Effects Of FoxO1 On Mitochondrial Autophagy In Renal Tubular Epithelial Cells Induced By High Glucose And Its Mechanism

BackgroundDiabetic nephropathy(Diabetes nephropathy,DN)is one of the most common microvascular complications of diabetes mellitus.At present,the pathogenesis of DN is complicated,and the mechanism is still not clear.It is thought that glomerular injury is the most important pathological change during the development of diabetic nephropathy to end-stage renal disease(ESRD).However,renal tubules and renal interstitium account for 90% of the total renal volume,and renal tubular cell damage plays an equally important role in the pathogenesis of DN.More and more evidence that in normal glomerular filtration and tubular proteinuria can be detected in the urine,which shows the change in the pathogenesis of DN in glomerular independent of renal tubules,renal tubular cell damage is the original DN change early,blocking the proximal renal tubular epithelial cell injury is a therapeutic target for more close to the source.Autophagy(autophagy)is the process of cell autophagy.It is the process of cell degradation of organelles and macromolecules by lysosome,and plays an important role in renal diseases.Autophagy can be divided into non selective autophagy and selective autophagy.Through selective autophagy,the organism clears organelles such as mitochondria,endoplasmic reticulum and peroxisomes,which are called autophagy,endoplasmic reticulum autophagy,and peroxisomal autophagy.Normally,autophagy is at the basal level in order to maintain normal physiological functions of cells.Abnormal autophagy may lead to the development and development of kidney disease.At present,the molecular biological mechanism of regulating mitochondrial autophagy in renal tubular epithelial cells is not clear.Forkhead transcription factor O1(Fox O1)plays an important role in antioxidant stress,transcription,translation,glucose and lipid metabolism.Previous studies in this group have found that Fox O1 can modulate mitochondrial autophagy and reduce excessive release of ROS,thereby improving oxidative stress in diabetic foot cells.In renal tubular cells,high glucose can lead to the decline of autophagy in renal tubular epithelial cells,which may be closely related to the pathogenesis of DN.BNIP3 belongs to the BH3-only subfamily of the Bcl-2 family proteins,it was found that BNIP3 could activate mitochondrial autophagy,previous studies have found that in acute kidney injury in renal proximal tubule cells,Fox Os can inhibit m TORC1 through the downstream target gene Bnip3,start autophagy,apoptosis.We conclude that the Fox O1/Bnip3 pathway may also play an important role in the renal tubules of diabetic nephropathy.Fox O1 can activate mitochondrial autophagy and reduce damage by up regulation of Bnip3 expression.ObjectiveBased on the previous research,combined with the latest research achievements at home and abroad,proposed by in vivo and in vitro under high glucose condition,regulating the expression of Fox O1 on mitochondrial autophagy in renal tubular injury and the influence,and to explore whether expression through activation of downstream BNIP3 play a role in,to provide new ideas for the prevention and treatment of DN.MethodIn the animal experiment,the diabetic mice model was induced by STZ using transgenic mice overexpressing male C57BL/6 wild mice and the same type Fox O1.The mice were divided into 4 groups: normal mouse control group(NG),transgenic mice normal group(TG),normal diabetic mice(DM),and transgenic diabetic mice TG-DM.Each group has 8.At the end of the 12 mice from heart blood serum creatinine urea nitrogen;metabolic cages to collect 24 h urine and 24 h urine total protein;mouse tail vein blood glucose after anesthesia,quickly stripped out of kidney in 4%,poly formaldehyde fixed for HE examination in 4% glutaraldehyde for cooling electronic microscopy.Fresh kidney frozen in liquid nitrogen,Western blot detection of Fox O1 expression levels and phosphorylation status(p-Fox O1),BNIP3,LC3,P62 protein expression;immunohistochemical detection of FOXO1 and BNIP3 in renal tissue of rats.The pathological changes of kidney were observed by transmission electron microscope.Overexpression / knockdown of FoxO1 in renal tubular epithelial cells using CRISPR/Cas9 technique was performed using BNIP3 knockout plasmid transfection.The experimental groups were as follows: 1.Normal glucose concentration group(NG,5.6mmol/L),high glucose(HG,25mmol/L),high glucose +Fox O1 up-regulated group(HG+KI),and The Bnip3 transfection group(si RNA HG+KIBnip3).Each group was treated with real-time quantitative PCR(q RT-PCR)to detect the expression levels of Fox O1 and m RNA in each group.The expression levels of Fox O1 and phosphorylation status(p-Fox O1),protein levels of BNIP3,LC3 and P62 were detected by Western and blot,and the production rate of ROS in each group was detected by flow cytometry.Result1.Fox O1 mice of renal tubular epithelial cells in expression and activity changes,compared with the NG group,no statistically significant differences between the Fox O1 m RNA and DM protein levels in mice kidney in renal tubular epithelial cells(P>0.05),p-Fox O1/ increased the protein level of Fox O1(P<0.05),compared with the DM group,TG-DM group and Fox O1 m RNA protein.The level of p-Fox O1/(P<0.05),lower the level of Fox O1(P<0.05).This indicated that high glucose could induce the decrease of Fox O1 transcriptional activity.2.Effect of Fox O1 on the BNIP3 pathway in animal test,compared with the NG group,DM group,BNIP3 protein level decreased,LC3 and P62 protein levels were significantly increased(P<0.05),compared with the DM group,TG+DM group increased the protein level of BNIP3,LC3,P62 protein levels were decreased(P<0.05).HE and scanning electron microscopy showed that the pathological changes of renal tubules in group TG-DM were lower than those in group DM.3.The changes of Fox O1 expression and activity in the cell groups: compared with group NG,the Fox O1,m RNA and protein levels in the HG group did not change significantly,and the levels of p-Fox O1/ and Fox O1 increased(P<0.05).In group HG+KI,the levels of Fox O1,m RNA and protein increased,and the levels of p-Fox O1/ and Fox O1 decreased(P<0.05)compared with those in group HG.This indicated that high glucose could induce the decrease of Fox O1 transcriptional activity,and overexpression of Crispr in renal tubular epithelial cells and knockdown of Fox O1 were successful.4.Effect of Fox O1 cells in the BNIP3 pathway: compared with NG group,HG group,BNIP3 m RNA and protein levels decreased,LC3,P62 and m RNA protein levels were elevated(P<0.05),compared with the HG group,HG+KI group increased BNIP3 and protein levels of m RNA,LC3,P62,m RNA and protein level were decreased(P<0.05).After transfection of BNIP3 knockout plasmids,the effect of up regulation of Fox O1 on these parameters was blocked.5.compared with the NG group,HG group significantly increased ROS levels in renal tubular epithelial cells(P<0.05),compared with the HG group,HG+KI group can significantly inhibit high glucose(P<0.05),transfection of BNIP3 knockout plasmid,effect of upregulation of Fox O1 on the above indexes was blocked.Conclusion:(1)high glucose can reduce the activity of Fox O1 expression in renal tubular epithelial cells,lead to mitochondrial dysfunction and increase intracellular ROS.(2)overexpression of Fox O1 can significantly increase mitochondrial autophagy level and decrease mitochondrial damage induced by high glucose in HK-2 cells under high glucose,the mechanism may be achieved by up regulation of BNIP3.