Experimental Study On The Protective Effect And Mechanism Of Mitochondrial Acetaldehyde Dehydrogenase 2 On Post-contrast Acute Kidney Injury And Evaluation Value Of Functional Magnetic Resonance Imaging

Contrast induced nephropathy(CIN)or Post-Contrast Acute Kidney Injury(PC-AKI)refers to the newly occurring renal dysfunction or the aggravation of the original renal dysfunction after intravenous or intra-arterial administration of contrast media(CM).It is classically defined as an increase in serum creatinine(Scr)of at least25% or 44 μmol/L(5mg/L)within 72 hours after CM administration in absence of an alternative aetiology.In recent years,with the development of medical imaging technology,the utilization rate of CM has increased gradually.In addition,with the development of interventional therapy for cardio-cerebrovascular disease and tumor treatment,a large number of CM have been applied in clinical practice,leading to PC-AKI become the third most common cause of hospital acquired renal failure.At the same time,PC-AKI mostly occurs in elderly patients with cardio-cerebrovascular disease or chronic kidney disease(CKD),which often brings a series of serious adverse consequences to patients.Moreover,long-term negative outcomes,such as increased risk of death and progression to chronic kidney disease(CKD)/end-stage renal disease(ESRD),have been documented by a number of observational studies.Therefore,the study of PC-AKI has attracted extensive attention.The pathophysiological of PC-AKI is complex and involves many different mechanisms.Currently,several mechanisms including oxidative stress,renal microcirculation changes,renal hypoxia,cytotoxic effect of iodine contrast agent on renal tubules and endothelial cells have been proposed.In particular,Oxidative stress plays a critical role in PC-AKI.Regarding Reactive oxygenspecies(ROS)generation,CM can either cause excessive ROS production or reduce antioxidant enzyme activity,resulting in increased oxidative stress and leading to impaired renal function.The increase of endothelin and decrease of Nitric oxide and prostaglandin can cause vasoconstriction and diastolic disorder,resulting in renal ischemia-hypoxia(especially in the medullary region)by altering renal microcirculation,and then ROS is produced by the way of xanthine oxidase.On the other,CM can also lead to ROS production by reducing the activity of antioxidant enzymes.Finally,increased ROS will lead to lipid peroxidation of polyunsaturated fatty acids,resulting in the production of aldehydes.These aldehydes are significantly cytotoxic and more stable than ROS,dispersing throughout tissues and organs and amplifying oxidative damage.Therefore,it is of great clinical significance to find an effective intervention strategy that can block the renal oxidative stress response caused by contrast agents.Mitochondrial acetaldehyde dehydrogenase 2(ALDH2),as a tetramer enzyme,has a tissue-specific distribution,and it is widely expressed in the kidney,liver,heart.ALDH2 is the key enzyme for the metabolism of reactive aldehydes.By metabolizing aldehydes,ALDH2 can produce anti-oxidative stress and anti-apoptosis.Alda-1,as a specific agonist of ALDH2,can specifically activate ALDH2 to play a protective role.In the kidney,studies have found that preconditioning with physiological levels of ethanol protect kidney against ischemia/reperfusion injury by modulating oxidative stress.Increased expression of ALDH2 can reduce reduces renal cell apoptosis during ischemia/reperfusion injury after hypothermic machine perfusion.However,inhibition of ALDH2 expression can aggravate renal injury in a rat sepsis syndrome model.In addition to its application in kidney,Bai found that ALDH2 could inhibit the production of ROS and reduce the expression of caspase-3 protein,and thus attenuate neurotoxicity induced by 4-hydroxynonenal in cultured primary hippocampal neurons.In cardioprotection,ALDH2 was found to inhibit ROS production and apoptosis induced by acetaldehyde and ethanol.Preconditioning with Alda-1 and ethanol before ischemia reduced the accumulation of 4-HNE and reduced the size of myocardial infarction by60%.Therefore,ALDH2 is currently considered as an important endogenous protective factor,which plays an vital role in anti-oxidative stress,and is expected to become a new target for the prevention of PC-AKI.With the development of magnetic resonance technology,functional magnetic resonance imaging(f MRI),as a non-invasive means of examination,provides a powerful method for the early detection and diagnosis of many diseases.It can make diagnosis of renal function abnormalities before