REGULATION OF GLOMERULAR FILTRATION RATE BY CANONICAL TRANSIENT RECEPTOR POTENTIAL (TRPC) 6 IN MESANGIAL CELLS

Purpose: Canonical transient receptor potential (TRPC) 6 affects glomerular filtration of proteins. Whether this protein also regulates other glomerular functions is not known. The present study was conducted to test a hypothesis that TRPC6 regulated glomerular filtration rate (GFR) by regulating the contractile function of glomerular mesangial cells (MCs). Methods: GFR in mice was estimated using a two-compartment clearance model. 5% FITC-inulin (3.74 µl/g body weight) was retroorbitally injected and blood samples were periodically collected from the tail vein under the conscious state. GFR in rats was evaluated based on the plasma inulin clearance during intravenous infusion of 1% FITC-inulin at a rate of 1 ml/h/100 g body weight. Blood samples were collected from the left common carotid artery and urine samples were collected from either the bladder or the urether under the anesthetized state (sodium pentobarbital, 50 mg/kg body weight, i.p). In vivo knockdown of TRPC6 in rat kidneys was achieved by injection of shRNA constructs against rat TRPC6 via the left renal artery. Ca2+ imaging in combination of molecular and pharmacological tools were employed to determine a role of TRPC6 in Ca2+ signaling in MCs. Furthermore, MC contractile response was assessed by measuring a change in the planar surface area of cultured MCs in response to Ang II stimulation. Results: We found that GFR in TRPC6 knockout mice was remarkably elevated compared to wild type mice. In rats, acute infusion of Ang II significantly reduced GFR. However, the Ang II-induced GFR decrease was abolished by local knockdown of TRPC6 in kidneys. Immunohistochemistry showed that MCs were the predominantly transfected glomerular cells. Consistently, the GFR of the left kidney (the transfected kidney) significantly decreased by Ang II when transfected with control vectors, but did not have a significant change when transfected with shRNA against TRPC6. There were no significant differences in arterial blood pressure and renal blood flow between TRPC6-knocked down and their control rats. Ca2+ imaging experiments showed that the Ang II-stimulated Ca2+ entry was significantly inhibited in MCs isolated from TRPC6 knockout mice and in human MCs with TRPC6 knocked down. Moreover, the Ang II-stimulated contraction was significantly suppressed in TRPC6 deficient mouse MCs. Conclusions: These results suggest that TRPC6 channel is an important component regulating GFR by modulating MC Ca2+ signaling and contraction.