LncRNA MALAT1 Drives Diabetic Kidney Injury via Nrf2 Suppression in Glomerular Endothelium

LncRNA MALAT1 Drives Diabetic Kidney Injury via Nrf2 Suppression in Glomerular Endothelium | 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 LncRNA MALAT1 Drives Diabetic Kidney Injury via Nrf2 Suppression in Glomerular Endothelium Li-rong Yang, Hui Wang, Li-rui Jia, Li-ting Cao, Hong-yan Luo, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8249151/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 16 You are reading this latest preprint version Abstract Objective: To investigate the role of LncRNA MALAT1 in insulin resistance (IR) and and oxidative stress injury in glomerular endothelial cells (HGECs) under high glucose and insulin (HG/Ins) conditions, and to elucidate its regulatory mechanism involving the Nrf2 pathway. HGECs were exposed to 25 mM glucose and 100 nM insulin for 48 h to induce insulin resistance. MALAT1 was silenced by siRNA, with knockdown efficiency verified via qPCR. Groups included control, ML385, and SFN. Protein expression, ROS, apoptosis, and Nrf2 translocation were assessed by Western blot, flow cytometry, and immunofluorescence. Following IR induction in HGECs (25 mM glucose + 100 nM insulin, 48 h) and siRNA-mediated MALAT1 knockdown, effects on proteins, ROS, apoptosis, and Nrf2 translocation were assessed in control and modulator groups. Conclusion: MALAT1 exacerbates IR and oxidative stress-induced injury in HGECs by inhibiting Nrf2 nuclear translocation. Targeting the MALAT1-Nrf2 axis may serve as a novel strategy for diabetic nephropathy management. Biological sciences/Cell biology Health sciences/Diseases Biological sciences/Molecular biology Health sciences/Nephrology LncRNA MALAT1 Insulin resistance Oxidative stress Diabetic nephropathy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Diabetic kidney disease (DKD), a major microvascular complication of diabetes, represents the leading cause of end-stage renal disease worldwide. Its rising prevalence poses a significant public health burden, particularly in China [1-3] . Glomerular endothelial cell (GEC) dysfunction is a pivotal early event in DKD, contributing to glomerulosclerosis through mechanisms such as endothelial dysfunction and endothelial-mesenchymal transition [4-6] . Among the key drivers of GEC injury are high glucose-induced insulin resistance (IR) and oxidative stress, which form a self-amplifying vicious cycle that aggravates renal damage [7-8] . Long non-coding RNAs (lncRNAs), including the highly conserved MALAT1, have emerged as important regulators of gene expression in various pathologies [9-10] . MALAT1 is upregulated in diabetic complications and is implicated in vascular endothelial injury [11-12] . In DKD patients, elevated MALAT1 levels in serum and urinary exosomes correlate with albuminuria and renal function decline, highlighting its potential role as a biomarker and pathogenic mediator [13-14] . Although MALAT1 promotes renal fibrosis and endothelial damage in DKD through miRNA-dependent mechanisms [15-16] , its function in the context of glomerular endothelial IR and oxidative stress remains poorly defined. The Keap1-Nrf2-ARE pathway serves as a central regulator of cellular antioxidant responses. Under oxidative stress, Nrf2 dissociates from Keap1 and translocates into the nucleus, activating cytoprotective genes such as SOD2 and GPX1 to counteract ROS accumulation [17-18] . Impaired Nrf2 signaling is closely linked to DKD progression, while its activation alleviates renal injury in diabetic models [19] . Moreover, Nrf2 dysfunction exacerbates IR, creating a feed-forward loop of metabolic and oxidative damage [20] . Although MALAT1 has been shown to regulate the Keap1-Nrf2 axis in diabetic retinopathy [21] , its role in modulating Nrf2 activity in glomerular endothelial cells under diabetic conditions remains unknown. In this study, we established a high glucose/insulin-induced IR model in human glomerular endothelial cells (HGECs) to investigate whether MALAT1 influences insulin sensitivity and oxidative injury via the Nrf2 pathway. Our findings reveal a novel MALAT1-Nrf2 regulatory axis, offering new insights into DKD pathogenesis and potential therapeutic strategies. Results 1.Establishment and Validation of the IR Model HGECs were treated with 25 mM glucose and 100 nM insulin for 48 hours to induce insulin resistance (IR). As shown in Fig. 1A, glucose consumption was significantly reduced in the IR group compared with the normal control group, as measured by the GOD-POD method. Consistent with this, Western blot analysis revealed markedly decreased phosphorylation levels of key insulin signaling proteins, including IRS-1 and Akt, in the IR group (Fig. 1B–C). These findings collectively demonstrate the successful establishment of the IR model in HGECs. 2. Effects of LncRNA MALAT1 Knockdown on Oxidative Stress Levels 2.1 Validation of MALAT1 Knockdown and Its Impact on Insulin Signaling qPCR analysis confirmed efficient MALAT1 silencing, with its expression reduced by over 70% in the IR+MKD group compared to the IR group (Fig. 2A). Furthermore, MALAT1 knockdown rescued the impaired phosphorylation of IRS-1 and Akt in IR-HGECs, indicating restoration of insulin signaling (Fig. 2B-I). 2.2 Regulation of Mitochondrial ROS and Antioxidant Defense by MALAT1 Flow cytometry revealed a significant increase in mitochondrial ROS in IR-HGECs, while total ROS levels were unaltered. MALAT1 knockdown markedly attenuated mitochondrial ROS production(Fig. 3J). Correspondingly, Western blot analysis showed that the protein levels of the antioxidant enzymes SOD2 and GPx1, which were downregulated in the IR group, were upregulated by MALAT1 knockdown (Fig. 3B-F). 2.3 MALAT1 Knockdown Attenuates Apoptosis in IR-HGECs The IR group exhibited elevated apoptosis, as evidenced by increased Cleaved caspase-3 levels and a higher Bax、Bcl-2 expression ratio. MALAT1 knockdown significantly reversed these pro-apoptotic changes, indicating a protective role against IR-induced apoptosis (Fig. 3K-L). 3. High-throughput data simulation of the binding capacity between Lnc MALAT1 and Nrf2 Building on our previous work, we simulated the binding activity between Lnc MALAT1 and Nrf2 using high-throughput data. The ligand establishes stable interactions within the protein binding pocket (Fig. 3A): conventional hydrogen bonds with Gln26 (3.24 Å) and Ile501 (2.38 Å), a π-cation interaction with Lys508 (3.90 Å), and π-alkyl/π-σ interactions with Ile474 (4.42/4.62 Å) and Arg504 (5.01 Å). The 3D structure of the complex (Fig. 3B) reveals complementary spatial fitting between the ligand and key residues (Arg456, Thr455, Gln26, Ile501, Lys508, Ile474), which provides a structural basis for binding affinity and specificity . 