NR5A2 promotes epithelial-to-mesenchymal transition in renal fibrosis by targeting MMP25 transcription

NR5A2 promotes epithelial-to-mesenchymal transition in renal fibrosis by targeting MMP25 transcription | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article NR5A2 promotes epithelial-to-mesenchymal transition in renal fibrosis by targeting MMP25 transcription wang xiao, Guang Chen, Weimin Shan, Yuanzhe Chen, Wei Wang, Xiaowei Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4422061/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Epithelial-to-mesenchymal transition (EMT) is crucial for the progression of renal tubulointerstitial fibrosis, typically leading to end-stage renal failure. The role of Nuclear receptor subfamily 5 group A member 2 (NR5A2) in renal fibrosis is not yet fully understood. This research assessed NR5A2 expression in human renal fibrosis and in mouse models with unilateral ureteral obstruction (UUO). We further investigated NR5A2's function in TGF-β1-driven renal tubular EMT. Our findings demonstrated a significant elevation in NR5A2 and profibrogenic agents like collagen-I(Col-Ⅰ), alpha-smooth muscle actin (α-SMA), and fibronectin (FN) in the UUO-induced renal fibrosis mouse model. Suppressing NR5A2 and blocking it with ML180 diminished the TGF-β1-induced expression of E-cadherin, α-SMA, Col-Ⅰ , and FN in HK2 cells. Functionally, NR5A2 boosted MMP25 expression in HK2 cells after TGF-β1 activation. Luciferase and Chromatin Immunoprecipitation (ChIP) assays verified NR5A2's attachment to a distinct motif on the MMP25 promoter, initiating transcription. Crucially, silencing MMP25 countered the NR5A2-induced elevation of profibrogenic factors in HK2 cells following TGF-β1 activation. These insights reveal that NR5A2 orchestrates TGF-β1-prompted EMT in renal tubular cells via MMP25, highlighting a novel avenue for curbing renal fibrosis. Health sciences/Biomarkers Health sciences/Diseases Health sciences/Molecular medicine Health sciences/Nephrology NR5A2 MMP25 renal fibrosis EMT Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Full Text Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4422061","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":311440240,"identity":"85d333c0-e9ba-41de-8062-63d8882b6b4a","order_by":0,"name":"wang xiao","email":"","orcid":"","institution":"Fuyang People's Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"wang","middleName":"","lastName":"xiao","suffix":""},{"id":311440241,"identity":"5ce26e88-872f-4e9b-b3cf-db60cf57dbcb","order_by":1,"name":"Guang Chen","email":"","orcid":"","institution":"Fuyang People's Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Guang","middleName":"","lastName":"Chen","suffix":""},{"id":311440242,"identity":"087a95bc-6330-479d-a8b3-d93e03c62009","order_by":2,"name":"Weimin Shan","email":"","orcid":"","institution":"Fuyang People's Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Weimin","middleName":"","lastName":"Shan","suffix":""},{"id":311440243,"identity":"361f2dfd-8fb1-450f-a8d2-452ad5906e57","order_by":3,"name":"Yuanzhe Chen","email":"","orcid":"","institution":"Fuyang People's Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuanzhe","middleName":"","lastName":"Chen","suffix":""},{"id":311440244,"identity":"9b4a94d6-3e1d-4a63-8cbb-92dfc98890f1","order_by":4,"name":"Wei Wang","email":"","orcid":"","institution":"The First Affiliated Hospital of Anhui Medical University, Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Wang","suffix":""},{"id":311440245,"identity":"6c09c501-4ebc-412b-b131-b2f0c2da7a65","order_by":5,"name":"Xiaowei Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA70lEQVRIiWNgGAWjYBACPnbmBgiLvbHh8I8KBhk2QlrYmBmhWngOH3zMcIaBhwQtEmnJxoxtDDwEHQbU0ibN23Y4ccOBHDPpwnl2PHwSOWYPGGpsovFoaTaccQak5YyZ9MxtyTxsEjnmBgzH0nIbcGtpfPCh4nbutoM9ZhK825hBWswkGBsO49PScCDBAKjlMA9Qy5x6orRAbTnGlmzM23CYKC0gv/yv33+G+eDDGceO87DxPCuTSMDjF3725mPAEEszlpz/sOHAh5pqOfn25G0SH2pscGrBAgQSGBgSiFcOtvgAaepHwSgYBaNg2AMAJ/xV4PtqnwUAAAAASUVORK5CYII=","orcid":"","institution":"Fuyang People's Hospital of Anhui Medical University","correspondingAuthor":true,"prefix":"","firstName":"Xiaowei","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2024-05-15 02:33:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4422061/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4422061/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":57882449,"identity":"271d0e95-0760-4374-987b-3cb600346c2c","added_by":"auto","created_at":"2024-06-07 03:02:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1947039,"visible":true,"origin":"","legend":"\u003cp\u003eElevated NR5A2 Expression in Renal Fibrosis\u003c/p\u003e\n\u003cp\u003e(A) Analysis of GSE121190 dataset reveals that NR5A2 expression is significantly\u003c/p\u003e\n\u003cp\u003ehigher in the kidneys of mice with Unilateral Ureteral Obstruction (UUO) compared\u003c/p\u003e\n\u003cp\u003eto controls.\u003c/p\u003e\n\u003cp\u003e(B) Hematoxylin and Eosin (HE) and Masson trichrome staining show increased renal\u003c/p\u003e\n\u003cp\u003etubular damage and excessive extracellular matrix deposition (indicated in blue) in\u003c/p\u003e\n\u003cp\u003ehuman fibrotic kidneys relative to normal kidneys. Immunohistochemistry (IHC)\u003c/p\u003e\n\u003cp\u003eanalysis indicates elevated NR5A2 levels, predominantly in the tubular cells of\u003c/p\u003e\n\u003cp\u003efibrotic human kidneys.\u003c/p\u003e\n\u003cp\u003e(C) \u0026amp; (D) Quantitative Polymerase Chain Reaction (qPCR) and Western Blot (WB)\u003c/p\u003e\n\u003cp\u003eanalyses, along with quantitative data analysis, demonstrate significantly higher\u003c/p\u003e\n\u003cp\u003eNR5A2 levels in hydronephrotic kidneys compared to normal kidneys.\u003c/p\u003e\n\u003cp\u003e(E) In the UUO mouse model, HE and Masson trichrome staining reveal pronounced\u003c/p\u003e\n\u003cp\u003erenal tubular damage and increased extracellular matrix deposition (blue) at both 7\u003c/p\u003e\n\u003cp\u003edays (UUO7d) and 14 days (UUO14d) post-UUO, compared to normal mouse\u003c/p\u003e\n\u003cp\u003ekidneys. IHC shows that NR5A2 expression peaks and is primarily located in the\u003c/p\u003e\n\u003cp\u003erenal tubular cells of the UUO7d model.\u003c/p\u003e\n\u003cp\u003e(F) Both mRNA and protein levels of NR5A2 are significantly upregulated in the\u003c/p\u003e\n\u003cp\u003erenal tissues of the UUO model, especially at UUO7d.\u003c/p\u003e\n\u003cp\u003eData are presented as representative images or as means ± SD from three independent\u003c/p\u003e\n\u003cp\u003ebiological replicates. Statistical analyses were performed using a two-tailed unpaired\u003c/p\u003e\n\u003cp\u003eStudent's t-test. Significance levels are indicated as follows: *P \u0026lt; 0.05, **P \u0026lt; 0.01,\u003c/p\u003e\n\u003cp\u003e***P \u0026lt; 0.001. Scale bars = 50 μm\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4422061/v1/5c705deb52833f793ea79dbd.png"},{"id":57882252,"identity":"0f20739c-d84b-485b-bb08-16ea6451a94c","added_by":"auto","created_at":"2024-06-07 02:54:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":178184,"visible":true,"origin":"","legend":"\u003cp\u003eNR5A2 Knockdown Alleviates TGF-β1-Induced EMT and Fibrosis in\u003c/p\u003e\n\u003cp\u003eHK-2 Cells\u003c/p\u003e\n\u003cp\u003e(A) Transfection of HK-2 cells with NR5A2-specific siRNA (siNR5A2) or scramble\u003c/p\u003e\n\u003cp\u003econtrol (siCon) for 6 hours, followed by 24-hour TGF-β1 (5 ng/ml) treatment, led to\u003c/p\u003e\n\u003cp\u003esignificant NR5A2 downregulation in siNR5A2-1 transfected cells, as demonstrated\u003c/p\u003e\n\u003cp\u003eby quantitative RT-PCR. For the subsequent experiment, we selected this specific\u003c/p\u003e\n\u003cp\u003esiRNA to achieve knockdown of NR5A2.