Tetrandrine Suppresses Transient Receptor Potential Cation Channel Protein 6 Overexpression- Induced Podocyte Damage via Blockage of RhoA/ROCK1 Signaling.

Podocyte damage is common in many renal diseases characterized by proteinuria. Transient receptor potential cation channel protein 6 (TRPC6) plays an important role in renal function through its regulation of intracellular Ca2+ influx and RhoA/ROCK pathways. Chinese herb Stephania tetrandra, with the main active component being tetrandrine, has been used for the treatment of various kidney diseases for several years and has shown a positive effect. This study aimed at investigating the effect and mechanism of tetrandrine in podocyte damage induced by high expression of TRPC6. Immortalized, differentiated murine podocytes, MPC5 were treated with valsartan (0-800 μM) and tetrandrine (0-40 μM) for 48 h. The maximum safe concentrations of valsartan and tetrandrine were selected using a cell viability assay. MPC5 podocytes stably expressing TRPC6 were constructed using a lentivirus packaging system, followed by treatment with valsartan, tetrandrine, and Y-27632 for 48 h and U73122 (10 μM) for 10 min. The RhoA/ROCK pathway and podocyte-specific proteins (nephrin and synaptopodin) levels were quantified. Podocyte apoptosis and intracellular Ca2+ concentration were measured. Maximum safe concentrations of 100 μM valsartan and 10 μM tetrandrine showed no observable toxicity in podocytes. MPC5 podocytes stably expressing TRPC6 had higher intracellular Ca2+ influx, apoptotic percentages, and expression of RhoA/ROCK proteins, but lower expression of nephrin and synaptopodin proteins. U73122 treatment for 10 min did not inhibit TRPC6, but suppressed RhoA/ROCK protein. Y-27632 decreased ROCK1 expression, but did not influence the expression of TRPC6 protein. Both 100 μM valsartan and 10 μM tetrandrine for 48 h significantly inhibited intracellular Ca2+ influx, apoptosis, and RhoA/ROCK pathway, and increased nephrin and synaptopodin proteins in podocytes stably expressing TRPC6. Elevated TRPC6 expression can lead to podocyte injury by inducing intracellular Ca2+ influx and apoptosis of podocytes, and this effect may be mediated by activation of the RhoA/ROCK1 pathway. Tetrandrine can alleviate podocyte injury induced by TRPC6 expression through inhibition of the RhoA/ROCK pathway, suggesting a protective role in podocyte damage.

Yu J ，Zhu C ，Yin J ，Yu D ，Wan F ，Tang X ，Jiang X ... -  《Drug Design Development and Therapy》

Tetrandrine Attenuates Podocyte Injury by Inhibiting TRPC6-Mediated RhoA/ROCK1 Pathway.

Tetrandrine (Tet), a compound found in a traditional Chinese medicine, presents the protective effect for kidney function. Our study is aimed at clarifying the efficacy and underlying mechanism of Tet on podocyte injury. In this study, podocyte injury was induced in rats with adriamycin (ADR), and MPC5 podocytes were constructed with TRPC6 overexpression. We found that Tet treatment reduced the levels of proteinuria, serum creatinine, and blood urea nitrogen and increased plasma albumin levels in ADR-induced rats. Tet reduced intracellular Ca2+ influx and apoptosis in MPC5 podocytes overexpressing TRPC6. Tet downregulated the expression of renal TRPC6, RhoA, and ROCK1 and upregulated the expression of synaptopodin; meanwhile, it reduced calcineurin activity in vivo and in vitro. In conclusion, Tet protects against podocyte by affecting TRPC6 and its downstream RhoA/ROCK1 signaling pathway.

Mao L ，Ding Y ，Yu D ，Yin J ，Yu J ... -  《-》

Tetrandrine alleviates podocyte injury via calcium-dependent calpain-1 signaling blockade.

