The NETs Contribute To Renal Ischemia-reperfusion Injury By Mediating The Dysfunction Of Peritubular Capillaries

Objective: Renal ischemia-reperfusion is an unavoidable stage in the process of renal transplantation.Necrosis of tubular capillaries and vasculitis of the peritubular capillaries are the pathological features of renal ischemia-reperfusion injury.In our previous studies,we found that renal ischemia-reperfusion can cause neutrophils to release proteins and DNA chains outside of their cells,called neutrophil extracellular traps(NETs).As NETs production is blocked or NETs structure is degraded,renal tubules are protected from damage and renal function can be protected.As a major component of renal microcirculation,peritubular capillary(PTC)plays an important role in maintaining the normal physiological function of renal tubules and promoting repair after injury.NETs may impact peritubular capillary function and,consequently,renal tubule repair,but its effect is uncertain.Therefore,the purpose of this study is to investigate the effect and mechanism of NETs on peritubular capillaries during renal ischemia-reperfusion.Methods: Eighteen healthy male SPF Wistar rats were selected,aged about 8 weeks and weighing about 220 g.The rats were randomly assigned to 3 groups,labeled as the Sham group,IR group,and IR+PAD inhibitor Cl-amidine group(IR+PAD i).In the IR group,the free bilateral renal pedicles were clamped with artery clamps for 45 min to make the model.IR+PAD i group was pretreated by intraperitoneal injection of Cl-amidine at 20mg/kg 1 hour before anesthesia and then underwent anesthesia operation for modeling.In the Sham group,only bilateral renal pedicles were exposed without clipping.The rats were sacrificed 24 h after reperfusion,and blood and kidney tissue samples were collected from each group.Renal function was assayed for serum creatinine(Scr)and urea levels.The right kidney tissue was cut open along the coronal plane,and part of the tissue was fixed in 4% paraformaldehyde,then paraffin embedding was performed,PAS staining was performed to evaluate the renal tubule injury,PTC permeability change was observed by HE staining,apoptosis level of tissues was marked by TUNEL staining,and the peritubular capillary number was observed by CD34 staining.In the other part,Western Blot was used to quantify the proteins of Cit-H3,MPO,and MMP2 in kidney tissue,and q RT-PCR was used to detect the m RNA expression levels of IL-1,CXCL-1,CXCL-2,and HIF-1α.Results:(1)Compared with the Sham group,the expression levels of Cit-H3 and MPO protein in renal tissue of the IR group were significantly increased(P<0.05);Compared with the IR group,the levels of Cit-H3 and MPO in the IR+PAD i group were decreased(P<0.05).(2)Compared with the Sham group,serum Scr,and UREA levels in the IR group were significantly increased(P<0.05);Compared with the IR group,Scr level in IR+PAD i group was significantly decreased(P<0.05),there was an overall decreasing trend of UREA level,but it did not reach statistical difference(P>0.05).(3)Renal tissue PAS staining: compared with the IR group,the degree of renal tissue damage was significantly reduced in IR+PAD i group,and the semi-quantitative results of renal tubular damage score were lower than those in the IR group(P<0.05).(4)CD34 immunohistochemical staining: compared with the Sham group,the area of CD34 staining in the IR group was significantly decreased(P<0.05);Compared with the IR group,CD34 staining area in IR+PAD i group was significantly increased(P<0.05).(5)HE staining of renal tissue: compared with the Sham group,the number of abnormal leakages of erythrocytes in renal interstitial was significantly increased in the IR group;Compared with the IR group,the number of leakages erythrocytes from renal interstitial was decreased in IR+PAD i group.(6)TUNEL staining: Compared with the IR group,the number of apoptotic cells in the IR+PAD i group was significantly reduced(P<0.05).(7)q RT-PCR results indicated that m RNA expression levels of inflammatory chemokines IL-1,CXCL-1,and CXCL-2 in the IR+PAD i group were significantly lower than those in IR group(P<0.05),increased m RNA levels of angiogenic factor HIF-1α(P<0.05).(8)Western Blot results indicated that compared with the Sham group,the expression level of MMP2 protein in the IR group was increased(P<0.05);Compared with the IR group,the expression of MMP2 protein in the IR+PAD i group was significantly decreased(P<0.05).Conclusion:(1)The production of NETs is related to the dysfunction and number of peritubular capillaries.(2)The NETs induced peritubular capillary injury by regulating peritubular capillary inflammatory phenotype transformation and abnormal formation of blood vessels.(3)Cl-amidine,as a broad-spectrum PAD inhibitor,effectively blocks the production of NETs,thereby maintaining peritubular capillary homeostasis and protecting the kidney from ischemiareperfusion injury.