| Objective: Acute Kidney Injury(AKI)refers to a series of clinical syndromes caused by rapid decline of renal function in a short period of time caused by various reasons,which can gradually progress to chronic kidney disease.Folic acid(FA)-induced kidney injury is characterized by tubular damage and progressive interstitial fibrosis caused by oxidative stress.Renal tubular epithelial cell death caused by the production of a large number of reactive oxygen species and the decline in antioxidant capacity is a well-known mechanism of oxidative stress-mediated kidney damage.Ferroptosis,which is caused by decreased antioxidant capacity and iron overload,is iron-dependent lipid peroxidation.It is one of the main causes of AKI.At present,there are no effective treatments to prevent the progress of AKI.Therefore,effective preventive medication is of great significance for improving the prognosis of AKI.Roxastat(FG-4592),a HIF stabilizer,is an inhibitor of hypoxia inducible factor(HIF)prolyl hydroxylase.Recent studies have found that HIF1α has antioxidant activity and has a certain protective effect in ischemic heart disease and spinal cord injury.In this study,FA-induced renal injury model was used to investigate the effect of pretreatment with FG-4592 on renal injury and its possible mechanism.Methods:1.Establish a model of FA-induced renal injury: the experimental group was administered FG-4592 for prevention.This study took 6 to 8 weeks of male C57 BL /6 mice as the research object,and used FA intraperitoneal injection to construct an acute renal tubular injury and renal fibrosis model.FG-4592 was given once 2 days before the FA administration,and the protective effect of FG-4592 on renal injury was observed.2.Test the model and evaluate the renal function: hematoxylin-eosin staing(HE)staining was to evaluate the histopathology of renal injury,preliminary proof of the establishment of a mouse model of acute tubular injury induced by FA,Serum creatinine(Scr)and blood urea nitrogen(BUN)kits were used to detect kidney function in each group of mice,renal injury molecule 1(KIM-1)levels were assessed by Western blot to evaluate renal tubular injury.3.Detection of inflammation-related indicators: Tumor necrosis factor α(TNF-α),interlukin 1β(IL-1β)were detected by immunohistochemical staining and Western blot.And macrophage biomarker F4 /80 was detected in tissue sections.4.Detection of ferroptosis-related indicators: immunohistochemical staining,Western blot and related kits were used to quantify the tubular lipid peroxidation metabolite 4-hydroxynonenal(4-HNE)and malondialdehyde(MDA)expression.Prussian blue iron staining and iron kit were used to detect the iron content in the kidney tissue of each group of mice.TUNEL staining was used to detect ferroptosis of renal tubular epithelial cells in mice.5.Detection of anti-ferroptosis indicators: Western blot and immunofluorescence were used to detect nuclear factor erythroid 2-related factor 2(Nrf2)protein expression and nuclear transcription levels.Realtime PCR,Western blot and immunohistochemical staining were used to detect the expression of related genes and proteins in the Nrf2/ARE pathway.Western blot was used to detect the expression of GSH antiporter protein(SLC7A11)and Ferroportin(Fpn)in mouse kidney tissues.The glutathione(GSH)kit was used to detect the level of GSH in the kidney tissue of mice in each group.6.Indicators for detecting anti-ferroptosis mechanism: Western blot was used to detect the expression of HIF-1α protein,Keap1 protein and PI3K/Akt signaling pathway related proteins.The PI3K/Akt signaling pathway inhibitor Wortmannin was used to detect changes in PI3K/Akt signaling pathway-related proteins in the kidney tissue in each group of mice.7.Detection of fibrosis-related indicators: Masson’s staining(Masson)was assessed renal interstitial fibrosis,and preliminary evidence of the establishment of FA-induced renal fibrosis in mice.Immunohistochemical staining and Western blot were used to detect the expression of fibronectin(Fn),collagen IV(Col IV),and vimentin to further evaluate the degree of renal fibrosis.Results:1.FG-4592 pretreatment alleviated FA-induced acute tubular injury in miceMice were harvested 2 days after FA injection.HE staining showed that renaltubules in the FA group were severely damaged,showing swelling and vacuolar degeneration of renal tubular epithelial cells,narrowing of the lumen,visible exfoliated cells and transparent casts in the lumen,and a large number of inflammatory cells infiltrated around cortical vessels,suggesting that successful acute renal tubular injury modeling was established.In addition,serum creatinine,urea nitrogen,and KIM-1(markers of renal tubular injury)were significantly increased in the FA group,while serum creatinine and urea nitrogen levels in the FA + FG-4592 group were significantly decreased,consistent with this result,the renal tubular injury was improved.The renal function and renal morphology of the control group and FG-4592 group were normal.2.FG-4592 pretreatment reduced FA-induced renal inflammation in miceTwo days after FA injection,immunohistochemical staining showed that the expression of TNF-α and IL-1β in renal tubular epithelial cells in the FA group was significantly increased.Western blot results also showed that the expression of TNF-αand IL-1β in the FA group was increased compared with the control group.In addition,immunohistochemical staining showed that tubulointerstitial macrophage infiltration increased in the FA group,while as compared to the FA group,the expressions of TNF-α and IL-1β and macrophage infiltration in the FA + FG-4592 group were significantly reduced.3.FG-4592 pretreatment reduced FA-induced ferroptosis in renal tubular epithelial cellsIn the FA group,expression of 4-HNE increased in both Western blot and immunohistochemical staining,the changes were reversed in the FA + FG-4592 group,indicating that lipid peroxidation of renal tubular epithelial cells was reduced with FG-4592 pretreatment.In addition,serial sections of immunohistochemical staining and Prussian blue iron staining showed that MDA protein on membrane expression and intracellular iron content in renal tubular epithelial cell in the FA + FG-4592 group were significantly reduced compared with the FA group,indicating that FG-4592 preventive medication reduced ferroptosis in renal tubular epithelial cells.4.Fer-1 treatment further proved ferroptosis existed in FA-induced renaltubular injury in miceFA-induced mice were treated with Fer-1.As compared to the FA group,histologic analysis showed noticeably alleviated renal injury in the FA + Fer-1 group.Similarly,serum levels of BUN and creatinine decreased in the FA + Fer-1 group,immunostaining showed decreased levels of MDA and 4-HNE,and TUNEL staining showed decreased cell death.The above results prove that FA-induced mouse renal tubular epithelial cells exist ferroptosis and can be effectively inhibited by Fer-1.5.FG-4592 pretreatment can reverse FA-induced reduction of mouse kidney HIF-1α proteinWestern blot results showed that the expression of HIF-1α protein in the kidney of FA mice was significantly reduced compared with the control group,while the expression of HIF-1α in the FG-4592 group and the FA + FG-4592 group was higher than that in the FA group,of which FG-4592 group had the most expression.6.FG-4592 pretreatment activated Nrf2/ARE pathway in mouse kidneyWestern blot showed that the expression of Nrf2 protein in kidney nucleus and cytoplasm of mice in the FA group was significantly reduced compared with the control group,while the expression of Nrf2 in the FA + FG-4592 group was increased compared with the FA group,but the Nrf2 expression in the FG-4592 group was the highest in all groups.Immunofluorescence staining also showed that compared with the control group,Nrf2 showed extensive nuclear transcription in the FG-4592 group,while FA + FG-4592 group reversed the decrease Nrf2 nuclear transcription in the FA group.Immunohistochemical staining showed that compared with the control group,the protein expressions of GPX4 and HO-1 downstream of Nrf2 in the FA group were significantly reduced,and the protein levels of GPX4 and HO-1 were reversed in the FA + FG-4592 group.Consistent with the results of immunohistochemistry,the results of Realtime PCR and Western blot showed that compared with the control group,the levels of HO-1 and GPX4 mRNA and protein in the FG-4592 group increased,while compared with the control group,the FA group significantly decreased.The levels of HO-1 and GPX4 mRNA and protein were increased in the FA + FG-4592 group as compared to the FA group.7.FG-4592 pretreatment increased GSH content and the protein expressions of SLC7A11 and Ferroportin in mice kidneyThe GSH kit results showed that compared with the control group,the GSH content of the FG-4592 group increased,the GSH content of the FA group decreased,and the FA + FG-4592 group reversed the reduced GSH content.Western blot results showed that compared with the control group,the downstream target proteins SLC7A11 and Ferroportin of Nrf2 in the FA group were significantly down-regulated.Compared with the FA group,the protein expressions of SLC7A11 and Ferroportin in the FA + FG-4592 group were up-regulated,consistent with an increase in glutathione levels and a decrease in iron content.8.FG-4592 pretreatment reduced ferroptosis through Akt/GSK-3β/Nrf2pathwayWestern blot results showed that compared with the control group,the levels of p-Akt/Akt and p-GSK-3β/GSK-3β in the FA group were significantly reduced,and compared with the FA group,p-Akt/Akt and p-GSK-3β/GSK-3β increased significantly in the FG-4592 group and FA + FG-4592 group.The PI3 K inhibitor Wortmannin was used to inhibit the PI3 K / Akt signaling pathway.Western blot results showed that compared with the FA group,the expressions of p-Akt,p-GSK-3β,Nrf2,GPX4,and HO-1 in the FA + Wortmannin group were reduced.Consistently,compared with the FA + FG-4592 group,the expression of p-Akt,p-GSK-3β,Nrf2,GPX4,and HO-1 was reduced in the FA +FG-4592 + Wortmannin group,that is,Wortmannin reduced that the FG-4592 pretreatment activated Nrf2.9.FG-4592 pretreatment delayed renal interstitial fibrosis 14 days after FA injectionMasson staining showed a significant increase in renal tubulointerstitial collagen fibers in the FA group,and immunohistochemical staining showed a significant increase in the deposition of renal interstitial fibronectin and type IV collagen in the FA group.Western blot results also showed that fibronectin and type IV collagen levels of the kidneys in the FA group were significantly higher than those in the control group.These results indicate that we successfully established a model of renalfibrosis in mice 14 days after FA injection.Compare with the FA group,the expression of collagen fibers,fibronectin and IV collagen in the FA + FG-4592 group was significantly reduced.Conclusion:1.FG-4592 pretreatment ameliorated acute tubular injury and inflammation,which delayed the renal interstitial fibrosis induced by FA in mice.2.FG-4592 pretreatment reduced FA-induced ferroptosis in renal tubular epithelial cells in mice through Akt/GSK3β/Nrf2 pathway. |
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