Mitochondrial DNA Release Activates cGAS-STING Signaling in Membranous Nephropathy: Therapeutic Attenuation by Pathway Inhibition

Mitochondrial DNA Release Activates cGAS-STING Signaling in Membranous Nephropathy: Therapeutic Attenuation by Pathway Inhibition | 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 Research Article Mitochondrial DNA Release Activates cGAS-STING Signaling in Membranous Nephropathy: Therapeutic Attenuation by Pathway Inhibition Halinuer Shadekejiang, Guoqiang Zhu, Ting Xia, Mingzhu Liang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8685107/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 Background Membranous nephropathy (MN) is a chronic kidney disease mediated by autoimmunity, but its molecular mechanisms remain incompletely understood. Studies have suggested that mitochondrial damage leading to mitochondrial DNA (mtDNA) leakage may contribute to the development of autoimmune diseases by activating the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, but this mechanism has not yet been explored in MN. Methods In this study, an MN animal model was established in Balb/c mice using cationic bovine serum albumin (cBSA) induction. Concurrently, an in vitro podocyte injury model was generated by stimulating cultured podocytes with Zymosan. Mitochondrial damage was assessed by quantifying the relative abundance of mtDNA in total cellular and cytosolic fractions via quantitative PCR (qPCR). Interventions were performed using the cGAS inhibitor RU.521 and STING inhibitor C-176. The therapeutic effects of inhibiting the cGAS-STING pathway were systematically evaluated through in vivo and in vitro assessments of urinary protein levels, glomerular immune complex deposition, podocyte injury markers, and mitochondrial functional parameters. Results Integrated in vivo and in vitro experimental results confirm that MN induces mitochondrial dysfunction and triggers mtDNA leakage into the cytosol, thereby activating the cGAS-STING signaling pathway. Intervention with the cGAS-specific inhibitor RU.521 or the STING inhibitor C-176 demonstrated significant renoprotective effects in both animal and cellular models. In the cBSA-induced MN mouse model, the treatment groups exhibited significantly reduced urinary protein levels, decreased glomerular IgG and C3 deposition, and significant downregulation of inflammatory cytokines (IL-1β, IL-6, TNF-α). Similarly, in vitro podocyte experiments showed that inhibitor treatment reversed the Zymosan-induced reduction in mitochondrial membrane potential and decreased mitochondrial superoxide levels. These findings collectively demonstrate the therapeutic potential of targeting the cGAS-STING pathway in MN. Conclusion Our study unveils a critical role for the cGAS-STING signaling pathway in the development and progression of MN. Targeted inhibition of this pathway confers remarkable renal protection, highlighting its potential as a novel therapeutic strategy for managing MN. Membranous nephropathy cGAS-STING pathway Podocyte injury Inflammation Mitochondrial damage Figures Figure 1 Figure 2 Figure 3 Figure 4 1 Introduction Membranous nephropathy (MN) is a leading cause of chronic kidney disease, accounting for approximately 30% of nephrotic syndrome cases in adults. Globally, the incidence of MN is estimated at 8–10 cases per million population, with a significantly higher prevalence of 23.4% in China—second only to IgA nephropathy—and has shown a steady annual increase in recent years[1–4]. Despite advances in immunosuppressive therapy, adverse effects and treatment intolerance remain unresolved, highlighting an urgent need for novel therapeutic targets[5]. MN is an autoimmune glomerular disease characterized by subepithelial immune complex deposition and diffuse glomerular basement membrane (GBM) thickening. Its pathogenesis involves autoantibodies (predominantly IgG) binding to podocyte antigens, which form immune complexes that deposit in a subepithelial location. This subsequently activates the complement system, leading to the formation of the membrane attack complex (MAC). MAC induces glomerular filtration barrier injury and proteinuria by disrupting podocyte foot processes, downregulating slit diaphragm proteins (e.g., Nephrin), and promoting podocyte apoptosis[6, 7]. While the initiating mechanisms of immune complex deposition and complement activation are well established, the key innate immune pathways driving sustained inflammatory amplification remain elusive. The cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) axis, a central signaling hub for cytosolic DNA sensing, is activated through the following mechanism: cytosolic double-stranded DNA (dsDNA) binds to cGAS, triggering a conformational change that activates its cyclase activity to synthesize the second messenger 2'3'-cyclic GMP-AMP (cGAMP). Acting as a molecular switch, cGAMP then binds to endoplasmic reticulum-resident STING, inducing a conformational opening and translocation to the Golgi apparatus, which in turn recruits and activates the kinase TBK1. TBK1 phosphorylates downstream transcription factors IRF3 and NF-κB, initiating the cascaded expression of type I interferons (e.g., IFN-β1) and proinflammatory cytokines (e.g., IL-6, TNF-α), ultimately regulating inflammation-immune homeostasis in antiviral immunity and autoinflammatory conditions[8–11]. Recent studies have highlighted the critical pathological role of this pathway in kidney diseases[12, 13]. As a highly metabolically active organ, the kidney is vulnerable to mitochondrial injury induced by hypoxia and oxidative stress, leading to the release of mitochondrial DNA (mtDNA) that activates cGAS-STING signaling. This activation drives podocyte damage, tubular inflammation, and fibrotic progression. In models of acute kidney injury[14, 15], diabetic nephropathy[16], and Alport syndrome[8], pathway inhibition has markedly improved renal function and histopathology, underscoring its potential as a therapeutic target. However, its role in MN remains unreported. Based on the MN’s pathological features and the pathway characteristics, we hypothesize that immune complex deposition activates complement C5b-9, leading to podocyte membrane injury and apoptosis. The released mtDNA and nuclear DNA fragments then serve as endogenous ligands to activate the cGAS-STING pathway. Downstream IFN-β1 and proinflammatory cytokines further exacerbate podocyte damage and inflammation, forming a vicious cycle of “ immune complexes → cGAS-STING activation → inflammation amplification ”. Excessive pathway activation may contribute to podocyte loss by impairing mitochondrial function. To validate this hypothesis, we systematically investigated the role of the cGAS-STING pathway in MN and discovered: (i) First evidence confirming that MN induces mtDNA leakage, with significant upregulation of key molecules in the cGAS-STING pathway (cGAS, p-STING, p-TBK1, p-IRF3); (ii) Specific inhibitors improved renal function and alleviated glomerular pathological damage; (iii) The protective mechanism is synergistically achieved through three pathways: suppressing the IRF3-IFN-β inflammatory axis to block inflammatory cascades, maintaining podocyte structural integrity, and ameliorating mitochondrial dysfunction. This study reveals that the cGAS-STING pathway is a critical driver of MN progression and clarifies its novel mechanism in regulating MN pathology through the "inflammation-mitochondria-podocyte" axis, thus providing a theoretical basis and potential strategy for the targeted therapy of MN. 2 MATERIALS AND METHODS 2.1 Animals Eight-week-old male BALB/c mice (SPF grade, body weight 22 ± 5 g) were obtained from the Animal Experiment Center of The First Affiliated Hospital of Xinjiang Medical University [License No: SCXK (Xin) 2018-0003]. All experimental protocols were approved by the Institutional Animal Care and Use Committee of Xinjiang Medical University (Approval No: IACUC-20230627-19) and strictly adhered to the "3R" principles (Reduction, Refinement, Replacement). Blinding procedures were implemented during key stages of the experiment, including allocation, intervention, outcome assessment, and data analysis. Animals were housed in individually ventilated cages (≤ 5 mice/cage) containing autoclaved pinewood bedding changed every 48 hours. Mice were maintained under controlled conditions (temperature: 23 ± 3℃; relative humidity: 40%-60%; 12-hour light/dark cycle) with ad libitum access to irradiated SPF-grade feed and autoclaved water. Daily health monitoring was performed, with individuals showing abnormal signs immediately isolated. After a 1-week acclimation period, mice were randomly assigned by computer-generated random number table method into the following groups: Control group (CON), MN group, MN + RU-521 group, and MN + C-176 group ( n = 6)). At the endpoint, the mice were given 3% isoflurane for anesthesia induction and 1.5% isoflurane for maintenance. Cardiac puncture was immediately performed for whole blood collection, followed by cervical dislocation under sustained anesthesia for euthanasia. Whole blood was clotted at room temperature for 30 min before centrifugation (3,000 × g, 10 min, 4℃) to obtain serum supernatant for storage. Following bilateral nephrectomy, one renal aliquot was immediately flash-frozen in liquid nitrogen for subsequent protein analyses (including Western blotting), with long-term storage at -80℃. The contralateral kidney was immersion-fixed in 4% paraformaldehyde for 24 hours at 4℃ before histological processing. 2.2 cBSA-Induced Murine Model of Membranous Nephropathy Based on previous studies with modifications[17, 18], the detailed modeling procedure was as follows: Cationic bovine serum albumin (cBSA) (#9058, Chondrex, USA) was emulsified with an equal volume of Freund’s complete adjuvant (#7008, Chondrex, USA). Mice in the MN group received a subcutaneous injection of 0.1 mL of the emulsion (0.1 mg cBSA) at the tail base for initial immunization. Two weeks later, the MN group mice received tail base injections of cBSA: an initial dose of 100 µg, followed by subsequent injections every other day at an increased dose of 10 mg/kg. Injections were administered three times per week for six consecutive weeks. Mice in the control group received an equal volume of physiological saline on the same schedule. Twenty-four-hour urine samples were collected every two weeks using metabolic cages. Model success was ultimately determined based on renal function and pathological alterations. 2.3 Cell culture and treatment The mouse podocyte cell line (MPC-5) was originally obtained from iCell Bioscience Inc. (Shanghai, China) and cultured in DMEM medium supplemented with 10% fetal bovine serum. The membrane attack complex C5b-9 was prepared according to previously established methods. Specifically, it was generated from normal mouse serum treated with zymosan A (#Z4250, Sigma-Aldrich, USA) as described in earlier studies[19–21]. The detailed procedure was as follows: zymosan was mixed with normal saline (10 mg/mL), boiled for 1 hour, and cooled to room temperature, followed by centrifugation to discard the supernatant. The pellet was resuspended in normal mouse serum to a concentration of 10 mg/mL and incubated for 1 hour in a water bath at 37℃. Zymosan particles were then removed by centrifugation (14,000 ×g, 4 ℃, 5 min ) and filtration through a 0.8 µm filter. The resulting zymosan-activated mouse serum (ZAS) was collected. MPC-5 cells were treated for 24 hours with culture medium containing 10% ZAS, with heat-inactivated mouse serum used as the control. Model success was ultimately verified by C5b-9 immunofluorescence results. 2.4 Inhibitor Administration To inhibit cGAS activity, mice received daily intraperitoneal injections of RU.521 (5 mg/kg; Cat# HY-114180, MedChemExpress, USA) dissolved in a vehicle consisting of 5% DMSO (Cat# HY-Y0320C, MedChemExpress, USA) and 95% corn oil (Cat# HY-Y1888, MedChemExpress, USA) for one week before sacrifice[22, 23]. For STING inhibition, mice were administered C-176 (750 nM, 200 µL; Cat# HY-112906, MedChemExpress, USA), prepared in the same vehicle, via daily intraperitoneal injection for three weeks before sacrifice. Control mice received an equivalent volume of the vehicle alone on the same schedule to account for potential non-specific effects of the injection procedure or solvent[8]. In the cellular model, podocytes were pretreated with 5 µM RU.521 or C-176 (final DMSO, 0.05%) for 1 hour before exposure to ZAS. Control cells were treated with an equal volume of DMSO. 2.5 Renal function analysis To evaluate the renal function in mice, this study measured urinary protein, serum creatinine (SCr), blood urea, and a series of lipid parameters. Urinary protein and urinary creatinine levels were assessed using commercial kits (Elabscience, China), and the urinary protein-to-creatinine ratio (UPCR) was calculated. The measurements of SCr, UREA, and lipid profiles (including total cholesterol and triglycerides) were conducted using an automated biochemical analyzer at the Animal Experiment Center of Xinjiang Medical University. 