The Mechanisms Of Diabetic Nephropathy Glomerulosclerosis Comparative Proteomics Research

Objective:Diabetic nephropathy is the leading cause of end stage renal disease, and is associated with increased cardiovascular mortality. The major renal pathological changes of diabetic nephropathy both developed from type 1 and type 2 diabetes are glomerular sclerosis. The major mechanism of diabetic nephropathy is not clear up to now, although oxidative stress, polyol pathway activation, advanced glycation end products, hemodynamics and various cytokines are presumed to be the main culprits of development of diabetic nephropathy. To explore the pathophysiologic mechanisms of diabetic nephropathy in depth, and improve our understanding of DN pathogenesis, proteomics strategy was performed to screen for dynamic proteomics profile of mesangial cells in response to high glucose condition and global changes of renal cortex protein expression in OLETF and LETO rats.Methods:1. Differential proteomics study of spontaneous type 2 diabetes model of OLETF and LETO rats.Spontaneous diabetic rat of OLETF and healthy control rats with the same genetic background LETO rats were used in this experiment. OLETF rats developed hyperglycemia, progressive increase of urinary protein excretion and glomeruloscerosis. OLETF and LETO rats were sacrificed at the age of 36 weeks, and the renal cortical total membrane and cytosolic protein were respectively extracted, and separated by. Two-dimensional gel electrophoresis and stained by colloidal Coomassie brilliant blue method. The gels were scanned with Lab Scan III system and analyzed by Imagemaster software 6.0. The determinant criterion of differential protein was statistical significance of the volume% of the same protein spot between the two groups of various time points (i.e. P value was less than 0.05 which obtained by t-test of heterogeneity of variance and then adjusted by false discovery rate (FDR)), and difference of volume% exceeding twofold compared with NC group. The differentially expressed proteins were subject to MALDI-TOF-MS and Mascot search engine analysis to get the molecular weight and pI value of the protein. Gene Ontology (GO) classification and over expression analysis of the differential proteins were conducted using GoMinerTM and BIGO software. Homology and conservation of phosphorylations sites of Ephx2 were further analyzed.2. Dynamic proteomic analysis of high glucose cultured rat glomerular mesangial cells.Primary glomerular mesangial cells of the 5th generation were cultured. Cells were cultured in 5.4 mM normal glucose (NG) and 30 mM high glucose (HG) for 0,8,16,72 hour and 25 day respectively. Cell total protein of both groups at different time point were extracted and separated by two-dimensional gel electrophoresis. The differential proteins were analyzed and identified by the same methods mentioned above. Protein interaction network were imported from IntAct Web Service Client, illustrated by cytoscape and analyzed by MCODE.Results:1. Compared with LETO rats:in the cytosolic fractions,9 protein spots (identified as Gapdh, Qdpr, Fah, similar to Protein C14orf159 mitochondrial precursor, Ephx2, Alb, Acadsb, Clybl) were down-regulated, and 8 (identified as Gapdh, Qdpr, Fah, similar to Protein C14orfl59 mitochondrial precursor, Ephx2, Alb, Acadsb, Clybl) were up-regulated (identified as Qdpr, Ephx2, Fthfd, Tf, Naprtl, Idh1, Fah, Cryab); In the membrane fractions,11 protein spots (identified as Uqcrfs1, Nrn11, Pbld, LOC683519, Calb1, Acadsb, Dmgdh, Etfb) were down-regulated and 10 (LOC683519, Qdpr, GMPK, Etfb, Uqcrfsl, Fah, Dmgdh, Gpx3, cytokeratin 8 polypeptide) up-regulated. The differential expressed proteins of OLETF and LETO rats were mainly located in the cytosol, mitochondria and cyto plasmic vesicle. Their molecular functions were mainly related to ion, vitamin and protein transportation, with the biological functions of oxidation-reduction reaction, electron carrier, transporter and antioxidant, and were primarily involved in cell communication, cell apoptosis, response to stress and cell metabolic process such as aromatic compound, oxygen and reactive oxygen species. GO analysis verified biology process related to those proteins were oxidation reduction, aromatic compound metabolic process, carboxylic acid metabolic process, oxygen and reactive oxygen species metabolic process, acute inflammatory response, fatty acid metabolic process, monocarboxylic acid metabolic process. MALDI-TOF-MS results suggested Qdpr,Ephx2,LOC683519,GMPK in different spots stand for different phosphorylation states. Homology of ephx2 was high, and Thr-50,Thr-59,Ser-70 of ephx2 were much conserved in rat, mouse and human.2.28 differential protein spots were found between NC and HG group. Among those in 8, 16h,2 (Rcn2, Hspb1) up regulated, and 24 (Tcpl, Wdrl, Cct2, Enol, Adss, Eeflg, Idhl, Pcbpl, Akrlbl, rCG53488, Pgaml, Psma6, Psmb7, Prdx6, Gstpl, Stmn1, Sodl, Krt2-7, Hmgb1, Serpinb9) were down regulated. In 72 h,2 (Psma6, Hmgbl) were down regulated. At the 25th day of culture,2 were up regulated (Pdia3 and Idh3a) over twofold. The differentially expressed proteins were mainly located in the cytosol, nucleus, cytoskeleton, mitochondria and endoplasmic reticulum. They could be bound with proteins, nucleic acids, ions. Their molecular function were related with antioxidant, oxidation-reduction, hydrolytic enzyme and isomerase, were primarily involved in the biological process such as cell communication, cell cycle, apoptosis, proliferation and metabolic process of carbohydrate, DNA, RNA, protein, oxygen and active oxygen. Gene ontology(GO) over expression and protein interaction analysis revealed that those protein were not only linked with already known diabetic nephropathy process such as glucose metabolism, oxidative stress and molecular chaperones, but also related to protein degradation.Conclusions:1.Some of differentially expressed proteins or their modification transform like Qdpr,Ephx2,Fah,Fthfd and p55 protein, which found in OLETF, were newly linked with pathogenesis of type 2 diabetic nephropathy. It can be concluded that type2 DN is not only associated with protein abundance changes but also modification changes. Especially we found ephx2 which perform important function in body, can be phosphorylated and its phosphorylation is changed in DN seems much more prospective. Those found would provide new clues about the etiopathogenisis of diabetic nephropathy.2.PCBP1, HMGB1, Psma6, Psmb7, Prdx6, Rcn2 and Serpinb9 are newly found to be dynamically changed in the mesangial cells response to high glucose conditions. Through bioinformatics analysis, we found Proteasomes dysfunction might play an important role in proliferation and hypertrophy of mesangial cells in response to high glucose conditions which might provide new clues of the pathogenesis of diabetic nephropathy.