TRPC6 effects on albumin permeation, nephrin shedding, and apoptosis in podocytes: Role of calcineurin and metalloproteases

TL;DR

This study explores how TRPC6 activation affects podocyte function, focusing on albumin permeation, nephrin shedding, and cell death over different time periods.

Contribution

The study reveals the time-dependent effects of TRPC6 activation on podocyte permeability, nephrin shedding, and apoptosis, and identifies calcineurin and metalloproteases as key mediators.

Findings

TRPC6 activation for 24 h increases nephrin shedding and MMP-7 levels, blocked by calcineurin and metalloprotease inhibitors.

TRPC6 activation for 72 h induces apoptosis, which is inhibited by SAR-7334 and cyclosporin A.

TRPC6 activation does not induce pyroptosis, as indicated by unchanged cleaved gasdermin D levels.

Abstract

The effects of TRPC6 activation for various periods of time on three classes of functional outputs were examined in cultured podocytes: albumin permeation across a confluent layer; changes in nephrin dynamics; and cell death. Albumin permeation in transwell assays was significantly increased within 1 h in response to the activation of formyl peptide receptors (FPR), but the TRPC6 inhibitor SAR‐7334 had no effect on this response, and 1 h or 24 h exposures to the TRPC6 activator PPZ2 did not increase albumin permeation. Direct TRPC6 activation for 24 h evoked an increase in shedding of nephrin ectodomains into the surrounding media, accompanied by an increase in matrix metalloprotease‐7 (MMP‐7). These effects were blocked by the calcineurin inhibitor cyclosporin A (CsA), as well as by Batimastat, a broad‐spectrum inhibitor of metalloproteinases including MMP‐7. TRPC6 activation for 24 h also evoked an increase in occludin abundance but had no effect on the abundance of podocin. Finally, TRPC6 activation for 72 h, but not for 24 h, evoked an increase in apoptotic cell death based on increases in cleaved caspase‐3. This effect was blocked by both SAR‐7334 and CsA. TRPC6 activation did not induce pyroptosis based on the measurement of cleaved gasdermin D.

Certain ligands of pattern recognition receptors can induce a rapid increase in albumin permeability across a podocyte monolayer that can be seen in 1 h, but activation of TRPC6 for up to 24 h does not mimick this process. However, continuous TRPC6 activation for 24 h results in nephrin ectodomain shedding that calcineurin activation and metalloproteinases and that is is accompanied by an increase in overall abundance of occludin and matrix metalloprotreinase‐7. Finally, continuous TRPC6 activation for 72 h induces apoptosis as reported earlier, but does not induce pyroptosis.

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