V-ATPase promotes transforming growth factor-β-induced epithelial-mesenchymal transition of rat proximal tubular epithelial cells
V-ATPase promotes transforming growth factor-β-induced epithelial-mesenchymal transition of rat proximal tubular epithelial cells
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DOI：
10.1152/ajprenal.00278.2011
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发表时间：
2012-05-01
影响因子：
4.2
通讯作者：
Yu, Xueqing
中科院分区：
文献类型：
--
作者：
Cao, Xueqin;Yang, Qiongqiong;Yu, Xueqing
曹旭，杨强，秦静，赵S，李旭，范静，陈伟，周勇，毛宏，余欣。V-ATPase促进转化生长因子-β诱导的大鼠近端肾小管上皮细胞向间质转化。AM J Physiol Renal Physiol 302：F1121-F1132,2012。2011年11月30日首次出版；doi：10.1152/ajprenal.00278.2011。-无处不在的液泡H+-ATPase(V-ATPase)，一种多亚单位质子泵，对细胞器内酸化是必不可少的。在此，我们假设V-ATPase参与了肾小管间质纤维化的发病机制。我们首先检测了其在单侧输尿管梗阻(UUO)大鼠肾纤维化模型和转化生长因子(TGF)-β1介导的大鼠近端肾小管上皮细胞(NRK52E)上皮向间充质转化(EMT)中的表达。免疫荧光实验显示，UUO导致肾小管间质损伤区域V-ATPase亚单位(B2、E、c)和α-平滑肌肌动蛋白(α-SMA)表达显著上调。通过实时定量聚合酶链式反应和免疫印迹分析，我们进一步观察到，转化生长因子-β1(10 ng/ml)以时间依赖的方式导致NRK52E的EMT(上调α-SMA和下调E-钙粘蛋白)，并在48h后显著上调V-ATPase B2和c亚基，在24 h后显著上调E亚基。用ATP/NADH偶联实验检测，经48h的转化生长因子-β1处理后，细胞的ATP水解酶活性增加。用snarf-4F指示剂测量细胞内pH，48h的转化生长因子-β1处理的细胞在NH4Cl脉冲后，Na+非依赖性的pH恢复明显快于对照组。此外，V-ATPase抑制剂巴非霉素A1对EMT有部分保护作用。转化生长因子-β1诱导细胞表面B2亚基表达增加，小干扰RNA介导的B2亚基基因敲除部分降低了V-ATPase活性，减轻了转化生长因子-β1诱导的EMT。这些结果表明，V-ATPase可能通过其亚基的过度表达或重新分布而增加其活性，从而促进EMT和慢性肾小管间质纤维化。
Cao X, Yang Q, Qin J, Zhao S, Li X, Fan J, Chen W, Zhou Y, Mao H, Yu X. V-ATPase promotes transforming growth factor-beta-induced epithelial-mesenchymal transition of rat proximal tubular epithelial cells. Am J Physiol Renal Physiol 302: F1121-F1132, 2012. First published November 30, 2011; doi:10.1152/ajprenal.00278.2011.-The ubiquitous vacuolar H+-ATPase (V-ATPase), a multisubunit proton pump, is essential for intraorganellar acidification. Here, we hypothesized that V-ATPase is involved in the pathogenesis of kidney tubulointerstitial fibrosis. We first examined its expression in the rat unilateral ureteral obstruction (UUO) model of kidney fibrosis and transforming growth factor (TGF)-beta 1-mediated epithelial-to-mesenchymal transition (EMT) in rat proximal tubular epithelial cells (NRK52E). Immunofluorescence experiments showed that UUO resulted in significant upregulation of V-ATPase subunits (B2, E, and c) and alpha-smooth muscle actin (alpha-SMA) in areas of tubulointerstitial injury. We further observed that TGF-beta 1 (10 ng/ml) treatment resulted in EMT of NRK52E (upregulation of alpha-SMA and downregulation of E-cadherin) in a time-dependent manner and significant upregulation of V-ATPase B2 and c subunits after 48 h and the E subunit after 24 h, by real-time PCR and immunoblot analyses. The ATP hydrolysis activity tested by an ATP/NADH-coupled assay was increased after 48-h TGF-beta 1 treatment. Using intracellular pH measurements with the SNARF-4F indicator, Na+-independent pH recovery was significantly faster after an NH4Cl pulse in 48-h TGF-beta 1-treated cells than controls. Furthermore, the V-ATPase inhibitor bafilomycin A1 partially protected the cells from EMT. TGF-beta 1 induced an increase in the cell surface expression of the B2 subunit, and small interfering RNA-mediated B2 subunit knockdown partially reduced the V-ATPase activity and attenuated EMT induced by TGF-beta 1. Together, these findings show that V-ATPase may promote EMT and chronic tubulointerstitial fibrosis due to increasing its activity by either overexpression or redistribution of its subunits.