AARS1-Mediated Lactylation of Akt/p65 Drives Their Activation and Glycolytic Reprogramming in Diabetic Kidney Disease

Background: Lysine lactylation, a post-translational modification, is critical in kidney disease pathophysiology. In diabetic kidney, the ability to metabolize lactate is impaired, leading to lactate accumulation. Alanyl-tRNA synthetase 1 (AARS1), a newly identified lactylation writer, can sense lactate and transfer it to lysine residues in histones and nonhistone substrates to modulate gene expression and protein function. However, the role of AARS1 in DKD remains unknown. Methods: To investigate the role of AARS1 in DKD, we generated AARS1fl/fl:Ksp-Cre mice and induced diabetes with streptozotocin (STZ). We test the effect of an AARS1 inhibitor, β-alanine, in DKD mouse model. We identify novel AARS1 substrates and target genes by co-immunoprecipitation and CUT&Tag analysis in CRISPR/Cas9 mediated AARS1 knockout HK-2 cells. Results: We found that the expression of AARS1 was increased in high glucose stimulated HK-2 cells and DKD kidneys. Genetic deletion and pharmacological inhibition of AARS1 with β-alanine ameliorated renal injury as seen by decreased blood urea nitrogen (BUN) levels, urinary albumin excretion, interstitial fibrosis examined with PAS and Masson's trichrome staining, and macrophage infiltration examined by F4/80 staining, reflecting a reduction of renal inflammation. We identified that Akt and the p65 subunit of NF-kB are novel non-histone substrates of AARS1, which lactylates Akt at K14 and p65 at K310 determined by site-directed mutagenesis, leading to increase their phosphorylation. The activated Akt further promoted AARS1 mediated p65 lactylation and phosphorylation. Our ChIP assay indicated that p65 binds to the promoter of AARS1 to regulate its transcription, supporting a positive feedback loop between AARS1 and NF-kB to reinforce AARS1 transcription and lactylation activity. With CUT&Tag analysis, we identified novel AARS1 target genes in HK-2 cells, including two glycolysis genes, HK2 and PFKP. The binding of AARS1 on the promoters of HK2 and PFKP was confirmed by ChIP assay and their expression was decreased upon AARS1 knockout or pharmacological inhibition. Conclusion: AARS1 regulates DKD progression through the lactylation of its novel substrates, Akt and NF-kB, and its novel target genes, such as HK2 and PFKP, to promote glycolytic reprogramming. Targeting AARS1 is a promising therapeutic strategy for DKD treatment. Funding: NIDDK Support