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)

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

EARLY CKD AS A RISK FACTOR FOR CARDIOVASCULAR DISEASE

0 0

VASCULAR PATHOPHYSIOLOGY IN CHRONIC KIDNEY DISEASE

Increased CV risk

Cardiac damage

Oxidative stress

Genes

CKD

Ambient

Microinflammation

Atherosclerosis

Increased CV risk

Metabolicalterations

Hemodynamicdisturbances

MONOCYTES/MACROPHAGES IN ATHEROSCLEROTIC LESIONS

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

NADPH

gp91 p22

p47

p67

rac

·O2-

O2H+

NADP+

EC

IC

Cathcart MKRegulation of superoxide anion productionby NADPH oxidase in monocytes/macrophages.Contributions to atherosclerosis.Arterioscler Thromb Vasc Biol 2004;24:23-28

EXPRESSION OF NADPH OXIDASE IN HUMAN CORONARY ARTERIES

Nonatheroscleroticarteries

Atheroscleroticarteries

Advancedatherosclerotic

lesions

Hematoxylin-eosin Anti-p22phox Negative controls

(Azumi H et al, Circulation 1999;100:1494-1498)

PHAGOCYTIC NADPH OXIDASE ACTIVITY AND ATHEROSCLEROSISIN ASYMPTOMATIC SUBJECTS

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

·O2-

pro

du

ctio

n (

coun

ts/s

)

Carotid IMT quartiles

0

25

20

15

10

5

30

P < 0.05

q1 q2 q3 q4

HYPOTHESIS AND GOALS

Early stages of CKD are associated with phagocytic NADPH oxidase overactivity

To assess NADPH oxidase-mediated ·O2-

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

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

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 483 57±2 * 63±10 *

GFR, ml/min/1.73 m2 904 914 50±8 * †

U alb. : U creat., mg/g 3.60.4 33.82.4 * 46.6±4.7 * †

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

DETERMINATION OF NADPH OXIDASE ACTIVITY IN HUMAN PHAGOCYTIC CELLS

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

Lu

cige

nin

-en

han

ced

lu

min

esce

nce

(R

LU

/s)

PMA

0

25

20

15

10

5

30

P < 0.05

Basal Control DPI Apocynin SOD

35

BASAL NADPH OXIDASE ACTIVITY IN PHAGOCYTIC CELLSFROM PATIENTS WITH CKD

(Fortuño A et al, submitted)

·O2-

pro

du

ctio

n (

RL

U/s

)

Controls Stage 1-2 Stage 30

2.5

2.0

1.5

1.0

0.5

3.0

N.S.

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

(Fortuño A et al, submitted)

·O2-

pro

du

ctio

n (

RL

U/s

)

Controls Stage 1-2 Stage 30

10.0

8.0

6.0

4.0

2.0

12.0

P < 0.02

(NADPH oxidase overactivitywas not associated with age)

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

MECHANISMS OF PHAGOCYTIC NADPH OXIDASE OVERACTIVTY INPATIENTES WITH STAGE 1-2 CKD

The clinical context

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

·O2-

pro

du

ctio

n (

RL

U/s

)

Basal PMA Basal PMA0

15.0

12.0

9.0

6.0

3.0

18.0

P < 0.05

P < 0.02 P < 0.01

Normotensive Hypertensive

HEMODYNAMIC CHARACTERISTICS OF PATIENTS WITH STAGE 1-2 CKD

(Fortuño A et al, submitted)

Controls Patients with stage

1-2 CKD

SBP, mmHg 1102 1414 *

DBP, mmHg 722 893 *

MAP, mm Hg 84±3 106±5 *

PP, mm Hg 30±2 51±4 *

Hypertensive, % 0 82

( * P < 0.05 compared with controls)

MECHANISMS OF PHAGOCYTIC NADPH OXIDASE OVERACTIVTY INPATIENTES WITH STAGE 1-2 CKD

Environmental factors

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

NAD(P)Hox

HORMONESAng II, ET-1, Insulin...

CYTOKINES TNF, IFNγ…

METABOLITESGlucose, LDL, oxLDL...

GROWTH FACTORS TGF1, PDGF…

PPARsα ,γ

-

++

+

+

Controls Patients with stage

1-2 CKD

BMI, kg/m2 25.50.6 29.50.9 *

Glucose, mg/dL 912 992 *

Total Cholesterol, mg/dL 22012 23011

LDL-cholesterol, mg/dL 1 5111 1549

HDL-cholesterol, mg/dL 522 4510 *

Triglycerides, mg/dL 834 13010 *

Obesity

Diabetes % 0 36

Met. Synd.

