Exploring the Role of TRAF6-TAK1 Pathway in Podocyte Pyroptosis and Its Implications for Primary Membranous Nephropathy Therapy

Abstract

Primary membranous nephropathy (PMN) is a prevalent renal disorder characterized by immune-mediated damage to the glomerular basement membrane, with recent studies highlighting the significant role of pyroptosis in its progression. In this study, we investigate the molecular mechanisms underlying PMN, focusing on the role of Tumor necrosis factor receptor-associated factor 6 (TRAF6) in promoting disease advancement. Specifically, we examine how TRAF6 facilitates PMN progression by inducing the ubiquitination of Transforming growth factor-beta-activated kinase 1 (TAK1), which in turn activates the Gasdermin D (GSDMD)/Caspase-1 axis, leading to podocyte pyroptosis. Utilizing transcriptomic data from the gene expression omnibus database, we identified key regulatory factors involved in pyroptosis and validated these findings through the establishment of a C3a-induced podocyte injury model and a Sprague–Dawley (SD) rat model of PMN. Our findings reveal that TRAF6 is significantly upregulated in PMN, and its interaction with TAK1 is crucial for the activation of the GSDMD/Caspase-1 axis, ultimately driving podocyte pyroptosis. Further biochemical and molecular analyses confirmed the pivotal role of the TRAF6/TAK1 signaling pathway in the pathogenesis of PMN. These results underscore the importance of TRAF6-mediated signaling in the progression of PMN and suggest that targeting the TRAF6/TAK1/GSDMD/Caspase-1 axis may offer a novel therapeutic strategy for the treatment of this debilitating renal disease.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10753-025-02249-w.

Introduction

Primary membranous nephropathy (PMN) is a common chronic glomerular disease, primarily characterized by thickening of the glomerular basement membrane and severe podocyte injury [1]. Patients with PMN often exhibit significant proteinuria, hypoalbuminemia, and edema, which severely impact their quality of life and prognosis [2]. In recent years, substantial progress has been made in understanding the pathogenesis of PMN, particularly regarding the identification of podocyte-targeting autoantibodies [3]. Anti-phospholipase A2 receptor (PLA2R) and anti-thrombospondin type 1 domain-containing 7A (THSD7A) antibodies have emerged as the most significant pathogenic factors for PMN. These antibodies are detected in approximately 70–80% of PMN cases, with PLA2R antibodies being widely regarded as a key marker of the disease [4, 5]. The discovery of anti-PLA2R and anti-THSD7A antibodies has significantly advanced the diagnosis and treatment of PMN. Despite the symptomatic relief offered by current treatments, such as glucocorticoids and immunosuppressants, these therapies often come with substantial side effects and limited efficacy [6, 7]. Treatment options are particularly scarce for refractory and recurrent PMN. Therefore, there is an urgent need for further insights into PMN pathogenesis to develop more effective therapeutic strategies.

Pyroptosis is a programmed cell death pathway mediated by inflammatory proteins, characterized by cell membrane rupture and release of inflammatory mediators [8]. Cell pyroptosis typically involves the activation of Caspase-1 and cleavage of Gasdermin D (GSDMD), leading to pore formation, cell content leakage, and intense inflammatory responses [9]. In recent years, increasing evidence indicates the significant role of pyroptosis in various kidney diseases, such as diabetic nephropathy [10] and acute kidney injury [11]. However, the specific mechanisms of pyroptosis in PMN have not been thoroughly investigated.

TRAF6 (TNF receptor-associated factor 6) is a multifunctional signaling molecule that plays a crucial role in regulating inflammatory responses and immune reactions [12, 13]. It can participate in various cellular processes by mediating signaling pathways like NF-κB and MAPK pathways [14, 15]. TAK1 is an important downstream molecule of TRAF6, also playing a key role in cellular stress and inflammatory responses [16, 17]. The activation of TAK1 typically depends on its ubiquitin modification, which can be mediated by TRAF6 [18, 19]. Studies have suggested that abnormal activation of TAK1 is closely associated with various inflammatory diseases and cancers [20–22]. However, the specific mechanisms of TRAF6 and TAK1 in PMN are not yet clear. This study aims to investigate the role of the TRAF6/TAK1 axis in pyroptosis to elucidate its specific mechanisms in the progression of PMN.

In the present study, We hypothesize that TRAF6 activates TAK1 via ubiquitin modification, thereby activating the GSDMD/Caspase-1 axis to induce pyroptosis and promote the progression of PMN. To validate this hypothesis, we first analyzed transcriptomic data related to PMN using bioinformatics methods and identified the potential key role of TRAF6 in the progression of PMN. Subsequently, through in vitro and in vivo experiments, we further investigated the specific molecular mechanisms of the TRAF6/TAK1 axis in pyroptosis.

Materials and Methods

Public Data Retrieval and Collection of Gene Sets Related to PMN

In order to acquire transcriptome datasets related to PMN, transcriptome data searches were conducted by accessing the gene expression omnibus of the National Center for Biotechnology Information (NCBI). The search used the keywords "PMN" or "Idiopathic Membranous Nephropathy." The selected dataset identifier was GSE216841, which comprised samples from 12 PMN patients and 8 healthy controls, all kidney tissue samples analyzed using the Affymetrix Human Genome U133 Plus 2.0 Array (Affymetrix, USA) for gene expression profiling.

Differential Analysis

Differential analysis was conducted using the R package "limma", with healthy samples as the control group and PMN samples as the experimental group. The sequencing data was analyzed for differential gene expression using the "limma" package in R software, with the criteria of │logFC│ > 1 and p-value < 0.05 indicating differential gene expression. The results of the differential analysis were visualized using the "ggplot2" package in R, generating volcano plots.

Key genes related to pyroptosis were retrieved by searching the GeneCards online database. The relevant genes obtained were intersected using the "VennDiagram" package in R software and illustrated in a Venn diagram.

