1. ADVANCED CKD AS A RISK FACTOR FOR CARDIOVASCULAR DISEASE (Foley RN et al, Am J Kidney Dis 1998;32:S112-S119)

2. 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)

3. (Vanholder R et al, Nephrol Dial Transplant 2005;20:1048-1056) EARLY CKD AS A RISK FACTOR FOR CARDIOVASCULAR DISEASE 0 0

4. VASCULAR PATHOPHYSIOLOGY IN CHRONIC KIDNEY DISEASE Increased CV risk Cardiac damage Oxidative stress Genes CKD Ambient Microinflammation Atherosclerosis Increased CV risk Metabolic alterations Hemodynamic disturbances

5. MONOCYTES/MACROPHAGES IN ATHEROSCLEROTIC LESIONS

6. NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (NADPH) OXIDASE IN MONOCYTES/MACROPHAGES NADPH gp91 p22 p47 p67 rac ·O 2 - O2O2 H+H+ NADP + EC IC Cathcart MK Regulation of superoxide anion production by NADPH oxidase in monocytes/macrophages. Contributions to atherosclerosis. Arterioscler Thromb Vasc Biol 2004;24:23-28

7. 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)

8. PHAGOCYTIC NADPH OXIDASE ACTIVITY AND ATHEROSCLEROSIS IN ASYMPTOMATIC SUBJECTS (Zalba G et al, Arterioscler Thromb Vasc Biol 2005;APP April 28) ·O 2 - production (counts/s) Carotid IMT quartiles 0 25 20 15 10 5 30 P < 0.05 q1 q2 q3 q4

9. HYPOTHESIS AND GOALS Early stages of CKD are associated with phagocytic NADPH oxidase overactivity To assess NADPH oxidase-mediated ·O 2 - production 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

10. 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 42 patients with CKD subjects (22 stage 1-2, 20 stage 3) Carotid Measurement of Biochemical arteries NADPH oxidase & hormonal ultrasonography in PMN cells determinations

11. DEMOGRAPHIC AND RENAL CHARACTERISTICS OF STUDIED SUBJECTS (Fortuño A et al, submitted) Controls Patients with CKD stage 1-2 stage 3 Gender, m/f 14/7 19/3 16/4 Age, years 48  3 57±2 * 63±10 * GFR, ml/min/1.73 m 2 90  4 91  4 50±8 * † U alb. : U creat., mg/g 3.6  0.4 33.8  2.4 * 46.6±4.7 * † ( * P < 0.05 compared with controls, † P < 0.05 compared with stage 1+2)

12. DETERMINATION OF NADPH OXIDASE ACTIVITY IN HUMAN PHAGOCYTIC CELLS (Fortuño A et al, J Hypertens 2004;22:2169-2175) Lucigenin-enhanced luminescence (RLU/s) PMA 0 25 20 15 10 5 30 P < 0.05 Basal Control DPI Apocynin SOD 35

13. BASAL NADPH OXIDASE ACTIVITY IN PHAGOCYTIC CELLS FROM PATIENTS WITH CKD (Fortuño A et al, submitted) ·O 2 - production (RLU/s) Controls Stage 1-2 Stage 3 0 2.5 2.0 1.5 1.0 0.5 3.0 N.S.

14. PMA-STIMULATED NADPH OXIDASE ACTIVITY IN PHAGOCYTIC CELLS FROM PATIENTS WITH CKD (Fortuño A et al, submitted) ·O 2 - production (RLU/s) Controls Stage 1-2 Stage 3 0 10.0 8.0 6.0 4.0 2.0 12.0 P < 0.02 (NADPH oxidase overactivity was not associated with age)

15. Abnormally high activity Normal activity (Fortuño A et al, submitted) PMA-STIMULATED NADPH OXIDASE ACTIVITY IN PHAGOCYTIC CELLS FROM PATIENTS WITH CKD 5% 48% 52% 53% 47% 95% Controls Stage 1-2 CKD Stage 3 CKD P < 0.05

16. MECHANISMS OF PHAGOCYTIC NADPH OXIDASE OVERACTIVTY IN PATIENTES WITH STAGE 1-2 CKD The clinical context (Fortuño A et al, J Hypertens 2004;22:2169-2175) ·O 2 - production (RLU/s) Basal PMA 0 15.0 12.0 9.0 6.0 3.0 18.0 P < 0.05 P < 0.02P < 0.01 Normotensive Hypertensive

17. HEMODYNAMIC CHARACTERISTICS OF PATIENTS WITH STAGE 1-2 CKD (Fortuño A et al, submitted) Controls Patients with stage 1-2 CKD SBP, mmHg 110  2 141  4 * DBP, mmHg 72  2 89  3 * MAP, mm Hg 84±3 106±5 * PP, mm Hg 30±2 51±4 * Hypertensive, % 0 82 ( * P < 0.05 compared with controls)

