The Effect And Mechanism Of VDR On The Oxidative Stress Of Ischemic Kidney Injury

Background:Ischemic injury is a common clinical cause of acute kidney injury(AKI),which occurs after trauma,major cardiac surgery,and kidney transplantation.Severe ischemic injury results in irreversible damage to renal structure,which affects the prognosis and outcome of these disease.Kidneys are organs with high oxygen consumption.The continuous ischemia and hypoxia and subsequent blood flow reperfusion lead to mitochondrial dysfunction,and the increase of free radical generation destroys the redox balance and induces oxidative stress state.Glutathione peroxidase family(GPXs)is an important component of the body’s antioxidant defense system,in which GPX3 is an important internal antioxidant synthesized and secreted by renal tubular epithelial cells,which is closely related to the occurrence and development of a variety of kidney diseases.Nuclear transcription factor VDR participates in many physiological and pathological processes of kidney by regulating the expression of target genes,but its effect on oxidative stress in ischemic kidney injury has not been reported.In the early stage of this research,transcriptome sequencing results of renal cortex of Vdr knockout mice showed that the expression of antioxidant gene Gpx3 was down-regulated,suggesting that VDR has a potential regulatory effect on Gpx3.Based on this,this study aims to explore the role of VDR on oxidative stress in ischemic kidney injury and elucidate its transcriptional regulation mechanism on the antioxidant gene GPX3.Methods:1.Serum and kidney biopsy specimens from patients clinically diagnosed with AKI were collected,and serum specimens from healthy people and paracancerous normal kidney tissues from patients undergoing renal cancer surgery were used as control group.The levels of NGAL,KIM-1 and GPX3 in serum and the expression of VDR and GPX3 in renal tissues were examined.2.The mouse model of acute renal injury induced by ischemia-reperfusion was established using wild-type C57BL/6J mice,Vdr gene knockout mice(VDR-KO)and VDR-OE mice constructed in the background of C57BL/6J mice.The changes of renal function,serum NGAL and GPX3 and the pathological changes of renal tissues were detected in each group.Reactive oxygen species(ROS)generation and oxidative stress indexes such as MDA and 8-OHDG were detected by fluorescence probe.The distribution and expression of VDR and GPX3 in renal tissues of each group were detected by immunohistochemistry and immunofluorescence.The expression levels of VDR and GPX3 in renal cortex of mice were detected by RT-q PCR and western-blot.3.Human renal tubular epithelial cells(HK-2)were used to establish H/R induced acute cell damage model,and were pretreated with VDR agonist paricalcitol.Morphological changes of cells in each group were observed by phase contrast microscope.The production of intracellular reactive oxygen species(ROS)and the expression of MDA and 8-OHDG were detected by fluorescence probe.The apoptosis of different groups of cells was detected by flow cytometry.The expression levels of VDR and GPX3 in cells were detected by western-blot.4.The Gpx3 overexpressed HK-2 cell line and the Gpx3 knockdown HK-2 cell line were constructed by genetic engineering and the hypoxic-reoxygenation cell damage models were established.Morphological changes of different groups of cells were observed by phase contrast microscopy and apoptosis was detected by flow cytometry.The production of intracellular reactive oxygen species(ROS)and the expression of MDA and 8-OHDG were detected by FLUORESCENCE probe.Western Blot was used to detect the protein changes of intracellular GPX3.5.Bioinformatics analysis and prediction of possible VDR binding sites(VDRE)in the GPX3 promoter region and the VDR binding sites were further verified by chromatin immunoprecipitation(Ch IP)and q PCR.Luciferase reporter plasmid and VDR overexpression plasmid containing GPX3 promoter sequence were constructed respectively,and the transcriptional activity of GPX3 by VDR was determined by dual-luciferase reporter experiment.Results:1.The expression of VDR and GPX3 in renal biopsy tissues of AKI patients decreased compared with the control group;The serum GPX3 level was higher than that of the normal group and the trend was consistent with the changes of NGAL and KIM-1.2.In the model of ischemia-reperfusion induced acute kidney injury(I/R-AKI)in wild-type C57BL/6J mice,the renal function,serum NGAL and GPX3 in AKI group were significantly higher than those in sham group,and the renal tubule interstitial injury was aggravated,cell apoptosis was increased,and oxidative stress level was significantly increased.These biochemical and pathological changes were more pronounced in VDR-KO AKI mouse models.Pretreatment with VDR agonist paricalcitol or overexpression of VDR could partially reverse these changes.The expression of GPX3 in mouse kidney tissue decreased after I/R treatment,and the expression of GPX3 was up-regulated after paricalcitol pretreatment.The expression of GPX3 in renal tissues of VDR-KO mice was down-regulated,and the decrease was more obvious after I/R treatment than that in the control group,while the overexpression of VDR up-regulated the expression of GPX3 in renal tissues.3.After H/R treatment,the level of intracellular oxidative stress increased,the number of cell apoptosis increased and the expression of GPX3 decreased in HK-2 cells,which could be partially reversed by pretreatment with paricalcitol.Likewise,Overexpression of GPX3 in renal tubular epithelial cells can partially antagonize oxidative stress injury and apoptosis induced by H/R.However,there was no significant difference in the oxidative stress level and apoptosis of cells in the paricalcitol pretreatment with H/R group and the control with H/R group after GPX3 was knocked-down;4.The JASPAR predicted that there were multiple VDR binding sites in the GPX3 promoter,and the Ch IP-q PCR results showed that the promoter of GPX3 contained three VDR binding motifs.The results of the dual-luciferase reporter gene assay showed that the luciferase activity of the VDR overexpressed plasmid group was significantly increased compared with that of the wild-type GPX3 reporter gene group and reduced in the mutant GPX3 reporter gene group.Conclusion:1.The abnormal expression of VDR and GPX3 in serum and renal tissue is closely related to the degree of renal injury in ischemic AKI.2.VDR can inhibit oxidative stress injury induced by hypoxia and reoxygenation partly by up-regulating the expression of renal GPX3,thus exerting a renoprotective role in ischemic AKI injury.