HOMA 1.60.1 4.50.5 *

METABOLIC CHARACTERISTICS OF PATIENTS WITH STAGE 1-2 CKD

(Fortuño A et al, submitted)

( * P < 0.05 compared with controls)

(Fortuño A et al, submitted)

Plasma insulin (U/L)

50403020100

30

20

10

0·O

2- p

rod

ucti

on (

RL

U/s

)

25

15

5

r = 0.441P < 0.05

Pla

sma

insu

lin

(U/L

)

Controls Stage 1-2

0

20

15

10

5

P < 0.05

INSULIN AND PMA-STIMULATED NADPH OXIDASE ACTIVITY IN PHAGOCYTIC CELLS

EFFECT OF INSULIN ON HUMAN PHAGOCYTIC NADPH OXIDASE

(Fortuño A et al, submitted)

0

4

3

2

1

Basal Insulin

Fol

d in

crea

se c

ompa

red

wit

h b

asal

Insulin Apocynin

Insulin BIS I

P < 0.05

NADPH

gp91 p22

p47

p67

rac

·O2-

O2H+

NADP+

EC

IC

MECHANISMS OF PHAGOCYTIC NADPH OXIDASE OVERACTIVTY INPATIENTES WITH STAGE 1-2 CKD

The molecule itself

Critical subunitfor activity

p22

ph

ox :

β-a

ctin

(A

DU

)

0

15

10

5

Controls Normal Increased oxidase activity oxidase activity

(Fortuño A et al, submitted)

p22phox

-actin

MECHANISMS OF PHAGOCYTIC NADPH OXIDASE OVERACTIVTY INPATIENTES WITH STAGE 1-2 CKD

The molecule itself

Patients

P < 0.01

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

MECHANISMS OF PHAGOCYTIC NADPH OXIDASE OVERACTIVTY INPATIENTES WITH STAGE 1-2 CKD

The molecule itself *

* The -930 A/G polymorphismof the human p22phox gene

ATHEROGENIC MECHANISMS IN CHRONIC KIDNEY DISEASEThe 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

Increasedphagocytic-mediated

pro-oxidant activity

Renal disease

< NO availability LDL oxidation VSMC apoptosis

Endothelial Plaque Rupture & activation formation thrombosis

Oxidative stress

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

(Fortuño A et al, submitted)

Oxi

diz

ed L

DL

(U

/L)

0

80

60

40

20

Controls Patients

P < 0.05

Car

otid

IM

T (

mm

)

0

0.80

0.60

0.40

0.20

Controls Patients

P < 0.05

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

(Fortuño A et al, submitted)

Oxidized LDL (U/L)

1209060300

Car

otid

IM

T (

mm

)

0.90

0.85

0.80

0.75

0.70

0.65

0.60

r = 0.393 P < 0.005

Oxi

diz

ed L

DL

(U/L

)

·O2- production (RLU/s)

r = 0.349 P < 0.005

100806040200

140

120

100

80

60

40

20

VASCULAR PATHOPHYSIOLOGY IN EARLY CHRONIC KIDNEY DISEASEProposal

Increased CV risk

Oxidative stress

Genes

EarlyCKD

Ambient

Microinflammation

Atherosclerosis

Increased CV risk

Metabolicalterations

Hemodynamicdisturbances

Oxidant stress is aresult of NADPH oxidase-mediated

·O2- overproduction

by phagocytic cells

Cardiac damage

VASCULAR PATHOPHYSIOLOGY IN EARLY CHRONIC KIDNEY DISEASEConsequence

Increased CV risk

Oxidative stress

Genes

EarlyCKD

Ambient

Microinflammation

Atherosclerosis

Increased CV risk

Metabolicalterations

Hemodynamicdisturbances

It is necessaryto explore the

beneficial effectsof antioxidantmeasures with

proven efficacy

Cardiac damage

Universidad deUniversidad de NavarraNavarra

Cliniciens (CUN)Oscar BeloquiAlberto Benito

Inmaculada Colina

Biochemists (CIMA)Ana FortuñoUjué Moreno

Gorka San JoséGuillermo Zalba

Techniciens (CIMA)Ana MontoyaRaquel Ros