\u003c/p\u003e\n\u003cp\u003e(B,C-F) NR5A2 silencing notably counteracted the TGF-β1-induced enhancement in\u003c/p\u003e\n\u003cp\u003efibronectin,Col-Ⅰ, and α-SMA mRNA expressions, while inversely modulating\u003c/p\u003e\n\u003cp\u003eE-cadherin mRNA levels.\u003c/p\u003e\n\u003cp\u003e(G, H) Western blot analyses revealed that TGF-β1 exposure markedly increased\u003c/p\u003e\n\u003cp\u003eFN,Col-Ⅰ, α-SMA, and NR5A2 protein levels. In contrast, siNR5A2 transfection\u003c/p\u003e\n\u003cp\u003esubstantially diminished these protein expressions, opposite to the observed trend in\u003c/p\u003e\n\u003cp\u003eE-cadherin levels.\u003c/p\u003e\n\u003cp\u003e(I, J) In a similar vein, Western blot analysis revealed significant upregulation of\u003c/p\u003e\n\u003cp\u003efibronectin (FN),Col-Ⅰ, α-smooth muscle actin (α-SMA), and NR5A2 in HK-2 cells\u003c/p\u003e\n\u003cp\u003etreated with TGF-β. In contrast, treatment with the NR5A2 inhibitor ML-180\u003c/p\u003e\n\u003cp\u003epredominantly suppressed these increases, while E-cadherin expression exhibited an\u003c/p\u003e\n\u003cp\u003einverse pattern.\u003c/p\u003e\n\u003cp\u003eData represent means ± SD from three independent biological replicates. Statistical\u003c/p\u003e\n\u003cp\u003esignificance was determined using two-tailed unpaired Student's t-tests, with *P\u0026lt; 0.05,\u003c/p\u003e\n\u003cp\u003e**P\u0026lt; 0.01, and ***P\u0026lt; 0.001 denoting significance levels.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4422061/v1/deee470f781e1995d41a43f8.png"},{"id":57882251,"identity":"6c2989e4-7e5d-4237-b883-495f6929c3b0","added_by":"auto","created_at":"2024-06-07 02:54:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3682291,"visible":true,"origin":"","legend":"\u003cp\u003eR5A2 inhibitor ML-180 can alleviated EMT progression in mice with\u003c/p\u003e\n\u003cp\u003eUUO\u003c/p\u003e\n\u003cp\u003e(A, B) Hematoxylin and Eosin (HE) staining of kidneys from sham-operated mice,\u003c/p\u003e\n\u003cp\u003eUUO mice, UUO mice treated with ML-180, and sham-operated mice treated with\u003c/p\u003e\n\u003cp\u003eML-180 (negative control), revealed morphological alterations. Compared to sham,\u003c/p\u003e\n\u003cp\u003ethe renal injury index in UUO7d mice was significantly elevated but showed marked\u003c/p\u003e\n\u003cp\u003eimprovement upon ML-180 treatment. Masson trichrome staining displayed enhanced\u003c/p\u003e\n\u003cp\u003eextracellular matrix deposition in UUO7d mice, which ML-180 treatment effectively\u003c/p\u003e\n\u003cp\u003ereversed.\u003c/p\u003e\n\u003cp\u003e(C, D) Immunohistochemistry (IHC) identified increased NR5A2 expression in\u003c/p\u003e\n\u003cp\u003eUUO7 mice, notably decreased in ML-180-treated UUO7 mice .\u003c/p\u003e\n\u003cp\u003e(E, F) Fibronectin (FN) andCol-Ⅰ expressions were elevated in UUO7 mice compared\u003c/p\u003e\n\u003cp\u003eto sham but were significantly reduced following ML-180 treatment in UUO7 mice .\u003c/p\u003e\n\u003cp\u003e(G, H) IHC analysis revealed heightened α-smooth muscle actin (α-SMA) expression\u003c/p\u003e\n\u003cp\u003ein UUO7 mice, which ML-180 treatment substantially decreased. Conversely,\u003c/p\u003e\n\u003cp\u003eE-cadherin expression showed an inverse pattern.