Diabetic nephropathy is considered one of the most common microvascular complications of diabetes and the pathophysiology involves multiple factors. Progressive diabetic nephropathy is believed to be related to the structure and function of the tubular epithelial cells in the kidney. However, the role of lysine acetylation in lesions of the renal tubular epithelial cells arising from hyperglycemia is poorly understood. Consequently, in this study, we cultured mouse renal tubular epithelial cells in vitro under high glucose conditions and analyzed the acetylation levels of proteins by liquid chromatography-high-resolution mass spectrometry. We identified 48 upregulated proteins and downregulated 86 proteins. In addition, we identified 113 sites with higher acetylation levels and 374 sites with lower acetylation levels. Subcellular localization analysis showed that the majority of the acetylated proteins were located in the mitochondria (43.17%), nucleus (28.57%) and cytoplasm (16.19%). Enrichment analysis indicated that these acetylated proteins are primarily associated with oxidative phosphorylation, the citrate cycle (TCA cycle), metabolic pathways and carbon metabolism. In addition, we used the MCODE plug-in and the cytoHubba plug-in in Cytoscape software to analyze the PPI network and displayed the first four most compact MOCDEs and the top 10 hub genes from the differentially expressed proteins between global and acetylated proteomes. Finally, we extracted 37 conserved motifs from 4915 acetylated peptides. Collectively, this comprehensive analysis of the proteome reveals novel insights into the role of lysine acetylation in tubular epithelial cells and may make a valuable contribution towards the identification of the pathological mechanisms of diabetic nephropathy.

Diabetic nephropathy (DN) is the main cause of end-stage renal disease (ESRD), which is characterized by a series of abnormal changes such as glomerulosclerosis, podocyte loss, renal tubular atrophy and excessive deposition of extracellular matrix. Simultaneously, the occurrence of inflammatory reaction can promote the aggravation of DN-induced kidney injury. The most important processes in the canonical inflammasome pathway are inflammasome activation and membrane pore formation mediated by gasdermin family. Converging studies shows that pyroptosis can occur in renal intrinsic cells and participate in the development of DN, and its activation mechanism involves a variety of signaling pathways. Meanwhile, the activation of the NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome can not only lead to the occurrence of inflammatory response, but also induce pyroptosis. In addition, a number of drugs targeting pyroptosis-associated proteins have been shown to have potential for treating DN. Consequently, the pathogenesis of pyroptosis and several possible activation pathways of NLRP3 inflammasome were reviewed, and the potential drugs used to treat pyroptosis in DN were summarized in this review. Although relevant studies are still not thorough and comprehensive, these findings still have certain reference value for the understanding, treatment and prognosis of DN.

New Findings What is the central question of this study?Activation of the glycogen synthase kinase 3 β (GSK‐3β)–hypoxia‐inducible factor 1 α (HIF‐1α) pathway results in stimulation of pyroptosis under high glucose, and exerts actions in a number renal diseases: does this pathway have a role in renal tubular epithelial cells? What is the main finding and its importance?Down‐regulation of GSK‐3β can inhibit pyroptosis of renal tubular epithelial cells induced by high glucose and this may be related to down‐regulation of HIF‐1α. This role of the GSK‐3β–HIF‐1α pathway has not previously been reported and identifies a potential new therapeutic target in diabetic nephropathy. Abstract Diabetic nephropathy (DN) is not only one of the main complications of diabetes, but also has a high incidence rate and a high mortality rate. Glycogen synthase kinase 3 β (GSK‐3β) and hypoxia‐inducible factor 1 α (HIF‐1α) have been demonstrated to influence DN by regulating pyroptosis. This study aimed to investigate the effect of the GSK‐3β–HIF‐1α pathway on pyroptosis of high‐glucose (HG)‐induced renal tubular cells. Mouse renal proximal tubular epithelial cells (TKPT cells) were induced by HG to simulate DN cell and we transfected TKPT cells with GSK‐3β knockdown lentivirus. Western blot analysis confirmed the transfection effects and detected the expression of GSK‐3β, HIF‐1α, Nod‐like receptor protein 3 (NLRP3), cleaved‐caspase‐1, pro‐caspase‐1, gasdermin D (GSDMD) and GSDMD‐N. The expression of GSDMD‐N and HIF‐1α were also verified by immunofluorescence. The levels of interleukin (IL)‐1β and IL‐18 were measured by enzyme linked immunosorbent assay. Flow cytometric analysis determined the apoptosis rate. Results showed that HIF‐1α expression was increased in HG‐induced TKPT cells, and GSK‐3β knockdown could decrease the levels of NLRP3, cleaved‐caspase‐1, GSDMD‐N and HIF‐1α, verified by immunofluorescence. Moreover, GSK‐3β knockdown suppressed the expression of IL‐1β and IL‐18, and reduced the apoptosis rate. Lithium chloride (LiCl) interference could cause the same changes as GSK‐3β knockdown for HG‐induced TKPT cells, and dimethyloxallyl glycine could reverse the effect of GSK‐3β‐knockdown interference. Our studies definitively demonstrate that the GSK‐3β–HIF‐1α signalling pathway mediates HG‐stimulated pyroptosis in renal tubular epithelial cells and that down‐regulation of GSK‐3β inhibited HG‐induced pyroptosis by inhibiting the expression of HIF‐1α. These findings suggest a new potential target for the treatment of DN.