Immune-mediated kidney diseases, particularly membranous nephropathy (MN), have emerged as a major global health challenge [1, 2]. These conditions not only impact patients’ quality of life but can also lead to end-stage renal disease and renal failure. The main pathological features of MN include deposition of immune complexes in the glomerular basement membrane, with 70% to 80% of cases exhibiting autoantibodies against the phospholipase A2-receptor (PLA2R) [2, 3]. Immunosuppressive therapies that are most often employed clinically have demonstrated partial effectiveness in delaying the disease; however, they are frequently not sufficient to prevent renal damage and deterioration of renal function. The etiology of MN remains unclear and may be associated with environmental factors [4], genetic predisposition [5], or autoimmunity [6, 7].

The gut microbiota is a complex community of microorganisms living in the human gut, consistings of beneficial bacteria, neutral bacteria, and harmful bacteria. The gut microbiota is a multifaceted microecosystem that forms a symbiotic relationship with the host, establishing a complex ecological network that supports homeostasis [8, 9]. It is becoming increasingly evident that the gut microbiota is closely linked to the pathogenesis of numerous diseases, including inflammatory conditions, obesity [10], diabetes [11], and Parkinson’s disease [12]. Research has confirmed that the gut microbiota coordinates systemic immune and metabolic homeostasis by promoting chronic inflammatory responses, regulating microbial metabolic activity, and altering the intestinal microenvironment [13, 14, 15, 16].

A clinical study found that patients with MN often exhibit alterations in the structure of their gut microbiota [17]. Additionally, specific changes in the gut microbiota composition of patients with MN may promote the formation of anti-PLA2R antibodies through immune mechanisms such as epitope spreading or self-antigen modification [18]. Nevertheless, current research mainly uses 16S ribosomal RNA (rRNA) sequencing technology to investigate gut microbiota composition, which primarily provides taxonomic data but lacks the ability to analyze community function and detailed metabolic activities. Li et al. [19] employed 16S rRNA sequencing to compare gut microbiome differences between MN and other chronic kidney disease (CKD) subtypes. However, its taxonomic resolution is largely limited to the genus/family level, and functional profiling mainly relies on indirect inference rather than direct gene- and pathway-level annotation, thereby restricting the detection of species/strain signatures and the systematic characterization of MN-related functional pathway shifts.

By contrast, shotgun metagenomic sequencing enables species- and strain-level profiling and provides a gene-resolved view of microbial functional capacity through the direct annotation of microbial genes, pathways, and metabolic modules [20]. This higher-resolution framework facilitates the identification of MN-associated taxa together with their functional repertoires (e.g., pathways related to amino acid metabolism, energy metabolism, and pathogen-associated functions) and supports integrative analyses linking microbial features to clinical phenotypes. Moreover, metagenomics can capture clinically relevant genetic elements such as virulence factors and antibiotic resistance genes that are inaccessible to 16S rRNA gene sequencing approaches [21], thereby offering a more mechanistically informative characterization of MN-associated gut microbiome alterations beyond prior 16S rRNA sequencing-based studies.

This research utilized metagenomic technology to analyze the gut microbiome in patients with MN to better understand its functional role and possible mechanisms in disease progression. These findings may provide potential insights for the diagnosis and treatment of MN and support the development of therapeutic strategies targeting the microbiome.

Gut microbiota diversity and abundance are crucial for sustaining normal physiological function [16, 22]. This study employed metagenomic sequencing to comprehensively characterize the gut microbial community of patients with MN. Our results revealed substantial differences in microbial diversity, taxonomic composition, and functional potential across patient groups and normal controls.

