Protective Effect Of FG-4592 On Contrast-induced Acute Kidney Injury

Research background:Contrast-Induced Acute Kidney Injury(CI-AKI)is the third most common cause of acute kidney injury(AKI)in hospitalized patients[1].However,detailed pathogenesis and effectual remedy remain elusive.Studies have shown that contrast agents mainly cause renal hemodynamic changes and cytotoxic effects,leading to renal ischemia/hypoxia,oxidative stress reaction,promote the damage and apoptosis of epithelial cells in the thyroid tube of the kidneys,causing a sharp decline in the glomerular filtration rate of the kidney[2].Numerous experimental studies have shown that oxidative stress is a key link in cell apoptosis.The endoplasmic reticulum stress(ERS)pathway is one of the important pathways of oxidative stress-mediated cell apoptosis,which can regulate kinase(PERK)/eukaryotic starting factor 2 alpha(eIF2 alpha)/C-EBP homologous protein(CHOP)pathway through non-folding protein reaction and calcium ion starting signal,resulting in apoptosis.But the exact molecular mechanism has not been fully clarified.Studies have shown that contrast agents can induce apoptosis in rat renal tubular epithelial cells by activating this signaling pathway[3],but the role of this signaling pathway in the pathogenesis of CI-AKI and its regulatory mechanism still need to be further studied.Rozadustat(FG-4592)is a new type of Hypoxia Inducible Factor(HIF)-Prolyl Hydroxylase(PH)inhibitor,which can regulate the molecular stability of HIF gene,Improve antioxidant capacity and reduce oxidative stress[4].In addition,studies have shown that FG-4592 can significantly reduce ischemia damage and apoptosis[5].Therefore,we speculate whether FG-4592 may play a protective effect on CI-AKI by reducing oxidative stress,regulating the PERK/eIF2α/CHOP signaling pathway through the ERS response pathway,and reducing the apoptosis of renal tubular epithelial cells.Purpose:In this study,iopromide 370 was used to construct rat CI-AKI model and human renal tubular epithelial cell injury model,and FG-4592 was administered to intervene the modals.Try to explore the effect of FG-4592 on CI-AKI and Its related molecular mechanism.Method:(1)CI-AKI model was constructed by iopromide 370(20mg/kg)injected to SD rats after 48 hour’s intervention of low,medium and high dose of FG-4592(5mg/kg·d,10mg/kg·d,20mg/kg·d)respectively.Blood was collected 24 hours later to detect Serum Creatinine(Scr)levels.ELISA was used to detect Kidney Injury Molecule 1 levels in kidney tissues.Kidney tissue sections were stained with HE and immunohistochemistry to observe the pathological changes of kidney tissue and the expression of Bax,Bcl-2 and HIF-1α.Real-Time Fluorescent Quantitative PCR(RT-PCR)and Western blotting(Western Blot,WB)wered used to detect the expression levels of Bax,Bcl-2 and HIF-1α in kidney tissues.The content of ROS was detected,and PERK,eIF2α and CHOP protein expression in kidney tissues were detected by WB.To explore the protective effect of FG-4592 on CI-AKI,the influence on oxidative stress and the regulation of apoptosis pathway.(2)Iopromide 370 at 76.89mg/ml induces human renal tubular epithelial cells(HK-2)injury,and at the same time,different doses of FG-4592(5μM,10μM,20μM)are given to intervene the cells for 48h,and the apoptosis is detected by flow cytometry.RT-PCR and WB were used to detect the expression levels of Bax,Bcl-2 and HIF-1α,and WB was used to detect the expression levels of PERK/eIF2α/CHOP protein,and to detect the intracellular ROS content.To explore the possible protective mechanism of FG-4592 on oxidative stress and human renal tubular epithelial cell apoptosis.Result:(1)Compared with the control group,the serum creatinine of the CI-AKI model group was significantly increased(P<0.01);compared with the CI-AKI model group,the serum creatinine of the FG-4592 intervention group at each dose was significantly decreased(P<0.05).