1. Renal lesions aggravation are prevented by low doses of sitagliptin in a rat model of type 2 diabetes Cristina Mega, Helena Vala, Jorge Oliveira, Rosa Fernandes, Filipa Mascarenhas-Melo, Belmiro Parada, Rui Pinto, Frederico Teixeira, Flávio Reis, Edite Teixeira de Lemos

2. Macrovascular Microvascular Heine RJ, Spijkerman AM. 2006. Major complications of Type 2 Diabetes Mellitus (T2DM) Cardiovascular diseases Neuropathy Retinopathy Nephropathy

3. Diabetic nephropathy is a major microvascular complication of T2DM Incidence of T2DM is rapidly increasing, as well as the prevalence of chronic kidney disease (CKD), resulting from these diabetic complications In different regions of the World, it accounts for almost one-third of all cases of end-stage renal disease (ESRD) Diabetic Nephropathy – A Major Diabetic Complication Diabetes remains the single most important cause of kidney failure

4. Prevention of Diabetes-Induced dysmetabolism and associated vascular complications Blood Glucose levelsInsulin secretion/action Therapeutic Goals in Type 2 Diabetes Mellitus (T2DM)

5. Until now, anti-diabetic drugs were able to Hyperglycaemia None were able to preserve β-cells Therapeutic Goals in Type 2 Diabetes Mellitus (T2DM) Anti-diabetic drugs

6.  incretin defect is a major contributor to β-cell dysfunction  incretins stimulate release of insulin by β-cells  incretin inhibits glucagon release by α-cells  and that in T2DM the incretin effect is decreased Sitagliptin Knowing that New anti-diabetic drugs, focus on the increase of incretin levels in diabetic patients, like Sitagliptin a dipeptidyl peptidase-4 (DPP-4) inhibitor and one of the best known incretin enhancers

7. Sitagliptin targets 2 physiologic glucose-lowering actions with a single oral agent Improves 24 h glycaemic control Incretin hormones GLP-1 and GIP are released by the intestine throughout the day Their levels increase in response to a meal and are found to be reduced in Diabetes Inactive GIP DPP-4 enzyme Inactive GLP-1 Beta cells  Insulin (GLP-1 and GIP) Glucose dependent  Glucagon (GLP-1) Alpha cells Glucose uptake by peripheral Glucose uptake by peripheraltissues Glucose production by liver SITAGLIPTIN allows for a longer circulation life for incretins X 2 - 4 minutes BLOOD GLUCOSE GIPGLP-1

8. Preserving pancreatic function Inhibition of DPP-4 activity, by incretin enhancers, such as Sitagliptin (Known Effects) but the real impact of low-dose sitagliptin treatment on diabetic nephropathy remains to be elucidated Improve glycaemic control, in diabetic patients by prolonging the actions of incretin hormones

9. Prevention of Diabetes-Induced dysmetabolism and microvascular complications – Diabetic Nephropathy Assess the effects SITAGLIPTIN Evaluate the effects of chronic (6 weeks) inhibition of DPP-4 by low doses of sitagliptin, in the ZDF rat, an animal model of T2DM, on progression of renal lesions AIM:

10. Animals and experimental design: Divided in 2 subgroups (n = 8 rats) treated with: - Sitagliptin 10 mg/kg/BW/day or or - Vehicle (orange juice) SID (6:00 PM), for 6 weeks ♂ Zucker Diabetic Fatty (ZDF fa/fa) (n=16) Age: 20 weeks Controls: their lean littermates (ZDF +/+) (n=8) Age: 20 weeks

11. Sample Collection and Preparation Blood and tissues - collected at 20 weeks (Ti) and at 26 weeks (Tf) Renal specimens were paraffin-embedded and the 3 µ m thick sections stained for routine histopathological diagnosis with haematoxylin and eosin (HE) and Periodic Acid of Schiff (PAS) All samples were examined by light microscopy using a Zeiss Axioplan 2 microscope

12. Arteriolar hyalinosis Arteriosclerosis. Inflammation Hyaline cylinders Tubular basement membrane irregularity Tubular calcification Interstitial fibrosis/tubular atrophy (IFTA) Histomorphological Evaluation Mesangial expansion GBM thickening Capsule of Bowman thickening Nodular sclerosis Glomerulosclerosis Atrophy Hyalinosis of the vascular pole R E N A L E S I O N S