Enrichment Analysis

In our study of gene set functional enrichment analysis, gene ontology (GO) annotations from the R package org.Hs.eg.db (version 3.1.0) were employed as the background to map genes to the background set. Enrichment analysis was subsequently performed using the R package clusterProfiler (version 3.14.3) to ascertain the enrichment of gene sets. The minimum gene set size was set to 5 and the maximum gene set size was set to 5000. The GO analysis encompassed biological processes (BP), molecular function (MF), and cellular component (CC) analysis, culminating in identifying the cellular functions, signaling pathways significantly influenced by candidate target genes, and pathways enriched with disease-associated differentially expressed genes. For gene set functional enrichment analysis, the latest gene annotations for KEGG Pathway were retrieved using the KEGG REST API as the background to mapping genes to the background set. Again, the R package clusterProfiler (version 3.14.3) was utilized to analyze the enrichment of the gene set. Notably, a p-value of < 0.05 was deemed a significant condition for enrichment.

Construction of Protein–Protein Interaction (PPI) Networks

The filtered PPI network among factors was established using the STRING database (version 11.0, https://string-db.org/), aiming to identify crucial cellular signaling axes. Initially, selected cell death-related factors were used as query inputs, with the confidence threshold set at 0.7 to ensure the accuracy of interaction information within the network. Topological analysis of the network revealed that TRAF6 holds a central position in the PPI network, suggesting its potential role as a key signaling axis in the process of cell death.

Establishment of a PMN Model

A total of 36 female SD rats (8 weeks old) were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. (Experimental Animal Production License Number: SCXK (Beijing) 2021–0006). The rats were housed in specific pathogen-free level animal facilities in individual cages with a humidity of 60% to 65% and a temperature of 22 to 25 °C. Following one week of acclimatization, the experimental procedures were initiated after assessing the rats' health status. The experimental protocol and animal usage scheme received approval from the relevant ethical committee.

For immunization, rats were emulsified with an equal volume of complete Freund's adjuvant (F5881, Sigma) containing 1 mg of cBSA (9058, Nanjing Wobo Biological Co., Ltd.). Two weeks post-immunization, rats were intravenously injected with cBSA at a dose of 16 mg/kg, administered three times a week at a one-day interval, and designated as the PMN group. After 4 weeks, the animals were euthanized, and we collected blood, urine, and kidney samples for analysis [23].

Animal Model Grouping and Treatment

A Slow Virus Treatment was conducted on a PMN rat model that was constructed through a 4-week cBSA treatment. The commercial TRAF6 silencing slow virus (sc-156004-V, Santa Cruz Biotechnology) was titrated to 10⁹ TU/mL. The slow virus was administered via tail vein injection, with the PMN + sh-TRAF6 group rats receiving 5 × 10⁶ TU (Transduction Units) on the first day after modeling, and the animals were euthanized 14 days post-modeling. Blood, urine, and kidney samples were collected for analysis and subsequent experiments.

Based on the known TAK1 sequence in NCBI, Shanghai Hanheng Biotechnology Co., Ltd. (Shanghai, China) was commissioned to construct oe-NC, oe-TAK1 into the slow virus vector pHBLV-CMV-MCS-EF1-Puromycin, with a slow virus titer of 1 × 10⁸ TU/ml. The sh-TRAF6 and oe-TAK1 slow viruses were mixed in proportion, and the PMN + sh-TRAF6 + oe-TAK1 group rats received a mixture of sh-TRAF6 and oe-TAK1 slow viruses via tail vein injection on the first day after modeling, with each rat receiving 5 × 10⁶ TU. The animals were euthanized 14 days post-modeling, and samples were collected for analysis and further processing for subsequent experiments.

VX765 was purchased from MCE (HY-13205, MCE), dissolved in DMSO to prepare a stock solution with a concentration of 10 mg/mL, stored at −20 °C, and then added to physiological saline to achieve a concentration of 100 mg/kg for intragastric administration. In the PMN + sh-TRAF6 + oe-TAK1 group rats on the first day post-modeling success, the animals were treated by daily intragastric administration for 14 days before being euthanized, and blood, urine, and kidney samples were collected for analysis.

The rats were grouped as follows: 1) Normal group (rats without any treatment); 2) PMN group (rats induced with cBSA through tail vein injection to induce membranous nephropathy); 3) PMN + sh-NC + oe-NC group (rats in the PMN group infected with oe-NC, sh-NC slow viruses); 4) PMN + sh-TRAF6 + oe-NC group (rats in the PMN group infected with oe-NC, sh-TRAF6 slow viruses); 5) PMN + sh-TRAF6 + oe-TAK1 group (rats in the PMN group infected with oe-TAK1, sh-TRAF6 slow viruses); 6) PMN + sh-TRAF6 + oe-TAK1 + VX765 group (rats in the PMN group infected with oe-TAK1, sh-TRAF6 slow viruses and then stimulated with VX765). Each group consisted of six rats.

Biochemical Indicators Analysis

The urinary protein levels in rats were detected using the Coomassie Bright Blue assay (P0006, Beyotime). Rat serum was separated from fresh blood using an automated biochemical analyzer (7180 Autoanalyzer; Hitachi, Tokyo, Japan) for biochemical testing, including serum creatinine (SCr), blood urea nitrogen (BUN), albumin (ALB), total cholesterol (T-CHOL), triglycerides (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL).

