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Kidney Int. 2014 May;85(5):1225-37. doi: 10.1038/ki.2013.422. Epub 2013 Nov 6.

Subfractionation, characterization, and in-depth proteomic analysis of glomerular membrane vesicles in human urine.

Author information

1
Division of Nephrology and Hypertension, Department of Internal Medicine, Mayo Clinic, Rochester, Minnesota, USA.
2
Mayo Clinic Proteomics Core, Department of Biochemistry and Molecular Biology, Medical Sciences Building, Mayo Clinic, Rochester, Minnesota, USA.
3
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.
4
Mayo Electron Microscopy Core Laboratory, Mayo Clinic, Rochester, Minnesota, USA.
5
Department of Biomedical Statistics and Informatics, Mayo Clinic, College of Medicine, Rochester, Minnesota, USA.

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.

PMID:
24196483
PMCID:
PMC4008663
DOI:
10.1038/ki.2013.422
[Indexed for MEDLINE]
Free PMC Article

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