The Impact Of ACBP On Ectopic Lipid Deposition And Lipophagy In Diabetic Nephropathy

Background: Ectopic lipid deposition and lipotoxic renal injury have been confirmed to play a very important role in the progress of diabetic nephropathy(DN).Recent studies have shown that lipophagy,a selective autophagy,can selectively recognize and degrade lipid and plays a key role in regulating lipid metabolism,maintaining lipid homeostasis and preventing excessive lipid accumulation.Acyl Co A binding protein(ACBP)is a lipogenic factor that can not only stimulate food intake,promote lipid synthesis,and inhibit lipid oxidation,but also relys on autophagy to be released and held back autophagy.However,whether it participates in lipophagy in DN is still unclear.Therefore,this study explores the role of ACBP in renal lipid deposition and lipophagy in DN renal tubules and its possible mechanisms.Method: Part 1.In vivo experiment: The experimental mice were randomly divided into four groups: db/m,db/db,db/db+virus control,db/db+ACBP sh RNA.Blood and urine samples were taken to detect blood creatinine,urine microalbumin,urine creatinine and other indicators,and routine pathological staining was used to evaluate the renal injury score.Detection of ectopic fat deposition in renal tubules by oil red O staining,immunohistochemistry,and immunofluorescence detection of ADRP expression on frozen sections of renal tissue.The degree of co-localization of lipid related protein ADRP and lysosomal marker molecule LAMP2 was observed by immunofluorescence multiple staining to detect the lipophagy in renal tubule.Part 2.In vitro experiment: HK-2 cells were cultured with high glucose(30m M glucose)to simulate the environment of diabetes in vitro,and ACBP si RNA and ACBP overexpression plasmid were given for intervention.Ectopic lipid deposition in proximal renal tubular epithelial cells was detected by oil red O staining,Western blot and immunofluorescence detection of ADRP expression.Observing the degree of co-localization between lipid dye BODIPY and lysosomal dye Lyso Tracker through immunofluorescence multiple staining,and detecting the expression level of related proteins such as LC3 by Western blot to detect intracellular lipophagy.Part 3.In vitro and in vivo experiments were performed to explore the underlying mechanism:(1)Immunohistochemistry and Western blot were used to detect the expression of DNA Damage Induced Transcript 4(DDIT4)in renal tissues of DN patients and db/db mice.(2)HK-2 cells were cultured with high glucose(30m M glucose)to simulate the environment of diabetes in vitro,and DDIT4 si RNA was given for intervention.Ectopic lipid deposition in cells was detected by oil red O staining and detection of ADRP expression by Western blot.(3)Western blot and immunohistochemistry were used to detect the expression of DDIT4 and p-mTORC1 in the kidney tissues of four experimental mice:db/m,db/db,db/db+virus control,db/db+ACBP sh RNA.HK-2 cells were cultured with high glucose(30m M glucose)to simulate the environment of diabetes in vitro,and ACBP si RNA was given for intervention.Western blot was used to detect the expression levels of DDIT4,p-mTORC1,LC3,p62 and other related proteins.(4)HK-2 cells were cultured with high glucose(30m M glucose)to simulate the environment of diabetes in vitro,and were intervened with ACBP si RNA,DDIT4 si RNA,ACBP si RNA and DDIT4 si RNA.Ectopic fat deposition in proximal renal tubular epithelial cells was detected by oil red O staining,detection of ADRP expression by western blot and immunofluorescence Observing the degree of co-localization between lipid dye BODIPY and lysosomal dye Lyso Tracker through immunofluorescence multiple staining to detect intracellular lipophagy.Result: Part 1.In the renal tissues of DN patients and db/db mice,the expression of ACBP was significantly increased,mainly located in the proximal renal tubules.Compared to db/db mice,db/db mice with knockout of the ACBP gene showed significant reductions in blood glucose and body weight,improved urinary protein levels,reduced renal tubulointerstitial damage,and significantly reduced lipid deposition.The degree of co-localization of lipid related protein ADRP and lysosomal marker LAMP2 significantly increased,indicating enhanced lipophagyPart 2.Compared with the high glucose group,the ACBP si RNA intervention group showed a significant decrease in lipid droplet deposition and a significant decrease in ADRP protein expression,indicating that knocking down ACBP reduced lipid deposition.The co-localization degree of lipid dye BODIPY and lysosomal dye Lyso Tracker was significantly increased,and the expression of autophagy indicator LC3II/I increased,indicating that knocking down ACBP enhances lipophagy.The intervention of ACBP overexpression plasmid produced the opposite results.Part 3.DDIT4 expression was significantly reduced in kidney tissue of DN patients and db/db mice.After DDIT4 si RNA intervention in HK-2 cells and high glucose stimulation,the expression of lipid related protein ADRP was significantly increased,and oil red staining showed a significant increase in lipid droplets.The intervention of ACBP si RNA in HK-2 cells and high glucose stimulation inhibited the DDIT4/p-mTORC1 pathway,increased the expression of LC3II/I,and decreased the expression of p62.The oil red staining of the HK-2 high glucose stimulation group showed a significant increase in lipid droplet deposition and a significant increase in ADRP protein expression,indicating an increase in lipid deposition.In addition,the autophagy index LC3II/I was significantly reduced,and the co-localization of lipid droplet dye BODIPY and lysosomal Lyso Tracker was significantly reduced,indicating impaired lipophagy.The ACBP si RNA intervention improved these injuries,and DDIT4 si RNA reversed the enhanced intracellular lipid autophagy caused by ACBP si RNA,partially reversing the reduced lipid deposition caused by ACBP si RNA.Conclusion: Lipophagy is weakened in DN renal tissue,ectopic lipid deposition worsens,and renal tubular damage is evident.Knocking out ACBP can enhance lipophagy in renal tissue of DN mice,significantly improve ectopic fat deposition,and have a significant renal protective effect.The DDIT4/p-mTORC1 pathway mediates the effect of ACBP on lipophagy and ectopic lipid deposition in renal tubular cells under high glucose conditions.ACBP may become a new target for preventing and treating ectopic lipid deposition in DN kidneys.