Renal Tissue Electron Microscopy Examination

Renal tissue pieces of approximately 1 mm³ from each animal were fixed in 2.5% glutaraldehyde (Sigma-Aldrich, USA) at 4 °C for over 4 h. The samples were then washed three times with phosphate-buffered saline (PBS) and post-fixed in 1% osmium tetroxide (catalog number 1.24505, Sigma-Aldrich, USA) at room temperature for 1 h. Subsequently, staining with uranyl acetate (CAS number 6159–44-0, Shanghai Magi Biological Co., Ltd) and lead citrate (CAS number 512–26-5, Beijing Hyde Biological Technology Co., Ltd) was carried out before preparing ultrathin Sects. (70 nm thick). The stained specimens were observed after drying, and transmission electron micrographs were captured using an H-7500 transmission electron microscope (Hitachi Ltd., Tokyo, Japan). The average width of foot processes was calculated using the DigitalMicrograph software (Gatan, Inc., Pleasanton, CA, USA) as (π/4) × (total length of glomerular basement membrane (GBM) / total number of foot processes).

Western Blot Analysis

Cells and tissues were lysed on ice for 30 min in RIPA buffer (P0013B, Beyotime, Shanghai, China) with 1% PMSF, followed by centrifugation at 14,000 × g at 4 °C. Protein concentration was determined using the BCA method (P0012S, Beyotime, Shanghai, China). After denaturing 50 µg of protein at 100 °C for 10 min, electrophoresis and transfer to a PVDF membrane were performed. The membrane was blocked with 5% non-fat milk for 1 h and incubated overnight at 4 °C with primary antibodies (TRAF6, TAK1, Ubiquitin, Cleaved Caspase-1, GSDMD-N, β-Tublin). After washing, an HRP-conjugated secondary antibody was applied, and signal detection was performed using the ECL system (32209, Thermo Fisher Scientific). Grayscale analysis was conducted using ImageJ software. The experiment was repeated three times.

Quantitative Real-Time PCR (qRT-PCR)

Total RNA from cells and kidney tissue was extracted using Trizol reagent (Invitrogen, USA) and assessed for concentration and purity using a Nanodrop 2000 spectrophotometer. RNA was reverse transcribed into cDNA using the PrimeScript RT reagent kit (Takara, Japan). qRT-PCR was performed with the Fast SYBR Green PCR kit (Takara) on an ABI PRISM 7300 system, with GAPDH as the reference gene. The 2^−ΔΔCt method was used to analyze relative gene expression levels, and each reaction had three technical replicates. The experiment was repeated three times. Primers were synthesized by Shanghai Shenggong Biological Company (Table S1).

Differentiation and Validation of Human Podocytes

The human podocyte line was obtained from Xiamen Yimo Biological Technology Co., Ltd. (IMP-H062). The cells were seeded onto culture plates coated with type I collagen and cultured in RPMI-1640 medium (11875093, USA) supplemented with 10% FBS (26140079, USA), 100 U/ml penicillin, and 100 μg/ml streptomycin (15140122, USA). Cells were maintained in a proliferative state at 33 °C with 5% CO₂. Upon transfer to 37 °C, cell proliferation ceased, and differentiation began. Under the 33 °C culture conditions, the medium was changed every two days. Once cells reached 70% confluency, they were transferred to 37 °C with 5% CO₂ to undergo differentiation for 10–12 days. Differentiation status was confirmed by immunofluorescence staining for podocalyxin (1:200, Abcam, UK). For human podocyte lines, ITS (C0341-10 ml, Beyotime) was added at a 1:100 ratio to the standard culture medium. Podocytes were treated with 50 nM C3a (HY-P7862, MCE) for 1 h to establish a complement-induced podocyte injury model (PMN group) [24, 25].

Commercial TRAF6 silencing lentivirus (sc-36717-V) was purchased from Santa Cruz Biotechnology (Shanghai) and titrated to 10⁹ TU/mL. Podocytes were seeded at 1 × 10⁶ cells per well in a 6-well plate, incubated for 24 h, infected with the lentivirus, and further experiments were conducted 72 h post-infection.

Based on the known TAK1 sequence in NCBI, Shanghai Hanheng Biotechnology Co., Ltd. (Shanghai, China) constructed oe-NC and oe-TAK1 into the lentiviral vector pHBLV-CMV-MCS-EF1-Puromycin and stably knocked down TRAF6 in podocytes seeded at 1 × 10⁵ cells per well in a 6-well plate. After 24 h of routine culture, when the cell confluence reached around 75%, the medium containing an appropriate amount of packaged lentivirus (MOI = 10, working titer approximately 5 × 10⁶ TU/mL) and 5 μg/mL polybrene (Merck, TR-1003, USA) was added for infection. The lentiviral titer was 1 × 10⁸ TU/ml. The medium was replaced with a medium containing 4 μg/mL puromycin (Invitrogen, A1113803) 72 h later, and cells were cultured for at least 14 days. Puromycin-resistant cells were expanded in a medium containing 2 μg/mL puromycin (Invitrogen, A1113803) for 9 days before transferring to a puromycin-free medium to obtain stable podocytes overexpressing TAK1 [26].

VX765 was purchased from MCE (HY-13205, MCE), dissolved in DMSO to prepare a 10 mg/mL stock solution, stored at −20 °C, and added to the cell culture medium at a concentration of 20 μM to stimulate podocytes for 48 h before subsequent experiments.

The cells were divided into the following groups: 1) Control group (normal podocytes); 2) PMN group (podocytes treated with C3a); 3) PMN + sh-NC + oe-NC group (PMN group cells overexpressing the negative control and infected with shRNA negative control); 4) PMN + sh-TRAF6 + oe-NC group (PMN group cells overexpressing the negative control and infected with shTRAF6); 5) PMN + sh-TRAF6 + oe-TAK1 group (PMN group cells overexpressing TAK1 and infected with shTRAF6); 6) PMN + sh-TRAF6 + oe-TAK1 + VX765 group (PMN group cells overexpressing TAK1 and infected with shTRAF6, followed by stimulation with VX765).

