Department of Nephrology, Guangxing Hospital Affiliated to ZheJiang Chinese Medical University (Key Laboratory of Zhejiang Province, Management of Kidney Disease), Hangzhou 310007, People's Republic of China.
Drug Des Devel Ther. 2020 Jan 28;14:361-370. doi: 10.2147/DDDT.S234262. eCollection 2020.
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 Ca influx and RhoA/ROCK pathways. Chinese herb , 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 Ca 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 Ca 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 Ca 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 Ca 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.
足细胞损伤常见于多种以蛋白尿为特征的肾脏疾病。瞬时受体电位阳离子通道蛋白 6（TRPC6）通过调节细胞内 Ca 内流和 RhoA/ROCK 通路在肾功能中发挥重要作用。中药，主要活性成分为汉防己甲素，已用于治疗多种肾脏疾病多年，并显示出积极的效果。本研究旨在探讨汉防己甲素在高表达 TRPC6 诱导的足细胞损伤中的作用及机制。
用缬沙坦（0-800 μM）和汉防己甲素（0-40 μM）处理永生化、分化的鼠足细胞 MPC5 48 h。采用细胞活力测定法选择缬沙坦和汉防己甲素的最大安全浓度。采用慢病毒包装系统构建稳定表达 TRPC6 的 MPC5 足细胞，然后用缬沙坦、汉防己甲素和 Y-27632 处理 48 h，用 U73122（10 μM）处理 10 min。定量检测 RhoA/ROCK 通路和足细胞特异性蛋白（nephrin 和 synaptopodin）水平。测量足细胞凋亡和细胞内 Ca 浓度。
100 μM 缬沙坦和 10 μM 汉防己甲素的最大安全浓度在足细胞中无明显毒性。稳定表达 TRPC6 的 MPC5 足细胞内 Ca 内流增加，凋亡率和 RhoA/ROCK 蛋白表达增加，但 nephrin 和 synaptopodin 蛋白表达降低。U73122 处理 10 min 不能抑制 TRPC6，但能抑制 RhoA/ROCK 蛋白。Y-27632 降低了 ROCK1 的表达，但不影响 TRPC6 蛋白的表达。48 h 时，100 μM 缬沙坦和 10 μM 汉防己甲素均可显著抑制稳定表达 TRPC6 的足细胞内 Ca 内流、凋亡和 RhoA/ROCK 通路，并增加足细胞中 nephrin 和 synaptopodin 蛋白的表达。
升高的 TRPC6 表达可通过诱导足细胞内 Ca 内流和凋亡导致足细胞损伤，这种作用可能是通过激活 RhoA/ROCK1 通路介导的。汉防己甲素通过抑制 RhoA/ROCK 通路减轻由 TRPC6 表达引起的足细胞损伤，提示其在足细胞损伤中具有保护作用。
