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

Marie C Hogan, Kenneth L. Johnson, Roman M. Zenka, M. Cristine Charlesworth, Benjamin J. Madden, Doug W. Mahoney, Ann L Oberg, Bing Q. Huang, Alexey A. Leontovich, Lisa L. Nesbitt, Jason L. Bakeberg, Daniel J Mc Cormick, Harold Robert (Bob) III Bergen, Christopher J. Ward

Research output: Contribution to journalArticle

51 Scopus citations

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 A 2 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.

Original languageEnglish (US)
Pages (from-to)1225-1237
Number of pages13
JournalKidney International
Volume85
Issue number5
DOIs
StatePublished - 2014

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Keywords

  • Actin cytoskeleton
  • Exosome
  • Glomerular disease
  • Podocyte
  • Proteomics
  • Q-exactive mass spectrometer

ASJC Scopus subject areas

  • Nephrology

Cite this

Hogan, M. C., Johnson, K. L., Zenka, R. M., Cristine Charlesworth, M., Madden, B. J., Mahoney, D. W., Oberg, A. L., Huang, B. Q., Leontovich, A. A., Nesbitt, L. L., Bakeberg, J. L., Mc Cormick, D. J., Bergen, H. R. B. III., & Ward, C. J. (2014). Subfractionation, characterization, and in-depth proteomic analysis of glomerular membrane vesicles in human urine. Kidney International, 85(5), 1225-1237. https://doi.org/10.1038/ki.2013.422