Mechanism Of Neutrophil Extracellular Traps Exacerbating CI-AKI Via Glomerular And Peritubular Capillary Injury

Part I.Correlation between the severity of contrast induced acute kidney injury and the release of neutrophil extracellular traps in mice Objective:To investigate the correlation between neutrophil extracellular traps(NETs)and kidney injury in mice by constructing a contrast induced acute kidney injury(CI-AKI)mouse model.Methods:In this part,an in vivo model of CI-AKI in mice was constructed using iodixanol and taken at different time points or after different doses of contrast agent intervention.The mouse CI-AKI model was constructed by doing ELISA experiments on sera.The kidney injury and the presence of neutrophil extracellular trap network in mice were detected by doing pathology,transmission electron microscopy and immunofluorescence histochemistry experiments on kidney paraffin sections.The expression of NETs products in mouse kidney was detected by doing q RT-PCR and WB experiments on kidney tissues.Results:1.A mouse CI-AKI model was successfully constructed.The kidneys of model mice had obvious acute kidney injury as observed by HE staining,blood creatinine level and transmission electron microscopy.2.The mouse CI-AKI model was related to both time and contrast agent dose.By taking mice at different time points after contrast injection,it was found that kidney injury peaked at 24 h and gradually decreased at 48 h and after.In contrast,higher doses of contrast agent caused more kidney damage in mice than lower doses.3.NETs were found in the kidneys of CI-AKI mice for the first time,and the process of NETosis in neutrophils was captured in the kidneys of diseased mice by immunohistochemistry and fluorescence,which confirmed the involvement of NETs in the mechanism of CI-AKI injury in mice.4.NETs mainly accumulated in the glomeruli and peritubular capillaries of CI-AKI mice.By immunofluorescence co-staining technique,NETs products were successfully localized to be enriched around vascular endothelial cells.5.The expression level of NETs was positively correlated with the severity of CI-AKI.The detection of serum MPO and creatinine levels in CI-AKI mice showed that the NETs products correlated with blood creatinine levels.Analysis of m RNA and protein expression in kidney tissues by q RT-PCR and WB showed that the level of NETs was positively correlated with kidney injury in CI-AKI mice.Conclusion:In the mouse CI-AKI model,NETs were present in the diseased kidneys and mainly accumulated in the glomeruli and peritubular capillaries.The expression level of NETs was positively correlated with the degree of kidney injury in mice,indicating that NETs were involved in the injury mechanism of CI-AKI.Part II.Inhibition of neutrophil extracellular traps attenuates contrast induced acute kidney injury in mice Objective:To investigate the protective effects and mechanisms of blocking NETs accumulation on mouse kidney by pretreating CI-AKI mice to inhibit NETs formation or degrade NETs products.Methods:In this section,mice were divided into four groups: control group,CI-AKI group,CI-AKI+GSK484 group,and CI-AKI+DNase Ⅰ group.Among them,GSK484 is an inhibitor of PADI4,which inhibits the release of NETs;DNase Ⅰ disrupts the DNA structure and can degrade the NETs products.The expression of NETs markers was detected by immunohistochemical fluorescence and WB to evaluate the effect of inhibitors.The level of improvement of glomerular and peritubular capillary damage was evaluated by detecting renal injury by pathological tests,ELISA,and immunofluorescence.Results:1.Drug pretreatment blocked the expression of renal NETs in CI-AKI mice.Both inhibitors of PADI4 and degrading enzymes of NETs products can reduce the expression of NETs in CI-AKI mice.2.Blocking the accumulation of NETs could reduce the pathological kidney injury in CI-AKI mice.By detecting blood creatinine level and doing HE and PAS glycogen staining on kidneys,it was confirmed that reducing NETs could reduce blood creatinine and renal tubular injury score.And reduce the level of inflammation in CI-AKI mice.3.Blocking the accumulation of NETs could reduce the damage of renal vascular endothelium.The expression of markers was reduced in CI-AKI mice by immunofluorescence and histochemical detection of glomerular and peritubular capillary markers,and inhibition of NETs attenuated renal vascular endothelial cell injury.4.Blocking the accumulation of NETs could reduce renal apoptosis.Significant apoptosis was seen in the kidneys of CI-AKI mice,and reducing the level of NETs could attenuate the level of apoptosis.Conclusion:The expression of NETs was significantly increased in CI-AKI mice,which mainly accumulated in glomeruli and peritubular capillaries of the kidney and damaged vascular endothelial cells.After inhibition of NETs formation or degradation of NETs products,the pathological injury of kidney was reduced and the damage of vascular endothelial cells was significantly alleviated.It is suggested that NETs promote the development of CI-AKI and blocking NETs has an ameliorative effect on the injury.