RESULTS Snail1 depletion ameliorates UUO-induced fibrosis Supplementary Fig. 1), impedes Snail1 expression in Cadherin-16 positive cells. Snail1 expression is silent in the adult kidney but it is reactivated after UUO in the cortex and in the medulla (Supplementary Fig. 1c). During renal development, Snail1 is a strong Cadh16 repressor 5 , and it is only when Snai1 is downregulated that renal cells express Cadherin-16 and epithelialization occurs. As Snail1 is silenced in the adult, preventing Snail1 activation in Cadherin-16 positive cells in the kidney does not have any impact in healthy mice. As expected, while Snail1 was highly reactivated in the kidney upon UUO, this was not the case in recombined renal epithelial cells in SFKC mice (Fig. 1a-c). We analyzed overall morphology, collagen deposition and expression of alpha smooth muscle actin ( -SMA) and vimentin 7 or 15 days after UUO and found that kidneys from SFKC mice were protected from the development of overt fibrosis, although signs of tubular distension resulting from the obstruction 5 were readily evident (Fig. 1d,e and Supplementary Fig. 2a,b). We confirmed the attenuation of fibrosis by analysis of the expression of epithelial and mesenchymal markers and this was particularly evident 2 weeks after UUO (Fig. 1f). Quantification of Sirius Red staining (Fig. 1g) showed a 35% reduction in fibrosis in the cortex of obstructed kidneys from SFKC mice when compared to obstructed kidneys from WT or control (Ksp1.3-Cre-only) mice ( Fig. 1g and Supplementary Fig. 3a,b). In our SFKC model, Snail1 reactivation was also prevented in the collecting ducts, and we observed a much better preserved morphology and lower collagen deposition in the medulla compared to WT animals ( Supplementary Fig. 3c) indicating that the overall protection from fibrotic degeneration is higher than that reflected by the quantification of SiriusRed in the cortex.We next assessed the contribution of epithelial cells to the interstitium after UOO in our system. We generated a mouse model harboring Cre-loxP mediated expression of the Tomato fluorescent protein driven by the Ksp 1.3 promoter (Ksp1.3-Cre; Rosa-LSL-TdTomato). These mice allow the visualization and fate mapping of renal epithelial cells. Notably, we could not detect tdTomato+ cells in the interstitium 7 days after obstruction ( Supplementary Fig. 4a) and found less than 1% after 15 days (Fig. 2a). One of those rare cells leaving the tubule is shown in Supplementary Fig. 4b. Thus, renal epithelial cells do not delaminate from the tubules to contribute to myofibroblasts or other interstitial cells, also in keeping with the observation that the total number of tubular cross sections did not differ between kidneys from WT or SFKC mice 15 days after UUO (Fig. 2b), indicative of tubular integrity. 6Snail1 expression was reactivated after UUO in over 80% of tubular cells and also in activated interstitial cells as reported in cancer models 16 (Fig. 2c).Although tubular cells did not significantly contribute to myofibroblasts, UUO pr...

SummaryAlthough TGF- suppresses early stages of tumour development, it later contributes to tumour progression when cells become resistant to its suppressive effects. In addition to circumventing TGF--induced growth arrest and apoptosis, malignant tumour cells become capable of undergoing epithelial-to-mesenchymal transition (EMT), favouring invasion and metastasis. Therefore, defining the mechanisms that allow cancer cells to escape from the suppressive effects of TGF- is fundamental to understand tumour progression and to design specific therapies. Here, we have examined the role of Snail1 as a suppressor of TGF--induced apoptosis in murine non-transformed hepatocytes, rat and human hepatocarcinoma cell lines and transgenic mice. We show that Snail1 confers resistance to TGF--induced cell death and that it is sufficient to induce EMT in adult hepatocytes, cells otherwise refractory to this transition upon exposure to TGF-. Furthermore, we show that Snail1 silencing prevents EMT and restores the cell death response induced by TGF-. As Snail1 is a known target of TGF- signalling, our data indicate that Snail1 might transduce the tumour-promoting effects of TGF-, namely the EMT concomitant with the resistance to cell death.

Although TGF- suppresses early stages of tumour development, it later contributes to tumour progression when cells become resistant to its suppressive effects. In addition to circumventing TGF--induced growth arrest and apoptosis, malignant tumour cells become capable of undergoing epithelial-to-mesenchymal transition (EMT), favouring invasion and metastasis. Therefore, defining the mechanisms that allow cancer cells to escape from the suppressive effects of TGF- is fundamental to understand tumour progression and to design specific therapies. Here, we have examined the role of Snail1 as a suppressor of TGF--induced apoptosis in murine non-transformed hepatocytes, rat and human hepatocarcinoma cell lines and transgenic mice. We show that Snail1 confers resistance to TGF--induced cell death and that it is sufficient to induce EMT in adult hepatocytes, cells otherwise refractory to this transition upon exposure to TGF-. Furthermore, we show that Snail1 silencing prevents EMT and restores the cell death response induced by TGF-. As Snail1 is a known target of TGF- signalling, our data indicate that Snail1 might transduce the tumour-promoting effects of TGF-, namely the EMT concomitant with the resistance to cell death.

In the version of this article initially published, the labels to the left of the two micrographs in Figure 2c are reversed. Also, in Figure 4g, MKi67, used as a cell proliferation marker, is misspelled. The errors have been corrected in the HTML and PDF versions of the article.