The Role And Mechanism Of SIRT3 In Contrast-Induced Acute Kidney Injury

Background:With the popularization of iodine-containing contrast media in clinical diagnosis and treatment,contrast-induced acute kidney injury(CIAKI)has become one of the most common hospital-acquired kidney injuries.Contrast-induced acute kidney injury is a common complication of iodine-containing contrast media in the use process.Serious cases can lead to acute renal failure,high mortality and poor prognosis.At present,there is no effective diagnosis and treatment for contrast-induced acute kidney injury.The pathogenesis of contrast-induced acute kidney injury is complex and has not been fully elucidated.At present,most viewpoints focus on the direct damage of iodine-containing contrast agent to renal tubular epithelial cells and renal medulla ischemia and hypoxia.Reactive oxygen species(ROS)play an important role in the occurrence of contrast-induced acute kidney injury,but its specific mechanism has not been fully elucidated.Iodine-containing contrast agents can cause direct damage to renal tubular epithelial cells by activating a series of ion channels and induce multiple apoptotic pathways.Activation leads to apoptosis of renal tubular epithelial cells.At present,a large number of studies have confirmed that contrast agents can produce a large number of ROS through a variety of ways,leading to the accumulation of oxygen free radicals in lesion tissues,resulting in local tissue damage.More and more attention has been paid to the role of oxidative stress in the pathogenesis of contrast-induced acute kidney injury.Relevant experimental studies have found that the activity and quantity of malondialdehyde and xanthine oxidase in kidney tissue homogenate of mice with contrast-induced acute kidney injury increased significantly,while the activity and quantity of superoxide dismutase and glutathione peroxidase decreased significantly.Apoptosis is a highly conservative process,which requires strict regulation of many genes and enzymes in the body.The initiation of this process is the opening and closing of a series of intracellular control switches after cells feel the corresponding external signal stimulation.Bcl-2 and Bax in Bcl-2 family proteins have opposite functions.The former can promote cell apoptosis,while the latter can inhibit the process of cell apoptosis.Caspase 3 is a cysteine-containing aspartate proteolytic enzyme,which is the key enzyme in the whole process of apoptosis.It degrades its substrate irreversibly and finitely.The process of apoptosis is the cascade amplification of the degradation process.Bcl-2 regulates Caspases by reducing the release of cytochrome C.SIRT3 is an important deacetylase in organisms.The deacetylation of SIRT3 plays an important role in important biological processes such as improving mitochondrial antioxidant stress and reducing apoptosis and metabolism.Current studies have confirmed that SIRT3 exists as a key regulator in many biological processes.It often plays an important role in many diseases by regulating the deacetylation of various substrates in the modification stage of protein synthesis.Many experimental studies have shown that SIRT3 is involved in many cell biological processes,such as transcription of various genes,metabolism and oxidative stress.Deacetylase SIRT3 can regulate oxidative stress and apoptosis in various diseases by regulating the deacetylation of substrates during post-translational modification of proteins.The kidney is an organ of high energy metabolism.There are a large number of mitochondria in kidney cells,especially in renal tubular epithelial cells,to meet the needs of high energy metabolism.Mitochondria are widely involved in biological processes such as energy metabolism,fatty acid oxidation,macromolecular biosynthesis,amino acid decomposition,electronic transmission and oxidative phosphorylation.While mitochondria are involved in these processes,they also create It becomes a serious accumulation of oxygen free radicals.SIRT3,a NAD+-dependent deacetylation protein,is located in mitochondria.It is the key enzyme for mitochondria to regulate the above biological processes.In these processes,many enzyme activities are regulated by its deacetylation modification.Nicotinamide riboside(NR)is a compound composed of ribose and nicotinamide.NR is found in mammalian milk,yeast and various common bacteria.After NR is transported to cells,it is broken down into single nucleotide by its kinase,and single nucleotide is broken down into NAD+by its adenosyltransferase,which improves the level of NAD in tissues and plays a variety of biological activities in the