The Role And Mechanism Of TIMP2 In Sepsis-induced Acute Kidney Injury

Objective Acute kidney injury(AKI)is a frequent complication of sepsis and contributes to increased morbidity and mortality.Urinary tissue inhibitor of metalloproteinases-2(TIMP2)has been recently recognized as an early biomarker to predict AKI in critically ill patients.Most prior studies have focused on the potential diagnostic value of TIMP2,particularly the diagnostic value of urinary TIMP2 in AKI.However,the biological functions of TIMP2 remain largely unknown.In this study,we try to investigate the role of TIMP2 in mediating inflammation and tubular cell apoptosis in AKI.We hypothesize that TIMP2 also plays important roles in the development of septic AKI.The present study therefore sought to identify the detailed functions of TIMP2 in lipopolysaccharide(LPS)injured human kidney 2(HK-2)cells,an in vitro model of septic AKI,and in a septic mice model by using cecal ligation and puncture(CLP).In addition,we tested whether underexpression of TIMP2 would benefit or harm in the setting of sepsis.Methods The AKI model of sepsis was constructed by Cecal Ligation and Puncture(CLP).Serum creatinine was detected by creatinine detection kit.TIMP2 in renal cortex was detected by Elisa kit,and the correlation between serum creatinine and TIMP2 concentration was calculated by Peason correlation analysis.The m RNA of TIMP2 and pro-inflammatory cytokines in HK-2 cells were detected by q RT-PCR after exposure to LPS.The TIMP2 and apoptotic related proteins were detected by Western Blot,and the intracellular localization of TIMP2 was determined by immunofluorescence.After the stimulation of HK-2 cells by,The cell cycle was analyzed by flow cytometry after exposure to human recombinant cytokines(IL-1β,IL-6,TNFα).The TIMP2 si RNA was transfected into HK-2 cells,the expression of TIMP2 was detected by q RT-PCR.The sequences which could knock down TIMP2 were screened,and then lentivirus was constructed to infect HK-2 cells.The effect of TIMP2 knockdown on apoptosis was analyzed by flow cytometry and TUNEL after LPS stimulation,and the cell viability was detected by CCK-8.In animal experiments,TIMP2 lentivirus was injected into mice kidney,immunofluorescence and immunohistochemistry was employed to show the expression of TIMP2.Serum creatinine,TUNEL and H&E staining were used to reveal the renal function,apoptosis ratio and renal pathology.Results In kidney tissue taken from mice exposed to cecal ligation and puncture(CLP)and in HK-2 cells exposed to lipopolysaccharide(LPS)in culture,TIMP2 expression was significantly upregulated.The expression of TIMP2 in the kidney tissue correlated with the severity of AKI in vivo.In cultured HK-2 cells,LPS challenge markedly induced cytokine release,and recombinant cytokines promoted TIMP2 expression and apoptosis.However,TIMP2 silencing ameliorated LPS-induced cytokine release,apoptosis,and cell injury.We further found that the effects of downregulation of TIMP2 on a suppression of release of inflammatory cytokines were mediated by p-P65.Stable,kidney-specific TIMP2 knockdown mice were transduced by injecting the TIMP2 knockdown lentiviral vector into kidney parenchyma.TIMP2 silencing ameliorated CLP-induced proinflammatory cytokines,kidney dysfunction as measured by serum creatinine level,and histopathological changes.Downregulation of TIMP2 showed renoprotective effects on endotoxin-induced AKI,which was associated with the anti-inflammatory activity through inhibition of the NF-κB pathway.Conclusion In the presence of endotoxin,high LPS levels induce the release of the TIMP2 protein,which results in the activation of the NF-κb pathway.Increased p-P65 level mediates cytokine(including IL-1β,IL-6 and TNF-α)release.The extracellular cytokines bind with their receptor and drive apoptosis.The augmented cytokine release further promotes TIMP2 synthesis and release.This,in turn,forms an inflammation loop leading to AKI.Collectively,our results indicate that TIMP2 plays an important role in mediating sepsis-induced AKI through regulation of NF-κB.These findings reveal the pathogenic role of TIMP2 in AKI and suggest a novel target for the treatment of AKI.