4. LncRNA MALAT1 Exerts Its Function by Regulating Nrf2 Nuclear Translocation Immunofluorescence staining revealed that Nrf2 nuclear translocation was significantly suppressed in the IR group, with a marked reduction in nuclear Nrf2 fluorescence intensity compared to the normal control (NC) group. In contrast, MALAT1 knockdown prominently promoted Nrf2 nuclear translocation, leading to substantial accumulation of Nrf2 in the nucleus. ML385 inhibited this promotional effect, while SFN mimicked the inductive effect of MALAT1 knockdown on Nrf2 nuclear translocation (Fig.3 A). Western blot(Fig4 A) and flow cytometry results indicated that MALAT1 knockdown reduced the apoptosis rate of IR-HGECs, and this anti-apoptotic effect was abrogated by ML385 treatment (Fig.3 B-C).Quantitative real-time polymerase chain reaction (qPCR) and Western blot (WB) analyses demonstrated that the mRNA and protein expression levels of Nrf2 downstream antioxidant genes (SOD2, GPx1, HO-1, NQO1) were downregulated in the IR group, whereas MALAT1 knockdown (IR+MKD group) significantly upregulated the expression of these genes(Fig 4 A-E). The Nrf2 inhibitor ML385 reversed this upregulatory effect, while the Nrf2 activator SFN recapitulated it. Meanwhile, flow cytometry analysis showed that the MALAT1 knockdown-mediated decrease in mitochondrial reactive oxygen species (ROS) levels was also blocked by ML385, confirming that MALAT1 regulates oxidative stress and cell survival in IR-HGECs through the Nrf2 pathway (Fig.4 F). Materials and Methods 1. Cell Culture ： Human glomerular endothelial cells (HGECs) were obtained from Procell and maintained in DMEM (Gibco) supplemented with 10% fetal bovine serum (Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C in a 5% CO₂ atmosphere. 2. Reagents and Antibodies ： MALAT1-targeting siRNA and its negative control were synthesized by Qingke. Total RNA was extracted and reverse-transcribed using kits from TaKaRa. The following primary antibodies from Proteintech were used for Western blotting: anti-p-IRS-1, anti-p-Akt ,anti-Nrf2, anti-SOD2, anti-GPX1, anti-Cleaved caspase-3, anti-Bax, anti-Bcl-2, and anti-β-actin. Mitochondrial ROS levels were measured using a detection kit from Elabscience. Apoptosis was assessed with an Annexin V-FITC/PI apoptosis detection kit (Beyotime). Cellular glucose consumption was determined by a GOD-POD assay kit (Beyotime). 3. Validation of the IR Model Measurement of Glucose Consumption: Glucose consumption was determined by measuring the glucose concentration in the cell culture supernatant using the GOD-POD method. The value was calculated by subtracting the residual glucose concentration after culture from the initial concentration in the fresh medium. Western Blot Analysis: Following SDS-PAGE and transfer, membranes were probed with primary antibodies overnight at 4°C and then with a secondary antibody. Protein bands were visualized by ECL and quantified with ImageJ. 4. Validation of MALAT1 Knockdown Efficiency Total RNA was extracted using Trizol and reverse-transcribed into cDNA. qPCR was performed with the following primers: MALAT1 (Forward: 5'-CATGACGCAGGGAGAATTGC-3', Reverse: 5'-GCCTTCCCGTACTTCTGTCTT-3'). The thermal cycling conditions consisted of initial denaturation at 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. Gene expression was quantified using the 2^(–ΔΔCt) method. 5. Detection of Oxidative Stress-Related Indicators Total and mitochondrial ROS levels were measured by incubating cells with 10 μM DCFH-DA for 30 min or 5 μM MitoSOX Red for 15 min at 37°C, respectively. Fluorescence intensity was quantified after PBS washing. The mRNA expression of SOD2, GPX1, HO-1, and NQO1 was analyzed by qPCR, and SOD2 and GPx1 protein levels were determined by Western blot. 6. Detection of Cell Apoptosis: Cell apoptosis was assessed by flow cytometry following Annexin V-FITC/PI staining. The expression of apoptosis-related proteins was analyzed by Western blot, and the Bax/Bcl-2 ratio was calculated. 7. Detection of Nrf2 Nuclear Translocation After treatment, cells were fixed, permeabilized, and blocked, then incubated overnight with anti-Nrf2 antibody (1:200) at 4°C, followed by a fluorescent secondary antibody (1:500) for 1 h at room temperature. Nuclei were counterstained with DAPI. Nrf2 localization was visualized by confocal microscopy, and nuclear fluorescence intensity was quantified with ImageJ. 7. Statistical Analysis: Data are expressed as mean ± SD and analyzed by one-way ANOVA with LSD post-hoc test using SPSS 22.0. A P value < 0.05 was considered statistically significant. Discussion Diabetic kidney disease (DKD) progression is closely associated with glomerular endothelial injury, driven largely by high glucose/insulin-induced insulin resistance (IR) and oxidative stress. This study reveals a novel role of the long non-coding RNA MALAT1 in regulating the Nrf2-mediated antioxidant pathway in HGECs under IR conditions. We established an IR model in HGECs using high glucose and insulin, observing impaired glucose consumption and insulin signaling, alongside significant MALAT1 upregulation. This aligns with reports of elevated MALAT1 in diabetic kidneys and DKD patient serum. The IR model exhibited specific elevation in mitochondrial ROS without total ROS changes, accompanied by reduced expression of antioxidant enzymes SOD2 and GPX1. MALAT1 knockdown effectively attenuated mitochondrial ROS, restored antioxidant enzyme levels, suppressed apoptosis, and improved insulin sensitivity. Mechanistically, Nrf2 nuclear translocation was suppressed in IR-HGECs, while MALAT1 knockdown promoted this process. The Nrf2 inhibitor ML385 abolished the protective effects of MALAT1 silencing, whereas the activator sulforaphane mimicked them, confirming Nrf2 dependency. Given evidence of direct MALAT1-Nrf2 binding, we propose that MALAT1 interferes with Nrf2 transcriptional activity, potentially by blocking its access to antioxidant gene promoters. Furthermore, improved insulin signaling following MALAT1 knockdown was Nrf2-dependent, consistent with Nrf2's known role in mitigating oxidative stress-induced insulin pathway impairment. In conclusion, IR-induced MALAT1 upregulation exacerbates HGEC injury by inhibiting Nrf2 nuclear translocation, leading to mitochondrial ROS accumulation, impaired antioxidant defense, and aggravated apoptosis and insulin resistance. Targeting the MALAT1-Nrf2 axis represents a potential therapeutic strategy for DKD, though in vivo validation remains necessary. Declarations Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author contributions Author Contributions Li-rong Yang: Writing – review & editing, Writing original draft. Hui Wang: Writing – review & editing, Writing – original draft. Li-rui Jia: Writing – original draft, Data curation. Li-ting Cao: Data curation, Visualization. Jia-mei Ding: Data curation, Resources. Yan-song Wang: Visualization. Yun-peng Tang: Visualization. Ya-bo Chen: Visualization. Xiao He: Visualization. Ya-li Zheng: Methodology. Funding This work was supported by Natural Science Foundation