Rac GTPase-mediated NADPH oxidase redox signaling in hyperhomocysteinemic glomerular injury

Hyperhomocysteinemia (hHcys) is emerging as a critical pathogenic factor in the progression of end stage renal disease (ESRD) and in the development of cardiovascular complications related to ESRD. Previous studies in our laboratory have demonstrated that Hcys acutely increases the production of superoxide (O2·-) via NADPH oxidase and thereby stimulates the formation of tissue inhibitor of metalloproteinase-1 (TIMP-1) in renal mesangial (MG) cells, ultimately resulting in the deposition of collagen. However, the mechanism by which Hcys activates NADPH oxidase in these glomercular cells and whether this signaling pathway contributes to glomerular injury induced by hHcys in vivo remains unknown. Ceramide is a critical signaling molecule that mediates the activation of NADPH oxidase in different cells. NADPH oxidase contains the small G protein, Rac GTPase, which possesses a high affinity for lipid binding and plays an important role in the regulation of this enzyme. In the present study, we performed a series of in vitro and in vivo experiments to test the hypothesis that Hcys-induced ceramide production activates Rac-GTPase and thereby enhances NADPH oxidase activity and O2·- production in the cultured mesangial cells and the glomeruli of hyperhomocysteinemic rats, consequently resulting in glomerulosclerosis.; Thin Layer Chromatography (TLC) analysis demonstrated that L-Hcys increased de novo production of ceramide in rat MG cells. L-Hcys and increased ceramide did not alter the amount of NADPH oxidase subunit p47 phox and p67phox in both membrane and cytosolic fractions from MG cells. However, L-Hcys or ceramide markedly increased Rac GTPase activity, which was accompanied by enhanced activity of NADPH oxidase. These Hcys or ceramide-induced actions were substantially inhibited by the Rac GTPase inhibitor, GDPbetaS and the de novo ceramide synthesis inhibitor, fumonisin B1 (FB1 ). These results indicate that Hcys activates NADPH oxidase by stimulating de novo ceramide synthesis and consequent activation of Rac GTPase in rat MG cells.; To determine the role of Rac GTPase-NADPH oxidase activity in the mechanisms of Hcys-induced reduction of MMP-1 activity in these cells, RNA interference by small inhibitory RNA (siRNA) duplex was employed to reduce Rac expression and activity in rat MG cells. (Abstract shortened by UMI.)...