\u003c/p\u003e\n\u003cp\u003e(I, J) Western blot analysis confirmed significant upregulation of FN,Col-Ⅰ, α-SMA,\u003c/p\u003e\n\u003cp\u003eand NR5A2 proteins in UUO7d mice, which ML-180 treatment in UUO7 mice\u003c/p\u003e\n\u003cp\u003esubstantially downregulated. E-cadherin expression followed an opposite trend.\u003c/p\u003e\n\u003cp\u003eData represent means ± SD from three independent biological replicates. Statistical\u003c/p\u003e\n\u003cp\u003eanalyses were conducted using two-tailed unpaired Student's t-tests, with *P\u0026lt; 0.05,\u003c/p\u003e\n\u003cp\u003e**P\u0026lt; 0.01, ***P \u0026lt; 0.001 denoting significance levels. Scale bars = 50 μm.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4422061/v1/855d5b0b837815373110c450.png"},{"id":57882450,"identity":"3201a62e-fd06-44f6-981e-fa6e842c190f","added_by":"auto","created_at":"2024-06-07 03:02:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1039582,"visible":true,"origin":"","legend":"\u003cp\u003ePositive Correlation Between NR5A2 and MMP25 in Fibrotic Kidneys\u003c/p\u003e\n\u003cp\u003e(A) A gene expression volcano plot reveals that MMP25 is upregulated in renal\u003c/p\u003e\n\u003cp\u003esamples from the UUO7 group compared to those treated with ML180 (U-M). Gene\u003c/p\u003e\n\u003cp\u003eOntology (GO) enrichment analysis highlights significant changes in biological\u003c/p\u003e\n\u003cp\u003eprocesses (BP), molecular functions (MF), and cellular components (CC) in the\u003c/p\u003e\n\u003cp\u003eUUO7 group versus the ML180-treated UUO7 group.\u003c/p\u003e\n\u003cp\u003e(B) The correlation of immunohistochemistry (IHC) scores for NR5A2 and MMP25\u003c/p\u003e\n\u003cp\u003eprotein expression in human hydronephrotic kidneys shows a significant positive\u003c/p\u003e\n\u003cp\u003erelationship (Spearman correlation coefficient r = 0.6439, p = 0.00222, n = 20).\u003c/p\u003e\n\u003cp\u003eAdditionally, qPCR analysis confirms a positive correlation between NR5A2 and\u003c/p\u003e\n\u003cp\u003eMMP25 expression in human hydronephrotic kidneys (Spearman correlation\u003c/p\u003e\n\u003cp\u003ecoefficient r = 0.6956, p = 0.007, n = 20).\u003c/p\u003e\n\u003cp\u003e(C) IHC indicates MMP25 expression in UUO7 mice, which is reduced in UUO7\u003c/p\u003e\n\u003cp\u003emice treated with ML-180.\u003c/p\u003e\n\u003cp\u003e(D) Western blot analysis demonstrates that siNR5A2 can downregulate the protein\u003c/p\u003e\n\u003cp\u003eexpression of NR5A2 and MMP25 in vitro.\u003c/p\u003e\n\u003cp\u003e(E) Western blot analysis further shows that ML-180 can reduce NR5A2 and MMP25\u003c/p\u003e\n\u003cp\u003eprotein expression both in vitro and in vivo.\u003c/p\u003e\n\u003cp\u003eData are presented as representative images or as mean ± SD from three independent\u003c/p\u003e\n\u003cp\u003ebiological replicates. Statistical analysis was performed using a two-tailed unpaired\u003c/p\u003e\n\u003cp\u003eStudent's t-test, with significance levels indicated as*P\u0026lt; 0.05, **P\u0026lt; 0.01, ***P \u0026lt;\u003c/p\u003e\n\u003cp\u003e0.001. Scale bars = 50 μm.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-4422061/v1/a24313b9342945c806ed88b2.png"},{"id":57882253,"identity":"0125b37d-fe7f-4455-9f23-7bb46ff329cd","added_by":"auto","created_at":"2024-06-07 02:54:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":124448,"visible":true,"origin":"","legend":"\u003cp\u003eMMP25 is a Direct Transcriptional Target of NR5A2\u003c/p\u003e\n\u003cp\u003e(A) Illustrative depiction of NR5A2 binding sites (BS1, BS2, BS3) on the MMP25\u003c/p\u003e\n\u003cp\u003epromoter, as predicted by the Jaspar database (https://jaspar.elixir.no/).