Study On The Mechanism Of The Effect Of Sidt2 Gene Deletion On Liver And Kidney Function Caused By Autophagy Disorder

Objective:To explore the changes in the autophagy process of mouse kidneys when the Sidt2 gene is deleted and its effect on the filtration function of mouse kidneys.Methods:In this experiment,a mouse model of systemic Sidt2 gene knockout was constructed by using Cre-Lox P recombinase system.The urine protein and urine creatinine of the two genotypes of mice were detected,the structure changes of the mouse kidneys were observed under electron microscope,and the changes of autophagy-related proteins were detected by western blot.Lentivirus was used to infect MPC5,SV40 MES 13 cells to obtain a cell model of Sidt2 gene knockout.Use Western blotting technology combined with autophagy inhibitory drug chloroquine,autophagy activator rapamycin drug experiment,real-time quantitative PCR,Ad-m Cherry-GFP-LC3B double labeling,immunofluorescence and other technologies to further explore the abnormal process of autophagy;Use Lyso Tracker and Lyso Sensor to detect the number of acid lysosomes and the internal environment;explore how the autophagy pathway of mouse kidney cells play a role when the Sidt2 gene is missing,and further explore the relationship between Sidt2,autophagy and kidney structure and filtration function.Results:1.Successfully constructed Sidt2 systemic knockout mice and constructed Sidt2knockout mouse kidney mesangial cells and podocyte lines;2.Morphological observation revealed that Sidt2^-/-mouse kidneys showed thickened basement membrane,foot process fusion,mitochondrial swelling,accumulation of autophagosomes and autophagolysosomes,and 24h urine protein,urine creatinine,urine protein/urine Increased creatinine;3.The expression of LC3-Ⅱ/I and P62 protein in mouse kidney tissues and cells removed by Sidt2increased,while the expression of autophagy-related proteins increased,and the expression of combined P62m RNA decreased,drug induction experiment,LC3B and LAMP1 fusion experiment,Ad-m Cherry-GFP-LC3B double-labeling experiment found that the increase of LC3-Ⅱ/Ⅰand P62 is due to the obstacle of autophagosome and lysosome fusion,which leads to the obstacle of autophagosome degradation;4.The number of acid lysosomes in kidney cells and podocytes of mice lacking Sidt2 decreased,the activity and expression of acid hydrolase in lysosomes decreased,the expression of transcription factor EB decreased,and the p H of lysosomes increased.Conclusion:The deletion of Sidt2 gene causes impaired lysosomal function and decreased number of acid lysosomes,leading to the formation and degradation of autophagic lysosomes,which ultimately manifests as abnormal kidney structure and function.Sidt2 is essential in maintaining the normal function of lysosomes and the physiological homeostasis of the kidney.Objective: To deepen the study of the relevant mechanism between Sidt2 and lipid metabolism and autophagy,and to clarify the close correlation between lysosomal membrane protein and lipid metabolism and autophagy.Methods: In the study of Sidt2 on TG transport,this experiment uses Sidt2-/-liver cells as an in vitro model,which is synthesized from TG under different conditions（normal,high-fat）,β-oxidation of fatty acids,and VLDL transport and secretion.Start with it and detect the expression status of related proteins.Lipin-1,SCD1,DGAT1,etc.related to TG synthesis.SCD1,a protein related to fatty acid beta oxidation,Proteins related to VLDL synthesis and transport,including Apo B,a structural protein directly related to VLDL synthesis,and MTP,which is necessary for fat transport,Proteins that help Apo B fold correctly in the ER cavity,such as PDI,Bi P,GRP94,and vesicle transport related proteins that play a major role in the transport and secretion of VLDL between ER and Golgi and cell membranes,such as ABCA1,CNX,GM130,etc.An important protein factor that affects the intracellular transport of VLDL-containing vesicles.In the study of Sidt2’s regulation of liver autophagy,the research is mainly based on the detection of the expression of key proteins LC3 and P62,and the detection of autophagy-lysosomal degradation pathways.Results: 1.Successfully constructed Sidt2 knockout human liver cell line（HL7702）;2.Under basic culture,the lack of Sidt2 in HL7702 cells leads to disorders of lipid metabolism,TG production decreases,fatty acid β oxidation increases,VLDL transport between the endoplasmic reticulum and Golgi is enhanced,and it mediates the transport of key proteins from the Golgi to the cell membrane.Decreased expression;3.In the environment of palmitic acid,the lack of Sidt2 leads to increased production of TG,decreased β-oxidation of fatty acids,and barriers to the transport and secretion of VLDL between the endoplasmic reticulum and the Golgi apparatus;4.The expression of LC3 and P62 proteins increased in the human liver cell line knocked out by Sidt2,the P62 entering the autophagolysosome degradation system was reduced,and the autophagolysosome degradation pathway was blocked;5.After Sidt2 gene deletion,autophagosomes and The disorder of lysosomal fusion is an important reason why the autophagy lysosomal degradation pathway is blocked.Conclusion: Under basic conditions,the deletion of Sidt2 gene inhibits the production of TG and the β-oxidation of fatty acids.The transport and secretion of TG between the endoplasmic reticulum and the Golgi are enhanced,and the key proteins that mediate the transport and secretion of the Golgi to the cell membrane are reduced.It is suggested that the transport of VLDL between the Golgi apparatus and the cell membrane is abnormal,and the formation and degradation of autophagolysosomes are blocked,leading to lipid accumulation in liver cells.Under the induction of a high-fat environment,the loss of Sidt2 leads to increased TG production and decreased β-oxidation of fatty acids.At the same time,the transport and secretion of VLDL between the endoplasmic reticulum and the Golgi are hindered,which leads to lipid accumulation.