The microRNA miR-433 promotes renal fibrosis by amplifying the TGF-β/Smad3-Azin1 pathway
2013; Elsevier BV; Volume: 84; Issue: 6 Linguagem: Inglês
10.1038/ki.2013.272
ISSN1523-1755
AutoresRong Li, Arthur C.K. Chung, Yuan Dong, Weiqin Yang, Xiang Zhong, Hui Y. Lan,
Tópico(s)Renal and related cancers
ResumoThe TGF-β/Smad3 pathway plays a major role in tissue fibrosis, but the precise mechanisms are not fully understood. Here we identified microRNA miR-433 as an important component of TGF-β/Smad3-driven renal fibrosis. The miR-433 was upregulated following unilateral ureteral obstruction, a model of aggressive renal fibrosis. In vitro, overexpression of miR-433 enhanced TGF-β1-induced fibrosis, whereas knockdown of miR-433 suppressed this response. Furthermore, Smad3, but not Smad2, bound to the miR-433 promoter to induce its expression. Delivery of an miR-433 knockdown plasmid to the kidney by ultrasound microbubble–mediated gene transfer suppressed the induction and progression of fibrosis in the obstruction model. The antizyme inhibitor Azin1, an important regulator of polyamine synthesis, was identified as a target of miR-433. Overexpression of miR-433 suppressed Azin1 expression, while, in turn, Azin1 overexpression suppressed TGF-β signaling and the fibrotic response. Thus, miR-433 is an important component of TGF-β/Smad3-induced renal fibrosis through the induction of a positive feedback loop to amplify TGF-β/Smad3 signaling, and may be a potential therapeutic target in tissue fibrosis. The TGF-β/Smad3 pathway plays a major role in tissue fibrosis, but the precise mechanisms are not fully understood. Here we identified microRNA miR-433 as an important component of TGF-β/Smad3-driven renal fibrosis. The miR-433 was upregulated following unilateral ureteral obstruction, a model of aggressive renal fibrosis. In vitro, overexpression of miR-433 enhanced TGF-β1-induced fibrosis, whereas knockdown of miR-433 suppressed this response. Furthermore, Smad3, but not Smad2, bound to the miR-433 promoter to induce its expression. Delivery of an miR-433 knockdown plasmid to the kidney by ultrasound microbubble–mediated gene transfer suppressed the induction and progression of fibrosis in the obstruction model. The antizyme inhibitor Azin1, an important regulator of polyamine synthesis, was identified as a target of miR-433. Overexpression of miR-433 suppressed Azin1 expression, while, in turn, Azin1 overexpression suppressed TGF-β signaling and the fibrotic response. Thus, miR-433 is an important component of TGF-β/Smad3-induced renal fibrosis through the induction of a positive feedback loop to amplify TGF-β/Smad3 signaling, and may be a potential therapeutic target in tissue fibrosis. Progressive renal fibrosis is the final common manifestation of chronic kidney diseases and leads to end-stage renal failure.1.Remuzzi G. Bertani T. Pathophysiology of progressive nephropathies.N Engl J Med. 1998; 339: 1448-1456Crossref PubMed Scopus (1150) Google Scholar, 2.Iwano M. Neilson E.G. Mechanisms of tubulointerstitial fibrosis.Curr Opin Nephrol Hypertens. 2004; 13: 279-284Crossref PubMed Scopus (206) Google Scholar, 3.Muller G.A. Zeisberg M. Strutz F. 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Recent studies demonstrate the emerging role of microRNAs in various diseases, and their therapeutic potential.13.Lan H.Y. Chung A.C. Transforming growth factor-beta and Smads.Contrib Nephrol. 2011; 170: 75-82Crossref PubMed Scopus (92) Google Scholar, 14.Zhong X. Chung A.C. Chen H.Y. et al.Smad3-mediated upregulation of miR-21 promotes renal fibrosis.J Am Soc Nephrol. 2011; 22: 1668-1681Crossref PubMed Scopus (347) Google Scholar, 15.Chung A.C. Huang X.R. Meng X. et al.miR-192 mediates TGF-beta/Smad3-driven renal fibrosis.J Am Soc Nephrol. 2010; 21: 1317-1325Crossref PubMed Scopus (309) Google Scholar, 16.Qin W. Chung A.C. Huang X.R. et al.TGF-{beta}/Smad3 signaling promotes renal fibrosis by inhibiting miR-29.J Am Soc Nephrol. 2011; 22: 1462-1474Crossref PubMed Scopus (452) Google Scholar, 17.Chung A.C. Dong Y. 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Blumenberg M. et al.Transforming growth factor-beta and microRNA:mRNA regulatory networks in epithelial plasticity.Cells Tissues Organs. 