Nitrated proteins were detected in both tissues, with
prominent bands visible at ,40 kDa, ,25 kDa in Wistar and
SHR medulla (Fig. 1A) and at ,38 KD only in SHR medulla
cytosol (Fig. 1A). Densitometry revealed a statistically signif-
icant difference in 3NT immunoreactivity between medulla
from the normotensive and hypertensive strains (Fig. 1C),
principally due to the presence of the extra band, but similar
analysis of Wistar and SHR medulla membrane, and cortical
membrane and cytosolic fractions exhibited no differential
immunoreactivity (data not shown). Our data suggest
enhanced nitrosative stress within the SHR medulla cytosol
compared to normotensive kidney. Although some studies
have focused on the dynamics of protein nitration in mito-
chondria [17, 38, 39], this may not feature as significantly in
the medulla due to the relatively low abundance of mito-
chondria in this tissue [40].
3.2 Proteomic analysis reveals differential anti-3NT
immunopositive cytosolic proteins in SHR and
Wistar medulla
Proteins from both SHR and Wistar medulla cytosolic frac-
tions, separated by 2-DE and probed with anti-3NT, revealed
enhanced nitration in the SHR medulla compared to nor-
motensive Wistar controls (Fig. 2). Immunopositive spots
common to both SHR (Fig. 2A) and Wistar (Fig. 2B), and
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics 2007, 7, 4555–4564
spots unique to the SHR group were matched by PDQUEST
and assessed for qualitative (spots unique to SHR) and
quantitative (p,0.01–0.05, greater intensity in SHR vs. Wis-
tar) differences. Examples of differences in 3NT immuno-
reactivity between SHR and Wistar samples are illustrated by
zoom boxes in Fig. 2E.
Sodium dithionite reduction eliminated anti-3NT
immunoreactivity in both BSA nitrated in vitro (Fig. 3A) and
in blots generated from SHR medulla cytosolic protein
(Fig. 3B), which indicates that 3NT residues were success-
fully converted to aminotyrosines, and that any nonspecific
binding of anti-3NT antibody is unlikely. In addition, LC-MS/
MS analysis of nitrated BSA revealed specific nitration of the
protein at only one of six possible tyrosines. A y1 series ion
was found at LGEY*GFQNALIVR (Tyr 424) with the correct
mass of 208 (Fig. 3C).
Analysis revealed 23 spots of interest; 12 spots were
unique to SHR medulla, and 11 spots exhibited greater
intensity in SHR than in Wistar. These were matched and
excised from a corresponding Coomassie-stained polyacryl-
amide gel, followed by in-gel tryptic digestion and identifi-
cation by LC-MS/MS. Database searching with the peptide
masses gave protein identifications for 23 of the spots (Table
1).
3.3 SHR medulla cytosol proteins exhibit more
carbonylation than Wistar
Protein carbonyl formation is another important marker of
oxidative stress, and results from the action of ROS on such
amino acids as lysine, arginine, threonine, and proline. Pro-
tein carbonyls readily react with the hydrazine moiety of
DNPH, facilitating detection with anti-DNP antibodies [34].
Figure 1. Representative exam-
ples of protein tyrosine nitration
(3-nitrotyrosine immuno-
reactivity) from SHR and Wistar
rat kidney medulla. Tyrosine
nitration was compared in both
SHR and Wistar medulla cyto-
solic (A), and ponceau S staining
of blots after transfer revealed
equivalent loading of total pro-
tein (B). Bands obtained from
three experiments were ana-
lysed by densitometry. Histo-
gram (C) represents mean OD of
total bands in lane from three
independent experiments. Data
are mean 6 SD. n = 3. *p,0.05.
