Citrate synthase lactylation promotes the transition fromacute kidney injury to chronic kidney disease by activating nlrp3 inflammasome

Abstract Background and Aims Renal interstitial inflammation has been postulated to play a critical role in the acute kidney injury (AKI) to chronic kidney disease (CKD) transition. Recent studies have shown that protein lactylation caused by lactate accumulation can regulate chronic organ injury. The purpose of this study was to investigated the role and mechanism of protein lactlylation in AKI-CKD transition. Method Severe renal ischemia-reperfusion(I/R) injury (severe AKI) was constructed by bilateral renal ischemia for 35min. The lactylation enhancer rotenone and the lactylation inhibitor oxamate were used to verify the effect of protein lactylation. Lactylated proteomics was used to detect the changes of lactylated proteins in renal cortex at different time points (day 0, 3, and 7), and the lactylated proteins related to kidney injury were screened for verification. Results The pathological staining results showed that the severe AKI mice were most severely injuried at day 3, partially recovered at day 7, and developed renal interstitial fibrosis at day 28. The Serum creatinine (SCr) levels did not return to baseline at day 28 after I/R injury. The levels of lactate and protein lactylation modification detected by Elisa and western blot analysis showed the same tendency as the above pathological injury, indicating that protein lactylation might contribute to the chronic transition of severe AKI. The renal injury score was significantly increased at day 3 and the percentage of renal interstitial fibrosis was significantly increased at day 28 after treatment with protein lactylation enhancer. IL-1b and IL-18 were found to be the most significantly enhanced inflammatory cytokines in the blood of mice, and the NLRP3 inflammasome activation was significantly enhanced in renal tissue at 3 days. The in vitro results further confirmed these findings. We found that protein lactylation might activate the NLRP3 inflammasome, therefore leading to AKI-CKD transition. Meanwhile, lactylation inhibitor had a protective effect on AKI-CKD transition. Lactylated proteins were detected by lactylated proteomics analysis at day 0, 3 and 7 after I/R, and the enrichment of differential lactylated proteins was analyzed. The results showed that the differential proteins with significant difference mainly existed in the tricarboxylic acid cycle. One of them is citrate synthase (CS), a key rate-limiting enzyme in the tricarboxylic acid cycle. Citrate synthase lactylation level increased significantly on day 3 after AKI. Among the three lactylation sites of CS, the further screening revealed that CS-K370 is the most significant. We constructed mutations of CS-K370 lactylation sites to verify its effects on the enzyme activity and function. Results showed in hypoxia/reoxygenation(H/R) model for mice renal tubular epithelial cells (mRTECs), compared with control group, the enzyme activity of CS in K370T group (modified status) decreased significantly, while in K370R group (unmodified status) did not decrease significantly. Moreover, the lactylation modification of CS-K370 can lead to the activation of NLRP3 inflammasome. The above results indicated that the lactylation of CS-K370 site inhibited the enzyme activity of CS and activated NLRP3 inflammasome. Conclusion Renal protein lactylation promotes activation of NLRP3 inflammasome, leading to AKI-CKD transition. Citrate synthase may be the key lactylated protein.