Protective Mechanisms Of ADAMTS13 In Acute Kidney Injury And Chronic Kidney Disease Induced By Ischemia/reperfusion

Renal ischemia/reperfusion(IR)occurred during infection,shock,cardiac surgery and renal transplantation and then resulted in acute kidney injury(AKI).Some AKI patients needed dialysis for replacement therapy to maintain fluid and electrolyte balance.Recent research indicated that IR-induced AKI could not fully recover.Some patients would progress to chronic kidney disease(CKD),even end stage renal disease(ESRD)which affected the quality of patients' life.The pathophysiology of IR was complex.Various systems,factors and pathway were involved.However,the rising incidence of AKI,the transition of AKI to CKD,and subsequent cardiovascular events indicated that the current strategies to prevent and treat IR were not effective.This study aims to find new therapeutic drug to protect against IR.Recent research confirmed recombinant human a disintegrin and metalloprotease with thrombospondin motifs 13(rhADAMTS13)was effective in congentital Thrombotic Thrombocytopenic Purpura(TTP).Acute renal failure is the clinical magnifestion of TTP.It was natural to understand rhADAMTS13 could treat acute renal failure in TTP patients.While whether rhADAMTS 13 could be used to treat other kidney injury was unknown.Clinical evidence showed that high von Willebrand factor(VWF)/ADAMTS13 ratio was associated with the decline of renal function.Animal experiments exhibited that ADAMTS13 retarded diabetic nephropathy by inhibiting intrarenal micro vascular thrombosis.Moreover,rhADAMTS13 could be used to treat ischemic brain and myocardial injury by its anti-inflammatory effect.So this study established IR-induced renal injury animal models to investigtate VWF level and ADAMTS13 activity and its correlation with oxidative stress.In addition,this study explored the protive role of rhADAMTS13 in AKI and CKD induced by IR and its underlying mechanisms.Part one:VWF and ADAMTS13 activity and its correlation with oxidative stress in renal ischemia/reperfusionAims:Ischemia/reperfusion-induced AKI and CKD animal models were established.VWF and AD AMTS 13 activity were detected at various time points.The correlations among VWF:ADAMTS13 ratio oxidative stress and inflammation were analyzed.Methods:1)Bilateral renal ischemia was used as AKI and CKD models in this study.Blood,urine and kidney were collected at 24 hours,48 hours,1 week,1 month and 3 months after reperfusion.2)The parameters of renal function,oxidative stress,inflammation and urinary protein and ADAMTS13 loss were dectected.3)Plasma and renal VWF,ADAMTS13 expression were detected.The correlation between the ratio of V WF:AD AMTS 13 and oxidative stress,inflammation associated indexes were analyzed.Results:1)Blood urea nitrogen(BUN),serum creatinine(Scr)and urinary protein were increased at 24 hours and 48 hours after reperfusion in mice subject to ischemia for 20 minutes.BUN and Scr were increased at 3 months after reperfusion in mice subject to ischemia for 18 minjutes.Increased plasma VWF level,reduced ADAMTS13 activity,high VWF:AD AMTS 13 ratio and urinary AD AMTS 13 loss were observed in IR mice at various time points.2)Malondialdehyde(MDA),tumor necrosis factor-a(TNF-a)and interleukin-6(IL-6)were increased,while superoxide dismutase(SOD)and catalase(CAT)activity was decreased in IR mice at different time points.3)Plasma VWF:ADAMTS13 ratio exhibited positive correlations with MDA and TNF-a.It showed negative correlations with SOD and CAT activities.An obvious correlation was observed between VWF:ADAMTS13 ratio and urinary protein.Conclusion:This study demonstrated that IR induced AKI and CKD.High VWF:AD AMTS 13 ratio companied by oxidative stress and inflammation activation were observed in IR mice at various time points.VWF:ADAMTS13 