Nephropathy 1 Formula alleviates kidney injury by ameliorating mitochondrial dysfunction and pyroptosis in diabetic nephropathy through the TMAO-mROS-NLRP3 axis

Nephropathy 1 Formula (N1F), a traditional Chinese medicine, has demonstrated promising clinical efficacy in diabetic nephropathy (DN). However, its underlying protective mechanisms remain insufficiently defined. In this study, we employed a type 2 diabetes mellitus (T2DM) mouse model induced by a high-fat diet (HFD) and streptozotocin (STZ). We simulated DN in vitro using high glucose (HG) and trimethylamine-N-oxide (TMAO) exposure in mouse glomerular mesangial cells (MES-13). To elucidate the mechanistic basis of N1F’s renoprotective effects, we integrated metabolomics, transcriptomics, and 16S rRNA sequencing. N1F treatment improved urinary albumin-to-creatinine ratio (UACR), preserved renal function, and attenuated histopathological damage and renal fibrosis in diabetic mice. Mechanistically, N1F modulated TMAO and energy metabolism, altered gut microbiota composition, and suppressed microbial production of TMAO-related metabolites. Under hyperglycemic conditions, TMAO induced excessive mitochondrial reactive oxygen species (mROS), impaired mitochondrial dynamics, and disrupted energy metabolism. In contrast, N1F normalized mROS levels, restored mitochondrial structure and function, enhanced oxidative phosphorylation (OXPHOS), increased ATP production, and decreased glycolytic dependency. Moreover, N1F suppressed expression of pyroptosis-related proteins, including NLRP3, N-GSDMD, cleaved-Casp1, IL1β, and IL18, in both in vivo and in vitro models, indicating that it mitigates pyroptosis by inhibiting the TMAO-mROS-NLRP3 signaling axis. Collectively, these findings reveal that N1F exerts protective effects against DN by targeting mitochondrial dysfunction and pyroptotic injury, supporting its potential as a therapeutic strategy for DN.