Podocytes have become a crucial target for interventions in proteinuric kidney diseases. Many studies have reported that overexpression of transient receptor potential cation channel protein 6 (TRPC6) in podocyte injury upregulates intracellular Ca2+ influx and stimulates Ca2+-dependent protease calpain-1 signaling. The traditional Chinese drug, tetrandrine, a nonselective Ca2+ channel blocker, has long been used to treat chronic kidney disease. This research aimed to explore the possible mechanisms underlying the anti-proteinuric properties of tetrandrine. We investigated the involvement of tetrandrine in Ca2+ dependent calpain-1 signaling in mouse podocytes and adriamycin-induced nephropathy rats. Cyclosporine A (CsA) and U73122 were used as positive controls. Cell viability, cytotoxicity, Ca2+ concentration, calpain activity, and mRNA and protein expression levels of calpain-1 signaling pathways were examined. The clinical and pathological changes were measured. Tetrandrine decreased intracellular Ca2+ influx in cultured TRPC6-overexpressing podocytes. In both in vitro and in vivo studies, the administration of tetrandrine downregulated calpain activity and the expression of calpain-1 and restored the expression of downstream Talin-1 and nephrin. Compared to CsA, tetrandrine treatment exhibited superior inhibitory effects on calpain activity and calpain-1 expression. Tetrandrine has therapeutic potential in podocyte damage by blocking Ca2+-dependent activation of the calpain-1 signaling pathway. Tetrandrine reduced proteinuria, improved renal function, and alleviate renal pathological damage.

Ding Y ，Tang X ，Wang Y ，Yu D ，Zhu C ，Yu J ... -  《BMC Complementary Medicine and Therapies》

Klotho Stabilizes the Podocyte Actin Cytoskeleton in Idiopathic Membranous Nephropathy through Regulating the TRPC6/CatL Pathway.

The aim of this study was to explore the renoprotective effects of Klotho on podocyte injury mediated by complement activation and autoantibodies in idiopathic membranous nephropathy (IMN). Rat passive Heymann nephritis (PHN) was induced as an IMN model. Urine protein levels, serum biochemistry, kidney histology, and podocyte marker levels were assessed. In vitro, sublytic podocyte injury was induced by C5b-9. The expression of Klotho, transient receptor potential channel 6 (TRPC6), and cathepsin L (CatL); its substrate synaptopodin; and the intracellular Ca2+ concentration were detected via immunofluorescence. RhoA/ROCK pathway activity was measured by an activity quantitative detection kit, and the protein expression of phosphorylated-LIMK1 (p-LIMK1) and p-cofilin in podocytes was detected via Western blotting. Klotho knockdown and overexpression were performed to evaluate its role in regulating the TRPC6/CatL pathway. PHN rats exhibited proteinuria, podocyte foot process effacement, decreased Klotho and Synaptopodin levels, and increased TRPC6 and CatL expression. The RhoA/ROCK pathway was activated by the increased phosphorylation of LIMK1 and cofilin. Similar changes were observed in C5b-9-injured podocytes. Klotho knockdown exacerbated podocyte injury, while Klotho overexpression partially ameliorated podocyte injury. Klotho may protect against podocyte injury in IMN patients by inhibiting the TRPC6/CatL pathway. Klotho is a potential target for reducing proteinuria in IMN patients.

Wang H ，Liu H ，Cheng H ，Xue X ，Ge Y ，Wang X ，Yuan J ... -  《-》

High glucose-induced apoptosis in cultured podocytes involves TRPC6-dependent calcium entry via the RhoA/ROCK pathway.

Increasing evidence indicates that podocyte apoptosis is a key event in the development of diabetic nephrology. However, the underlying mechanism of this apoptosis remains poorly understood. In this study, we report that high levels of glucose enhanced the expression of TRPC6 and TRPC6-dependent Ca(2+) influx, but glucose levels did not affect TRPC1 and TRPC5 expression. TRPC6 knockdown by siRNA interference attenuated the observed increase in glucose-induced podocyte apoptosis. High glucose levels also increased the generation of ROS; inhibition of ROS activity by N-acetyl-l-cysteine attenuated the high glucose-induced increase in TRPC6 expression and Ca(2+) influx. Exogenous treatment with H2O2 mimicked the high glucose response, resulting in an increase in TRPC6 expression and Ca(2+) influx. Taken together, these data suggest that high glucose levels induce ROS, thereby mediating TRPC6 expression and Ca(2+) influx. Because RhoA activity is increased following TRPC6 activation, we investigated whether TRPC6 is involved in high glucose-induced apoptosis via the RhoA/ROCK pathway. We report that high glucose levels produced an increase in RhoA activity, and this effect was abolished by the knockdown of TRPC6. Moreover, inhibition of the RhoA/ROCK pathway by a ROCK inhibitor, Y27632, also attenuated high glucose-induced apoptosis. We conclude that TRPC6 is involved in high glucose-induced podocyte apoptosis through the RhoA/ROCK pathway.

Yang H ，Zhao B ，Liao C ，Zhang R ，Meng K ，Xu J ，Jiao J ... -  《-》