body.NAD+is an important auxiliary factor of SIRT3 catalytic deacetylation.The level of NAD directly limits the deacetylation activity of SIRT3.Although SIRT3 plays an indispensable role in the pathological process of many diseases,the role of SIRT3 in contrast-induced acute kidney injury and its related mechanism are still unclear.Therefore,this study intends to use sirt3-ko mice to establish CIAKI mice model,and initially explore the role of SIRT3 in the pathological process and related mechanisms,so as to bring new ideas for clinical prevention and treatment of contrast-induced acute kidney injury,and provide theoretical support and experimental basis for the design of new drugs targeting SIRT3.Aims:1.To investigate the change of SIRT3 expression in a mouse model of contrast-induced acute kidney injury.2.To investigate the effect of SIRT3 on oxidative stress in mice with contrast-induced acute kidney injury.3.To investigate the effect of SIRT3 on the apoptosis of mouse models with contrast-induced acute kidney injury.Experiment and methods:1.Experimental animals and groups;129 wild-type mice(male,8 weeks old)were purchased from Institute of experimental animals,Peking University.SIRT3 knockout mice(SIRT3-KO)were purchased from Jackson laboratories in the United States.Its gene background is 129S6/SvEvTac.All the experiments were carried out in strict accordance with the regulations and rules of the Ministry of health of the people’s Republic of China on the management of experimental animals(Document No.55,2001),and were approved by the ethics committee of Qilu Hospital Affiliated to Shandong University.The first part of the in vivo experiment:129 wild-type mice and sirt3-ko mice were randomly selected,each of them was randomly divided into control group and CIAKI model group,8 mice in each group(n=8).The second part of in vivo experiment:129 wild-type mice(8 weeks old)and sirt3-ko mice(8 weeks old)were randomly selected to construct CIAKI animal model.In order to evaluate the role of SIRT3 activator nicotinamide riboside(NR)in CIAKI,based on the construction of CIAKI model,four groups were divided according to whether or not to add NR intervention,namely WT+Ioversol+saline group,SIRT3-KO+ Ioversol+ saline group,WT+Ioversol+NR group,SIRT3-KO+Ioversol+NR group.2.Establish an animal model of contrast-induced acute kidney injury:intravenous injection of indomethacin(10ug/g)into tail vein of mice;intravenous injection of N-nitro-L-arginine methyl ester(L-NAME,lOug/g)15 minutes later;intravenous injection of Ioversol(350 mg iodine/ml)at the dosage of 2 mg Iodine/g.In the control group,saline of equal volume was injected into tail vein.The mice were executed 24 hours after the establishment of the model.Blood was taken from the heart of the mice for the detection of serological indicators related to renal function.Two kidneys of the mice were taken for follow-up study of their kidney cortex,and urine was collected during the 24 hours of the establishment of the model.3.Add NR intervention treatment:NR intervention group mice were given NR(1mg/g)twice a day tail vein injection,control group tail vein injection of equal volume of saline,after 5 days of continuous tail vein injection,the mice were euthanized,blood was taken from the heart,the biochemical indicators of renal function were tested,the double kidneys of the mice were extracted,the renal cortex of the mice was further studied,and the urine of the mice was taken during 5 days of modeling.4.Tissue staining:After taking samples,paraffin-embedded kidney tissues were embedded.After slicing,hematoxylin and eosin(HE)staining was performed.Histopathological changes of kidney cortex were observed under light microscope.Immunohistochemical staining was used to observe the expression of oxidative stress and apoptosis-related proteins,and automatic image analysis was used to analyze the soft tissue.Image Pro Plus 6.0 was used for quantitative analysis.5.Detection of renal function indicators:After centrifuging the urine and blood of mice,the supernatant was taken and the levels of creatinine and protein in urine,serum creatinine(Scr)and urea nitrogen(BUN)were detected by ELISA.6.Protein immunoblot analysis(Western-blot):the kidney cortex of mice was fully lapping,and the kidneys were lysed by lysis solution.After SDS-PAGE electrophoresis,the membrane was transferred to the PVDF membrane by 90V gel electrophoresis.After the 5%milk powder was closed,the specific resistance 4 degree C shaking table was incubated overnight.Secondary antibody,dropping with chemiluminescent liquid,develops the target protein by chemiluminescent instrument,and detects and compares