Endocrine Abstracts

JOINT597

Synopsis: Diabetic kidney disease (DKD) is a major burden and a predictor of mortality in diabetes, characterized by clinical heterogeneity. Hyperglycemia enhances glycolysis-driven pyruvate-to-lactate conversion in the proximal tubules. Lysine lactylation (Kla), a novel post-translational modification induced by intracellular lactate, may contribute to this heterogeneity. Here, we used lactylome analysis and experiments to explore its impact on biological processes with redirection of glycolytic flux.

Purpose: DKD is associated with altered metabolic patterns. Enhanced glycolysis and pyruvate-to-lactate conversion are common early adaptive features in the proximal tubules. Here we investigate the metabolic consequences and present a global lactylome profiling of lactate dehydrogenase(LDH) inhibition to characterize the landscape of lactylation in the human renal proximal epithelial tubular cell line(HK-2) under hyperglycemic conditions.

Methods: HK-2 cells were treated with 25 mM high D-glucose(HG) for 7 days, followed by 40 mM oxamate, the LDH inhibitor, for 24 hours. This induced metabolic reprogramming without affecting cell growth¹. Global lactylome analysis using 4D label-free proteomics identified differentially lactylated proteins. Co-immunoprecipitation(Co-IP)and site-directed mutagenesis were used to verify modification sites. Respiratory measurements using Seahorse assay, alongside fluorescent probes, western blot, qPCR and cross-species sequence comparisons were performed.

Results: Integrative lactylome and proteome analysis identified 1, 149 Kla sites and 83 differentially expressed (DE) modified sites. Notely, three glycolytic enzymes contained DE sites: ATP-Dependent 6-Phosphofructokinase, Platelet Type (PFKP), Alpha-enolase (ENO1) and Aldolase (ALDOA). PFKP was the second most down-regulated DE site with P-value = 4. 86×10^-5and log2FC = -0. 66, and only one lactylated lysine residue in PFKP (K688) was identified (modified sequence: NFGTK(1)ISAR). Subsequently, the lactylation modification of PFKP was verified by Co-IP, while neither those of ENO1 nor ALDOA. The modification site of PFKP (K688) was further confirmed by site-directed mutagenesis. The modification level of PFKP (K688) was downregulated by oxamate. During this process, mitochondrial membrane potential significantly increased, while the oxygen consumption rate revealed a decrease in ATP-linked respiration, a reduction in maximum respiration, and an increase in proton leak. Cross-species sequence comparison reveals both the conservation and diversity of the PFKP gene across various organisms, with the K688 site being highly conserved throughout evolution.

Conclusion: PFKP lactylation revealed a feedback loop among glycolysis, lactate, and lactylation in hyperglycemic proximal tubular cells. The high conservation of the PFKP K688 site, along with its dynamic modifications, coupled with glycolytic reprogramming and their impact on mitochondrial respiration in diabetic proximal tubules, may provide valuable insights into the study of heterogeneity in DKD.