Abstract
Extracellular vesicles (ECV) reflect physiological or pathological conditions, emerging as potential biomarkers for disease. They can be obtained from a variety of body fluids, particularly urine that is an ideal source because it can be obtained in great quantities, recurrently and with minimal intervention. However, the characterization of urine ECV is challenging because the preparation is usually contaminated with soluble proteins, such as uromodulin (UMOD) or Tamm-Horsfall glycoprotein that forms large extracellular filaments co-sedimenting with ECV. We developed a method to obtain human urine ECV free of UMOD by the addition of ZnSO4 prior to vesicle isolation by differential centrifugation. Treatment with ZnSO4 did not affect the size and concentration of the vesicle preparation and preserved the storage of the samples at low temperatures. We did not observe a variation in the number of vesicles isolated during different times of the day or different days between different donors. The glycoprotein pattern of urine ECV was characterized by binding to concanavalin A (Con A) and mass spectroscopy. Several markers were found, including dipeptidyl peptidase IV (CD26), vacuolar protein sorting factor 4A (VPS4A) and dipeptidase 1 (DPEP1), and galectin 3 binding protein (G3-BP). The levels of VPS4A and DPEP1 were similar in ECV preparations obtained from several donors of both sexes. Con A binding pattern and monosaccharide composition were also comparable between subjects. In summary, our method for the isolation of highly pure ECV derived from human urine is likely to help in the use of these vesicles as potential biomarkers.
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References
Ahmed KA, Xiang J (2011) Mechanisms of cellular communication through intercellular protein transfer. J Cell Mol Med 15:1458–1473
Bokhove M (2016) A structured interdomain linker directs self-polymerization of human uromodulin. PNAS 113:1552–1557
Davis DM (2007) Intercellular transfer of cell-surface proteins is common and can affect many stages of an immune response. Nat Rev Immunol 7:238–243
De Maio A (2011) Extracellular heat shock proteins, cellular export vesicles, and the stress observation system: a form of communication during injury, infection, and cell damage. It is never known how far a controversial finding will go! Dedicated to Ferruccio Ritossa. Cell Stress Chaperones 16:235–249
De Maio A, Vega VL, Contreras JE (2002) Gap junctions, homeostasis, and injury. J Cell Physiol 191:269–282
Duijvesz D, Versluis CY, van der Fels CA, Vredenbregt-van den Berg MS, Leivo J, Peltola MT, Bangma CH, Pettersson KS, Jenster G (2015) Immuno-based detection of extracellular vesicles in urine as diagnostic marker for prostate cancer. Int J Cancer 137:2869–2878
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
Franzen CA, Blackwell RH, Foreman KE, Kuo PC, Flanigan RC, Gupta GN (2016) Urinary exosomes: the potential for biomarker utility, intercellular signaling and therapeutics in urological malignancy. J Urol 195:1331–1339
Gamez-Valero A, Lozano-Ramos SI, Bancu I, Lauzurica-Valdemoros R, Borras FE (2015) Urinary extracellular vesicles as source of biomarkers in kidney diseases. Front Immunol 6:6
Gonzales PA, Pisitkun T, Hoffert JD, Tchapyjnikov D, Star RA, Kleta R, Wang NS, Knepper MA (2009) Large-scale proteomics and phosphoproteomics of urinary exosomes. J Am Soc Nephrol 20:363–379
Harpole M, Davis J, Espina V (2016) Current state of the art for enhancing urine biomarker discovery. Expert Rev Proteomics 13:609–626
Keller S, Rupp C, Stoeck A, Runz S, Fogel M, Lugert S, Hager HD, Abdel-Bakky MS, Gutwein P, Altevogt P (2007) CD24 is a marker of exosomes secreted into urine and amniotic fluid. Kidney Int 72:1095–1102
Khurana R, Ranches G, Schafferer S, Lukasser M, Rudnicki M, Mayer G, Huttenhofer A (2017) Identification of urinary exosomal non-coding RNAs as novel biomarkers in chronic kidney disease. RNA 23:142–152
Lin SY, Chang CH, Wu HC, Lin CC, Chang KP, Yang CR, Huang CP, Hsu WH, Chang CT, Chen CJ (2016) Proteome profiling of urinary exosomes identifies alpha 1-antitrypsin and H2B1K as diagnostic and prognostic biomarkers for urothelial carcinoma. Sci Rep 6:34446
Musante L, Tataruch D, Gu D, Benito-Martin A, Calzaferri G, Aherne S, Holthofer H (2014) A simplified method to recover urinary vesicles for clinical applications, and sample banking. Sci Rep 4:7532
Oshikawa S, Sonoda H, Ikeda M (2016) Aquaporins in urinary extracellular vesicles (exosomes). Int J Mol Sci 17
Pisitkun T, Shen RF, Knepper MA (2004) Identification and proteomic profiling of exosomes in human urine. Proc Natl Acad Sci U S A 101:13368–13373
Puhka M, Nordberg ME, Valkonen S, Rannikko A, Kallioniemi O, Siljander P, Af Hallstrom TM (2017) KeepEX, a simple dilution protocol for improving extracellular vesicle yields from urine. Eur J Pharm Sci 98:30–39
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
Sharon N, Lis H (2001) The structural basis for carbohydrate recognition by lectins. Adv Exp Med Biol 491:1–16
Thery C, Ostrowski M, Segura E (2009) Membrane vesicles as conveyors of immune responses. Nat Rev Immunol 9:581–593
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
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We would like to thank Barbara Rho for her excellent editing of the manuscript.
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This study was supported by National Institutes of Health (NIH) under Grants R01 GM098455 and R01 GM11447.
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Liu, Z., Cauvi, D.M., Bernardino, E.M.A. et al. Isolation and characterization of human urine extracellular vesicles. Cell Stress and Chaperones 23, 943–953 (2018). https://doi.org/10.1007/s12192-018-0902-5
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DOI: https://doi.org/10.1007/s12192-018-0902-5