of Ningxia Province (Grant No. 2023A0138); Preliminary Research Project of the National Natural Science Foundation of China (Grant No. 2026GZRYSY007). References Zhang L, Wang Y, Li J, et al. Global prevalence and progression of diabetic kidney disease: A systematic review and meta-analysis[J]. Lancet Diabetes Endocrinol, 2020, 8(5): 423 - 432. Jha V, Garcia - Garcia G, Iseki K, et al. Chronic kidney disease: Global dimension and perspectives[J]. Lancet, 2013, 382(9891): 607 - 619. Zuo L, Li Y, Chen N, et al. Prevalence of chronic kidney disease in China: A cross - sectional survey[J]. Lancet, 2012, 379(9818): 815 - 822. Bakris GL, Weir MR. Diabetic kidney disease: Core curriculum 2020[J]. Am J Kidney Dis, 2020, 75(3): 409 - 421. Chinese guideline for the prevention and treatment of diabetic kidney disease (2021 edition)[J]. Chin Med J (Engl), 2022, 135 (17): 2021 - 2058. Nangaku M. Pathophysiology of diabetic nephropathy[J]. Clin Exp Nephrol, 2016, 20(1): 1 - 9. Schmidt AM, Warner FJ, Leroux F, et al. Glomerular endothelial cell dysfunction in diabetic kidney disease[J]. Cardiovasc Diabetol, 2019, 18(1): 148. Yu X, Chen Y, Li M, et al. Endothelial - to - mesenchymal transition contributes to glomerulosclerosis in diabetic nephropathy[J]. Am J Physiol Renal Physiol, 2018, 314(3): F452 - F463. Chen W, Huang Y, Xu C, et al. Vascular endothelial growth factor signaling in glomerular endothelial cell dysfunction of diabetic nephropathy[J]. Int J Mol Sci, 2020, 21(12): 4389. Evans JL, Goldfine ID, Maddux BA, et al. Oxidative stress and stress - activated signaling pathways: A unifying hypothesis of type 2 diabetes[J]. Endocr Rev, 2002, 23(5): 599 - 622. Brownlee M. The pathobiology of diabetic complications: A unifying mechanism[J]. Diabetes, 2005, 54(6): 1615 - 1625. Xu D, Liu J, Zhang Y, et al. Insulin resistance and oxidative stress: A vicious cycle in diabetic kidney disease[J]. Oxid Med Cell Longev, 2020, 2020: 8892745. Wang X, Li D, Zhao J, et al. High glucose - induced insulin resistance in glomerular endothelial cells via impaired insulin signaling pathway[J]. Mol Cell Endocrinol, 2019, 497: 110508. Jiang Y, Chen L, Zhang M, et al. Advanced glycation end products promote insulin resistance and oxidative stress in diabetic kidney disease[J]. J Cell Mol Med, 2021, 25(11): 5123 - 5134. Lay A, Lovatt C, Cook S, et al. Profiling of insulin-resistant kidney models and human biopsies reveals common and cell-type-specific mechanisms underpinning Diabetic Kidney Disease [J]. Kidney Int, 2024, 105 (6): 1129 - 1145. Esteller M. Non - coding RNAs in human disease[J]. Nat Rev Genet, 2011, 12(12): 861 - 874. Rinn JL, Chang HY. Genome regulation by long noncoding RNAs[J]. Annu Rev Biochem, 2012, 81: 145 - 166. Tripathi V, Ellis JD, Shen Z, et al. MALAT1, a noncoding RNA, regulates alternative splicing and tumor metastasis[J]. Genes Dev, 2010, 24(11): 1174 - 1189. Gutschner T, Diederichs S. The role of long non - coding RNAs in cancer[J]. Semin Cancer Biol, 2012, 22(3): 231 - 240. Zhang Y, Wang Z, Li C, et al. LncRNA MALAT1: A novel therapeutic target in kidney diseases[J]. Life Sci, 2022, 307: 120987. Chatterjee K, Das S. MALAT1: a therapeutic candidate for a broad spectrum of vascular and cardiorenal complications[J]. Nat Rev Nephrol, 2019, 15(12): 745 - 758. Liu C, Zhang J, Wang Y, et al. Urinary exosomal MALAT1 as a diagnostic biomarker for diabetic kidney disease[J]. Diabetes Care, 2023, 46(8): 1659 - 1667. Zhao L, Chen J, Li Y, et al. Upregulated MALAT1 correlates with albuminuria and renal dysfunction in diabetic kidney disease[J]. Int J Biochem Cell Biol, 2021, 136: 105912. Wang H, Zhang X, Liu M, et al. MALAT1 promotes endothelial dysfunction in diabetic nephropathy via regulating miR - 124 - 3p/TLR4 axis[J]. Cell Death Dis, 2022, 13(5): 432. Li S, Guo Y, Han J, et al. LncRNA MALAT1 exacerbates diabetic kidney injury by inducing renal fibrosis[J]. J Diabetes Invest, 2020, 11(6): 1389 - 1398. Zhou Y, Chen F, Huang X. Knockdown of MALAT1 attenuates high - glucose - induced angiogenesis and inflammation via endoplasmic reticulum stress in human retinal vascular endothelial cells[J]. Biochem Biophys Res Commun, 2020, 529(4): 987 - 993. Xue Y, Wang L, Li M, et al. Urinary exosomal lncRNAs MALAT1 and PVT1 as potential biomarkers for diabetic kidney disease[J]. Diabetes, 2023, 72(Suppl 1): A456. Kensler TW, Wakabayashi N, Biswal S. Cell survival responses to environmental stresses via the Keap1 - Nrf2 - ARE pathway[J]. Annu Rev Pharmacol Toxicol, 2007, 47: 89 - 116. Suzuki T, Motohashi H, Yamamoto M. Toward clinical application of the Keap1 - Nrf2 pathway[J]. Trends Pharmacol Sci, 2019, 40(1): 68 - 83. Liu Z, Liu J, Li Y, et al. Activation of Nrf2 ameliorates diabetic kidney disease by inhibiting ferroptosis[J]. Adv Sci, 2024, 11(3): 2303245. Lee HJ, Kim JH, Park JS, et al. Keap1 - Nrf2 pathway in diabetic kidney disease: A potential therapeutic target[J]. J Endocrinol, 2020, 247(3): R157 - R170. Zhang M, Chen L, Jiang Y, et al. LncRNA MALAT1 regulates Keap1 - Nrf2 - antioxidant defense in diabetic retinopathy[J]. Free Radic Biol Med, 2021, 172: 123 - 135. Additional Declarations No competing interests reported. Supplementary Files 20260104.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 09 Feb, 2026 Reviewers agreed at journal 02 Feb, 2026 Reviews received at journal 02 Feb, 2026 Reviews received at journal 29 Jan, 2026 Reviewers agreed at journal 28 Jan, 2026 Reviews received at journal 27 Jan, 2026 Reviewers agreed at journal 27 Jan, 2026 Reviewers agreed at journal 27 Jan, 2026 Reviewers agreed at journal 26 Jan, 2026 Reviewers agreed at journal 26 Jan, 2026 Reviewers agreed at journal 26 Jan, 2026 Reviewers invited by journal 19 Jan, 2026 Editor assigned by journal 19 Jan, 2026 Editor invited by journal 16 Jan, 2026 Submission checks completed at journal 07 Jan, 2026 First submitted to journal 07 Jan, 2026 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8249151","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":577496217,"identity":"6ce026c9-c2ff-409b-9401-be89b1445ed5","order_by":0,"name":"Li-rong Yang","email":"","orcid":"","institution":"People’s Hospital of Ningxia Hui Autonomous Region","correspondingAuthor":false,"prefix":"","firstName":"Li-rong","middleName":"","lastName":"Yang","suffix":""},{"id":577496218,"identity":"e88ad49a-9968-4185-8902-50bd30d6fb5e","order_by":1,"name":"Hui Wang","email":"","orcid":"","institution":"People’s Hospital of Ningxia Hui Autonomous Region","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Wang","suffix":""},{"id":577496219,"identity":"f93861c9-f532-4101-a51e-61c920577916","order_by":2,"name":"Li-rui Jia","email":"","orcid":"","institution":"People’s Hospital of Ningxia Hui Autonomous Region","correspondingAuthor":false,"prefix":"","firstName":"Li-rui","middleName":"","lastName":"Jia","suffix":""},{"id":577496220,"identity":"3688a624-b91c-4e10-9599-aac3b2c8c884","order_by":3,"name":"Li-ting