ratio had a positive correlation with oxidative stress,proinflammatory factor and urinary protein.It had a negative correlation with the activities of anti-oxidative stress related enzymes.Part two:The protective role and mechanisms of rhADAMTS13 in acute kidney injury induced by ischemia/reperfusionAims:This study aimed to explore the protective of rhADAMTS13 in IR-induced AKI and its underlying mechanisms.Methods:1)Bilateral renal ischemia for 20 minutes was used as AKI models.Prophylactic infusion of different doses of rhADAMTS13(1.3?g/kg,2.6?g/kg,5.2?g/kg)or rhADAMTS 13(2.6 ?ig/kg)post surgery through tail vein.Blood and kidneys were collected to detect biochemical parameters.2)BUN and Scr were measured by commercial kits.Kidney injury molecule-1(KIM-1),Cystatin C(CysC)and Neutropil gelatinase-associated lipocalin(NGAL)were measured by ELISA.Renal KIM-1 and NGAL mRNA expression were measured by RT-qPCR.3)PAS and Immunohistochemistry were employed to estimate tubular injury and apoptosis.4)Oxidative stress-related molecules were dectected by RT-qPCR,Western blot and commericial kits.The prophylactic administration of superoxide dismutase(SOD)mimic 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl(tempol)50mg/kg by intraperitoneal injection was used as positive control to confirm the protective role of rhADAMTS13.rhADAMTS 13-treated IR mice were given VWF(80?g/kg)at the same time.Paramters of oxidative stress and renal function were measured to explore if the protective role of rhADAMTS 13 were dependent on the cleavage of VWF.5)Inflammatory indicators were tested to explore the anti-inflammatory effect of rhADAMTS 13 in IR mice and its possible mechanisms.GW9662(2mg/kg),Peroxisome Proliferator-Activated Receptor y(PPARy)inhibitor were administrated one day prior surgery by intraperitoneal injection to confirm the function of PPARy pathway.6)Acetylcholine(ACh)/sodium nitroprusside(SNP)-induced decline of blood pressure was observed.ACh/SNP-induced vasodilation of afferent arterioles by microperfusion was adopted to evaluate microvascular endothelial function.Hypoxia/reoxygenation(H/R)of renal afferent arterioles with or without preincubation of rhADAMTS 13(3750ng/ml)to explore the protective role of rhADAMTS13 in microvascular endothelial cell in vitro.7)The fluorescence of afferent arterioles was detected with fluorescent probe to estimate nitric oxide(NO)and reactive oxygen species(ROS)production.ACh-induced vasodiatory responses of afferent arterioles were observed with pre-incubation by NG-Nitro-L-arginine Methyl Ester,Hydrochloride(L-NAME),tempol and hydrogen peroxide(H2O2).8)Renal Akt and endothelial nitric oxide synthase(eNOS)expression were measured by Western blot.rhADAMTS-treated IR mice were given Wortmannin(Akt pathway inhibitor)(50nmol/kg)through tail vein to confirm the role of Akt pathway.Renal function and endothelial cell function were detected to explore if the improvement of rhAD AMTS 13 in endothelial cell dyfunction was via Akt/eNOS pathway.Results:1)rhADAMTS 13 limited the increased VWF levels,reduced AD AMTS 13 activity and altered the abnormal renal ADAMTS13 distribution in IR mice.2)rhADAMTS13 limited BUN,Scr and proteinuria in IR mice and decreased the necrosis and apoptosis of tubular epithelial cell.The protective role of rhADAMTS13 was further confirmed by positive control groups:IR mice given tempol.3)rhADAMTS13 up-regulated Nuclear factor-erythroid-2-related factor 2(Nrf2)/Heme oxygenase-1(HO-1)pathway,enhanced the anti-oxidant enzymatic activity and then inhibited ROS and MDA production.4)rhADAMTS13 inhibited the activation of p38/ERK/(Cyclooxygenase-2)COX-2/Prostaglandin E2(PGE2)pathway and enhanced the expression of PPARy.Then it inhibited the inhibition of proinflammatory factors and inflammatory cell infiltration.PPARy