High glucose modifies transient receptor potential canonical type 6 channels via increased oxidative stress and syndecan-4 in human podocytes

Transient receptor potential canonical (TRPC) channels type 6 play an important role in the function of human podocytes. Diabetic nephropathy is characterized by altered TRPC6 expression and functions of podocytes. Thus, we hypothesized that high glucose modifies TRPC6 channels via increased oxidative stress and syndecan-4 (SDC-4) in human podocytes. Human podocytes were exposed to control conditions (5.6 mmol/L D-glucose), high glucose (30 mmol/L D-glucose or L-glucose), 100 mu mol/L peroxynitrite, or high glucose and the superoxide dismutase mimetic tempol (100 mu mol/L). TRPC6 and SDC-4 transcripts and protein expression were measured using RT-PCR and in-cell Western assay. Intracellular reactive oxygen species (ROS) and cytosolic calcium were measured using fluorescent dye techniques. High D-glucose increased TRPC6 transcripts to 8.66 +/- 4.08 (p < 0.05) and TRPC6 protein expression to 1.44 +/- 0.07 (p < 0.05) without altering SDC-4 transcripts or protein expression. The D-glucose induced increase of TRPC6 expression was blocked by tempol. Increased oxidative stress using peroxynitrite significantly increased TRPC6 transcripts to 4.29 +/- 1.26 (p < 0.05) and TRPC6 protein expression to 1.28 +/- 0.05 (p < 0.05) without altering SDC-4 transcripts or protein expression. In human podocytes transfected with scrambled siRNA, high D-glucose increased ROS after 90 min to 3.55 +/- 0.08 arbitrary units while 5.6 mmol/L D-glucose increased ROS to 2.49 +/- 0.09 (p < 0.001) only. The increase in ROS was inhibited by tempol and by SDC-4 knockdown. High glucose modifies TRPC6 channels and ROS production via SDC-4 in human podocytes. (C) 2014 Elsevier Inc. All rights reserved.