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• W2011143322 abstract "Article9 November 2006free access Snail activation disrupts tissue homeostasis and induces fibrosis in the adult kidney Agnès Boutet Agnès Boutet Instituto de Neurociencias de Alicante, CSIC-UMH, Sant Joan d'Alacant, Alicante, Spain Search for more papers by this author Cristina A De Frutos Cristina A De Frutos Instituto de Neurociencias de Alicante, CSIC-UMH, Sant Joan d'Alacant, Alicante, Spain Search for more papers by this author Patrick H Maxwell Patrick H Maxwell Department of Nephrology, Imperial College London, Hammersmith Campus, London, UK Search for more papers by this author M José Mayol M José Mayol Department of Anatomopathology and Urology, Sant Joan d'Alacant University Hospital, Sant Joan d'Alacant, Spain Search for more papers by this author J Romero J Romero Department of Anatomopathology and Urology, Sant Joan d'Alacant University Hospital, Sant Joan d'Alacant, Spain Search for more papers by this author M Angela Nieto Corresponding Author M Angela Nieto Instituto de Neurociencias de Alicante, CSIC-UMH, Sant Joan d'Alacant, Alicante, Spain Search for more papers by this author Agnès Boutet Agnès Boutet Instituto de Neurociencias de Alicante, CSIC-UMH, Sant Joan d'Alacant, Alicante, Spain Search for more papers by this author Cristina A De Frutos Cristina A De Frutos Instituto de Neurociencias de Alicante, CSIC-UMH, Sant Joan d'Alacant, Alicante, Spain Search for more papers by this author Patrick H Maxwell Patrick H Maxwell Department of Nephrology, Imperial College London, Hammersmith Campus, London, UK Search for more papers by this author M José Mayol M José Mayol Department of Anatomopathology and Urology, Sant Joan d'Alacant University Hospital, Sant Joan d'Alacant, Spain Search for more papers by this author J Romero J Romero Department of Anatomopathology and Urology, Sant Joan d'Alacant University Hospital, Sant Joan d'Alacant, Spain Search for more papers by this author M Angela Nieto Corresponding Author M Angela Nieto Instituto de Neurociencias de Alicante, CSIC-UMH, Sant Joan d'Alacant, Alicante, Spain Search for more papers by this author Author Information Agnès Boutet1, Cristina A De Frutos1, Patrick H Maxwell2, M José Mayol3, J Romero3 and M Angela Nieto 1 1Instituto de Neurociencias de Alicante, CSIC-UMH, Sant Joan d'Alacant, Alicante, Spain 2Department of Nephrology, Imperial College London, Hammersmith Campus, London, UK 3Department of Anatomopathology and Urology, Sant Joan d'Alacant University Hospital, Sant Joan d'Alacant, Spain *Corresponding author. Instituto de Neurociencias de Alicante, CSIC-UMH, Apartado 18, Sant Joan d'Alacant, Alicante 03550, Spain. Tel.: +34 96 591 92 43; Fax: +34 96 591 95 61; E-mail: [email protected] The EMBO Journal (2006)25:5603-5613https://doi.org/10.1038/sj.emboj.7601421 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info During embryonic development, the kidney epithelium originates from cells that undergo a mesenchymal to epithelial transition (MET). The reverse process, epithelium to mesenchyme transition (EMT), has been implicated in epithelial tumor progression and in the fibrosis that leads to end-stage kidney failure. Snail transcription factors induce both natural and pathological EMT, but their implication in renal development and disease is still unclear. We show that Snail genes are downregulated during the MET that occurs during renal development and that this is correlated with Cadherin-16 expression. Snail suppresses Cadherin-16 via the direct repression of the kidney differentiation factor HNF-1β, a novel route by which Snail disrupts epithelial homeostasis. Indeed, Snail activation is sufficient to induce EMT and kidney fibrosis in adult transgenic mice. Significantly, Snail is also activated in patients with renal fibrosis. Thus, Snail expression is suppressed during renal development and it must remain silent in the mature kidney where its aberrant activation leads to fibrosis. Introduction During embryonic development, epithelial and mesenchymal phenotypic transitions are necessary for the correct formation of different tissues. However, these processes are not only involved in development but they are also thought to be implicated in