2.6 Histology For histological analysis, renal tissue samples were fixed in 4% paraformaldehyde for 24–48 hours. The tissues were then dehydrated through a graded ethanol series, cleared in xylene, and embedded in paraffin. Serial sections of 4 µm thickness were cut using a microtome, mounted on glass slides, and dried overnight in a 60°C oven. The sections were subjected to Hematoxylin and Eosin (H&E) staining for general morphological observation, Periodic Acid–Schiff (PAS) staining for highlighting basement membranes and glycogen deposits, and Periodic Acid–Silver Methenamine (PASM) staining for detailed visualization of glomerular basement membranes and mesangial matrix. PASM staining was performed according to the manufacturer's instructions (Solarbio, China). 2.7 Immunohistochemical staining For IHC analysis, paraffin-embedded renal tissue sections were first subjected to heat-induced antigen retrieval using EDTA or citrate buffer. Following this, endogenous peroxidase activity was blocked with 3% hydrogen peroxide, and non-specific binding sites were blocked with 5% bovine serum albumin (BSA) at room temperature. After blocking, the sections were incubated with primary antibodies at 4°C overnight. The next day, the sections were incubated with corresponding species-specific horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature, followed by color development using a 3,3'-diaminobenzidine (DAB) substrate kit. The percentage of positively stained area for the target proteins within the glomeruli was quantified using ImageJ software. Primary antibodies used included: anti-STING (#19851-1-AP, Proteintech), and anti-WT-1 (#ab89901, Abcam). 2.8 Immunofluorescence For IF analysis, cell samples were first washed with phosphate-buffered saline (PBS) and then fixed with 4% paraformaldehyde for 30 minutes at room temperature. Paraffin-embedded tissue sections underwent antigen retrieval using citrate or EDTA buffer. Subsequently, all samples were permeabilized with 0.3% Triton X-100 (in PBS) for 10 minutes and blocked with 5% bovine serum albumin (BSA) for 30 minutes at room temperature to prevent non-specific binding. After blocking, the samples were incubated with diluted primary antibodies at 4°C overnight. The next day, the samples were incubated with appropriate species-specific secondary antibodies (such as Alexa Fluor 488-conjugated goat anti-rabbit IgG, #ab150077, Abcam) for 1 hour at room temperature in the dark. Finally, the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 5–10 minutes at room temperature. All fluorescent images were acquired using a Leica confocal laser scanning microscope and subjected to quantitative analysis. The primary antibodies used included: anti-C5b-9 (#ab55811, Abcam), anti-C3 (#ab321966, Abcam), and anti-IgG (#ab6785, Abcam). 2.9 Western blot An appropriate amount of protein sample was mixed with loading buffer and denatured by boiling, followed by separation via SDS-PAGE gel electrophoresis. The proteins were then transferred from the gel onto a PVDF membrane. After transfer, the membrane was blocked with 5% BSA in TBST for 1 hour at room temperature. Subsequently, the membrane was incubated sequentially with diluted primary antibodies (overnight at 4°C) and secondary antibodies (1–2 hours at room temperature). Following each antibody incubation, the membrane was washed three times with TBST. Finally, the blots were visualized using an ECL chemiluminescence substrate, and images were captured using an imaging system. The intensity of target protein bands was quantified using grayscale analysis ​​with​​ ImageJ software. The primary antibodies used included: anti-cGAS (#31659, Cell Signaling Technology), anti-STING (#13647, Cell Signaling Technology), anti-IRF3 (#4302, Cell Signaling Technology), anti–p-IRF3 (#29047, Cell Signaling Technology), anti-TBK1 (#3504, Cell Signaling Technology), anti-p-TBK1 (#5483, Cell Signaling Technology), anti–IFN-β1 (#97450, Cell Signaling Technology), anti-GAPDH (#60004-1-Ig, Proteintech), and anti-Nephrin (#ab216341, abcam). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (#ab205718, Abcam) was used as the secondary antibody.​ 2.10 Detection of mtDNA by Quantitative PCR (qPCR) Cells were collected and centrifuged at 400 × g for 5 min. The cell pellet was resuspended in ice-cold PBS and equally divided into two aliquots for the extraction of total cellular DNA and cytoplasmic DNA, respectively. Total DNA Extraction: Total DNA was isolated using the FastPure Cell/Tissue DNA Isolation Mini Kit (#DC102, Vazyme) according to the manufacturer's instructions. Briefly, the cell pellet was resuspended in 220 µL PBS containing 10 µL RNase Solution and 20 µL Proteinase K, followed by incubation at room temperature for ≥ 15 min. Subsequently, 250 µL Buffer GB was added, and the mixture was incubated at 65 ℃ for 15–30 min for lysis. After adding 180 µL absolute ethanol, the lysate was directly loaded onto a gDNA adsorption column and centrifuged at 13,400 × g for 1 min. The column was sequentially washed with Washing Buffer A and Washing Buffer B. Residual ethanol was removed by spinning the empty column. DNA was finally eluted using 30 µL of pre-warmed (70 ℃) Elution Buffer. Cytoplasmic DNA Extraction: The cell pellet was resuspended in 250 µL of cytoplasmic lysis buffer (150 mM NaCl, 50 mM HEPES [pH 7.4], 25 µg/mL digitonin) and incubated at room temperature for 10 min. The lysate was centrifuged at 1,000 × g for 10 min to pellet nuclei and cellular debris. The resulting supernatant was further centrifuged at 17,000 × g for 30 min at 4℃ to isolate the cytoplasmic fraction. DNA was then extracted from this cytoplasmic supernatant using the same protocol described for total DNA extraction. mtDNA quantification by quantitative PCR (qPCR): Target Gene: Mitochondrial cytochrome c oxidase subunit 1 (mtCOI / mtCOX2) gene Reference Gene: 18S ribosomal RNA (18S rDNA) gene Primer Sequences: 18S rDNA: Forward: 5′-TGTGTTAGGGGACTGGTGGACA-3′ Reverse: 5′-CATCACCCACTTACCCCCAAAA-3′ mtCOX2: Forward: 5′-ATAACCGAGTCGTTCTGCCAAT-3′ Reverse: 5′-TTTCAGAGCATTGGCCATAGAA-3′ Data Analysis: Relative mtDNA copy number was calculated by normalizing mtCOX2 quantification cycle (Cq) values to those of 18S rDNA and compared between experimental groups. 2.11 Enzyme-Linked Immunosorbent Assay (ELISA) Serum levels of inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6), were quantified using commercial ELISA kits (Jianglai Bioengineering, Shanghai, China) according to the manufacturer’s protocols. Briefly, serum samples were diluted and added to the pre-coated plates. After incubation and washing, biotinylated detection antibodies and then enzyme conjugates were added. Following another incubation and washing step, the substrate solution (TMB) was added for color development, which was stopped with the stop solution. The absorbance was immediately measured at 450 nm using a microplate reader. Cytokine concentrations were calculated based on standard curves generated from the standard samples provided in the kits. 2.12 ATP measurement Cellular ATP levels were measured using an ATP Assay Kit (S0026, Beyotime, China). Briefly, after cell lysis, the supernatant was collected and mixed with the ATP detection solution. The luminescence was immediately measured using a luminescence microplate reader (Thermo Fisher Scientific, USA). The ATP concentration of each sample was calculated based on a standard curve and normalized to the total protein concentration determined by a BCA protein assay kit. The results are expressed as micromoles per gram of protein (µmol/g protein). 2.13 Mitochondrial transmembrane potential The mitochondrial membrane potential (MMP) was detected using the JC-1 fluorescent probe (#C2006, Beyotime, China). Briefly, after incubating the cells with the JC-1 working solution, fluorescence signals were observed under a confocal microscope (Leica, Germany). JC-1 forms aggregates that emit red fluorescence in mitochondria with normal membrane potential, whereas it remains in a monomeric state and emits green fluorescence when the membrane potential is decreased. Changes in MMP were quantitatively assessed by measuring the ratio of red to green fluorescence intensity. All experiments were strictly performed in accordance with the manufacturer's instructions. 2.14 Mitochondrial Superoxide Detection Methodology Mitochondrial superoxide levels in MPC-5 were detected using the MitoSO™ Red mitochondrial superoxide indicator (Beyotime Biotechnology, Cat# S0061). Briefly, cells were incubated with the MitoSO™ Red Working Solution according to the manufacturer's instructions. Following incubation and washing, real-time changes in mitochondrial superoxide were visualized and analyzed using laser scanning confocal microscopy (Leica Microsystems). 2.15 Transmission Electron Microscopy (TEM) Renal tissues were collected from MN and control mice. Fresh tissue blocks (not exceeding 1mm³) were rapidly dissected to minimize mechanical damage and immediately immersed in prechilled electron microscopy fixative (containing 4% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4) for 24 hours at 4℃. After fixation, samples were rinsed three times with 0.1 M phosphate buffer (pH 7.4) and post-fixed in 1% osmium tetroxide for 2 hours at room temperature in the dark. The samples were dehydrated through a graded ethanol and acetone series, infiltrated, and embedded stepwise using mixtures of acetone and EMBed-812 resin at 37°C, followed by polymerization at 65°C for 48 hours. Ultrathin sections (60–80 nm) were cut using an ultramicrotome (Leica), collected onto 150-mesh copper grids, and sequentially stained with 2% uranyl acetate in saturated alcohol solution and 2.6% lead citrate, with thorough rinsing after each staining step. Sections were observed under a transmission electron microscope (HT7800/HT7700, Hitachi). 2.16 Bioinformatic Analysis To address the lack of clinical data on STING in human MN, we first explored the expression characteristics of the STING gene using public databases. Data for this study were sourced from the GSE108113 dataset (GPL19983 platform) within the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/ ). Principal component analysis (PCA) and hierarchical clustering for data quality control resulted in the inclusion of 43 MN patients and 6 healthy controls for subsequent analysis. All data processing and analysis were performed in the R language environment (v4.4.0). After preprocessing raw expression data with background correction, log2 transformation, and quantile normalization, differential expression analysis was conducted using the limma package. We first compared glomerular STING gene expression levels between the MN group and the control group. Differentially expressed genes (DEGs) were subsequently screened using thresholds of |log₂FC| > 0.6 and adjusted P value (adj.P.Val) < 0.01, with results visualized via volcano plots. 2.17 Statistical analysis All quantitative data are presented as mean ± standard error of the mean (SEM). Statistical analyses were conducted using GraphPad Prism 9.0 (GraphPad Software, San Diego, CA). The normality of data distribution and homogeneity of variances were evaluated using the Shapiro–Wilk test and Bartlett’s test, respectively. For datasets satisfying both assumptions, two-group comparisons utilized unpaired two-tailed Student’s t-tests, while multiple comparisons employed one-way ANOVA with Tukey’s post-hoc test. Non-parametric datasets were analyzed by the Kruskal–Wallis test followed by Dunn’s test. Statistical significance was defined as P < 0.05. 3 RESULTS 3.1 Successful establishment of mouse and cellular models of MN To investigate the role of the cGAS-STING signaling pathway in MN, an MN mouse model was established in vivo by cBSA immunization (Fig. 1 A). Compared to control mice, the MN model exhibited characteristic nephrotic syndrome phenotypes: UPCR progressively increased starting at week 2 post-induction, becoming significantly elevated by week 6 (p < 0.001; Fig. 1 B). Concurrently, Scr and urea levels were significantly increased (p < 0.001; Fig. 1 C, D). Dyslipidemia was evident, with significant elevations in total cholesterol (TC), triglycerides (TG), and low-density lipoprotein (LDL), accompanied by a significant reduction in serum albumin (ALB) (p < 0.001; Fig. 1 E-H). Histopathological analysis of kidney tissues revealed diffuse thickening of the GBM and expansion of the mesangial matrix in MN mice. Characteristic spike-like formations were observed on PASM staining. Immunofluorescence analysis demonstrated granular deposition of immunoglobulin G (IgG) and complement C3 along the glomerular capillary walls. Further confirmation by TEM revealed uneven GBM thickening, subepithelial electron-dense deposits, and extensive effacement of podocyte foot processes (Fig. 1 B-G). Collectively, these findings confirm the successful establishment of the cBSA-induced MN model. In vitro, a podocyte injury model mimicking the MN microenvironment was generated using MPC-5 cells stimulated with zymosan to activate the complement system (Fig. 1 K). Successful assembly of the membrane attack complex C5b-9 on the podocyte surface following zymosan stimulation was confirmed by immunofluorescence (p < 0.001; Fig. 1 L, M), validating the establishment of the injury model. 