TGFβ modulates cell-to-cell communication in early epithelial-to-mesenchymal transition

Abstract

Aims/hypothesis A key pathology in diabetic nephropathy is tubulointerstitial fibrosis. The condition is characterised by increased deposition of the extracellular matrix, fibrotic scar formation and declining renal function, with the prosclerotic cytokine TGF-β1 mediating many of these catastrophic changes. Here we investigated whether TGF-β1-induced epithelial-to-mesenchymal transition (EMT) plays a role in alterations in cell adhesion, cell coupling and cell communication in the human renal proximal tubule. Methods Whole-cell and cell compartment abundance of E-cadherin, N-cadherin, snail, vimentin, β-catenin and connexin-43 was determined in human kidney cell line (HK)2 and human proximal tubule cells with or without TGF-β1, using western blotting and immunocytochemistry, followed by quantification by densitometry. The contribution of connexin-43 in proximal tubule cell communication was quantified using small interfering RNA knockdown, while dye-transfer was used to assess gap junctional intercellular communication (GJIC). Functional tethering was assessed by single-cell force spectroscopy with or without TGF-β1, or by immunoneutralisation of cadherin ligation. Results High glucose (25 mmol/l) increased the secretion of TGF-β1 from HK2 cells. Analysis confirmed early TGF-β1-induced morphological and phenotypical changes of EMT, with altered levels of adhesion and adherens junction proteins. These changes correlated with impaired cell adhesion and decreased tethering between coupled cells. Impaired E-cadherin-mediated adhesion reduced connexin-43 production and GJIC, these effects being mimicked by neutralisation of Ecadherin ligation. Upregulation of N-cadherin failed to restore adhesion or connexin-43-mediated GJIC. Conclusions/interpretation We provide compelling evidence that TGF-β1-induced EMT instigates a loss of Ecadherin, cell adhesion and ultimately of connexinmediated cell communication in the proximal tubule under diabetic conditions; these changes occur ahead of overt signs of renal damage.

Arg tyrosine kinase modulates TGF-β1 production in human renal tubular cells under high-glucose conditions

“…However, what is the main cellular type responsible for extracellular matrix deposition is still a matter of debate. Some studies have shown that tubular cells might undergo an in vitro epithelialmesenchymal transition (EMT) and have a direct role in tubulointerstitial fibrosis development (Lee and Han, 2010;Hills et al, 2012;Gu et al, 2013). However, conclusive evidence of a full EMT process in vivo is a controversial point.…”

Fasudil Inhibits Epithelial-Myofibroblast Transdifferentiation of Human Renal Tubular Epithelial HK-2 Cells Induced by High Glucose

“…14) TGF-β 1 and CTGF have been found to be up-regulated in both experimental and human DN, and to be associated with EMT, 7,10,11,[15][16][17][18][19][20][21] supporting a key role for these signaling molecules. A previous study in diabetic rats showed that using fasudil as a treatment also decreased EMT.…”

Maternal diabetes modulates kidney formation in murine progeny: the role of hedgehog interacting protein (HHIP)

“…It is wellknown that TGF-β1 decreases renal branching morphogenesis via its negative feedback mechanisms [35,36]. Compelling evidence suggests that TGF-β1 plays a key role in glomerular endothelial cell damage [37,38], production of extracellular matrix and development of renal interstitial fibrosis in diabetic nephropathy [39][40][41]. In studies in vitro, we further demonstrated that the increase of HHIP expression either by overexpression of Hhip or by the induction of a high-glucose milieu might target TGF-β1 signalling, which, in turn, may lead to events such as phosphorylation of Smad2/3 and decreases in p27…”