Immunoprecipitation Assay

For immunoprecipitation, the Pierce™ Co-Immunoprecipitation Kit (26,149, USA) was used following the manufacturer's instructions. Pre-cleaning of lysates containing 1 μg total protein was performed using a suitable isotype IgG antibody (ab6721, diluted at 1:2000). Subsequently, the pre-cleared lysates were mixed with 2 mg of anti-TRAF6 antibody (ab137452, Abcam) and gently shaken at 41 °C overnight. Protein G agarose beads (Thermo) were added to each tube, followed by further incubation at 41 °C with gentle shaking. The immunocomplexes were washed with cold radioimmunoprecipitation assay buffer, and the antibody-selected proteins were eluted from the agarose beads by boiling in SDS sample buffer (0.1 M Tris–HCl, 10% glycerol, 2% SDS, 0.05% bromophenol blue, and 0.1 M DTT) for 5 min. Similar conditions using the IgG antibody (ab6721) were employed for each gel's control lane. Subsequently, each sample was resolved on a 10% SDS-PAGE gel, and Western blotting with anti-TAK1 antibody (PA5-17507, Thermo Fisher Scientific) was conducted to confirm the physical interaction between the two proteins in the immunocomplex.

ELISA Experiment

Kidney tissues were extracted from rats and homogenized in 5–10 ml pre-chilled PBS. The resulting homogenate was further processed by sonication or repeated freeze–thaw cycles, followed by centrifugation at 850 × g for 15 min at 4 °C to collect the supernatant for subsequent experiments. For cell preparation, cells were initially digested with trypsin, harvested by centrifugation, and sonicated. After centrifuging at 1500 × g for 10 min at 4 °C, the supernatant was collected for later use. Following the instructions provided in the rat IL-6 ELISA kit (PI326) and rat TGF-β1 ELISA kit (PT878), as well as the human IL-6 ELISA kit (PI330) and human TGF-β1 ELISA kit (PT880), monoclonal antibodies against IL-6 and TGF-β1 were coated on a 96-well microplate. The plate was then incubated overnight at 4 °C, followed by a 1-h blocking step at room temperature, and washed with PBS. Subsequent steps were carried out according to the kit instructions. The optical density (OD) values were measured at a wavelength of 450 nm using an enzyme immunoassay reader (A51119500C, Thermo Fisher, USA).

Cell Counting Kit-8 (CCK-8) Experiment

The CCK-8 (C0037, Beyotime) was utilized in this experiment. Cells were seeded in a 96-well plate. After a 48-h incubation period, 10 μL of CCK-8 solution was added to each well. Subsequently, the cells were further incubated for 1–2 h, followed by the measurement of cell optical density at a wavelength of 450 nm. The experiment was repeated thrice.

Immunofluorescence Staining

Differentiated and undifferentiated podocytes were fixed on glass slides using 4% formaldehyde (F1635, Sigma-Aldrich) at room temperature for 15 min. Immunofluorescence staining was performed using a specific antibody against the podocyte differentiation marker PODXL (PA1-46169, Thermo Fisher). Cells treated with the PODXL antibody were then incubated with Alexa Fluor® 488-conjugated secondary antibody (ab150077, Abcam, UK). Nuclear staining was performed using DAPI (C1005, Beyotime). The specimens were imaged at 400 × magnification using an inverted Olympus FV1000 laser scanning confocal microscope (Olympus).

Reactive Oxygen Species (ROS) and Cell Viability Assay

Hoechst 33342 and PI Staining: Cells were cultured at 37 °C and stained with propidium iodide (PI) (3.34 μg/mL, HY-D0815, MCE) for 20 min, followed by incubation with Hoechst 33342 (10 μg/mL, HY-15559, MCE) for 20 min for nuclear staining at 37 °C. Microscopic images were captured using a confocal microscope (LSM710; Carl Zeiss Meditec AG, Oberkochen, Germany).

ROS Detection: Cell ROS levels were assessed using a 2',7'-dichlorofluorescin diacetate (DCFH-DA) probe (HY-D0940, MCE, USA). A stock solution of H2DCFDA was prepared at 10 mM in DMSO and further diluted before use. Cells were incubated with PBS solution containing 5 μM H2DCFDA at 37 °C in the dark for 30 min, followed by PBS wash and the addition of fresh culture medium. Fluorescence microscopy images were captured immediately.

Immunohistochemistry of Human Tissue Samples

Human renal tissue samples, including 10 cases each of PMN and healthy controls, were collected and fixed in 4% paraformaldehyde at room temperature for 24 h. The samples were dehydrated using an ethanol gradient (75%, 85%, 95%, and absolute ethanol, each for 1 min), followed by two 1-min immersions in xylene. The tissues were embedded in molten paraffin, sectioned into 3–4 µm thick slices, and subsequently deparaffinized and hydrated in xylene and ethanol. Antigen retrieval was performed by immersing the samples in 10 mM sodium citrate buffer (pH 6.0) and heating at 92 °C for 40 min. After washing twice with PBS, the sections were blocked with 5% BSA at 37 °C for 30 min. Primary antibodies, including rabbit anti-TRAF6 (ab33915, Abcam, USA), anti-TAK1 (PA5-17507, Thermo Fisher), anti-Ubiquitin (E4I2J, 43124S, Cell Signaling Technology), anti-Cleaved Caspase-1 (PA5-99390, Thermo Fisher), and anti-GSDMD-N (AF4012, Affinity Bioscience), were incubated at room temperature for 1 h. Following PBS-T washing, HRP-conjugated goat anti-rabbit IgG (ab205718, Abcam, UK) was applied as the secondary antibody. Staining was visualized using a DAB substrate kit (Beyotime, P0203), and the tissue sections were imaged using the EVOS cell imaging system.

