Neutrophil Extracellular Traps Are Triggered By C3 And Contribute To Acute Kidney Injury

Objective: Ischemia reperfusion(IRI)injury is a common cause of acute kidney injury(AKI),and characterized by sharp inflammatory responses,including neutrophil infiltration and complement activation.Infiltration neutrophils can be stimulated to formed neutrophil extracellular trap(NETs),which may lead to renal injury.Complement C3 play a prominent role in inflammatory processes,its activation exacerbates acute kidney injury.But the relationship between the formation NETs and activation C3 in ischemia-reperfusion injury-induced AKI was not clear.However,the relationship between NETs and complement C3 has not been elucidated.Methods: C57BL/6 wild type mice(WT)were subjected to renal ischemia-reperfusion injury-induced AKI model by clamping both renal pedicle for 45 minutes.Mice were sacrificed after 6,12,24,48 hours.To deplete neutrophil,mice were treated with intraperitoneal injection of anti-Ly6 G Ig G(1A8)or control Ig G 24 and 2 hours before bilateral IR injury.To test the effect of C3,C3 KO mice were subjected to renal IR injury.The renal function were measured by serum creatinine(Scr)and blood urea nitrogen(BUN).The level of renal neutrophils were evaluated by myeloperoxidase(MPO),lymphocyte antigen 6G(LY6G)and intercellular adhesion molecule-1(ICAM-1)immunohistochemistry.NETs formation was defined by the colocalization of diffused DAPI nuclear materials,Ly6 G and citullinated histone-H3(Cit H3)signal by immunofluorescence and protein level of Cit H3 by western blot.The expression of C3 in renal were estimated by immunofluorescence,real-time fluorescence quantitative and ELISA.In vitro,neutrophil were isolated from normal individuals and were assessed by Wright-Giemsa stain.Neutrophils in RPMI were simulated with 25 n M phorbol myristate acetate(PMA,positive control),RPMI(negative control),0.01 μM,0.1 μM and 1 μM C3 a.Results: In vivo,renal IR injury produced a significant increase in serum BUN and Scr level.The expression of neutrophil,NETs and C3 of each temporal point after IR increased obviously,the peak point of neutrophil and NETs was at the 24 hours.At 24 hours,postischemic kidneys represent positivity for DAPI,Cit H3 and Ly6 G colocalizing of NETs in the outer medulla.Injection of 1A8 suppressed the increase in serum BUN and Scr 24 hours after renal IR injury,with a concomitant reduction of neutrophils infiltration and NETs formation,while there were no significant differences in the expression of C3 in mice with and without neutrophil depletion.Compared with WT-sham group,C3 KO can ameliorate the accumulation of neutrophil and protect renal against IR injury.Kidney sections from C3 KO mice contained less NETs formation with WT mice,corroborating the Cit H3 western blot.In vitro,neutrophils were assessed to be >90% pure.After 4 hours of incubation,0.1 μM C3 a simulated the formation of NETs whi ch reflected by amorphous extracellular DNA structures that colocalized with Cit H3 and MPO.Positive control PMA altered neutrophil phenotype with decondensed chromatin,while C3 a remained intact neutrophil phenotype with lobulated nuclei.Our data reveal that C3 may be a critical determinant for releasing NETs.C3 KO may be crucial for minimizing NETs related I/R injury.Conclusion: C3 activation can stimulate neutrophil motivation and lead to the formation of NETs in renal ischemia-reperfusion injury.Targeting C3 activation may be a new therapeutic strategy to ameliorate the necroinflammation of NETs.