VEGF regulates TRPC6 channels in podocytes
VEGF regulates TRPC6 channels in podocytes
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DOI：
10.1093/ndt/gfr457
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发表时间：
2012-03-01
影响因子：
6.1
通讯作者：
Tepel, Martin
中科院分区：
文献类型：
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作者：
Thilo, Florian;Liu, Ying;Tepel, Martin
背景血管内皮生长因子（VEGF）的血浆浓度增加和足细胞中瞬时受体电位典型6型（TRPC 6）通道表达增加均与蛋白尿性肾病相关。现在，我们研究了VEGF调节足细胞中TRPC 6的假设。TRPC 6信使RNA（mRNA）和TRPC 6蛋白表达进行了分析，在培养的足细胞给予VEGF 165后，使用定量实时逆转录聚合酶链反应和免疫印迹，分别。使用共聚焦激光扫描显微镜分析足细胞中YFP标记的TRPC 6。TRPC 6相关的钙内流荧光测定。采用免疫荧光和免疫组织化学方法对糖尿病患者和对照组的肾组织进行检测。给予足细胞VEGF 165显著增加TRPC 6 mRNA表达和TRPC 6蛋白水平。VEGF 165的作用呈剂量依赖性，并可被磷酸肌醇-3-激酶抑制剂阻断。在放线菌酮（蛋白质生物合成的抑制剂）的存在下，我们没有观察到VEGF对TRPC 6蛋白质水平的影响，表明需要从头蛋白质合成。VEGF 165显著增加TRPC 6介导的足细胞钙内流。使用针对TRPC 6的siRNA进行基因敲低后，足细胞中的钙内流显著降低。免疫组化显示，与对照组相比，糖尿病肾病患者的足细胞中TRPC 6通道蛋白和VEGF受体2型（VEGFR-2）蛋白均增加。人肾皮质中VEGFR-2 mRNA和TRPC 6 mRNA的表达存在显著相关性（n = 48; r（2）= 0.585; P < 0.0001）。VEGF调节足细胞中的TRPC 6。
Background. Both, increased plasma concentrations of vascular endothelial growth factor (VEGF) and increased expression of transient receptor potential canonical type 6 (TRPC6) channels in podocytes have been associated with proteinuric kidney diseases. Now, we investigated the hypothesis that VEGF regulates TRPC6 in podocytes.Methods. TRPC6 messenger RNA (mRNA) and TRPC6 protein expression were analyzed in cultured podocytes after administration of VEGF165 using quantitative real-time reverse transcription-polymerase chain reaction and immunoblotting, respectively. YFP-tagged TRPC6 in podocytes was analyzed using confocal laser scanning microscopy. TRPC6-associated calcium influx was measured fluorometrically. Both, immunofluorescence and immunohistochemistry were performed in renal tissue from patients with diabetes mellitus and controls.Results. Administration of VEGF165 to podocytes significantly increased TRPC6 mRNA expression and TRPC6 protein levels. The effects of VEGF165 were dose dependent and could be blocked by phosphoinositide-3-kinase inhibitors. In the presence of cycloheximide, an inhibitor of protein biosynthesis, we did not observe an effect of VEGF on TRPC6 protein levels, indicating the requirement of de novo protein synthesis. VEGF165 significantly increased TRPC6-mediated calcium influx in podocytes. Calcium influx was significantly lower in podocytes after gene knockdown using siRNA against TRPC6. Immunohistochemistry showed both increased TRPC6 channel protein and VEGF receptor type 2 (VEGFR-2) protein in podocytes from patients with diabetic nephropathy compared to control subjects. There was a significant association between VEGFR-2 mRNA and TRPC6 mRNA (n = 48; r(2) = 0.585; P < 0.0001) in human renal cortex.Conclusion. VEGF regulates TRPC6 in podocytes.