2.2.1 Experimental design
These studies were carried out in 16 week-old, male obese ZSF1 rats (ZSF1-LeprfaLeprcp/Crl; strain code 378) and lean ZSF1 rats (strain code 379) obtained from Charles River Laboratories. The ZSF1 rat is a cross between Zucker Diabetic Fatty (ZDF) female and Spontaneously Hypertensive Heart Failure (SHHF) male rats. These rats are known to be an excellent model of diabetic nephropathy (Bilan et al., 2011).
As we have described in a recent study, at 16 weeks of age the obese ZSF1 rats were diabetic, hypertensive, and had renal injury compared to the age-matched lean ZSF1 rats (Hye Khan et al., 2018). Diabetes (plasma glucose >250mg/dL), hypertension (SBP >150mmHg), and proteinuria (>150mg/d) were confirmed in the obese ZSF1 rats. The rats were randomized into four groups (n=6-8/group), Group 1: Lean ZSF1 rats treated with vehicle; Group 2: obese ZSF1 rats treated with vehicle; Group 3: obese ZSF1 rats treated with PTUPB (10mg/kg/d i.p.), and Group 4: obese ZSF1 rats treated with enalapril (10mg/kg/d p.o.). PTUPB administration was carried out using an intra-peritoneal implanted osmotic pump (ALZET® osmotic pump, DURECT Corporation,Cupertino, CA). All rats were weighed, and systolic blood pressure was measured by tail-cuff plethysmography (IITC Life Science Inc., Woodland Hills, CA, USA) during the 8 week treatment protocol.
At baseline, intraperitoneal glucose tolerance tests, blood pressure measurements, and urine collections were conducted. At the end of the 8 week treatment protocol parameters were measured and rats were euthanized for plasma and tissue collection. Urine, blood, plasma, kidney, and liver samples were frozen and stored at -800C for biochemical assays and other analysis. The kidney and liver tissue samples were either immersion fixed in 10% neutral buffered formalin for paraffin embedding or processed for frozen section for use in histopathology and immunohistopathological experiments.