In recent years, growing attention has been directed toward the GM as a pivotal modulator of host immune regulation and metabolic homeostasis, with substantial evidence implicating its involvement in the occurrence, progression, prevention, and therapeutic management of KSD [9]. Earlier MR-based studies have revealed important causal links between specific gut microbial taxa (particularly Oxalobacter and Haemophilus) and susceptibility to KSD [22]. Such findings have provided critical mechanistic insights into KSD pathophysiology and offered a basis for interventions focused on microbiota modulation or immune regulation. Nonetheless, the possibility that immune cells mediate the causal interactions between GM and KSD remains unexplored. Therefore, the current study comprehensively evaluated the causal relationships among GM, immune cell characteristics, and KSD risk, identifying potential immune-mediated pathways through which particular microbial taxa influence kidney stone formation. Our results highlight Prevotella, Phascolarctobacterium, and Ruminococcaceae as protective genera, while Clostridiales and Bacteroides were associated with increased KSD risk. In addition, we identified multiple immune cell subsets significantly linked to KSD, particularly regulatory T cells (Tregs) and monocyte subtypes. Through mediation analysis, we propose the hypothesis that the protective effect of Prevotella on KSD was partially mediated by CD28 on CD39 + CD4 + T cells, providing novel causal evidence for the “gut–immune–kidney” axis.

Recent studies have emphasized the crucial role of metabolic processes in the pathogenesis of KSD. The GM maintains a complex and interactive relationship with host physiology, actively contributing to multiple metabolic pathways, especially nutrient absorption and utilization. Increasing evidence indicates that GM dysbiosis significantly contributes to kidney stone development [23]. Certain gut bacterial taxa, including Oxalobacter formigenes, Enterobacter, Dorea, and multiple Lactobacillus species, possess oxalate-degrading capabilities and have consistently shown associations with calcium oxalate (CaOx) stone formation [24,25,26,27]. Besides CaOx stones, alterations in GM composition have been linked to other stone types. For example, fecal Bacteroides abundance in patients suffering from uric acid stones has a positive correlation with serum uric acid concentration [28], and patients with stones exhibit significantly elevated Bacteroides levels relative to healthy controls [29, 30]. The MR analyses conducted in the present study yielded robust and previously unreported evidence indicating that increased abundance of Bacteroides is causally linked to elevated KSD susceptibility. Although earlier literature reported decreased Subdoligranulum levels within the gut microbiota of uric acid nephrolithiasis patients [31], the positive correlation between Subdoligranulum abundance and KSD risk identified here aligns closely with findings from another recent MR-based study [22]. Such an inconsistency highlights the necessity of further research, either through larger microbiome sequencing cohorts or mechanistic experiments, to precisely define Subdoligranulum’s function in the pathogenesis of KSD. In addition, the observation from our analysis regarding Phascolarctobacterium abundance negatively correlating with KSD risk further supports conclusions drawn from prior study [32].

In the present study, we identified Ruminococcaceae and Clostridiales act as protective and risk factors for KSD, respectively, opposing the conclusions of an earlier MR study [33]. The discrepancy likely stems from population-specific variations in GM composition derived from distinct GWAS datasets, influenced by geographic, genetic, and lifestyle factors that differentially modulate KSD pathogenesis. Notably, emerging evidence from a fecal microbiota transplantation (FMT) study demonstrates that restoration of Ruminococcaceae_UCG-014 and its metabolic functions significantly reduces hyperoxaluria-induced urinary oxalate excretion and renal CaOx deposition [34]. This study further provides mechanistic support for our observation of Ruminococcaceae’s protective role in KSD. Future studies should combine strain-level MR analyses, multi-ethnic cohorts, and gnotobiotic model validation, complemented by metabolomic profiling and microbial genetic fine-mapping to fully elucidate host-microbiome-disease interaction mechanisms in KSD.

Beyond its well-established role in regulating host metabolism, the GM also contributes to the pathogenesis of KSD through immune modulation within the local microenvironment. Excessive inflammation during stone formation may induce urinary tract damage and alter urine composition [5]. Indeed, nearly all major chronic inflammatory biomarkers exhibit elevated levels in KSD patients [35]. Renal crystal deposition, particularly CaOx, frequently triggers localized immune responses in kidney tissues [36], reinforcing the role of immunological pathways in kidney stone development. Unlike prior research predominantly centered around oxalate metabolism or isolated metabolic processes, the current study underscores immune cells as potential mediators within the GM–KSD causal pathway. Our MR analyses indicate that Prevotella influences KSD susceptibility partly through the mediation of CD28 expression on CD39 + CD4 + T cells, representing a mediation effect of approximately − 9.019%. This finding suggests a negative feedback mechanism in the “gut–immune–kidney” axis: increased Prevotella abundance may trigger immune responses that diminish its protective effects against KSD, while reduced Prevotella levels may attenuate pro-inflammatory immune activity. Although this immune-mediated pathway accounts for a relatively small portion of the overall causal relationship, immune cells are key regulators in complex biological networks [37], pointing to the involvement of additional mechanisms such as metabolic disturbances and oxidative stress [38].

