Snail1-induced partial epithelial-to-mesenchymal transition drives renal fibrosis in mice and can be targeted to reverse established disease

Abstract

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...

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