Cao","email":"","orcid":"","institution":"People’s Hospital of Ningxia Hui Autonomous Region","correspondingAuthor":false,"prefix":"","firstName":"Li-ting","middleName":"","lastName":"Cao","suffix":""},{"id":577496221,"identity":"4ef5a3cd-6be1-433e-b61e-8c709700987a","order_by":4,"name":"Hong-yan Luo","email":"","orcid":"","institution":"People’s Hospital of Ningxia Hui Autonomous Region","correspondingAuthor":false,"prefix":"","firstName":"Hong-yan","middleName":"","lastName":"Luo","suffix":""},{"id":577496222,"identity":"c4a57eb1-270b-4f2d-9021-79bb06220d09","order_by":5,"name":"Jia-mei Ding","email":"","orcid":"","institution":"Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jia-mei","middleName":"","lastName":"Ding","suffix":""},{"id":577496223,"identity":"4d8a3f0d-892f-4f3f-a71e-99554383892a","order_by":6,"name":"Yan-song Wang","email":"","orcid":"","institution":"Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yan-song","middleName":"","lastName":"Wang","suffix":""},{"id":577496224,"identity":"9dc92477-a0c6-4ef9-bdf0-63eeb0ae38b4","order_by":7,"name":"Yun-peng Tang","email":"","orcid":"","institution":"Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yun-peng","middleName":"","lastName":"Tang","suffix":""},{"id":577496225,"identity":"f10bea0e-350d-4891-b710-64b305cd2f74","order_by":8,"name":"Ya-bo Chen","email":"","orcid":"","institution":"Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ya-bo","middleName":"","lastName":"Chen","suffix":""},{"id":577496226,"identity":"bfe2d43c-e0ee-4a57-8b33-e64258bf4b4b","order_by":9,"name":"Xiao He","email":"","orcid":"","institution":"Ningxia Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"","lastName":"He","suffix":""},{"id":577496227,"identity":"c1152462-aa86-46b5-822b-d7bfb13c2ed1","order_by":10,"name":"Xin Tao","email":"","orcid":"","institution":"People’s Hospital of Ningxia Hui Autonomous Region","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Tao","suffix":""},{"id":577496230,"identity":"9ac3768f-fc73-4110-b055-78a0def9514d","order_by":11,"name":"Ya-li Zheng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2UlEQVRIiWNgGAWjYDACCQglw8beQKIWHjaeA6RqYZBIIFKH/OzmYw+/ttnx8Em+PSZdmXM4sYH98NEN+LQwzjmWbizblszDJp2XbHh2G1ALT1raDXxamCVyzKQl25iBWnIMHzaCtEjwmOHVwiaR/w2opZ6HTfKMwUGitPBI5LBJfmw7zMMmwUOkLRISaWbSDOeOAwM5x9iwcVu6cRshv8jPSH4m+aOsWk6+/YyZZOM2a9l+9sPH8GoBAWZeNji72bGNkHIQYPzxB86usydGxygYBaNgFIwsAADfQUTOx8YbugAAAABJRU5ErkJggg==","orcid":"","institution":"People’s Hospital of Ningxia Hui Autonomous Region","correspondingAuthor":true,"prefix":"","firstName":"Ya-li","middleName":"","lastName":"Zheng","suffix":""}],"badges":[],"createdAt":"2025-12-01 10:23:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8249151/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8249151/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":100782095,"identity":"3c5f6443-9ea5-45f4-b86b-0166df083622","added_by":"auto","created_at":"2026-01-21 11:45:09","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1529714,"visible":true,"origin":"","legend":"","description":"","filename":"LncRNAMALAT1DrivesDiabeticKidneyInjuryviaNrf2SuppressioninGlomerularEndothelium.docx","url":"https://assets-eu.researchsquare.com/files/rs-8249151/v1/cf06f4561c4ccfb26ca4670e.docx"},{"id":100782046,"identity":"47655316-cbba-4864-876b-4279bb6a4ceb","added_by":"auto","created_at":"2026-01-21 11:44:44","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":11485,"visible":true,"origin":"","legend":"","description":"","filename":"4b90874c3ffe48d8957eeaef72a1c075.json","url":"https://assets-eu.researchsquare.com/files/rs-8249151/v1/d26e695a695cd335d12f0344.json"},{"id":100782182,"identity":"3c328513-85e8-4002-9829-1550307b1253","added_by":"auto","created_at":"2026-01-21 11:45:27","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":27698983,"visible":true,"origin":"","legend":"","description":"","filename":"20260104.docx","url":"https://assets-eu.researchsquare.com/files/rs-8249151/v1/245f4da66e96f8271d9637bb.docx"},{"id":100782196,"identity":"a2098030-05aa-417e-8546-983291fa9c16","added_by":"auto","created_at":"2026-01-21 11:45:32","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":63182,"visible":true,"origin":"","legend":"","description":"","filename":"4b90874c3ffe48d8957eeaef72a1c0751enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8249151/v1/dfe91a599e2ca90f3070d86a.xml"},{"id":100781929,"identity":"ba59077a-a1a6-4103-a400-0968eadf0a87","added_by":"auto","created_at":"2026-01-21 11:44:34","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":11073,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8249151/v1/bcf588e4707518f4e0e8fbdf.png"},{"id":100781879,"identity":"6d16e1d1-5059-481a-9659-cc33af90ac0c","added_by":"auto","created_at":"2026-01-21 11:44:33","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":101477,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8249151/v1/5cef4e293cb16dde0a8e5aa0.png"},{"id":100782194,"identity":"fe95be8d-265e-4766-9908-35952be2dcfd","added_by":"auto","created_at":"2026-01-21 11:45:31","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":89578,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8249151/v1/3c43065bd0f4cc82fbaf0959.png"},{"id":100782068,"identity":"f3e152f5-dd77-45e7-84bf-e68f5540d8aa","added_by":"auto","created_at":"2026-01-21 11:44:56","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":109377,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8249151/v1/16c1a3ccace037a3112af09b.png"},{"id":100782324,"identity":"364725bf-9f70-489c-8d0a-4c501200352e","added_by":"auto","created_at":"2026-01-21 11:46:05","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":60888,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8249151/v1/57a71d5e1b154d3c667556db.png"},{"id":100782080,"identity":"75987eda-a4bc-4dce-8988-90f54e19e8b1","added_by":"auto","created_at":"2026-01-21 11:45:00","extension":"xml","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":60284,"visible":true,"origin":"","legend":"","description":"","filename":"4b90874c3ffe48d8957eeaef72a1c0751structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8249151/v1/172397f19a0e7936d80557d0.xml"},{"id":100781965,"identity":"ca8eae75-54e0-4b25-a58a-53de553bae44","added_by":"auto","created_at":"2026-01-21 11:44:38","extension":"html","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":72351,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8249151/v1/07f77e4a4089a0fc8d862a75.html"},{"id":100782241,"identity":"27a5461b-59a2-419f-a3b3-8b829567646d","added_by":"auto","created_at":"2026-01-21 11:45:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":51306,"visible":true,"origin":"","legend":"\u003cp\u003eValidation of the insulin resistance (IR) model. A: Detection of glucose consumption; B-C: Western blot analysis of the expression levels of p-AKT and p-IRS, key proteins in the insulin signaling pathway.Data are shown as the mean ± SEM. (n = 3) \u003cem\u003e*P \u0026lt; 0.05, **P \u0026lt; 0.01 \u003c/em\u003eversus control group;\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8249151/v1/cbb565ac619207052c24fe5f.png"},{"id":100781820,"identity":"5e442f64-db20-4dc1-8b7f-623f45beaa09","added_by":"auto","created_at":"2026-01-21 11:44:24","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":444761,"visible":true,"origin":"","legend":"\u003cp\u003eMALAT1 knockdown mitigates insulin resistance, oxidative stress, and apoptosis in IR-HGECs.