ADVANCED CKD AS A RISK FACTOR FOR CARDIOVASCULAR DISEASE

(Foley RN et al, Am J Kidney Dis 1998;32:S112-S119)

EARLY CKD AS A RISK FACTOR FOR CARDIOVASCULAR DISEASE

(Manjunath G et al, J Am Coll Cardiol 2003;41:47-65)

(Manjunath G et al, Kidney Int 2003;63:1121-1129)

EARLY CKD AS A RISK FACTOR FOR CARDIOVASCULAR DISEASE

(Vanholder R et al, Nephrol Dial Transplant 2005;20:1048-1056)

VASCULAR PATHOPHYSIOLOGY IN CHRONIC KIDNEY DISEASE

Increased CV risk

Cardiac damage
Hemodynamic disturbances

Genes
Metabolic alterations

CKD
Ambient
Microinflammation

Oxidative stress

Atherosclerosis Increased CV risk

MONOCYTES/MACROPHAGES IN ATHEROSCLEROTIC LESIONS

NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (NADPH) OXIDASE IN MONOCYTES/MACROPHAGES

O2O2 gp91 NADPH rac p67 p47 NADP+
Cathcart MK Regulation of superoxide anion production by NADPH oxidase in monocytes/macrophages. Contributions to atherosclerosis. Arterioscler Thromb Vasc Biol 2004;24:23-28

H+
EC

p22
IC

EXPRESSION OF NADPH OXIDASE IN HUMAN CORONARY ARTERIES

Nonatherosclerotic arteries

Atherosclerotic arteries

Advanced atherosclerotic lesions

Hematoxylin-eosin Anti-p22phox Negative controls
(Azumi H et al, Circulation 1999;100:1494-1498)

PHAGOCYTIC NADPH OXIDASE ACTIVITY AND ATHEROSCLEROSIS IN ASYMPTOMATIC SUBJECTS

P < 0.05
30

O2- production (counts/s)

25 20 15 10 5 0

q1

q2

q3

q4

Carotid IMT quartiles

(Zalba G et al, Arterioscler Thromb Vasc Biol 2005;APP April 28)

HYPOTHESIS AND GOALS

Early stages of CKD are associated with phagocytic NADPH oxidase overactivity

To assess NADPH oxidase-mediated O2production in peripheral blood monocytes and lymphocytes from patients with stage 1-2 and 3 CKD

&
To assess associations of NADPH oxidase activity with systemic oxidative parameters and atherosclerosis in the same patients

SUBJECTS AND DESIGN

Subjects who attended for routine medical examination

No known history of renal disease and atherosclerosis

Complete medical work-up after informed consent 21 healthy subjects 42 patients with CKD
(22 stage 1-2, 20 stage 3)

Carotid arteries ultrasonography

Measurement of NADPH oxidase in PMN cells

Biochemical & hormonal determinations

DEMOGRAPHIC AND RENAL CHARACTERISTICS OF STUDIED SUBJECTS

Controls

Patients with CKD stage 1-2 stage 3 19/3 572 * 914 33.82.4 * 16/4 6310 * 508 * 46.64.7 *

Gender, m/f Age, years GFR, ml/min/1.73 m2 U alb. : U creat., mg/g

14/7 483 904 3.60.4

( * P < 0.05 compared with controls, P < 0.05 compared with stage 1+2)

(Fortuo A et al, submitted)

DETERMINATION OF NADPH OXIDASE ACTIVITY IN HUMAN PHAGOCYTIC CELLS

P < 0.05
35 30

Lucigenin-enhanced luminescence (RLU/s)

25 20 15 10 5 0

Basal

Control

DPI

Apocynin PMA

SOD

(Fortuo A et al, J Hypertens 2004;22:2169-2175)

BASAL NADPH OXIDASE ACTIVITY IN PHAGOCYTIC CELLS FROM PATIENTS WITH CKD

N.S.
3.0

O2- production (RLU/s)

2.5 2.0 1.5 1.0 0.5 0

Controls

Stage 1-2

Stage 3

(Fortuo A et al, submitted)

PMA-STIMULATED NADPH OXIDASE ACTIVITY IN PHAGOCYTIC CELLS FROM PATIENTS WITH CKD

P < 0.02
12.0

O2- production (RLU/s)

10.0 8.0 6.0 4.0 2.0 0

Controls
(NADPH oxidase overactivity was not associated with age)

Stage 1-2

Stage 3

(Fortuo A et al, submitted)

PMA-STIMULATED NADPH OXIDASE ACTIVITY IN PHAGOCYTIC CELLS FROM PATIENTS WITH CKD

5%

95%

Controls

48%

52%

Stage 1-2 CKD

P < 0.05

53%

47%

Stage 3 CKD

Abnormally high activity Normal activity

(Fortuo A et al, submitted)

MECHANISMS OF PHAGOCYTIC NADPH OXIDASE OVERACTIVTY IN PATIENTES WITH STAGE 1-2 CKD The clinical context
P < 0.05 P < 0.02
18.0

P < 0.01

O2- production (RLU/s)

15.0 12.0 9.0 6.0 3.0

Basal

PMA

Basal

PMA

Normotensive
(Fortuo A et al, J Hypertens 2004;22:2169-2175)

Hypertensive

HEMODYNAMIC CHARACTERISTICS OF PATIENTS WITH STAGE 1-2 CKD

Controls

Patients with stage 1-2 CKD 1414 * 893 * 1065 * 514 * 82

SBP, mmHg DBP, mmHg MAP, mm Hg PP, mm Hg Hypertensive, %

1102 722 843 302 0

( * P < 0.05 compared with controls)