\u003c/p\u003e\n\u003cp\u003e(B) Evaluation of NR5A2 overexpression on MMP25 promoter activity in HK-2 cells\u003c/p\u003e\n\u003cp\u003evia dual-luciferase reporter assays revealed significant promoter activation by NR5A2\u003c/p\u003e\n\u003cp\u003e(#P\u0026lt;0.001).\u003c/p\u003e\n\u003cp\u003e(C) Generation of pGL3 reporter constructs containing varying lengths of the MMP25\u003c/p\u003e\n\u003cp\u003epromoter to investigate promoter activity.\u003c/p\u003e\n\u003cp\u003e(D) Analysis of MMP25 promoter deletion constructs exposed differential regulatory\u003c/p\u003e\n\u003cp\u003eeffects by NR5A2, with notable activity alterations observed in specific constructs,\u003c/p\u003e\n\u003cp\u003eemphasizing the critical role of the BS1 region (#P\u0026lt;0.001 compared to M-930;\u003c/p\u003e\n\u003cp\u003e*P\u0026gt;0.05 compared to M-whole).\u003c/p\u003e\n\u003cp\u003e(E,F) Chromatin immunoprecipitation (ChIP) followed by agarose gel electrophoresis\u003c/p\u003e\n\u003cp\u003eand ChIP-qPCR in HK-2 cells confirmed NR5A2's binding to the BS1 site of the\u003c/p\u003e\n\u003cp\u003eMMP25 promoter, with significant BS1 site enrichment in NR5A2-associated DNA\u003c/p\u003e\n\u003cp\u003esegments. Conversely, siRNA-mediated knockdown of NR5A2 attenuated this\u003c/p\u003e\n\u003cp\u003einteraction, reducing the affinity of NR5A2 for the MMP25 promoter(**P\u0026lt;0.01).\u003c/p\u003e\n\u003cp\u003eData represent means ± SD from three independent experiments. Statistical\u003c/p\u003e\n\u003cp\u003esignificance was determined using a two-tailed unpaired Student's t-test.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-4422061/v1/2d2d836703d729aa11fdb8a2.png"},{"id":57882256,"identity":"b5040c54-ef29-4c20-936a-1cd44a2e24f2","added_by":"auto","created_at":"2024-06-07 02:54:22","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":199014,"visible":true,"origin":"","legend":"\u003cp\u003eContribution of NR5A2 to Renal Fibrosis via MMP25 Regulation\u003c/p\u003e\n\u003cp\u003e(A) HK-2 cells were transfected with siRNA targeting MMP25 (siMMP25) or a\u003c/p\u003e\n\u003cp\u003escramble control (siCon) for 6 hours, followed by a 24-hour TGF-β1 (5 ng/ml)\u003c/p\u003e\n\u003cp\u003etreatment. Quantitative RT-PCR analysis identified siMMP25-1 as the most effective\u003c/p\u003e\n\u003cp\u003esiRNA in reducing MMP25 expression in HK-2 cells.\u003c/p\u003e\n\u003cp\u003e(B) Quantitative RT-PCR results demonstrated that MMP25 knockdown attenuated\u003c/p\u003e\n\u003cp\u003ethe TGF-β1-induced FN and α-SMA mRNA levels in HK-2 cells.\u003c/p\u003e\n\u003cp\u003e(C) Western blot analysis revealed that MMP25 knockdown attenuated the\u003c/p\u003e\n\u003cp\u003eTGF-β1-induced FN and α-SMA mRNA levels in HK-2 cells.\u003c/p\u003e\n\u003cp\u003e(D)Western blot analysis revealed increased expression of α-SMA, fibronectin,\u003c/p\u003e\n\u003cp\u003eNR5A2, and MMP25 in cells transfected with an NR5A2 overexpression plasmid.\u003c/p\u003e\n\u003cp\u003eConversely, siMMP25 significantly downregulated the expression of FN, -SMA and\u003c/p\u003e\n\u003cp\u003eMMP25.\u003c/p\u003e\n\u003cp\u003eStatistical significance was determined using appropriate tests, with p-values\u003c/p\u003e\n\u003cp\u003eindicated as *P\u0026lt; 0.05, **P\u0026lt; 0.01, ***P \u0026lt; 0.001 .