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Proteomics 2007, 7, 4555–4564
A greater level of carbonylation was observed in the SHR
medulla cytosolic extract when compared with Wistar (Fig. 4)
indicating that proteins in hypertensive animals are subject
to enhanced oxidative stress. Immunoblotting of DNPH-
treated SHR medulla samples revealed significantly greater
carbonylation in the 3NT-postive proteins- Eno 1 protein,
dihydrolipoamide dehydrogenase, catalase, carbonic anhy-
drase II (CA II), and predicted: similar to aldehyde dehy-
drogenase family 7 member A1 than in Wistar medulla (data
not shown). In this case it would seem nitration can occur in
concert with carbonylation, but the latter may not necessarily
be a prerequisite for the former. This is an important con-
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Animal Proteomics 4559
Figure 2. Representative 2-D immunoblots of
nitrated proteins from medulla cytosolic fraction
of (A) SHR and (B) Wistar (100 mg per gel) and
corresponding nitrocelluose membranes (SHR
medulla, C; Wistar Medulla, D) stained with
BLOT-FastStain. Zoom boxes (E) illustrate exam-
ples of differences in spot immunoreactivity be-
tween SHR and Wistar blots. All qualitatively or
quantitatively different immunoreactive spots
are indicated by arrows. Numbers correspond to
those in Table 1.
sideration when protein loss/gain of function is attributed
solely to nitration but where the protein may be subject to
other types of oxidative modification.
4 Discussion
The medulla of SHR kidneys is the principal site of nitrosa-
tive stress, and a nitroproteome of 23 protein spots exhibiting
differential immunoreactivity, representing at least 19 pro-
teins, has been identified. Nitrated proteins were most pre-
valent in medulla cytosol, consistent with the observation
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4560 R. Tyther et al.
that NO metabolism/signalling is more significant in
medulla [24, 25] and suggesting that SHR medulla may be
subject to enhanced nitrosative stress. Comparable immu-
nohistochemical studies of angiotensin II-infused rats also
found prominent anti-3NT staining in the renal inner
medulla [20]. Greater protein carbonylation was detected in
SHR medulla than in that of normotensive Wistars, but we
only observed enhanced carbonylation in five of the proteins
immunopositive for 3NT. This suggests that nitration and
carbonylation are not necessarily mutually inclusive, and
similar studies have arrived at the same conclusion [41, 42].
The amino acids subject to carbonylation are far more abun-
dant in proteins than tyrosines, but protein structure, sub-
cellular location and what type of RNS/ROS the local cellular
environment gives rise to, may all be factors in what residues
are modified.
CA II is the predominant CA isoform found in kidney,
where it facilitates H1 secretion by catalysing formation of
HCO32 from OH2 in the presence of CO2. CA isoforms have
previously been identified as nitration targets in inflamma-
tory disease models [43], asthma [21] and as a sensitive marker
of oxidative stress [42]. In kidney, CA II is involved in main-
taining acid–base and fluid balance, but it is unclear what
consequences its nitration may have. Acidosis may favour
generation of RNS by acidification of nitrite and nitrate. This
route to 3NT formation has primarily been considered in
relation to intragastric compartments with low pH, but may
contribute in other acidic compartments such as lysosomes,
or in regions where local pH may be low (e.g. adjacent to H1
pumps). Conversely, inhibition of CA II may mitigate against
3NT formation if lowering cytosolic pH reduces the stability of
ONOO2, or reduces the CO2 concentration, which appears to
be a key requirement for 3NT formation [10].
Catalase is a key peroxisomal antioxidant enzyme cata-
lyzing decomposition of H2O2 to water and oxygen. It has
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics 2007, 7, 4555–4564
Figure 3. Assessment of 3NT
antibody specificity. Nitrated
and untreated BSA (2 mg) were
immunoblotted for 3NT, with
and without treatment with
100 mM sodium dithionite,
which reduces nitrotyrosine to
aminotyrosine (A). 2-D immu-
noblots of SHR medulla cyto-
solic protein fraction (100 mg)
treated with 100 mM sodium
dithionite prior to incubation
with 3NT antibody (B). MS/MS
spectrum of the 3NT-containing
peptide L421-R433 from nitrated
BSA, showing nitration at posi-
tion Y424 (C).
been identified as a nitration target in nitrosative stress [21],
and, when coadministered in the kidney medulla with the
SOD mimetic Tempol, it can prevent blood pressure
increases in SHR [44], suggesting a role in managing hyper-
tension. Attempts have been made to exploit the therapeutic
potential of catalase in lung transplantation [45], and a simi-
lar strategy may prove beneficial in hypertension.