2007; 185: 157-161Crossref PubMed Scopus (141) Google Scholar Some recent reports demonstrate that TGF-β1 is capable of inducing miR-21, miR-382, miR-192, miR-216a, and miR-377, but it reduces the expression of miR-200 and miR-29 families.13.Lan H.Y. Chung A.C. Transforming growth factor-beta and Smads.Contrib Nephrol. 2011; 170: 75-82Crossref PubMed Scopus (92) Google Scholar, 14.Zhong X. Chung A.C. Chen H.Y. et al.Smad3-mediated upregulation of miR-21 promotes renal fibrosis.J Am Soc Nephrol. 2011; 22: 1668-1681Crossref PubMed Scopus (347) Google Scholar, 15.Chung A.C. Huang X.R. Meng X. et al.miR-192 mediates TGF-beta/Smad3-driven renal fibrosis.J Am Soc Nephrol. 2010; 21: 1317-1325Crossref PubMed Scopus (309) Google Scholar, 16.Qin W. Chung A.C. 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Wang M. et al.TGF-beta activates Akt kinase through a microRNA-dependent amplifying circuit targeting PTEN.Nat Cell Biol. 2009; 11: 881-889Crossref PubMed Scopus (499) Google Scholar However, another recent study demonstrates that TGF-β1 suppresses miR-192 expression in human tubular epithelial cells (TECs).27.Krupa A. Jenkins R. Luo D.D. et al.Loss of MicroRNA-192 promotes fibrogenesis in diabetic nephropathy.J Am Soc Nephrol. 2010; 21: 438-447Crossref PubMed Scopus (303) Google Scholar These findings suggest that microRNAs may have a role in TGF-β-induced fibrosis, but more studies are required to confirm how TGF-β regulates the microRNA expression. In this study, we hypothesized that miR-433 regulated the progression of renal fibrosis in response to TGF-β. We used both in vitro and in vivo studies to examine whether miR-433 expression was elevated in TECs in response to TGF-β1 and the fibrotic kidneys. More importantly, we performed both prevention and intervention studies to determine whether blocking miR-433 in vivo was able to attenuate renal fibrosis in a mouse model of obstructive nephropathy. We then determined the role of Smads in regulating miR-433 expression. Finally, we determined whether targeting antizyme inhibitor 1 (Azin1) might be a mechanism by which miR-433 regulates renal injury. Microarray-based analysis demonstrated that miR-433 was significantly upregulated from mouse kidneys after unilateral ureteral obstruction (UUO) for 5 days, and this upregulation was abrogated in UUO kidneys of Smad3 KO mice in which renal fibrosis was inhibited.15.Chung A.C. Huang X.R. Meng X. et al.miR-192 mediates TGF-beta/Smad3-driven renal fibrosis.J Am Soc Nephrol. 2010; 21: 1317-1325Crossref PubMed Scopus (309) Google Scholar These results were further validated by quantitative real-time PCR (Figure 1a). Similarly, real-time PCR analysis demonstrated that renal miR-433 was elevated in anti-glomerular basement membrane glomerulonephritis (day 14) in wild-type (WT) mice, and deletion of Smad3 in mice blocked the elevation of renal miR-433 (Figure 1b). Renal miR-433 expression was also induced in diseased kidneys of a mouse model of adriamycin nephropathy (Figure 1c). These results suggest a close link between renal injury and miR-433 expression. To examine whether TGF-β1 contributed to the elevated expression of miR-433 in the fibrotic kidneys, we determined miR-433 expression in rat TECs, NRK52E cells, after treatment with TGF-β1. Real-time PCR confirmed that TGF-β1, but not the control, upregulated miR-433 expression in a time- and dose-dependent manner, peaking at 24h with an optimal dose at 2ng/ml (Figure 1d and e). The suppression of this upregulation by a neutralizing anti-TGF-β antibody implied that TGF-β signaling was required for miR-433 expression (Figure 1d). All these results strongly suggested that TGF-β signaling regulates miR-433 expression. We next determined whether miR-433 had a role in renal fibrosis in vitro by transient transfection of miR-433 mimic or anti-miR-433 into TECs. As shown in Supplementary Figure S1A and B online, the expression levels of collagen I and α-smooth muscle actin (α-SMA) induced by TGF-β1 were significantly enhanced by miR-433 mimic treatment, but inhibited by anti-miR-433 treatment. To confirm these results, we used NRK52E cell lines stably expressing vectors for miR-433 overexpression or miR-433 small hairpin RNA (shRNA; Figure 1f). The knockdown (KD) efficiency of miR-433 expression almost reached 40%, whereas overexpression of miR-433 caused a threefold elevation of the expression of miR-433. Knockdown of miR-433 significantly attenuated TGF-β1-induced