Effect Of Forkhead Box O1 On Endotoxin-induced Acute Kidney Injury In Renal Tubular Epithelial Cells

BackgroundSepsis-associated acute kidney injury(SA-AKI)is a life-threatening organ dysfunction caused by a dysregulated host response to infection.Endotoxin,also known as lipopolysaccharide(LPS),plays a key role in SA-AKI.Damage to renal tubular epithelial cells(RTECs)caused by endotoxemia and its triggered inflammatory factor cascade effect is an important cause of SA-AKI,but its specific molecular mechanism is still unclear.Forkhead box O1(FOXO1),as a transcription factor,can bind to reactive elements on DNA,and play a key role in many processes such as DNA damage/repair,stress,angiogenesis,sugar metabolism and tumorigenesis.Previous studies have found that FOXO1 is widely expressed in various renal parenchymal cells and participates in many kidney diseases such as diabetic nephropathy,renal fibrosis and lupus nephritis.However,whether FOXO1 participates in endotoxin-induce AKI in RTECs and related mechanism remained unclear.AimTo investigate the role of FOXO1 on endotoxin-induced acute kidney injury and related mechanism.Methods1)In vivo,the mouse model of endotoxin-induced AKI was induced by intra-abdominal injection of LPS(10 mg/kg).The expression of FOXO1 and PGC1-? in mouse kidney were determined by qPCR,western blot and immunofluorescence staining.Then we established a mouse model of renal overexpression of FOXOl by in situ injection of FOXO1 adeno-related virus in renal cortex before intra-abdominal injection of LPS.Renal function was detected.2)In vitro,human proximal tubular epithelial(HK-2)cells were cultured and stimulated with LPS(40?g/ml),then infected with FOXO1 overexpression adenoviruses.The cell viability was measured by MTT assay.The morphological changes of mitochondria were observed using mitotracker staining.Mito-SOX was used to detect the changes of mitochondrial superoxide content and the expression of FOXO1,Peroxisome proliferator-activated receptor y-coactivator 1-?(PGC1-?)and Bax was dectected in order to observe the damage of oxidative phosphorylation of mitochondria.Results1)In vivo experiment:(1)Compare with the control group,serum crestinine(SCr)and blood urine nitrogen(BUN)were significantly increased in mice of LPS group.The expression of FOXO1 and PGC1-? were decreased in the mice with endotoxin-induced AKI compared with the normal control,as well as mitochondrial complex I(Ndufsl)and-V(Atp5o)mRNA.PAS staining showed hypertrophy and vacuolar degeneration of RTECs,and stenosis of renal tubular lumen.The ultrastructure of RTECs,observed by transmission electron microscopy showed mitochondrial swelling,crest disappearance and fusion.(2)The expression of FOXO1 and PGC1-? were upregulated after injection of FOXO1 AAV.Meanwhile,the SCr and BUN level were down-regulated in the LPS+FOXO1 group,and the mitochondrial damage was alleviated,compared with the LPS group.2)In vitro experiment:(1)Compare with the control group,LPS stimulation could cause HK-2 cell viability decrease,mitochondrial fragmentation and increase of mitochondrial superoxide content,which could be attenuated by overexpression of FOXO1.(2)At the molecular level,FOXO1,PGC1-? was downregulated,pro-apoptotic protein Bax was upregulated,and mitochondrial complex I(Ndufb8),-V(Atp5g1)mRNA transcription level were downregulated induced by LPS,compare with the control group.ConclusionsIn endotoxin-induced AKI,the expression of FOXO1 were down-regulated.Over-expression of FOXO1 reduces renal injury and protects the structure and function of mitochondria,which proves that transcription factor FOXO1 plays a protective role in endotoxin-induced AKI.