MO620: A LC-MS/MS Analysis of Proteins from Urinary Exosomes in Diabetic Nephropathy

Abstract

Abstract BACKGROUND AND AIMS Urinary exosomes are small extracellular vesicles mainly secreted by epithelial cells of urinary system, reflecting the composition and characteristics of bioactive molecules in physiology and disease conditions of kidney. Diabetic nephropathy (DN) has become the leading cause of CKD and ESRD in the world. The purpose of this study was to describe the proteomic characteristics by LC-MS/MS, and clarify the protein composition and functional changes of urinary exosomes in DN. METHOD A total of 50 mL of first morning urine were collected from 24 subjects, including healthy controls group (Ctrl, n = 8), type 2 diabetes mellitus (T2DM, n = 8) and renal biopsy confirmed DN (n = 8). Exosomes were isolated by differential centrifugation and proteins were extracted and analyzed by LC-MS/MS. The types and expression abundance of proteins in urinary exosome were identified. Differential expression proteins (DEPs) were screened, and gene ontology (GO) and Reactome pathway enrichment analysis were performed to reveal the composition and function changes of DEPs in urinary exosomes of DN. RESULTS Under transmission electron microscope, scattered vesicles with diameters of 40–100 nm were observed in the urine. Nanoparticle tracking analysis showed that the number of urine exosomes was about 10^ 11 to 10^ 12 in 24-h urine with a peak diameter of about 100 nm. Western blot showed the expression of CD63, TSG101 and CD9, which were regarded as the markers of exosome. An average of 3110 (2506∼3380) proteins were identified in the urinary exosomes of the 24 subjects. In each group, the numbers of co-expressed proteins in urinary exosomes of eight subjects were 1680, 1309 and 1416, respectively. GO Cellular Component (CC) analysis showed that extracellular exosomes ranked first in the functional annotation of co-expressed proteins. Subcellular localization showed that the proportions of proteins annotated as extracellular exosome in the three groups were 77%, 81% and 76%, respectively. In upregulated proteins of urinary exosome in DN, GOCC annotation showed that ‘Extracellular exosome’ ranks first. GO molecular function (MF) enrichment analysis showed that the top 10 terms identified that pathways associated with complement activation such as ‘Regulation of Complement activation’, ‘Complement activation,’ ‘Complement activation, classical Pathway’ and ‘Complement activation, alternative Pathway’. Reactome pathway analysis showed that ‘Complement cascade’ and ‘Regulation of complement cascade’ were significantly activated in the top 10 pathways. CONCLUSION Exosomes can be steadily isolated from urine of healthy adults, T2DM and DN, and about 2506 to 3380 proteins can be identified by LC-MS/MS technique, of which about 76%–83% are related to the secretion and composition of exosomes. DEP analysis showed significant enrichment of exosome production-related annotations in urinary exosomes in DN patients compared with HC or T2DM; GOMF and Reactome pathway enrichment analysis showed that pathways related to complement activation were significantly enriched in the urinary exosomes of DN. This study provides proteomics characters of urinary exosome for healthy adults, T2DM and DN patients, suggesting that the production of urinary exosome is increased during DN, and complement activation may be one of the main characteristics of urinary exosome in DN, of significance of reference for studying the pathogenesis and developing new biomarkers of DN.