The C3a/C3aR Axis Mediates NETs Releasing To Exacerbate Acute Kidney Injury

Background Acute kidney injury(AKI)is a serious clinical emergency with rapid onset,rapid progression and poor prognosis.Ischemia/reperfusion(I/R)renal injury is the main cause of AKI,and the pathophysiology of I/R is reactive oxygen species production,neutrophil infiltration and complement activation.In our previous study,we found that inhibition of complement C3 reduced the infiltration of neutrophils,formation of neutrophil extracellular traps(NETs),and improved acute kidney injury.In this study,we further investigated the effects of C3 a R and NETs components on I/R kidney injury,and the signalling pathways through which C3 a regulates the release of NETs.Objective To investigate the effects of C3 a R and NETs on ischemia-reperfusion acute kidney injury,and to explore the possible mechanisms of C3a-induced NETs release.Methods Part Ⅰ: Study the role of C3 a R in ischemia-reperfusion injury1.1 Effect of I/R on renal injuryA mouse model of bilateral renal clamping was used to simulate ischaemia-reperfusion acute kidney injury.Renal function was assessed by measuring blood creatinine and urea nitrogen;renal histopathology was observed by hematoxyln-eosin(HE)staining;renal apoptosis was detected by Td T-mediated d UTP Nick End Labeling(TUNEL);immunofluorescence and Western blot to detect C3 a R expression.1.2 Effect of C3 a R inhibition or knockdown on renal I/RThe effect of C3 a R inhibition or deletion on I/R was observed by blocking the expression of C3 a R using the C3 a R antagonist SB290157 or knocking down C3 a R using CRISPR/Cas9 gene editing technology.The renal function was assessed by measuring blood creatinine and urea nitrogen;the renal histopathology was observed by HE;apoptosis was detected by TUNEL;C3a expression was detected by enzyme-linked immunosorbent assay(ELISA);C3aR expression was detected by immunofluorescence and Western blot;and C3 a R expression was detected by immunofluorescence and Western blot.expression;immunofluorescence and western blot for C3 a R expression;immunofluorescence and western blot for NETs expression.1.3 Effect of C3 a on neutrophilsHealthy human peripheral blood neutrophils were isolated and purified and cultured in a hypoxic incubator;intervention with C3 a in the presence or absence of C3 a RA;immunofluorescence double staining and Western blot to detect the expression levels of NETs.Part Ⅱ Effect of NETs on renal I/R damage1.1 Effect of inhibition of individual components of NETs on renal I/RThe inhibitor of NETs production,GSK 484,and the inhibitors of NETs components,DNaseI and NEi,were injected before I/R surgery,and the expression levels of NETs were measured by immunofluorescence double staining and Western blot.Blood creatinine and urea nitrogen were measured to assess renal function;HE staining to observe renal histopathology;TUNEL method to detect renal tubular injury;immunofluorescence and Western blot to detect the expression of C3 a R.1.2 Effect of C3 a and NETs on renal tubular epithelial cellsRenal tubular epithelial cells were hypoxic for 24 h.The apoptosis of renal tubular epithelial cells was observed at different reoxygenation times,and the time points with more severe injury were selected.C3 a and purified NETs were added respectively,and the apoptotic level of renal tubular epithelial cells was detected by flow cytometry and TUNEL staining.Part Ⅲ Exploration of the mechanism of C3a-induced NETs releaseTo further explore the molecular mechanisms by which C3 a regulates the release of NETs from neutrophils,Western blot examined the levels of ERK1/2phosphorylation and histone guanylation upon C3 a stimulation of neutrophils.Flow cytometry was used to detect levels of reactive oxygen species.Further pretreatment with p38 MAPK pathway inhibitor(SB203580,20 μM),reactive oxygen species inhibition(DPI,10 μM)and ERK pathway inhibitor(U0126,10 μM)was used to observe the changes on C3a-induced neutrophil guanylation levels;Western blot was used to detect the expression levels of Cit H3 and Histone.ResultsPart Ⅰ.C3a/C3 a R axis is involved in renal I/R injuryC3aR inhibition or knockdown improved renal function,reduced neutrophil infiltration in renal tissue,attenuated tubular injury and decreased the release of neutrophil extracellular trap network.Neutrophils released extracellular trap networks under hypoxic conditions and the expression of C3 a R gradually increased.C3 a intervention caused neutrophils to release NETs,which could be blocked by C3 a RA.Part Ⅱ NETs aggravate I/R kidney tissue damageInhibition of PAD4 using GSK 484 reduces NETs formation in renal tissue,attenuates tubular injury and improves renal function.The use of inhibitors of the NETs component,DNaseI and NEi,had no significant effect on the formation of NETs in renal tissue and renal function.In vitro purified NETs,co-incubated with renal tubular epithelial cells,damaged renal tubular epithelial cells;after degradation of NETs using GSK 484,renal tubular epithelial cell damage was reduced.Part Ⅲ The mechanism of C3 a mediated neutrophils to release NETsC3a induced increased phosphorylation of ERK1/2 and reactive oxygen species in neutrophils,and C3 a RA inhibited phosphorylation of ERK1/2 and the release of reactive oxygen species in neutrophils.The use of the ERK pathway inhibitor U0126 and the reactive oxygen species inhibitor DPI reduced the guanylation of histones,with the use of U0126 reducing ERK phosphorylation while reducing reactive oxygen species levels,and the use of DPI reducing reactive oxygen species levels but having no effect on ERK phosphorylation levels,indicating that ERK is located upstream of reactive oxygen species.Conclusions1.The C3a-C3 a R axis is involved in I/R renal tissue injury.2.NETs formation exacerbates I/R renal tissue injury.3.C3 a regulates the release of NETs from neutrophils via the ERK/ROS pathway.