\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-4422061/v1/f4383309976bb42eb2cc9021.png"},{"id":57882254,"identity":"e6c4ad49-0d78-4cff-8eda-f201318a52e2","added_by":"auto","created_at":"2024-06-07 02:54:22","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":37234,"visible":true,"origin":"","legend":"\u003cp\u003eNR5A2 promotes epithelial-to-mesenchymal transition in renal fibrosis\u003c/p\u003e\n\u003cp\u003eby targeting MMP25 transcription\u003c/p\u003e\n\u003cp\u003eThe schematic model illustrates that NR5A2 exacerbates renal fibrosis by directly\u003c/p\u003e\n\u003cp\u003eenhancing MMP25 transcription, contributing to the progression of fibrosis.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-4422061/v1/46407363e4c0649ef224e2c2.png"},{"id":79269135,"identity":"2f74dcf8-69f5-416a-9538-a07299845353","added_by":"auto","created_at":"2025-03-26 10:46:59","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2046221,"visible":true,"origin":"","legend":"","description":"","filename":"manuscriptscientificreports0525.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4422061/v1_covered_537facd4-a413-46d1-b8b7-94aa45d8ebaf.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"NR5A2 promotes epithelial-to-mesenchymal transition in renal fibrosis by targeting MMP25 transcription","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"NR5A2, MMP25, renal fibrosis, EMT","lastPublishedDoi":"10.21203/rs.3.rs-4422061/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4422061/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Epithelial-to-mesenchymal transition (EMT) is crucial for the progression of renal tubulointerstitial fibrosis, typically leading to end-stage renal failure. The role of Nuclear receptor subfamily 5 group A member 2 (NR5A2) in renal fibrosis is not yet fully understood. This research assessed NR5A2 expression in human renal fibrosis and in mouse models with unilateral ureteral obstruction (UUO). We further investigated NR5A2's function in TGF-β1-driven renal tubular EMT. Our findings demonstrated a significant elevation in NR5A2 and profibrogenic agents like collagen-I(Col-Ⅰ), alpha-smooth muscle actin (α-SMA), and fibronectin (FN) in the UUO-induced renal fibrosis mouse model. Suppressing NR5A2 and blocking it with ML180 diminished the TGF-β1-induced expression of E-cadherin, α-SMA, Col-Ⅰ , and FN in HK2 cells. Functionally, NR5A2 boosted MMP25 expression in HK2 cells after TGF-β1 activation. Luciferase and Chromatin Immunoprecipitation (ChIP) assays verified NR5A2's attachment to a distinct motif on the MMP25 promoter, initiating transcription. Crucially, silencing MMP25 countered the NR5A2-induced elevation of profibrogenic factors in HK2 cells following TGF-β1 activation. These insights reveal that NR5A2 orchestrates TGF-β1-prompted EMT in renal tubular cells via MMP25, highlighting a novel avenue for curbing renal fibrosis.","manuscriptTitle":"NR5A2 promotes epithelial-to-mesenchymal transition in renal fibrosis by targeting MMP25 transcription","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-07 02:54:18","doi":"10.21203/rs.3.rs-4422061/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4c6bbdc7-7a2d-471e-869d-79af38067aa0","owner":[],"postedDate":"June 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":32917864,"name":"Health sciences/Biomarkers"},{"id":32917865,"name":"Health sciences/Diseases"},{"id":32917866,"name":"Health sciences/Molecular medicine"},{"id":32917867,"name":"Health sciences/Nephrology"}],"tags":[],"updatedAt":"2025-03-26T10:38:45+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-07 02:54:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4422061","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4422061","identity":"rs-4422061","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}