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TRPC家族在TGF-β1诱导足细胞钙离子内流中的表达及作用

黄海庭, 林栩^*, 郭鹏威, 庞君, 马静, 何林檩, 郑心彤

右江民族医学院附属医院，百色 533000

摘要

瞬时受体电位阳离子通道蛋白(transient receptor potential cation channel, TRPC)是哺乳动物重要的阳离子通道蛋白，参与细胞多种生理和病理过程。本研究组前期研究发现转化生长因子-β1 (transforming growth factor-β1, TGF-β1)可提高肾小球足细胞TRPC6的表达，为进一步明确TRPC家族其他成员在足细胞损伤中的作用，本研究探讨TGF-β1对足细胞TRPC家族表达的影响以及TRPC家族在TGF-β1诱导足细胞内钙离子浓度变化中的作用。体外培养条件永生化肾小球足细胞株MPC5，通过TGF-β1构建足细胞损伤模型，qRT-PCR和Western blot法检测TGF-β1干预对足细胞TRPC家族mRNA和蛋白表达水平的影响。通过基因干扰siRNA技术和药理学方法分别抑制TRPC家族表达后，荧光探针Fluo-3/AM静态检测足细胞游离钙水平，动态高速钙离子成像系统检测足细胞内钙离子浓度动态变化。结果显示，与正常对照组相比，TGF-β1可使足细胞TRPC1/3/6蛋白表达增多，对TRPC4蛋白表达无影响，对TRPC5/7蛋白表达分别只在4 ng/mL和8 ng/mL时有显著影响。TGF-β1可使足细胞TRPC1/3/6 mRNA表达增多，对TRPC4/5/7 mRNA表达无明显影响。TGF-β1干预可显著提高足细胞内钙离子浓度，TRPC1/4/5/7低表达对TGF-β1干预下足细胞内钙离子浓度无明显影响，TRPC3/6低表达可降低TGF-β1干预下足细胞内钙离子浓度。TRPC6低表达组足细胞内钙离子浓度与SKF96365 (TRPC通道广泛抑制剂)组相当，TRPC3低表达组钙离子浓度高于SKF96365组。以上结果提示，TGF-β1可使足细胞TRPC1/3/6表达升高，TGF-β1提高足细胞内钙离子浓度与TRPC3/6有关，TRPC6对足细胞内钙离子浓度的影响程度可能大于TRPC3。

关键词： 转化生长因子-β1; 足细胞; 瞬时受体电位阳离子通道蛋白; 钙离子

Expression and role of the TRPC family in TGF-β1-induced calcium influx in podocytes

HUANG Hai-Ting, LIN Xu^*, GUO Peng-Wei, PANG Jun, MA Jing, HE Lin-Lin, ZHENG Xin-Tong

Affiliated Hospital of Youjiang Medical University for Nationalities, Baise 533000, China

Abstract

The TRPC family consists of multiple important cationic channels in mammals that participate in a variety of physiological and pathological processes. Our previous studies have shown that transforming growth factor-β1 (TGF-β1) increases the expression of TRPC6 in podocytes, but the roles of other members of the TRPC family in podocytes require further investigation. In this study, we investigated the effect of TGF-β1 on the expression of the TRPC family and the role of the TRPC family in the changes of the intracellular Ca2+ concentration ([Ca2+]i) in podocytes induced by TGF-β1. The model of podocyte injury was established by treatment with TGF-β1 in immortalized glomerular podocytes (MPC5) in vitro. qRT-PCR and Western blot were used to detect the effect of TGF-β1 on the mRNA and protein expression of each TRPC family member. After the expression of each TRPC family member was knocked down by a siRNA-based approach and blocked by SKF96365, respectively, free cytosolic Ca2+ was measured using the fluorescent Ca2+ indicator Fluo-3/AM, and the dynamic change of [Ca2+]i in podocytes was detected by a dynamic high-speed calcium imaging system. The results showed that TGF-β1 increased the protein expression of TRPC1/3/6 in podocytes, but had no effects on the protein expression of TRPC4. The protein expression levels of TRPC5/7 were only affected by 4 ng/mL and 8 ng/mL TGF-β1, respectively. TGF-β1 increased TRPC1/3/6 mRNA levels in podocytes, however had no effects on TRPC4/5/7 mRNA. TGF-β1 significantly increased [Ca2+]i in podocytes. Knockdown of TRPC1/4/5/7 in podocytes had no significant effect on the [Ca2+]i induced by TGF-β1, but TRPC3/6 knockdown significantly decreased the [Ca2+]i. There was no significant difference in the [Ca2+]i between the TRPC6 siRNA-treated group and SKF96365-treated group, but the [Ca2+]i of the TRPC3 siRNA-treated group was significantly higher than that of SKF96365-treated group. These results demonstrate that TGF-β1 increases the expression of the TRPC1/3/6 in podocytes. TGF-β1 increases [Ca2+]i in podocytes, which is dependent on the TRPC3/6 expression. Our results also suggest that the effect of TRPC6 on [Ca2+]i in podocytes may be greater than that of TRPC3.

Key words: transforming growth factor-β1; podocyte; transient receptor potential cation channel; calcium ion

收稿日期：　　录用日期：

通讯作者：林栩　　E-mail: 00528@ymcn.edu.cn

引用本文：

黄海庭, 林栩, 郭鹏威, 庞君, 马静, 何林檩, 郑心彤. TRPC家族在TGF-β1诱导足细胞钙离子内流中的表达及作用[J]. 生理学报 2022; 74 (6): 1005-1013.

HUANG Hai-Ting, LIN Xu, GUO Peng-Wei, PANG Jun, MA Jing, HE Lin-Lin, ZHENG Xin-Tong. Expression and role of the TRPC family in TGF-β1-induced calcium influx in podocytes. Acta Physiol Sin 2022; 74 (6): 1005-1013 (in Chinese with English abstract).