pathological conditions such as cancer and fibrosis (Nieto, 2002; Kalluri and Neilson, 2003; Liu, 2004; Thiery and Sleeman, 2006). One striking example is in kidney development and disease. During development, mesenchymal cells are initially formed by epithelium to mesenchyme transition (EMT) and subsequently, some of these cells undergo mesenchymal to epithelial transition (MET) to form the epithelia of the pronephros, mesonephros and metanephros (Dressler, 2002). In the adult, the reverse process (EMT) is implicated in the progress of carcinomas and in organ fibrosis. Fibrosis is regarded as the main event leading to end-stage kidney failure in most progressive renal diseases (Liu, 2004). Thus, while kidney formation involves reciprocal transformations, suppression of this plasticity in the adult is critical to maintain normal tissue architecture and homeostasis. The Snail gene family of transcription factors is best known for its ability to trigger EMT, converting epithelial cells into mesenchymal cells with migratory properties. Snail genes influence both tissue formation during embryonic development and the acquisition of invasive properties in epithelial tumors (Barrallo-Gimeno and Nieto, 2005). Of the three vertebrate Snail family members, Snail and Slug (recently been renamed as Snail1 and Snail2, respectively; Barrallo-Gimeno and Nieto, 2005) are functionally equivalent (del Barrio and Nieto, 2002; Bolós et al, 2003). Prompted by our observation that Snail1 is a strong suppressor of kidney-specific cadherin (Cadherin-16) expression in cell culture, we have studied the role of Snail genes in the kidney. We found that Snail genes are repressed during kidney differentiation and identified that they regulate Cadherin-16 expression by repressing HNF-1β. To determine to what extent plasticity may be retained in the adult, we developed a system allowing us to reactivate Snail expression. We show that Snail activation induces renal fibrosis in transgenic mice and report the presence of pathological Snail expression in human fibrotic kidneys. Hence, the activation of Snail in the adult has profound effects on epithelial homeostasis in the kidney, which can be considered as a process of reverse embryogenesis likely to be pivotal in the development of renal fibrosis. Results To gain insight into the mechanisms used by Snail to repress the epithelial phenotype, we established an in vitro model of Snail1-induced EMT in the mouse mammary gland-derived epithelial cell line NMuMG. These cells typically display a cobblestone-like phenotype in culture (Figure 1A), yet they acquire a mesenchymal phenotype when stably transfected with Snail1 (Figure 1B). This morphological change is accompanied by the loss of E-Cadherin and a reorganization of the actin cytoskeleton (Supplementary Figure 1). Likewise, Snail1 transfectants become motile and acquire invasive properties (Supplementary Figure 1). Indeed, the transcription of many epithelial genes was strongly repressed in Snail1 transfectants, including that of E-Cadherin (Figure 1C) and other known Snail1 targets such as mucin-1, claudins and occludins (data not shown and Barrallo-Gimeno and Nieto, 2005; Huber et al, 2005). Thus, this cell-culture system provides a potent tool to investigate EMT driven by Snail. Figure 1.Snail1 represses the kidney epithelial Cadherin-16 both in cell culture and in the embryo. (A, B) Phase-contrast images and (C) Snail1, E-Cadherin and Cadherin-16 expression in stable mock- and Snail1-transfected cells. Snail1 expression represses E-cadherin and Cadherin-16 transcription. GAPDH levels are shown as a control. (D–X) ISH for Cadherin-16, Snail1 and Snail2 in whole-mount mouse embryos and transverse sections taken at the mid (E, H, K) and posterior (F, I, L) trunk levels. Cadherin-16 is expressed in the newly formed nephric duct epithelium (nd, E) that no longer expresses Snail genes (H, K insets). Snail transcripts are observed in the undifferentiated anterior (H, K) and posterior (I, L) nephrogenic mesenchyme (nm). Dissected urogenital system (see M, inset) or gelatin sections (M–R) hybridized with Cadherin-16 and Snail probes. Cadherin-16 is expressed in the collecting duct epithelia (M) and their ureteric tips (ut, N) of the developing metanephros