Oxidative stress is an important factor in the pathogenesis of diabetic nephropathy (PDN). One of the consequences of oxidative injury is activation of the nuclear enzyme, poly(ADP-ribose) polymerase (PARP). It is triggered by free radical and oxidant-induced DMA single-strand breakage and leads to 1) energy failure; 2)down-regulation of glyceraldehyde 3-phosphate dehydrogenase with resulting activation of three pathways contributing to diabetic complications i.e. non-enzymatic glycation, protein kinase C and hexosamine pathway; and 3) changes in transcriptional regulation and gene expression. Evidence for the role for PARP activation in the pathogenesis of diabetic complications e.g., endothelial dysfunction, peripheral and autonomic neuropathy, and retinopathy, is emerging; however, the role for PARP in diabetic nephropathy remains unexplored. The overall hypothesis of this proposal is that PARP activation is an important mechanism in diabetic renal disease.
The specific Aims are 1) to determine whether two structurally unrelated PARP inhibitors counteract development of diabetic nephropathy in streptozotocin-diabetic rats; 2) to compare severity of diabetic nephropathy in PARP-1 deficient (PARP-/-) mice and the wild-type mice with normal PARP content; and 3) to identify contribution of two enzymes of the sorbitol pathway, aldose reductase and sorbitol dehydrogenase, and individual reactive oxygen species to high glucose-induced PARP activation in human mesangial cells. In animal experiments, we will determine PARP activity in glomerular and tubular compartments of the renal cortex, as well as the following markers of diabetic nephropathy: urinary albumin excretion, glomerular volume, renal endothelin-1 content and endothelin-1 and endothelin receptor mRNA expression, renal vascular endothelial growth factor expression, glomerular transforming growth factor-beta and collagen alpha1(IY) immunoreactivities, plasma creatinine levels, glomerular mesangial matrix expansion and glomerulosclerosis. In cell culture studies, we will evaluate intracellular oxidative stress (DCF fluorescence test/flow cytometry), PARP activation (CELISA) and sorbitol pathway intermediates (enzymatic spectrofluorometric assays).The studies will generate novel information regarding the role for PARP in the pathogenesis of diabetic nephropathy and will provide rationale for development of PARP inhibitors for prevention and treatment of this devastating complication of diabetes mellitus.
