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, 26.10.2012 fannibanki@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). fannibanki@yahoo.com

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. fannibanki@yahoo.com

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 fannibanki@yahoo.com

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. fannibanki@yahoo.com

Mean arterial blood pressure (MAP) and heart rate Control Diabetes (D) D+ Enalapril D+ Losartan D+ Spironolactone D+ Eplerenone MAP (mmHg) Before treatment 105 + 17 117 ± 20 103 ± 33 110 ± 18 118 ± 22 118 ± 25 MAP (mmHg) After treatment 100 + 15 103 ± 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 fannibanki@yahoo.com

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 fannibanki@yahoo.com

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 fannibanki@yahoo.com

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

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 fannibanki@yahoo.com

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. fannibanki@yahoo.com

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