(A) Validation of MALAT1 knockdown efficiency.(B)Representative Western blots of key proteins in insulin signaling (p-IRS-1, p-Akt), antioxidant defense (SOD2, GPx1), and apoptosis (Cleaved caspase-3, Bcl-2, Bax). (C-I) Quantitative analysis of protein levels. (J) Quantification of mitochondrial ROS (K-L) and flow cytometry analysis. Data are mean ± SD (n=3). *p \u0026lt; 0.05, **p \u0026lt; 0.01 vs. NC; *p \u0026lt; 0.05, **p \u0026lt; 0.01 vs. IR; * p \u0026lt; 0.05, **p \u0026lt; 0.01 vs. IR+MKD.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8249151/v1/73d81c77684a776b3349c485.jpeg"},{"id":100781994,"identity":"bd930e84-b36f-4ecf-9c49-6031c0108ae0","added_by":"auto","created_at":"2026-01-21 11:44:41","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":336203,"visible":true,"origin":"","legend":"\u003cp\u003eA: Schematic of key interactions between ligand and protein binding pocket B: 3D spatial arrangement of ligand-protein complex\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8249151/v1/a6c11cf7870666e457e9f418.jpeg"},{"id":100781649,"identity":"cb815c0a-b55d-4dd3-842b-78d3db4f7b65","added_by":"auto","created_at":"2026-01-21 11:44:07","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":553383,"visible":true,"origin":"","legend":"\u003cp\u003eA: Immunofluorescence (IF) staining for Nrf2 nuclear translocation; B-C: Flow cytometry analysis of cell apoptosis.Data are mean ± SD (n=3). *p \u0026lt; 0.05, **p \u0026lt; 0.01 vs. NC; *p \u0026lt; 0.05, **p \u0026lt; 0.01 vs. IR; * p \u0026lt; 0.05, **p \u0026lt; 0.01 vs. IR+MKD.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8249151/v1/0ecb1d5c1919b890a79f729c.jpeg"},{"id":100781692,"identity":"f7661d3c-83c3-4202-b50d-58be09adac35","added_by":"auto","created_at":"2026-01-21 11:44:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":369619,"visible":true,"origin":"","legend":"\u003cp\u003eA-B: Western blot analysis of the expression levels of apoptosis-related proteins (Bax, Bcl-2, cleaved-Caspase-3) and key proteins in the insulin signaling pathway (SOD2, p-AKT1, p-IRS); C-E:qPCR analysis of NQO1, HO-1, and GPX1 mRNA expression levels; F: Flow cytometry detection of mitochondrial ROS levels. Data are mean ± SD (n=3). *p \u0026lt; 0.05, **p \u0026lt; 0.01 vs. NC; *p \u0026lt; 0.05, **p \u0026lt; 0.01 vs. IR; * p \u0026lt; 0.05, **p \u0026lt; 0.01 vs. IR+MKD.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8249151/v1/380b5196d14e49a3cd7c4f7d.png"},{"id":100785543,"identity":"30c35c88-9587-49d9-aa96-00ef031934f4","added_by":"auto","created_at":"2026-01-21 11:56:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2208420,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8249151/v1/0684f1e3-2bc9-43a9-9ddd-445d8d239819.pdf"},{"id":100782180,"identity":"742afdbd-9ecf-4f5f-9efb-50dac51fbc8d","added_by":"auto","created_at":"2026-01-21 11:45:25","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":27698983,"visible":true,"origin":"","legend":"","description":"","filename":"20260104.docx","url":"https://assets-eu.researchsquare.com/files/rs-8249151/v1/55dcad1dd9201a3d89e0fb26.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"LncRNA MALAT1 Drives Diabetic Kidney Injury via Nrf2 Suppression in Glomerular Endothelium","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDiabetic kidney disease (DKD), a major microvascular complication of diabetes, represents the leading cause of end-stage renal disease worldwide. Its rising prevalence poses a significant public health burden, particularly in China \u003csup\u003e[1-3]\u003c/sup\u003e. Glomerular endothelial cell (GEC) dysfunction is a pivotal early event in DKD, contributing to glomerulosclerosis through mechanisms such as endothelial dysfunction and endothelial-mesenchymal transition \u003csup\u003e[4-6]\u003c/sup\u003e. Among the key drivers of GEC injury are high glucose-induced insulin resistance (IR) and oxidative stress, which form a self-amplifying vicious cycle that aggravates renal damage \u003csup\u003e[7-8]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eLong non-coding RNAs (lncRNAs), including the highly conserved MALAT1, have emerged as important regulators of gene expression in various pathologies \u003csup\u003e[9-10]\u003c/sup\u003e. MALAT1 is upregulated in diabetic complications and is implicated in vascular endothelial injury \u003csup\u003e[11-12]\u003c/sup\u003e. In DKD patients, elevated MALAT1 levels in serum and urinary exosomes correlate with albuminuria and renal function decline, highlighting its potential role as a biomarker and pathogenic mediator \u003csup\u003e[13-14]\u003c/sup\u003e. Although MALAT1 promotes renal fibrosis and endothelial damage in DKD through miRNA-dependent mechanisms \u003csup\u003e[15-16]\u003c/sup\u003e, its function in the context of glomerular endothelial IR and oxidative stress remains poorly defined.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe Keap1-Nrf2-ARE pathway serves as a central regulator of cellular antioxidant responses. Under oxidative stress, Nrf2 dissociates from Keap1 and translocates into the nucleus, activating cytoprotective genes such as SOD2 and GPX1 to counteract ROS accumulation \u003csup\u003e[17-18]\u003c/sup\u003e. Impaired Nrf2 signaling is closely linked to DKD progression, while its activation alleviates renal injury in diabetic models \u003csup\u003e[19]\u003c/sup\u003e. Moreover, Nrf2 dysfunction exacerbates IR, creating a feed-forward loop of metabolic and oxidative damage \u003csup\u003e[20]\u003c/sup\u003e. Although MALAT1 has been shown to regulate the Keap1-Nrf2 axis in diabetic retinopathy \u003csup\u003e[21]\u003c/sup\u003e, its role in modulating Nrf2 activity in glomerular endothelial cells under diabetic conditions remains unknown. In this study, we established a high glucose/insulin-induced IR model in human glomerular endothelial cells (HGECs) to investigate whether MALAT1 influences insulin sensitivity and oxidative injury via the Nrf2 pathway. Our findings reveal a novel MALAT1-Nrf2 regulatory axis, offering new insights into DKD pathogenesis and potential therapeutic strategies.