expression of collagen I, fibronectin, and α-SMA in NRK-52E cells in both RNA and protein levels (Figure 1g and h and Supplementary Figure S1C online). Overexpression of miR-433 further aggravated the TGF-β1 effects (Figure 1i and j and Supplementary Figure S1D online). All these results provide strong evidence to support the role of miR-433 in TGF-β1-mediated renal fibrosis and are a compelling rationale for a therapeutic approach targeting miR-433 to suppress renal fibrosis by delivering a KD construct into fibrotic kidneys. Download .jpg (.13 MB) Help with files Supplementary Figure 1 To explore the therapeutic potential of miR-433 in renal fibrosis, we used shRNA to knock down renal miR-433 expression in a UUO model. First, we tested whether a single treatment of miR-433 shRNA was capable of reducing renal miR-433 expression. We delivered miR-433 KD plasmid into kidneys via a tail vein injection. This was followed by ultrasound transducer treatment directly onto the kidneys of normal mice, as described in our previous studies in mice and rats.14.Zhong X. Chung A.C. Chen H.Y. et al.Smad3-mediated upregulation of miR-21 promotes renal fibrosis.J Am Soc Nephrol. 2011; 22: 1668-1681Crossref PubMed Scopus (347) Google Scholar, 16.Qin W. Chung A.C. Huang X.R. et al.TGF-{beta}/Smad3 signaling promotes renal fibrosis by inhibiting miR-29.J Am Soc Nephrol. 2011; 22: 1462-1474Crossref PubMed Scopus (452) Google Scholar, 17.Chung A.C. Dong Y. Yang W. et al.Smad7 suppresses renal fibrosis via altering expression of TGF-beta/Smad3-regulated microRNAs.Mol Ther. 2013; 21: 388-398Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 19.Zhong X. Chung A.C. Chen H.Y. et al.miR-21 is a key therapeutic target for renal injury in a mouse model of type 2 diabetes.Diabetologia. 2013; 56: 663-674Crossref PubMed Scopus (286) Google Scholar, 28.Ng Y.Y. Hou C.C. Wang W. et al.Blockade of NFkappaB activation and renal inflammation by ultrasound-mediated gene transfer of Smad7 in rat remnant kidney.Kidney Int Suppl. 2005; : S83-S91Abstract Full Text Full Text PDF PubMed Google Scholar, 29.Lan H.Y. Mu W. Tomita N. et al.Inhibition of renal fibrosis by gene transfer of inducible Smad7 using ultrasound-microbubble system in rat UUO model.J Am Soc Nephrol. 2003; 14: 1535-1548Crossref PubMed Scopus (322) Google Scholar, 30.Hou C.C. Wang W. Huang X.R. et al.Ultrasound-microbubble-mediated gene transfer of inducible Smad7 blocks transforming growth factor-beta signaling and fibrosis in rat remnant kidney.Am J Pathol. 2005; 166: 761-771Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 31.Ka S.M. Huang X.R. Lan H.Y. et al.Smad7 gene therapy ameliorates an autoimmune crescentic glomerulonephritis in mice.J Am Soc Nephrol. 2007; 18: 1777-1788Crossref PubMed Scopus (108) Google Scholar As shown in Supplementary Figure S2 online, delivery of miR-433 KD plasmid was able to rapidly reduce miR-433 at day 1 after transfer (P<0.01). Although renal miR-433 expression gradually recovered, miR-433 expression at day 21 after gene transfer was still 20% below normal levels at day 0 (P<0.05), implying that our gene transfer system was sufficient to suppress miR-433 expression for 14 days. Download .jpg (.05 MB) Help with files Supplementary Figure 2 We then performed both prevention and intervention studies to determine the therapeutic potential of miR-433 in renal fibrosis. For the prevention study, miR-433 would be knocked down at the same time of the initiation of renal fibrosis. The results from the prevention study should provide the evidence whether miR-433 was able to suppress the renal fibrosis. After the ligation of the left ureter, we delivered miR-433 KD-expressing plasmids into kidneys, shortly followed by ultrasound transducer treatment directly onto the left kidney (Figure 2a). Upregulation of renal expression of TGF-β1 and miR-433 was observed at day 7 after UUO, confirming that miR-433 might have a role in renal injury (Figure 2b). Delivery of miR-433 KD plasmids into the ligated kidneys reduced renal expression of both TGF-β1 and miR-433 (Figures 2b–d). Results from in situ hybridization showed that miR-433 expression was mainly located in the tubulointerstitial area where fibrosis occurred (Figure 2d). This induction was attenuated by ∼30–40% in the presence of miR-433 shRNA treatment (Figure 2c and d). No effect was