3.2 MN Podocyte Model Reveals Significant Increase in Cytosolic mtDNA Leakage The total cellular mtDNA relative abundance in glomerular cells of the MN group was significantly lower than that in the CON group (P < 0.01), suggesting abnormal mitochondrial quantity reduction or mtDNA replication inhibition in MN glomerular cells (Fig. 1 N). In stark contrast, the relative abundance of cytosolic mtDNA in the MN group was significantly higher than that in the CON group (P < 0.001), directly reflecting abnormal leakage of mtDNA from the mitochondrial matrix into the cytoplasm (Fig. 1 O). In glomerular cells of MN, mitochondrial membrane damage or decreased mitochondrial membrane potential compromises mitochondrial membrane integrity, leading to mtDNA escaping into the cytoplasm through membrane gaps. The combined results reveal a dual signature of mitochondrial dysfunction in MN glomerular cells: on one hand, reduced total mtDNA indicates a decline in overall mitochondrial quantity or replication capacity, reflecting diminished functional reserve; on the other hand, increased cytosolic mtDNA serves as a direct marker of structural damage (abnormal membrane permeability). This imbalance between "quantity/replication deficiency" and "structural damage" may lead to dysregulated mtDNA metabolism—impaired mitochondria fail to be effectively repaired or compensated, instead releasing damage-associated molecular patterns (DAMPs) through leaked mtDNA, which activates cGAS-STING pathway and contributes to glomerular inflammatory responses in the pathological process. 3.3 Bioinformatics analysis reveals upregulation of STING in MN patients To verify whether mitochondrial DNA leakage activates the cGAS-STING pathway, we first analyzed clinical transcriptomic data. The MN dataset GSE108113 was retrieved from the GEO database. Principal component analysis (PCA) confirmed sample grouping reliability (Fig. 2 A). Using thresholds of |log₂FC| > 0.6 and adj. P < 0.01, we identified 2,629 differentially expressed genes (DEGs), including 1,486 upregulated and 1,143 downregulated genes (Fig. 2 B). Focusing on innate immunity regulatory genes revealed significantly elevated expression of STING1 (stimulator of interferon genes) in the MN group (log₂FC = 0.87, P < 0.001), suggesting aberrant activation of the cGAS-STING signaling pathway may contribute to MN pathogenesis (Fig. 2 C). 3.4 Mouse model of MN reveals pathological overactivation of cGAS-STING pathway To systematically validate cGAS-STING pathway overactivation in MN, protein analysis of murine renal tissues demonstrated significant activation of the renal cGAS-STING innate immune axis under MN pathological conditions. Key findings revealed: (1) upregulation of pattern recognition receptor cGAS (P < 0.01) during pathway initiation; (2) concurrent elevation of both total and phosphorylated STING (p-STING, P < 0.01) indicating core adaptor activation; (3) phosphorylation-mediated signal transduction where activated STING induced TBK1 phosphorylation (p-TBK1), subsequently phosphorylating IRF3 (p-IRF3) to establish a complete cGAS→STING→TBK1→IRF3 phosphorylation cascade. Crucially, unchanged total TBK1 and IRF3 levels confirmed pathway activation primarily depended on enhanced post-translational modifications rather than protein synthesis, with this signaling axis further driving type I interferon responses through IRF3 activation as evidenced by significantly elevated interferon-β1 (IFN-β1) expression in renal cortex (P < 0.01), collectively demonstrating cGAS-STING overactivation and its inflammatory mediation in MN kidneys(Fig. 2 ). 3.5 Inhibition of the cGAS-STING pathway attenuates cBSA-induced renal injury To validate the pivotal role of the cGAS-STING pathway in MN pathogenesis, targeted inhibition using specific inhibitors (C-176 for STING, RU.521 for cGAS) significantly suppressed pathway activation compared to untreated MN controls, evidenced by reduced cGAS protein expression (P < 0.01, RU.521 only), decreased total STING and phosphorylated STING (p-STING) levels (P < 0.05), and marked inhibition of downstream p-TBK1 and p-IRF3 expression (P < 0.01), confirming effective blockade of the cGAS-STING axis (Fig. 2 D-J). Functionally, pathway inhibition ameliorated renal injury and systemic metabolic dysregulation: renal parameters showed significantly reduced UPCR(P < 0.0001) alongside decreased Scr and urea ( P < 0.001); lipid profiling revealed C-176 reduced TC, TG, and LDL (P < 0.05) while RU.521 only lowered TG (P < 0.05) without affecting TC and LDL (Fig. 1 B-H); renal pathology demonstrated attenuated IgG and C3 deposits along glomerular capillaries via IF (P < 0.001, Fig. 3 A-D); and TEM analysis exhibited thinner glomerular basement membranes approaching normal uniformity, reduced foot process effacement, and absence of subepithelial electron-dense deposits (Fig. 3 I). 3.6 Inhibition of the cGAS-STING Pathway Preserves Podocyte Integrity To investigate the impact of the cGAS-STING pathway on podocytes, we examined podocyte-specific marker expression. Western blot analysis revealed significantly reduced levels of the key functional protein Nephrin in renal cortex of MN model mice (P < 0.05), with this downregulation being markedly reversed by RU.521 or C-176 intervention (P < 0.05) (Fig. 3 F, G). IHC staining further confirmed substantially decreased WT-1-positive cell counts in glomeruli of the MN group versus controls (P < 0.001), while inhibitor treatment significantly restored WT-1-positive cell numbers (P < 0.05) (Fig. 3 E, H) 3.7 Inhibition of the cGAS-STING Pathway Attenuates Inflammatory Responses in MN Activation of the cGAS-STING pathway drives robust inflammatory responses; to evaluate its pro-inflammatory effects in the MN model, ELISA analysis of serum cytokines revealed significantly elevated concentrations of classic pro-inflammatory cytokines—interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α)—in MN mice versus controls (P < 0.01), whereas treatment with RU.521 or C-176 markedly reversed this aberrant upregulation, significantly reducing the levels of these inflammatory factors compared to the MN group (P < 0.05) (Fig. 3 J-L). 3.8 Inhibition of the cGAS-STING Pathway Ameliorates Mitochondrial Dysfunction in MN To investigate whether cGAS-STING pathway activation contributes to mitochondrial damage in podocytes during MN, transmission electron microscopy revealed significant ultrastructural abnormalities in MN podocytes: mitochondrial membrane blurring, swelling, partial matrix dissolution, cristae fragmentation or disappearance, and vacuolation; whereas RU.521 or C-176 intervention markedly improved mitochondrial morphology, reducing swelling and partially restoring cristae integrity (Fig. 4 A). Functional analyses confirmed direct regulatory effects: (1) Mitochondrial ATP production: Podocyte ATP levels significantly decreased in MN versus controls (P < 0.01), reversed by inhibitors to near-normal levels (P < 0.01) (Fig. 4 B); (2) Mitochondrial membrane potential JC-1 probe analysis showed a significantly reduced aggregate (red)/monomer (green) fluorescence ratio in MN podocytes (P < 0.001), indicating depolarization, while inhibitor treatment increased this ratio (P < 0.001), demonstrating ΔΨm restoration (Fig. 4 C, D); (3) Mitochondrial ROS (mtROS): MitoSOX™ Red fluorescence quantification indicated elevated mtROS in MN glomeruli (P < 0.01), effectively suppressed by pathway inhibitors (P < 0.01) (Fig. 4 E, F). Discussion This study systematically investigated the pathogenesis of MN by integrating the cBSA-induced mouse model and the zymosan-stimulated podocyte model. The cBSA model successfully recapitulates the characteristic clinical manifestations of human MN, including significant proteinuria, thickening of the GBM, and deposition of IgG and complement C3, by mimicking the anti-PLA2R/THSD7A antibody-mediated pathological processes. Concurrently, the zymosan model, through specific activation of the TLR2/6-Dectin-1 receptor pathway, provides an ideal platform for in-depth investigation into the innate immune mechanisms involving alternative complement pathway activation and inflammasome activation in MN[17, 19, 24]. Within these two complementary model systems, we observed significant mitochondrial dysfunction in podocytes affected by MN, characterized by three distinct alterations: 1)Ultrastructural Examination: Revealed abnormal mitochondrial changes including blurred membrane structure, swelling, matrix dissolution, cristae fragmentation or even disappearance, with some mitochondria exhibiting vacuolar degeneration. 2) Functional Assessment: Detected a significant decrease in mitochondrial membrane potential concurrently with a marked elevation in mitochondrial reactive oxygen species levels. 3) Molecular Analysis: Demonstrated a reduction in total cellular mtDNA abundance accompanied by abnormal accumulation of cytosolic mtDNA. These seemingly paradoxical yet interconnected phenomena collectively indicate a severe disruption of mitochondrial homeostasis in MN podocytes. Of particular note, the significant increase in cytosolic mtDNA levels provides direct evidence for mtDNA leakage from damaged mitochondria. Current research has unequivocally established that such leaked mtDNA, acting as a crucial Damage-Associated Molecular Pattern (DAMP), serves as the key trigger for activating the cGAS-STING innate immune signaling pathway[25–27]. To further validate this mechanism, we first analyzed public databases of MN patients and found that STING gene expression was significantly upregulated in renal tissues from MN patients. Subsequent protein-level detection confirmed the activation of the cGAS-STING signaling pathway in MN models, with its downstream signaling primarily dependent on the IRF3-IFNβ axis. To investigate the functional significance of this pathway, we conducted intervention studies using the specific inhibitors RU.521 and C-176. The experimental results were compelling: both inhibitors significantly improved renal function parameters in MN mice, including reduced UPCR levels and ameliorated SCr and blood urea levels. Concurrently, they restored the normal structure of the GBM, alleviated podocyte injury. Furthermore, levels of the inflammatory cytokines IL-6, IL-1β, and TNF-α were significantly decreased. These results are not only consistent with previous research but, more importantly, confirm that the cGAS-STING pathway in MN follows the classical cGAS-STING-IRF3 signaling cascade. This supports the conclusion that the observed phenotypic improvements primarily stem from the specific inhibition of the cGAS-STING pathway, thereby establishing its significant value as a potential therapeutic target for MN. Of particular note, regarding lipid metabolism, the STING inhibitor exhibited a comprehensive regulatory effect, significantly lowering TC, LDL, and TG levels. In contrast, the cGAS inhibitor only reduced TG levels. Drawing on the findings of Yu et al.