Inhibition of TRPC6 by protein kinase C isoforms in cultured human podocytes

Abstract

Transient receptor potential canonical‐6 (TRPC6) ion channels, expressed at high levels in podocytes of the filtration barrier, are recently implicated in the pathogenesis of various forms of proteinuric kidney diseases. Indeed, inherited or acquired up‐regulation of TRPC6 activities are suggested to play a role in podocytopathies. Yet, we possess limited information about the regulation of TRPC6 in human podocytes. Therefore, in this study, we aimed at defining how the protein kinase C (PKC) system, one of the key intracellular signalling pathways, regulates TRPC6 function and expression. On human differentiated podocytes, we identified the molecular expressions of both TRPC6 and several PKC isoforms. We also showed that TRPC6 channels are functional since the TRPC6 activator 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG) induced Ca2+‐influx to the cells. By assessing the regulatory roles of the PKCs, we found that inhibitors of the endogenous activities of classical and novel PKC isoforms markedly augmented TRPC6 activities. In contrast, activation of the PKC system by phorbol 12‐myristate 13‐acetate (PMA) exerted inhibitory actions on TRPC6 and suppressed its expression. Importantly, PMA treatment markedly down‐regulated the expression levels of PKCα, PKCβ, and PKCη reflecting their activation. Taken together, these results indicate that the PKC system exhibits a ‘tonic’ inhibition on TRPC6 activity in human podocytes suggesting that pathological conditions altering the expression and/or activation patterns of podocyte‐expressed PKCs may influence TRPC6 activity and hence podocyte functions. Therefore, it is proposed that targeted manipulation of certain PKC isoforms might be beneficial in certain proteinuric kidney diseases with altered TRPC6 functions.

Human podocytes express functional thermosensitive TRPV channels

“…The human podocyte cell line provided by Prof. Hermann Pavenstädt (University Hospital of Münster, Münster, Germany) was established and cultured as described previously (Saleem et al, ; Ambrus et al, ). In brief, cells were cultured in ‘permissive’ condition in RPMI medium (PAA Laboratories GmbH, Pasching, Austria) supplemented with 10% fetal bovine serum (Invitrogen, Paisley, UK), 50 U·mL −1 penicillin, 50 μg·mL −1 streptomycin, 1.25 μg·mL −1 Fungizone (both from PPA Laboratories GmbH) and insulin‐transferrin‐selenium (1:100; Invitrogen) at 33°C to maintain proliferation.…”

“…Differentiation was induced by switching to ‘non‐permissive’ condition transferring cells to 37°C and kept in culture for 7 days. The process of differentiation was evaluated using Western blotting and immunocytochemistry by determining the expression of the podocyte‐specific marker podocin and the differentiation marker synaptopodin as shown in our previous work (Ambrus et al, ). HEK293T cells were cultured in DMEM medium (PAA Laboratories GmbH) supplemented with 10% fetal bovine serum (Invitrogen), 50 U·mL −1 penicillin, 50 μg·mL −1 streptomycin, 1.25 μg·mL −1 Fungizone (both from PPA Laboratories GmbH) and non‐essential aminoacids (Sigma‐Aldrich, St Louis, MO, USA).…”

Pharmacological inhibition of focal segmental glomerulosclerosis‐related, gain of function mutants of TRPC6 channels by semi‐synthetic derivatives of larixol

“…Compatible with the author's description of a twofold elevation of TRPC6 mRNA levels in glomeruli from this mouse and of low abundance of the TRPC6 protein (Krall et al, 2010), a mild podocyte-specific increase in the expression of TRPC6 channels may still not suffice to augment TRPC6-like Ca 2+ influx signal above detection thresholds. Of note, all studies that demonstrate a robust OAG-mediated Ca 2+ influx in podocyte cultures used immortalized mouse or human podocyte cell lines rather than primary cultures (Möller et al, 2007;Nijenhuis et al, 2011;Yang et al, 2013;Sonneveld et al, 2014;Ambrus et al, 2015).…”

Mechanism of Regulation of Big-Conductance Ca2+-Activated K+ Channels by mTOR Complex 2 in Podocytes

“…Next, we used GF109203X (bisindolylmaleimide I), a PKC inhibitor, to test whether PKCα takes part in regulating BK channel protein expression in podocytes (Lee et al, 2006; Ambrus et al, 2015; Yao et al, 2015). We treated podocytes with 0.5, 1, 2, 4, or 8 μM GF109203X for 24 h. We found GF109203X decreased the expression of p-PKCα S657/Y658, but had no influence on the BK channel protein levels (Figure 7C).…”