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003e1.Establishment and Validation of the IR Model\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHGECs were treated with 25 mM glucose and 100 nM insulin for 48 hours to induce insulin resistance (IR). As shown in Fig. 1A, glucose consumption was significantly reduced in the IR group compared with the normal control group, as measured by the GOD-POD method. Consistent with this, Western blot analysis revealed markedly decreased phosphorylation levels of key insulin signaling proteins, including IRS-1 and Akt, in the IR group (Fig. 1B\u0026ndash;C). These findings collectively demonstrate the successful establishment of the IR model in HGECs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2. Effects of LncRNA MALAT1 Knockdown on Oxidative Stress Levels\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.1 Validation of MALAT1 Knockdown and Its Impact on Insulin Signaling\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eqPCR analysis confirmed efficient MALAT1 silencing, with its expression reduced by over 70% in the IR+MKD group compared to the IR group (Fig. 2A). Furthermore, MALAT1 knockdown rescued the impaired phosphorylation of IRS-1 and Akt in IR-HGECs, indicating restoration of insulin signaling (Fig. 2B-I).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.2 Regulation of Mitochondrial ROS and Antioxidant Defense by MALAT1\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFlow cytometry revealed a significant increase in mitochondrial ROS in IR-HGECs, while total ROS levels were unaltered. MALAT1 knockdown markedly attenuated mitochondrial ROS production(Fig. 3J). Correspondingly, Western blot analysis showed that the protein levels of the antioxidant enzymes SOD2 and GPx1, which were downregulated in the IR group, were upregulated by MALAT1 knockdown (Fig. 3B-F).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.3 MALAT1 Knockdown Attenuates Apoptosis in IR-HGECs\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe IR group exhibited elevated apoptosis, as evidenced by increased Cleaved caspase-3 levels and a higher Bax、Bcl-2 expression ratio. MALAT1 knockdown significantly reversed these pro-apoptotic changes, indicating a protective role against IR-induced apoptosis (Fig. 3K-L).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3. High-throughput data simulation of the binding capacity between Lnc MALAT1 and Nrf2\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBuilding on our previous work, we simulated the binding activity between Lnc MALAT1 and Nrf2 using high-throughput data. The ligand establishes stable interactions within the protein binding pocket (Fig. 3A): conventional hydrogen bonds with Gln26 (3.24 \u0026Aring;) and Ile501 (2.38 \u0026Aring;), a\u0026nbsp;\u0026pi;-cation interaction with Lys508 (3.90 \u0026Aring;), and\u0026nbsp;\u0026pi;-alkyl/\u0026pi;-\u0026sigma;\u0026nbsp;interactions with Ile474 (4.42/4.62 \u0026Aring;) and Arg504 (5.01 \u0026Aring;). The 3D structure of the complex (Fig. 3B) reveals complementary spatial fitting between the ligand and key residues (Arg456, Thr455, Gln26, Ile501, Lys508, Ile474), which provides a structural basis for binding affinity and specificity\u003cstrong\u003e\u003cem\u003e.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e4. LncRNA MALAT1 Exerts Its Function by Regulating Nrf2 Nuclear Translocation\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunofluorescence staining revealed that Nrf2 nuclear translocation was significantly suppressed in the IR group, with a marked reduction in nuclear Nrf2 fluorescence intensity compared to the normal control (NC) group. In contrast, MALAT1 knockdown prominently promoted Nrf2 nuclear translocation, leading to substantial accumulation of Nrf2 in the nucleus. ML385 inhibited this promotional effect, while SFN mimicked the inductive effect of MALAT1 knockdown on Nrf2 nuclear translocation (Fig.3 A). Western blot(Fig4 A) and flow cytometry results indicated that MALAT1 knockdown reduced the apoptosis rate of IR-HGECs, and this anti-apoptotic effect was abrogated by ML385 treatment (Fig.3 B-C).Quantitative real-time polymerase chain reaction (qPCR) and Western blot (WB) analyses demonstrated that the mRNA and protein expression levels of Nrf2 downstream antioxidant genes (SOD2, GPx1, HO-1, NQO1) were downregulated in the IR group, whereas MALAT1 knockdown (IR+MKD group) significantly upregulated the expression of these genes(Fig 4 A-E). The Nrf2 inhibitor ML385 reversed this upregulatory effect, while the Nrf2 activator SFN recapitulated it. Meanwhile, flow cytometry analysis showed that the MALAT1 knockdown-mediated decrease in mitochondrial reactive oxygen species (ROS) levels was also blocked by ML385, confirming that MALAT1 regulates oxidative stress and cell survival in IR-HGECs through the Nrf2 pathway (Fig.4 F).\u0026nbsp;\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cem\u003e1. Cell Culture\u003c/em\u003e\u003cem\u003e：\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eHuman glomerular endothelial cells (HGECs) were obtained from Procell and maintained in DMEM (Gibco) supplemented with 10% fetal bovine serum (Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C in a 5% CO₂ atmosphere.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2.\u0026nbsp;\u003cem\u003eReagents and Antibodies\u003c/em\u003e\u003cem\u003e：\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMALAT1-targeting siRNA and its negative control were synthesized by Qingke. Total RNA was extracted and reverse-transcribed using kits from TaKaRa. The following primary antibodies from Proteintech were used for Western blotting: anti-p-IRS-1, anti-p-Akt ,anti-Nrf2, anti-SOD2, anti-GPX1, anti-Cleaved caspase-3, anti-Bax, anti-Bcl-2, and anti-β-actin. Mitochondrial ROS levels were measured using a detection kit from Elabscience. Apoptosis was assessed with an Annexin V-FITC/PI apoptosis detection kit (Beyotime). Cellular glucose consumption was determined by a GOD-POD assay kit (Beyotime).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3. Validation of the IR Model\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMeasurement of Glucose Consumption: Glucose consumption was determined by measuring the glucose concentration in the cell culture supernatant using the GOD-POD method. The value was calculated by subtracting the residual glucose concentration after culture from the initial concentration in the fresh medium.\u003c/p\u003e\n\u003cp\u003eWestern Blot Analysis: Following SDS-PAGE and transfer, membranes were probed with primary antibodies overnight at 4°C and then with a secondary antibody. Protein bands were visualized by ECL and quantified with ImageJ.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4. Validation of MALAT1 Knockdown Efficiency\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted using Trizol and reverse-transcribed into cDNA. qPCR was performed with the following primers: MALAT1 (Forward: 5'-CATGACGCAGGGAGAATTGC-3', Reverse: 5'-GCCTTCCCGTACTTCTGTCTT-3'). The thermal cycling conditions consisted of initial denaturation at 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. Gene expression was quantified using the 2^(–ΔΔCt) method.