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。
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 Ca^(2+)concentration([Ca^(2+)]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 Ca^(2+)was measured using the fluorescent Ca^(2+)indicator Fluo-3/AM,and the dynamic change of[Ca^(2+)]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[Ca^(2+)]i in podocytes.Knockdown of TRPC1/4/5/7 in podocytes had no significant effect on the[Ca^(2+)]i induced by TGF-β1,but TRPC3/6 knockdown significantly decreased the[Ca^(2+)]i.There was no significant difference in the[Ca^(2+)]i between the TRPC6 siRNA-treated group and SKF96365-treated group,but the[Ca^(2+)]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
作者
黄海庭
林栩
郭鹏威
庞君
马静
何林檩
郑心彤
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)
出处
《生理学报》
CAS
CSCD
北大核心
2022年第6期1005-1013,共9页
Acta Physiologica Sinica
基金
supported by the Youth Fund of Guangxi Natural Science Foundation Program of China(No.2019GXNSFBA245090)