observed when control plasmids were used (Figure 2b–d). Consistent with the expectation from the results of renal TGF-β1 expression, the typical histological features of hydronephrosis, that is, fibrosis in the ligated kidney, were reduced in mice treated with miR-433 shRNA when compared with normal mice (Figure 2e). We next investigated the effect of knockdown of miR-433 on renal fibrosis, and we found that knockdown of miR-433 in UUO kidneys significantly decreased the severity of tubulointerstitial fibrosis, as demonstrated by Masson’s trichrome staining (Figure 2e). When examining fibrotic markers in the ligated kidney, we found that only minimal accumulation of collagen I, fibronectin protein, and α-SMA+ cells was observed in the normal kidney (Figure 2e and Supplementary Figure S3A and B online). At day 7 after UUO, a substantial amount of collagen I and fibronectin, as well as α-SMA+ cells, was accumulated in the ligated kidneys. However, this accumulation was significantly reduced after knocking down the renal miR-433 expression (Figure 2e and Supplementary Figure S3A and B online). The suppressive effects of miR-433 shRNA on mRNA and protein expression of fibrotic markers were further confirmed by the results from real-time PCR and western blot analyses (Figure 3a–c). These findings implied that knocking down miR-433 expression could effectively attenuate renal fibrosis by reducing ECM synthesis and deposition in the prevention study. Download .jpg (.19 MB) Help with files Supplementary Figure 3 As knockdown of miR-433 was able to suppress renal fibrosis, we next performed an intervention study to investigate whether miR-433 shRNA was able to produce a therapeutic effect on established renal fibrosis. This study was achieved by delivering miR-433 KD plasmids directly into mice at day 4 after UUO, a time when active fibrogenesis occurs (Figure 4a).14.Zhong X. Chung A.C. Chen H.Y. et al.Smad3-mediated upregulation of miR-21 promotes renal fibrosis.J Am Soc Nephrol. 2011; 22: 1668-1681Crossref PubMed Scopus (347) Google Scholar,16.Qin W. Chung A.C. Huang X.R. et al.TGF-{beta}/Smad3 signaling promotes renal fibrosis by inhibiting miR-29.J Am Soc Nephrol. 2011; 22: 1462-1474Crossref PubMed Scopus (452) Google Scholar In addition, at this time, elevation of renal expression of TGF-β1 and miR-433 had already occurred (Figure 4b and c), revealing the initiation of obstructive nephropathy. However, the elevation of expression of TGF-β1 and miR-433 was reduced after treatment with miR-433 shRNA plasmids into the ligated kidneys (Figure 4b and c). No effect was observed when control plasmids were used (Figure 4b and c). As shown in Figure 4d, tubular atrophy, loss and dilation, and proliferation of fibroblastic cells were prominent in the UUO kidneys at day 10 after UUO in comparison with mice at day 4 after UUO. More importantly, treatment with miR-433 shRNA ameliorated these pathologies in the mice. Compared with mice at day 4 after UUO, the mice also developed severe tubulointerstitial fibrosis at day 10 after UUO, as evidenced by Masson’s trichrome staining. Immunohistochemical staining also showed an abundant accumulation of collagen I and fibronectin with numerous α-SMA+ cells in UUO kidneys at day 10 (Figure 4d and Supplementary Figure S3C online). Knockdown of miR-433 in UUO kidneys markedly decreased tubulointerstitial fibrosis and reduced the expression of fibrotic markers (Figure 4d). Compared with the ligated kidney at day 4 after UUO, there was a 20–30% increase in mRNA and protein expression of fibrotic markers at day 10 after UUO (Figure 5a–c), whereas treatment with miR-433 shRNA reduced both the mRNA and protein levels of these markers to the levels obtained at day 4 after UUO (Figure 5a–c). These findings suggested that the knockdown of miR-433 expression had a therapeutic effect on renal diseases by inhibiting the progression of renal fibrosis, as demonstrated by a downregulation of ECM synthesis and the deposition and number of α-SMA+ cells. As TGF-β1 is a fibrogenesis-inducing and propagating cytokine and TGF-β1 is able to induce miR-433 expression, we continued to investigate the signaling mechanisms by which TGF-β1 regulated miR-433 expression. An inducible doxycycline (Dox)-regulated Smad7-expressing TEC line that suppresses the TGF-β/Smad3 activation32.Li J.H. Zhu H.J. Huang X.R. et al.Smad7 inhibits fibrotic effect of TGF-Beta on renal tubular epithelial cells by blocking Smad2 activation.J Am Soc Nephrol. 