(Fortuo A et al, submitted)

MECHANISMS OF PHAGOCYTIC NADPH OXIDASE OVERACTIVTY IN PATIENTES WITH STAGE 1-2 CKD Environmental factors

HORMONES Ang II, ET-1, Insulin... GROWTH FACTORS METABOLITES TGF-b1, PDGF Glucose, LDL, oxLDL... +

+
NAD(P)Hox

+
+
CYTOKINES TNF, IFN

PPARs ,
(Lassgue B, Am J Physiol Regul Integr Comp Physiol 2003;285:R277-R297)

METABOLIC CHARACTERISTICS OF PATIENTS WITH STAGE 1-2 CKD

Controls

Patients with stage 1-2 CKD

29.50.9 * 992 * 23011 1549 4510 * 13010 *

BMI, kg/m2 Glucose, mg/dL Total Cholesterol, mg/dL LDL-cholesterol, mg/dL HDL-cholesterol, mg/dL Triglycerides, mg/dL Obesity Diabetes Met. Synd. HOMA

25.50.6 912 22012 1 5111 522 834

36

1.60.1
( * P < 0.05 compared with controls)

4.50.5 *

(Fortuo A et al, submitted)

INSULIN AND PMA-STIMULATED NADPH OXIDASE ACTIVITY IN PHAGOCYTIC CELLS

20

P < 0.05
30

r = 0.441 P < 0.05

O2- production (RLU/s)

Plasma insulin (U/L)

15

25 20 15 10 5

10

0 Controls Stage 1-2

10

20

30

40

50

Plasma insulin (U/L)

(Fortuo A et al, submitted)

EFFECT OF INSULIN ON HUMAN PHAGOCYTIC NADPH OXIDASE

P < 0.05
4

Fold increase compared with basal

Basal

Insulin

Insulin Apocynin

Insulin BIS I

(Fortuo A et al, submitted)

MECHANISMS OF PHAGOCYTIC NADPH OXIDASE OVERACTIVTY IN PATIENTES WITH STAGE 1-2 CKD The molecule itself
Critical subunit for activity

O2O2 H+
EC

gp91

p22
IC

NADPH
rac p67 NADP+

p47

MECHANISMS OF PHAGOCYTIC NADPH OXIDASE OVERACTIVTY IN PATIENTES WITH STAGE 1-2 CKD The molecule itself
p22phox

b-actin

P < 0.01
15

p22phox : -actin (ADU)

10

Controls

Normal oxidase activity

Increased oxidase activity

(Fortuo A et al, submitted)

Patients

MECHANISMS OF PHAGOCYTIC NADPH OXIDASE OVERACTIVTY IN PATIENTES WITH STAGE 1-2 CKD The molecule itself *

* The -930 A/G polymorphism

of the human p22phox gene
(San Jos et al, Hypertension 2004;44:163-169)

ATHEROGENIC MECHANISMS IN CHRONIC KIDNEY DISEASE The role of oxidative stress

Renal disease

Increased phagocyticmediated pro-oxidant activity

Reduced anti-oxidant systems

< NO availability

LDL oxidation

VSMC apoptosis

Oxidative stress
Endothelial activation Plaque formation Rupture & thrombosis

(Locatelly F et al, Nephrol Dial Transplant 2003;18:1272-1280; Moldinger PS et al, Semin Nephrol 2004;24:354-365)

LDL OXIDATION AND CAROTID ATHEROSCLEROSIS IN PATIENTS WITH STAGE 1+2 CKD

P < 0.05
80 0.80

P < 0.05

Oxidized LDL (U/L)

60

Carotid IMT (mm)

0.60

40

0.40

20

0.20

Controls

Patients

Controls

Patients

(Fortuo A et al, submitted)

ASSOCIATIONS OF PHAGOCYTIC NADPH OXIDASE ACTIVITY, OXIDIZED LDL AND CAROTID INTIMA-MEDIA THICKNESS

140 120 100 80 60 40 20 0 20 40 60 80 100 r = 0.349 P < 0.005

0.90 0.85

Carotid IMT (mm)

Oxidized LDL (U/L)

0.80 0.75 0.70 0.65 0.60 0 30 60 90 120

r = 0.393 P < 0.005

O2- production (RLU/s)

Oxidized LDL (U/L)

(Fortuo A et al, submitted)

VASCULAR PATHOPHYSIOLOGY IN EARLY CHRONIC KIDNEY DISEASE Proposal

Increased CV risk Cardiac damage

Hemodynamic disturbances

Genes
Metabolic alterations

Early CKD
Ambient
Microinflammation

Oxidative stress

Oxidant stress is a result of NADPH oxidase-mediated O2- overproduction by phagocytic cells

Atherosclerosis Increased CV risk

VASCULAR PATHOPHYSIOLOGY IN EARLY CHRONIC KIDNEY DISEASE Consequence

Increased CV risk Cardiac damage

Hemodynamic disturbances

Genes
Metabolic alterations

Early CKD
Ambient
Microinflammation

Oxidative stress

It is necessary to explore the beneficial effects of antioxidant measures with proven efficacy

Atherosclerosis

Increased CV risk

Cliniciens (CUN) Oscar Beloqui Alberto Benito Inmaculada Colina Biochemists (CIMA) Ana Fortuo de Navarra Uju Moreno Gorka San Jos Guillermo Zalba Techniciens (CIMA) Ana Montoya Raquel Ros

Universidad