Abstract
Urinary exosomes are membranous vesicles 40–100 nm in size containing proteins that are characteristic of every renal tubule epithelial cell type. In this chapter, we describe a methodology to isolate and analyze urinary exosomes proteome by 2-DE and LC–MS/MS, in the search for biomarkers of vascular and associated kidney diseases. We describe an isolation methodology by serial (ultra)centrifugation steps compatible with 2-DE and LC–MS/MS analysis. Exosome purity is confirmed by electron microscopy and Western blot.
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References
Azevedo LC, Pedro MA, Laurindo FR (2007) Circulating microparticles as therapeutic targets in cardiovascular diseases. Recent Pat Cardiovasc Drug Discov 2:41–51
Street JM, Birkhoff W, Menzies RI, Webb DJ, Bailey MA, Dear JW (2011) Exosomal transmission of functional aquaporin 2 in kidney cortical collecting duct cells. J Physiol 589:6119–6127
Rood IM, Deegens JK, Merchant ML, Tamboer WP, Wilkey DW, Wetzels JF, Klein JB (2010) Comparison of three methods for isolation of urinary microvesicles to identify biomarkers of nephrotic syndrome. Kidney Int 78:810–816
Sonoda H, Yokota-Ikeda N, Oshikawa S, Kanno Y, Yoshinaga K, Uchida K, Ueda Y et al (2009) Decreased abundance of urinary exosomal aquaporin-1 in renal ischemia-reperfusion injury. Am J Physiol Renal Physiol 297:F1006–F1016
Goligorsky MS, Addabbo F, O’Riordan E (2007) Diagnostic potential of urine proteome: a broken mirror of renal diseases. J Am Soc Nephrol 18:2233–2239
van Balkom BW, Pisitkun T, Verhaar MC, Knepper MA (2011) Exosomes and the kidney: prospects for diagnosis and therapy of renal diseases. Kidney Int 80:1138–1145
Zhou H, Cheruvanky A, Hu X, Matsumoto T, Hiramatsu N, Cho ME, Berger A et al (2008) Urinary exosomal transcription factors, a new class of biomarkers for renal disease. Kidney Int 74:613–621
Arscott WT, Camphausen KA (2011) Analysis of urinary exosomes to identify new markers of non-small-cell lung cancer. Biomark Med 5:822
Conde-Vancells J, Rodriguez-Suarez E, Gonzalez E, Berisa A, Gil D, Embade N et al (2010) Candidate biomarkers in exosome-like vesicles purified from rat and mouse urine samples. Proteomics Clin Appl 4:416–425
Mitchell PJ, Welton J, Staffurth J, Court J, Mason MD, Tabi Z, Clayton A (2009) Can urinary exosomes act as treatment response markers in prostate cancer? J Transl Med 7:4
Duijvesz D, Luider T, Bangma CH, Jenster G (2011) Exosomes as biomarker treasure chests for prostate cancer. Eur Urol 59:823–831
Lu Q, Zhang J, Allison R, Gay H, Yang WX, Bhowmick NA, Frelix G, Shappel l S, Chen YH (2009) Identification of extracellular delta-catenin accumulation for prostate cancer detection. Prostate 69:411–418
Welton JL, Khanna S, Giles PJ, Brennan P, Brewis IA, Staffurth J, Mason MD, Clayton A (2010) Proteomics analysis of bladder cancer exosomes. Mol Cell Proteomics 9:1324–1338
Barratt J, Topham P (2007) Urine proteomics: the present and future of measuring urinary protein components in disease. CMAJ 177:361–368
Gonzales PA, Zhou H, Pisitkun T, Wang NS, Star RA, Knepper MA, Yuen PS (2010) Isolation and purification of exosomes in urine. Methods Mol Biol 64:89–99
Zhou H, Yuen PS, Pisitkun T, Gonzales PA, Yasuda H, Dear JW, Gross P, Knepper MA, Star RA (2006) Collection, storage, preservation, and normalization of human urinary exosomes for biomarker discovery. Kidney Int 69:1471–1476
Fernandez-Llama P, Khositseth S, Gonzales PA, Star RA, Pisitkun T, Knepper MA (2010) Tamm-Horsfall protein and urinary exosome isolation. Kidney Int 77:736–742
Acknowledgments
This work was supported by grants from FIS CP09/229, FIS PI11/01401, and FIS PI08/0970.
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Zubiri, I., Vivanco, F., Alvarez-Llamas, G. (2013). Proteomic Analysis of Urinary Exosomes in Cardiovascular and Associated Kidney Diseases by Two-Dimensional Electrophoresis and LC–MS/MS. In: Vivanco, F. (eds) Vascular Proteomics. Methods in Molecular Biology, vol 1000. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-405-0_16
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DOI: https://doi.org/10.1007/978-1-62703-405-0_16
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Publisher Name: Humana Press, Totowa, NJ
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