RENIN-ANGIONTENSIN-ALDOSTERONE-SYSTEM (RAAS) BLOCKERS IN DIABETIC NEPHROPATHY (DN)

RENIN-ANGIONTENSIN-ALDOSTERONE-SYSTEM (RAAS)

BLOCKERS IN DIABETIC NEPHROPATHY (DN)

Nóra Fanni Bánki

SE-MTA “Lendulet” Diabetes Research Group, 1st Dep. of Pediatrics, Academic Research Group for Pediatrics and Nephrology,

Semmelweis University, Budapest

2012 V4 ACADEMIES FORUM

Mátraháza, 26.10.2012

[email protected]@yahoo.com

Introduction• By 2035 the number of diabetic patients will reach

approximately 400 million (IDF – 2011).

• 35-40% of diabetic patients develop DN within 15-20 years after the diagnosis (USRDS – 2010).

• The 2012 American Diabetes Association protocol recommends the use of ACE inhibitors or ARBs in the case of microalbuminuria (ADA – 2012).

• Renal RAAS is activated in diabetes and angiotensin II (AngII) level is increased (Ribiero et al – 2008).


Sigma-1 receptor (Sigma-1R)

• The Sigma-1R is expressed in several tissues and organs (Pontén, 2009).

• Renal localization and function are yet unknown.

• The activation of Sigma-1R induces the Akt – endothelial nitric oxide synthase (eNOS) pathway

– protective against hypoxic injury in the heart and brain (Bhuiyan, 2011).

– preserves the Na/K ATPase (NKA) in its physiological location (Lei, 2011).

Previous experiments• In Streptozotocin (STZ) induced diabetic rats:

– elevated expression and mislocation of renal NKA.

– exogenly given AngII causes further progression of DN.

• Sigma-1R agonsits are renoprotective against ischemia-reperfusion injury.


AimTo investigate the effect of different RAAS blockers on the pathophysiology of DN and the Sigma-1R – Akt - NKA system.

Angiotensinogen AngI AngII ANG Receptor

ACE Aldosterone

enalapril

losartan

spironolactone

eplerenone


Methods• After 5 weeks of STZ-induced (60 mg/kg iv.) diabetes, Wistar rats

were treated daily p.o. for 2 weeks with

a. enalapril (40 mg x kg-1 x day-1; n=6), b. losartan (20 mg x kg-1 x day-1; n=6), c. spironolactone (50 mg x kg-1 x day-1; n=6),d. epleronone (50 mg x kg-1 x day-1; n=6),e. saline (n=6).

• Blood pressure was monitored non-invasively before and after treatment with a CODA tail-cuff system.

• Serum and urine parameters were measured and histological scanning of the excised kidney was performed.

• Protein levels and intrarenal localization of Sigma-1R-Akt-NKA were evaluated.


ControlDiabetes

(D)D+

EnalaprilD+

LosartanD+

SpironolactoneD+

Eplerenone

MAP(mmHg)

Before treatment

105 + 17 117 ± 20 103 ± 33 110 ± 18 118 ± 22 118 ± 25

After treatment

100 + 15 103 ± 21 116 ± 29 109 ± 17 108 ± 19 125 ± 11

Heart rate(/min)

Before treatment

443 ± 48 366 ± 47* 346 ± 41* 334 ± 39* 349 ± 32* 366 ± 42*

After treatment

422 ± 70 350 ± 49* 356 ± 26* 362 ± 43 400 ± 20§ 389 ± 35§

Mean arterial blood pressure (MAP) and heart rate

* p<0,05 vs. Control; § p<0,05 vs. D; n=6


Parameter Control Diabetes (D) D+Enalapril D+Losartan D+Spironolactone D+Eplerenone

Body weight(g) 342 ± 2 260 ± 5* 256 ± 7 250 ± 11 349 ± 5§ 273 ± 9

Se glucose (mmol/L) 11.6 ± 0.5 43.6 ± 1.2* 35.6 ± 2.3§ 36.1 ± 2.6§ 33.2 ± 0.9§ 38.5 ± 1.8§