Statistical Analysis Methods

The data were obtained from at least three independent experiments, and the results are presented as mean ± standard deviation (Mean ± SD). A two-sample independent t-test was used to compare the two groups. For comparisons involving three or more groups, a one-way analysis of variance (ANOVA) was conducted, followed by hoc Tukey's HSD test. For data not following a normal distribution or exhibiting inhomogeneous variances, either the Mann–Whitney U test or the Kruskal–Wallis H test was employed. All statistical analyses were conducted using GraphPad Prism 9 (GraphPad Software, Inc.) and the R programming language. The significance level for all tests was set at 0.05, and a two-tailed p-value less than 0.05 was considered statistically significant.

Results

Cell Necroptosis as a Key Pathway Influencing the Progression of PMN

To investigate metabolic changes during the progression of PMN and identify therapeutic targets, we analyzed a PMN-related transcriptome dataset obtained from a public database. Through gene differential analysis, we identified 2910 genes showing significant differential expression in PMN, with 1444 genes upregulated and 1466 genes downregulated (Fig. 1A). Furthermore, to reveal the differential changes in metabolic pathways during the progression of PMN, we conducted enrichment analysis on all differentially expressed genes related to metabolic pathways. Through GO enrichment analysis and KEGG enrichment analysis (Fig. 1B, Figure S1A-C), we discovered that BP in cells was altered during PMN progression, particularly changes in the MAPK signaling pathway. We noted that the MAPK signaling pathway may play a crucial role in PMN, as it regulates cell proliferation, differentiation, and apoptosis. This suggests that the onset of nephropathy is closely related to changes in cellular states. Therefore, changes in cellular states appear to be a key influencing pathway in the progression of PMN.

Fig. 1.

Enrichment Analysis Reveals Key Metabolic Pathways Influencing PMN. Note: A Volcano plot showing differentially expressed genes in kidney tissue between the Normal group and PMN patients, where red represents upregulated genes, blue represents downregulated genes, and gray represents genes with no significant difference; B Enriched results from KEGG pathway enrichment analysis of differentially expressed genes. Normal group n = 8, PMN group n = 12

TRAF6 as a Key Gene Regulating Cell Pyroptosis in the Progression of PMN

In order to further identify key factors mediating cell pyroptosis in the progression of PMN, we utilized the GeneCards database to search for "pyroptosis" and retrieved 886 cell pyroptosis regulatory factors. By intersecting them with genes differentially expressed in PMN, we identified 83 significant cell pyroptosis regulatory factors in PMN (Fig. 2A). Subsequently, to narrow down the crucial regulatory factors, we explored the PPI of candidate factors using the String online platform and visualized their network regulation (Fig. 2B). The connections between proteins represent their regulatory relationships. To pinpoint the core regulatory factors, we conducted a statistical analysis on the number of interconnected nodes for all genes, revealing that CASP3, IL1B, and TRAF6 were the top three genes based on the number of connection nodes (Fig. 2C).

Fig. 2.

Constructing a PPI Network to Identify Core Regulators of Cell Pyroptosis. Note: A Venn diagram showing the intersection of differentially expressed genes in PMN with cell pyroptosis-related factors from the GeneCards database; B PPI network of differentially expressed cell pyroptosis genes, with connecting lines indicating regulatory relationships between genes; C Bar graph illustrating the number of connections for each gene node, displaying only the top 10 nodes, where a higher count signifies a greater core significance; D Differential expression analysis of TRAF6. Normal group = 8, PMN group = 12

Studies have shown that TRAF6 activates cell pyroptosis by ubiquitinating TAK1, thereby contributing to cell demise [27, 28]. Therefore, we chose TRAF6 as the focus of our subsequent studies, with transcriptome data revealing significant upregulation of TRAF6 in the PMN group (Fig. 2D).

Collectively, these findings highlight TRAF6 as a key gene regulating cell pyroptosis in the progression of PMN.

Upregulation of the TRAF6/TAK1 Signaling Axis in a Rat Model and Significant Increase in TAK1 Ubiquitin Levels

To investigate the expression changes of TRAF6 in the SD rat model of membranous nephropathy, we successfully established the SD rat model of membranous nephropathy by tail vein injection of C-BSA. Biochemical analysis results revealed that compared to the Normal group, rats in the PMN group showed a significant decrease in serum TP and ALB levels, while T-CHOL, TG, Scr, and BUN levels were significantly elevated. Additionally, the quantification of 24-h urinary protein by the Bradford method further confirmed the successful establishment of the membranous nephropathy model, with significantly higher urinary protein excretion in the PMN group rats compared to the normal group (Fig. 3A).

Fig. 3.

Analysis of the Expression of TRAF6/TAK1 Signaling Axis and Related Cell Pyroptosis in a Rat Model of PMN. Note: A Detection of significant differences in serum levels of TP, ALB, T-CHOL, TG, Scr, BUN, and 24 h-UPRO levels between the PMN group and normal group rats. N = 6; B Kidney tissue electron microscopy examination of glomerular basement membrane thickening and level of immune complex deposition, scale bar = 2 μm, N = 6; C qRT-PCR analysis of mRNA expression levels of TRAF6, TAK1, Caspase-1, and GSDMD in rat glomerular tissue comparing the PMN group with the normal group, N = 6; D Western blot analysis comparing the protein expression levels of TRAF6, TAK1, Caspase-1, and GSDMD in rat glomerular tissue between the PMN group and normal group; E Detection of ubiquitination status of TRAF6, N = 6; *p < 0.05, **p < 0.01

Renal tissue electron microscopy revealed significant thickening of the glomerular basement membrane in the PMN group rats, along with a marked increase in the deposition of immune complexes compared to the normal group, with electron-dense material deposited in the capillary loops (Fig. 3B). These ultrastructural changes provide direct evidence of the pathological characteristics of PMN. Furthermore, as reported, TRAF6 facilitates the formation of TRAF2-TAK1-TAB or TRAF6-TAK1-TAB complexes, mediating the K63-linked polyubiquitination of TAK1, leading to the activation of TAK1 and subsequently influencing cell apoptosis [29]. Therefore, Western blot and qRT-PCR analyses of TRAF6, TAK1, and TAK1 ubiquitin levels in kidney tissues revealed significantly higher expression of TRAF6 and TAK1, as well as TAK1 ubiquitin levels in the PMN group rats compared to the normal group. Further analysis of apoptosis-related protein expression in kidney tissues of the PMN group rats showed a significant upregulation of Cleaved Caspase-1 and GSDMD-N, consistent with the observed cellular apoptosis by electron microscopy (Fig. 3C-E). Additionally, we collected kidney tissue samples from PMN patients and healthy controls for immunohistochemistry analysis. The results demonstrated higher expression levels of TRAF6 and TAK1, as well as increased TAK1 ubiquitination in PMN patients. The expression of Cleaved Caspase-1 and GSDMD-N was also significantly upregulated (Figure S2).

