Study On The Effect And Mechanism Of Arginase 2 Facilitated Nitrosative Stress Of Renal Tubular Epithelial Cells On Iodine Contrast-induced Acute Kidney Injury

Background:Iodinated contrast medium has been widely used in the diagnostic and interventional procedures to enhance the accuracy of imaging.11%～50%patients who used contrast medium have been found to develop contrast-induced acute kidney injury(CI-AKI),which is a well-established important clinical problem with a tremendous individual and socioeconomic burden.Until recently,no effective strategies have been reported to prevent or treatment CI-AKI.As reported,old age,diabetes mellitus,and chronic kidney disease(CKD)are risk factors of CI-AKI.Especially for CKD,patients with different renal function stages may have different incidence of CI-AKI.However,many studies have found that none of the published methods can precisely estimate the renal function in Asian CKD patients.Consequently,developing a novel approach to accurately estimate glomerular filtration rate(GFR)for Chinese CKD patients is important to clear the confusion of incidence of CI-AKI in those patients.The mechanism of CI-AKI remains to be elucidated.After injection,appropriately all the contrast mediums can be excreted in the urine.However,contrast medium can be reabsorbed by proximal tubule,leading to high concentration of contrast medium within and surrounding the epithelial cell of renal tubule.Direct or indirectly toxicity of contrast medium can precipitate an abrupt loss of renal function.To elucidate the mechanism of CI-AKI,proteomics and transcriptomics have been performed using the renal tissue of CI-AKI mice and the high expression of arginase2(ARG2)is found in present study.ARG2 is predominantly expressed in the kidney of mice and human,which can hydrolyze L-arginine into urea and L-ornithine.Although the physiological function of ARG2 is not clear,abnormal ARG2 expression has been found in various renal disease models,such as diabetic nephropathy and ischemia-reperfusion induced AKI.Proximal renal tubules are the major sites for extrahepatic synthesis of L-arginine.Under physiological conditions,both ARG2 and nitric oxide synthase(NOS)use L-arginine as substrate.Abnormally elevated ARG2 competes with NOS for a common substrate,promoting the production of many free oxygen radicals(O2.-),which rapidly combines with NO to form peroxynitrite anoin(ONOO-),leading to the nitrosation of protein tyrosine and then inducing nitrosative stress.However,it has not been reported whether ARG2 plays an important role in CI-AKI by mediating nitrosative stress in renal tubular epithelial cells.Heme oxygenase-1(HO-1)is an important antioxidant enzyme that can catalyze the hydrolysis of heme to ferrous iron,carbon monoxide and biliverdin,then removing the peroxynitrite and playing an anti-nitrifying role.Elevated kidney HO-1 expression can reduce the nitrosative stress in ischemia-reperfusion or cisplatin-induced AKI.Proteomic analysis of present study found that HO-1 was significantly negatively correlated with ARG2,and silencing ARG2 expression could reverse the down-regulated protein expression of HO-1 in Iohexol treated HK-2 cells.Therefore,HO-1 may play an important role in ARG2 mediated nitrosative stress in renal tubular epithelial cells.Based on all the above,present study proposed that contrast medium may promote the transcription and protein expression of ARG2 in renal tubular epithelial cells,and then facilitate nitrosative stress through down-regulating the expression of HO-1 in CI-AKI.Thus,present study will be performed as follows:(1)analyzing the incidence of CI-AKI in patients with different comorbidities or different combined drugs;developing a more accurate renal function estimation formula for Chinese CKD patients and finding whether the incidence of CI-AKI is different for different CKD stages classified by different formulas;investigating the risk factors of CI-AKI;finding the potential strategies to prevent CI-AKI;(2)confirming the important role of ARG2 in the Iohexol induced nitrosative stress in renal tubular epithelial cells;and analyzing the potential role of ARG2 to be a diagnostic biomarker of CI-AKI in mice and patients;(3)determining whether ARG2 can facilitate nitrosative stress