Marked Dysfunction in Gating Properties and Surface Expression of Podocyte TRPC6 Channels in Experimental Models of Primary and Secondary Focal and Segmental Glomerulosclerosis (FSGS) In Vivo and In Vitro
Authors: Dryer Stuart E , Kim Eun Young , Roshanravan Hila 
Published: November 2023

FSGS is one of the most common forms of glomerular disease leading to endstage renal failure. Relatively rare genetic forms of FSGS are caused by mutations in TRPC6 (leading to gain of function of TRPC6 channels). However, the majority of FSGS is not genetic. Primary FSGS, for example, is thought to be induced by a circulating factor, whereas secondary forms of FSGS occur in response to any condition that leads to loss of podocytes. We implemented the chronic puromycin aminonucleoside (PAN) model in Sprague‐Dawley rats to study secondary FSGS. After 90 days, when albuminuria and FSGS was well established, we made whole‐cell recordings from podocytes in acutely isolated glomeruli. We observed that TRPC6 currents evoked by mechanical stimuli (hypoosmotic stretch) were nearly an order of magnitude greater in chronic PAN nephrosis compared to control. By contrast, responses to a diacylglycerol (DAG) analog were almost completely abolished. We have previously reported similar changes in podocyte TRPC6 after podocin knockdown [1]. Total glomerular TRPC6 was markedly increased, whereas podocin expression was markedly decreased in PAN nephrosis in rats. To study primary FSGS, differentiated cells of the mouse MPC‐5 podocyte cell line were treated for 24 hr with serum or plasma from patients with FSGS that recurred after they received a kidney transplant. The FSGS serum samples tested caused large increases in mechanically‐evoked TRPC6 accompanied by loss of responses to a DAG analog. Steady‐state surface abundance of TRPC6 was increased. Most but not all of the FSGS samples caused a loss of podocin. In two cases where we had multiple samples from the same patient at different times, the effects on TRPC6 and podocin correlated with disease severity and were substantially more severe when patients were in relapse. The soluble urokinase/plasminogen activator receptor (suPAR) has been proposed to mediate primary forms of FSGS [2]. We observed that suPAR treatment (10 ng/ml) for 24 hr also caused marked increased in mechanical activation of podocyte TRPC6, loss of responsiveness to DAG, an increase in surface abundance of TRPC6, and a loss of podocin. The effects of a recurrent FSGS plasma were reduced but were not eliminated by a suPAR‐neutralizing antibody that abolished responses to exogenous suPAR. The effects of FSGS plasma and suPAR on surface TRPC6 were inhibited by cilengitide, a blocker of outside‐in signaling through αvβ3 integrin. Podocytes, especially foot processes, are always present in a mechanically dynamic environment in vivo. We propose that loss of podocin and resulting increase in mechanical activation of TRPC6 may be a common pathway to chronic Ca2+‐overload in most forms of TRPC6, including genetic forms associated with mutations in NPHS2 and TRPC6, secondary forms that occur in late stages of other kidney diseases, and in many cases of primary FSGS that occur in response to circulating factors. We further propose that recurrent FSGS is heterogeneous, and that more than one circulating factor may be driving changes in TRPC6. If these hypotheses are true, agents that inhibit mechanical activation of TRPC6, or that block pathways leading to TRPC6 function may be useful agents for treatment of FSGS of multiple etiologies.

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