the occurrence of renal pathological structure abnormalities.Magnetic Resonance Imaging-Blood Oxygenation Level Dependence(MRI-BOLD)technique is based on the ratio of oxygenated and deoxygenated hemoglobin in tissues.At present,some studies have reported that MRI-BOLD can be used for early evaluation of renal injury induced by contrast agents in patients with diabetes,and can be used for noninvasive evaluation of dynamic changes in renal oxygen content during PC-AKI.Intravoxel incoherent motion(IVIM)can simultaneously detect the diffusion of water molecules and perfusion of capillary.It has been reported that IVIM technology can not only dynamically observe the diffusion of water molecules,but also effectively monitor the constriction and dilation of renal vessels and the increase of fluid load in renal injury caused by contrast agent.Therefore,the combination of BLOD and IVIM imaging technology can be used to evaluate and monitor the protective effect of ALDH2 on PC-AKI.Part 1 ALDH2 protects against Post-Contrast Acute Kidney Injury via inhibition of oxidative stress and evaluation value of Functional Magnetic Resonance ImagingObjective: To investigate the expression changes and protective effect of ALDH2 in post-contrast acute kidney injury(PC-AKI)model,and to evaluate the value of BLOD combined with IVIM imaging technology in monitoring the protective effect of ALDH2 in PC-AKI.Methods: Twenty-four rabbits were randomly divided into four groups: saline–treated control group,Alda-1 alone-treated group,contrast medium-treated group(PC-AKI),and contrast medium plus Alda-1 treated group(A+PC-AKI),with 6 rabbits in each group.Alda-1 group and A+PC-AKI group were infused with Alda-1,an agonist of ALDH2,by gavage at 5 mg/Kg/d for 7 days,On the 8th day,rabbits in each group were intravenously injected with normal saline or iodohexol.After 24 h,MRI-BOLD and IVIM were performed after the rabbits were fully anesthetized.Blood biomarkers,renal histology,TUNEL,Malondialdehyde(MDA)content were assessed.The expression of ALDH2,4-HNE,superoxide dismutase 2(SOD-2),catalase(CAT),Cleaved-Caspase3 were measured by Western Blot.Thiobarbituric acid method(TBA)was used to determine MDA content.For in vitro experiments,firstly,HK-2 cells were incubated for 1h,12 h,24h,and 48 h in the medium of iohexol at concentrations of 0mg I/ml,5mg I/ml,10 mg I/ml,20 mg I/ml,40 mg I/ml,80 mg I/ml,and 160 mg I/ml,respectively.The proliferation inhibition rate of HK-2 cells was detected by using a microplate reader.Then,HK-2 cells with good growth status were incubated in medium containing half inhibition concentration of iohexol,and then cultured for 1h,12 h,24h and 48 h,and the expressions of ALDH2 and 4-HNE were detected by Western Blot.In addition,HK-2 cells were treated with Alda-1 or Daidzin,an ALDH2 inhibitor,before treatment with iohexol.The expression of ALDH2,4-HNE,SOD-2,CAT,Cleaved-Caspase3 were measured by western blotting.The content of MDA and reactive oxygen species(ROS)and DAPI were assessed according to the instructions.Results: The PC-AKI model were successful established.The levels of serum creatinine,urea nitrogen increased in PC-AKI group,and the difference was statistically significant compared with the control group(P<0.001,P=0.001).Compared with PC-AKI group, the levels of serum creatinine and urea nitrogen in A+PC-AKI group decreased,the difference was statistically significant(P=0.027,P=0.035).Functional magnetic resonance results showed that both in the renal cortex and medulla,R2* value increased and D,D* and F values decreased in the PC-AKI group,and the differences were statistically significant compared with the control group(P<0.001,P<0.001,P<0.001,P<0.001).In the cortical area,R2* decreased and D value increased in A+PC-AKI group compared with PC-AKI group,and the difference of the parameters between the two groups showed a statistically significant(P<0.001,P<0.001).In the medulla region,R2* values were decreased and D and f values were increased in A+PC-AKI group compared with PC-AKI group,and the differences were statistically significant(P<0.001,P<0.001,P<0.001).Histopathology and TUNEL showed no significant difference between Alda-1 group and control group(P=0.019,P=0.601).Compared with the control group,the pathological injury score and apoptosis rate were increased in PC-AKI group,and the differences between the two groups were statistically significant Compared with PC-AKI group,the degree of renal injury and apoptosis were reduced in A+PC-AKI group.WB results further verified that ALDH2 could reduce apoptosis of renal tubular epithelial cells,and the expression of