the relative gray value with Image J software.7.Detection of intracellular superoxide anion level:Dihydroethidium(DHE)dissolves in DM SO and is prepared into appropriate mother liquor.The fluorescent probes were loaded with appropriate solution containing 2 micromoles per liter of DHE and tissue sections incubated at 37℃ for about 30 minutes,and then washed properly for direct detection by fluorescence microscopy.8.Detection of antioxidant stress kinase activity:(1)250 mmol/1,5 mmol/1 H2O2 solution was prepared first,and then color working fluid was prepared.Dissolve the chromogenic substrates in ice bath,separate them properly and use them again.Prepare the samples according to the instructions.Dilute the buffer with hydrogen peroxide enzyme 400 times before use,and then carry on the follow-up determination.Firstly,the standard curve was determined,and then the activity of hydrogen peroxidase in the sample was calculated.(2)Prepare the samples according to the instructions,prepare WST-8/enzyme working fluid and reaction start-up working fluid,incubate for 30 minutes at 37 ℃,determine the absorbance at 450 nm,and finally calculate the total SOD activity in the samples.9.TUNEL staining:fixed kidney tissue,embedding,slicing,dewaxing to water,dripping 20 ug/ml of protease K for 30 minutes,adding 50 ugl TUNEL detection solution to the sample,incubating in dark for 60 minutes,observing and counting apoptotic cells under fluorescence microscope after sealing,calculating the apoptotic index of tubular epithelial cells.10.Statistical Processing:Quantitative data were expressed by standard error of mean addition and subtraction(X±S).All statistical data were analyzed by GraphPad Prism 6 software.Independent sample t-test was used to compare the two groups,and t-test was used to compare the model group,P<0.05 was considered to have significant difference.Results:1.The expression of SIRT3 in renal cortex was elevated in mice with contrast-induced acute kidney injuryWestern blot analysis showed that SIRT3 expression in renal cortex of CIAKI mice was higher than that of control group.The results suggest that SIRT3 may play a potential role in the CIAKI process.2.Endogenous SIRT3 deficiency aggravates pathological damage of renal cortex in CIAKI miceTissue staining showed that two kinds of mice(wild type and SIRT3-KO type)CIAKI model group had typical renal pathological damage,including renal interstitial edema,large renal tubular epithelial cell cytoplasmic vacuolation,brush like edge of tubular epithelial cells falling off,protein tube type formation,which was more serious in SIRT3-KO model group.These results suggest that endogenous SIRT3 deficiency aggravates the pathological damage of renal cortex in CIAKI mice.After the treatment with SIRT3 activator NR,the pathological changes of renal cortex in the wild mouse model group were significantly reduced,while those in the SIRT3-KOmouse model group were little changed.These findings suggest that the activation of SIRT3 can reduce the histopathological changes of CIAKI3.Endogenous SIRT3 deficiency aggravates CIAKI and NR intervention can improve acute renal dysfunction of contrast medium in wild miceThe first part of the in vivo experiment:after the comparison of the renal function test indexes,it was found that the renal function damage of the two kinds of mice CIAKI model group was obvious,while the renal function damage of SIRT3-KO mice was more serious.These results suggest that endogenous SIRT3 deficiency aggravates CIAKI in mice.The second part of the in vivo experiment:the comparison of renal function test indexes showed that:(1)compared with the blank control group,the level of serum creatinine and urea nitrogen in the wild mouse model decreased significantly after adding NR stimulation,but this decline trend did not appear in the SIRT3-KO mouse model group;(2)in the two groups of NR intervention mice,the level of wild-type Cr and BUN was significantly lower than that of SIRT3-KO mice Rat.These results suggest that SIRT3 may play a protective role in the pathological process of CIAKI4.Loss of endogenous SIRT3 enhances oxidative stress in renal cortex of mice with contrast-induced acute kidney injuryDihydroethidium was used to detect the level of superoxide anion.When the level of superoxide anion in renal cortex was high,more ethidium was produced.The results showed that the red fluorescence of SIRT3-KO mice model group was significantly stronger than that of 129 wild mice model group.These findings suggest that the absence of endogenous SIRT3 enhances oxidative stress in renal cortex of mice with contrast-induced acute kidney injury.5.SIRT3 regulates