in renal tubular epithelial cell through down-regulating HO-1 in CI-AKI.Purpose:(1)investigating the incidence of CI-AKI in patients with different comorbidities or different combined drugs and the risk factors of CI-AKI;and finding the potential strategies to prevent CI-AKI;(2)confirming the important role of nitrosative stress in renal tubular epithelial cells in CI-AKI,and evaluating whether ARG2 could be used as a diagnostic biomarker;(3)elucidating that contrast medium may promote the expression of ARG2,and then facilitate nitrosative stress in renal tubular epithelial cell through down-regulating HO-1 in CI-AKI.Methods:Methods for the first part:The incidence and risk factors of CI-AKIA cohort of patients who used contrast medium from 2007 to 2021 was retrospectively screened and constructed.Demographic information and clinical diagnostic and treatment data were collected for each patient.The incidences of CI-AKI in different groups were compared.Furthermore,a novel approach based on a multi-center,multi-ethnic Chinese large CKD population was developed.And then,the novel approach was compared to other formulas in the renal function stage classification and incidence of CI-AKI in CKD patients.Univariate and multi-variate logistic regressions were then performed to find the risk factors of CI-AKI.Methods for the second part:The effect of ARG2 mediated nitrosative stress in renal tubular epithelial cells in CI-AKI1.The role of nitrosative stress in renal tubular epithelial cells in CI-AKIFeTPPS(positive drug of anti-nitrosative stress)was pretreated in HK-2 cells treated with Iohexol,respectively.MTT assay was used to test the cell viability.Immunofluorescence was used to determine the levels of peroxynitrite,and mitochondrial membrane permeability pore(mPTP)opening.Apoptosis was detected by TUNEL.Western blot analysis of 3-NT,Cleaved caspase-3,Caspase-3 were performed.Mice were pretreated with FeTPPS before Iohexol treatment.Orbital blood was collected 24h after modeling to detect serum creatinine and urea to evaluate renal function.Renal tissue was collected for HE staining to assess tubular injury.2.ARG2 mediates CI-AKI by inducing nitrosative stress in renal tubular epithelial cells(1)The expression of ARG2 in HK-2 cells and mice treated with IohexolThe renal tissue of CI-AKI mice was collected 24h after Iohexol treatment.Transcriptome and proteomic analysis were performed.The tissue and sub-tissue localization of ARG2 were determined by immunofluorescence co-localization.The expression of ARG2 in HK-2 cells were detected by Real-time PCR and Western blot.(2)To determine whether ARG2 mediates CI-AKI through inducing nitrosative stress in renal tubular epithelial cellsHK-2 cells,ARG2 gene silencing or overexpression HK-2 cells were treated with Iohexol in L-arginine free medium or not,respectively.MTT assay was used to test the cell viability.Immunofluorescence was used to determine the levels of mPTP opening,peroxynitrite and superoxide anion.Apoptosis was detected by TUNEL.Western blot analysis of ARG2,3-NT,Cleaved caspase-3,Caspase-3 were performed.The activity of ARG2 was tested.Immunofluorescence was used to determine the activity of NOS.Western blot analysis of iNOS,nNOS,and eNOS were performed.Mice,and ARG2 KO mice were pretreated with nor-NOHA,the activity inhibitor of ARG2,before treated with Iohexol.Urine was collected for 24h,and urine volume was recorded.Orbital blood was collected 24h after modeling to detect serum creatinine and urea to evaluate renal function.Renal tissue was collected for HE staining to assess tubular injury.Serum ARG2 level was detected.Western blot analysis of ARG2 was performed.