The Mechanism Of FXR Inducing Tubular Epithelial Cell Apoptosis In Renal Ischemia-reperfusion Injury

ObjectivesIn this study,we established an ischemia-reperfusion injury(IR)model in wild-type mice and farnesoid X receptor(FXR)knockout mice at the same time,and explored the role and mechanism of FXR in renal IR injury.We also established a Hypoxic-reoxygenation(HR)model in human renal tubular epithelial cells(HK-2)to verify the role of FXR in HR and its mechanism.This study will explore the mechanism of acute kidney injury and provide new therapeutic targets.Methods1.Establishment of IR model of right nephrectomy and left renal clippingThe C57BL/6 background FXR knockout mice from Jackson Laboratory,USA,was introduced and compared with wild type(WT)C57BL/6 mice.Nine male fxr^-/-mice aged 8-10 weeks plus nine male wild-type mice aged 8-10 weeks were selected.The experimental groups were as follows:1.WT sham group:right nephrectomy in wild type mice;2.WT IR group:right nephrectomy left kidney 20 or 25 minute-clipping in wild type mice was established;3.fxr^-/-sham group:right nephrectomy in fxr^-/-mice;4.fxr^-/-IR group:right nephrectomy left kidney 20 or 25 minute-clipping in fxr^-/-mice.After 24 hours of reperfusion,abdominal aorta blood was collected under microscope and left kidney tissue was collected.Biochemical methods were used to detect creatinine and urea nitrogen in mice;PAS staining was used to observe histological changes of kidney;myeloperoxidase immunohistochemical staining was used to observe neutrophil infiltration;Tunel fluorescence staining was used to observe apoptotic cells in kidney tissue;Western blot was used to detect the expression of FXR protein and caspase-3/cleaved caspase-3 protein in kidney tissue.2.Bone marrow transplantation modelMale fxr^-/-mice aged 8-10 weeks and wild type mice were selected as recipients.6-8hours after 8.0Gy X-ray irradiation,male fxr-/-mice and wild type mice were selected as donors.1*10⁶ bone marrow cells were extracted and transfused back to recipient mice by tail veins.Thirty days after bone marrow transplantation,recipient mice were established renal IR model.The experimental groups were:1.WT?WT:bone marrow cells from WT mice were transplanted to WT mice;2.fxr^-/-?WT:fxr^-/-bone marrow cells from WT mice were transplanted to WT mice;3.WT?fxr^-/-:WT mice were transplanted to fxr-/-mice;4.fxr^-/-?fxr^-/-:fxr^-/-bone marrow from WT mice was transplanted to fxr^-/-mice.Four groups of mice were established IR model with right nephrectomy left kidney 20 or 25 minute-clipping.After 24 hours of reperfusion,abdominal aorta blood was collected under microscope and left kidney tissue was collected.PCR gel electrophoresis was used to verify the successful establishment of the bone marrow transplantation model.Biochemical methods were used to detect creatinine and urea nitrogen.PAS staining was used to observe the histological changes of the kidney.Neutrophil infiltration was observed by myeloperoxidase immunohistochemical staining,and apoptotic cells in the renal tissues were observed by Tunel fluorescence staining.3.HK-2 cell culture and hypoxia-reoxygenation modelHK-2 cells were inoculated with DMEM/F12 medium containing 10%fetal bovine serum and cultured in 37?and 5%CO₂ incubator.The FXR in HK-2 cells was silenced by si RNA small interference technique.Western Blot technique was used to verify the silencing effect of target gene FXR.Control si RNA normoxia culture group:HK-2 cells were cultured in normoxia incubator(5%CO₂,21%O₂ and 74%N₂);FXR si RNA normoxia culture group:FXR si RNA HK-2 cells were cultured in normoxia incubator(5%CO₂,21%O₂ and 74%N₂);3.Control si RNA hypoxia culture group:HK-2 