[28], which demonstrated that STING deficiency ameliorates dyslipidemia in mice with high-fat-diet-induced nonalcoholic steatohepatitis, we postulate that STING-targeted inhibition strategies may indirectly influence lipid metabolism by modulating systemic inflammatory responses. However, the precise mechanisms underlying this effect require further elucidation. Compared to the role of the cGAS-STING pathway in other kidney diseases, this study ystematically elucidate the activation mechanism and pathological significance of this pathway in MN. Although previous studies have reported the involvement of this pathway in podocyte injury in diabetic nephropathy[16] and Alport syndrome[8], the unique value of this study lies in: 1) revealing the pivotal role of the cGAS-STING pathway in the MN-specific pathological process of "autoantibody activation - complement activation - podocyte injury"; 2) reporting the regulatory effect of STING inhibition on lipid metabolism in MN; 3) clarifying the dominant role of the IRF3-IFNβ axis in the activation of this pathway. These findings provide a new perspective for understanding the pathogenesis of MN. From a therapeutic perspective, this study confirms that inhibiting the cGAS-STING pathway can simultaneously ameliorate two key pathological aspects of MN: First, reducing immune complex deposition and complement activation mediated by autoantibodies, specifically manifested as significantly decreased IgG and C3 deposition in glomeruli; Second, alleviating podocyte injury associated with mitochondrial dysfunction, including ultrastructural restoration and improvement of functional indicators. This "dual-action" therapeutic effect makes the cGAS-STING pathway a highly promising therapeutic target. Certainly, this study also has some limitations. While we have confirmed the critical role of mtDNA leakage in activating the cGAS-STING pathway, the specific mechanisms of mtDNA release in MN (such as whether it occurs through the BAX/BAK or VDAC pathways) remain incompletely elucidated. Additionally, the molecular basis underlying the improvement of lipid metabolism by STING inhibition requires more in-depth investigation. These will be key focus areas for our future research. Declarations Ethics approval and consent to participate This study was conducted in accordance with the International Guiding Principles for Biomedical Research Involving Animals, animal welfare regulations, and relevant laboratory operating procedures. All methods are reported in accordance with the ARRIVE guidelines. The study was approved by the Animal Ethics Committee of Xinjiang Medical University (IACUC-20230627-19). Consent for publication Not applicable. Competing interests The authors declare no competing interests. Funding This research was supported by the National Key Research and Development Program Cultivation Project (XYD2024ZX05), the Major Science and Technology Project of Xinjiang Uygur Autonomous Region (2022A03001), the Regional Collaborative Innovation Special Project of Xinjiang Uygur Autonomous Region (Shanghai Cooperation Organization Science and Technology Partnership Program and International Science and Technology Cooperation Program) (2023E01020), the Construction Project of the Wisdom Medical Innovation Center of Xinjiang Medical University (ZHYL-07), and the Scientific Research Innovation Platform Talent Team Support Program of the Xinjiang Talent Development Fund. Author Contribution H.S.: Conceptualized and designed the experiments, performed experiments and data analysis, and contributed to manuscript writing. G.Z., T.X., and M.L.: Conducted data verification and correction. Y.D., H.H., and X.G.: Performed literature search and participated in some experimental procedures. X.L.: Performed literature research and provided experimental guidance. C.L.: Participated in experimental design, provided experimental guidance, and revised the manuscript. Acknowledgement We sincerely thank the First Affiliated Hospital of Xinjiang Medical University and Xinjiang University for their contributions to this work. This This research was supported by the National Key Research and Development Program Cultivation Project (XYD2024ZX05), the Major Science and Technology Project of Xinjiang Uygur Autonomous Region (2022A03001), the Regional Collaborative Innovation Special Project of Xinjiang Uygur Autonomous Region (Shanghai Cooperation Organization Science and Technology Partnership Program and International Science and Technology Cooperation Program) (2023E01020), the Construction Project of the Wisdom Medical Innovation Center of Xinjiang Medical University (ZHYL-07), and the Scientific Research Innovation Platform Talent Team Support Program of the Xinjiang Talent Development Fund. Data Availability Data is provided within the manuscript or supplementary information files References Wang M, Yang J, Fang X, Lin W, Yang Y: Membranous nephropathy: pathogenesis and treatments . MedComm 2024, 5 (7):e614. Alsharhan L, Beck LH, Jr.: Membranous Nephropathy: Core Curriculum 2021 . 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Journal of the American Society of Nephrology : JASN 2022, 33 (12):2153–2173. Decout A, Katz JD, Venkatraman S, Ablasser A: The cGAS-STING pathway as a therapeutic target in inflammatory diseases . Nature reviews Immunology 2021, 21 (9):548–569. Chen C, Xu P: Cellular functions of cGAS-STING signaling . Trends in cell biology 2023, 33 (8):630–648. Zhang X, Bai XC, Chen ZJ: Structures and Mechanisms in the cGAS-STING Innate Immunity Pathway . Immunity 2020, 53 (1):43–53. Jiang A, Liu J, Wang Y, Zhang C: cGAS-STING signaling pathway promotes hypoxia-induced renal fibrosis by regulating PFKFB3-mediated glycolysis . Free radical biology & medicine 2023, 208 :516–529. Bai J, Liu F: cGAS‒STING signaling and function in metabolism and kidney diseases . Journal of molecular cell biology 2021, 13 (10):728–738. Maekawa H, Inoue T, Ouchi H, Jao TM, Inoue R, Nishi H, Fujii R, Ishidate F, Tanaka T, Tanaka Y et al : Mitochondrial Damage Causes Inflammation via cGAS-STING Signaling in Acute Kidney Injury . Cell reports 2019, 29 (5):1261–1273.e1266. Shi L, Zha H, Pan Z, Wang J, Xia Y, Li H, Huang H, Yue R, Song Z, Zhu J: DUSP1 protects against ischemic acute kidney injury through stabilizing mtDNA via interaction with JNK . Cell death & disease 2023, 14 (11):724. Zang N, Cui C, Guo X, Song J, Hu H, Yang M, Xu M, Wang L, Hou X, He Q et al : cGAS-STING activation contributes to podocyte injury in diabetic kidney disease . iScience 2022, 25 (10):105145. Chen JS, Chen A, Chang LC, Chang WS, Lee HS, Lin SH, Lin YF: Mouse model of membranous nephropathy induced by cationic bovine serum albumin: antigen dose-response relations and strain differences . Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association 2004, 19 (11):2721–2728. Wu HH, Chen CJ, Lin PY, Liu YH: Involvement of prohibitin 1 and prohibitin 2 upregulation in cBSA-induced podocyte cytotoxicity . Journal of food and drug analysis 2020, 28 (1):183–194. Jiang HX, Feng Z, Zhu ZB, Xia CH, Zhang W, Guo J, Liu BL, Wang Y, Liu YN, Liu WJ: Advances of the experimental models of idiopathic membranous nephropathy (Review) . Molecular medicine reports 2020, 21 (5):1993–2005. Li XJ, Wang YN, Wang WF, Nie X, Miao H, Zhao YY: Barleriside A, an aryl hydrocarbon receptor antagonist, ameliorates podocyte injury through inhibiting oxidative stress and inflammation . Frontiers in pharmacology 2024, 15 :1386604. Zheng R, Deng Y, Chen Y, Fan J, Zhang M, Zhong Y, Zhu R, Wang L: Astragaloside IV attenuates complement membranous attack complex induced podocyte injury through the MAPK pathway . Phytotherapy research : PTR 2012, 26 (6):892–898. Xu Q, Xiong H, Zhu W, Liu Y, Du Y: Small molecule inhibition of cyclic GMP-AMP synthase ameliorates sepsis-induced cardiac dysfunction in mice . Life sciences 2020, 260 :118315. An C, Sun F, Liu C, Huang S, Xu T, Zhang C, Ge S: IQGAP1 promotes mitochondrial damage and activation of the mtDNA sensor cGAS-STING pathway to induce endothelial cell pyroptosis leading to atherosclerosis . International immunopharmacology 2023, 123 :110795. Arya P, Kumar N, Bhandari U, Thapliyal S, Sharma V: Hidden attributes of zymosan in the pathogenesis of inflammatory diseases: A tale of the fungal agent . Iranian journal of basic medical sciences 2023, 26 (4):380–387. Zhou J, Zhuang Z, Li J, Feng Z: Significance of the cGAS-STING Pathway in Health and Disease . International journal of molecular sciences 2023, 24 (17). Li Q, Wu P, Du Q, Hanif U, Hu H, Li K: cGAS-STING, an important signaling pathway in diseases and their therapy . MedComm 2024, 5 (4):e511. Motwani M, Pesiridis S, Fitzgerald KA: DNA sensing by the cGAS-STING pathway in health and disease . Nature reviews Genetics 2019, 20 (11):657–674. Yu Y, Liu Y, An W, Song J, Zhang Y, Zhao X: STING-mediated inflammation in Kupffer cells contributes to progression of nonalcoholic steatohepatitis . The Journal of clinical investigation 2019, 129 (2):546–555. Additional Declarations No competing interests reported. Supplementary Files SupplementaryFile1WBrawFigure.pdf 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. We do this by developing innovative software and high quality services for the global research community. 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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-8685107","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":588529606,"identity":"9506b886-ce02-4d71-b957-c2293e699572","order_by":0,"name":"Halinuer Shadekejiang","email":"","orcid":"","institution":"The First Affiliated Hospital of Xinjiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Halinuer","middleName":"","lastName":"Shadekejiang","suffix":""},{"id":588529607,"identity":"d53c2e10-10f4-4cd2-acae-bbbd66758629","order_by":1,"name":"Guoqiang Zhu","email":"","orcid":"","institution":"The First Affiliated Hospital of Xinjiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Guoqiang","middleName":"","lastName":"Zhu","suffix":""},{"id":588529608,"identity":"2334bab0-f4a8-46c0-b196-29ee26e79ce6","order_by":2,"name":"Ting Xia","email":"","orcid":"","institution":"The First Affiliated Hospital of Xinjiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ting","middleName":"","lastName":"Xia","suffix":""},{"id":588529609,"identity":"473ac38f-5cb7-463a-8c98-172b120dcc04","order_by":3,"name":"Mingzhu Liang","email":"","orcid":"","institution":"The First Affiliated Hospital of Xinjiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Mingzhu","middleName":"","lastName":"Liang","suffix":""},{"id":588529610,"identity":"1abcfe4b-086b-4621-baf5-66ca5fe9ec11","order_by":4,"name":"Huan Hong","email":"","orcid":"","institution":"The First Affiliated Hospital of Xinjiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Huan","middleName":"","lastName":"Hong","suffix":""},{"id":588529614,"identity":"33cfd198-4e2d-4496-a37f-74d65fc74028","order_by":5,"name":"Xinyu Gan","email":"","orcid":"","institution":"Xinjiang University","correspondingAuthor":false,"prefix":"","firstName":"Xinyu","middleName":"","lastName":"Gan","suffix":""},{"id":588529617,"identity":"00a56c31-28aa-4312-bd8b-46e8093af18a","order_by":6,"name":"Xu Li","email":"","orcid":"","institution":"The First Affiliated Hospital of Xinjiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xu","middleName":"","lastName":"Li","suffix":""},{"id":588529621,"identity":"9b9d1581-ff9c-42d6-9b75-f5dddf783888","order_by":7,"name":"YANYA Duan","email":"","orcid":"","institution":"The First Affiliated Hospital of Xinjiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"YANYA","middleName":"","lastName":"Duan","suffix":""},{"id":588529624,"identity":"a1dd8d66-db10-4113-8b5a-a9e580439b7a","order_by":8,"name":"Chen Lu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtUlEQVRIiWNgGAWjYBACPgYGNhDNzM/MfPgBUVrYYFok29nSDEjSwmBwnkdBgjgtEsnPHnzccZjd+DAPgwFDjU00EVrSzA1nnkljNjvMe+ABw7G03AbCWnLYpHnbbIBa+BIMGBsOE6nlb5sEs3Ezj4EE8VoYgbYYMBOtheeZmWRvWxqzxGFgICcQ4xd+9uRnEj/bDifz9x8+/OBDjQ1hLTCQDCYTiFUOAnakKB4Fo2AUjIIRBgCqWzM9lFpcjgAAAABJRU5ErkJggg==","orcid":"","institution":"The First Affiliated Hospital of Xinjiang Medical University","correspondingAuthor":true,"prefix":"","firstName":"Chen","middleName":"","lastName":"Lu","suffix":""}],"badges":[],"createdAt":"2026-01-24 08:38:52","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8685107/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8685107/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102353307,"identity":"218b7899-80eb-413a-a893-bc56711b9be3","added_by":"auto","created_at":"2026-02-10 19:47:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3525583,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of experimental models for Membranous Nephropathy (MN). (A) Schematic of the experimental timeline and intervention in mice. (B) Urine protein-to-creatinine ratio (UPCR) results(n=6). (C-H) Serum biochemical parameters: (C) Serum Creatinine (SCr), (D) Urea, (E) Albumin, (F) Total Cholesterol, (G) Triglycerides, (H) Low-Density Lipoprotein (LDL) (n=6). (I) Representative renal histology images (HE, PAS, and PASM staining). (J) Transmission electron microscopy (TEM) images of glomeruli. (K) Schematic of the cell model and treatment protocol. (L, M) Immunofluorescence staining for C5b-9 (MAC) in podocytes(n=3). (N) Total cellular mtDNA relative abundance in podocytes (qPCR); (O) Cytosolic mtDNA relative abundance in podocytes (qPCR)(n=3). *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8685107/v1/cebdf731d615425c3a7cb4f7.png"},{"id":102398000,"identity":"c5e83727-fb4a-4aa7-a940-84358625b98e","added_by":"auto","created_at":"2026-02-11 10:20:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5160353,"visible":true,"origin":"","legend":"\u003cp\u003eActivation of the cGAS-STING pathway in membranous nephropathy. (A) Gene expression PCA. (B) DEGs volcano plot. (C)STING1 expression in MN patients based on GEO database (CON=6,MN=43). (D) Representative Western blot (WB) images of key pathway proteins in the renal cortex(n=4). (E-J)Quantitative analysis of Western blot results: (E) cGAS/GAPDH, (F) STING/GAPDH, (G) p-STING/STING, (H) p-TBK1/TBK1, (I) p-IRF3/IRF3. (J) IFN-ß1/GAPDH; (K) Immunohistochemical (IHC) staining for STING in mouse kidney sections(n=5). (L) Quantitative analysis of STING IHC staining. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8685107/v1/ecc3d2ce4b68a025f9ce0ddf.png"},{"id":102353310,"identity":"fd1909e7-ae86-4e73-86df-30f82a0fe900","added_by":"auto","created_at":"2026-02-10 19:47:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":10323851,"visible":true,"origin":"","legend":"\u003cp\u003eInhibition of the cGAS-STING pathway attenuates renal injury in membranous nephropathy. (A-B) Representative immunofluorescence (IF) images depicting (A)IgG and (B) C3 deposits in glomeruli.; (C-D)Quantitative analysis of (C) IgG, and (D) C3 immunofluorescence intensity(n=5).(E) Quantitative analysis of WT-1 positive cells (n=5); (F) Quantitative analysis of Nephrin(n=4); (G) Representative blot images of Nephrin; (H) Representative immunohistochemical (IHC) staining for WT-1. (I) Representative transmission electron microscopy (TEM) images of glomerular basement membrane ultrastructure. (J-L) Serum levels of pro-inflammatory cytokines measured by ELISA (n=6): (J) IL-6, (K) TNF-ɑ, (L) IL-1ß. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8685107/v1/8161c4a014be156da7c2d794.png"},{"id":102398055,"identity":"05c69318-c0c9-478b-b1af-8c5c390d4e4b","added_by":"auto","created_at":"2026-02-11 10:20:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6053653,"visible":true,"origin":"","legend":"\u003cp\u003eInhibition of the cGAS-STING pathway ameliorates mitochondrial dysfunction (A)Representative transmission electron microscopy (TEM) images of mitochondrial ultrastructure. (B)Intracellular ATP content(n=4). (C, D) Assessment of mitochondrial membrane potential (MMP): (C) Representative fluorescent images, (D) Quantitative analysis of MMP(n=5). (E, F) Measurement of mitochondrial superoxide (MitoSOX) levels: (E) Representative fluorescent images； (F) Quantitative analysis (n=5). *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8685107/v1/83f123ca65a639bd758d9964.png"},{"id":105565116,"identity":"37b7ed73-2f66-47a3-95a3-9bf4afe9b06a","added_by":"auto","created_at":"2026-03-27 12:52:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":39169563,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8685107/v1/dca2d2aa-2c95-4bad-bab3-43ae03bb2f87.pdf"},{"id":102353312,"identity":"96c9748c-5e31-4796-a7cb-88a7a618f552","added_by":"auto","created_at":"2026-02-10 19:47:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":27773724,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFile1WBrawFigure.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8685107/v1/fcf28163edee6b1264c4cff1.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mitochondrial DNA Release Activates cGAS-STING Signaling in Membranous Nephropathy: Therapeutic Attenuation by Pathway Inhibition","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eMembranous nephropathy (MN) is a leading cause of chronic kidney disease, accounting for approximately 30% of nephrotic syndrome cases in adults. Globally, the incidence of MN is estimated at 8\u0026ndash;10 cases per million population, with a significantly higher prevalence of 23.4% in China\u0026mdash;second only to IgA nephropathy\u0026mdash;and has shown a steady annual increase in recent years[1\u0026ndash;4]. Despite advances in immunosuppressive therapy, adverse effects and treatment intolerance remain unresolved, highlighting an urgent need for novel therapeutic targets[5].\u003c/p\u003e \u003cp\u003eMN is an autoimmune glomerular disease characterized by subepithelial immune complex deposition and diffuse glomerular basement membrane (GBM) thickening. Its pathogenesis involves autoantibodies (predominantly IgG) binding to podocyte antigens, which form immune complexes that deposit in a subepithelial location. This subsequently activates the complement system, leading to the formation of the membrane attack complex (MAC). MAC induces glomerular filtration barrier injury and proteinuria by disrupting podocyte foot processes, downregulating slit diaphragm proteins (e.g., Nephrin), and promoting podocyte apoptosis[6, 7]. While the initiating mechanisms of immune complex deposition and complement activation are well established, the key innate immune pathways driving sustained inflammatory amplification remain elusive.\u003c/p\u003e \u003cp\u003eThe cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) axis, a central signaling hub for cytosolic DNA sensing, is activated through the following mechanism: cytosolic double-stranded DNA (dsDNA) binds to cGAS, triggering a conformational change that activates its cyclase activity to synthesize the second messenger 2'3'-cyclic GMP-AMP (cGAMP). Acting as a molecular switch, cGAMP then binds to endoplasmic reticulum-resident STING, inducing a conformational opening and translocation to the Golgi apparatus, which in turn recruits and activates the kinase TBK1. TBK1 phosphorylates downstream transcription factors IRF3 and NF-κB, initiating the cascaded expression of type I interferons (e.g., IFN-β1) and proinflammatory cytokines (e.g., IL-6, TNF-α), ultimately regulating inflammation-immune homeostasis in antiviral immunity and autoinflammatory conditions[8\u0026ndash;11].\u003c/p\u003e \u003cp\u003eRecent studies have highlighted the critical pathological role of this pathway in kidney diseases[12, 13]. As a highly metabolically active organ, the kidney is vulnerable to mitochondrial injury induced by hypoxia and oxidative stress, leading to the release of mitochondrial DNA (mtDNA) that activates cGAS-STING signaling. This activation drives podocyte damage, tubular inflammation, and fibrotic progression. In models of acute kidney injury[14, 15], diabetic nephropathy[16], and Alport syndrome[8], pathway inhibition has markedly improved renal function and histopathology, underscoring its potential as a therapeutic target. However, its role in MN remains unreported.\u003c/p\u003e \u003cp\u003eBased on the MN\u0026rsquo;s pathological features and the pathway characteristics, we hypothesize that immune complex deposition activates complement C5b-9, leading to podocyte membrane injury and apoptosis. The released mtDNA and nuclear DNA fragments then serve as endogenous ligands to activate the cGAS-STING pathway. Downstream IFN-β1 and proinflammatory cytokines further exacerbate podocyte damage and inflammation, forming a vicious cycle of \u0026ldquo;\u003cem\u003eimmune complexes \u0026rarr; cGAS-STING activation \u0026rarr; inflammation amplification\u003c/em\u003e\u0026rdquo;. Excessive pathway activation may contribute to podocyte loss by impairing mitochondrial function.\u003c/p\u003e \u003cp\u003eTo validate this hypothesis, we systematically investigated the role of the cGAS-STING pathway in MN and discovered: (i) First evidence confirming that MN induces mtDNA leakage, with significant upregulation of key molecules in the cGAS-STING pathway (cGAS, p-STING, p-TBK1, p-IRF3); (ii) Specific inhibitors improved renal function and alleviated glomerular pathological damage; (iii) The protective mechanism is synergistically achieved through three pathways: suppressing the IRF3-IFN-β inflammatory axis to block inflammatory cascades, maintaining podocyte structural integrity, and ameliorating mitochondrial dysfunction.\u003c/p\u003e \u003cp\u003eThis study reveals that the cGAS-STING pathway is a critical driver of MN progression and clarifies its novel mechanism in regulating MN pathology through the \"inflammation-mitochondria-podocyte\" axis, thus providing a theoretical basis and potential strategy for the targeted therapy of MN.\u003c/p\u003e"},{"header":"2 MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Animals\u003c/h2\u003e \u003cp\u003eEight-week-old male BALB/c mice (SPF grade, body weight 22\u0026thinsp;\u0026plusmn;\u0026thinsp;5 g) were obtained from the Animal Experiment Center of The First Affiliated Hospital of Xinjiang Medical University [License No: SCXK (Xin) 2018-0003]. All experimental protocols were approved by the Institutional Animal Care and Use Committee of Xinjiang Medical University (Approval No: IACUC-20230627-19) and strictly adhered to the \"3R\" principles (Reduction, Refinement, Replacement). Blinding procedures were implemented during key stages of the experiment, including allocation, intervention, outcome assessment, and data analysis. Animals were housed in individually ventilated cages (\u0026le;\u0026thinsp;5 mice/cage) containing autoclaved pinewood bedding changed every 48 hours. Mice were maintained under controlled conditions (temperature: 23\u0026thinsp;\u0026plusmn;\u0026thinsp;3℃; relative humidity: 40%-60%; 12-hour light/dark cycle) with ad libitum access to irradiated SPF-grade feed and autoclaved water. Daily health monitoring was performed, with individuals showing abnormal signs immediately isolated. After a 1-week acclimation period, mice were randomly assigned by computer-generated random number table method into the following groups: Control group (CON), MN group, MN\u0026thinsp;+\u0026thinsp;RU-521 group, and MN\u0026thinsp;+\u0026thinsp;C-176 group ( n\u0026thinsp;=\u0026thinsp;6)).\u003c/p\u003e \u003cp\u003eAt the endpoint, the mice were given 3% isoflurane for anesthesia induction and 1.5% isoflurane for maintenance. Cardiac puncture was immediately performed for whole blood collection, followed by cervical dislocation under sustained anesthesia for euthanasia. Whole blood was clotted at room temperature for 30 min before centrifugation (3,000 \u0026times; g, 10 min, 4℃) to obtain serum supernatant for storage. Following bilateral nephrectomy, one renal aliquot was immediately flash-frozen in liquid nitrogen for subsequent protein analyses (including Western blotting), with long-term storage at -80℃. The contralateral kidney was immersion-fixed in 4% paraformaldehyde for 24 hours at 4℃ before histological processing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 cBSA-Induced Murine Model of Membranous Nephropathy\u003c/h2\u003e \u003cp\u003eBased on previous studies with modifications[17, 18], the detailed modeling procedure was as follows: Cationic bovine serum albumin (cBSA) (#9058, Chondrex, USA) was emulsified with an equal volume of Freund\u0026rsquo;s complete adjuvant (#7008, Chondrex, USA). Mice in the MN group received a subcutaneous injection of 0.1 mL of the emulsion (0.1 mg cBSA) at the tail base for initial immunization. Two weeks later, the MN group mice received tail base injections of cBSA: an initial dose of 100 \u0026micro;g, followed by subsequent injections every other day at an increased dose of 10 mg/kg. Injections were administered three times per week for six consecutive weeks. Mice in the control group received an equal volume of physiological saline on the same schedule. Twenty-four-hour urine samples were collected every two weeks using metabolic cages. Model success was ultimately determined based on renal function and pathological alterations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Cell culture and treatment\u003c/h2\u003e \u003cp\u003eThe mouse podocyte cell line (MPC-5) was originally obtained from iCell Bioscience Inc. (Shanghai, China) and cultured in DMEM medium supplemented with 10% fetal bovine serum. The membrane attack complex C5b-9 was prepared according to previously established methods. Specifically, it was generated from normal mouse serum treated with zymosan A (#Z4250, Sigma-Aldrich, USA) as described in earlier studies[19\u0026ndash;21]. The detailed procedure was as follows: zymosan was mixed with normal saline (10 mg/mL), boiled for 1 hour, and cooled to room temperature, followed by centrifugation to discard the supernatant. The pellet was resuspended in normal mouse serum to a concentration of 10 mg/mL and incubated for 1 hour in a water bath at 37℃. Zymosan particles were then removed by centrifugation (14,000 \u0026times;g, 4 ℃, 5 min ) and filtration through a 0.8 \u0026micro;m filter. The resulting zymosan-activated mouse serum (ZAS) was collected. MPC-5 cells were treated for 24 hours with culture medium containing 10% ZAS, with heat-inactivated mouse serum used as the control. Model success was ultimately verified by C5b-9 immunofluorescence results.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Inhibitor Administration\u003c/h2\u003e \u003cp\u003eTo inhibit cGAS activity, mice received daily intraperitoneal injections of RU.521 (5 mg/kg; Cat# HY-114180, MedChemExpress, USA) dissolved in a vehicle consisting of 5% DMSO (Cat# HY-Y0320C, MedChemExpress, USA) and 95% corn oil (Cat# HY-Y1888, MedChemExpress, USA) for one week before sacrifice[22, 23].\u003c/p\u003e \u003cp\u003eFor STING inhibition, mice were administered C-176 (750 nM, 200 \u0026micro;L; Cat# HY-112906, MedChemExpress, USA), prepared in the same vehicle, via daily intraperitoneal injection for three weeks before sacrifice. Control mice received an equivalent volume of the vehicle alone on the same schedule to account for potential non-specific effects of the injection procedure or solvent[8].