(3)To evaluate the potential of ARG2 as a diagnostic biomarker in CI-AKI mice and CI-AKI patientsPatients with high-risk of CI-AKI were enrolled in a cohort.Propensity score matching was used to match the control group that did not use contrast medium or group that used contrast medium but without CI-AKI by 1:1:1.Blood samples were collected from the patients within 72h of Iohexol treatment.Serum creatinine,KIM-1,NGAL and ARG2 levels were measured.Differences between groups were compared.The correlation between serum ARG2 and serum creatinine,KIM-1 or NGAL were analyzed.Methods for the third part:The effect and mechanism of HO-1 in ARG2 mediated CI-AKI1.The effect of HO-1 on ARG2 mediated nitrosative stress in renal tubular epithelial cellsCoPP(HO-1 agonist)and HO-1 siRNA was pretreated in HK-2 cells treated with Iohexol.MTT assay was used to test the cell viability.Immunofluorescence was used to determine the levels mPTP opening,and superoxide anion.Western blot analysis of HO-1 was performed.Correlation analysis of proteomic analysis was performed.Western blot analysis of HO-1 in the renal tissue was performed.2.To study the regulatory relationship between ARG2 and HO-1HK-2 cells,ARG2 gene silencing or overexpression HK-2 cells were treated with Iohexol,respectively.Real-time PCR or western blot analysis of HO-1 was performed.HK-2 cells,or HO-1 gene silencing HK-2 cells were treated with Iohexol,respectively.Western blot analysis of ARG2 was performed.Mice were pretreated with nor-NOHA,the inhibitor of ARG2,before treated with Iohexol.Western blot analysis of HO-1 was performed.HK-2 cells,or ARG2 gene silencing HK-2 cells were pretreated with cycloheximide(CHX)for 0h,2h,6h,8h,and 10h,respectively,before treated with Iohexol.Western blot analysis of HO-1 was performed.Results:Results for the first part:The incidence and risk factors of CI-AKIFive kinds of contrast medium,Urografin,Iopamidol,Iohexol,Iodixanol and Ioversol,were used between 2007-2021,showing a gradual increase.Among the five kinds of contrast agents,non-ionic dimer isotonic iodine contrast medium(iodixanol)had the highest annual increase of 2.4 times.In our hospital,a total of 76 648 patients who used contrast medium were retrospectively enrolled,and the overall incidence of CI-AKI was about 9.69%.Patients with CI-AKI were older and had higher level of basal creatinine.The incidence of CI-AKI was 26.19%in patients admitted to intensive care units and 14.18%in patients with CKD,respectively.The incidence of CI-AKI in patients who received diuretics,furosemide,NSAIDs,glycopeptide antibiotics,quinolones or third generation cephalosporins was 14.19%,14.58%,12.24%,15.28%,12.84%and 11.09%,respectively,which was significantly higher than in those who did not.The incidence of CI-AKI was significantly lower in patients using ACEI/ARB,statins,or vitamin C(7.88%,7.23%and 8.13%,respectively)than in patients not using these drugs.The P30 of Xiangya-s was 79.61%,78.45%and 79.05%in the Third Xiangya Hospital of Central South University,the second Xiangya Hospital of Central South University and the First Affiliated Hospital of Xinjiang Medical University,respectively,which was significantly higher than that of the 12 published renal function estimation formulas.P30 in Uygur population was 80.41%.To sum up,Xiangya-s formula could estimate GFR more accurately when validated in the multi-center,multi-ethnic cohort of Chinese CKD patients.Renal function stages classified by different equations,including Xiangya-s formula,MDRD formula and CKD-EPI formula,could affect the incidence of CI-AKI.Multivariate Logistic regression analysis showed that the level of urea or triglyceride,and combined use with furosemide were independent risk factors for CI-AKI,while combined use with vitamin C might be a protective factor for CI-AKI.Results for the second part:The effect of ARG2 mediated nitrosative stress in renal tubular epithelial cells in CI-AKI1.The role of nitrosative stress in renal tubular epithelial cells in CI-AKIFeTPPS can significantly improve cell viability in Iohexol treated HK-2 cells,and significantly reverse Iohexol-induced increase in the levels of peroxynitrite,mPTP opening and apoptosis.3-NT and Cleaved caspase-3/Caspase-3 protein expression were significantly downregulated.FeTPPS pretreatment can significantly improve renal function in CI-AKI mice.2.ARG2 mediates CI-AKI by inducing nitrosative stress in renal tubular epithelial cellsProteomics and transcriptome analysis of renal tissue of CI-AKI mice showed that expressions of ARG2 protein and mRNA were significantly up-regulated.The results of immunofluorescence co-location indicated that the renal ARG2 significantly upregulated in renal tubules of CI-AKI mice.Real-time PCR and Western blot results showed that Iohexol could significantly up-regulate the mRNA and protein expression of ARG2 in HK-2 cells.ARG2 gene silencing