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e5. Detection of Oxidative Stress-Related Indicators\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTotal and mitochondrial ROS levels were measured by incubating cells with 10 μM DCFH-DA for 30 min or 5 μM MitoSOX Red for 15 min at 37°C, respectively. Fluorescence intensity was quantified after PBS washing. The mRNA expression of SOD2, GPX1, HO-1, and NQO1 was analyzed by qPCR, and SOD2 and GPx1 protein levels were determined by Western blot.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e6. Detection of Cell Apoptosis:\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eCell apoptosis was assessed by flow cytometry following Annexin V-FITC/PI staining. The expression of apoptosis-related proteins was analyzed by Western blot, and the Bax/Bcl-2 ratio was calculated.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e7. Detection of Nrf2 Nuclear Translocation\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAfter treatment, cells were fixed, permeabilized, and blocked, then incubated overnight with anti-Nrf2 antibody (1:200) at 4°C, followed by a fluorescent secondary antibody (1:500) for 1 h at room temperature. Nuclei were counterstained with DAPI. Nrf2 localization was visualized by confocal microscopy, and nuclear fluorescence intensity was quantified with ImageJ.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e7. Statistical Analysis:\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eData are expressed as mean ± SD and analyzed by one-way ANOVA with LSD post-hoc test using SPSS 22.0. A P value \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eDiabetic kidney disease (DKD) progression is closely associated with glomerular endothelial injury, driven largely by high glucose/insulin-induced insulin resistance (IR) and oxidative stress. This study reveals a novel role of the long non-coding RNA MALAT1 in regulating the Nrf2-mediated antioxidant pathway in HGECs under IR conditions. We established an IR model in HGECs using high glucose and insulin, observing impaired glucose consumption and insulin signaling, alongside significant MALAT1 upregulation. This aligns with reports of elevated MALAT1 in diabetic kidneys and DKD patient serum. The IR model exhibited specific elevation in mitochondrial ROS without total ROS changes, accompanied by reduced expression of antioxidant enzymes SOD2 and GPX1. MALAT1 knockdown effectively attenuated mitochondrial ROS, restored antioxidant enzyme levels, suppressed apoptosis, and improved insulin sensitivity. Mechanistically, Nrf2 nuclear translocation was suppressed in IR-HGECs, while MALAT1 knockdown promoted this process. The Nrf2 inhibitor ML385 abolished the protective effects of MALAT1 silencing, whereas the activator sulforaphane mimicked them, confirming Nrf2 dependency. Given evidence of direct MALAT1-Nrf2 binding, we propose that MALAT1 interferes with Nrf2 transcriptional activity, potentially by blocking its access to antioxidant gene promoters. Furthermore, improved insulin signaling following MALAT1 knockdown was Nrf2-dependent, consistent with Nrf2's known role in mitigating oxidative stress-induced insulin pathway impairment. In conclusion, IR-induced MALAT1 upregulation exacerbates HGEC injury by inhibiting Nrf2 nuclear translocation, leading to mitochondrial ROS accumulation, impaired antioxidant defense, and aggravated apoptosis and insulin resistance. Targeting the MALAT1-Nrf2 axis represents a potential therapeutic strategy for DKD, though in vivo validation remains necessary.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthor Contributions Li-rong Yang: Writing – review \u0026amp; editing, Writing \u0026nbsp; original draft. Hui Wang: Writing – review \u0026amp; editing, Writing – original draft. Li-rui Jia: Writing – original draft, Data curation. Li-ting Cao: Data curation, Visualization. Jia-mei Ding: Data curation, Resources. Yan-song Wang: Visualization. Yun-peng Tang: Visualization. Ya-bo Chen: Visualization. Xiao He: Visualization. Ya-li Zheng: Methodology.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Natural Science Foundation of Ningxia Province (Grant No. 2023A0138); Preliminary Research Project of the National Natural Science Foundation of China (Grant No. 2026GZRYSY007).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZhang L, Wang Y, Li J, et al. Global prevalence and progression of diabetic kidney disease: A systematic review and meta-analysis[J]. Lancet Diabetes Endocrinol, 2020, 8(5): 423 - 432.\u003c/li\u003e\n\u003cli\u003eJha V, Garcia - Garcia G, Iseki K, et al. Chronic kidney disease: Global dimension and perspectives[J]. Lancet, 2013, 382(9891): 607 - 619.\u003c/li\u003e\n\u003cli\u003eZuo L, Li Y, Chen N, et al. Prevalence of chronic kidney disease in China: A cross - sectional survey[J]. Lancet, 2012, 379(9818): 815 - 822.\u003c/li\u003e\n\u003cli\u003eBakris GL, Weir MR. Diabetic kidney disease: Core curriculum 2020[J]. Am J Kidney Dis, 2020, 75(3): 409 - 421.\u003c/li\u003e\n\u003cli\u003eChinese guideline for the prevention and treatment of diabetic kidney disease (2021 edition)[J]. Chin Med J (Engl), 2022, 135 (17): 2021 - 2058.\u003c/li\u003e\n\u003cli\u003eNangaku M. Pathophysiology of diabetic nephropathy[J]. Clin Exp Nephrol, 2016, 20(1): 1 - 9.\u003c/li\u003e\n\u003cli\u003eSchmidt AM, Warner FJ, Leroux F, et al. Glomerular endothelial cell dysfunction in diabetic kidney disease[J]. Cardiovasc Diabetol, 2019, 18(1): 148.\u003c/li\u003e\n\u003cli\u003eYu X, Chen Y, Li M, et al. Endothelial - to - mesenchymal transition contributes to glomerulosclerosis in diabetic nephropathy[J]. Am J Physiol Renal Physiol, 2018, 314(3): F452 - F463.\u003c/li\u003e\n\u003cli\u003eChen W, Huang Y, Xu C, et al. Vascular endothelial growth factor signaling in glomerular endothelial cell dysfunction of diabetic nephropathy[J]. Int J Mol Sci, 2020, 21(12): 4389.\u003c/li\u003e\n\u003cli\u003eEvans JL, Goldfine ID, Maddux BA, et al. Oxidative stress and stress - activated signaling pathways: A unifying hypothesis of type 2 diabetes[J]. Endocr Rev, 2002, 23(5): 599 - 622.\u003c/li\u003e\n\u003cli\u003eBrownlee M. The pathobiology of diabetic complications: A unifying mechanism[J]. Diabetes, 2005, 54(6): 1615 - 1625.\u003c/li\u003e\n\u003cli\u003eXu D, Liu J, Zhang Y, et al. Insulin resistance and oxidative stress: A vicious cycle in diabetic kidney disease[J]. Oxid Med Cell Longev, 2020, 2020: 8892745.\u003c/li\u003e\n\u003cli\u003eWang X, Li D, Zhao J, et al. High glucose - induced insulin resistance in glomerular endothelial cells via impaired insulin signaling pathway[J]. Mol Cell Endocrinol, 2019, 497: 110508.\u003c/li\u003e\n\u003cli\u003eJiang Y, Chen L, Zhang M, et al. Advanced glycation end products promote insulin resistance and oxidative stress in diabetic kidney disease[J]. J Cell Mol Med, 2021, 25(11): 5123 - 5134.