13. 0 = absent 1 = mild 2 = moderate 3 = severe 0 = < 25% 1 = 25 - 50% 2 = 50 - 75% 3 = > 75 % Scored in single blind fashion Severity Extension Semi-quantitative scoring of lesions (Except IFTA) All lesions were evaluated on the total tissue on the slide. +

14. Arteriolar hyalinosis 0 = absent 1 = one arteriole with hyalinosis was present 2 = more than one arteriole was observed Arteriosclerosis 0 = no intimal thickening was present 1 = intimal thickening was < than media thickness 2 = intimal thickening > than media thickness 0 = absent 1 = < 25%, 2 = 25 - 50% 3 = > 50% (of affected area) IFTA Scoring of lesions VASCULAR

15. RESULTS

16. Sitagliptin Reduced Kidney Oxidative Stress (Positive Impact on Lipid Peroxidation Levels in renal tissues) Tf (26 wks) Ti (20 wks) Treated Diabetic Rats

17. Sitagliptin Reduced Kidney Dysfunction (Decrease of Blood Urea Nitrogen – BUN)

18. Effect of Sitagliptin on Glomerular Lesions Diabetic ZDF (Sita-treated vs Untreated) UNTREATED SITA-TREATED RATS 50µm 25µm

19. Effect of Sitagliptin on Glomerular Lesions Diabetic ZDF (Sita-treated vs Untreated) UNTREATED SITAGLIPTIN-TREATED 50µm 25µm mnng

20. Effect of Sitagliptin on Glomerular Lesions Diabetic ZDF (Sita-treated vs Untreated)

21. SITAGLIPTIN-TREATED Effect of Sitagliptin on Tubulointersticial Lesions Diabetic ZDF (Sita-treated vs Untreated) 50µm 25µm UNTREATED 50µm

22. All Tubulointerstitial Lesions improved Diabetic ZDF (Sita-treated vs Untreated)

23. 25µm The Improvement of Glomerular & Tubulointerstitial Lesions Diabetic ZDF (Sita-treated vs Untreated)

24. Effect of Sitagliptin on Vascular Lesions Diabetic ZDF (Sita-treated vs Untreated) UNTREATED SITA-TREATED ANIMALS 25µm

25. Effect of Sitagliptin on Vascular Lesions Diabetic ZDF (Sita-treated vs Untreated) UNTREATED SITAGLIPTIN-TREATED 25µm

26. Effect of Sitagliptin on Vascular Lesions Diabetic ZDF (Sita-treated vs Untreated) UNTREATED SITAGLIPTIN-TREATED 25µm

27. Effect of Sitagliptin on Vascular Lesions Diabetic ZDF (Sita-treated vs Untreated) UNTREATED SITAGLIPTIN-TREATED 25µm

28. Effect of Sitagliptin on Vascular Lesions Diabetic ZDF (Sita-treated vs Untreated)

29. Conclusions In an animal model of T2DM, a 6 week once daily treatment with a low dose of Sitagliptin promoted : - Glomerulosclerosis - Tubulointerstitial lesions - Vascular lesions IMPROVEMENT OF DIABETIC NEPHROPATHY REGULATION OF DIABETIC DISMETABOLISM Hyperglycaemia Hypertriglyceridaemia RENAL FUNCTION Oxidative stress Blood Urea Nitrogen Has the effect in

30. Conclusions Sitagliptin was able to delay the development of diabetic nephropathy in this model of T2DM, viewed by reduction of renal lesions This effect might be, at least, partially due, to its benefits on correction of diabetes dysmetabolism (hyperglicaemia, dyslipidaemia and insulin production/sensitivity), as well as due to a favorable impact on kidney lipid peroxidation The prevention of diabetic nephropathy evolution might represent a key step forward in the management of T2DM and these serious complications

31. This presentation was supported by the Polytechnic Institute of Viseu, Portugal Muito obrigada! Cristina Mega, Helena Vala, Jorge Oliveira, Rosa Fernandes, Filipa Mascarenhas-Melo, Belmiro Parada, Rui Pinto, Frederico Teixeira, Flávio Reis, Edite Teixeira de Lemos Thank you very much!Paljon kiitoksia