A second peroxisomal protein identified in this study was
hydroxyacid oxidase 3 (HAOX3), a member of the HAOX
family, which catalyse oxidation of a-hydroxy acids, a-amino
acids and glycolate with production of H2O2. HAOX3 was
originally reported as a pancreatic HAOX [46], but was mis-
identified as murine a-hydroxyacid oxidase 2 (HAOX2). This
suggests that we have identified the rat kidney isoform
HAOX2. Due to co-compartmentalisation with catalase,
H2O2 generated by HAOXs should be rapidly decomposed
but, in rat liver, HAOX1 is down-regulated during oxidative
stress [47]. Nitration of HAOX2 may cause a similar down-
regulation of activity in kidney, and could be an adaptive re-
sponse to diminish ROS generation. Furthermore, differ-
ential gene expression analysis identified the HAOX2 gene
as a qualitative trait locus for blood pressure in hypertensive
Dahl-salt-sensitive rats, suggesting HAOX2 may restrict
vasodilation though NOS inhibition [48].
We identified various aldo-keto-reductases (AKR). A
growing body of evidence implicates these proteins in both
protection against, and propagation of, oxidative and nitrosa-
tive stress. The AKR superfamily comprises approximately
60 proteins, all monomeric NADPH-dependent oxido-
reductases, with broad tissue distribution and substrate spec-
ificity for aldehydes and ketones. AKR metabolise a range of
toxic aromatic and aliphatic carbonyl compounds including
lipid peroxidation byproducts [49]. The ability to protect
against diseases like vasculitis [50] has earned AKR a nominal
cytoprotective role but, under certain conditions, they
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Proteomics 2007, 7, 4555–4564 Animal Proteomics 4561

Table 1. Differentially nitrated proteins in SHR medulla

Spot Protein Accession 3NT 3NT MW (kDa)/pI Sequence MASCOT Peptides Functional grouping
no. no. qualitative quantitative predicted– coverage score matched
difference difference observed (%)

1 CA II AAH65577 p,0.01 29/6.9–29/7.13 53 472 18 CO2 metabolism

2 Heat shock protein 8 NP_077327 p,0.05 71/5.4–69.19/5.3 44 1383 42 Chaperone/stress response
3 Albumin AAH85359 p,0.05 70/6.1–70.7/5.6 71 2419 67 Transport/homoeostasis
4 Eno 1 protein AAH81847 3 51/7.2–49.3/6 74 1849 58 Gluconeogeneis/glycolysis
8 Eno 1 protein AAH81847 3 51/7.2–49.3/5.48 60 1429 37 Gluconeogeneis/glycolysis
5 Glycerol-3-phosphate NP_071551 p,0.05 38/6.2–38/6.16 53 812 22 Gluconeogeneis/glycolysis
dehydrogenase
13 p,0.01 38/6.2–38/6.04 51 806 24
6 Malate dehydrogenase NP_150238 p,0.05 37/6.2–38.4/5.9 62 801 31 Gluconeogeneis/glycolysis
7 AKR family 1, member A1 NP_112262 p,0.05 37/6.8–38.6/6.7 59 1048 37 Oxidoreductase/antioxidant
defence
20 Afar protein AAH78872 3 39/6.8–48.7/6.68 49 705 28 Oxidoreductase/antioxidant
defence
23 AKR family 1, member A1 NP_112262 p,0.01 37/6.8–45/6.5 36 407 11 Oxidoreductase/antioxidant
defence
9 DDAH1 NP_071633 3 32/5.8–37/5.6 54 888 23 NOS regulation
10 Ornithine aminotransferase NP_071966 3 49/6.3–48.9/5.96 48 1092 25 Urea cycle
11 Mitochondrial aldehyde AAS75814 3 56/6.7–63/5.91 39 988 26 Aldehyde metabolism
dehydrogenase
precursor
12 Hypothetical protein NP_001034120 p,0.05 38/6.2–67.2/7.2 51 806 24 Unknown: homologous to
LOC361730 dihydroxyacetone kinase
14 PDI A3 precursor P11598 3 57/5.9–65.7/5.55 41 791 21 Chaperone/disulphide
bridge formation
