title: V-ATPase promotes transforming growth factor-β-induced epithelial-mesenchymal transition of rat proximal tubular epithelial cells.
abstract: 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)-β1-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 α-smooth muscle actin (α-SMA) in areas of tubulointerstitial injury. We further observed that TGF-β1 (10 ng/ml) treatment resulted in EMT of NRK52E (upregulation of α-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-β1 treatment. Using intracellular pH measurements with the SNARF-4F indicator, Na(+)-independent pH recovery was significantly faster after an NH(4)Cl pulse in 48-h TGF-β1-treated cells than controls. Furthermore, the V-ATPase inhibitor bafilomycin A1 partially protected the cells from EMT. TGF-β1 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-β1. 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.
authors: Department of Nephrology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China.
authors: Xueqin
authors: X
authors: Cao
authors: Qiongqiong
authors: Q
authors: Yang
authors: Jing
authors: J
authors: Qin
authors: Shili
authors: S
authors: Zhao
authors: Xiaoyan
authors: X
authors: Li
authors: Jinjin
authors: J
authors: Fan
authors: Wenfang
authors: W
authors: Chen
authors: Yi
authors: Y
authors: Zhou
authors: Haiping
authors: H
authors: Mao
authors: Xueqing
authors: X
authors: Yu
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects*; D005355:Fibrosis; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D007687:Kidney Tubules, Proximal / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D018942:Macrolides / Q000494:pharmacology; D008297:Male; D023421:Models, Animal; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*; D014517:Ureteral Obstruction / Q000150:complications; D025262:Vacuolar Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D000199:Actins; D004791:Enzyme Inhibitors; D018942:Macrolides; D034741:RNA, Small Interfering; D053773:Transforming Growth Factor beta1; C057620:bafilomycin A; D025262:Vacuolar Proton-Translocating ATPases