1. Academic Validation
  2. Cytosolic mtDNA-cGAS-STING axis contributes to sepsis-induced acute kidney injury via activating the NLRP3 inflammasome

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

  • Clin Exp Nephrol. 2024 Jan 19. doi: 10.1007/s10157-023-02448-5.
Xi Luo # 1 Yang Zhao # 1 2 Yunpeng Luo # 1 3 Jian Lai 1 Jiemei Ji 1 Jiao Huang 1 Yuanyuan Chen 1 Ziru Liu 1 Jingchen Liu 4
Affiliations

Affiliations

  • 1 Department of Anesthesiology, The First Affiliated Hospital of Guangxi Medical University, 6 Shuangyong Road, Nanning, 530021, Guangxi, China.
  • 2 Department of Anesthesiology, Affiliated Hospital of North Sichuan Medical College, No.1 Maoyuan South Road, Nanchong, 637000, Sichuan, China.
  • 3 Department of Anesthesiology, Guizhou Provincial People's Hospital, Guiyang, 550002, Guizhou, China.
  • 4 Department of Anesthesiology, The First Affiliated Hospital of Guangxi Medical University, 6 Shuangyong Road, Nanning, 530021, Guangxi, China. jingchenliunn@163.com.
  • # Contributed equally.
Abstract

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.

Keywords

Acute kidney injury; Mitochondrial DNA release; NLRP3 inflammasome; Sepsis; cGAS–STING.

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