Study Of Lipid Expression In Renal Tubular Epithelial Cells And Their Exosomes Stimulated By High Glucose

Objective: The disorder of lipid metabolism is closely related to the development of diabetic nephropathy(DN).The disorder of lipid metabolism in inherent cells of the kidney is an important pathophysiological basis for the occurrence of DN.However,due to the heterogeneity of lipid composition and the limitation of previous detection methods,little is known about the overall composition of lipid in renal cells and the changes in diabetic state.Besides,exosomes can cooperate with autophagy-lysosome pathway,relieve intracellular stress and maintain intracellular homeostasis.Exosomes can also be used as a carrier for intercellular material transmission and information exchange.The lipid species,abundance and function of exosomes from different cell sources are different.There are few studies on the lipid composition of exosomes in diabetes and DN,compared with the attention of proteins and RNA.Understanding the differential lipidomics of renal cells and their exosomes are of great significance to elucidate the pathogenesis of DN and find new therapeutic targets.Methods: 1.Differential expression of lipid composition in renal tubular epithelial cells(HK-2)under high glucose stimulation.(1)The cells of normal control group NG)and high glucose group(HG)were cultured for 48 hours.(2)Quantitative analysis of lipid composition and differential expression under high glucose stimulation in HK-2 cells by isotope labeling combined with tandem mass spectrometry(n=3).2.Extraction and identification of exosomes.(1)The supernatant of both groups cells was obtained and the exosomes were extracted by Differential ultracentrifugation.(2)Identification exosomes by transmission electron microscopy,nanoparticle tracking(NTA)and Western blot.3.Isotope labeling and tandem mass spectrometry were used to quantitatively analyze the lipid composition of HK-2 exosomes and the differential expression under high glucose stimulation.4.Effect of lipid changes in HK-2 cells and their exosomes on related functions under high glucose stimulation.(1)The changes of multivesiclar bodies(MVBs)in HK-2 cells were observed by TEM.(2)Determination of exosome protein concentration by BCA method,The exosome secretion of the two groups was compared.(3)Autophagy-related protein LC3 and P62 were tested by Western blot.(4)PKH67fluorescent labeled exosomes,and Laser confocal microscopy was used to observe the uptake of HK-2 exocrine by glomerular mesangial cells.Results: 1.Differential expression of lipid composition in renal tubular epithelial cells(HK-2)under high glucose stimulation.(1)Quantitative detection of 421 lipid species in21 classes.(2)Obtained the average content of each lipid and the results showed that the PC average content was the highest in both groups.(3)GM3 d18:1/22:0,GM3d18:1/16:0,GM3 d18:0/16:0,GM3 d18:1/22:1 increased significantly in HG,LPE18:1,LPE,CL66:4(16:1),BMP36:3,CL70:7(16:1),CL74:8(16:1)decreased significantly in HG.(4)Quantitative detection of phosphoinositide(PIPs)expression in HK-2 cells,and PI3/5P 、 PI45P2 、 PI4 P expression increased in HG,but there was no significantly statistical difference.2.Extraction and identification of exosomes.(1)The microvesicles with double-layer membrane structure are relatively uniform in size.(2)The size of the extract was detected by NTA,and the results showed that the diameter of the microvesicles in the NG group was about 94±4nm,and the diameter of the microvesicles in the HG group was about 98±4nm(n=3).(3)The expression of CD63 and Alix which are markers of exosome were positive and the expression Calnexind which is endoplasmic reticulum chaperone molecule was negative.It was proved that the extracts were exosomes.3.Differential expression of lipid composition in exosomes secreted by HK-2 under high glucose stimulation.(1)Quantitative detection of 218 lipid species in21 classes.(2)Obtained the average content of each lipid and the results showed that the Cho average content was the highest in both groups.(3)PG36:1,FFA22:5,PC38:3,SM d18:1/16:1,CE-16:1,CE-18:3,CE-20:5,CE-22:6 increased significantly in HG,GM3d18:1/24:1,GM3 decreased significantly in HG.(4)Compared with HK-2 mother cells,TAG,PC,CL lipid species were decreased in exosomes.4.Effect of lipid changes in HK-2 cells and their exosomes on related functions under high glucose stimulation.(1)In HG group,both the MVBs volume and the number of intraluminal vesicles(ILVs)were increased in HK-2 cells.(2)A significant increase in exosome protein was observed in HG group by BCA method which means the production of exosome increased stimulated by high glucose.(3)Western blot results showed a significant increase in LC3 expression and decrease in P62 in HK-2 cells stimulated by high glucose for 48 hours.(4)Laser confocal microscopy results showed that PKH67 fluorescent labeled exosomes secreted by high glucose-stimulated HK-2 cells were obviously distributed around the nucleus and in the plasma of glomerular mesangial cells.Conclusions: 1.Quantitative detection of 421 lipid species in 21 classes in HK-2 cells.The average content of PC was the highest,and the identified TAG species were the most.GM3 d18:1/22:0,GM3 d18:1/16:0,GM3 d18:0/16:0,GM3 d18:1/22:1 increased significantly in HG,and LPE18:1,LPE,CL66:4(16:1),BMP36:3,CL70:7(16:1),CL74:8(16:1)decreased significantly in HG.2.Quantitative detection of 218 lipid species in 21 classes in exosomes secreted by HK-2 cells.The average content of Cho was the highest,and the identified PC species were the most.PG36:1,FFA22:5,PC38:3,SM d18:1/16:1,CE-16:1,CE-18:3,CE-20:5,CE-22:6 increased significantly in HG,and GM3 d18:1/24:1,GM3 decreased significantly in HG.3.Under high glucose stimulation,the number of intraluminal vesicles in MVBs of HK-2 cells increased,the production of exosomes increased significantly,and autophagy was activated,but there was no clear correlation with PIPs in renal tubular epithelial cells.4.Paracrine uptake of exosomes secreted by high glucose-stimulated HK-2 cells by glomerular mesangial cells is increased,and this may be associated with changes in exosomal lipid composition under high glucose stimulation.