The Role And Mechanisms Of Interleukin-35 On Acute Kidney Injury

1.Identification of Mouse Single Chain Interleukin-35 Fusion Gene and detection of Expression kineticsObjective: To identificate mouse single chain interleukin-35 fusion gene and to detect its expression kinetics in vitro and in vivo.Methods: The recombinant plasmid pc DNA3.1-IL35 was analyzed by digestion fo restriction endonuclease and was identified by gene sequencing.The pc DNA3.1-IL35 plasmid was transfected into 293 T cells and detected its expressionin vitro.The pc DNA3.1-IL35 plasmid was injected into the mice by hydrodynamic gene transfer and detected its tissue distribution and dynamic expression in vivo.Results: Gel electrophoresis showed that a specific band of about 1328 bp was amplified by RT-PCR.DNA sequencing analysis showed that the splicing order,sequence,orientation and series of EBI3,IL-12p35 and linker in mouse IL-35 fusion gene were completely correct.Pc DNA3.1-IL35 plasmid was transfected into 293 T cells and it can correctly expression in vitro by ELISA detection.The high level of IL-35 expression was detected in the liver,heart,spleen and kidney after injection of pc DNA3.1-IL35 plasmid.Conclusion: Mouse IL-35 single-stranded fusion gene and linker was completely correct.pc DNA3.1-IL35 plasmid continuously expressedin mice liver,spleen,kidney and other organs,which will be helpful for the further research on the role of IL-35 in acute renal injury.2.Protective effect and mechanisms of IL-35 on acute kidney injury in endotoxemia.Objective: To explore the role of IL-35 in acute renal injury in endotoxemia and its mechanisms.Methods: Establish a kind of endotoxemia acute kidney injury mouse model.SPF grade C57BL/6 mice were randomly assigned to two groups:the experiment group mice IL-35 was injected into the mice by hydrodynamic gene transfer with plasmid(pc DNA3.1-IL35,100?g / 2ml PBS);the control group was injected into the mice by hydrodynamic gene transfer injected with empty plasmid(pc DNA3.1,100?g / 2ml PBS).Each group of mice was given intraperitoneal injection of LPS(10mg/Kg)to establish endotoxemia acute kidney injury model 7 days after plasmid injection.Mice renal function were examined at 12 h,24h and 48 h after LPS injection.Pathological,cytokine expression and inflammatory signaling pathway were detected in renal tissues.The proportion of Th1,Th2,Th17 and Treg cells in spleen of mice were detected by flow cytometry.Results:IL-35 can improve the survival in endotoxemia acute kidney injury mice and the difference was significant(P <0.002)compared with those of control mice.Renal function and renal pathology suggests that IL-35 has a protective effect on acute kidney injury in endotoxemia.The serum creatinine and urea nitrogen in the experimental group were lower than those in the control group at each time point,the difference was significant(P <0.05).Renal pathology suggests that IL-35 pretreatment can significantly reduce inflammatory cell infiltration and renal tubular vacuolar degeneration.The expression of TNF-?,IL-6 and IL-1? of serum and renal tissue were significantly decreased in the experimental group,there was significant difference between the two groups(P <0.05),but there was no significant difference on the expression of antiinflammatory cytokine IL-10.Compared with the control group,IL-35 pretreatment could inhibit the phosphorylation and expression of renal tissue NF-?B signaling pathway P65 and ikk(P <0.05)in endotoxemia acute kidney injury.Conclusion: IL-35 pretreatment can significantly improve the survival and the degree of mice acute kidney injury in endotoxemia.The mechanism is mainly through reducing the infiltration of renal inflammatory cells and the production of proinflammatory cytokines TNF-?,IL-6,IL-1? viainhibition NF-?B signaling pathway.3.The role and mechanisms of IL-35 in renal ischemia and reperfusion injury.Objective: To explore the role of IL-35 in renal ischemia and reperfusion injury and its mechanisms.Methods: Establish renal ischemia and reperfusion injury mouse model by bilateral renal pedicle clipping.SPF grade C57BL/6 mice were randomly assigned to two groups:the experiment group mice IL-35 plasmidwas injected into the mice by