Investigation Of Renal Injury Induced By Iodinated Contrast Agents With Different Viscosity On Rabbits Kidney And The Protective Effects Of Hydration And Atorvastatin Using Functional MRI

Objective:With the continuous development of interventional techniques and radiodiagnostic techniques,iodine contrast agents are widely used,and the incidence of contrast-induced nephropathy(CIN)also increases.The pathogenesis of iodine contrast agents has also become popular.The non-ionic dimer contrast agent with osmotic pressure close to the osmotic pressure of plasma,is called isotonic contrast agent.The viscosity increased compare to non-ionic monomer contrast agent.The relationship between viscosity and CIN has become a research hotspot after osmotic pressure.However,in previous studies,viscosity was not the only controllable variable,and the osmotic pressure of contrast agent was also a variable,which was a confounding factor affecting the experimental results.Due to the gradual population aging in our country,diabetes,especially type II diabetes,is particularly prominent.Diabetic patients receiving iodine contrast agents will also increase,and diabetic nephropathy is an independent risk factor for contrast-induced nephropathy.Studying the influence of contrast agent's viscosity on the structure of diabetic kidneys will help to clarify the different damage of contrast agent on diabetic kidney damage and normal kidney damage so as to formulate the iodine contrast agent medicine suitable for diabetes to protect the kidneys of diabetic patients.There is no effective treatment for contrast-induced nephropathy.Intravenous hydration is the only proven and effective measure for prevention.However,hydration does not apply to all patients,such as patients with cardiac insufficiency,the need to strictly control the liquid input,so in addition to hydration,various contrast agent nephropathy prophylaxis has also become a research focus,especially statins.A lot of meta-analysis,and a large number of clinically RCT research on statin prevention of contrast-induced nephropathy,affirmed the preventive effect of statins on contrast-induced nephropathy and concluded that short-term and high-dose atorvastatin works best.Although there are numerous studies on hydration and statins,the assessment of renal function in these studies is mostly based on clinical laboratory parameters,mainly changes in creatinine levels,which are less stable.At the same time,most of these studies use only one kind of contrast agent.There is no clear explanation for changes in kidney micro-changes,especially in the absence of CIN.Previous studies have shown that parameters of functional oxygenation level dependent imaging(BOLD),diffusion weighted imaging(DWI),diffusion tensor Imaging(diffusion tensor imaging,DTI)could detect the changes in renal microstructure noninvasively.The purpose of this study was to investigate the effects of non-ionic isotonic contrast agent viscosity on oxygen content and dispersion of renal tissue in normal and diabetic nephropathy rabbits by BOLD,DWI and DTI imaging.Through histological examination and immunohistochemistry HIF1-a and AQP-1 to observe the microscopic changes of the kidney to verify the diagnostic value of BOLD,DWI,DTI.At the same time,assess the prevention value of hydration and atorvastatin on non-ionic dimer iodine contrast agent-induced diabetic nephropathy Rabbit kidney injury,so as to provide a theoretical basis for patients with diabetic nephropathy using contrast agent.Methods:1.New Zealand rabbits,male,about 2kg,all animal experiments are carried out under anesthesia.In the experiment,all animals required fasting 6h before scanning.Iodixanol contrast agents were selected at concentrations of 270,320,and 350,with viscosity of 5.7,11.1,and 17.3 respectively.2.Effect of contrast medium viscosity on normal rabbit kidney:63 healthy male rabbits,3 were killed without any intervention and got normal right kidney tissue.The remaining 60 patients were randomly divided into 4groups:control group(saline),low viscosity group,medium viscosity group and high viscosity group.The baseline BOLD,DWI and DTI-MRI scans were performed.The experimental groups were injected with 270,320 and 350 iodixanol respectively.The control group was injected with the same volume of saline.BOLD,DWI and DTI-MRI scans were performed at 2 hours,1 day,2 days,3 days and 7 days after injection of contrast agent or saline.Observe the changes of functional magnetic resonance parameters R2*,ADC and FA in renal cortex,outer stripe of outer medulla,inner stripe of outer medulla,and inner medulla.Three rabbits were killed at each time point after injection,and the right kidney was removed for pathological staining and immunohistochemistry.3.Effect of contrast medium viscosity on DN rabbit kidney:63healthy male New Zealand rabbits.The inducation of type II diabetic nephropathy model:Insulin resistance was induced by diet.And ALX(alloxan)was used to destroy the islet cells of rabbits after 2 weeks of adaptive feeding.After 12 weeks,the rabbits blood was