the expression of oxidative stress-related proteins in contrast-induced acute kidney injuryImmunohistochemical staining results showed that compared with 129 wild mice,the expression level of NOX4 in SIRT3-KO mice model group increased significantly,indicating that the expression of oxidative stress-related protein increased after SIRT3 gene knockout,indicating the high level of ROS in vivo.The same results were confirmed by Western blot.The expression level of acetyl-MnSOD K68,which was used to mark SIRT3 activity in the normal mice model group,decreased significantly,while the activity of SIRT3 increased,while the decrease trend of this protein acetylation level did not appear in the SIRT3 knockout mice model group.The second part of the experiment in vivo:after NR stimulation,SIRT3 activity was enhanced and the expression level of acetyl-MnSOD K68 was significantly decreased in the wild mice model group,but the decreased trend of this protein acetylation level did not appear in SIRT3 knockout mice model group.These results suggest that SIRT3 regulates the expression of oxidative stress-related protein in the renal cortex of mice with contrast-induced acute kidney injury.6.Loss of endogenous SIRT3 decreases antioxidant stress kinase activity in contrast-induced acute kidney injuryTwo kinds of mice(wild type and SIRT3-KO type)were compared with the control group respectively.The intervention of contrast agent significantly inhibited the activity of antioxidant kinase Catalase and MnSOD,and SIRT3 knockout further reduced the activity of antioxidant kinase.When the intervention of SIRT3 activator nicotinamide riboside was applied,the activity of Catalase and MnSOD in the wild mouse model group was significantly enhanced after adding NR stimulation However,this enhancement trend did not appear in SIRT3-KO mice model group.These results suggest that the absence of endogenous SIRT3 can reduce the activity of antioxidant kinase and the ability of scavenging oxygen free radicals,while the activation of SIRT3,NR,can up regulate SIRT3 in wild mice and significantly reduce the inhibition of contrast agents on the activity of antioxidant kinase Catalase and MnSOD.7.Loss of endogenous SIRT3 aggravates cell apoptosis in mice with contrast-induced acute kidney injuryTUNEL staining showed that apoptotic cells appeared in both groups compared with the control group,and the apoptotic cells in SIRT3-KO mice model group were more serious than those in the control group.These findings suggest that the loss of endogenous SIRT3 aggravates cell apoptosis in mice with contrast-induced acute kidney injury.8.SIRT3 regulates the expression of apoptosis-related proteins in contrast-induced acute kidney injuryImmunohistochemical staining showed that compared with the 129 wild mice model group,the expression of pro-apoptotic molecule Bax and anti-apoptotic molecule Bcl-2 were significantly up-regulated and the ratio of Bcl-2 to Bax was down-regulated in SIRT3-KO mice model group.At the same time,the up-regulated expression of Caspase 3,a key enzyme in the process of apoptosis,also indicated that the expression of apoptotic-related protein increased after SIRT3 gene knockout.Western blot analysis also showed that the expression of pro-apoptotic molecule Bax and anti-apoptotic molecule Bcl-2 were up-regulated,the ratio of Bcl-2 to Bax was down-regulated and the expression of Caspase 3 was up-regulated in SIRT3-KO mice.These results suggest that SIRT3 regulates the expression of apoptosis-related proteins in contrast-induced acute kidney injury.Conclusions:1.The expression of SIRT3 was increased in mice with contrast-induced acute kidney injury.2.Loss of endogenous SIRT3 aggravates contrast-induced acute kidney injury.3.SIRT3 may regulate contrast-induced acute kidney injury by regulating oxidative stress and apoptosis.Background:At present,with the rapid development of medical diagnosis and treatment methods such as enhanced CT examination and arterial interventional therapy in various medical institutions,contrast-induced acute kidney injury has become one of the main causes of hospital-acquired acute kidney injury.Contrast-induced acute kidney injury not only significantly prolongs the length of hospital stay,increases the cost of diagnosis and treatment,but also significantly increases the mortality of patients.Up to now,the pathogenesis of contrast-induced acute kidney injury has not been fully elucidated.At present,there are no effective preventive measures to prevent and treat contrast-induced acute kidney injury except hydro chemotherapy.Therefore,contrast-induced acute kidney injury has become a common concern of experts