The Effect Of High Glucose And Hyperlipemia On Human Mesangial Cells By Proteomics Analysis

As a result of dramatic change in the dietary structure, the increasedincidences of diabetes mellitus and hyperlipemia have caused more thanever before the renal lesions. Studies have indicated the contributive roleof renal hemodynamics, non-enzyme glycosylation of proteins,activation of polyalcohol channel, proliferation and apoptosis of cells inthe occurence and development of diabetic nephropathy (DN). Inaddition lipid deposition, a major risk leading to compromisedmovement and contraction of HMC facilitates the advancement of DN.Though intensive investigations have been made on the role ofmesangial cells in the renal lesions associated with diabetes mellitus andhyperlipemia, the exact mechanism of the disease still remains unknown.The proliferation of mesangial cells and mesangial matrix pose to be thefundamental pathological changes, however it still remains unknownwhich signal transduction pathways, ion channels and proteins areinvolved in such complicated changes. Especially the interrelationshipand network between the contributing factors remain poorly understood,and we think it necessary to establish a comprehensive study of thesefactors. Genomics and Proteomics researches, a best acceptedapproaches to health study, focus from a general perspective on thefactors affecting human health thus avoid the discordance between thephenotype and genotype.In our study, two-dimensional gel electrophorograms obtained from human mesangial cells (HMC) affected by high glucose, Ox-LDL andhigh glucose plus Ox-LDL respectively presented different protein spots,a further mass chromatographic analysis of which identified 15 proteins.Our study provides a new vision to reveal the pathogenesis of diabeticnephropathy.Objective: To investigate proteomics of HMC under the effects ofhigh glucose, Ox-LDL and high glucose plus Ox-LDL respectively.Methods:a) Human mesangial cells were cultured in vitro in different mediums asof normal glucose level(NC group), high glucose level(HG group),Ox-LDL(OX group), high glucose plus Ox-LDL(HO group) andManicol(OC group) for 48 hours.The total proteins were extractedrespectively through cell spallation and were gathered. All thesamples were analyzed using two-dimensional gelelectrophoresis.The first electrophoresis was carried by immobilized pH gradient(IPG) isoelectric focusing system, the second electrophoresis wasprogressed by vertical SDS-PAGE system, then the gels were stainedby coomas brilliant blue.b) The two type stained gels were analyzed, the protein spots werematched and compared by PDQuest image analysis software. Thestatistical method was applied to calculate the 2DE results.c) The procedure was duplicated, optimized and improved.d) Twenty dominant differential protein spots in 2DE gels were chosen and digested by trypsin. After digestion the amino acid sequences andpeptide fingerprints were analyzed by matrix-assisted laserdesorption/ionization time of flight mass spectrometry (MALDI-TOF-MS).e) The amino acid sequences and peptide fingerprints were search in web from MASCOT database for identification the proteins.f) The function of the identified proteins were searched in PDB-Protein Data Bank and SWISS-PROT database.Results:1. Reproducible results were acquired by using the optimized 2DEtechnique.2. 15 Separation gel were obtained by 2-DE (there were three gel pergroup).They were analyzed by PDQuest image softward. The resultsshowed the average spots of 3 gels in NC group were 896±51,733±34 spots were matched with an average matching rate of 81.9％;the average spots of 3 gels in HG group were791±86, 651±74 spotswere matched with an average matching rate of 83.5％; the averagespots of 3 gels in OX group were 698±69, 591±54 spots werematched with an average matching rate of 84.7％; the average spotsof 3 gels in HO group were 659±56, 531±42 spots were matched withan average matching rate of 80.6％; the average spots of 3 gels in OCgroup were 787±63, 657±63 spots were matched with an averagematching rate of 83.5％. The average position deviation of matched spots in IEF direction in NC group was 1.08±0.21 mm, and1.78±0.64 mm in SDS-PAGE direction;. the average positiondeviation of matched spots in IEF direction in HG group was1.04±0.13mm, and 2.05±0.64mm in SDS-PAGE direction; theaverage position deviation of matched spots in IEF direction in OXgroup was 1.25±0.25mm, and 1.89±0.5164mm in SDS-PAGEdirection; the average position deviation of matched spots in IEFdirection in HO group was 1.32±0.21mm, and 2.21±0.76mm inSDS-PAGE direction; the average position deviation of matchedspots in IEF direction in OC group was 1.12±0.17mm, and2.12±0.37mm in SDS-PAGE direction. Among the differentiallyexpressed 42 spots between NC and HG group, 24 spots wereexpressed only in NC group and 18 spots were highly expressed inHG group. Among the differentially expressed 33 spots between NCand OX group, 22 spots were expressed only in NC group and 10spots were highly expressed with 1 spot lowly expressed in OXgroup. Among the differentially expressed 35 spots between NC andOC group, 27 spots were only expressed in NC group and 8 spotswere highly expressed in OC group. Among the differentiallyexpressed 26 spots between NC and HO group, 8 spots were onlyexpressed in NC group and 13 spots were highly expressed with 5spots lowly expressed in HO group. 20 dominant differential proteinswere analyzed by peptide mass finger(PMF) and 15 proteins were identified. These proteins were involved in cytoskeleton, cellularjunction, metabolism, signal transduction and nucleic acidmetabolism.Conclusion:1. Proteomics analysis reveals that high glucose promotes proliferationof HMC and accumulation of mesangial matrix which may be theresult of changed expression of cytoskeleton protein, connexins andthe proteins associated with nucleic acid metabolism. Ox-LDLcaused higher expression of vimentin and PKP4 (a member ofconnexin family) and lower expression of protein associated withnucleic acid metabolism, that may play a contributive role tomesangial cell apoptosis. The expressions of the proteins of HMCimplicated with cytoskeleton, MAPK signal transduction channel andnucleic acid metabolism were changed when exposed to theenvironment of high glucose and Ox-LDL, which may accelerate theadvancement of diabetic nephropathy by altering the normal proteinsynthesis, differentiation and apoptosis of HMC, mesangial matrixdegradation. Diabetic patients complicated with hyperlipidemiausually suffer a more severe renal impairment.2. Among the 15 identified proteins, The expression of PolypeptideN-acetylgalacto Saminyltransferase 9 in renal tissue wasauthenticated in our study; moreover PKP4 and hemidesmosomalplaque protein belong toβ-Catenin family found in human mesangial cells in our study had only been reported before in endothelia.3. Manicol, as a classic high osmolar control, can cause abnormalprotein expressions in HMC.