Neutrophil Extracellular Trap Stimulated By HMGB1 Exacerbates Kidney Ischemia Reperfusion Injury

Background:HMGB1 released from damaged kidney cells can activate My D88-dependent MAPK pathway by acting on TLR4 receptor in renal tissue cells,especially renal tubular epithelial cells,activation of NF-ΚB produces a lot of pro-inflammatory factors,which aggravates inflammation and tissue injury.Neutrophil extracellar traps（NETs）is a hot topic in the study of the mechanism of ischemia-reperfusion injury.It has been proved that the formation of NETs aggravates renal injury in ischemia-reperfusion injury,but the specific mechanism of the formation of NETs,the upstream and downstream,is not clear.Our previous work showed that the level of NETs in the kidney after ischemia-reperfusion was higher than that in the normal group.After the intervention of recombinant DNase-1,the degree of NETs induction and renal tubular injury was alleviated obviously.Based on this,we intended to explore the specific molecular mechanism and role of HMGB1 and NETs in renal IRI and speculated that HMGB1 was the upstream inducer of NETs during renal ischemia reperfusion.We proposed the hypothesis that HMGB1 enhanced renal IRI by regulating the formation of NETs,which promoted renal tubular epithelial cell apoptosis and renal platelet aggregation.Methods:In vivo:The model of renal ischemia-reperfusion was established in rats by ligation of bilateral renal pedicle for 45 minutes then releasing.Blood and kidney were collected at a specific time point after reperfusion.To study how the content of NETs and degree of renal injury change in the short term after reperfusion,rats were divided into sham operation group,6 h group,12 h group,24 h group,48 h group and 72 h group.To study the relationship between HMGB1 and NETs in renal IRI,they were divided into control group,glycyrrhizic acid group,recombinant HMGB1 protein group,recombinant HMGB1 and Cl-amidine group.PAS staining of the pathological sections of kidney were made to evaluate the degree of kidney injury.The contents and localization of NETs in kidney were detected by immunofluorescence and western blotting.Apoptosis was detected by Tunel staining,and platelet aggregation was detected by immunohistochemistry.In vitro:HK2 cells were subjected to hypoxia under5%CO₂ and 1%O₂ conditions for 12 hours and reoxygenation under 5%CO₂ and 21%O₂for 6 hours.Neutrophil were isolated by standard density gradient centrifugation from fresh peripheral blood of healthy volunteers.NETs were detected in neutrophil cultured by conditional media of HK2 cells after hypoxia and reoxygenation.The relationship among HMGB1,TLR4 and NETs was investigated by adding HMGB1 antibody and TLR4 inhibitor into different conditional media.The neutrophils were stimulated with the conditional media of Necrotic HK2 source,cultured for 3-4 hours,then replaced with fresh RPMI medium.The bottom NETs was collected to culture HK2 cells,and cell apoptosis was detected by flow cytometry after 24 hours culture.HMGB1 antibody was added to NETs media to investigate the role of HMGB1 contained in NETs.Results:Compared with the sham-operated Group,Cit H3 was detected at 6 hours after 45 minutes of Renal Ischemia reperfusion,and further increased at 12 hours,reached the peak at 24 hours and decreased at 48 hours,72 hours down to low level（P<0.05）.Compared with the vehicle,the content of Cit H3 in the kidney of rats in Glycyrrhizin acid group decreased,in which recombinant HMGB1 group increased（P<0.05）;while Cit H3 content of recombinant HMGB1 and Cl-amidine groups was lower than recombinant HMGB1 group（P<0.05）.The pathological injury of kidney after ischemia appeared at 6 hours,aggravated at 12 hours,further worsen at24 hours,alleviated at 48 hours,and returned to near normal state at 72 hours（P<0.05）.Compared with the vehicle,it was alleviated in glycyrrhizic acid group,aggravated in recombinant HMGB1 group（P<0.05）;in which recombinant HMGB1 and Cl-amidine group slighter than recombinant HMGB1 group.The degree of platelet aggregation in kidney after ischemia compared with the vehicle,Glycyrrhizic acid group decreased,recombinant HMGB1 group aggravated;recombinant HMGB1 and Cl-amidine group slighter than recombinant HMGB1 group.Compared with the normal group,the content of Cit H3 in HK2 hypoxia-reoxygenation media-induced group increased significantly（P<0.05）,while compared with hypoxia-reoxygenation condition media group,the content of anti-HMGB1 antibody group and TLR4 inhibitor group decreased（P<0.05）.Compared with the normal media group,the apoptosis in the NETs culture group was significantly increased（P<0.05）,and the apoptosis in the anti-HMGB1 antibody group was reduced compared with the NETs group（P<0.05）.Conclusion:After Renal ischemia-reperfusion,there were NETs induced in post-ischemia tissues,and the changes of NETs content were consistent with the changes of kidney injury in time.HMGB1 released from injured tissue after reperfusion can induce NETs formation,promoting the apoptosis of Tubular epithelial cells and aggregation of platelets in the kidney,which aggravates kidney injury.TLR4 is involved in the NETs induction during renal ischemia reperfusion injury.Furthermore,HMGB1 is involved in the apoptosis of epithelial tubules as a toxic mediator in NETs.