| Acute kidney injury(AKI),a clinical common critical disease involved with multiple systems,is the cause of acute kidney function decline,with or without oliguria or anuria of a group of clinical syndromes.The case fatality rate of AKI is high,and AKI plays an vital role in the occurrence and progress of end-stage renal disease(ESRD),which is a serious threaten to the life safety and the surival quality of patients.As a consequence,intensive study about the reasons and the mechanisms of acute kidney injury has important significance to the early prevention of acute kidney injury and the control of chronic kidney disease evolvement.However,we known few signal pathway about acute kidney injury in vivo.For the past few years,the role of mitochondrial dysfunction in nephropathy has be received much concern.Mitochondria,energy supply factory of living organism,is the occasion where ATP synthesis,electron transfer,oxidative phosphorylation happened.Kidney needs to consume vast energy to maintain normal function.Any trouble in the step of mitochondrial function may result in renal dysfunction.As existing literature reports,mitochondrial dysfunction is bound up with quite extensive disease,and play an inportant role in the the occurrence and progress of renal disease.Researches show that mitochondrial dysfunction can be found in acute kidney injury,including morphological abnormality,mitochondrial DNA mutation and the reduce of mitochondrial copy,and eventually lead to apoptosis.Our research group found that mitochondrial dysfunction is an early event of many kidney disease model including acute kidney injury,and participate in the the occurrence and progress disease.Blocking-up mitochondrial dysfunction may protect kidney.Therefore,finding the way to control mitochondrial dysfunction may be the key to cure renal disease.With the deepening research and development of life science by people,the biological function of non-coding RNA are discovered increasingly.As a member of non-coding RNA,microRNAs are taken seriously gradually and intensively researched in various fields because of its wide role in living body.MicroRNAs(miRNAs)are a class of endogenous non-coding small RNAs,molecules of 22 nucleotides,that modulate gene expression via binding to 3'-untranslated region(3'-UTR)of target mRNAs and thereby induce mRNA degradation or block protein translation at post-transcriptional level.Evidence proves that miRNAs play important roles in pathological and physiological process,including cell proliferation,differentiation,apoptosis and oxidative stress.In the previous study of our group,Male C57BL/6J mice were chosen to induce kidney injury with implanting pellets containing aldosterone subcutaneously,then take a miRNA microarray assay to survey a genome-wide miRNA profile of the renal cortex tissue of aldosterone infused group and the Sham group.The research found that the expression of miR-709 in aldosterone induced kidney injury model significantly increased compare with Sham group.In the mouse renal tubular epithelial cell injury model in vitro induced by cisplatin,our group found that miR-709 can be induced by cisplatin dose and time dependently,and down-regulated the expression of miR-709 can alleviate the cisplatin induced damage of renal tubular epithelial cells through targeting TFAM,a important gene of mitochondrial transcription.However,the effect of miR-709 in the acute kidney injury model in vivo remains to be further researched.Therefore,further exploration the function and mechanism of miR-709 in acute kidney injury will bring the great promising future of prevention and treatment measure for acute kidney injury and kidney diseases.As a consequence,we have done the following research.Object:To investigate the expression of miR-709 in the model of acute kidney injury induced by cisplatin,and the role of down-regulating miR-709 in the model of acute kidney injury and mitochondrial dysfunction induced by cisplatin.Methods:Cisplatin was used to establish the experimental animal model of renal involvement to investigate the expression of miR-709.Take the SPF C57BL/6J healthy adult male mice(8 to 12 weeks),intraperitoneal inject with miR-709 antigomir,control antigomir and cisplatin.The mice were divided into four group:Anti-control group,Anti-miR-709 group,Anti-control+cisplatin group and Anti-miR-709+cisplatin group.The mice were