could significantly improve the cell viability and decrease the arginase activity of Iohexol treated HK-2 cells,while ARG2 overexpression could aggravate the decreased HK-2 cell viability induced by Iohexol.ARG2 gene silencing reversed Iohexol-induced increased mPTP opening,peroxynitrite and superoxide anion levels,and apoptosis,and significantly down-regulated Cleaved caspase-3/caspase-3 and 3-NT protein expression in HK-2 cells treated with Iohexol.In L-arginine free medium,ARG2 gene silencing reversed Iohexol-induced increases in superoxide anion production,and significantly down-regulated 3-NT protein expression.Western blot results showed that iNOS was the main subtype of NOSs in HK-2 cells.The protein expression of iNOS,nNOS and eNOS and NOS enzyme activity in HK-2 cells were not affected by Iohexol treatment or ARG2 gene silencing.ARG2 inhibitor down-regulated renal ARG2 expression,reduced serum ARG2 level,and improved renal function in CI-AKI mice.ARG2 KO mice had no significant decrease in renal function when constructing CI-AKI model.The serum ARG2 levels of CI-AKI mice and clinical patients were significantly higher than that of non-CI-AKI group.The serum ARG2 levels of the control group without contrast medium treatment was the lowest after matching with clinical characteristics.The serum levels of KIM-1 and NGAL in patients with CI-AKI were significantly higher than those in patients without CI-AKI.ARG2 significantly positively correlated with KIM-1 and NGAL.Results for the third part:The effect and mechanism of HO-1 in ARG2 mediated CI-AKI1.The effect of HO-1 on ARG2 mediated nitrosative stress in renal tubular epithelial cellsProteomic correlation analysis showed that HO-1 was negatively correlated with ARG2.Iohexol can significantly down-regulate the expression of HO-1 protein in HK-2 cells.CoPP,a HO-1 agonist,significantly reversed declined cell viability,mPTP opening and superoxide anion production in HK-2 cells treated with Iohexol.HO-1 gene silencing significantly accelerated Iohexol-induced decline in HK-2 cell viability and mPTP opening.2.The regulatory relationship between ARG2 and HO-1ARG2 gene silencing could significantly reverse Iohexol-induced down-regulation of HO-1 expression in HK-2 cells.ARG2 overexpression could down-regulate the HO-1 mRNA expression in HK-2 cells,and further aggravate the Iohexol-induced down-regulation of HO-1 protein expression in HK-2 cells.ARG2 inhibitor,nor-NOHA,significantly improved the downregulation of HO-1 expression in the renal tissue of CI-AKI mice.HO-1 gene silencing did not affect ARG2 protein expression in HK-2 cells and Iohexol-treated HK-2 cells.ARG2 induced down-regulation of HO-1 protein expression may not be related to reduced protein degradation.Conclusions:1.Iodixanol had the highest annual increase of 2.4 times.The incidence of CI-AKI was 9.69%.The incidence of CI-AKI was higher in CKD patients and those admitted to intensive care units.Patents with combined treatment of diuretics,furosemide,NSAIDs,glycopeptide antibiotics,quinolones or third generation cephalosporins were related to higher incidence of CI-AKI than those who did not.The incidence of CI-AKI was significantly lower in patients using ACEI/ARB,statins,or vitamin C than in patients not using these drugs.Urea,triglyceride,and combined use with furosemide were independent risk factors for CI-AKI,while combined use with vitamin C might be a protective factor for CI-AKI.2.Xiangya-s formula could accurately estimate GFR when validated in the multi-center,multi-ethnic cohort of Chinese CKD patients.Renal function stages classified by different equations,including Xiangya-s formula,MDRD formula and CKD-EPI formula,could affect the incidence of CI-AKI.3.ARG2 protein and mRNA was significantly up-regulated in the renal tubules of CI-AKI mice.The serum ARG2 levels of CI-AKI mice and clinical patients were significantly higher than that of non-CI-AKI group and control group without contrast medium treatment.Furthermore,ARG2 was significantly positively correlated with KIM-1 and NGAL.ARG2 may be a potential diagnostic biomarker of CI-AKI.4.Iohexol can up-regulate the expression of ARG2,and then facilitate nitrosative stress in renal tubular epithelial cells through down-regulating HO-1 in CI-AKI.