[The mechanism of β-hydroxybutyrylation in G6PDX regulated by β-hydroxybutyric acid on alleviating podocyte injury induced by high glucose].

<b>Objective:</b> To explore the mechanism of β-hydroxybutyric acid (β-HB) mediated β-hydroxybutyrylation in glucose-6-phosphate dehydrogenation (G6PDX) on reducing the damage of podocyte induced by high glucose. <b>Methods:</b> Mouse podocytes (MPC-5) were stimulated with high glucose to establish a podocyte injury model of diabetic nephropathy. The cells were divided into three groups: the control group, the high glucose group (stimulated with 30 mmol/L glucose for 48 hours), and the high glucose+β-HB group (stimulated with 30 mmol/L glucose plus 20 mmol/L β-HB for 48 hours). The content of β-HB, cell viability, and the expressions of podocin, Wilms tumor protein 1 (WT-1), and synaptopodin in MPC-5 cells of each group were measured. Liquid chromatography-tandem mass spectrometry was employed to screen for potential protein sites undergoing β-hydroxybutyrylation, and the expression of G6PDX was detected. A mutant vector was transfected to mutate the lysine at position 432 of G6PDX to arginine. The cells were then divided into three groups: the high glucose group (stimulated with 30 mmol/L glucose for 48 hours), the high glucose+β-HB group (stimulated with high glucose and 20 mmol/L β-HB for 48 hours), and the high glucose+β-HB+site mutation group (subjected to high glucose, β-HB, and transfection of a plasmid for lysine site mutation). The cell viability and G6PDX expression in each subgroup were determined. <b>Results:</b> Compared with the control group, the cell viability in the high glucose group was significantly decreased (34.8%±2.8% vs 100.0%±8.5%, <i>P</i><0.001). In contrast, the cell viability in the high glucose+β-HB group was significantly higher than that in the high glucose group (72.7%±1.9% vs 34.8%±2.8%, <i>P</i><0.001). Moreover, β-HB could ameliorate podocyte injury induced by high glucose, as evidenced by the increased expressions of podocin (0.63±0.02 vs 0.39±0.03, <i>P</i><0.001), WT-1 (0.75±0.04 vs 0.50±0.03, <i>P</i><0.001), and synaptopodin (0.74±0.02 vs 0.56±0.02, <i>P</i><0.001). Liquid chromatography-tandem mass spectrometry analysis revealed an increased β-hydroxybutyrylation modification of the lysine at position 432 of G6PDX after β-HB treatment (fold change>1.5). The results of Western blotting (0.68±0.05 vs 0.38±0.01, <i>P</i><0.001), PCR (0.70±0.10 vs 0.15±0.03, <i>P</i><0.001), and immunofluorescence all indicated an upregulation of G6PDX expression. After the site mutation, the restoration of cell viability under β-HB treatment was restricted (171.7%±7.1% vs 259.7%±14.6%, <i>P<</i>0.001). Additionally, the results of Western blotting (1.43±0.04 vs 2.22±0.09, <i>P</i><0.001) and PCR (2.33±0.16 vs 3.60±0.34, <i>P</i><0.001) showed a decrease in G6PDX expression compared with that with simple β-HB treatment. <b>Conclusion:</b> β-HB mediated β-hydroxybutyrylation of G6PDX can alleviate podocyte injury induced by high glucose.