Effects Of Insulin-like Growth Factor Binding Protein-7 On Mitochondrial Function And Their Potential Mechanisms In Sepsis Associated Acute Kidney Injury

Part1 The effects of IGFBP7 on mitochondrial function in LPS-induced HK-2sepsis acute kidney injury modelBackground:Sepsis acute kidney injury is one of the common diseases in ICU,its pathogenesis is not totally clearly,which could be related to mitochondrial dysfunction and injury.Insulin-like growth factor binding protein 7 is an early biomarker for the diagnosis of acute kidney injury,but its role in acute kidney injury remain obscure.Therefore,to research the relation between IGFBP7 and mitochondrial dysfunction,we constructed the LPS induced sepsis acute kidney injury model.Methods: We divided HK-2 cells into four groups:LPS,LPS+IGFBP7siRNA,LPS+NC 和 CTR.Lipo 3000 were used to transferred IGFBP7 siRNA into HK-2 cells of group LPS+IGFBP7siRNA,then added 500μg/ml LPS to groups except group CTR.Collecting samples after 6 h.The IL-6 and TNF-a of cell supernatant were detected by ELISA;CCK8 was used to measured cell viability;and we used qRT-PCR to measure the expression of IGFBP7 、 ATP-5a 、PGC-1α and mtDNA;used Western blot to measure the expression of protein ATP-5a、CPT1A、PGC-1α、MFN2、DRP1;flow cytometry was also used to detect the production of ROS.Results:1.Compared with CTR group,the levels of IL-6 and TNF-a in LPS group and LPS +NC group were significantly increased,and the cell viability was decreased.Compared with the LPS + NC group,the IL-6 and TNF-a in the supernatant of the LPS + IGFBP7 siRNA group were significantly reduced,and the cell viability was increased.2.Compared with CTR group,IGFBP7 and ATP-5a gene showed up-regulation in group LPS and LPS+NC,but down-regulation in group LPS+IGFBP7siRNA,with the comparation of LPS+NC group;the expression of PGC-1α gene was opposite—down-regulation in group LPS and LPS+NC,but up-regulation in group LPS+IGFBP7siRNA.3.The copy numbers of mtDNA decreased in group LPS and LPS+NC,compared with CTR group;but increased in group LPS+IGFBP7siRNA,compared with LPS+NC group.4.The protein expression of ATP-5a and CPT1 A in LPS group and LPS + NC group was higher than that in CTR group;the protein expression of ATP-5a and CPT1 A in LPS + IGFBP7 siRNA group was lower than that in LPS + NC group.5.Compared with CTR group,the expression of PGC-1α and MFN2 protein decreased and the expression of DRP1 protein increased in LPS group and LPS + NC group.Compared with LPS + NC group,the expression of PGC-1α and MFN2 protein in LPS + IGFBP7 siRNA group increased,and the expression of DRP1 protein decreased.6.The intracellular ROS in LPS group and LPS + NC group was higher than that in CTR group;the intracellular ROS in LPS + IGFBP7 siRNA group was lower than that in LPS + NC group.Conclusions:1.IGFBP7 silencing can alleviate renal inflammation and injury in S-AKI.2.IGFBP7 silencing can alleviate mitochondrial dysfunction caused by S-AKI and reduce aerobic oxidation.Part 2 IGFBP7 affects mitochondrial function by regulating mTOR signaling.Background: The mTOR signaling pathway is one of the important signaling pathways in the regulation of mitochondrial function.Its main role is to inhibit aerobic metabolism,increase anaerobic metabolism,and promote mitochondrial biogenesis.In the last part,we demonstrated that insulin-like growth factor binding protein 7 can affect mitochondrial function,which can mainly increase anaerobic metabolism,promote mitochondrial occurrence,that same as mTOR signaling.We will further explore whether the effect of IGFBP7 on mitochondrial function is achieved through the mTOR signaling pathway.Methods: We divided HK-2 cells into six groups :LPS,LPS+IGFBP7siRNA,LPS+IGFBP7siRNA+AZD,LPS+AZD,LPS+NC and CTR.Group LPS+IGFBP7siRNA+AZD and LPS+AZD were treated by 500μM mTOR inhibitor before added LPS.Then we used qRT-PCR to measure the expression of ATP-5a、PGC-1α and mtDNA;used Western blot to measure the expression of protein ATP-5a、CPT1A、PGC-1α、MFN2、 DRP1 and mTOR;flow cytometry was also used to detect the production of ROS.Results:1.Compared with CTR group,p-mTOR / mTOR decreased in LPS group and LPS +NC group;compared with LPS + NC group,p-mTOR / mTOR in LPS +IGFBP7siRNA group increased;compared with LPS + IGFBP7 siRNA group,pmTOR / mTOR in LPS + IGFBP7 siRNA + AZD group and LPS + AZD group decreased.2.The expression of ATP-5a gene was high,but PGC-1α gene was low,and copy number of mtDNA reduced in LPS and LPS+NC,with the comparation of CTR group;and compared with LPS+NC,group LPS+IGFBP7siRNA showed high expression of PGC-1α,increased copy number of mtDNA and low expression of ATP-5a;group LPS+IGFBP7siRNA+AZD and LPS+AZD showed down-regulation of PGC-1α,decreased copy number of mtDNA and up-regulation of ATP-5a,with the comparation of group LPS+IGFBP7siRNA.3.The expression of ATP-5a,CPT1 A and DRP1 protein in LPS group and LPS + NC group was higher than that in CTR group;the expression of PGC-1α and MFN2 protein decreased,and the expression of ATP-5a,CPT1 A and DRP1 protein in LPS +IGFBP7 siRNA group was lower than that in LPS + NC group.Compared with LPS +IGFBP7siRNA group,the expression of ATP-5a and CPT1 A protein in LPS + AZD group increased.The expression of PGC-1α,MFN2 and DRP1 protein decreased.4.Compared with CTR group,the mitochondrial oxygen consumption rate of LPS group and LPS + NC group increased;compared with LPS + NC group,the mitochondrial oxygen consumption rate of LPS + IGFBP7 siRNA group decreased;compared with LPS + IGFBP7 siRNA group,the mitochondrial oxygen consumption rate of LPS + IGFBP7 siRNA + AZD group and LPS + AZD group also increased.5.Compared with CTR group,ROS in mitochondria was up-regulated in LPS group and LPS + NC group.Compared with LPS + NC group,ROS in mitochondria was down-regulated in LPS + IGFBP7 siRNA group.Compared with LPS +IGFBP7siRNA group,ROS in mitochondria of LPS + IGFBP7 siRNA + AZD group and LPS + AZD group was also up-regulated.Conclusions:1.IGFBP7 regulate mTOR negatively.2.In early S-AKI,IGFBP7 can alter mitochondrial metabolism by inhibiting mTOR signaling.3.IGFBP7 can reduce mitochondrial biogenesis by inhibiting mTOR.4.The change of fusion regulated by IGFBP7 has little relation.5.IGFBP7 can aggravate mitochondrial dysfunction via inhibiting mTOR signaling.