Activation of TRPC6 by AngII Induces Podocyte Injury and Participates in Proteinuria of Nephrotic Syndrome

Background: Nephrotic syndrome (NS) is a common glomerular disease, and podocyte injury is the character of primary NS, usually caused by minimal change disease and membranous nephropathy. Podocytopathy is primarily associated with glomerular proteinuria. Losartan, an angiotensin receptor blocker (ARB), is commonly used in the treatment of NS, and the AngiotensinII (AngII)-transient receptor potential ion channel 6 (TRPC6) axis has been reported to act on podocytes to regulate proteinuria in NS. Therefore, the purpose of this study was to explore the relationship in between AngII-TRPC6, podocyte injury, and proteinuria based on the adriamycin (ADR) NS rat model.Method: All male rats were divided into three groups: control group, model group, and ARB group. The rats in the model group were induced by ADR, and the rats in the ARB group received losartan after induction of renal injury for 4 weeks. The changes in parameters related to renal dysfunction, and glomerular and podocyte structural damage, such as AngII, AngII type I receptor (AT1R), TRPC6, CaN, Caspase-3, Nephrin, and Podocin, were analyzed. Furthermore, the kidneys were isolated for study via transmission electron microscopy (TEM), immunohistochemistry, and western blot (WB) after the rats were sacrificed. In vitro, immortalized mouse MPC5 podocytes were used to investigate the regulatory effect of flufenamic acid (Flu) and SAR7334 (SAR) on the AngII-TRPC6 signaling axis. Flow cytometry and WB were conducted to determine the relationship between podocyte injury and AngII-TRPC6.Results: In vivo results showed that NS rats developed massive albuminuria and abnormal renal function, accompanied by abnormally increased levels of AngII, TRPC6, AT1R, and CaN and a decreased expression of actin molecules in podocytes, extensive fusion of foot processes (FP), loss of glomerular structural integrity, collapse of podocyte structure, and skeletal reorganization. In vitro experiments indicated that both AngII and Flu (the specific agonist of TRPC6) stimulated the expressions of TRPC6, AT1R, and Caspase-3 in podocytes. The AngII receptor-blocker losartan and TRPC6-specific inhibitor SAR blocked the overexpression of the aforementioned proteins. In addition, SAR also attenuated the degradation of podocyte structural proteins and inhibited the fluorescence intensity of intracellular calcium (Ca2+) and cell apoptosis.Conclusion: The involvement of AngII in the occurrence of NS proteinuria may be related to podocyte injury induced by activated TRPC6.