The Role Of Aryl Hydrocarbon Receptor In Omeprazole Induced Renal Injury

BackgroundOmeprazole is a kind of proton pump inhibitors(PPIs),which can specifically and non-competitively bind to gastric parietal cells and inhibit gastric acid secretion.Omeprazole is the first choice for the treatment of gastric acid-related diseases.The renal adverse reactions of omeprazole have been reported more and more in recent years.Clinical retrospective analysis found that the most common pathological type of renal injury caused by omeprazole is acute interstitial nephritis(AIN).Recent epidemiological studies have shown that there is a significant correlation between long-term use of omeprazole and the increased risk of Chronic kidney disease(CKD)in patients and is related to the worse renal outcome.However,the rodent model of CKD induced by omeprazole is yet to be established,and the mechanism behind the CKD in omeprazole users remains largely obscure.Aryl hydrocarbon receptor(AHR)is a ligand-activated transcription factor that mediates cell metabolic responses to environmental toxins and carcinogens.AHR can be activated by a series of compounds with different structures,such as environment,microflora,drugs and body metabolites,which indicates that AHR has a very complex ligand binding site.Recent studies have shown that AHR can be activated by a variety of endogenous ligands and induce the expression of downstream target genes.AHR regulates a variety of physiological and pathological events,including cell proliferation,differentiation,apoptosis,adhesion and migration.Recent clinical studies have shown that the activation of AHR in vivo is associated with cardiovascular disease,chronic kidney disease and renal cell carcinoma.omeprazole is a benzimidazole derivative in chemical structure.Prior studies have shown that AHR can be activated by omeprazole in liver and lung tissues.However,the relationship between AHR and omeprazole-induced kidney injury remains completely unknown.ObjectiveWe aim to observe the changes in mice renal function and mice kidney morphology alternation after continuous intraperitoneal injection of omeprazole.This study attempts to develop the omeprazole-induced CKD mouse model and elucidate the role of the AHR-CYP1A1/CYP1A2 pathway in the progression of omeprazole-induced renal injury.Method1.Observation of omeprazole-induced renal injury in vivo:Six to eight-week-old male C57BL/6 mice underwent unilateral nephrectomy and recovery for a week,omeprazole,was injected intraperitoneally with the same volume of normal saline in the control group for 2 months.During this period,the changes of early renal injury markers KIM-1 and NGAL in mice urine were detected by ELISA kit.When the renal injury indicators in urine were significantly changed,the early renal injury molecules and epithelial-interstitial transition related proteins in renal tissue were detected by immunoblotting and immunohistochemical method.At the same time,the levels of creatinine and urea nitrogen in mice were detected and histological staining was performed.The effect of omeprazole on renal injury was observed and we had established the CKD mouse model induced by omeprazole.2.In vitro experiment:human proximal tubular epithelial(HK-2)cells were stimulated with different concentrations of omeprazole for 6h,12h and 24h.First of all,CCK-8 cytotoxicity kit was used to detect the cell activity after stimulation,and the 50%inhibitory concentration(IC50)of HK-2 cell damage caused by omeprazole was calculated for following experiments.Secondly,Western blotting was used to detect the changes of renal tubular injury markers(KIM-1/NGAL),the expression of AHR in nucleus and cytoplasm,and the levels of downstream molecules of AHR(CYP1A1/CYP1A2)after omeprazole stimulation for 6h,12h and 24h.The process of activation and nuclear translocation of AHR was further verified by immunofluorescence.Finally,in order to explore the regulatory mechanism of AHR in OME-induced renal tubular epithelial cell injury,AHR antagonists CH223191 and OME were cotreated to HK-2 cells,and the control group was given the same volume of normal saline.Western blotting was used to detect the expression of AHR in nucleus and cytoplasm,AHR downstream molecules(CYP1A1,CYP1A2)and renal tubular injury markers(KIM-1,NGAL)at 6h/12h and 24h respectively.Results1.First of all,the early renal injury indexes KIM-1 and NGAL in urine and renal tissue of mice treated with omeprazole for 4 weeks were significantly increased(P<0.05).Pathological staining of renal tissue slide showed local renal tubular atrophy and scattered lymphocyte infiltration in renal tubulointerstitium,but the levels of creatinine and urea nitrogen in mice were barely increased.Secondly,after 8 weeks of continuous action of omeprazole in mice,we established an omeprazole-induced CKD mouse model.The levels of creatinine and urea nitrogen in mice increased significantly(P<0.05).Renal tissue staining showed that renal tubular necrosis and atrophy,with a large number of lymphocyte infiltration in omeprazole group at the 6th week,and renal tubular necrosis and atrophy and interstitial fibrosis were still appeared at the 8th week.Finally,we found that the expression of α-SMA and Vimentin in renal tubular epithelial cells increased significantly during omeprazole-induced renal injury.The expression level of E-cadherin decreased significantly.Which indicated that renal tubular epithelial cells undergo epithelial-mesenchymal transition(EMT).2.To investigate the mechanism underlying the toxic effect of omeprazole on tubular cells,we established an in vitro cytotoxic model using human tubule epithelial cells(HK-2)Immunoblotting and cellular immunofluorescence staining showed that after 24h of omeprazole stimulation,firstly,the expression levels of renal injury markers KIM-1 and NGAL were increased in a time-dependent manner.At the same time,AHR was activated and transferred into the nucleus,and the expression of CYP1A1 and CYP1A2 in HK-2 cells was significantly up-regulated.Western blotting and immunofluorescence staining further showed that AHR antagonist could significantly reduce OME-induced AHR activation and nuclear translocation,and significantly inhibit the expression of AHR downstream molecules(CYP1A1,CYP1A2).At the same time,the levels of renal injury markers(KIM-1,NGAL)in HK-2 cells were significantly decreased(P<0.05).ConclusionIn this study,firstly,the omeprazole-induced CKD mouse model was developed.Secondly,in the process of omeprazole-induced renal injury,AHR activation was involved in the tubular damage and eventually promoted the development of CKD.The toxic renal effect of omeprazole can be ameliorated via inhibiting AHR-CYP1A1/CYP1A2 pathway.Our results suggest that AHR could be a potential role in omeprazole-mediated renal disease.