hydrodynamic gene transfer with plasmid(pc DNA3.1-IL35,100?g / 2ml PBS);the control group was injected into the mice by hydrodynamic gene transfer injected with empty plasmid(pc DNA3.1,100?g /2ml PBS).Mice was performed renal ischemia and reperfusion injury model 7 days after plasmid injection.Mice renal function were examined at 24 h and 48 h after reperfusion.Pathological,cytokine expression in renal tissues was analysised and proportion of Th1,Th2,Th17 and Treg cells in spleen of mice were detected by flow cytometry.Results: IL-35 can improve the survival in renal ischemia and reperfusion and the difference was significant(P <0.001)compared with those of control mice.Renal function and renal pathology suggests that IL-35 has a protective effect.The serum creatinine and urea nitrogen in the experimental group were lower than those in the control group at each time point,the difference was significant(P <0.05).Renal pathology suggests that IL-35 pretreatment can significantly reduce renal tubular epithelial cell necrosis and infiltration of mononuclear cells and macrophages.The expression of TNF-?,IL-6 and IL-1? of renal tissue were significantly decreased in the experimental group,there was significant difference between the two groups(P <0.05).Compared with the control group,IL-35 pretreatment could reduce the apoptosis of renal tubular epithelial cells in renal ischemia and reperfusion injury.IL-35 could inhibit the secretion of IFN-? and IL-17 in CD4 + T cells of the spleen,and the difference was statistically significant(P <0.05)?Conclusion: IL-35 pretreatment can significantly improve the survival and the degree of mice renal ischemia and reperfusion injury.The mechanism is mainly through reducing the infiltration of renal inflammatory cells and the production of proinflammatory cytokines TNF-?,IL-6,IL-1? and the apoptosis of renal tubular epithelial cells.Meanwhile,IL-35 can effectively inhibit CD4 + T cells secrete IFN-? and IL-17 in the spleen,thereby reducing inflammatory cell infiltration.4.The Role of Inflammatory Cytokines in Early Allograft Injuryof kidney transplant patients.Objective: To explore the role of inflammatory cytokines in early allograft injury in patients with kidney transplantation.Methods: There were 45 patients who received renal transplantation in Department of Urology,First Affiliated Hospital of Soochow University from September 2015 to March 2017,including 31 males and 14 females,The median age was 37 years old.Patients were divided into two groups: the delayed graft function group(DGF group)and the immediate graft function group(IGF group)according to the postoperative renal function recovery and whether need for hemodialysis.The clinical characteristics,risk factors and prognosis of DGF were analyzed.The blood and urine samples were taken from patients,the content of IL-35,NGAL was detected and inflammatory cytokines was detected by flow cytometry CBA in order to analyze the correlation with DGF.Results: All patients acheieved a successful renal transplantation surgery.Among them,13(28.9%)patients developed delayed graft function(DGF group)and 32(71.1%)patients developed immediatelygraft function(IGF group).There were no differences in gender,age,BMI,dialysis method and time,induction drug and Immunosuppressive agents,HLA mismatches,and cold ischemic time.The proportion of acute rejection in DGF group was significantly higher than that in IGF group(23.1% vs 9.3%)within one month after renal transplantation.We found that cold ischemia time,donor age donor terminal serum creatinine is independent risk factors of DGF.The glomerular filtration rate of DGF group were lower than those of IGF group(P <0.05),the difference between the two groups was significant(P <0.05).The levels of serum and urine IL-35 in DGF group were significantly lower than those in IGF group(P <0.05),while the levels of proinflammatory cytokines IL-6,IL-1? and TNF-? in urine were significantly higher than IGF group(P <0.05).Conclusion: The dynamic balancebetween proinflammatory and anti-inflammatory cytokines,could be the indicators of early renal injury and allograft progression.It is expected to become and indicators acute kidney injury after renal transplantation.