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.