2002; 13: 1464-1472Crossref PubMed Scopus (235) Google Scholar was used. Although TGF-β1 induced miR-433 expression in TECs, overexpression of Smad7 in NRK52E cells virtually abolished this induction (Figure 6ai). Similarly, delivery of Smad7 overexpression plasmids into UUO kidneys suppressed miR-433 expression (Figure 6aii). In contrast, disruption of Smad7 gene in mice demonstrated opposite results that miR-433 expression was further enhanced in UUO kidneys of Smad7 KO (Smad7ΔE1) mice (Figure 6aiii), implying that activation of Smad may be necessary for TGF-β1 to induce miR-433 expression. By using TEC cell lines in which Smad2 or Smad3 expressions were specifically knocked down,33.Zhou L. Fu P. Huang X.R. et al.Mechanism of chronic aristolochic acid nephropathy: role of Smad3.Am J Physiol Renal Physiol. 2010; 298: F1006-F1017Crossref PubMed Scopus (106) Google Scholar,34.Yang F. Chung A.C. 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Huang X.R. Chung A.C. et al.Smad2 protects against TGF-{beta}/Smad3-mediated renal fibrosis.J Am Soc Nephrol. 2010; 21: 1477-1487Crossref PubMed Scopus (271) Google Scholar Smad3 KO mice were protected against renal tubulointerstitial fibrosis, but deletion of the Smad2 gene in kidneys promoted tubulointerstitial fibrosis after UUO. Consistent with the results from in vitro studies, miR-433 expression was suppressed in UUO kidneys from Smad3 KO mice but was further enhanced in UUO kidneys from conditional Smad2 KO mice (Figures 1a and 6bii), demonstrating that miR-433 is positively regulated by Smad3 but negatively by Smad2. To determine whether Smad3 specifically binds the miR-433 promoter in response to TGF-β1, we searched for potential Smad-binding sites (SBSs) in the miR-433 promoter (rVista 2.0, http://rvista.dcode.org/)37.Loots G.G. Ovcharenko I. rVISTA 2.0: evolutionary analysis of transcription factor binding sites.Nucleic Acids Res. 2004; 32: W217-W221Crossref PubMed Scopus (349) Google Scholar and we found two conserved SBSs at -1845bp and -582bp in the 5′-end of miR-433 in human, mouse, and rat genomes (Figure 6c). To identify which SBS was functional, we constructed reporter plasmids carrying various genomic sequences around the transcriptional start site of miR-433, and subjected them to luciferase assays. After treatment with TGF-β1 for 24h, results showed that the region from -995 to -65bp (SBS2), not -2840 to -976bp (SBS1), had a high promoter activity in TECs after TGF-β1 treatment, which was comparable to that of the longest region from -2840 to -65bp (Figure 6di). Luciferase assays using mutated constructs further revealed that SBS2, not SBS1, is critically required for the miR-433 promoter activity in response to TGF-β1 (Figure 6dii). To confirm this finding, we used a chromatin immunoprecipitation assay to determine the interaction of Smad3 with the miR-433 promoter region in TECs. As shown in Figure 6e, the antibody against Smad3 could successfully immunoprecipitate the DNA fragments from TECs containing the potential SBS2 but not SBS1 (Figure 6e). Smad3 binding on SBS2 in the miR-433 promoter was further confirmed by using mouse embryonic fibroblasts. All these results support the fact that Smad3 could physically interact with the miR-433 promoter region and induce the transcription of miR-433. Four microRNA target prediction programs (TargetScan, miRDB, miRanda, and Diana Lab) were used to identify the potential targets of miR-433 in human, mouse, and rat transcripts.38.Grimson A. Farh K.K. 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This approach minimized the number of possible false positives. Using this approach, we identified five potential targets, including Azin1, DNAJC5, NR2F6, SLC17A5, and TRIP12 (Figure 7a). We selected Azin1 for further study, as Azin1 is recently reported to have a role in regulating ECM expression in liver fibrosis.42.Paris A.J. Snapir Z. Christopherson C.D. et al.A polymorphism that delays fibrosis in hepatitis C promotes alternative splicing of AZIN1, reducing fibrogenesis.Hepatology. 2011; 54: 2198-2207Crossref PubMed Scopus (23) Google Scholar Treatment of TGF-β1 in TECs indeed reduced Azin
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