title: Subfractionation, characterization, and in-depth proteomic analysis of glomerular membrane vesicles in human urine.
abstract: Urinary exosome-like vesicles (ELVs) are a heterogenous mixture (diameter 40-200 nm) containing vesicles shed from all segments of the nephron including glomerular podocytes. Contamination with Tamm-Horsfall protein (THP) oligomers has hampered their isolation and proteomic analysis. Here we improved ELV isolation protocols employing density centrifugation to remove THP and albumin, and isolated a glomerular membranous vesicle (GMV)-enriched subfraction from 7 individuals identifying 1830 proteins and in 3 patients with glomerular disease identifying 5657 unique proteins. The GMV fraction was composed of podocin/podocalyxin-positive irregularly shaped membranous vesicles and podocin/podocalyxin-negative classical exosomes. Ingenuity pathway analysis identified integrin, actin cytoskeleton, and Rho GDI signaling in the top three canonical represented signaling pathways and 19 other proteins associated with inherited glomerular diseases. The GMVs are of podocyte origin and the density gradient technique allowed isolation in a reproducible manner. We show many nephrotic syndrome proteins, proteases, and complement proteins involved in glomerular disease are in GMVs and some were only shed in the disease state (nephrin, TRPC6, INF2 and phospholipase A2 receptor). We calculated sample sizes required to identify new glomerular disease biomarkers, expand the ELV proteome, and provide a reference proteome in a database that may prove useful in the search for biomarkers of glomerular disease.
authors.affiliation: Division of Nephrology and Hypertension, Department of Internal Medicine, Mayo Clinic, Rochester, Minnesota, USA.
authors.forename: Marie C
authors.initials: MC
authors.lastname: Hogan
authors.affiliation: Mayo Clinic Proteomics Core, Department of Biochemistry and Molecular Biology, Medical Sciences Building, Mayo Clinic, Rochester, Minnesota, USA.
authors.forename: Kenneth L
authors.initials: KL
authors.lastname: Johnson
authors.affiliation: Mayo Clinic Proteomics Core, Department of Biochemistry and Molecular Biology, Medical Sciences Building, Mayo Clinic, Rochester, Minnesota, USA.
authors.forename: Roman M
authors.initials: RM
authors.lastname: Zenka
authors.affiliation: Mayo Clinic Proteomics Core, Department of Biochemistry and Molecular Biology, Medical Sciences Building, Mayo Clinic, Rochester, Minnesota, USA.
authors.forename: M Cristine
authors.initials: MC
authors.lastname: Charlesworth
authors.affiliation: Mayo Clinic Proteomics Core, Department of Biochemistry and Molecular Biology, Medical Sciences Building, Mayo Clinic, Rochester, Minnesota, USA.
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Madden
authors.affiliation: 1] Department of Health Sciences Research, Mayo Clinic, College of Medicine, Rochester, Minnesota, USA [2] Department of Biomedical Statistics and Informatics, Mayo Clinic, College of Medicine, Rochester, Minnesota, USA.
authors.forename: Doug W
authors.initials: DW
authors.lastname: Mahoney
authors.affiliation: 1] Department of Health Sciences Research, Mayo Clinic, College of Medicine, Rochester, Minnesota, USA [2] Department of Biomedical Statistics and Informatics, Mayo Clinic, College of Medicine, Rochester, Minnesota, USA.
authors.forename: Ann L
authors.initials: AL
authors.lastname: Oberg
authors.affiliation: Mayo Electron Microscopy Core Laboratory, Mayo Clinic, Rochester, Minnesota, USA.
authors.forename: Bing Q
authors.initials: BQ
authors.lastname: Huang
authors.affiliation: Department of Biomedical Statistics and Informatics, Mayo Clinic, College of Medicine, Rochester, Minnesota, USA.
authors.forename: Alexey A
authors.initials: AA
authors.lastname: Leontovich
authors.affiliation: Division of Nephrology and Hypertension, Department of Internal Medicine, Mayo Clinic, Rochester, Minnesota, USA.
authors.forename: Lisa L
authors.initials: LL
authors.lastname: Nesbitt
authors.affiliation: Division of Nephrology and Hypertension, Department of Internal Medicine, Mayo Clinic, Rochester, Minnesota, USA.
authors.forename: Jason L
authors.initials: JL
authors.lastname: Bakeberg
authors.affiliation: Mayo Clinic Proteomics Core, Department of Biochemistry and Molecular Biology, Medical Sciences Building, Mayo Clinic, Rochester, Minnesota, USA.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: McCormick
authors.affiliation: Mayo Clinic Proteomics Core, Department of Biochemistry and Molecular Biology, Medical Sciences Building, Mayo Clinic, Rochester, Minnesota, USA.
authors.forename: H Robert
authors.initials: HR
authors.lastname: Bergen
authors.affiliation: Division of Nephrology and Hypertension, Department of Internal Medicine, Mayo Clinic, Rochester, Minnesota, USA.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Ward
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000595:Amino Acid Sequence; D015415:Biomarkers / Q000652:urine; D016022:Case-Control Studies; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D055354:Exosomes / Q000737:chemistry*; D005260:Female; D050533:Glomerular Basement Membrane / Q000737:chemistry*; D006801:Humans; D007674:Kidney Diseases / Q000175:diagnosis / Q000652:urine*; D008297:Male; D008969:Molecular Sequence Data; D050199:Podocytes / Q000737:chemistry*; D011507:Proteinuria / Q000175:diagnosis / Q000652:urine*; D040901:Proteomics / Q000379:methods*; D016482:Urinalysis; D014556:Urine / Q000737:chemistry*; D055815:Young Adult
chemical_list: D015415:Biomarkers