Prevotella, a core GM (approximately 4.81% relative abundance), plays essential physiological roles in dietary fiber fermentation to generate butyrate [39] and oxalate metabolism regulation [40]. Current research demonstrates significant associations between Prevotella abundance variations and multiple pathological conditions, including urolithiasis [29], metabolic syndromes [41], autoimmune diseases [42], and neuropsychiatric disorders [43], with its colonization dynamics being substantially influenced by geographical distribution and nutritional patterns. Butyrate, a short-chain fatty acid, is a primary energy source for colonocytes and strengthens intestinal mucosal integrity [44]. It also modulates immune responses through mechanisms such as activating PPARα [45] and inhibiting NF-κB activation [46], ultimately suppressing inflammatory cytokine production. CD28 on CD39 + CD4 + T cells are a regulatory T cell (Treg) subset with immunosuppressive potential. CD28 provides co-stimulatory signals crucial for T cell activation [47], while CD39 hydrolyzes extracellular ATP to adenosine, which has anti-inflammatory effects. In tumor microenvironments, CD39 + CD4 + T cells have been implicated in immune suppression and tumor progression [48]. During renal inflammation, CD4 + T cells are recruited to sites of inflammation, enhancing macrophage activity and dendritic cell antigen presentation through IFN-γ and TNF-α secretion [49], thereby establishing a pro-inflammatory environment conducive to stone formation.

These results suggest that Prevotella-associated immune responses may involve complex biological mechanisms, and that modulating specific gut microbes or immune cell subpopulations could offer novel therapeutic strategies for KSD. Moreover, our study identified various monocyte subtypes, including CD14 + CD16 + monocytes, that are associated with KSD risk. Some of these subtypes appear to have bidirectional effects, implying that inflammation in stone formation is not exclusively promotive—certain immune cells may exert protective effects at specific stages of disease progression. These findings merit further investigation through experimental validation.

Recent advances from GWAS have considerably deepened our understanding of KSD’s genetic underpinnings. However, most previous GWAS research has not distinguished among kidney stone subtypes such as CaOx, cystine, or uric acid stones. A prominent trans-ancestry GWAS conducted by Hao et al. identified 59 loci linked to kidney stone susceptibility (including 13 novel loci), demonstrating conserved genetic mechanisms across European and East Asian populations involving pathways of calcium metabolism (e.g., RGS14), vitamin D regulation (e.g., CASR), and urate transport (SLC17A3) [50]. While candidate genes such as AGXT (oxalate metabolism) and SLC3A1/SLC7A9 (cystine transport) suggest subtype-specific genetic associations [51]. Future studies incorporating detailed stone composition analyses and functional validation are warranted to elucidate precise genotype-phenotype correlations and facilitate personalized prevention strategies.

From a methodological viewpoint, this investigation utilized a two- stage MR framework integrated with IVW estimation, heterogeneity assessment, pleiotropy detection, and leave-one-out sensitivity analysis to enhance robustness in causal inference. Additionally, mediation MR approaches quantified the proportion of GM’s impact on KSD risk attributable to immune cell mediation, offering enhanced depth and interpretability relative to traditional MR analyses. Nevertheless, several limitations must be acknowledged. Primarily, all datasets were exclusively derived from cohorts of European ancestry, potentially constraining generalizability and introducing ethnic bias into the findings. Second, although the immune-mediated effects were statistically significant, their absolute magnitudes were relatively modest, suggesting the presence of additional biological pathways—possibly metabolic or oxidative—that warrant further investigation. Future research should incorporate longitudinal, multi-ethnic cohorts, multi-omics approaches such as metagenomics and single-cell immunomics, and mechanistic studies using animal models and in vitro systems to comprehensively elucidate the “gut–immune–kidney” axis in KSD. Moreover, integrated interventions involving targeted probiotics and immune-modulating therapies should be explored in high-risk populations.