\u003c/p\u003e \u003cp\u003eIn the cellular model, podocytes were pretreated with 5 \u0026micro;M RU.521 or C-176 (final DMSO, 0.05%) for 1 hour before exposure to ZAS. Control cells were treated with an equal volume of DMSO.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Renal function analysis\u003c/h2\u003e \u003cp\u003eTo evaluate the renal function in mice, this study measured urinary protein, serum creatinine (SCr), blood urea, and a series of lipid parameters. Urinary protein and urinary creatinine levels were assessed using commercial kits (Elabscience, China), and the urinary protein-to-creatinine ratio (UPCR) was calculated. The measurements of SCr, UREA, and lipid profiles (including total cholesterol and triglycerides) were conducted using an automated biochemical analyzer at the Animal Experiment Center of Xinjiang Medical University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Histology\u003c/h2\u003e \u003cp\u003eFor histological analysis, renal tissue samples were fixed in 4% paraformaldehyde for 24\u0026ndash;48 hours. The tissues were then dehydrated through a graded ethanol series, cleared in xylene, and embedded in paraffin. Serial sections of 4 \u0026micro;m thickness were cut using a microtome, mounted on glass slides, and dried overnight in a 60\u0026deg;C oven. The sections were subjected to Hematoxylin and Eosin (H\u0026amp;E) staining for general morphological observation, Periodic Acid\u0026ndash;Schiff (PAS) staining for highlighting basement membranes and glycogen deposits, and Periodic Acid\u0026ndash;Silver Methenamine (PASM) staining for detailed visualization of glomerular basement membranes and mesangial matrix. PASM staining was performed according to the manufacturer's instructions (Solarbio, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Immunohistochemical staining\u003c/h2\u003e \u003cp\u003eFor IHC analysis, paraffin-embedded renal tissue sections were first subjected to heat-induced antigen retrieval using EDTA or citrate buffer. Following this, endogenous peroxidase activity was blocked with 3% hydrogen peroxide, and non-specific binding sites were blocked with 5% bovine serum albumin (BSA) at room temperature. After blocking, the sections were incubated with primary antibodies at 4\u0026deg;C overnight. The next day, the sections were incubated with corresponding species-specific horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature, followed by color development using a 3,3'-diaminobenzidine (DAB) substrate kit. The percentage of positively stained area for the target proteins within the glomeruli was quantified using ImageJ software. Primary antibodies used included: anti-STING (#19851-1-AP, Proteintech), and anti-WT-1 (#ab89901, Abcam).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Immunofluorescence\u003c/h2\u003e \u003cp\u003eFor IF analysis, cell samples were first washed with phosphate-buffered saline (PBS) and then fixed with 4% paraformaldehyde for 30 minutes at room temperature. Paraffin-embedded tissue sections underwent antigen retrieval using citrate or EDTA buffer. Subsequently, all samples were permeabilized with 0.3% Triton X-100 (in PBS) for 10 minutes and blocked with 5% bovine serum albumin (BSA) for 30 minutes at room temperature to prevent non-specific binding. After blocking, the samples were incubated with diluted primary antibodies at 4\u0026deg;C overnight. The next day, the samples were incubated with appropriate species-specific secondary antibodies (such as Alexa Fluor 488-conjugated goat anti-rabbit IgG, #ab150077, Abcam) for 1 hour at room temperature in the dark. Finally, the nuclei were stained with 4\u0026prime;,6-diamidino-2-phenylindole (DAPI) for 5\u0026ndash;10 minutes at room temperature. All fluorescent images were acquired using a Leica confocal laser scanning microscope and subjected to quantitative analysis. The primary antibodies used included: anti-C5b-9 (#ab55811, Abcam), anti-C3 (#ab321966, Abcam), and anti-IgG (#ab6785, Abcam).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Western blot\u003c/h2\u003e \u003cp\u003e An appropriate amount of protein sample was mixed with loading buffer and denatured by boiling, followed by separation via SDS-PAGE gel electrophoresis. The proteins were then transferred from the gel onto a PVDF membrane. After transfer, the membrane was blocked with 5% BSA in TBST for 1 hour at room temperature. Subsequently, the membrane was incubated sequentially with diluted primary antibodies (overnight at 4\u0026deg;C) and secondary antibodies (1\u0026ndash;2 hours at room temperature). Following each antibody incubation, the membrane was washed three times with TBST. Finally, the blots were visualized using an ECL chemiluminescence substrate, and images were captured using an imaging system. The intensity of target protein bands was quantified using grayscale analysis ​​with​​ ImageJ software. The primary antibodies used included: anti-cGAS (#31659, Cell Signaling Technology), anti-STING (#13647, Cell Signaling Technology), anti-IRF3 (#4302, Cell Signaling Technology), anti\u0026ndash;p-IRF3 (#29047, Cell Signaling Technology), anti-TBK1 (#3504, Cell Signaling Technology), anti-p-TBK1 (#5483, Cell Signaling Technology), anti\u0026ndash;IFN-β1 (#97450, Cell Signaling Technology), anti-GAPDH (#60004-1-Ig, Proteintech), and anti-Nephrin (#ab216341, abcam). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (#ab205718, Abcam) was used as the secondary antibody.​\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Detection of mtDNA by Quantitative PCR (qPCR)\u003c/h2\u003e \u003cp\u003eCells were collected and centrifuged at 400 \u0026times; g for 5 min. The cell pellet was resuspended in ice-cold PBS and equally divided into two aliquots for the extraction of total cellular DNA and cytoplasmic DNA, respectively.\u003c/p\u003e \u003cp\u003eTotal DNA Extraction: Total DNA was isolated using the FastPure Cell/Tissue DNA Isolation Mini Kit (#DC102, Vazyme) according to the manufacturer's instructions. Briefly, the cell pellet was resuspended in 220 \u0026micro;L PBS containing 10 \u0026micro;L RNase Solution and 20 \u0026micro;L Proteinase K, followed by incubation at room temperature for \u0026ge;\u0026thinsp;15 min. Subsequently, 250 \u0026micro;L Buffer GB was added, and the mixture was incubated at 65 ℃ for 15\u0026ndash;30 min for lysis. After adding 180 \u0026micro;L absolute ethanol, the lysate was directly loaded onto a gDNA adsorption column and centrifuged at 13,400 \u0026times; g for 1 min. The column was sequentially washed with Washing Buffer A and Washing Buffer B. Residual ethanol was removed by spinning the empty column. DNA was finally eluted using 30 \u0026micro;L of pre-warmed (70 ℃) Elution Buffer.\u003c/p\u003e \u003cp\u003eCytoplasmic DNA Extraction: The cell pellet was resuspended in 250 \u0026micro;L of cytoplasmic lysis buffer (150 mM NaCl, 50 mM HEPES [pH 7.4], 25 \u0026micro;g/mL digitonin) and incubated at room temperature for 10 min. The lysate was centrifuged at 1,000 \u0026times; g for 10 min to pellet nuclei and cellular debris. The resulting supernatant was further centrifuged at 17,000 \u0026times; g for 30 min at 4℃ to isolate the cytoplasmic fraction. DNA was then extracted from this cytoplasmic supernatant using the same protocol described for total DNA extraction.\u003c/p\u003e \u003cp\u003emtDNA quantification by quantitative PCR (qPCR):\u003c/p\u003e \u003cp\u003eTarget Gene: Mitochondrial cytochrome c oxidase subunit 1 (mtCOI / mtCOX2) gene\u003c/p\u003e \u003cp\u003eReference Gene: 18S ribosomal RNA (18S rDNA) gene\u003c/p\u003e \u003cp\u003ePrimer Sequences:\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e18S rDNA:\u003c/h3\u003e\n\u003cp\u003eForward: 5\u0026prime;-TGTGTTAGGGGACTGGTGGACA-3\u0026prime;\u003c/p\u003e \u003cp\u003eReverse: 5\u0026prime;-CATCACCCACTTACCCCCAAAA-3\u0026prime;\u003c/p\u003e \u003cp\u003emtCOX2:\u003c/p\u003e \u003cp\u003eForward: 5\u0026prime;-ATAACCGAGTCGTTCTGCCAAT-3\u0026prime;\u003c/p\u003e \u003cp\u003eReverse: 5\u0026prime;-TTTCAGAGCATTGGCCATAGAA-3\u0026prime;\u003c/p\u003e \u003cp\u003eData Analysis: Relative mtDNA copy number was calculated by normalizing mtCOX2 quantification cycle (Cq) values to those of 18S rDNA and compared between experimental groups.\u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Enzyme-Linked Immunosorbent Assay (ELISA)\u003c/h2\u003e \u003cp\u003e Serum levels of inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6), were quantified using commercial ELISA kits (Jianglai Bioengineering, Shanghai, China) according to the manufacturer\u0026rsquo;s protocols. Briefly, serum samples were diluted and added to the pre-coated plates. After incubation and washing, biotinylated detection antibodies and then enzyme conjugates were added. Following another incubation and washing step, the substrate solution (TMB) was added for color development, which was stopped with the stop solution. The absorbance was immediately measured at 450 nm using a microplate reader. Cytokine concentrations were calculated based on standard curves generated from the standard samples provided in the kits.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.12 ATP measurement\u003c/h2\u003e \u003cp\u003eCellular ATP levels were measured using an ATP Assay Kit (S0026, Beyotime, China). Briefly, after cell lysis, the supernatant was collected and mixed with the ATP detection solution. The luminescence was immediately measured using a luminescence microplate reader (Thermo Fisher Scientific, USA). The ATP concentration of each sample was calculated based on a standard curve and normalized to the total protein concentration determined by a BCA protein assay kit. The results are expressed as micromoles per gram of protein (\u0026micro;mol/g protein).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Mitochondrial transmembrane potential\u003c/h2\u003e \u003cp\u003eThe mitochondrial membrane potential (MMP) was detected using the JC-1 fluorescent probe (#C2006, Beyotime, China). Briefly, after incubating the cells with the JC-1 working solution, fluorescence signals were observed under a confocal microscope (Leica, Germany). JC-1 forms aggregates that emit red fluorescence in mitochondria with normal membrane potential, whereas it remains in a monomeric state and emits green fluorescence when the membrane potential is decreased. Changes in MMP were quantitatively assessed by measuring the ratio of red to green fluorescence intensity. All experiments were strictly performed in accordance with the manufacturer's instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.14 Mitochondrial Superoxide Detection Methodology\u003c/h2\u003e \u003cp\u003eMitochondrial superoxide levels in MPC-5 were detected using the MitoSO\u0026trade; Red mitochondrial superoxide indicator (Beyotime Biotechnology, Cat# S0061). Briefly, cells were incubated with the MitoSO\u0026trade; Red Working Solution according to the manufacturer's instructions. Following incubation and washing, real-time changes in mitochondrial superoxide were visualized and analyzed using laser scanning confocal microscopy (Leica Microsystems).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.15 Transmission Electron Microscopy (TEM)\u003c/h2\u003e \u003cp\u003eRenal tissues were collected from MN and control mice. Fresh tissue blocks (not exceeding 1mm\u0026sup3;) were rapidly dissected to minimize mechanical damage and immediately immersed in prechilled electron microscopy fixative (containing 4% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4) for 24 hours at 4℃. After fixation, samples were rinsed three times with 0.1 M phosphate buffer (pH 7.4) and post-fixed in 1% osmium tetroxide for 2 hours at room temperature in the dark. The samples were dehydrated through a graded ethanol and acetone series, infiltrated, and embedded stepwise using mixtures of acetone and EMBed-812 resin at 37\u0026deg;C, followed by polymerization at 65\u0026deg;C for 48 hours. Ultrathin sections (60\u0026ndash;80 nm) were cut using an ultramicrotome (Leica), collected onto 150-mesh copper grids, and sequentially stained with 2% uranyl acetate in saturated alcohol solution and 2.6% lead citrate, with thorough rinsing after each staining step. Sections were observed under a transmission electron microscope (HT7800/HT7700, Hitachi).