18. MECHANISMS OF PHAGOCYTIC NADPH OXIDASE OVERACTIVTY IN PATIENTES WITH STAGE 1-2 CKD Environmental factors (Lassègue B, Am J Physiol Regul Integr Comp Physiol 2003;285:R277-R297) NAD(P)Hox HORMONES Ang II, ET-1, Insulin... CYTOKINES TNF , IFNγ… METABOLITES Glucose, LDL, oxLDL... GROWTH FACTORS TGF  1, PDGF… PPARs α,γ - + + + +

19. Controls Patients with stage 1-2 CKD BMI, kg/m2 25.5  0.6 29.5  0.9 * Glucose, mg/dL 91  2 99  2 * Total Cholesterol, mg/dL 220  12 230  11 LDL-cholesterol, mg/dL 1 51  11 154  9 HDL-cholesterol, mg/dL 52  2 45  10 * Triglycerides, mg/dL 83  4 130  10 * Obesity Diabetes % 0 36 Met. Synd. HOMA 1.6  0.1 4.5  0.5 * METABOLIC CHARACTERISTICS OF PATIENTS WITH STAGE 1-2 CKD (Fortuño A et al, submitted) ( * P < 0.05 compared with controls)

20. (Fortuño A et al, submitted) Plasma insulin (U/L) 50403020100 30 20 10 0 ·O 2 - production (RLU/s) 25 15 5 r = 0.441 P < 0.05 Plasma insulin (U/L) Controls Stage 1-2 0 20 15 10 5 P < 0.05 INSULIN AND PMA-STIMULATED NADPH OXIDASE ACTIVITY IN PHAGOCYTIC CELLS

21. EFFECT OF INSULIN ON HUMAN PHAGOCYTIC NADPH OXIDASE (Fortuño A et al, submitted) 0 4 3 2 1 BasalInsulin Fold increase compared with basal Insulin Apocynin Insulin BIS I P < 0.05

22. NADPH gp91 p22 p47 p67 rac ·O 2 - O2O2 H+H+ NADP + EC IC MECHANISMS OF PHAGOCYTIC NADPH OXIDASE OVERACTIVTY IN PATIENTES WITH STAGE 1-2 CKD The molecule itself Critical subunit for activity

23. p22phox : β-actin (ADU) 0 15 10 5 Controls Normal Increased oxidase activity oxidase activity (Fortuño A et al, submitted) p22phox  -actin MECHANISMS OF PHAGOCYTIC NADPH OXIDASE OVERACTIVTY IN PATIENTES WITH STAGE 1-2 CKD The molecule itself Patients P < 0.01

24. (San José et al, Hypertension 2004;44:163-169) 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

25. ATHEROGENIC MECHANISMS IN CHRONIC KIDNEY DISEASE The role of oxidative stress (Locatelly F et al, Nephrol Dial Transplant 2003;18:1272-1280; Moldinger PS et al, Semin Nephrol 2004;24:354-365) Reduced anti-oxidant systems Increased phagocytic- mediated pro-oxidant activity Renal disease < NO availability LDL oxidation VSMC apoptosis Endothelial Plaque Rupture & activation formation thrombosis Oxidative stress

26. LDL OXIDATION AND CAROTID ATHEROSCLEROSIS IN PATIENTS WITH STAGE 1+2 CKD (Fortuño A et al, submitted) Oxidized LDL (U/L) 0 80 60 40 20 Controls Patients P < 0.05 Carotid IMT (mm) 0 0.80 0.60 0.40 0.20 Controls Patients P < 0.05

27. ASSOCIATIONS OF PHAGOCYTIC NADPH OXIDASE ACTIVITY, OXIDIZED LDL AND CAROTID INTIMA-MEDIA THICKNESS (Fortuño A et al, submitted) Oxidized LDL (U/L) 120906030 0 Carotid IMT (mm) 0.90 0.85 0.80 0.75 0.70 0.65 0.60 r = 0.393 P < 0.005 Oxidized LDL (U/L) ·O 2 - production (RLU/s) r = 0.349 P < 0.005 100806040200 140 120 100 80 60 40 20

28. VASCULAR PATHOPHYSIOLOGY IN EARLY CHRONIC KIDNEY DISEASE Proposal Increased CV risk Oxidative stress Genes Early CKD Ambient Microinflammation Atherosclerosis Increased CV risk Metabolic alterations Hemodynamic disturbances Oxidant stress is a result of NADPH oxidase-mediated ·O 2 - overproduction by phagocytic cells Cardiac damage

29. VASCULAR PATHOPHYSIOLOGY IN EARLY CHRONIC KIDNEY DISEASE Consequence Increased CV risk Oxidative stress Genes Early CKD Ambient Microinflammation Atherosclerosis Increased CV risk Metabolic alterations Hemodynamic disturbances It is necessary to explore the beneficial effects of antioxidant measures with proven efficacy Cardiac damage

30. Universidad deNavarra Universidad de Navarra Cliniciens (CUN) Oscar Beloqui Alberto Benito Inmaculada Colina Biochemists (CIMA) Ana Fortuño Ujué Moreno Gorka San José Guillermo Zalba Techniciens (CIMA) Ana Montoya Raquel Ros