N-acetyltransferase 10 exacerbates renal tubular epithelial cell pyroptosis in diabetic kidney disease via N4-acetylcytidine modification of NLRP3 mRNA

Diabetic kidney disease (DKD) remains a leading cause of end-stage renal disease worldwide. Renal tubular injury, particularly pyroptosis and subsequent inflammation, is now recognized as a critical driver of DKD progression. Although the NLRP3 inflammasome is a well-established mediator of pyroptosis, the upstream regulatory mechanisms that control its aberrant expression in tubular epithelial cells (TECs) under diabetic conditions are poorly understood. The role of epitranscriptomic regulation, specifically the RNA modification N4-acetylcytidine (ac4C) and its sole writer enzyme N-acetyltransferase 10 (NAT10), in DKD pathogenesis is entirely unknown.

We employed a multi-faceted approach including analysis of human renal transcriptomic datasets (Nephroseq), scRNA-seq data from DKD patients, and two distinct murine DKD models (HFD/STZ-induced and db/db mice). In vitro studies utilized TECs under high glucose (HG) stimulation. Loss-of-function studies were conducted via proximal tubule-specific NAT10 knockout mice, CRISPR/Cas9-mediated gene editing in vitro, and pharmacological inhibition with Remodelin. The molecular mechanism was delineated using RNA sequencing, ac4C RNA immunoprecipitation (acRIP-qPCR), chromatin immunoprecipitation (ChIP), luciferase reporter assays, RNA stability assays, and polysome profiling.

We first identified a significant upregulation of NAT10 expression and global ac4C modification in TECs from DKD patients and experimental models. This induction was transcriptionally driven by the hyperglycemia-responsive factor EGR-1, which directly binds to the NAT10 promoter. Functionally, NAT10 overexpression potentiated HG-induced NLRP3 inflammasome assembly, Caspase-1 activation, Gasdermin-D cleavage, and IL-1β/IL-18 maturation, thereby promoting pyroptosis. Conversely, genetic ablation or pharmacological inhibition of NAT10 robustly suppressed this inflammatory cascade. Mechanistically, we identified NLRP3 mRNA as a key downstream target. NAT10 directly binds to NLRP3 mRNA and installs ac4C modifications at a conserved site, which enhances NLRP3 mRNA stability and promotes its translation efficiency, leading to amplified NLRP3 protein synthesis. In vivo, proximal tubule-specific NAT10 knockout or Remodelin treatment in diabetic mice significantly attenuated renal dysfunction, tubular injury, macrophage infiltration, and NLRP3-driven inflammasome activation, without altering systemic metabolism. Most importantly, in clinical DKD specimens, the intrarenal levels of NAT10 were positively correlated with NLRP3 expression and negatively correlated with eGFR.

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Our study delineates a novel and complete pathogenic axis in DKD: hyperglycemia induces EGR-1, which transcriptionally upregulates NAT10, leading to ac4C-dependent post-transcriptional enhancement of NLRP3 expression, which ultimately triggers TEC pyroptosis and renal inflammation. These findings not only unveil the NAT10/ac4C/NLRP3 axis as a fundamental epitranscriptomic mechanism in DKD but also firmly establish NAT10 as a central regulator of tubular injury and a highly promising therapeutic target for intervention.