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.16 Bioinformatic Analysis\u003c/h2\u003e \u003cp\u003eTo address the lack of clinical data on STING in human MN, we first explored the expression characteristics of the STING gene using public databases. Data for this study were sourced from the GSE108113 dataset (GPL19983 platform) within the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/geo/\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/geo/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Principal component analysis (PCA) and hierarchical clustering for data quality control resulted in the inclusion of 43 MN patients and 6 healthy controls for subsequent analysis. All data processing and analysis were performed in the R language environment (v4.4.0). After preprocessing raw expression data with background correction, log2 transformation, and quantile normalization, differential expression analysis was conducted using the limma package. We first compared glomerular STING gene expression levels between the MN group and the control group. Differentially expressed genes (DEGs) were subsequently screened using thresholds of |log₂FC| \u0026gt; 0.6 and adjusted P value (adj.P.Val)\u0026thinsp;\u0026lt;\u0026thinsp;0.01, with results visualized via volcano plots.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e2.17 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll quantitative data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Statistical analyses were conducted using GraphPad Prism 9.0 (GraphPad Software, San Diego, CA). The normality of data distribution and homogeneity of variances were evaluated using the Shapiro\u0026ndash;Wilk test and Bartlett\u0026rsquo;s test, respectively. For datasets satisfying both assumptions, two-group comparisons utilized unpaired two-tailed Student\u0026rsquo;s t-tests, while multiple comparisons employed one-way ANOVA with Tukey\u0026rsquo;s post-hoc test. Non-parametric datasets were analyzed by the Kruskal\u0026ndash;Wallis test followed by Dunn\u0026rsquo;s test. Statistical significance was defined as \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 RESULTS","content":"\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Successful establishment of mouse and cellular models of MN\u003c/h2\u003e \u003cp\u003eTo investigate the role of the cGAS-STING signaling pathway in MN, an MN mouse model was established in vivo by cBSA immunization (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Compared to control mice, the MN model exhibited characteristic nephrotic syndrome phenotypes: UPCR progressively increased starting at week 2 post-induction, becoming significantly elevated by week 6 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Concurrently, Scr and urea levels were significantly increased (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D). Dyslipidemia was evident, with significant elevations in total cholesterol (TC), triglycerides (TG), and low-density lipoprotein (LDL), accompanied by a significant reduction in serum albumin (ALB) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-H).\u003c/p\u003e \u003cp\u003eHistopathological analysis of kidney tissues revealed diffuse thickening of the GBM and expansion of the mesangial matrix in MN mice. Characteristic spike-like formations were observed on PASM staining. Immunofluorescence analysis demonstrated granular deposition of immunoglobulin G (IgG) and complement C3 along the glomerular capillary walls. Further confirmation by TEM revealed uneven GBM thickening, subepithelial electron-dense deposits, and extensive effacement of podocyte foot processes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-G). Collectively, these findings confirm the successful establishment of the cBSA-induced MN model.\u003c/p\u003e \u003cp\u003eIn vitro, a podocyte injury model mimicking the MN microenvironment was generated using MPC-5 cells stimulated with zymosan to activate the complement system (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK). Successful assembly of the membrane attack complex C5b-9 on the podocyte surface following zymosan stimulation was confirmed by immunofluorescence (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL, M), validating the establishment of the injury model.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.2 MN Podocyte Model Reveals Significant Increase in Cytosolic mtDNA Leakage\u003c/h2\u003e \u003cp\u003eThe total cellular mtDNA relative abundance in glomerular cells of the MN group was significantly lower than that in the CON group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), suggesting abnormal mitochondrial quantity reduction or mtDNA replication inhibition in MN glomerular cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eN). In stark contrast, the relative abundance of cytosolic mtDNA in the MN group was significantly higher than that in the CON group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), directly reflecting abnormal leakage of mtDNA from the mitochondrial matrix into the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eO). In glomerular cells of MN, mitochondrial membrane damage or decreased mitochondrial membrane potential compromises mitochondrial membrane integrity, leading to mtDNA escaping into the cytoplasm through membrane gaps.\u003c/p\u003e \u003cp\u003eThe combined results reveal a dual signature of mitochondrial dysfunction in MN glomerular cells: on one hand, reduced total mtDNA indicates a decline in overall mitochondrial quantity or replication capacity, reflecting diminished functional reserve; on the other hand, increased cytosolic mtDNA serves as a direct marker of structural damage (abnormal membrane permeability). This imbalance between \"quantity/replication deficiency\" and \"structural damage\" may lead to dysregulated mtDNA metabolism\u0026mdash;impaired mitochondria fail to be effectively repaired or compensated, instead releasing damage-associated molecular patterns (DAMPs) through leaked mtDNA, which activates cGAS-STING pathway and contributes to glomerular inflammatory responses in the pathological process.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Bioinformatics analysis reveals upregulation of STING in MN patients\u003c/h2\u003e \u003cp\u003eTo verify whether mitochondrial DNA leakage activates the cGAS-STING pathway, we first analyzed clinical transcriptomic data. The MN dataset GSE108113 was retrieved from the GEO database. Principal component analysis (PCA) confirmed sample grouping reliability (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Using thresholds of |log₂FC| \u0026gt; 0.6 and adj. P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, we identified 2,629 differentially expressed genes (DEGs), including 1,486 upregulated and 1,143 downregulated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Focusing on innate immunity regulatory genes revealed significantly elevated expression of STING1 (stimulator of interferon genes) in the MN group (log₂FC\u0026thinsp;=\u0026thinsp;0.87, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), suggesting aberrant activation of the cGAS-STING signaling pathway may contribute to MN pathogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Mouse model of MN reveals pathological overactivation of cGAS-STING pathway\u003c/h2\u003e \u003cp\u003eTo systematically validate cGAS-STING pathway overactivation in MN, protein analysis of murine renal tissues demonstrated significant activation of the renal cGAS-STING innate immune axis under MN pathological conditions. Key findings revealed: (1) upregulation of pattern recognition receptor cGAS (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) during pathway initiation; (2) concurrent elevation of both total and phosphorylated STING (p-STING, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) indicating core adaptor activation; (3) phosphorylation-mediated signal transduction where activated STING induced TBK1 phosphorylation (p-TBK1), subsequently phosphorylating IRF3 (p-IRF3) to establish a complete cGAS\u0026rarr;STING\u0026rarr;TBK1\u0026rarr;IRF3 phosphorylation cascade. Crucially, unchanged total TBK1 and IRF3 levels confirmed pathway activation primarily depended on enhanced post-translational modifications rather than protein synthesis, with this signaling axis further driving type I interferon responses through IRF3 activation as evidenced by significantly elevated interferon-β1 (IFN-β1) expression in renal cortex (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), collectively demonstrating cGAS-STING overactivation and its inflammatory mediation in MN kidneys(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Inhibition of the cGAS-STING pathway attenuates cBSA-induced renal injury\u003c/h2\u003e \u003cp\u003eTo validate the pivotal role of the cGAS-STING pathway in MN pathogenesis, targeted inhibition using specific inhibitors (C-176 for STING, RU.521 for cGAS) significantly suppressed pathway activation compared to untreated MN controls, evidenced by reduced cGAS protein expression (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, RU.521 only), decreased total STING and phosphorylated STING (p-STING) levels (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and marked inhibition of downstream p-TBK1 and p-IRF3 expression (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), confirming effective blockade of the cGAS-STING axis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-J). Functionally, pathway inhibition ameliorated renal injury and systemic metabolic dysregulation: renal parameters showed significantly reduced UPCR(P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) alongside decreased Scr and urea ( P\u0026thinsp;\u0026lt;\u0026thinsp;0.001); lipid profiling revealed C-176 reduced TC, TG, and LDL (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) while RU.521 only lowered TG (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) without affecting TC and LDL (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-H); renal pathology demonstrated attenuated IgG and C3 deposits along glomerular capillaries via IF (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-D); and TEM analysis exhibited thinner glomerular basement membranes approaching normal uniformity, reduced foot process effacement, and absence of subepithelial electron-dense deposits (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Inhibition of the cGAS-STING Pathway Preserves Podocyte Integrity\u003c/h2\u003e \u003cp\u003eTo investigate the impact of the cGAS-STING pathway on podocytes, we examined podocyte-specific marker expression. Western blot analysis revealed significantly reduced levels of the key functional protein Nephrin in renal cortex of MN model mice (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with this downregulation being markedly reversed by RU.521 or C-176 intervention (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, G). IHC staining further confirmed substantially decreased WT-1-positive cell counts in glomeruli of the MN group versus controls (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while inhibitor treatment significantly restored WT-1-positive cell numbers (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, H)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Inhibition of the cGAS-STING Pathway Attenuates Inflammatory Responses in MN\u003c/h2\u003e \u003cp\u003eActivation of the cGAS-STING pathway drives robust inflammatory responses; to evaluate its pro-inflammatory effects in the MN model, ELISA analysis of serum cytokines revealed significantly elevated concentrations of classic pro-inflammatory cytokines\u0026mdash;interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α)\u0026mdash;in MN mice versus controls (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), whereas treatment with RU.521 or C-176 markedly reversed this aberrant upregulation, significantly reducing the levels of these inflammatory factors compared to the MN group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ-L).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e3.8 Inhibition of the cGAS-STING Pathway Ameliorates Mitochondrial Dysfunction in MN\u003c/h2\u003e \u003cp\u003eTo investigate whether cGAS-STING pathway activation contributes to mitochondrial damage in podocytes during MN, transmission electron microscopy revealed significant ultrastructural abnormalities in MN podocytes: mitochondrial membrane blurring, swelling, partial matrix dissolution, cristae fragmentation or disappearance, and vacuolation; whereas RU.521 or C-176 intervention markedly improved mitochondrial morphology, reducing swelling and partially restoring cristae integrity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Functional analyses confirmed direct regulatory effects: (1) Mitochondrial ATP production: Podocyte ATP levels significantly decreased in MN versus controls (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), reversed by inhibitors to near-normal levels (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB); (2) Mitochondrial membrane potential JC-1 probe analysis showed a significantly reduced aggregate (red)/monomer (green) fluorescence ratio in MN podocytes (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), indicating depolarization, while inhibitor treatment increased this ratio (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), demonstrating ΔΨm restoration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D); (3) Mitochondrial ROS (mtROS): MitoSOX\u0026trade; Red fluorescence quantification indicated elevated mtROS in MN glomeruli (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), effectively suppressed by pathway inhibitors (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study systematically investigated the pathogenesis of MN by integrating the cBSA-induced mouse model and the zymosan-stimulated podocyte model. The cBSA model successfully recapitulates the characteristic clinical manifestations of human MN, including significant proteinuria, thickening of the GBM, and deposition of IgG and complement C3, by mimicking the anti-PLA2R/THSD7A antibody-mediated pathological processes. Concurrently, the zymosan model, through specific activation of the TLR2/6-Dectin-1 receptor pathway, provides an ideal platform for in-depth investigation into the innate immune mechanisms involving alternative complement pathway activation and inflammasome activation in MN[17, 19, 24].