The composition and functions of the gut microbiota may change due to diet and geographic variations, as well as disease progression [23, 24]. To minimize the impact of dietary and lifestyle differences on the study results, we enrolled normal controls who lived in the same area with similar lifestyles. Our results indicated that fecal microbial richness of patients with MN was significantly reduced in comparison to normal controls, which was consistent with earlier studies [19, 25]. Taxonomic analyses also revealed that patients with MN had a distinct form of microbiota dysbiosis in the gut, characterized by an expansion in possible pathobionts and a reduction in health-related commensals. Specifically, the relative abundances of Proteobacteria (e.g., E. coli, K. pneumoniae), C. perfringens, Enterococcus spp., and M. funiformis were increased. E. coli and K. pneumoniae are intestinal opportunistic pathogens and their overproliferation is typically associated with intestinal barrier dysregulation, lipopolysaccharide (LPS)-induced hypersecretion, and systemic immune enhancement [26, 27]. Previous studies based on 16S rRNA sequencing have reported enrichment of Escherichia in patients with MN, with further linking these taxa to disease stage or immunopathological features [19]. However, increased intestinal levels of E. coli have also been reported in other kidney diseases, including IgA nephropathy [28] and diabetic nephropathy [29]. These findings suggest that the expansion of Escherichia may represent a shared feature associated with renal dysfunction or chronic inflammatory states rather than a microbiota alteration unique to MN. C. perfringens secretes a variety of toxins that disrupt intestinal epithelial integrity, leading to intestinal inflammation and metabolic imbalances [30]. Enterococcus spp. are highly drug-resistant pathogens capable of producing extracellular proteases and bile salt hydrolases, thereby enhancing intestinal permeability and inducing immune activation [31], which may exacerbate systemic inflammation and renal injury.

Moreover, the increase of pathogenic and pro-inflammatory bacteria species in patients with MN was accompanied by considerable changes in the microbial metabolic pathways. KEGG annotation-based functional prediction indicated that bacterial genes related to redox metabolism, tryptophan metabolism, and pathogenicity-associated mechanisms were highly upregulated in the MN group. Enhanced NADH:quinone oxidoreductase activity and cytochrome c oxidase pathways suggest increased bacterial oxidative phosphorylation and redox imbalance, which could result in excessive generation of ROS and facilitate the development of mucosal oxidative stress. Such oxidative stress reportedly disrupts the intestinal tight junction, exacerbating epithelial integrity and bacterial translocation [32].

Notably, the enrichment of the tryptophan metabolism pathway in patients with MN may represent a change in microbial catabolism toward the generation of immunomodulatory metabolites, including indole derivatives, kynurenine, and indoxyl sulfate. These substances may act as ligands to aryl hydrocarbon receptor (AhR) and other immune signaling receptors, affecting mucosal immune homeostasis and systemic inflammation [33]. Miao et al. [34] reported that gut microbiota–derived tryptophan metabolites are closely linked to intrarenal activation of the AhR in MN, with alterations in tryptophan metabolite profiles accompanying enhanced AhR signaling and increased renal immune activation. Potential mechanisms suggest that disrupted tryptophan metabolism may interfere with immune cell differentiation (particularly affecting the balance between pro-inflammatory Th17 cells and regulatory T cells [Tregs]) and modulate B-cell activation [35]. These immunological alterations may be linked to pathogenic autoantibody responses against podocyte antigens, including PLA2R. Furthermore, indoxyl sulfate is produced by microbial tryptophan metabolism and reportedly correlates with oxidative stress, endothelial dysfunction, and renal fibrosis, all of which contribute to the progression of CKD [36, 37].

By contrast, the gut microbiota of normal controls showed functional features that supported host metabolic and redox homeostasis. Pathway enrichment mainly involved amino acid and cofactor metabolic processes, such as homoprotocatechuate degradation, arginine succinyltransferase activity, pyrimidine breakdown, and the biosynthesis of phylloquinone (vitamin K₁) and heme. These metabolic pathways are essential in amino acid catabolism, antioxidant synthesis, and cofactor reuse, and have thus been suggested to maintain energy supply and redox balance in intestinal epithelial cells, thereby promoting mucosal repair and barrier function [33, 34]. Moreover, the normal group showed increased enrichment of symbiotic bacteria, such as Faecalibacterium prausnitzii and Roseburia spp., which synthesize short‑chain fatty acids (SCFAs), particularly butyrate. Butyrate activates epithelial cell metabolism, increases tight junction protein expression, and induces Treg differentiation, thereby producing anti-inflammatory and barrier-protective effects [38, 39, 40]. These butyrate-producing bacteria were significantly decreased in MN patients, potentially impairing the energy provision and immune tolerance of the intestinal mucosa, aggravating inflammatory reactions and disrupting the immune balance.

