AARS1 Mediates Lactylation of STAT3 and NF-kB Promotes Cyst Growth in ADPKD

Background: Metabolic reprogramming, including enhanced glycolysis and lactate accumulation, has been highlighted in ADPKD. Lactate is involved in the regulation of physiological and pathological processes through lactylation. Alanyl-tRNA synthetase 1 (AARS1) can sense lactate and act as lactyltransferases that lactylates lysine residues of histone and nonhistone substrates to promote cancer progression. However, the role and mechanisms of AARS1 in ADPKD remain unknown. Methods: To understand the role of AARS1, we generated Pkd1 and AARS1 double conditional knockout Pkd1fl/fl:AARS1fl/fl:Ksp-Cre mice and tested the effect of AARS1 inhibitor, β-alanine, in two ADPKD mouse models. To identify the novel AARS1 substrates and target genes involved in cystogenesis, we performed co-immunoprecipitation and CUT&Tag analysis. Results: We show that the expression of AARS1 is increased in Pkd1 mutant cells and kidneys. Knockout of AARS1 and inhibition of AARS1 delayed cyst growth as seen by decreased cystic index, kidney weight /body weight ratios, blood urea nitrogen levels, cyst lining epithelial cell proliferation, and increased cyst lining epithelial cell apoptosis in Pkd1 mutant mice (all p < 0.01). In addition, Pkd1 and AARS1 double knockout Pkd1fl/fl:AARS1fl/fl:Ksp-Cre mice lived longer, to a mean age of 24.6 days, while Pkd1 knockout mice died at a mean age of 18.2 days. We identified that STAT3 and the p65 subunit of NF-kB are novel substrates of AARS1, which lactylates STAT3 at K140 and p65 at K310 determined by site-directed mutagenesis, leading to their activation to regulate cell proliferation and survival. Our ChIP assay indicated that STAT3 and p65 bind to the promoter of AARS1 to regulate its transcription. Treatment with IL-6 or TNF-α increased the expression of AARS1 in mouse IMCD3 cells in a time-dependent manner, supporting the existence of two positive feedback loops: AARS1/IL-6/STAT3/AARS1 and AARS1/TNF-α/NF-κB/AARS1 in ADPKD kidneys. AARS1 also catalyzed the lactylation of H3K14. With CUT&Tag analysis, we identified novel AARS1 target genes, and one of those genes, ZEB1 regulates renal fibrosis in ADPKD kidneys. Conclusion: AARS1 regulates cyst growth through the lactylation of its novel substrates and its novel target genes. Targeting AARS1 is a promising therapeutic strategy for ADPKD treatment. Funding: NIDDK Support