but it is absent from the newly forming renal vesicle (pink star, rv; N). Expression is also detected in the sexual ducts and in the tubules of the transient mesonephros (sd and ms; M, inset). Snail1 and Snail2 expression is restricted to the metanephric mesenchyme and to the deep stroma (mm and ds, O–R). Nephrons (n) appear after the complete epithelialization of the metanephric mesenchyme. The nephron epithelia and the collecting ducts (cd) strongly express Cadherin-16 (S, T), whereas Snail1 and Snail2 expression disappears when the mesenchyme transforms into epithelia (U–X). nt, neural tube. Scale bar, 100 μm. (Y) Snail proteins repress the Cadherin-16 promoter. Schematic representation of the mouse Cadherin-16 promoter showing the regions of high similarity between mouse and human, and the location of the consensus Snail-binding sequences (white boxes). Luciferase reporter constructs carrying the wild-type mouse Cadherin-16 promoter (−1268) or deletions in the two E-boxes were assayed in the NMuMG cells together with either the mouse Snail1 or Snail2 expression vectors or an empty vector as a control (pcDNA3). Luciferase activity was measured 24 h after transfection and the activity is expressed relative to that of the wild-type construct. The results are the mean values±S.E. of duplicates from four independent experiments. Deletions of the Snail-binding sites do not relieve the repression of the Cadherin-16 promoter activity. Download figure Download PowerPoint Mock-transfected NMuMG cells were found to contain a high level of Cadherin-16 transcripts, and its expression was completely downregulated in stable Snail1 transfectants (Figure 1C). Cadherin-16 is a kidney-specific cadherin expressed in renal tubular epithelial cells and collecting ducts of the differentiating and mature kidney (Thomson et al, 1995). Hence, Cadherin-16 expression was unexpected in a mammary gland-derived cell line and indeed, it was not expressed in the mouse mammary gland or in human mammary gland-derived cell lines (Supplementary Figure 2), and we concluded that Cadherin-16 expression was merely a peculiarity of NMuMG cells. Given the importance of MET and EMT in renal development, this result prompted us to determine whether Snail genes regulate Cadherin-16 expression in the kidney. Inverse correlation between Cadherin-16 and Snail gene expression during kidney development To determine whether Snail1 could repress Cadherin-16, we first examined the expression of each gene at different stages during murine kidney development. As Snail2 also represses E-Cadherin and it is considered functionally equivalent to Snail1 (del Barrio and Nieto, 2002; Bolós et al, 2003), we also analyzed its expression in the kidney. Cadherin-16 expression commences during the differentiation of the developing kidney, concomitant with the MET that gives rise to the different renal epithelial cell populations (Wertz and Herrmann, 1999). The first renal epithelium to form is the nephric duct, derived from the intermediate mesoderm in the caudal part of the trunk region. Although the early mesoderm expresses Snail1 in mouse embryos, the expression of the Snail1 and 2 genes becomes more complex as the mesoderm segregates into distinct populations. As such, when Cadherin-16 is not yet expressed in the embryo (not shown), Snail2 expression is upregulated in the intermediate and lateral mesoderms (Sefton et al, 1998). Once the nephric duct became epithelialized, Snail genes expression was downregulated and Cadherin-16 transcripts could be detected (Figure 1E, H and K). The mesenchyme adjacent to the nephric duct (nm, nephrogenic mesenchyme) expresses both Snail genes (Figure 1H and K) and later epithelializes to form the transient mesonephros. The resulting mesonephric tubules express high levels of Cadherin-16 (Figure 1M, inset). In the posterior embryo, Snail transcripts are expressed in the nephrogenic mesenchyme where the metanephros will form (Figure 1I and L). Strong Cadherin-16 expression is observed in the collecting ducts of the metanephros that arise from the branching of the nephric duct, whereas Snail genes expression is maintained in the deep stroma (Figure 1M, O and Q). The ureteric tips of the collecting ducts also express Cadherin-16 unlike the