TGF-β1 induces epithelial–mesenchymal transition but not myofibroblast transdifferentiation in primary cultures of human epithelial renal tubular cells

Introduction  Although the origin of renal interstitial myofibroblasts is still a matter of debate (Powell et al. 1999), emerging evidences suggest that they may derive from tubular epithelial cells. Since the well-known mesenchymal origin of human tubular epithelial cells (HUTECs), our working hypothesis is that in renal fibrogenesis, fibrogenetic cytokines could reactivate the mesenchymal program [epithelial–mesenchymal transition (EMT)], switched-off during renal nephrogenesis, leading to differentiation into MF. TGF-β1 is a key modulator of the myofibroblast phenotype in fibroblasts during the process of wound healing and in mesangial cells in culture. TGF-β1 is also able to induce EMT in a variety of cell types. However, most of these studies including the recent one on SV40-transformed HKC-8 human tubular cells (Yang & Liu 2001) have been carried out in immortalized cells.

Materials and methods  Primary HUTEC cultures were established from histologically normal human renal cortexes, obtained from surgical biopsies performed following informed consent in paediatric patients undergoing surgery because of extrinsic pyelouretheral obstruction. HUTECs were cultured for 4 and 6 days on plastic or type-I collagen-coated plates with or without 1, 5, 10 and 50 ng/ml TGF-β1. Time-course experiments (24 h to 6 days) with 1 ng/ml TGF-β1 were also performed on primary human dermal fibroblasts, used as control mesenchymal cells, and HUTECs. Control conditions were represented by cells maintained for 4 and 6 days in 1% serum without TGF-β1. The EMT process was monitored by morphology and immunophenotyping for α-SMA, cytokeratin 8–18, E-cadherin, vimentin and Ki67. Quantitative comparative RT/PCR evaluated the expression of collagen-III and -V, fibronectin, tenascin, MMP-2, CTGF, E-cadherin and cadherin 11 genes. TGF-β1 regulation of α-SMA was investigated at both transcriptional and translational (Western blot) levels.

Results and discussion  TGF-β1-driven EMT was documented morphologically, biochemically and molecularly. HUTEC morphology changes following TGF-β1 exposure were already evident at 24 h and with as little as 1 ng/ml of TGF-β1; HUTECs acquired a spindle shape with front-end/back-end polarity, like fibroblasts. The transition was characterized by drastic up-regulation of all the mesenchymal markers studied, including CTGF, and down-regulation of cytokeratin and E-cadherin expression. These phenomena were dose-dependent and favuored by growth on collagen-I. TGF-β1 treatment did not induce MF conversion, because it induces neither de novo expression of α-SMA gene nor the myofibroblast phenotype. We demonstrate that the TGF-β1-driven EMT is characterized by the re-appearance of developmental gene networks which, in incomplete or uncoordinate fashion, could contribute to the pathogenesis of renal fibrosis.