biochemically examined.When blood glucose>16mmol/L,total cholesterol>5.2mmol/L,triglyceride>1.8mmol/L,serum creatinine>140?mol/L and urea nitrogen>7.2?mol/L,the modeling was successed.3 rabbits induced to be type II diabetic nephropathy were killed without any intervention and their right kidney tissues were obtained.The remaining 60 rabbits were randomly divided into 4 groups:saline group,low viscosity group,medium viscosity group and high viscosity group.The remaining steps were same as the normal rabbit group.4.Hydration and atorvastatin on diabetic nephropathy rabbit kidney injury prevention study:81 induced type II diabetic nephropathy rabbits were randomly divided into three groups,namely hydration group,atorvastatin group,hydration group and combined group,given hydration,atorvastatin and combination respectively before contrast medium injection.Each group was divided into three subgroups:low viscosity group,medium viscosity group and high viscosity group.The control group in each subgroup was the experimental groups of the DN rabbits.5.Magnetic Resonance Scanning and Postprocessing:The GE-3.0 Clinical Magnetic Resonance Scanner(Twin Speed Scanner,General Electric Medical Systems,Milwaukee,WI,USA),cardiac coil.After anesthesia,sacn the coronal slice of right kidney with supine,head first positon.GEadw4.4 workstation for post-processing,select the region of interest(ROI)from the right kidney,including renal CO,OSOM,ISOM and IM.R2*value,ADC,FA values were measured and the data were statistically analyzed.6.Histological analysis:routine dehydration,paraffin embedding.slice,paraffin sections conventional dewaxing,hematoxylin staining,distilled water,1%hydrochloric acid(differentiation),distilled water,eosin staining dehydration,transparent,sealing.The morphological changes of cytoplasm vacuoles,accumulation of intraluminal debris and luminal dilatation were observed after injection of contrast medium in rabbits.Semi-quantitative scores of each pathological manifestation were observed.All pathological findings scores of each renal anatomical stripe were added together to arrive at the total score of kidney damage in the anatomic zone.Each anatomical zone randomly selected five fields,taking the mean as the final score of the band.7.Immunohistochemistry:The expression of HIF-1?,AQP-1 was detected by streptavidin-biotin-peroxide technique.5?m thick paraffin sections were dewaxed,hydrated followed by microwave heating antigen retrieval,3%hydrogen peroxide at room temperature,normal goat serum closed,primary antibody incubation,secondary antibody incubation,horseradish peroxidase labeling,diaminobenzidine significant Color and hematoxylin counterstain.The expression of HIF-1?and membrane AQP-1 in the nucleus of renal tubular epithelial cells were observed and scored.8.Statistical Analysis:The data are expressed as mean±standard deviation,using SPSS20.0 and Graph Pad prism 6.0 statistics.For the repetitive R2*,ADC and FA functional MRI parameters,normal distribution was followed by one-way ANOVA and repeated measures analysis of variance(ANOVA)for comparison between groups and within groups.For non-normal distribution,Kruskal-Wallis test and Friedman test for inter-group and intra-group comparison.For histological and immunohistochemical scores,Kruska l-Wallis test was used for both inter-group and intro-group comparison.Spearman correlation analysis was used to analyze the correlation between the scores of renal tissue injury and the expression of immunohistochemistry and fMRI parameters.P<0.05 was assumed statistically significant.Results:1.The R2*values in the four anatomical zones of the kidneys rose rapidly after contrast injection,reached a peak at 24 hours,then decreased gradually and returned to baseline levels on day 7,whereas R2*did not show significant changes before and after saline injection Variety.Compared with saline group,R2*was significantly increased in low,medium and high viscosity groups at 2,24,48 and 72 hours,respectively,with significant difference from baseline.The higher the viscosity,the greater the R2*value rose.2.The FA values in the four anatomical zones of the kidneys dropped rapidly after contrast injection,reaching the lowest level at 24 hours,then gradually increasing and returning to baseline level on day 7,while FA did not change significantly before and after saline injection.At 2 h,24 h and 48 h,the FA values of each anatomic zone were significantly different from those of saline group.At 24 h,the higher the contrast agent viscosity,the smaller the FA value in the four anatomical bands.3.ADC values in the four anatomical zones of the kidneys dropped rapidly after contrast injection,reaching the lowest value at 24 hours,then rising and gradually returning to the baseline level,while there was no significant change in ADC before and after saline injection.At 24 h,in all anatomical zones of the kidney,the ADC was significantly lower in the experimental groups than in the saline group,and the higher the contrast agent viscosity,the smaller the ADC value was.4.The changing trend