Study On The Mechanism Of Insulin Alleviating Mitochondrial Dysfunction In Septic Acute Kidney Injury

BackgroundIntensive insulin therapy was reported to decrease new-onset kidney injury in critical ill patients,but the mechanisms remain to be fully elucidated.Objective1.To examine the therapeutic effects of insulin on sepsis-induced acute renal injury(AKI)and mitochondrial dysfunction2.To explore the mechanisms of insulin on renal mitochondrial dysfunction3.To explore the mechanisms of insulin on renal oxidative stress4.To test whether upregulation of UCP2 is the main mechanism of insulin alleviating mitochondrial dysfunction in septic AKIMethodsSepsis was mimicked by cecal ligation and puncture(CLP).Rats were randomly divided into five groups:control group,CLP group,sham surgery group,CLP +insulin group(the insulin group)and CLP + insulin + genipin group(the genipin group).1.Renal function,histopathology,mitochondrial function and ultrastructure were compared among the first four groups.2.The inflammation,oxidative stress,tyrosine kinase and phosphatase signaling pathway were compared among the first four groups.3.The antioxidant enzyme/protein,UCP2,mitophagy and mitochondrial biogenesis were compared among the first four groups.4.UCP2 level,oxidative stress,mitochondrial function and ultrastructure,renal function,histopathology was compared between the last two groups.Statistical analysisMeasurement data from different groups were analyzed by SPSS 17.0 software package using one-way analysis of variance.Statistical significance was accepted at P<0.05.ResultsPart 11.CLP increased BUN and serum CRE at 24 h and serum NGAL at 12 h and 24 h respectively(P<0.05)which was significantly decreased by insulin treatment at the same time point(P<0.05).2.Insulin attenuated CLP-induced renal pathological damage.3.Compared with the control group,CLP group had lower intrarenal ATP content and mitochondria membrane potential(MMP)but higher ROS production at 12 h and 24 h respectively(P<0.05),which were all significantly improved by 24-hour insulin therapy(P<0.05).4.CLP elevated the mRNA level of caspase-9 at 24 h(P<0.05)as well as apoptosis rate and the activity of caspase-9 at 12 h and 24 h respectively(P<0.05).Insulin treatment significantly reduced above parameters at the same time point(P<0.05).Similar changes of caspase-9 were observed in immunohistochemistry and western immunoblot analysis.5.Insulin restored mitochondrial morphology in septic rats.Part 21.CLP resulted in a significant increase in serum TNF-a and IL-1? concentration at 12 h and 24 h respectively(P<0.05),as well as intrarenal TNF-a and IL-1?content at 24 h(P<0.05)which were significantly reduced by insulin administration(P<0.05).2.Compared with the control group,the activity of induced nitric oxide synthase(iNOS)and the production of ROS and malondialdehyde were significantly increased in the CLP group at 12 and 24 h respectively(P<0.05)which were all significantly reduced by 24-hour insulin administration(P<0.05).Insulin normalized the overproduction of NO in CLP rats at 12 h and 24 h respectively(P<0.05).3.The CLP group and insulin group had equal mRNA level of tyrosine phosphatase SHP-2 but were both lower than that of the control group(P<0.05).There was no significant difference in the mRNA level of tyrosine kinase Src among the first four groups.Part 31.CLP did not increase the mRNA level of SOD 1 or SOD 2.Compared with the CLP group and control group,insulin treatment upregulated the mRNA level of SOD 2 at 24 h(P<0.05).The SOD activity decreased in the CLP group at 12 h and 24 h respectively(P<0.05)but was elevated by insulin administration at 24 h(P<0.05).2.The CLP group had lower expression of glutathione and transcription of glutathione synthetase(GSS)than the control group at 12 h and 24 h respectively(P<0.05).Insulin did not upregulate the transcription of GSS.Although intrarenal glutathione content in the insulin group was higher than that of the CLP group at 12 h(P<0.05)but was lower than that of the control group at 24 h(P<0.05).3.The mRNA level of UCP2 elevated in the insulin group and the CLP group compared with the control group at 24 h(P<0.05).The UCP2 expression increased in the CLP group at 24 h as well as in the insulin group at 12 h and 24 h respectively(P<0.05).Moreover,the insulin group had higher level of UCP2 than the CLP group at 24 h(P<0.05).Similar changes of UCP2 were observed in immunohistochemistry analysis.4.The mRNA level of PINK1(mitophagy related protein)upregulated in the CLP group at 24 h compared with the control group and the insulin group(P<0.05).Similar changes of UCP2 were observed in immunohistochemistry analysis.There was no significant difference in the mRNA level of PGC-1?(mitochondrial biogenesis related protein)among the first four groups.Part 41.Genipin inhibited the expression of UCP2 and SOD2 which was upregulated by insulin administration in CLP rats at 12 h and 24 h(P<0.05).2.Genipin increased serum NGAL concentration at 12 h and 24 h respectively(P<0.05)and exacerbated renal morphology which was restored by insulin treatment.3.Compared with the insulin group,the genipin group had higher renal content of ROS,iNOS at 24 h and malonaldehyde and NO at 12 h and 24 h respectively(P<0.05).4.The intrarenal ATP content and MMP of the genipin group was lower at 24 h and mitochondrial ROS production was higher than that of the insulin group at 12 h and 24 h respectively(P<0.05).The genipin group showed severer mitochondrial injury than the insulin group.5.Apoptosis was more severe in the genipin group than the insulin group.Conclusion1.Insulin ameliorated AKI and mitochondrial dysfunction,reduced inflammation and renal oxidative stress,upregulated intrarenal expression of UCP2 and SOD2 in septic rats.2.Upregulation of UCP2,reduction of oxidative status,restoration of mitochondria,improvement of renal function and morphology associated with insulin administration in septic rats was inhibited by genipin.3.Upregulation of UCP2 might be the main mechanism of insulin alleviating mitochondrial dysfunction in septic AKI.