This study found that certain gut microbial genera are associated with renal function indicators in patients with MN. Fusicatenibacter showed a negative correlation with PLA2R, suggesting that this butyrate-producing bacterium may exert immunomodulatory effects on membranous nephropathy or mitigate PLA2R-mediated autoimmune-related immune activation. Conversely, Bacillus_A had a significant positive correlation with CR and PCR, suggesting that its enrichment may reflect impaired renal filtration or increased proteinuria. Some Bacillus species can produce endotoxins or pro-inflammatory metabolites [41, 42], which may exacerbate renal injury and inflammatory responses.

Our findings indicate that a combination of four specific fecal bacterial genera (Dialister, Enterococcus_C, Fusicatenibacter, and Clostridium_P) can accurately distinguish patients with MN from healthy controls. However, future studies are crucial for confirming their reliability as diagnostic and prognostic biomarkers.

Together, our findings indicate that gut dysbiosis in patients with MN manifests not only as alterations in compositional structure but also involves functional reprogramming, leading to a shift in their metabolic state toward pro-inflammatory, oxidative stress, and uremic conditions. These altered microbial profiles may be associated with increased intestinal permeability and systemic inflammation and may relate to renal injury through the gut–kidney axis. The sample size in this study was relatively small (10 MN patients and 10 controls), highlighting the need for further validation. Future research should integrate metaproteomics, metabolomics, immunological profiling and should employ multicenter, large-sample, longitudinal studies with independent validation cohorts to elucidate the causal relationships among microbial metabolites, immune responses, and renal pathology in MN.

This study had several limitations. First, the sample size was small (10 MN patients and 10 controls), which may have limited statistical power, potentially leading to false-positive or false-negative results and affecting the robustness and reproducibility of the findings. Second, the cross-sectional observational design precludes causal inference, and the results should be interpreted as associations only. Third, although appropriate statistical methods were applied, differences in several clinical parameters between groups, including BMI, blood pressure, CR, and BUN, may represent potential confounding factors. Renal function impairment and altered metabolic status are known to influence gut microbiota composition and microbial metabolic activity, and therefore may have partially contributed to the observed microbiota alterations. Future research should include multicenter, large-sample, longitudinal studies with independent validation cohorts to confirm the reproducibility of the identified taxa, functional pathways, and the proposed microbial signature model.

In this exploratory study, we observed distinct gut microbial profiles in patients with MN compared with healthy controls. These findings suggest a potential association between MN and gut microbiome dysbiosis and provide preliminary evidence for candidate taxa that may be involved in MN-related host–microbe interactions. Given the limited sample size, these associations warrant further validation in larger, well-characterized cohorts. Collectively, our results provide a foundation for future studies to confirm these microbial signatures and to further investigate the mechanistic relationships between gut microbiota alterations and MN.

All data generated or analyzed during this study are included in this published article. Raw data are available from the corresponding author upon reasonable request.

YL: Writing—original draft, Investigation, Data curation. HG: Writing—original draft, Investigation, Data curation. ZL: Investigation. ZC: Data analysis. ZS: Writing—review& editing, Data analysis. WX: Writing—review& editing, Data analysis. YD: Conceptualization. WL: Supervision, Resources, Conceptualization. SL: Supervision, Resources, Conceptualization. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

This research was approved by the ethics committee of Shenzhen Longhua District Central Hospital (Approval No. 2025-108-01) and was carried out in accordance with the principles of the Declaration of Helsinki (1975). Informed consent was obtained from all participants.

We acknowledge the use of BioRender for creating the graphical abstract and confirm that appropriate copyright permissions have been obtained.

This work was supported by grants from Shenzhen Foundation of Science and Technology (Grant/Award Number: JCYJ20240813153002004, JCYJ20250604191024032); Guangdong Basic and Applied Basic Research Foundation (Grant/Award Number: 2025A1515012512); Shenzhen Longhua District Healthcare Institutions Scientific Research Project (Grant/Award Number: 2022041); Key Medical Discipline Construction Fund of Shenzhen Longhua District; Guangdong Yiyang Healthcare Charity Foundation (Grant/Award Number: JZ2025107).

The authors declare no conflict of interest.