These results suggest that TRAF6 may play a crucial role in the development of PMN by activating TAK1 through an ubiquitin-regulated mechanism and promoting its expression.

TRAF6 Interaction with TAK1 Promotes Pyroptosis

To validate the interaction between TRAF6 and TAK1 in promoting podocyte apoptosis, we successfully cultured human kidney podocytes and obtained differentiated, mature podocytes. Immunofluorescence staining of podocalyxin (PODXL) in podocytes confirmed their successful differentiation by comparing the staining with undifferentiated podocytes (Fig. 4A).

Fig. 4.

The Interaction between TRAF6 and TAK1 Promotes Pyroptosis and Its Role in the Progression of PMN. Note: A Immunofluorescence detection of PODXL protein expression levels in human podocytes before and after induced differentiation to assess podocyte differentiation status. Scale bar = 25 μm. B Co-immunoprecipitation confirmed the interaction between TRAF6 and TAK1 in podocytes. C Western blot analysis detected the ubiquitination status of TAK1 in oe-TRAF6 and oe-NC groups in podocytes. D qRT-PCR examined the expression levels of TRAF6, TAK1, Caspase-1, and GSDMD in podocytes across different groups. E Western blot analysis compared the expression of TRAF6, TAK1, Cleaved Caspase-1, and GSDMD-N in podocytes among various groups. F CCK-8 assay assessed the viability of podocytes at 48 h in different groups. G ELISA measured the levels of IL-6 and TNF-β in the podocyte culture medium of the various groups. H Representative images of Hoechst 33,342 and PI double fluorescent staining of treated podocytes showing PI-positive cells percentage. Arrows indicate PI-positive cells, scale bar = 25 μm. I H2DCFDA probe staining was used to detect ROS levels, scale bar = 25 μm. All cellular experiments were conducted in triplicate, *p < 0.05, **p < 0.01

Next, to simulate the damaged podocyte environment in PMN in vitro, we induced podocyte injury by supplementing C3a, creating the PMN group. Initially, to confirm the potential formation of a complex between TRAF6 and TAK1 leading to the activation of TAK1 ubiquitination, we conducted co-immunoprecipitation experiments on cells in the PMN group, demonstrating the formation of a complex between TRAF6 and TAK1 (Fig. 4B). Subsequently, Western blot analysis revealed an elevation in the levels of TAK1 ubiquitination in the oe-TRAF6 group compared to the oe-NC group, suggesting that TRAF6 may potentially enhance TAK1 ubiquitination, thus impacting cell pyroptosis (Fig. 4C).

Additionally, we used qPCR to detect the expression of TRAF6, TAK1, Caspase-1, and GSDMD in podocytes across different groups. The results showed that the expression levels of these molecules were significantly elevated in the PMN group compared to the Control group. After inhibiting TRAF6, the expression levels of TAK1, Caspase-1, and GSDMD were significantly reduced. Upon further overexpression of TAK1, the expression levels of Caspase-1 and GSDMD increased significantly. Treatment with the Caspase-1 inhibitor VX765 led to decreased expression of Caspase-1 and GSDMD, suggesting that TRAF6 promotes podocyte pyroptosis through its interaction with TAK1 (Fig. 4D). Furthermore, we detected the protein expression of TRAF6, TAK1, Cleaved Caspase-1, and GSDMD-N in podocytes using Western blot, and the results were consistent with the qRT-PCR findings. These results indicate that TRAF6 may interact with TAK1 to promote podocyte pyroptosis (Fig. 4E).

The CCK-8 assay revealed a significant decrease in podocyte viability in the PMN group compared to the Control group, while the PMN + sh-TRAF6 + oe-NC group showed a marked increase in viability. Subsequent oe-TAK1 treatment reduced viability, and VX765 administration reversed this effect, significantly increasing viability (Fig. 4F). ELISA results indicated elevated IL-6 and TGF-β1 levels in the PMN group, with reductions observed in the PMN + sh-TRAF6 + oe-NC group. After oe-TAK1 treatment, these cytokine levels increased again, but VX765 significantly reduced them (Fig. 4G).

To assess podocyte apoptosis, Hoechst 33342 and PI staining showed a significant increase in PI-positive cells and ROS levels in the PMN group, while the PMN + sh-TRAF6 + oe-NC group showed a marked reduction. Subsequent oe-TAK1 treatment increased PI-positive cells and ROS levels, but VX765 administration significantly reduced both in the PMN + sh-TRAF6 + oe-TAK1 + VX765 group compared to the PMN + sh-TRAF6 + oe-TAK1 group (Fig. 4H-I). These findings reveal that TRAF6 and TAK1 exacerbate foot cell damage and death by promoting oxidative stress, while the intervention with VX765 effectively mitigates this damaging process, offering a potential new strategy for the treatment of PMN.