in the field of radiation,cardiovascular disease and kidney disease.The pathogenesis of contrast-induced acute kidney injury has not been fully elucidated.Most viewpoints hold that the main pathogenesis of contrast-induced acute kidney injury includes reactive oxygen species(ROS),direct toxicity of contrast agent to renal tubular epithelial cells and renal medulla ischemia and hypoxia.Studies have shown that contrast agents can inhibit the activity of antioxidant stress kinase,directly lead to a large number of oxygen free radicals,aggravate the accumulation of oxygen free radicals in kidney tissue,and the large increase of oxygen free radicals directly cause toxic damage to renal tubular epithelial cells and lead to kidney damage.Experimental studies have shown that oxidative stress can induce apoptosis of renal tubular epithelial cells by affecting mitochondrial permeability,which strongly suggests the close relationship between oxidative stress and apoptosis.Apoptosis,as a basic biological phenomenon of tissue cells,is an active process.It involves the activation,expression and regulation of a series of genes.It is a process of death that organisms actively strive for to better adapt to the living environment.Caspase 3 is the key enzyme in the whole process of apoptosis.It degrades its substrate irreversibly and finitely.The process of apoptosis is the cascade amplification of this process.Bax is a protein that promotes apoptosis.When its stable binding state with Ku70 is broken,it can activate Caspase,a key apoptotic enzyme,to promote apoptosis.Bcl-2 is an important negative regulator of apoptosis.By reducing the release of cytochrome C,Bcl-2 regulates Caspases,a key apoptotic enzyme,and then inhibits apoptosis.SIRT3 is one of the important members of NAD+dependent deacetylase family.Its localization in cells is mitochondrial matrix.As the most widely studied deacetylase in Sirtuins family,SIRT3 plays an extremely important role in mitochondrial energy metabolism and mitochondrial function.A large number of experimental studies have confirmed that SIRT3 regulates many mitochondrial activities through its deacetylation activity,including information transmission,inhibiting genomic variation,energy generation,cell differentiation and cell stress protection,and plays an important role in mitochondrial activities.SIRT3 can deacetyl ate Fox03 alpha and induce up-regulation of antioxidant stress-related enzymes,such as manganese superoxide dismutase and catalase.It has been proved that SIRT3 can protect stress-induced cardiomyocytes,which is also confirmed in cisplatin-induced Cardiomyopathy Animal models.In the pathogenesis of CIAKI,oxidative stress and apoptosis are dominant in renal tubular epithelial cells,while SIRT3 can affect mitochondrial oxidative stress process,and then affect many biological processes in cells.Therefore,this study aims to explore the role of SIRT3 in the oxidative stress and apoptosis of HK-2(renal tubular epithelial cells)in the process of contrast agent acute injury,and to preliminarily explore the role of SIRT3 in the process of HK-2 injury and related mechanisms,so as to bring new ideas for clinical prevention and treatment of CIAKI,and provide theoretical support and experimental basis for the design of new drugs targeting SIRT3.Aims:1.To investigate the expression of SIRT3 in HK-2 cells injured by contrast medium.2.To investigate the effect of SIRT3 on contrast-mediated oxidative stress in HK-2 cells.3.To investigate the effect of SIRT3 on contrast-mediated apoptosis of HK-2 cells.Experiment and methods:1.Cell culture,treatment and grouping:The resuscitated human renal tubular epithelial cell line(HK-2)was inoculated into low-sugar DMEM medium.The cells were cultured routinely in incubator at 37 C and 5%C02.The starving cells were synchronized and transfected with SIRT3 small interfering RNA.The cells were divided into HK-2 and SIRT3-siRNA HK-2.Cells were divided into four groups:HK-2+Vehicle group,HK-2 siRNA+Vehicle group,HK-2+Ioversol group and HK-2 siRNA+Ioversol group according to whether or not ioversol was added.The model group was stimulated by ioversol for 30 minutes and then incubated in cell incubator for 24 hours.2.Protein immunoblot analysis(Western-blot):HK-2 cells were fully lapping,SDS-PAGE gel was used,after 90V electrophoresis,the wet film transfer method was transferred to the PVDF membrane,and after 5%milk powder was closed,a specific anti 4 degree C shaking table was incubated overnight and two days later.Dropping chemiluminescent liquid,developing the target protein by chemiluminescent instrument,and using Image J software to detect and compare the relative gray value.3.Detection of antioxidant stress kinase activity:(1)250 mmol/1,5 mmol/1 H202 solution was prepared first,and then color rendering working fluid was prepared.Dissolve the chromogenic substrates in ice bath,separate them properly and use them again.Prepare the samples according to the instructions.Dilute the buffer with hydrogen peroxide enzyme 400 times before use,and then carry on the follow-up determination.Firstly,the standard curve was determined,and then the activity of hydrogen peroxidase in the sample was calculated.