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

Abstract

Nephropathy 1 Formula (N1F), a traditional Chinese medicine (TCM), has demonstrated promising clinical efficacy in diabetic nephropathy (DN). However, its underlying protective mechanisms remain insufficiently defined. In this study, a type 2 diabetes mellitus (T2DM) mouse model was established using a high-fat diet (HFD) and streptozotocin (STZ). Additionally, DN was simulated in vitro via exposure of mouse glomerular mesangial cells (MES-13) to high glucose (HG) and trimethylamine-N-oxide (TMAO). To elucidate the mechanistic basis of N1F’s renoprotective effects, an integrative approach combining metabolomics, transcriptomics, and 16S ribosomal ribonucleic acid (rRNA) gene sequencing was employed. N1F treatment reduced the urinary albumin-to-creatinine ratio (UACR), preserved renal function, and attenuated histopathological damage and renal fibrosis in diabetic mice. Mechanistically, N1F modulated systemic TMAO levels 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 cellular energy metabolism. In contrast, N1F normalized mROS levels, restored mitochondrial structure and function, enhanced oxidative phosphorylation (OXPHOS), increased ATP production, and reduced glycolytic dependency. Furthermore, N1F downregulated the expression of key pyroptosis-related proteins—including NOD-like receptor family pyrin domain-containing 3 (NLRP3), N-terminal gasdermin D (GSDMD), cleaved-Casp1, interleukin-1β (IL-1β), and IL-18—in both in vivo and in vitro models, indicating suppression of pyroptosis via inhibition of the TMAO-mROS-NLRP3 signaling axis. Collectively, these findings demonstrate that N1F exerts protective effects against DN by targeting mitochondrial dysfunction and pyroptotic injury, supporting its potential as a therapeutic strategy for DN.

Keywords

Diabetic nephropathy / Trimethylamine-N-oxide (TMAO) / Mitochondrial dysfunction / Pyroptosis

Funding

This work was supported by the Science and Technology Department Research and Development Project of Zhejiang Province, China (No. 2022C03160) and the Traditional Chinese Medicine of Zhejiang Provincial Science and Technology Program Project (No. GZY-ZJ-KJ-24037).

Declaration of competing interest

The authors declare that they have no known competingfin-ancial interests or personal relationships that could haveap-peared to influence the work reported in this paper.

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