TIMP2 Regulates Mitochondrial Dynamics Promoting Sepsis-induced Acute Kidney Injury

OBJECTIVE In this study,we intended to verify the expression of TIMP2(Tissue inhibitor of metalloproteinase 2)in the kidney in a mouse model of sepsis,and to identify whether TIMP2 is implicated in the process of sepsis-associated acute kidney injury(S-AKI).Simultaneously,we aimed to demonstrate the role of mitochondrial dynamics perturbation in S-AKI.The relationship between TIMP2 and mitochondrial damage would be investigated,and the molecular mechanism would be explored in the human kidney(HK-2)cell line.METHODS C57BL/6 mice were randomly divided into two groups for sham operation or cecal puncture and ligation surgery(CLP)respectively.A sepsis model was established using CLP surgery,and the expressions of TIMP2 as well as mitochondrial dynamics-related proteins were detected by Western blot and immunostaining.Kidney-specific TIMP2 knockout mice were generated to assess the biological function of TIMP2 in sepsis-associated AKI in vivo.The renal function of wildtype(WT)or TIMP2-deficient mice was examined;renal tissue injury was observed by H&E staining;and apoptosis of renal tubular epithelial cells was detected by TUNEL staining.In the experiments in vitro,lipopolysaccharide(LPS)was applied to stimulate HK-2 cells.Then TIMP2 was knockdown with lentiviral particles,and the mitochondrial fission inhibitor Mdivi-1 was aslo used.The expression of proteins which are involved in mitochondrial dynamics and apoptosis were detected by Western blot and immunofluorescence staining.The apoptosis of HK-2 cells was evaluated by CCK-8(Cell Counting Kit-8)cell viability assay,TUNEL staining and flow cytometry.To investigate whether TIMP2 mediates mitochondrial damage,we measured cellular reactive oxygen species(ROS)and ATP production,along with the mitochondrial membrane potential.In addition,TIMP2 was overexpressed in HK-2cells,and the influence on expressions of mitochondrial dynamics-related proteins and mitochondrial function were analyzed.Finally,the activation of ERK1/2 pathway was evaluated by Western blot both in vivo and in vitro,and its implication in mitochondrial dynamics and dysfunction was explored with the application of a specific inhibitor named SCH772984.RESULTS Expressions of TIMP2 and mitochondrial fission-associated proteins were increased in the kidney of septic mice,accompanied by the condition of mitochondrial damage.Kidney-specific TIMP2 deletion protected from renal dysfunction and apoptosis induced by sepsis,while mitochondrial dynamics perturbation and mitochondrial damage were also alleviated.In HK-2 cells,TIMP2 knockdown effectively inhibited LPS-induced apoptosis,mitochondrial dynamic perturbation and mitochondrial damage,which was consistent with the effects of mitochondrial fission inhibitors in the condition of LPS challenge.Meanwhile,we found that mitochondrial fission inhibitors also alleviated mitochondrial dynamic disorders and mitochondrial damage caused by TIMP2 overexpression.Finally,we demonstrated the activation of ERK1/2 pathway in sepsis-associated renal injury,and TIMP2 deletion could inhibit the activation of ERK1/2.Besides,TIMP2 overexpression led to mitochondrial dynamic disorder and mitochondrial injury,which could be alleviated by the use of ERK1/2 pathway inhibitor.CONCLUSION In S-AKI,TIMP2 causes phosphorylation of the mitochondrial fission protein Drp1 through the activation of ERK1/2 pathway,which promotes mitochondria toward fission machinery,leading to mitochondrial dynamic perturbation.The dynamic disturbance ultimately results in mitochondrial damage and renal cell apoptosis,which contribute to acute renal dysfunction.