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

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-beta 1 mediating many of these catastrophic changes. Here we investigated whether TGF-beta 1-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. Whole-cell and cell compartment abundance of E-cadherin, N-cadherin, snail, vimentin, beta-catenin and connexin-43 was determined in human kidney cell line (HK)2 and human proximal tubule cells with or without TGF-beta 1, 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-beta 1, or by immunoneutralisation of cadherin ligation. High glucose (25 mmol/l) increased the secretion of TGF-beta 1 from HK2 cells. Analysis confirmed early TGF-beta 1-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 E-cadherin ligation. Upregulation of N-cadherin failed to restore adhesion or connexin-43-mediated GJIC. We provide compelling evidence that TGF-beta 1-induced EMT instigates a loss of E-cadherin, cell adhesion and ultimately of connexin-mediated cell communication in the proximal tubule under diabetic conditions; these changes occur ahead of overt signs of renal damage.