\u003c/p\u003e \u003cp\u003eWithin these two complementary model systems, we observed significant mitochondrial dysfunction in podocytes affected by MN, characterized by three distinct alterations: 1)Ultrastructural Examination: Revealed abnormal mitochondrial changes including blurred membrane structure, swelling, matrix dissolution, cristae fragmentation or even disappearance, with some mitochondria exhibiting vacuolar degeneration. 2) Functional Assessment: Detected a significant decrease in mitochondrial membrane potential concurrently with a marked elevation in mitochondrial reactive oxygen species levels. 3) Molecular Analysis: Demonstrated a reduction in total cellular mtDNA abundance accompanied by abnormal accumulation of cytosolic mtDNA. These seemingly paradoxical yet interconnected phenomena collectively indicate a severe disruption of mitochondrial homeostasis in MN podocytes. Of particular note, the significant increase in cytosolic mtDNA levels provides direct evidence for mtDNA leakage from damaged mitochondria. Current research has unequivocally established that such leaked mtDNA, acting as a crucial Damage-Associated Molecular Pattern (DAMP), serves as the key trigger for activating the cGAS-STING innate immune signaling pathway[25\u0026ndash;27].\u003c/p\u003e \u003cp\u003eTo further validate this mechanism, we first analyzed public databases of MN patients and found that STING gene expression was significantly upregulated in renal tissues from MN patients. Subsequent protein-level detection confirmed the activation of the cGAS-STING signaling pathway in MN models, with its downstream signaling primarily dependent on the IRF3-IFNβ axis.\u003c/p\u003e \u003cp\u003eTo investigate the functional significance of this pathway, we conducted intervention studies using the specific inhibitors RU.521 and C-176. The experimental results were compelling: both inhibitors significantly improved renal function parameters in MN mice, including reduced UPCR levels and ameliorated SCr and blood urea levels. Concurrently, they restored the normal structure of the GBM, alleviated podocyte injury. Furthermore, levels of the inflammatory cytokines IL-6, IL-1β, and TNF-α were significantly decreased.\u003c/p\u003e \u003cp\u003eThese results are not only consistent with previous research but, more importantly, confirm that the cGAS-STING pathway in MN follows the classical cGAS-STING-IRF3 signaling cascade. This supports the conclusion that the observed phenotypic improvements primarily stem from the specific inhibition of the cGAS-STING pathway, thereby establishing its significant value as a potential therapeutic target for MN.\u003c/p\u003e \u003cp\u003eOf particular note, regarding lipid metabolism, the STING inhibitor exhibited a comprehensive regulatory effect, significantly lowering TC, LDL, and TG levels. In contrast, the cGAS inhibitor only reduced TG levels. Drawing on the findings of Yu et al.[28], which demonstrated that STING deficiency ameliorates dyslipidemia in mice with high-fat-diet-induced nonalcoholic steatohepatitis, we postulate that STING-targeted inhibition strategies may indirectly influence lipid metabolism by modulating systemic inflammatory responses. However, the precise mechanisms underlying this effect require further elucidation.\u003c/p\u003e \u003cp\u003eCompared to the role of the cGAS-STING pathway in other kidney diseases, this study ystematically elucidate the activation mechanism and pathological significance of this pathway in MN. Although previous studies have reported the involvement of this pathway in podocyte injury in diabetic nephropathy[16] and Alport syndrome[8], the unique value of this study lies in: 1) revealing the pivotal role of the cGAS-STING pathway in the MN-specific pathological process of \"autoantibody activation - complement activation - podocyte injury\"; 2) reporting the regulatory effect of STING inhibition on lipid metabolism in MN; 3) clarifying the dominant role of the IRF3-IFNβ axis in the activation of this pathway. These findings provide a new perspective for understanding the pathogenesis of MN. From a therapeutic perspective, this study confirms that inhibiting the cGAS-STING pathway can simultaneously ameliorate two key pathological aspects of MN: First, reducing immune complex deposition and complement activation mediated by autoantibodies, specifically manifested as significantly decreased IgG and C3 deposition in glomeruli; Second, alleviating podocyte injury associated with mitochondrial dysfunction, including ultrastructural restoration and improvement of functional indicators. This \"dual-action\" therapeutic effect makes the cGAS-STING pathway a highly promising therapeutic target.\u003c/p\u003e \u003cp\u003eCertainly, this study also has some limitations. While we have confirmed the critical role of mtDNA leakage in activating the cGAS-STING pathway, the specific mechanisms of mtDNA release in MN (such as whether it occurs through the BAX/BAK or VDAC pathways) remain incompletely elucidated. Additionally, the molecular basis underlying the improvement of lipid metabolism by STING inhibition requires more in-depth investigation. These will be key focus areas for our future research.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003e This study was conducted in accordance with the International Guiding Principles for Biomedical Research Involving Animals, animal welfare regulations, and relevant laboratory operating procedures. All methods are reported in accordance with the ARRIVE guidelines. The study was approved by the Animal Ethics Committee of Xinjiang Medical University (IACUC-20230627-19).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003e This research was supported by the National Key Research and Development Program Cultivation Project (XYD2024ZX05), the Major Science and Technology Project of Xinjiang Uygur Autonomous Region (2022A03001), the Regional Collaborative Innovation Special Project of Xinjiang Uygur Autonomous Region (Shanghai Cooperation Organization Science and Technology Partnership Program and International Science and Technology Cooperation Program) (2023E01020), the Construction Project of the Wisdom Medical Innovation Center of Xinjiang Medical University (ZHYL-07), and the Scientific Research Innovation Platform Talent Team Support Program of the Xinjiang Talent Development Fund.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eH.S.: Conceptualized and designed the experiments, performed experiments and data analysis, and contributed to manuscript writing. G.Z., T.X., and M.L.: Conducted data verification and correction. Y.D., H.H., and X.G.: Performed literature search and participated in some experimental procedures. X.L.: Performed literature research and provided experimental guidance. C.L.: Participated in experimental design, provided experimental guidance, and revised the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe sincerely thank the First Affiliated Hospital of Xinjiang Medical University and Xinjiang University for their contributions to this work. This This research was supported by the National Key Research and Development Program Cultivation Project (XYD2024ZX05), the Major Science and Technology Project of Xinjiang Uygur Autonomous Region (2022A03001), the Regional Collaborative Innovation Special Project of Xinjiang Uygur Autonomous Region (Shanghai Cooperation Organization Science and Technology Partnership Program and International Science and Technology Cooperation Program) (2023E01020), the Construction Project of the Wisdom Medical Innovation Center of Xinjiang Medical University (ZHYL-07), and the Scientific Research Innovation Platform Talent Team Support Program of the Xinjiang Talent Development Fund.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData is provided within the manuscript or supplementary information files\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWang M, Yang J, Fang X, Lin W, Yang Y: \u003cb\u003eMembranous nephropathy: pathogenesis and 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\u003cb\u003e20\u003c/b\u003e(11):657–674.\u003c/li\u003e\n\u003cli\u003eYu Y, Liu Y, An W, Song J, Zhang Y, Zhao X: \u003cb\u003eSTING-mediated inflammation in Kupffer cells contributes to progression of nonalcoholic steatohepatitis\u003c/b\u003e. \u003cem\u003eThe Journal of clinical investigation\u003c/em\u003e 2019, \u003cb\u003e129\u003c/b\u003e(2):546–555.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"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":"Membranous nephropathy, cGAS-STING pathway, Podocyte injury, Inflammation, Mitochondrial damage","lastPublishedDoi":"10.21203/rs.3.rs-8685107/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8685107/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eMembranous nephropathy (MN) is a chronic kidney disease mediated by autoimmunity, but its molecular mechanisms remain incompletely understood. Studies have suggested that mitochondrial damage leading to mitochondrial DNA (mtDNA) leakage may contribute to the development of autoimmune diseases by activating the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, but this mechanism has not yet been explored in MN.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eIn this study, an MN animal model was established in Balb/c mice using cationic bovine serum albumin (cBSA) induction. Concurrently, an in vitro podocyte injury model was generated by stimulating cultured podocytes with Zymosan. Mitochondrial damage was assessed by quantifying the relative abundance of mtDNA in total cellular and cytosolic fractions via quantitative PCR (qPCR). Interventions were performed using the cGAS inhibitor RU.521 and STING inhibitor C-176. The therapeutic effects of inhibiting the cGAS-STING pathway were systematically evaluated through in vivo and in vitro assessments of urinary protein levels, glomerular immune complex deposition, podocyte injury markers, and mitochondrial functional parameters.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIntegrated in vivo and in vitro experimental results confirm that MN induces mitochondrial dysfunction and triggers mtDNA leakage into the cytosol, thereby activating the cGAS-STING signaling pathway. Intervention with the cGAS-specific inhibitor RU.521 or the STING inhibitor C-176 demonstrated significant renoprotective effects in both animal and cellular models. In the cBSA-induced MN mouse model, the treatment groups exhibited significantly reduced urinary protein levels, decreased glomerular IgG and C3 deposition, and significant downregulation of inflammatory cytokines (IL-1β, IL-6, TNF-α). Similarly, in vitro podocyte experiments showed that inhibitor treatment reversed the Zymosan-induced reduction in mitochondrial membrane potential and decreased mitochondrial superoxide levels. These findings collectively demonstrate the therapeutic potential of targeting the cGAS-STING pathway in MN.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur study unveils a critical role for the cGAS-STING signaling pathway in the development and progression of MN. Targeted inhibition of this pathway confers remarkable renal protection, highlighting its potential as a novel therapeutic strategy for managing MN.\u003c/p\u003e","manuscriptTitle":"Mitochondrial DNA Release Activates cGAS-STING Signaling in Membranous Nephropathy: Therapeutic Attenuation by Pathway Inhibition","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-10 19:47:04","doi":"10.21203/rs.3.rs-8685107/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":"615f0c66-792c-4aed-86dc-96df365ffd01","owner":[],"postedDate":"February 10th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-25T09:13:43+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-10 19:47:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8685107","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8685107","identity":"rs-8685107","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}