Pathogenesis of diabetic nephropathy (DN) involves multiple mechanisms initiated by hyperglycemia. Excessive oxidative stress, growth factor signaling related extracellular matrix expansion and renal cell apoptosis contributeto the progression of DN.
Key enzymes in the maintenance of oxidative balance are the nitric oxide synthases (NOS) including endothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS). This study measuredNOSmRNAs in kidneys of Zucker rats, an established model for obesity-related type 2 diabetes, comparing age, diet and gender effects. The data showed that in the early stage of DNeNOS and nNOSwere higher than in the late stage, suggesting an early physiological attempt to neutralize the increased oxidative stress induced by hyperglycemia. An antioxidant-fortified diet(AOdiet) loweredeNOS and nNOS in malesbut increased both in females, suggesting a beneficial response in females. Male rats had the tendency to have higher eNOS and nNOS than females in the younger age groups while in older rats the effect was the opposite, showing male rats had a greater early response and female gender was protective against early oxidative stress.
Previous studies have shown that vascular endothelial growth factor (VEGF) signaling played a role in the progression of DN. In this study, VEGF and VEGF receptor (Flt_1) did not show anydifferences between groups, indicating VEGF signaling may not be a major factor in DN in theZucker rat model. However, nephrin and podocin, indicators of healthiness of the renal filtration system, changed in different age, diet and gender groups. Rats of 20 weeks had a lower level ofpodocin compared to the 6 week rats, which was consistent with loss of the beneficial effect of podocytes. AO diet fed rats had higher levels of podocin compared to ratson regular (REG) diet, suggesting the AO diet may reduce the podocyte apoptosis. Female rats had higher levels of podocin and nephrin mRNA, showing again the protective effect of female gender.
Preoptic Area Regulatory Factor-2 (PORF-2) is a RhoGAP domain-containing protein that has been shown to inhibit proliferation and enhance apoptosis in neuronal stem cells. It is expressed in renal cortex and medulla, but the role of PORF-2 in DN has not been previously investigated. In this study, Zucker rats were used to examine the role of PORF-2 in DN in vivo as well as the Fischer ratthyroid cell line (FRTL-5) to study the expression regulation pathways for PORF-2in vitro.The results showed female rats on an AO diet had higher PORF-2 at 6 weeks of age than at 20 weeks. At 6 weeks, females on the REG diet had lower levels of PORF-2 mRNA than females on the AO diet,while at the age of 20 weeks, females on the REG diet had higher PORF-2 mRNA compared to females on the AO diet. At age of 6 weeks, females fed with the AO diet had higher levels of PORF-2 than their male counterparts. In vitro, it was demonstrated that both insulin and insulin like growth factor-1 (IGF-1) were able to suppress expression of PORF-2 mRNA. Inhibitors of Phosphoinositide 3-Kinases (PI3K), Protein Kinase B (AKT), Raf and Mitogen-Activated Protein Kinase Kinase(MAPKK) partially reversed the suppression effect of IGF-1 on PORF-2. In conclusion, PORF-2 plays a role in DN and its expression is regulated through both AKT and MAPK pathways.