condensing mesenchyme that gives rise to the renal vesicle and is still devoid of transcripts (Figure 1N). In the metanephric mesenchyme that surrounds the renal vesicle and that gives rise to the nephrons, Snail2 and weaker Snail1 expression was detected (Figure 1P and R). Again, the epithelialization of this tissue was correlated with Snail downregulation (Figure 1M–R). At 17.5 days post-coitum (dpc), the nephron displayed robust Cadherin-16 expression (Figure 1S and T), having downregulated Snail genes by this stage (Figure 1U–X). This situation was maintained throughout adulthood (Supplementary Figure 3). Thus, the expression of the Snail and Cadherin-16 genes is complementary during kidney ontogenesis and tissues that express Cadherin-16 are derived from Snail1- and Snail2-expressing mesenchyme. These results are compatible with Snail genes acting as repressors of Cadherin-16 in vivo. Direct repression of HNF-1β transcription by Snail genes prevents Cadherin-16 expression Given the precedent of E-Cadherin (Batlle et al, 2000; Cano et al, 2000), we examined whether Snail proteins could directly repress Cadherin-16 transcription. Two consensus E-boxes for Snail binding were identified at −581 and −746 bp in the mouse Cadherin-16 promoter (Figure 1Y). A construct containing these two boxes within sequences able to recapitulate the physiological expression of Cadherin-16 in the developing kidney (Whyte et al, 1999; Shao et al, 2002) was cotransfected with either Snail1 or Snail2 constructs. Promoter activity decreased to 61 and 56%, respectively, indicating that Snail proteins do indeed repress Cadherin-16 transcription. However, the modification or deletion of the individual E-boxes (Supplementary Figure 4) or simultaneous deletion of both boxes did not impair this repression (Figure 1Y), indicating that Snail1 and 2 do not directly downregulate Cadherin-16 expression. As Snail proteins act as transcriptional repressors (Nieto, 2002), we speculated that they might downregulate an activator of Cadherin-16. The HNF-1β transcription factor promotes the epithelial phenotype in the kidney by strongly activating Cadherin-16 expression (Bai et al, 2002). Thus, we compared HNF-1β expression with that of Cadherin-16 during kidney development. HNF-1β was expressed in the differentiated epithelia of the anterior nephric duct (Figure 2B), where Snail genes had been downregulated and Cadherin-16 transcripts were found at 10.5 dpc (Figure 1E, H and K, insets). At this stage, HNF-1β transcripts were also detected in the differentiating posterior nephric duct (dnd, white star; Figure 2C), where Cadherin-16 was not yet expressed (white star; Figure 1F). In the cortex of the metanephros at 13.5 dpc, HNF-1β expression was observed in the ureteric tips of the collecting ducts and in the forming renal vesicles that were still devoid of Cadherin-16 transcripts (Figures 1N and 2D and G). However, Cadherin-16 was expressed strongly in the previously differentiated collecting ducts (Figure 2E), while the tips of the collecting ducts and the renal vesicles are surrounded by Snail2-expressing cells yet to condense (Figure 2F). Accordingly, HNF-1β transcripts were detected in the areas of epithelializing mesenchyme (Figure 2D and G), some of which do not yet express Cadherin-16 (compare Figures 1F with 2C, white stars, and Figures 1N with 2G, pink stars). The differentiated nephrons that give rise to functional epithelial structures of the definitive kidney express both HNF-1β (Figure 2H and Cadherin-16 (Figure 1S and T). Thus, the expression of HNF-1β is compatible with it acting as an activator of Cadherin-16. Figure 2.HNF-1β expression precedes that of Cadherin-16 in the developing kidney epithelia. (A–H) ISH of embryos or dissected kidneys. HNF-1β expression was observed in the epithelia at all stages of kidney development. At 10.5 dpc, HNF-1β transcripts are seen in the nephric duct (nd, B) and in the condensing mesenchyme of the differentiating posterior nephric duct (dnd, C), which is still devoid of Cadherin-16 transcripts (white star in Figure 1F). At 13.5 dpc, in addition to the collecting duct (cd), the ureteric tips of the ducts (ut) and the renal vesicle (rv) contain HNF-1β transcripts (D, G), some of which remain negative for