Preparation of Urinary Exosomes: Methodological Issues for Clinical Proteomics

  • Marina PittoEmail author
  • Samuele Corbetta
  • Francesca Raimondo
Part of the Methods in Molecular Biology book series (MIMB, volume 1243)


Urinary exosomes are small (<100 nm) vesicles secreted into urine from renal epithelial cells. They are coated with lipid bilayer, they contain an array of membrane and cytosolic proteins, and selected RNA species, reflecting the molecular composition of their cell of origin. Thus, urinary exosomes have received considerable attention as potential biomarker source, as their proteomic analysis could lead to the discovery of new non-invasive site-specific biomarkers for renal diseases. Here, we describe a robust method for urinary exosome preparation, additional protocols for their biochemical characterization and for the quantitation of different preparations, to be used for comparative proteomic studies.

Key words

Urinary exosomes Proteomics Isolation Differential centrifugation Ultracentrifugation 


  1. 1.
    Cocucci E, Racchetti G, Meldolesi J (2009) Shedding microvesicles: artefacts no more. Trends Cell Biol 19:43–51PubMedCrossRefGoogle Scholar
  2. 2.
    Prunotto M, Farina A, Lane L et al (2013) Proteomic analysis of podocyte exosome-enriched fraction from normal human urine. J Proteomics 82:193–229PubMedCrossRefGoogle Scholar
  3. 3.
    Goligorsky MS, Addabbo F, O’Riordan E (2007) Diagnostic potential of urine proteome: a broken mirror of renal diseases. J Am Soc Nephrol 18:2233–2239PubMedCrossRefGoogle Scholar
  4. 4.
    Hoorn EJ, Pisitkun T, Zietse R et al (2005) Prospects for urinary proteomics: exosomes as a source of urinary biomarkers. Nephrology 10:283–290PubMedCrossRefGoogle Scholar
  5. 5.
    Gonzales PA, Pisitkun T, Hoffert JD et al (2009) Large-scale proteomics and phosphoproteomics of urinary exosomes. J Am Soc Nephrol 20:363–379PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Pisitkun T, Shen RF, Knepper MA (2004) Identification and proteomic profiling of exosomes in human urine. Proc Natl Acad Sci U S A 101:13368–13373PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Raimondo F, Morosi L, Chinello C et al (2011) Advances in membranous vesicle and exosome proteomics improving biological understanding and biomarker discovery. Proteomics 11:709–720PubMedCrossRefGoogle Scholar
  8. 8.
    Cheruvanky A, Zhou H, Pisitkun T et al (2007) Rapid isolation of urinary exosomal biomarkers using a nanomembrane ultrafiltration concentrator. Am J Physiol Renal Physiol 292:F1657–F1661PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Mathivanan S, Lim JWE, Tauro BJ et al (2010) Proteomics analysis of A33 immunoaffinity-purified exosomes released from the human colon tumor cell line LIM1215 reveals a tissue-specific protein signature. Mol Cell Proteomics 9:197–208PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Alvarez ML, Khosroheidari M, Ravi RK et al (2012) Comparison of protein, microRNA, and mRNA yields using different methods of urinary exosome isolation for the discovery of kidney disease biomarkers. Kidney Int 82:1024–1032PubMedCrossRefGoogle Scholar
  11. 11.
    Raimondo F, Morosi L, Corbetta S et al (2013) Differential protein profiling of renal cell carcinoma urinary exosomes. Mol Biosyst 9:1220–1233PubMedCrossRefGoogle Scholar
  12. 12.
    Raimondo F, Corbetta S, Morosi L et al (2013) Urinary exosomes and diabetic nephropathy: a proteomic approach. Mol Biosyst 9:1139–1146PubMedCrossRefGoogle Scholar
  13. 13.
    Schaeffer C, Santambrogio S, Perucca S et al (2009) Analysis of uromodulin polymerization provides new insights into the mechanisms regulating ZP domain-mediated protein assembly. Mol Biol Cell 20:589–599PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Lau WH, Leong WS, Ismail Z et al (2008) Qualification and application of an ELISA for the determination of Tamm Horsfall Protein (THP) in human urine and its use for screening of Kidney Stone Disease. Int J Biol Sci 4:215–222PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Dear JW, Street JM, Bailey MA (2013) Urinary exosomes: a reservoir for biomarker discovery and potential mediators of intrarenal signalling. Proteomics 13:1572–1580PubMedCrossRefGoogle Scholar
  16. 16.
    Zhou H, Yuen PST, Pisitkun T et al (2006) Collection, storage, preservation, and normalization of human urinary exosomes for biomarker discovery. Kidney Int 69:1471–1476PubMedCentralPubMedGoogle Scholar
  17. 17.
    Daffara R, Botto L, Beretta E et al (2004) Endothelial cells as early sensors of pulmonary interstitial edema. J Appl Physiol 97:1575–1583PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Marina Pitto
    • 1
    Email author
  • Samuele Corbetta
    • 1
  • Francesca Raimondo
    • 1
  1. 1.Department of Health SciencesUniversity of Milano-BicoccaMonzaItaly

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