Xi Luo, Yang Zhao, Yunpeng Luo, Jian Lai, Jiemei Ji, Jiao Huang, Yuanyuan Chen, Ziru Liu, Jingchen Liu
{"title":"细胞膜mtDNA-cGAS-STING轴通过激活NLRP3炎症小体导致脓毒症诱发的急性肾损伤。","authors":"Xi Luo, Yang Zhao, Yunpeng Luo, Jian Lai, Jiemei Ji, Jiao Huang, Yuanyuan Chen, Ziru Liu, Jingchen Liu","doi":"10.1007/s10157-023-02448-5","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>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.</p><p><strong>Methods: </strong>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.</p><p><strong>Results: </strong>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.</p><p><strong>Conclusion: </strong>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.</p>","PeriodicalId":10349,"journal":{"name":"Clinical and Experimental Nephrology","volume":null,"pages":null},"PeriodicalIF":2.2000,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Cytosolic mtDNA-cGAS-STING axis contributes to sepsis-induced acute kidney injury via activating the NLRP3 inflammasome.\",\"authors\":\"Xi Luo, Yang Zhao, Yunpeng Luo, Jian Lai, Jiemei Ji, Jiao Huang, Yuanyuan Chen, Ziru Liu, Jingchen Liu\",\"doi\":\"10.1007/s10157-023-02448-5\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Background: </strong>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.</p><p><strong>Methods: </strong>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.</p><p><strong>Results: </strong>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.</p><p><strong>Conclusion: </strong>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.</p>\",\"PeriodicalId\":10349,\"journal\":{\"name\":\"Clinical and Experimental Nephrology\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":2.2000,\"publicationDate\":\"2024-05-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Clinical and Experimental Nephrology\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.1007/s10157-023-02448-5\",\"RegionNum\":4,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2024/1/19 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"Q2\",\"JCRName\":\"UROLOGY & NEPHROLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Clinical and Experimental Nephrology","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1007/s10157-023-02448-5","RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/1/19 0:00:00","PubModel":"Epub","JCR":"Q2","JCRName":"UROLOGY & NEPHROLOGY","Score":null,"Total":0}
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.
期刊介绍:
Clinical and Experimental Nephrology is a peer-reviewed monthly journal, officially published by the Japanese Society of Nephrology (JSN) to provide an international forum for the discussion of research and issues relating to the study of nephrology. Out of respect for the founders of the JSN, the title of this journal uses the term “nephrology,” a word created and brought into use with the establishment of the JSN (Japanese Journal of Nephrology, Vol. 2, No. 1, 1960). The journal publishes articles on all aspects of nephrology, including basic, experimental, and clinical research, so as to share the latest research findings and ideas not only with members of the JSN, but with all researchers who wish to contribute to a better understanding of recent advances in nephrology. The journal is unique in that it introduces to an international readership original reports from Japan and also the clinical standards discussed and agreed by JSN.