Se cholesterole (mmol/L) 1.72 ± 0.19 4.1 ± 0.88* 3.13± 0.62 2.72 ± 0.41 1.64 ± 0.12§ 2.28 ± 0.2§

LDL-cholesterole (mmol/L)

UD 1.63 ± 0.74* 0.65 ± 0.38 0.48 ± 0.24 UD§ UD§

Se triglyceride (mmol/L) 1.32 ± 7 4.94 ± 1.4* 4.54 ± 1.95 2.1 ± 0.68 0.79 ± 0.11§ 2.16 ± 0.52§

Kidney/bodyweight x 100 0.42 ± 0.01 0.67 ± 0.01* 0.53 ± 0.02§ 0.54 ± 0.01§ 0.56 ± 0.01§ 0.58 ± 0.01§

Se creatinine (μmol/L) 55.6 ± 0.9 64.6 ±1.1* 57.8 ± 1.6 54.8 ± 1.2§ 56.7 ± 0.7§ 69.3 ± 1.2

BUN (mmol/L) 7.12 ± 0.05 15 ± 0.5* 11.9 ± 0.4 11.93 ± 0.2 8.61 ± 0.2§ 11.4 ± 0.4§

Se Potassium (mmol/L) 5.78 ± 0.07 7.26 ± 0.14* 7.24 ± 0.2 6 ± 0.08§ 5.34 ± 0.2§ 6.12 ± 0.01§

Se Sodium (mmol/L) 154 ± 1 135 ± 0.5* 137 ± 0.5 138 ± 0.2 141 ± 0.3§ 140 ± 0.3§

Laboratory parameters

* p<0,05 vs. Control; § p<0,05 vs. D; n=6; Se – serum, BUN – blood urea nitrogen; LDL – low density lipoprotein


Control Diabetes (D)

D + Enalapril D + Losartan D + Spironolactone D + Eplerenone

Mesangial matrix expansion

Renal histology

* p<0,05 vs. Control; § p<0,05 vs. D; n=6; PAS staining; 40x magnification; scalebar: 50 μm

Arterial hyalinisation


Renal Sigma-1R, pAkt, NKA

* p<0,05 vs. Control; ** p<0,01 vs. Control; § p<0,05 vs. D; n=6

Renal Sigma-1R and NKA localization

Green – NKA, Red – S1R, Blue – nuclei, 63x magnification

Summary

ParaméterDiabetes (D) vs. Control

Enalapril vs. D

Losartan vs. D

Spironolactone vs. D

Eplerenone vs. D

MAP - - - - -

Heart rate ↓ - - ↑ ↑

Serum glucose ↑ ↓ ↓ ↓ ↓

Serum lipids ↑ - - ↓ -/↓

Kidney/body weight ↑ ↓ ↓ ↓ ↓

Renal function ↓ -/↑ ↑ ↑ -/↑

Renal structure ↓ ↑ ↑ ↑ ↑

Renal Sigma-1R - - - - -Renal pAkt/Akt ↓ ↑ ↑ ↑ ↑

Renal NKA ↑ - ↓ ↓ ↓

NKA localization ↓ - ↑ ↑ ↑


Conclusion• RAAS inhibitor treatment can be used to prevent the

progression of DN in these doses without blood pressure lowering side effects in rats.

• Aldosterone antagonist monotherapy could be beneficial in the prevention of STZ-induced DN.

• The renal Sigma-1R – Akt – NKA pathway may play a role in the pathophysiology of DN and could serve as a new therapeutic target of RAAS inhibitors.


Plans for the future

• Introduction of type 2 diabetic animal models (Zucker rats, db/db mice).

• Use of Sigma-1R agonists (antidepressant fluvoxamine), antagonists and other RAAS inhibitors (ramipril ect.).

• Investigation of depressive behavior with the forced swim test ect.

• Evaluation of the NOS system.

• In vivo visualisation with multiphoton microscopy. * p<0,05 vs. Control; § p<0,05 vs. D; n=6

RENIN-ANGIONTENSIN-ALDOSTERONE-SYSTEM (RAAS) BLOCKERS IN DIABETIC NEPHROPATHY (DN)

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