TRAF6 Stabilizes TAK1 Through Ubiquitination to Promote PMN Progression

To investigate whether TRAF6 promotes the progression of PMN in a rat model by upregulating TAK1 expression, we utilized specific lentiviral vectors to knock down and overexpress TRAF6 and TAK1 genes in the SD rat model of membranous nephropathy established on the basis of CBSA. Biochemical analysis revealed a significant increase in serum TP and ALB levels and a decrease in T-CHOL, TG, Scr, and BUN levels, along with a notable reduction in urinary protein excretion in the PMN + sh-TRAF6 + oe-NC group compared to the control group. Conversely, the sh-TRAF6 + oe-TAK1 group exhibited opposite outcomes. Furthermore, compared to the sh-TRAF6 + oe-TAK1 group, the PMN + sh-TRAF6 + oe-TAK1 + VX765 group demonstrated a significant increase in serum TP and ALB levels and a decrease in T-CHOL, TG, Scr, BUN levels, and urinary protein excretion (Fig. 5A).

Fig. 5.

The Role of TRAF6 and TAK1 in the Membranous Nephropathy Model in SD Rats and Mechanistic Investigation via the Pyroptosis Pathway. Note: A Biochemical analysis of TP, ALB, T-CHOL, BUN, SCr, and 24 h-UPro levels in each group of treated rats, n = 6. B ELISA detection of IL-6 and TNF-β levels in the serum of each group of rats; n = 6. C qRT-PCR analysis of TRAF6, TAK1, Caspase-1, and GSDMD mRNA expression in the glomerular tissue of the rat groups, n = 6. D Western blot analysis of TRAF6, TAK1, Caspase-1, and GSDMD protein expression in the glomerular tissue of the treated rat groups, n = 6. E Transmission electron microscopy evaluation of the glomerular tissue in rats from each group, assessing the deposition of immune complexes. Scale bar = 2 μm, n = 6, *p < 0.05, **p < 0.01

Using ELISA, Western blot, and qRT-PCR techniques, we measured the levels of cytokines IL-6 and TGF-β1, as well as the expression levels of TRAF6, TAK1, and pyroptosis-related proteins. The results showed that, compared to the PMN + sh-NC + oe-NC group, the PMN + sh-TRAF6 + oe-NC group exhibited significantly reduced serum levels of IL-6 and TGF-β1. After overexpressing TAK1 (PMN + sh-TRAF6 + oe-TAK1 group), the expression levels of IL-6, TGF-β1, and pyroptosis-related molecules were significantly increased. Following treatment with the Caspase-1 inhibitor VX765, these levels decreased again (Fig. 5B-D). Electron microscopy of kidney tissues revealed that, compared to the PMN + sh-NC + oe-NC group, the PMN + sh-TRAF6 + oe-NC group showed a significant reduction in glomerular basement membrane thickness and a decrease in immune complex deposition, whereas the sh-TRAF6 + oe-TAK1 group showed the opposite pattern. Additionally, compared to the sh-TRAF6 + oe-TAK1 group, the sh-TRAF6 + oe-TAK1 + VX765 group exhibited a significant reduction in glomerular basement membrane thickness and a decrease in immune complex deposition (Fig. 5E).

These results further confirm the roles of TRAF6 and TAK1 in promoting the progression of membranous nephropathy, particularly through their involvement in inducing pyroptosis via the GSDMD/Caspase-1 axis.

Discussion

This study primarily delves into the molecular mechanism by which TRAF6 activates the TAK1 ubiquitin pathway to induce pyroptosis via the GSDMD/Caspase-1 axis, thereby promoting the progression of PMN. Through a series of bioinformatics analyses, in vitro cell experiments, and animal model studies, we identified the crucial role of the TRAF6/TAK1 signaling axis in the advancement of PMN. This discovery not only unveils new functions of TRAF6 in PMN but also elucidates the role of TAK1 ubiquitin in pyroptosis. Additionally, experimental validation showcased the interaction between TRAF6 and TAK1, leading to the activation of the GSDMD/Caspase-1 axis and subsequent promotion of pyroptosis. These findings offer a fresh perspective for further investigating the pathological mechanisms of PMN and potential therapeutic targets.

In the progression of PMN, pyroptosis plays a crucial role. Through pyroptosis, damaged kidney cells release large amounts of pro-inflammatory cytokines, such as IL-1β and IL-18, which not only exacerbate local inflammation but may also trigger systemic immune responses. Additionally, the release of intracellular contents due to pyroptosis can act as autoantigens, activating the immune system and promoting antibody deposition on the glomerular basement membrane, further aggravating kidney damage. Prolonged inflammation and immune activation may also drive renal fibrosis, leading to further deterioration of kidney function and clinical symptoms such as proteinuria [30]. Furthermore, studies have reported that activation of the MAPK pathway can affect intracellular iron levels and lipid peroxidation, thereby promoting pyroptosis [31]. Our results further demonstrate that pyroptosis plays a key role in regulating cell survival and death in PMN and may represent an important therapeutic target in the progression of PMN.

In addition, existing studies have shown that CASP3 is a key executioner enzyme in programmed cell death and belongs to the caspase family. It plays a central role in the execution phase of apoptosis, responsible for cleaving various substrate proteins, leading to the breakdown of cellular structures and the final execution of cell death [32]. IL-1β, a member of the IL-1 family, has a crucial impact on immune regulation, fever responses, and the development of various autoimmune and inflammatory diseases [33]. TRAF6 can activate multiple downstream signaling pathways, including the NF-κB and MAPK pathways. Studies suggest that TRAF6 may also directly activate MAPK to regulate the development, function, and homeostasis of the immune system, which are vital for cell survival, proliferation, and inflammatory responses [34]. Furthermore, TRAF6 functions as an E3 ubiquitin ligase, participating in various signal transduction pathways that play important roles in inflammation and immune responses.

