ABSTRACT: Podophyllotoxin (PPT) exhibits limited clinical utility due to its nephrotoxicity, and its underlying mechanisms remain poorly understood. This study employs the toxicological evidence chain (TEC) framework and integrated multi-omics analyses to investigate the potential involvement of the microbiota-gut-kidney (MGK) axis in PPT- induced nephrotoxicity in SD rats. Toxicity was systematically evaluated through longitudinal monitoring of body weight, general behavior, biochemical markers, intestinal barrier function, and histopathological alterations. In parallel, multi-omics analyses, encompassing microbiome, metabolomics, and transcriptomics, were conducted to delineate the mechanistic underpinnings. The results showed that PPT exposure induced pronounced renal and intestinal damage, manifesting as significant weight loss, diarrhea, elevated renal injury biomarkers, increased lipopolysaccharide (LPS) levels, and diamine oxidase (DAO), along with histopathological lesions and enhanced apoptosis in renal and colonic tissues. PPT exposure perturbed gut microbiota homeostasis, characterized by depletion of beneficial taxa (e.g., Lactobacillus) and enrichment of potentially pathogenic genera (e.g., Bacteroides and Aggregatibacter), concomitant with diminished short-chain fatty acid (SCFA) production and altered metabolite profiles in fecal, serum, and renal samples. Integrated multi- omics analysis further revealed activation of the JAK1/2-STAT3 signaling pathway, upregulation of pro-inflammatory mediators (TNF-α, IL-6, IL-1β, LPS, and TMAO), and suppression of anti-inflammatory cytokines (IL-10 and IL-4). These in vivo molecular and inflammatory patterns were partially reproduced in HK-2 cells co-cultured with fecal microbiota supernatant from PPT-treated rats. In addition, the JAK1/2 inhibitor ruxolitinib attenuated PPT-induced JAK1/2-STAT3 phosphorylation and inflammatory cytokine secretion in HK-2 cells. Correlation network analysis further identified associations between gut dysbiosis, systemic inflammation, and metabolic perturbations. Collectively, these findings support a mechanistic hypothesis that MGK- axis disruption and JAK1/2-STAT3 signaling may contribute to PPT-associated nephrotoxicity. However, in vivo interventional studies are required to establish definitive causality