Cadherin-16 (E and pink star in Figure 1N). Snail2 is expressed in the metanephric mesenchyme (mm) but not in the areas expressing HNF-1β. (H) At 17.5 dpc, the epithelia of the nephrons (n) and the collecting ducts express high levels of HNF-1β and both Snail genes have been downregulated (Figure 1U–X). nt, neural tube; scale bars, 100 μm. Download figure Download PowerPoint We detected HNF-1β transcripts in NMuMG cells, explaining the high levels of Cadherin-16 expression, but both genes were completely downregulated in stable Snail1 transfectants (not shown and Figure 1C). To determine if activating Snail was sufficient to repress the expression of these genes, we used a chimeric construct in which Snail1 could be activated by tamoxifen (Snail1-ER; see Materials and methods). Snail activation in stable transfectants triggered EMT within 24 h (Figure 3A) analyzed by the loss of E-cadherin, reorganization of the actin cytoskeleton and the acquisition of motile and invasive properties (not shown, but see Supplementary Figure 1). These phenotypic changes were concomitant with the downregulation of HNF-1β and Cadherin-16 (Figure 3C). Interestingly, within 6 h of Snail1 activation HNF-1β levels fell by 20%, whereas Cadherin-16 transcripts remained unaffected (not shown), indicative of the sequential downregulation of HNF-1β and Cadherin-16 and suggesting that Snail1 inhibits Cadherin-16 expression by repressing HNF-1β. Indeed, the two consensus sequences for Snail binding in the HNF-1β promoter indicate that Snail proteins could directly repress HNF-1β transcription. The most proximal of these was conserved and lies in a highly conserved region in mouse, rat and human (Figure 3D). Accordingly, both Snail proteins repressed HNF-1β promoter activity when assayed using a reporter construct that included both these boxes (Figure 3D). Moreover, while deletion of the distal nonconserved E-box did not affect repression, deleting the conserved box abrogated inhibition (Figure 3D). Figure 3.Snail genes directly repress HNF-1β transcription, which in turn impairs Cadherin-16 expression. (A) NMuMG cells stably transfected with an inducible Snail1 construct (Snail1-ER) or with the empty vector (Mock) were analyzed 24 h after induction. Note the phenotypic change of the Snail1-transfected cells upon 4′-OH-tamoxifen (4′-OH-TAM) administration. Scale bar, 25 μm. (B) Transgene expression visualized by RT–PCR in Mock and Snail1-ER transfectants (S-ER). (C) Quantitative RT–PCR for Cadherin-16 and HNF-1β 24 h after 4′-OH-tamoxifen administration. (D) Diagram of the 1 kb region upstream of the translational initiation site in the mouse HNF-1β gene showing regions highly conserved between mouse and human. Of the two consensus E-boxes for Snail binding, only one lies within the conserved region. Snail1 and Snail2 repressed the activity of the wild-type HNF-1β promoter (measured as described for the Cadherin-16 promoter), but they did not affect the promoter constructs in which the conserved E-box was deleted. (E) ChiP analyses show that Snail1 binds directly to the HNF-1β promoter. ChIP assays were carried out with anti-ER antibodies on Mock- and Snail1-ER cells 24 h after induction. As a positive control, the interaction of Snail with the E-pal element of the E-cadherin promoter is shown. Snail does not bind to the nonconserved (NC) E-box in the HNF-1β promoter. Amplifications of the indicated promoter regions in the input (1), nonimmunoprecipitated (2) and immunoprecipitated (3) fractions are shown. The data presented are representative of three independent experiments. Download figure Download PowerPoint Chromatin immunoprecipitation (ChiP) further demonstrated that Snail1 binds to the HNF-1β promoter (Figure 3E), repressing its transcriptional activity and surely accounting for the subsequent downregulation of Cadherin-16 expression. This interaction is specific for the fragment containing the conserved E-box, as HNF-1β promoter sequences were not recovered when a nonconserved promoter region was used. Indeed, Snail1 bound in a similar manner to the HNF-1β promoter sequences as to E-cadherin promoter sequences (Figure 3E). As NMuMG cells are derived from the mammary gland, we examined whether this repression also occurred in a kidney