Abundance of TRPC6 protein in glomerular mesangial cells is decreased by ROS and PKC in diabetes

Graham S, Gorin Y, Abboud HE, Ding M, Lee DY, Shi H, Ding Y, Ma R. Abundance of TRPC6 protein in glomerular mesangial cells is decreased by ROS and PKC in diabetes. Am J Physiol Cell Physiol 301: C304-C315, 2011. First published April 27, 2011; doi: 10.1152/ajpcell.00014.2011.-The present study was performed to investigate the underlying mechanism, particularly the roles of reactive oxygen species (ROS) and protein kinase C (PKC), in the diabetes-induced canonical transient receptor potential 6 (TRPC6) downregulation. We found that high glucose (HG) significantly reduced TRPC6 protein expression in cultured mesangial cells (MCs). TRPC6 protein was also significantly reduced in the glomeruli but not in the heart or aorta isolated from streptozotocin-induced diabetic rats. In the cultured MCs, H2O2 suppressed TRPC6 protein expression in a dose-and time-dependent manner, which emulated the HG effect. Catalase as well as superoxide dismutase were able to prevent the inhibitory effect of HG on TRPC6. The antioxidant effect observed in cultured cells was also observed in diabetic rats treated with tempol for 2 wk, which exhibited a preservation of TRPC6 in the glomeruli. Specific knockdown of Nox4, a component of NADPH oxidase, increased TRPC6 protein expression. Furthermore, the PKC activator phorbol 12-myristate 13-acetate (PMA), but not its analog 4 alpha-phorbol 12, 13-didecanoate (4 alpha-PDD), suppressed TRPC6 expression, and this PMA effect was not affected by catalase. Moreover, Go6976, but not LY333531, attenuated the negative effect of HG on TRPC6 expression. Go6976 also inhibited H2O2 effect on TRPC6. Furthermore, either knockdown of TRPC6 or HG treatment significantly decreased ANG II-stimulated MC contraction, and the HG-impaired MC contraction was rescued by overexpression of TRPC6. These results suggest that hyperglycemia in diabetes downregulated TRPC6 protein expression in MCs through a NADPH oxidase Nox4-ROS-PKC pathway, proving a mechanism for impaired MC contraction in diabetes.