(2)Prepare the samples according to the instructions,prepare WST-8/enzyme working fluid and reaction start-up working fluid,incubate for 30 minutes at 37C,determine the absorbance at 450 nm,and finally calculate the total SOD activity in the samples.4.The ROS level in HK-2 cells was measured by DCFH-DA method:DCFH-DA was diluted with serum-free medium at 1:1000.and the cell culture medium was removed.Serum-free medium containing 10 umol/1 DCFH-DA was added for 1 mL,and incubated in incubator of 5%C02 at 37 C for 20 minutes.The cells were washed three times with serum-free medium,stimulated with Iodophor alcohol of 100 mg I/ml for 30 minutes,then discarded the original medium,washed three times with PBS,and placed under confocal laser microscope to observe the fluorescence intensity of ROS and take photos for statistical analysis.5.Statistical Processing:Quantitative data were expressed by standard error of mean addition and subtraction(X+s).All statistical data were analyzed by GraphPad Prism6 software.Independent sample t-test was used to compare the two groups,and t-test was used to compare the model group,P<0.05 was considered to have significant difference.Results:1.The expression level of SIRT3 in contrast medium mediated HK-2 cell injury is increasedWestern blot results showed that the expression of SIRT3 in HK-2 cell intervention group was significantly higher than that in blank control group.Our results suggest that SIRT3 may play a potential role in contrast-mediated HK-2 cell injury.2.SIRT3 regulates the expression of oxidative stress-related proteins in HK-2 cells mediated by contrast mediaWestern blot results showed that after the inhibition of SIRT3 expression,the expression of Nox4 in HK-2 cells was significantly increased and ROS level in HK-2 cells was elevated.In normal cell intervention group,the expression of Acetyl-MnSOD k68,which was used to mark SIRT3 activity,was significantly decreased,while the activity of SIRT3 was increased,but the decreasing trend of acetylation level of SIRT3 protein did not appear in SIRT3 inhibition group.These results suggest that SIRT3 regulates the expression of oxidative stress-related proteins in HK-2 cells mediated by contrast media.3.SIRT3 regulates the expression of apoptosis-related proteins in HK-2 cells mediated by contrast mediaWestern blot analysis showed that after SIRT3 expression was inhibited,the expression of pro-apoptotic molecule Bax was up-regulated,the expression of anti-apoptotic molecule Bcl-2 was down-regulated,the ratio of Bcl-2/Bax was down-regulated and the expression of key enzyme Caspase 3 was up-regulated.These results suggest that SIRT3 regulates the expression of related proteins in HK-2 cell apoptosis mediated by contrast media.4.Low expression of SIRT3 decreases antioxidant stress kinase activity in contrast-mediated HK-2 cell injury Compared with the blank control group,the intervention of contrast agent significantly inhibited the activities of antioxidant stress kinase Catalase and MnSOD.In contrast agent-mediated HK-2 cell injury,the decrease of SIRT3 expression further reduced the activity of antioxidant stress kinase.These results suggest that the low expression of SIRT3 decreases the activity of antioxidant stress kinase and results in the decrease of oxygen free radical scavenging capacity of HK-2 cells.5.The low expression of SIRT3 enhances the oxidative stress level of HK-2 cells mediated by contrast mediaThe level of ROS in HK-2 cells was measured by DCFH-DA.The ROS level of HK-2 cells with low expression of SIRT3 was significantly higher than that of normal HK-2 cells in the process of HK-2 cell injury mediated by contrast medium.These findings suggest that the low expression of SIRT3 increases the oxidative stress level of HK-2 cells mediated by contrast media.Conclusions:1.The expression of SIRT3 increased in contrast medium-mediated HK-2 cell injury.2.The low expression of SIRT3 increased the oxidative stress level of HK-2 cells mediated by contrast media.3.SIRT3 regulates the expression of apoptosis-related proteins in HK-2 cells mediated by contrast media.