TRAF6 also plays a significant role in various kidney diseases. For example, in diabetic nephropathy and acute kidney injury, TRAF6 influences disease progression by regulating inflammatory responses and apoptosis [35, 36]. However, this study is the first to systematically explore the mechanism of TRAF6 in PMN. We found that TRAF6 is significantly upregulated in PMN patients and is a key gene regulating pyroptosis during the progression of PMN, with its activated TAK1 ubiquitination levels markedly increased. This finding is distinct from previous research, highlighting that TRAF6 not only plays a role in inflammation regulation but also influences podocyte pyroptosis by activating TAK1 ubiquitination, thereby promoting PMN progression. This novel mechanism provides new evidence regarding the multifunctionality of TRAF6 in kidney diseases.

TAK1 plays a critical role in cellular stress and inflammatory responses, with its activation predominantly relying on ubiquitin modification [16, 37]. Previous research has primarily focused on the role of TAK1 in inflammation and cancer, with limited studies on its specific mechanisms in cell pyroptosis [38–40]. Through a series of in vitro and in vivo experiments, this study unveils for the first time the specific mechanism of TAK1 ubiquitin in pyroptosis. We discovered that TRAF6 activates TAK1 ubiquitin, thereby activating the GSDMD/Caspase-1 axis to induce pyroptosis. This discovery broadens the scope of TAK1 functionality, revealing its new role in the progression of PMN and providing a reference for exploring the role of TAK1 in other diseases in the future.

GSDMD and Caspase-1 are key molecules in cell pyroptosis, with existing studies highlighting their crucial roles in various inflammatory diseases [41]. This study further confirms the specific mechanism of the GSDMD/Caspase-1 axis in pyroptosis. We found that TRAF6 activates TAK1 ubiquitin, leading to the activation of the GSDMD/Caspase-1 axis, inducing pyroptosis and thus promoting the progression of PMN. This discovery not only elucidates the role of GSDMD/Caspase-1 in PMN but also provides new evidence for understanding the widespread impact of cell pyroptosis in kidney diseases. Moreover, through an in-depth exploration of the GSDMD/Caspase-1 axis, we lay the theoretical groundwork for the future development of treatment strategies targeting this pathway.

The primary innovation of this study lies in systematically unraveling the role of the TRAF6/TAK1 axis in the progression of PMN. In comparison to previous research, we not only demonstrated the upregulation of TRAF6 and TAK1 in PMN but also proposed for the first time that TRAF6 activates TAK1 through ubiquitin to subsequently activate the GSDMD/Caspase-1 axis, leading to pyroptosis. This novel mechanism not only enriches our understanding of the pathogenesis of PMN but also provides a new direction for the development of novel therapeutic strategies. While previous studies have indicated the roles of TRAF6 and TAK1 in other renal diseases, this study systematically validates their specific mechanisms in PMN, further underscoring the potential of the TRAF6/TAK1 axis as a therapeutic target.

Multiple research methods were employed in this study, including bioinformatics analysis, in vitro cell experiments, and animal model studies, effectively elucidating the role of the TRAF6/TAK1 axis in PMN. Through analysis of the gene expression omnibus database, we identified TRAF6 as a key factor in the progression of PMN, which was subsequently validated through in vitro cell experiments and in vivo animal studies. The reliability of the experimental results was confirmed through multiple validations, including immunoprecipitation, ELISA, Western blot, and qRT-PCR techniques. These methods and experimental designs not only enhance the credibility of the research findings but also provide robust technical support for further investigating the role of the TRAF6/TAK1 axis in other diseases.

Despite the significant findings of this study, several limitations remain. First, the limited sample size may affect the generalizability of the results, and future studies should expand the sample size to validate the universality of these conclusions. Second, the control of experimental conditions and potential experimental errors require further optimization. Moreover, although this study revealed the mechanism of the TRAF6/TAK1 axis in PMN, further research is needed to explore its specific role in other kidney diseases. Additionally, this study primarily relies on in vitro cell models and animal experimental models, lacking direct validation with clinical samples. Although the role of the TRAF6/TAK1 axis in podocyte pyroptosis and the progression of PMN is supported by experimental data, whether it exhibits similar biological effects in clinical settings remains unconfirmed. Future research should incorporate clinical samples and use large-scale patient data and gene testing to further verify the role of this signaling pathway in PMN and assess its consistency and variation across different clinical subtypes.

Conclusion

This study identifies a novel mechanism in which TRAF6 promotes TAK1 ubiquitination, activating the GSDMD/Caspase-1 axis to induce pyroptosis, thereby advancing PMN progression (Fig. 6). Using transcriptomic analysis, machine learning, and in vitro and in vivo models, we confirmed that upregulated TRAF6 and TAK1 play pivotal roles in enhancing pyroptosis and inflammation in PMN. Additionally, the pyroptosis inhibitor VX765 mitigates TAK1-induced podocyte damage, suggesting a potential therapeutic approach for PMN. These findings lay the groundwork for targeting TRAF6 and TAK1 in future PMN treatments.

Fig. 6.

TRAF6 Promotes the TAK1-Mediated Ubiquitination to Accelerate the Progression of PMN through the Induction of Pyroptosis

Supplementary Information

Below is the link to the electronic supplementary material.

Author Contributions

Yaling Guo and Jingliang Min conceptualized and designed the study. Baochao Chang, Zheng Chen, and Weidong Chen conducted the experiments and analyzed the data. Yaling Guo drafted the manuscript with critical revisions from all authors. Jingliang Min and Yaling Guo supervised the study. All authors read and approved the final manuscript.

Funding

This study was supported by Natural Science Key Program of Bengbu Medical University (2023byzd056) and Anhui Province Scientific Research Programme Preparation Project (2024AH051269).

Data Availability

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethical Approval

All animal experiments were approved by the Animal Ethics Committee of the First Affiliated Hospital of Bengbu Medical University (No. [2024] No. 234).

Consent for Publication

Not applicable.

Competing Interests

The authors declare no competing interests.