Synaptopodin Limits TRPC6 Podocyte Surface Expression and Attenuates Proteinuria.

Gain-of-function mutations of classic transient receptor potential channel 6 (TRPC6) were identified in familial FSGS, and increased expression of wild-type TRPC6 in glomeruli is observed in several human acquired proteinuric diseases. Synaptopodin, an actin binding protein that is important in maintaining podocyte function, is downregulated in various glomerular diseases. Here, we investigated whether synaptopodin maintains podocyte function by regulating podocyte surface expression and activity of TRPC6. We show indirect interaction and nonrandom association of synaptopodin and TRPC6 in podocytes. Knockdown of synaptopodin in cultured mouse podocytes increased the expression of TRPC6 at the plasma membrane, whereas overexpression of synaptopodin decreased it. Mechanistically, synaptopodin-dependent TRPC6 surface expression required functional actin and microtubule cytoskeletons. Overexpression of wild-type or FSGS-inducing mutant TRPC6 in synaptopodin-depleted podocytes enhanced TRPC6-mediated calcium influx and induced apoptosis. In vivo, knockdown of synaptopodin also caused increased podocyte surface expression of TRPC6. Administration of cyclosporin A, which stabilizes synaptopodin, reduced LPS-induced proteinuria significantly in wild-type mice but to a lesser extent in TRPC6 knockout mice. Furthermore, administration of cyclosporin A reversed the LPS-induced increase in podocyte surface expression of TRPC6 in wild-type mice. Our findings suggest that alteration in synaptopodin levels under disease conditions may modify intracellular TRPC6 channel localization and activity, which further contribute to podocyte dysfunction. Reducing TRPC6 surface levels may be a new approach to restoring podocyte function.

Transient receptor potential channel 1 maintains adherens junction plasticity by suppressing sphingosine kinase 1 expression to induce endothelial hyperpermeability.

Stability of endothelial cell (EC) adherens junctions (AJs) is central for prevention of tissue edema, the hallmark of chronic inflammatory diseases including acute respiratory distress syndrome. Here, we demonstrate a previously unsuspected role of sphingosine kinase 1 (SPHK1) in the mechanism by which transient receptor potential channel 1 (Trpc1)-mediated Ca(2+) entry destabilizes AJs. Trpc1(-/-) monolayers showed a 2.2-fold increase in vascular endothelial (VE)-cadherin cell-surface expression above wild-type (WT) monolayers. Thrombin increased endothelial permeability (evident by a 5-fold increase in interendothelial gap area and 60% decrease in transendothelial electrical resistance) in WT but not Trpc1(-/-) ECs. Trpc1(-/-) mice resisted the hyperpermeability effects of the edemagenic agonists used and exhibited 60% less endotoxin-induced mortality. Because sphingosine-1-phosphate (S1P) strengthens AJs, we determined if TRPC1 functioned by inhibiting SPHK1 activity, which generates S1P. Intriguingly, Trpc1(-/-) ECs or ECs transducing a TRPC1-inactive mutant showed a 1.5-fold increase in basal SPHK1 expression compared with WT ECs, resulting in a 2-fold higher S1P level. SPHK1 inhibitor SK1-I decreased basal transendothelial electrical resistance more in WT ECs (48 and 72% reduction at 20 and 50 µM, respectively) than in Trpc1(-/-) ECs. However, SK1-I pretreatment rescued thrombin-induced EC permeability in Trpc1(-/-) ECs. Thus, TRPC1 suppression of basal SPHK1 activity enables EC-barrier destabilization by edemagenic agonists.