sacrificed in 72 hours after cisplatin treatment.The blood was transferred into heparinized tubes and centrifuged at 10,000 ×g for 10 min to separate the plasma,which is prepare for the level of serum creatinine.The pathological morphology change of kidney tissue was observed by light microscopy and scored by the improved Broekema blinded measure after PAS staining.The expression of miR-709 was detected by in situ hybridization immunofluorescence method.The expression of biomarker of AKI(NGAL,Kim-1)and miR-709 was detected by quantitive real time PCR and western bolt.The apoptosis rate of mouse renal tubular epithelial cell were measured by TUNEL staining.The expression of TFAM and mitochondrial DNA was detected by quantitive real time PCR.Mitochondrial superoxide was detected by GENMED purified mitochondrial oxidative stress reactive oxygen primary florescent kit and fluorescence microplate.The mitochondrial membrane potential(MMP,??m)in proximal tubular of kidney were determined by using the lipophilic cationic probe 5,5',6,6'-tetrachloro-1,1,'3,3'-tetraethyl-carbocyanine iodide(JC-1;Molecular Probes,Eugene,OR)and were examined by confocal microscopy.Results:(1)Obvious renal tubular atrophy and loss of brush border could be observed in the cisplatin group compare with control group after PAS staining,and the pathological score improved for 23.5 times(P<0.001).The apoptosis rate of renal tubular epithelial cell was raised to approximately 3.5 times(P<0.001)in the renal of mice which is intraperitoneal injected with cisplatin compare with control group.The level of serum creatinine was raised to approximately 7.3 times after cisplatin injection.The mRNA and protein level of NGAL and Kim-1 were increased after intraperitoneal inject with cisplatin for 72 hours,the mRNA and protein level of NGAL increased for 1632.4 times(P<0.001)and 3.7 times(P<0.05),and the mRNA and protein level of Kim-1 increased for 2.4 times and 7.5 times(P<0.05).(2)The expression of miR-709 was raised to approximately 1.7 times(P<0.05)in the renal of mice which is intraperitoneal inject with cisplatin(20mg/kg)after 72 hours compare with control group.Mainly miR-709 expresses in the nucleus of renal tubular epithelial cells,and a little in cytoplasm.(3)Intraperitoneal injection with miR-709 antigomir can downregulate miR-709 mRNA level for 99%(P<0.05).Down-regulation of miR-709 blocked the damage of mouse renal function and renal tubular.miR-709 antigomir decreased the level of serum creatinine and inhibited the apoptosis of renal tubular epithelial cells induced by cisplatin after TUNEL staining.Renal structure damage of Anti-miR-709+cisplatin group,which was observed under light microscope after PAS staining,was relieved after miR-709 antigomir treatment compare with Anti-control+cisplatin group.It showed the loss of tubular brush border and reduced tubular atrophy were significantly blocked.The pathological score was significantly reduced by 53%(P<0.05).The expression of NGAL and Kim-1 were reduced by intraperitoneal inject with miR-709 antigomir.The mRNA and protein level of NGAL decreased for 99%and 90%(P<0.05),and the mRNA and protein level of Kim-1 decreased for 51%and 73%(P<0.05).(4)The expression level of TFAM was downregulated(-60%,P<0.05)after cisplatin injection and mitochondrial dysfunction could be induced by cisplatin injection.The mitochondrial membrane potential(MMP,??m)in proximal tubular of kidney was reduced(-71%,P<0.05)after cisplatin treatment.The mitochondrial ROS was increased to 2.4 times(P<0.05)after intraperitoneal inject with cisplatin for 72 hours.The copies of mitochondrial DNA was reduced(-63%,P<0.05)after cisplatin injection.The expression level of TFAM was upregulated to 2.1 times(P<0.05)after cisplatin injection and down-regulation of the expression of miR-709 blocked the mitochondrial dysfunction of mouse renal proximal tubular,evidenced from the increased mitochondrial membrane potential to 2.1 times,reduced ROS production(-60%,P<0.05)and increased mtDNA copy number to 2.8 times(P<0.05).Conclusion:Cisplatin could induce the damage of renal tubular epithelial cell accompanied with the increase of miR-709 expression.Down-regulation the expression level of miR-709 in mouse renal plays a role of protection of acute renal injury and mitochondrial dysfunction induced by cisplatin. |
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