cell line. Indeed, HNF-1β and Cadherin-16 transcription was completely downregulated by Snail1 in MDCK cells (Supplementary Figure 5). Snail activation induces renal fibrosis in transgenic mice As Snail genes repress HNF-1β expression in culture and consequently the epithelial phenotype, we assessed whether they behave similarly in vivo. More importantly, we assessed whether they could induce full EMT in the kidney and thus, the loss of the epithelial homeostasis. A transgenic mouse expressing the same tamoxifen-inducible Snail1-ER construct was generated identifying a line that specifically expressed significant levels of transgenic protein in the kidney (Supplementary Figure 6). This inducible system is appropriate to analyze transcription factor activity since despite its constitutive expression, the exogenous protein is only active after nuclear translocation upon tamoxifen administration (Figure 4A–H). In these animals, nuclear exogenous Snail1 protein was observed after two weeks of subcutaneal tamoxifen administration (Figure 4B, D, F and H). Both HNF-1β and Cadherin-16 were downregulated in the cortex and the medulla of kidneys from tamoxifen-treated mice (Figure 4I–P and U). Interestingly, we also observed the induction of Snail2 expression (Figure 4Q–T and U), the family member whose expression is prominent during kidney development. These data are in agreement with our previous finding that Snail1 induces Snail2 transcription during neural crest development in Xenopus embryos (Aybar et al, 2003). Figure 4.Snail expression induces the loss of the epithelial character in postnatal transgenic kidneys. (A–H) Exogenous Snail1 protein was translocated to the nucleus upon tamoxifen (TAM) administration, as seen with an anti-hER antibody in both the medulla and the cortex. Note the absence of the transgenic protein in the glomeruli (yellow diamonds). (I–P) HNF-1β (I–L) and Cadherin-16 expression (M–P) in sections from 2-week-old transgenic kidneys show the loss of the two epithelial and differentiation markers. (Q–T) Snail1 activation also induces Snail2 expression but not in the glomeruli (yellow diamond in T). (U) Quantitative RT–PCR analysis of Snail1, HNF-1β, Cadherin-16 and Snail2 expression in wild-type and transgenic kidneys in the absence or presence of TAM. Transcription is normalized to GAPDH mRNA expression and the error bars represent the standard error of the mean. Download figure Download PowerPoint The downregulation of Cadherin-16 in vivo can be explained by Snail1 repressing HNF-1β transcription and the loss of both markers should be associated with the loss of epithelial characteristics. Indeed, upon Snail activation, collecting duct cells in the medulla seemed to acquire a fibroblast-like morphology (Figure 4F). The phenotype of the transgenic kidneys with active Snail1 was further analyzed and the collecting ducts in the medulla were clearly disorganized (Figure 5B). This disorganization correlated with the morphological changes characteristic of EMT (Figure 5B inset) and the repression of epithelial markers such as HNF-1β, Cadherin-16 (Figure 4) and also E-Cadherin (Figure 5C and D). In addition, cells expressed mesenchymal markers such as the intermediate filament vimentin (Figure 5E and F) and smooth muscle actin (SMA, Figure 5G and H). These histological changes are reminiscent of those observed after experimentally induced or pathological fibrosis (Li et al, 2003; Sato et al, 2003; Zeisberg et al, 2003; McMorrow et al, 2005; Slattery et al, 2005). The deposition of Collagen I is a hallmark of renal fibrosis (Alexakis et al, 2005) and focal deposition was observed after the activation of Collagen I transcription by Snail1 (Figure 5I–L). Epithelial disorganization was also observed in the cortex, where dilated tubules and disrupted basement membranes were readily apparent as well as Collagen I deposition (Figure 5M–T). The mice died after about 2 weeks of treatment, presumably of renal failure given the distorted morphology of the kidney and the high levels of urea in their serum (not shown). Figure 5.Snail expression induces EMT and features of fibrosis in postnatal transgenic mice. (A, B) Hematoxylin/eosin staining of sections from the kidneys shown in Figure 4. Note the depolarized and