of functional magnetic resonance imaging parameters of different viscosities on nephropathy rabbit's kidney is consistent with the trend of normal rabbits,but in the high viscosity group,renal injury did not recover on the 7^th day.5.Compared with non-intervention low viscosity group,the values of R2*in hydration group,atorvastatin group and combined group decreased at24h,ADC value and FA value increased,and in ISOM,the differences were Statistical significance.At 24 h,the R2*values of each anatomic zone in atorvastatin group were lower than those in hydration group,and FA and ADC values were higher than those in hydration group,but the difference was not statistically significant.At 24 hours,R2*in hydration+atorvastatin combined group was lower than that in hydration and atorvastatin group,FA value and ADC value were higher than the other two groups,and the difference was statistically significant.The parameters of each group of anatomical zones recovered on the third day.6.Compared with non-intervention medium viscosity group,the values of R2*in hydration group,atorvastatin group and combined group decreased at 24h,ADC value and FA value increased,and in ISOM,the differences were Statistical significance.At 24 h,the R2*values of each anatomic zone in atorvastatin group were lower than those in hydration group,and FA and ADC values were higher than those in hydration group,but the difference was not statistically significant.At 24hours,R2*in hydration+atorvastatin combined group was lower than that in hydration and atorvastatin group,FA value and ADC value were higher than the other two groups,and the difference was statistically significant.For hydration group and the combined group,the parameters of each anatomical zone recovered on the 3rd day.Each anatomical zone parameters of atorvastatin group recovered on the 7th day.7.Compared with the non-intervention high viscosity group,the values of R2*in hydration group,atorvastatin group and combined group decreased at 24h,ADC value and FA value increased,and in ISOM,the difference was statistical significance.At 24 h,the R2*values of each anatomic zone in atorvastatin group were lower than those in hydration group,and FA and ADC values were higher than those in hydration group.At 24 hours,R2*in hydration+atorvastatin combined group was lower than that in hydration and atorvastatin group,FA value and ADC value were higher than the other two groups,and the difference was statistically significant.The parameters of hydration group and combined group recovered on the 7th day.For atorvastatin group,each anatomical zone parameters did not recover on the 7th day.8.Histological analysis was performed on CO,OSOM and ISOM.In normal and diabetic nephropathy rabbits,compared with the saline group,atfte the three kinds of contrast agents injected,the tubular structure of the three anatomical bands in the kidneys were significantly expanded,proximal renal tubular epithelial cells vacuolar,a large number of debris accumulation were seen in the renal cortex and outer stripe of innermedulaa.The higher of the contrast agent viscosity induced heavier pathological change.At 24h,the damage is heaviest.Finally,in all groups of normal rabbits and low and medium gourps of DN rabbits,the damage recovered on the 7th day,while the high viscosity group of DN didn't recover on the 7th day.9.The expression of HIF-1a in renal tubular epithelial cells increased after injection of contrast agent,most obvious expression at 24h,and then gradually recovered.For nephropathy rabbits,the high viscosity group did not recover at on the 7^th day.10.After injection of contrast agent,the tubular epithelial cells AQP-1 expression decreased,reduced to a minimum on 24h,and then gradually recovered.For the DN rabbits,the AQP-1 expression of high viscosity group did not recover on the 7^th day.11.Sperman correlation analysis for the correlation between R2*value of inner stripe of outermedulla and HIF-1a score,P=0.000,r=0.78.Sperman correlation analysis for correlation between ADC value of inner stripe of outermedulla and AQP-1 score,P=0.000,r=0.91.Conclusion:1.For the normal and diabetic nephropathy rabbits,higher viscosity iodixanal caused more hypoxia and injury of kidney.2.After the contrast agent iodixanol injection,the kidney damage was most serious at 24h.3.For diabetic nephropathy rabbits,the high viscosity contrast agent significantly prolonged the time of kidney injury.4.The functional magnetic resonance parameters R2*,FA and ADC have important value in evaluating the oxygen content of renal tissue and the pathological changes of renal response.5.Hydration and atorvastatin have a prenventive effect on low and medium viscosity contrast agent-induced diabetic nephropathy kidney injury;atorvastatin can reduce the degree of injury,hydration can shorten the recovery time,the combination use had the best preventive effects.6.Combination of hydration and atorvastatin can reduce the high viscosity contrast agent-induced diabetic nephropathy kidney damage,but will not significantly shorten the recovery time.