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.

The Function of Vacuolar ATPase (V-ATPase) a Subunit Isoforms in Invasiveness of MCF10a and MCF10CA1a Human Breast Cancer Cells

“…TGF-␤1 can induce epithelial-to-mesenchymal transition in renal epithelial cells as well as increased expression of a number of V-ATPase subunits, including B2, E, and c (39). TGF-␤1 treatment also stimulates V-ATPase activity and plasma membrane localization in these cells, and inhibition of V-ATPase activity blocks the TGF-␤1 induced epithelial-to-mesenchymal transition (39). Although the effect of TGF-␤ stimulation on a3 expression has not been evaluated, one possibility is that a3 overexpression promotes invasion in MCF10a cells by a similar mechanism as TGF-␤ stimulation.…”

Membrane rafts–redox signalling pathway contributes to renal fibrosis via modulation of the renal tubular epithelial–mesenchymal transition

“…Furthermore, Cao et al . () demonstrated that Atp6v1e2‐mediated pH change was involved in TGF‐β1‐induced EMT in rat renal tubular cells, which also plays a critical role in MR formation (Xu et al . ).…”

Human renal tubular cells contain CD24/CD133 progenitor cell populations: Implications for tubular regeneration after toxicant induced damage using cadmium as a model

“…This feature requires the presence of adherens and tight junctions, cell structures that require the expression of cadherins, claudins and occludin proteins. Particularly important is the formation of the tight junction because this structure forms the boundary of two-dimensional diffusion of the apical and basal-lateral plasma membrane, which enables the polar distribution of uptake and efflux transporters (Cao et al, 2012). The current study assessed the expression of many members of these three classes of proteins.…”