(2)Compared with the control group,after HE staining of kidney tissue sections in the CI-AKI model group,the proximal curved renal tubules at the junction of the outer medulla are obviously damaged,with vacuolar degeneration,necrosis,brush border shedding,and tubular obstruction.The renal tubular injury in each dose of FG-4592 intervention group was improved in varying degrees;The renal tubular epithelial cell apoptosis,mitochondrial swelling,and mitochondrial cristae breakage can be found in CI-AKI model group under electron microscopy.The renal tubular injury in each dose of FG-4592 intervention group was improved in varying degrees.Kim-1 score was higher than that of the control group(P<0.01);compared with the CI-AKI model group,the Kim-1 score of the medium and high-dose FG-4592 intervention group decreased significantly(P<0.01).(3)RT-PCR and WB results:Compared with the control group,the expression level of Bcl-2 in the kidney tissue of the CI-AKI model group was significantly decreased(P<0.05),and the expression levels of Bax and HIF-1αwere significantly increased(P<0.05);Compared with the CI-AKI model group,the expression level of Bcl-2 and HIF-1α in renal tissue of each dose of FG-4592 intervention group were significantly increased(P<0.05),and the expression levels of Bax was significantly decreased(P<0.05).(4)Compared with the control group,the content of ROS in kidney tissue of the CI-AKI model group was significantly increased(P<0.05);compared with the CI-AKI model group,the content of ROS in kidney tissue of the intermediate and high-dose FG-4592 intervention group was significantly reduced(P<0.05).WB results showed that compared with the control group,the expression levels of PERK,eIF2α and CHOP protein in the kidney tissues of the CI-AKI model group were significantly up-regulated(P<0.05);compared with the CI-AKI model group,the middle and high-dose FG-4592 intervention group kidneys Tissue PERK,eIF2α and CHOP protein expression levels were significantly up-regulated(P<0.05).(5)Compared with the control group,the results of flow cytometry showed that,the apoptosis rate of HK-2 after induction of HK-2 with 76.89 mg/ml iopromide 370 increased significantly after 48 hours(P<0.01);at the same time,different doses of(5μM,10 μM)FG-4592 intervention treatment for 48h,the apoptosis rate decreased after the intervention group(P<0.05),especially in the 5μM FG-4592 intervention group(P<0.01).(6)RT-PCR and WB results showed that compared with the control group,76.89mg/ml iopromide 370 down-regulated HK-2’s Bcl-2 expression level(P<0.05),and up-regulated HK-2’s Bax and HIF-1α expression level(P<0.05);Compared with HK-2 cell injury model,the 5μM FG-4592 intervention group up-regulated the Bcl-2 and HIF-1α expression level of HK-2 cell injury model group(P<0.05),and down-regulated the expression levels of Bax(P<0.05).(7)Compared with the control group,the content of ROS in model group was significantly increased(P<0.05);the content of ROS decreased in the administration of 5μM FG-4592 to interfere with HK-2 cell injuy model group.(P<0.05).(8)WB test showed that compared with the control group,76.89mg/ml iopromide 370 can up-regulate PERK,eIF2α and CHOP protein expression levels:compared with HK-2 cell injury model(P<0.05),5μM FG-4592 can down-regulate PERK,eIF2α and CHOP protein expression levels(P<0.05).Conclusion:(1)The CI-AKI animal model was constructed by intravenous injection of 20mg/kg iopromide 370 after water deprivation.The pathological results showed that the renal tubular epithelial cells were degeneration,necrosis,mitochondrial swelling and spinal rupture,and blood creatinine and Kim-1 levels increased.FG-4592 can reduce the pathological damage of the kidney tissue in the CI-AKI animal model,decrease blood creatinine and Kim-1 levels,and play a role in protecting the kidneys.(2)76.89mg/ml iopromide 370 can induce human renal tubular epithelial cell damage and increase the apoptosis rate;5μM FG-4592 can decrease the apoptosis rate caused by iopromide 370.(3)FG-4592 can reduce the content of ROS,alleviate the oxidative stress response of kidney tissue or renal tubular epithelial cells in the CI-AKI model,and has the effect of anti-oxidative damaged.(4)FG-4592 can up-regulate the expression of Bcl-2 and HIF-1α mRNA and protein,and down-regulate the expression of Bax mRNA and protein,reduce the apoptosis of renal tissue or tubular epithelial cells in the CI-AKI model,and play a role in protecting the kidney.(5)FG-4592 can improve the oxidative stress state,and may reduce the damage of the kidney tissue or renal tubular epithelial cells in the CI-AKI model by regulating the PERK/eIF2α/CHOP pathway through the endoplasmic reticulum stress response pathway.