Antioxidant diet, gender and age affect renal expression of nitric oxide synthases in obese diabetic rats

Development of diabetic nephropathy (DN) is associated with decreased renal nitric oxide production and increased oxidative stress. We studied nitric oxide synthase (NOS) expression in kidney of obese Zucker fa/fa rats, a model of Type 2 obesity-related DN. Male and female rats received a regular (REG) or antioxidant-fortified (AO) diet starting at age 4weeks. Quantitative PCR and immunoblot analyses were performed on kidney cortex and medulla to determine levels of endothelial, neuronal and inducible NOS at 6, 13 and 20weeks of age. Multiple antibody-specific proteins were detected for each form. These may represent monomeric splice forms, post-translationally modified forms and their dimers, consistent with the known complexity of regulation of these enzymes. Levels of eNOS and nNOS are higher in males than females at 6weeks on the REG diet and 13weeks on either diet; the relationship is reversed in females at 6weeks on the AO diet. Levels of eNOS and nNOS are lower on the AO diet compared to REG, in males at 6 and 13weeks and females at 13weeks; the reverse is seen in 6week females and 20week males. All three isoforms show peak levels in the younger animals, at 6 or 13weeks. Better preservation of kidney function is associated with higher prevalence of dimers with potential to increase production of NO and lower levels of potentially harmful monomers. Differential expression of NOS isoforms may be linked to renal functional and histopathological changes in this rat model of DN.