title: Insulin Increases Expression of TRPC6 Channels in Podocytes by a Calcineurin-Dependent Pathway.
abstract: OBJECTIVE
  Insulin signaling to podocytes is relevant for the function of the glomerulus. Now, we tested the hypothesis that insulin increases the surface expression of canonical transient receptor potential canonical type 6 (TRPC6) channels in podocytes by a calcineurin-dependent pathway.
  
  
  METHODS
  We used quantitative RT-PCR, immunoblotting, immunofluorescence and fluorescence spectrophotometry in cultured podocytes. Activation of Nuclear Factor of Activated T-cells (NFATc1) was measured using a specific calorimetric assay.
  
  
  RESULTS
  Insulin increased the expression of TRPC6 transcripts and protein in podocytes. Insulin increased TRPC6 transcripts in a time and dose-dependent manner. The insulin-induced elevation of TRPC6 transcripts was blocked in the presence of tacrolimus, cyclosporine A, and NFAT-inhibitor (each p < 0.01 by ANOVA and Bonferroni's multiple comparison test). Transcripts of NOX4, another target gene of the calcineurin-NFAT pathway, were affected in a similar way. Immunoblotting showed that the administration of 100 nmol/L insulin increased TRPC6-proteins 2-fold within 48 hours. Insulin increased the activity of NFATc1 in nuclear extracts (p < 0.001) whereas tacrolimus, cyclosporine A, and NFAT-inhibitor blocked that insulin effect (p < 0.001; two way ANOVA). Immunofluorescence showed that insulin increased TRPC6-expression on the cell surface. Fluorescence-spectrophotometry and manganese quench experiments indicated that the increased TRPC6-expression after insulin administration was accompanied by an elevated transplasmamembrane cation influx. Insulin-stimulated surface expression of TRPC6 as well as transplasmamembrane cation influx could be reduced by pretreatment with tacrolimus.
  
  
  CONCLUSIONS
  Insulin increases the expression of TRPC6 channels in podocytes by activation of the calcineurin-dependent pathway.
authors: Odense University Hospital, Department of Nephrology, and University of Southern Denmark, Institute for Molecular Medicine, Cardiovascular and Renal Research, Institute of Clinical Research, Odense, Denmark.
authors: Shengqiang
authors: S
authors: Xia
authors: Ying
authors: Y
authors: Liu
authors: Xinming
authors: X
authors: Li
authors: Florian
authors: F
authors: Thilo
authors: Martin
authors: M
authors: Tepel
mesh_terms: D019703:Calcineurin / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D007328:Insulin / Q000378:metabolism* / Q000494:pharmacology; D050199:Podocytes / Q000187:drug effects / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D050052:TRPC Cation Channels / Q000032:analysis / Q000235:genetics*; D000074287:TRPC6 Cation Channel; D015533:Transcriptional Activation / Q000187:drug effects; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D007328:Insulin; D050052:TRPC Cation Channels; D000074287:TRPC6 Cation Channel; C117032:TRPC6 protein, human; D019703:Calcineurin