\u003c/li\u003e\n\u003cli\u003eLay A, Lovatt C, Cook S, et al. Profiling of insulin-resistant kidney models and human biopsies reveals common and cell-type-specific mechanisms underpinning Diabetic Kidney Disease [J]. Kidney Int, 2024, 105 (6): 1129 - 1145.\u003c/li\u003e\n\u003cli\u003eEsteller M. Non - coding RNAs in human disease[J]. Nat Rev Genet, 2011, 12(12): 861 - 874.\u003c/li\u003e\n\u003cli\u003eRinn JL, Chang HY. Genome regulation by long noncoding RNAs[J]. Annu Rev Biochem, 2012, 81: 145 - 166.\u003c/li\u003e\n\u003cli\u003eTripathi V, Ellis JD, Shen Z, et al. MALAT1, a noncoding RNA, regulates alternative splicing and tumor metastasis[J]. Genes Dev, 2010, 24(11): 1174 - 1189.\u003c/li\u003e\n\u003cli\u003eGutschner T, Diederichs S. The role of long non - coding RNAs in cancer[J]. Semin Cancer Biol, 2012, 22(3): 231 - 240.\u003c/li\u003e\n\u003cli\u003eZhang Y, Wang Z, Li C, et al. LncRNA MALAT1: A novel therapeutic target in kidney diseases[J]. Life Sci, 2022, 307: 120987. Chatterjee K, Das S. MALAT1: a therapeutic candidate for a broad spectrum of vascular and cardiorenal complications[J]. Nat Rev Nephrol, 2019, 15(12): 745 - 758.\u003c/li\u003e\n\u003cli\u003eLiu C, Zhang J, Wang Y, et al. Urinary exosomal MALAT1 as a diagnostic biomarker for diabetic kidney disease[J]. Diabetes Care, 2023, 46(8): 1659 - 1667.\u003c/li\u003e\n\u003cli\u003eZhao L, Chen J, Li Y, et al. Upregulated MALAT1 correlates with albuminuria and renal dysfunction in diabetic kidney disease[J]. Int J Biochem Cell Biol, 2021, 136: 105912.\u003c/li\u003e\n\u003cli\u003eWang H, Zhang X, Liu M, et al. MALAT1 promotes endothelial dysfunction in diabetic nephropathy via regulating miR - 124 - 3p/TLR4 axis[J]. Cell Death Dis, 2022, 13(5): 432.\u003c/li\u003e\n\u003cli\u003eLi S, Guo Y, Han J, et al. LncRNA MALAT1 exacerbates diabetic kidney injury by inducing renal fibrosis[J]. J Diabetes Invest, 2020, 11(6): 1389 - 1398.\u003c/li\u003e\n\u003cli\u003eZhou Y, Chen F, Huang X. Knockdown of MALAT1 attenuates high - glucose - induced angiogenesis and inflammation via endoplasmic reticulum stress in human retinal vascular endothelial cells[J]. Biochem Biophys Res Commun, 2020, 529(4): 987 - 993.\u003c/li\u003e\n\u003cli\u003eXue Y, Wang L, Li M, et al. Urinary exosomal lncRNAs MALAT1 and PVT1 as potential biomarkers for diabetic kidney disease[J]. Diabetes, 2023, 72(Suppl 1): A456.\u003c/li\u003e\n\u003cli\u003eKensler TW, Wakabayashi N, Biswal S. Cell survival responses to environmental stresses via the Keap1 - Nrf2 - ARE pathway[J]. Annu Rev Pharmacol Toxicol, 2007, 47: 89 - 116.\u003c/li\u003e\n\u003cli\u003eSuzuki T, Motohashi H, Yamamoto M. Toward clinical application of the Keap1 - Nrf2 pathway[J]. Trends Pharmacol Sci, 2019, 40(1): 68 - 83.\u003c/li\u003e\n\u003cli\u003eLiu Z, Liu J, Li Y, et al. Activation of Nrf2 ameliorates diabetic kidney disease by inhibiting ferroptosis[J]. Adv Sci, 2024, 11(3): 2303245.\u003c/li\u003e\n\u003cli\u003eLee HJ, Kim JH, Park JS, et al. Keap1 - Nrf2 pathway in diabetic kidney disease: A potential therapeutic target[J]. J Endocrinol, 2020, 247(3): R157 - R170.\u003c/li\u003e\n\u003cli\u003eZhang M, Chen L, Jiang Y, et al. LncRNA MALAT1 regulates Keap1 - Nrf2 - antioxidant defense in diabetic retinopathy[J]. Free Radic Biol Med, 2021, 172: 123 - 135.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"LncRNA MALAT1, Insulin resistance, Oxidative stress, Diabetic nephropathy","lastPublishedDoi":"10.21203/rs.3.rs-8249151/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8249151/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eObjective: To investigate the role of LncRNA MALAT1 in insulin resistance (IR) and and oxidative stress injury in glomerular endothelial cells (HGECs) under high glucose and insulin (HG/Ins) conditions, and to elucidate its regulatory mechanism involving the Nrf2 pathway. HGECs were exposed to 25 mM glucose and 100 nM insulin for 48 h to induce insulin resistance. MALAT1 was silenced by siRNA, with knockdown efficiency verified via qPCR. Groups included control, ML385, and SFN. Protein expression, ROS, apoptosis, and Nrf2 translocation were assessed by Western blot, flow cytometry, and immunofluorescence. Following IR induction in HGECs (25 mM glucose + 100 nM insulin, 48 h) and siRNA-mediated MALAT1 knockdown, effects on proteins, ROS, apoptosis, and Nrf2 translocation were assessed in control and modulator groups.\u003c/p\u003e\n\u003cp\u003eConclusion: MALAT1 exacerbates IR and oxidative stress-induced injury in HGECs by inhibiting Nrf2 nuclear translocation. Targeting the MALAT1-Nrf2 axis may serve as a novel strategy for diabetic nephropathy management.\u003c/p\u003e","manuscriptTitle":"LncRNA MALAT1 Drives Diabetic Kidney Injury via Nrf2 Suppression in Glomerular Endothelium","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-21 10:29:52","doi":"10.21203/rs.3.rs-8249151/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-09T14:01:48+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"76972605841888975856183773402673207603","date":"2026-02-02T21:08:00+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-02T08:56:18+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-29T11:45:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"112390325977048219559899473783764991253","date":"2026-01-28T16:36:51+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-27T11:50:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"267029354791523190222893644598855710117","date":"2026-01-27T10:36:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"171015473461966018925070887603632427966","date":"2026-01-27T09:50:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"60227310867171280676257605866227524869","date":"2026-01-26T23:05:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"325544574792383448105499999883001155366","date":"2026-01-26T15:05:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"167481096538736017155384859882471178720","date":"2026-01-26T11:29:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-19T11:07:16+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-19T06:52:41+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-16T07:51:09+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-07T06:25:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-01-07T06:14:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"edae639b-d27d-4d65-8b2f-b1d12fc16226","owner":[],"postedDate":"January 21st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":61440559,"name":"Biological sciences/Cell biology"},{"id":61440560,"name":"Health sciences/Diseases"},{"id":61440561,"name":"Biological sciences/Molecular biology"},{"id":61440562,"name":"Health sciences/Nephrology"}],"tags":[],"updatedAt":"2026-03-19T11:56:31+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-21 10:29:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8249151","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8249151","identity":"rs-8249151","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}