cells were cultured in hypoxia incubator(5%CO₂,1%O₂ and94%N₂);4.FXR si RNA hypoxia Culture Group Culture group:FXR si RNA HK-2cells were cultured in hypoxic incubator(5%CO₂,1%O₂ and 94%N₂).Western Blot was used to detect FXR protein and apoptosis-related protein caspase-3/cleaved caspase-3,p-BAD/BAD,Bcl-2 and Bax expression levels in cells;Tunel fluorescence staining was used to observe apoptotic cells in renal tissue.4.Intervention of BAD signaling pathway phosphorylation in HK-2 cellsPhosphatidylinositol 3-kinase inhibitor,Wortmannin was used to interfere with the phosphorylation of BAD protein in HK-2 cells.Control si RNA normoxic culture group:HK-2 cells were cultured in normoxic incubator,stimulated by 100 umol/L DMSO for 24 hours;2.FXR si RNA normoxic culture group:FXR si RNA HK-2 cells were cultured in normoxic incubator,stimulated by 100 umol/L DMSO for 24 hours;3.Control si RNA hypoxic culture group:HK-2 cells were cultured in hypoxia incubator,stimulated by 100 umol/L DMSO for 24 hours;4.FXR si RNA was low.Oxygen culture group:FXR si RNA HK-2 cells were cultured in hypoxia oxygen incubator,stimulated by 100 micromol/L DMSO for 24 hours;5.Control si RNA hypoxia culture+Wortmannin group:HK-2 cells were cultured in hypoxia incubator,stimulated by 100 micromol/L Wortmannin for 24 hours;6.FXR si RNA hypoxia culture+Wortmannin group:FXR si RNA HK-2 cells were cultured in normal oxygen incubator,stimulated by 100 micromol/L Wortmannin for 24 hours.Flow cytometry was used to detect apoptotic cells,and Tunel fluorescence staining was used to observe apoptotic cells in renal tissue.Results1.FXR knockout alleviates renal IR injury in miceAfter the establishment of IR model in mice,serum creatinine and urea nitrogen levels increased,reaching the peak in 24 hours,and the expression levels of FXR and cleaved caspase-3 protein in kidney tissue increased.After the establishment of IR model in FXR knockout mice,serum creatinine and urea nitrogen levels decreased,pathological changes decreased,neutrophil infiltration decreased,apoptotic cells decreased and cleaved caspase-3 protein expression decreased.2.FXR-mediated renal IR injury in renal tubular epithelial cellsAfter establishing bone marrow transplantation model,compared with WT?WT and fxr^-/-?WT groups,WT?fxr^-/-,fxr^-/-?fxr^-/-two groups of mice had significantly lower serum creatinine and urea nitrogen levels,less pathological damage and less neutrophil infiltration.There was no significant difference between WT?WT,fxr^-/-?WT and WT?fxr^-/-,fxr^-/-?fxr^-/-groups(p<0.05).Immunohistochemical staining of FXR indicated that FXR was mainly expressed in renal tubular epithelial cells.3.FXR gene silencing attenuates hypoxia-reoxygenation-induced HK-2 cell damageAfter 24 hours of hypoxia and reoxygenation,the activity of HK-2 cells decreased,the expression of FXR and cleaved capase-3 protein increased,and the difference was most obvious at 3 hours of reoxygenation.FXR si RNA significantly reduced the damage of HK-2 cells after hypoxia and reoxygenation and decreased cleaved capase-3 protein expression in HK-2 cells.4.FXR gene silencing promotes BAD phosphorylation in HK-2 cells induced by hypoxia and reoxygenationCompared with the Control si RNA group,the expression of p-BAD protein in FXR si RNA cells increased significantly after 24 hours of hypoxia and reoxygenation.Wortmannin,a phosphatidylinositol 3-kinase inhibitor,inhibited the phosphorylation of BAD,resulting in a marked increase in apoptotic cells.ConclusionsActivation of FXR in renal tubular epithelial cells aggravates renal IR injury,which may be achieved by inhibiting BAD phosphorylation and promoting apoptosis of renal tubular epithelial cells.