#395 Diabetic nephropathy evaluation in an non-genotypic animal model of type 2 diabetes

Abstract Background and Aims The pathogenesis of diabetic nephropathy (DN) is not completely known. This could be related with a lack of pre-clinical models. Current animal models, mostly genotypic and monogenic, do not fully reflect diabetic organ damage. In previous studies of our group we observed that Tacrolimus (TAC) induce a beta-cell toxicity in the context of obesity that resembles the pathogenesis of diabetes [1–3]. We proposed a new non-genotypic animal model of type 2 diabetes in which evaluate the DN. Method 26 Sprague Dawley male rats were fed with high fat diet to induce obesity and metabolic syndrome (MS) during 30 days. Then, animals were randomized to TAC or placebo during 6–9 months. The first 15 days animals were treated daily with a dose of 0.5 mg/kg. The next 30 days, animals were treated 3 times/week with 0.5 mg/kg of TAC. Then, until month 6 animals were treated 2 times/week with 0.5 mg/kg. At months 6 to 9, the dose used was 0.5 mg/kg 1 times/week. Animals continued with HFD until the end. Weight control was performed weekly. Blood levels of TAC were measured every month. Hyperglycemia and insulin resistance were analyzed regularly with fasting glucose, intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT). Glomerular filtration rate (GFR) was measured by iohexol-DBS plasma clearance. Urine at 24 hours was collected to analyze albuminuria and proteinuria. At the endpoint, renal morphology histology was analyzed (Fig 1A). Results HFD induced obesity and MS in all animals. Then, animals on TAC, compared with those in vehicle, developed diabetes at baseline and after glucose load since day 15 to 9 months. In the pancreas, diabetic animals on TAC showed characteristics similar to those observed in diabetic patients (e.g. decrease in islet number and area, fibrosis, disbalance in alpha/beta cells number) (data not shown). Blood levels of TAC at 24 h were 7.5 ± 2.4 ng/mL the first 15 days, 5.1 ± 1.8 ng/mL on month 1, 4.3 ± 1.4 ng/mL on month 3, 4 ± 1.6 ng/mL on month 6 and 0.9 ± 0.2 ng/mL on month 9. Animals increased the GFR after 30 days of HFD. Then, GFR continue to increase up to 6 months. From 6 months on, GFR started to decline. Animals on TAC showed higher GFR than control. In the kidney, diabetic animals on TAC showed increased glomerular area (TAC: 5260.6 ± 1922.8 pixel/µm vs. Control: 1910.6 ± 1051,7 pixel/µm, P ≤ 0.0001), mesangial expansion (P ≤ 0.0001) and fibrosis intraglomerular (TAC: 17.9 ± 16.5 % vs. Control: 8.8 ± 6 %, P ≤ 0.0002) and a higher proportion of glomeruli with nodular sclerosis (TAC: 34.8 ± 9.1 glomeruli (14%) vs. Control: 22.6 ± 4.3 glomeruli (6%), P ≤ 0.0043). Proteinuria was increased in diabetic animals (3932,8 ± 2872 mg/dL vs. 1257,7 ± 569,4 mg/dL, n.s.) (Fig. 1B). Conclusion in an animal model of chronic diabetes induced by Tacrolimus in a context of obesity and metabolic syndrome we observed markers of diabetic kidney disease: hyperfiltration and GFR decline, proteinuria, increased mesangial expansion and nodular sclerosis. This model can be used for studying the pathogenesis of DKD and as a platform for testing new drugs.