Insulin Increases Expression of TRPC6 Channels in Podocytes by a Calcineurin-Dependent Pathway.

BACKGROUND/AIMS: 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.

CONCLUSION

Insulin increases the expression of TRPC6 channels in podocytes by activation of the calcineurin-dependent pathway.