JDRF Albert Einstein Centre for Diabetes Complications, Diabetes and Metabolism Division, Baker Medical Research Institute, P.O. Box 6492, St. Kilda Rd., Central, Melbourne, Victoria, Australia. vicki.thallas@baker.edu.au
OBJECTIVE: Excessive production of reactive oxygen species (ROS) via NADPH oxidase has been implicated in the pathogenesis of diabetic nephropathy. Since NADPH oxidase activation is closely linked to other putative pathways, its interaction with changes in protein kinase C (PKC) and increased advanced glycation was examined. RESEARCH DESIGN AND METHODS: Streptozotocin-induced diabetic or nondiabetic Sprague Dawley rats were followed for 32 weeks, with groups randomized to no treatment or the NADPH oxidase assembly inhibitor apocynin (15 mg . kg(-1) . day(-1); weeks 16-32). Complementary in vitro studies were performed in which primary rat mesangial cells, in the presence and absence of advanced glycation end products (AGEs)-BSA, were treated with either apocynin or the PKC-alpha inhibitor Ro-32-0432. RESULTS; Apocynin attenuated diabetes-associated increases in albuminuria and glomerulosclerosis. Circulating, renal cytosolic, and skin collagen-associated AGE levels in diabetic rats were not reduced by apocynin. Diabetes-induced translocation of PKC, specifically PKC-alpha to renal membranes, was associated with increased NADPH-dependent superoxide production and elevated renal, serum, and urinary vascular endothelial growth factor (VEGF) concentrations. In both diabetic rodents and in AGE-treated mesangial cells, blockade of NADPH oxidase or PKC-alpha attenuated cytosolic superoxide and PKC activation and increased VEGF. Finally, renal extracellular matrix accumulation of fibronectin and collagen IV was decreased by apocynin. CONCLUSIONS: In the context of these and previous findings by our group, we conclude that activation of NADPH oxidase via phosphorylation of PKC-alpha is downstream of the AGE-receptor for AGE interaction in diabetic renal disease and may provide a novel therapeutic target for diabetic nephropathy.