Cytosolic mtDNA–cGAS–STING axis contributes to sepsis-induced acute kidney injury via activating the NLRP3 inflammasome.

Background: NLRP3 inflammasome activation is significantly associated with sepsis-induced acute kidney injury (S-AKI). Cytosolic DNA derived from damaged mitochondria has been reported to activate NLRP3 inflammasome via upregulating the cyclic GMP–AMP synthase (cGAS)—the stimulator of interferon genes (STING) axis in nucleus pulposus cell and cardiomyocytes. However, the regulatory effect of mitochondria DNA (mtDNA)–cGAS–STING axis on the NLRP3 inflammasome in S-AKI remains unclear. Methods: In the current study, we established an in vivo model of S-AKI by intraperitoneally injecting male C57BL/6 J mice with lipopolysaccharide (LPS). Next, selective cGAS inhibitor RU.521, and STING agonist DMXAA were intraperitoneally injected in the mice; then, blood urea nitrogen (BUN), serum creatinine (CRE), urinary kidney injury molecular-1 (KIM-1), pathological changes, and infiltrated neutrophils were detected to assess kidney injury. We also performed western blot and immunofluorescence assays to evaluate STING, cGAS, TBK-1, p-TBK-1, IRF3, p-IRF3, NF-kB, p-NF-kB, NLRP3, cleaved caspase-1, caspase-1, GSDMD-N, and GSDMD expression levels in kidney tissues. IL-18 and IL-1β in renal tissue were identified by ELISA. In vitro, we treated HK-2 cells with LPS to establish a cell model of S-AKI. Furthermore, ethidium bromide (EtBr) was administered to deplete mitochondria DNA (mtDNA). LPS-induced cytotoxicity was evaluated by LDH release assay. Protein expression of cGAS, STING, and NLRP3 in was quantified by western blot. Cytosolic mtDNA was detected by immunofluorescence and q-PCR. Released IL-1β and IL-18 in HK-2 supernatants were detected by ELISA. Results: LPS injection induced S-AKI in mice, as evidenced by neutrophil infiltration, tubular vacuolation, and increased levels of serum creatinine (CRE), blood urea nitrogen (BUN), and urinary KIM-1. In addition, LPS activated the cGAS–STING axis and NLRP3 inflammasome in vivo, illustrated by increased phosphorylation levels of TBK-1, IRF3, and NF-kB protein, increased ratio of cleaved caspase-1 to caspase-1 and GSDMD-N to GSDMD, and increased IL-1β and IL-18 levels. Moreover, the cGAS inhibitor RU.521 effectively attenuated NLRP3 inflammasome and S-AKI; however, these effects were abolished by treatment with the STING agonist DMXAA. Furthermore, cytosolic release of mtDNA and activation of the cGAS–STING–NLRP3 axis were observed in LPS-treated HK-2 cells. Inhibiting mtDNA replication by Ethidium Bromide (EtBr) treatment reduced cytosolic mtDNA accumulation and downregulated the cGAS–STING–NLRP3 axis, ameliorating the cytotoxicity induced by LPS. Conclusion: This study demonstrated that the cGAS–STING axis was triggered by cytosolic mtDNA and participated in the development of S-AKI by activating NLRP3 inflammasome. Reducing cytosolic mtDNA accumulation or inhibiting the cGAS–STING axis may be potential therapeutic targets for S-AKI. [ABSTRACT FROM AUTHOR]