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V-ATPase promotes transforming growth factor-β-induced epithelial-mesenchymal transition of rat proximal tubular epithelial cells.
Am J Physiol Renal Physiol. 2012 May 01; 302(9):F1121-32.AJ

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+Show Affiliations

Department of Nephrology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

22129967

Citation

Cao, Xueqin, et al. "V-ATPase Promotes Transforming Growth Factor-β-induced Epithelial-mesenchymal Transition of Rat Proximal Tubular Epithelial Cells." American Journal of Physiology. Renal Physiology, vol. 302, no. 9, 2012, pp. F1121-32.
Cao X, Yang Q, Qin J, et al. V-ATPase promotes transforming growth factor-β-induced epithelial-mesenchymal transition of rat proximal tubular epithelial cells. Am J Physiol Renal Physiol. 2012;302(9):F1121-32.
Cao, X., Yang, Q., Qin, J., Zhao, S., Li, X., Fan, J., Chen, W., Zhou, Y., Mao, H., & Yu, X. (2012). V-ATPase promotes transforming growth factor-β-induced epithelial-mesenchymal transition of rat proximal tubular epithelial cells. American Journal of Physiology. Renal Physiology, 302(9), F1121-32. https://doi.org/10.1152/ajprenal.00278.2011
Cao X, et al. V-ATPase Promotes Transforming Growth Factor-β-induced Epithelial-mesenchymal Transition of Rat Proximal Tubular Epithelial Cells. Am J Physiol Renal Physiol. 2012 May 1;302(9):F1121-32. PubMed PMID: 22129967.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - V-ATPase promotes transforming growth factor-β-induced epithelial-mesenchymal transition of rat proximal tubular epithelial cells. AU - Cao,Xueqin, AU - Yang,Qiongqiong, AU - Qin,Jing, AU - Zhao,Shili, AU - Li,Xiaoyan, AU - Fan,Jinjin, AU - Chen,Wenfang, AU - Zhou,Yi, AU - Mao,Haiping, AU - Yu,Xueqing, Y1 - 2011/11/30/ PY - 2011/12/2/entrez PY - 2011/12/2/pubmed PY - 2012/7/18/medline SP - F1121 EP - 32 JF - American journal of physiology. Renal physiology JO - Am J Physiol Renal Physiol VL - 302 IS - 9 N2 - 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. SN - 1522-1466 UR - https://www.unboundmedicine.com/medline/citation/22129967/V_ATPase_promotes_transforming_growth_factor_β_induced_epithelial_mesenchymal_transition_of_rat_proximal_tubular_epithelial_cells_ L2 - https://journals.physiology.org/doi/10.1152/ajprenal.00278.2011?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub=pubmed DB - PRIME DP - Unbound Medicine ER -