The Role Of Neutrophil Extracellular Trap In Glomerular Endothelial Cell Injury In Diabetic Kidney Disease

Background:Diabetic kidney disease（DKD）is the most common diabetic microvascular complication.Its prevalence is increasing year by year.It has become the leading cause of end-stage renal disease.More and more evidences show that DKD significantly increases the incidence of cardiovascular events and mortality in diabetics,which seriously damages patients’health and brings heavy burden to society.Glomerular endothelial cells are an important part of glomerular filtration barrier.Glomerular endothelial cells injury plays a key role in DKD,but its injury mechanism is not completely clear.Neutrophil extracellular traps（NETs）is a kind of network structure released by neutrophils after stimulation,which is composed of loose chromatin and embedded with multiple proteins,such as myeloperoxidase（MPO）and neutrophil elastase（NE）.NETs are mainly produced in the infected part of the body,which can capture and kill pathogens,quickly control infection and play an antibacterial role.Recent studies have reported that in addition to killing pathogens,NETs is also involved in autoimmune diseases such as systemic lupus erythematosus and inflammatory diseases such as atherosclerosis.Studies have found that the level of NETs in patients with diabetes increases significantly.Our previous study found that the level of the NETs marker,Cell-free DNA,in patients with DKD was associated with the progression of nephropathy,suggesting that NETs might play a role in the pathogenesis of DKD.However,the role and mechanism of NETs in the pathogenesis of DKD are not clearAim:The aim of this study was to investigate the role of NETs in the pathogenesis of diabetic nephropathy and its impact and mechanism of glomerular endothelial cell injury.Methods:Population study:We enrolled 70 non-DKD patients with type 2diabetes and 70 DKD patients.The level of NETs（MPO-DNA）in serum was detected by ELISA,and the level and location of NETs（MPO,NE）in renal tissue were detected by immunofluorescence.In vivo experiment:Wild type and NETs deficient(PAD4^-/-)diabetic animal models were constructed by low dose streptozotocin.The blood,urine and kidney tissues of mice were collected to detect the changes of renal function,nets level and renal injury.In vitro experiment:（1）High glucose intervened human neutrophils to detect the formation of NETs;Protein mass spectrometry detects the protein components of NETs induced by high glucose or phorbol-12-myristate-13-acetate（the classical inducer of NETs）.（2）Nets intervened human glomerular endothelial cells.The injury of glomerular endothelial cell injury was detected by transcriptome sequencing,scanning electron microscopy,cell flow cytometry,albumin filtration test,immunofluorescence staining,quantitative real-time PCR（q PCR）and Western blot（WB）.Results:Population study:Circulating NETs（MPO-DNA）in DKD patients was significantly higher than that in non-DKD diabetic patients.MPO-DNA level was positively correlated with urinary albumin creatinine ratio（UACR）.Immunofluorescence staining showed that NETs increased in the kidney of DKD patients,mainly located in the glomerulus and close to the glomerular endothelial cells.In vivo experiment:NETs increased in serum and kidney of diabetic mice and deposited in glomeruli.PAD4^-/-blocked the formation of NETs,significantly reduced the UACR in diabetic mice,alleviated glomerular enlargement,mesangial expansion,and glomerular endothelial cells and podocyte injury.In vitro experiment:（1）The effect of high glucose on the formation of NETs in neutrophils is mainly through reactive oxygen species dependent pathway;the protein components of NETs induced by high glucose and phorbol-12-myristate-13-acetate were significantly different.（2）Intervention of NETs induced by high glucose on human glomerular endothelial cells:a.CCK-8 results showed that NETs had a dose-dependent toxic effect on glomerular endothelial cells;b.NETs injured glomerular endothelial cells,the expression of endothelial intercellular associated protein（VE cadherin,CD31）decreased significantly,the expression of endothelial cell activation markers（VCAM-1,ET1）increased significantly,and endothelial cell albumin filtration increased;c.transcriptome sequencing showed that NETs mainly changed the expression of cell membrane protein related genes and cell membrane charge related genes;d.the results of scanning electron microscope showed that NETs made the cell membrane of glomerular endothelial cells appear spherical bulge and hole like changes;e.flow cytometry showed that NETs could induce endothelial cell death;f.The results of WB and q PCR suggested that NETs increased the expression of pyroptosis related proteins;g.intervening in cell surface charge（oleylamine increases cell surface charge）can reduce the pyroptosis of endothelial cells induced by NETs.Conclusion:NETs promotes the pyroptosis of glomerular endothelial cells through charge and participates in the pathogenesis of DKD.