The Role Of Extracellular ATP-P2X7R Signaling In Renal Ischemic-reperfusion Injury

Aims: We try to explore 1.the role of extracellular ATP in the pathological process of renal ischemia/reperfusion(I/R)injury;2.the mechanism of extracellular ATP induced renal inflammation,and the role of P2X7 R in this process;3.whether e ATP/P2X7 R signaling is involved in renal I/R injury through NLRP3 inflammasome.Methods: In vivo experiments: Male C57BL/6 mice and P2X7R(-/-)mice were randomly divided into Sham group and I/R group.Wildtype mice were injected or not with Apyrase or A438079 at 30 min after surgery.AKI model was established by unilateral nephrectomy combined with the lateral renal pedicle clamping for 35 min.The mice were sacrificed at 24 h after surgery,and the blood,urine,and kidneys were collected for analysis.In vitro experiments: Human renal tubular epithelial cells(HK2 cells)were subjected to 24 h hypoxia or not followed by 8h reoxygenation,with or without the treatment of Apyrase.HK2 cells were exposed to ATP 4m M for 8h in ATP associated experiments.P2X7 R si RNA or NLRP3 si RNA was transfected to test the role of P2X7 R and NLRP3 on the inflammatory injury induced by H/R or ATP.Serum creatinine(Scr)and blood urea nitrogen(BUN)were determined using biochemical methods.PAS was performed to assess the pathological damage of kidneys.The levels of ATP in urine and cell supernatant were detected by chemiluminescence assay.Immunohistochemical staining and immunofluorescence was performed to detect the location and expression of ATP5 a,ATP5b,IL-18,cleaved caspase-1,F4/80,IL-1 beta,CD68,P2X7 R,and NLRP3 proteins in kidneys.Real-time PCR and immunoblotting were used to detect the gene and protein expression of ATP5 a,ATP5b,P2X7 R,NLRP3 inflammasome,and IL-1 beta in kidneys,respectively.Results: In vivo experiments: The levels of urine ATP in I/R mice increased compared with those of Sham group.However,no difference was found in ATP synthase expression between two groups.I/R induced renal dysfunction and structural damage,increased the inflammatory factors and inflammatory cell infiltration number.These were ameliorated by Apyrase treatment.Compared with Sham mice,the expression of NLRP3 and cleaved caspase-1 in I/R mice increased,which are ameliorated by Apyrase or A438079 treatment.Moreover,P2X7 R gene knockout also ameliorated renal dysfunction and structure impairment induced by renal I/R.Compared with wild type I/R mice,the inflammatory factors and inflammatory cell infiltration number in P2X7 R gene knockout I/R mice decreased.In vitro experiments: H/R induced the release of ATP,increased the levels of IL-1 beta m RNA and IL-1 beta protein maturation.These were reduced by Apyrase treatment,or by transfection with P2X7 R si RNA or NLRP3 si RNA.Moreover,the expression of NLRP3,ASC,and cleaved caspase-1 proteins induced by H/R was ameliorated by transfection with P2X7 R si RNA.Conclusion: Reducing the level of extracellular ATP or P2X7 R gene knockout protects renal tubular epithelial cells from ischemic-reperfusion injury by targeting NLRP3 inflammasome.