inhibitor GW9662 increased BUN,Scr in rhADAMTS 13-treated mice.Moreover,it impaired ACh-induced vasodilatory response of afferent arterioles in rhADAMTS 13-treated IR mice.5)Basal blood pressure was similar among four groups.The decline of blood pressure induced by ACh was enhanced in rhADMTS13-treated IR mice.While the decline of blood pressure induced by SNP was similar among four groups.rhADAMTS 13 inhibited ROS production in afferent arterioles and improved microvascular diastolic dysfunction induced by oxidative stress.rhADAMTS 13 limited the down-regulation of Akt/eNOS pathway.Akt pathway inhibitor Wortmannin increased BUN,Scr and impaired ACh-induced vasodilation in rhADAMTS 13-treated mice.6)VWF increased BUN,Scr,MDA,H2O2 and reduced SOD,CAT activtiy in rhADAMTS 13-treated mice.Moreover,VWF impaired ACh-induced vasodilation in rhADAMTS 13-treated mice.Conclusion:rhADAMTS 13 inhibited oxidative stress in IR mice through the cleavage of VWF.And then it inhibited inflammation,cell death and microvascular endothelial cell dysfunction.rhADAMTS 13 protected against IR-induced AKI.Part three:The protective role and mechanisms of rhADAMTS13 in chronic kidney disease induced by ischemia/reperfusionAims:This study aimed to explore the protective role of rhADAMTS13 in IR-induced CKD mice and its possible underlying mechanisms.Methods:1)Bilateral renal ischemia for 18 minutes was employed in this study.rhADAMTS13(2.6?g/kg)were administrated for the subsequent 3 days once a day through tail vein.Blood,urine and kidneys were collected.2)BUN,Scr and urinary protein were measured by commercial kits.Plasma and urinary KIM-1 was detected by ELISA.Renal KIM-1 and NGAL mRNA expression were measured by RT-qPCR.3)PAS,MASSON and Sirius red were used to assess the injured tubules,glomerulosclerosis and interstitial fibrosis.4)Immunohistochemistry were used to assess tubular epithelial-mesenchymal transition and macrophage infiltration.5)Oxidative stress related molecules were detected by commercial kits.Renal proinflammatory and profibrotic molecules were detected by RT-qPCR and Western blot.6)ROS production was detected with fluorescent probe.Responses of afferent arteriles to angiotensin ?(Ang?)and ACh were observed by microperfusion.Results:1)Tubular injury,increased KIM-1/NGAL expression and urinary H2O2 were observed in IR mice at 1 weeek after reperfusion.rhADAMTS13 limited these changes.2)At 1 month after reperfusion,IR mice exhibited increased urinary protein and tubulointerstitial fibrosis.KIM-1,NGAL expression and urinary H2O2 remained high compared with sham-operated mice.rhADAMTS13 inhibited these changes.Blood pressure and the ratio of glomerulosclerosis were similar among four groups.3)At 3 months after IR,BUN and Scr increased slightly.IR mice exhibited elevated KIM-1/NGAL levels.Increased percentage of glomeruloscerosis and tubulointerstitial fibrosis were observed in IR mice.Moreover,increased MDA/H2O2 and reduced SOD/CAT activity were observerd in IR mice.Proinflammatory factors and macrophage infiltration were seen in IR mice.rhADAMTS 13 reversed these changes.rhADAMTS 13 up-regulated Nrf2/HO-1 expression and inhibited transforming growth factor-P(TGF-)/Smad3 pathway.rhADAMTS 13 limited oxidaitive stress,inflammation,renal fibrosis and glomerulosclerosis in IR mice.4)At 3 monts after IR,IR mice exhibited excessive ROS production in renal afferent arterioles.AngII-induced vasoconstriction was enhanced and ACh-induced vasodilation was impaired in IR mice.rhADAMTS 13 could limited ROS production and improved the imbalance of vasoconstriction and vasodilation.Conclusion:rhADAMTS 13 inhibited oxidative stress,inflammation in CKD induced by IR and then improved microvascular function.rhADAMTS 13 attenuated IR-induced CKD by inhibiting fibrosis and glomerosclerosis.