The differential role of Smad2 and Smad3 in the regulation of pro-fibrotic TGFβ1 responses in human proximal-tubule epithelial cells

In chronic renal diseases, progressive loss of renal function correlates with advancing tubulo-interstitial fibrosis. TGF beta 1-Smad (transforming growth factor-beta 1-Sma and Mad protein) signalling plays an important role in the development of renal tubulo-interstitial fibrosis. Secretion of CTGF (connective-tissue growth factor; CCN2) by PTECs (proximal-tubule epithelial cells) and EMT (epithelial-mesenchymal transdifferentiation) of PTECs to myofibroblasts in response to TGF beta are critical Smad-dependent events in the development of tubulo-interstitial fibrosis. In the present study we have investigated the distinct contributions of Smad2 and Smad3 to expression of CTGF, E-cadherin, alpha-SMA (alpha-smooth-muscle actin) and MMP-2 (matrix-metalloproteinase-2) in response to TGF beta 1 treatment in an in vitro culture model of HKC-8 (transformed human PTECs). RNA interference was used to achieve selective and specific knockdown of Smad2 and Smad3. Cellular E-cadherin, a-SMA as well as secreted CTGF and MMP-2 were assessed by Western immunoblotting. TGF beta 1 treatment induced a fibrotic phenotype with increased expression of CTGF, MMP-2 and alpha-SMA, and decreased expression of E-cadherin. TGF beta 1-induced increases in CTGF and decreases in E-cadherin expression were Smad3-dependent, whereas increases in MMP-2 expression were Smad2-dependent. Increases in alpha-SMA expression were dependent on both Smad2 and Smad3 and were abolished by combined knockdown of both Smad2 and Smad3. In conclusion, we have demonstrated distinct roles for Smad2 and Smad3 in TGF beta 1-induced CTGF expression and markers of EMT in human PTECs. This can be of therapeutic value in designing targeted anti-fibrotic therapies for tubulointerstitial fibrosis.