fibroblast morphology of the collecting duct cells in the medulla region of animals treated with tamoxifen (+TAM). (C, D) E-Cadherin expression is completely downregulated in the kidneys of transgenic mice upon Snail1 activation, confirming the loss of epithelial character. (E–H) Snail 1 activation also induces the expression of vimentin (E, F) and smooth muscle actin (SMA, G, H), both indicating the appearance of mesenchymal characteristics. (I–L) Collagen I transcripts (I, J) and Collagen fibrotic deposits (K, L) detected by Masson–Trichrome staining in the transgenic kidneys upon Snail1 activation. (M–T) Similar signs of epithelial disruption and fibrosis are also observed in the cortex of the transgenic kidneys from tamoxifen-treated mice. Hematoxylin/eosin staining (M, N) and collagen deposits (O, P). (Q–T) High-power images to better assess the dilation of renal tubules (asterisks) and the disruption of basement membranes upon Snail1 activation. Basement membranes that can be clearly observed in the untreated transgenic kidneys (arrows). Download figure Download PowerPoint As the postnatal kidney may be considered immature or prone to revert to the embryonic phenotype, it was important to check whether Snail1 activation alone could induce fibrosis in the adult kidney. When tamoxifen was administered for 2 months commencing 2 months after birth (Figure 6), the collecting ducts and renal tubules became disorganized and dilated (Figure 6A–D), and Collagen I deposits were formed (Figure 6E–H). A feature frequently associated with renal fibrosis is the appearance of cysts, a phenomenon that was incipient in the postnatal transgenic kidneys following Snail activation. As the young mice died very early, we could not follow the progress of this putative polycystic kidney disease. Nevertheless, we observed the appearance of scattered cysts in the adult kidneys when Snail1 was activated over several weeks (Figure 6D). Figure 6.Snail activation is sufficient to induce renal fibrosis in adult transgenic mice. (A–D) Hematoxylin/eosin staining of sections from 4-month-old transgenic mice kidneys. Tamoxifen treatment in mice (+TAM) was initiated 2 months after birth. Note the defective overall morphology including the presence of dilated tubules. (E–H) Fibrotic deposits can be observed by Masson–Trichrome staining in tamoxifen-treated mice. Fibrosis is also manifested by the presence of cysts (D). (I–P) Detection of the transgenic Snail1 protein with an anti-hER antibody (brown) in paraffin sections counterstained with hematoxylin (blue). Note the absence of transgenic protein in the glomeruli and in the interstitial cells (yellow arrows in O) and in some ducts in the medulla (yellow diamond in N). The yellow triangle in (N) indicates ducts in which the Snail1 protein has not been efficiently translocated to the nucleus (note the blue nuclei and brown cytoplasms). The ducts and tubules with Snail1 nuclear expression lost the epithelial character and present a completely disorganized structure (yellow star in N and P). Scale bars, 25 μm. Download figure Download PowerPoint The loss of epithelial features perfectly correlated with Snail activation since the transgenic protein was expressed in the renal tubules and the collecting ducts, but it was absent from the glomeruli and the interstitial cells (Figure 6O). Accordingly, Snail1 activation did not induce morphological changes or Snail2 expression in the glomeruli of postnatal animals (yellow diamond in Figure 4D, G and T) and adult mice (yellow arrow in Figure 6O). In the medulla of adult mice, the transgenic protein displayed a mosaic distribution. A tubular structure was maintained in ducts that did not express Snail1 or when the protein was not efficiently translocated to the nucleus upon tamoxifen administration (Figure 6N, yellow diamond and triangle, respectively). However, when Snail1 reached the nucleus, the tu" @default.
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• W2011143322 title "Snail activation disrupts tissue homeostasis and induces fibrosis in the adult kidney" @default.
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• W2011143322 doi "https://doi.org/10.1038/sj.emboj.7601421" @default.
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