1. Semmelweis University, Budapest
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,

2. Introduction
By 2035 the number of diabetic patients will reach approximately 400 million (IDF – 2011).
35-40% of diabetic patients develop DN within 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).

3. 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).

4. 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.

5. Aim
To 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
losartan
enalapril
spironolactone
eplerenone

6. Methods
After 5 weeks of STZ-induced (60 mg/kg iv.) diabetes, Wistar rats were treated daily p.o. for 2 weeks with
enalapril (40 mg x kg-1 x day-1; n=6),
losartan (20 mg x kg-1 x day-1; n=6),
spironolactone (50 mg x kg-1 x day-1; n=6),
epleronone (50 mg x kg-1 x day-1; n=6),
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.

7. Mean arterial blood pressure (MAP) and heart rate
Control
Diabetes (D)
D+ Enalapril
D+ Losartan
D+ Spironolactone
D+ Eplerenone
MAP (mmHg)
Before treatment
117 ± 20
103 ± 33
110 ± 18
118 ± 22
118 ± 25
MAP
(mmHg)
After treatment
103 ± 21
116 ± 29
109 ± 17
108 ± 19
125 ± 11
Heart rate
(/min)
443 ± 48
366 ± 47*
346 ± 41*
334 ± 39*
349 ± 32*
366 ± 42*
422 ± 70
350 ± 49*
356 ± 26*
362 ± 43
400 ± 20§
389 ± 35§
* p<0,05 vs. Control; § p<0,05 vs. D; n=6

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

9. Arterial hyalinisation
Renal histology
Control
Diabetes (D)
D + Enalapril
D + Losartan
D + Spironolactone
D + Eplerenone
Mesangial matrix expansion
Arterial hyalinisation
* p<0,05 vs. Control; § p<0,05 vs. D; n=6; PAS staining; 40x magnification; scalebar: 50 μm

10. Renal Sigma-1R, pAkt, NKA
* p<0,05 vs. Control; ** p<0,01 vs. Control; § p<0,05 vs. D; n=6

11. Renal Sigma-1R and NKA localization
Green – NKA, Red – S1R, Blue – nuclei, 63x magnification

12. Diabetes (D) vs. Control
Summary
Paraméter
Diabetes (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

13. 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.

14. 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