15 Actin–beta ATRTC 3 42/5.3–38.3/5.41 32 419 15 Structural
16 Catalase NP_036652 3 p,0.05 60/7.07–67.6/7.01 37 542 16 Antioxidant defence
17 Phosphoenolpyruvate NP_942075 3 70/6.1/–68.3/6.05 18 414 8 Gluconeogeneis/glycolysis
carboxykinase 1
18 Dihydrolipoamide NP_955417 3 55/7.96–66.3/6.35 32 440 12 Pyruvate dehydrogenase
dehydrogenase complex component
19 Predicted: similar to alde- XP_214535 3 59/7.9–63/6.5 37 697 16 Aldehyde dehydrogenase
hyde dehydrogenase superfamily
family 7 member A1
21 HAOX3 NP_114471 p,0.01 40/7.5–44.3/6.44 48 792 25 a-Hydroxy acid oxidation/
H2O2 generation
22 Predicted: similar to 3 XP_001053666 p,0.05 57/8.7–61.2/7.07 48 1015 29 Ketone body metabolism
oxoacid CoA transferase
1 isoform 2

Figure 4. Representative exam-

ples of carbonylated proteins in
both SHR and Wistar medulla
cytosol (A). Ponceau S staining
of blots after transfer revealed
equivalent loading of total pro-
tein. Bands obtained from at
least four experiments were
measured densitometrically.
Histogram (B) represents mean
OD of total bands per lane from
four experiments. Data are
mean 6 SD. n = 3. *p,0.05.

© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

4562 R. Tyther et al.
may contribute to pathology [51]. Having discovered that
AKR are susceptible to nitration, it was proposed that nitra-
tion of a conserved Tyr-55 residue in the catalytic site may
enhance rather than inhibit activity [38].
Ornithine aminotransferase (OAT) was also identified as
a member of the nitroproteome, but is generally associated
with mitochondria. This could have arisen through incom-
plete separation of the membrane/mitochondrial and cyto-
solic fractions, but there is an unusually high proportion of
cytosolic OAT in regions of the medulla [52] and mitochon-
dria are much less abundant in the inner medulla than in
other kidney regions [40]. OAT is a component of the urea
cycle catalyzing conversion of L-ornithine and a-ketogluta-
rate to glutamate and glutamate semialdehyde. The meta-
bolic fate of ornithine in kidney is linked to both the urea
cycle and glutamate synthesis. This, in turn, implicates
ornithine in glutathione biosynthesis and in the citric acid
cycle. It is not known if ornithine utilisation is perturbed in
SHR kidney, but it is interesting to consider possible con-
sequences of OAT modification. Enhanced OAT activity
might deplete the arginine pool utilised by NOS, thus
impairing NO signalling, whereas decreased OAT activity
could impair antioxidant defence through diminished gluta-
thione synthesis.
Nitration of dimethylarginine dimethylaminohydrolase
1, (DDAH1) may have more direct consequences for NO
bioavailability. DDAH1 was originally thought to be mainly
responsible for preventing bioaccumulation of the endoge-
nous arginine analogues, asymmetric dimethylarginine
(ADMA) and N-monomethylarginine, byproducts of protein
degradation. However, ADMA and N-monomethylarginine
are inhibitors of NOS [53]. DDAH1 is therefore understood to
regulate NOS activity through controlling the concentration
of these inhibitors. ADMA is eliminated by renal excretion
and the action of DDAH1. Inhibition of DDAH1 could cause
ADMA accumulation, inhibiting NOS and promoting vaso-
constriction over vasodilation. There is evidence that DDAH1
itself is regulated through S-nitrosylation, so alterations in
NO levels could have important activity implications [54].