Cleaved-Caspase3 protein was decreased in A+PC-AKI group,and the difference was statistically significant compared with that in PC-AKI group(P=0.005).Compared with control group,the ALDH2(P=0.002),SOD-2(P < 0.001)and CAT(P < 0.001)protein was decreased,the expression of 4-HNE(P<0.001)and the content of MDA(P<0.001)was increased in PC-AKI group.Moreover,compared to PC-AKI group,the expression of ALDH2(P<0.001),SOD-2(P=0.015)and CAT(P=0.008)were increased,the expression of 4-HNE(P=0.001)and the content of MDA(P=0.008)were decreased in A+PC-AKI group.In the vitro experiment,CCK-8 assay showed that with the increase of iohexol concentration and action time,the proliferation inhibition of HK-2 cells was enhanced,and with the extension of action time,the expression of ALDH2 protein was decreased,and the expression of 4-HNE protein was increased.Compared to Iohexol group,the content of ROS and MDA and the expression of 4-HNE(P=0.024),Cleaved-Caspase3(P=0.02)were increased in Alda-1 group(P<0.001,P=0.005),while the expression of ALDH2(P<0.001),SOD-2(P<0.001),CAT(P<0.001)protein were increased.DAPI results were consistent with the expression of Cleaved-Caspase3.Furthermore,the results were reversed after treating cells with the ALDH2 inhibitors Daidzin.Compared to control group,Daidzin reduced the expression of ALDH2(P=0.015),SOD-2(P<0.001)and CAT(P=0.004)and increased the expression of 4-HNE(P=0.002)and Cleaved-Caspase3(P < 0.001),the level of ROS and the content MDA was also increased(P<0.001).Conclusions: ALDH2 plays an anti-oxidative stress role in PC-AKI,by inhibiting the accumulation of 4-HNE and MDA or inhibiting the generation of ROS.BOLD combined with IVIM imaging technology can evaluate the protective effect of ALDH2 against PC-AKI.Part 2 ALDH2 plays protective roles in Post-Contrast Acute Kidney Injury Via regulating AMPK/FoxO1 pathway in rabbitObjective: To investigate whether ALDH2 is involved in the protective effect of anti-oxidative stress by regulating AMPK/ FoxO1 signaling pathway in PC-AKI.Methods: The cultured HK-2 cells were divided into 4 groups: the control group,Iohexol group,GSK621+ Iohexol group,and Compoud C+Iohexol group,the expression of AMPK,p AMPK,FoxO1,p FoxO1 in each group were detected by Western Blot.Then,the cultured HK-2 cells were divided into 5 groups: the control group,Iohexol group,Alda-1+Iohexol group,Daidzin+Iohexol group,Alda-1+Compoud C+Iohexol group(A+C),The expression of AMPK,p AMPK,FoxO1,p FoxO1,CAT and SOD-2 in each group were detected by Western Blot.The intensity of intracellular ROS fluorescence signal was detected by fluorescence microscope.Results: Compared with the control group,the expression of p AMPK,FoxO1 protein was decreased(P=0.014,P=0.03)and the expression of p FoxO1 protein was increased in iohexol group(P=0.012).Compared with the iohexol group,the expressions of p AMPK and FoxO1 in the GSK621 group were increased(P=0.019,P=0.034),and the expression of p Fox O was decreased(P=0.006),while the expressions of p AMPK and FoxO1 in the Compound C group were decreased,and the expression of p FoxO1 was increased,the difference was not statistically significant(P=0.080,P=0.966,P=0.524).In addition,compared with the iohexol group,the expression levels of p AMPK,FoxO1,CAT and SOD-2 in Alda-1 group were significantly increased,while the expression of p FoxO1 was decreased,and the difference was statistically significant(P=0.001,P=0.006,P=0.019,P=0.002,P=0.022).Daidzin could reverse the effect of Alda-1.However,after the combined intervention of AMPK inhibitor Compound C and Alda-1,the expression levels of FoxO1,CAT,and SOD-2 were decreased,while the expression of p FoxO1 were increased in A+C group,and the difference was statistically significant compared with that in Alda-1 group(P=0.018,P=0.001,P=0.02,P=0.018),but there was no statistically significant difference between A+C group and Iohexol group(P=0.986,P=0.563,P=0.522,P=0.925).ROS results were consistent with the results of protein expressions.The level of ROS in the iohexol group was observably higher than that in the control group(P<0.001).Compared with the iohexol group,the ROS level in Alda-1 group was significantly decreased,and the difference was statistically significant(P<0.001).The level of ROS in A+C group was significantly higher than that in Alda-1 group,and the difference was statistically significant(P < 0.001),but there was no statistical significance in comparison with iohexol group(P=0.499).Conclusions: AMPK/FoxO1 signaling pathway is involved in PC-AKI,and ALDH2 plays an anti-oxidative stress role in PC-AKI by regulating AMPK/FoxO1 signaling pathway.