RENIN-ANGIONTENSINALDOSTERONE-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
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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).
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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
physiological location (Lei, 2011).
(NKA)
in
its
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
ischemia-reperfusion injury.
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against
Aim
To investigate the effect of different RAAS
blockers on the pathophysiology of DN and
the Sigma-1R – Akt - NKA system.
Angiotensinogen
AngI
AngII
ACE
enalapril
Aldosterone
spironolactone
eplerenone
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ANG Receptor
losartan
Methods
• After 5 weeks of STZ-induced (60 mg/kg iv.) diabetes, Wistar rats
were treated daily p.o. for 2 weeks with
a.
b.
c.
d.
e.
•
•
•
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.
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Mean arterial blood pressure (MAP)
and heart rate
Before
MAP
treatment
(mmHg) After
treatment
Before
Heart
treatment
rate
After
(/min)
treatment
Control
Diabetes
(D)
D+
Enalapril
D+
Losartan
D+
Spironolactone
D+
Eplerenone
105 + 17
117 ± 20
103 ± 33
110 ± 18
118 ± 22
118 ± 25
100 + 15
103 ± 21
116 ± 29
109 ± 17
108 ± 19
125 ± 11
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
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Laboratory parameters
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§
0.67 ± 0.01*
0.53 ± 0.02§
0.54 ± 0.01§
0.56 ± 0.01§
0.58 ± 0.01§
Kidney/bodyweight x 100 0.42 ± 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
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Renal histology
Control
Diabetes (D)
D + Enalapril
D + Losartan
Mesangial matrix expansion
D + Spironolactone
Arterial hyalinisation
D + Eplerenone
* p<0,05 vs. Control; § p<0,05 vs. D; n=6; PAS staining; 40x magnification; scalebar: 50
μm
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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
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
↓
-
↑
↑
↑
Paraméter
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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
beneficial in the prevention of STZ-induced DN.
be
• 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.
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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
microscopy.
with
multiphoton
* p<0,05 vs. Control; § p<0,05 vs. D; n=6