Protein disulphide isomerase (PDI) and heat shock pro-
teins 70 isoforms (hsp70) have been identified in nitrosative
stress [21, 35], suggesting that ER-associated proteins are
also RNS targets. Under oxidative and nitrosative stress, both
increased degradation and accumulation of proteins have
been observed. The latter outcome may entail ER-associated
stress induced by peroxynitrite leading to accumulation of
misfolded proteins [55], a risk factor in kidney disease.
Another nitration target, albumin, which is involved in
osmotic pressure maintenance, has been identified in pre-
vious nitration studies and induces ER-associated stress in
renal proximal tubular cells [56].
In common with previous reports concerning oxidative/
nitrosative stress, enzymes involved in cellular energy pro-
duction and intermediary metabolism were identified in the
present study: enolase 1 [57]; malate dehydrogenase [21, 35,
57]; phosphoenolpyruvate carboxykinase 1 [39], dihy-
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics 2007, 7, 4555–4564
drolipoamide dehydrogenase and glycerol-3-phosphate
dehydrogenase. Restricting glycolysis may be a common
mechanism for preventing excessive ROS or RNS genera-
tion, but could compromise basic energy metabolism. Such a
strategy seems to be in place in the hypoxic regions of kidney
but may be perturbed in SHR. In our study, SHR medulla
exhibited enhanced carbonylation, and increased carbonyla-
tion and decreased antioxidant capacity are global features
within SHR kidney [58].
In our LC-MS/MS study 3NT peptides were not detected,
presumably because of being below detection limits. Miyagi
et al. [35] encountered a similar problem in their study of
endogenous 3NT formation in rat retinas, and other groups
have encountered difficulty in mapping 3NT formation due
to the low yield of nitrated proteins generated via endoge-
nous processes [36]. Individual 2-DE gel spots may contain
multiple protein isoforms, and the relative abundance of a
nitrated protein could be low within each spot. The 3NT
modification is both labile under strongly reducing condi-
tions and quite rare even in pathology. Peptides containing
3NT are susceptible to photodecomposition during MALDI,
which decreases the mass of the intact peptide, and causes
3NT-containing residues to be overlooked due to under-
estimation of their characteristic mass [59]. Groups have
attempted to address these problems with methods such as
residue-specific antinitrotyrosine antibodies [20, 60], dansyl
chloride labelling of 3NT residues [61], reduction to amino-
tyrosine followed by acetylation to enhance detection via MS
[59] and sample enrichment using a nitrotyrosine affinity
column [62].
Our observed increase in nitration was detected against a
background of extensive protein oxidation, suggesting that,
in addition to 3NT formation, oxidation of residue side-
chains to protein carbonyls could also contribute to alteration
of activity. Xu et al. [20] attributed a 50% decrease in Mn-SOD
activity to nitration of Tyr-34, but studies by Ghosh et al. [21]
into catalase inactivation found that tyrosine chlorination
was 20-fold greater than nitration.
The principal RNS involved in ONOO2 based protein
nitration under physiological conditions are not definite [63].
SHR kidney represents a singular scenario with respect to
nitrosative stress, given the presence of increased ROS in a
nominally hypoxic environment and the unusually high NO
generating potential of the medulla. This may create the
‘supraphysiological’ conditions necessary to cause enhanced
nitrosative stress. Further work will be necessary to explore
our findings’ deeper implications. Several of the identified
proteins have the potential to contribute to renopathy
observed in SHR but they could also be biomarkers rather
than effectors of the pathophysiology.
The authors would like to acknowledge the contribution of the
Proteomics Unit, University of Aberdeen, Scotland in preparing
this manuscript. Our laboratory (RT and DS) is funded by the
Higher Education Authority of Ireland Programme for Research
in Third Level Institutions, Cycle 3.
www.proteomics-journal.com
Proteomics 2007, 7, 4555–4564
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