Proc Natl Acad Sci U S A 101(36): 13368-13373

Identification and proteomic profiling of exosomes in human urine

National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-1603

^{To whom correspondence should be addressed at: National Institutes of Health, Building 10, Room 6N260, 10 Center Drive, MSC 1603, Bethesda, MD 20892-1603. E-mail:}vog.hin.iblhn@mreppenk.

Edited by William S. Sly, Saint Louis University School of Medicine, St. Louis, MO, and approved July 27, 2004

Received 2004 May 14

Abstract

Urine provides an alternative to blood plasma as a potential source of disease biomarkers. One urinary biomarker already exploited in clinical studies is aquaporin-2. However, it remains a mystery how aquaporin-2 (an integral membrane protein) and other apical transporters are delivered to the urine. Here we address the hypothesis that these proteins reach the urine through the secretion of exosomes [membrane vesicles that originate as internal vesicles of multivesicular bodies (MVBs)]. Low-density urinary membrane vesicles from normal human subjects were isolated by differential centrifugation. ImmunoGold electron microscopy using antibodies directed to cytoplasmic or anticytoplasmic epitopes revealed that the vesicles are oriented “cytoplasmic-side inward,” consistent with the unique orientation of exosomes. The vesicles were small (<100 nm), consistent with studies of MVBs and exosomes from other tissues. Proteomic analysis of urinary vesicles through nanospray liquid chromatography-tandem mass spectrometry identified numerous protein components of MVBs and of the endosomal pathway in general. Full liquid chromatography-tandem MS analysis revealed 295 proteins, including multiple protein products of genes already known to be responsible for renal and systemic diseases, including autosomal dominant polycystic kidney disease, Gitelman syndrome, Bartter syndrome, autosomal recessive syndrome of osteopetrosis with renal tubular acidosis, and familial renal hypomagnesemia. The results indicate that exosome isolation may provide an efficient first step in biomarker discovery in urine.

Abstract

Amajor goal in the field of clinical proteomics is to identify disease biomarkers in biological fluids that can be measured relatively inexpensively for early diagnosis of disease. An important challenge in this process is to develop a rational means of reducing the complexity of the proteome of body-fluid samples to enhance the detectability of relatively low-abundance proteins that may have special pathophysiological significance. Most of the focus thus far has been on proteomics of blood serum or plasma (1). Because urine can be collected noninvasively in large amounts, it provides an attractive alternative to blood plasma as a potential source of disease biomarkers (2).

The water channel aquaporin-2 (AQP2) is one biomarker that can be readily measured in urine (3) and that has been exploited in studies of various water-balance disorders (4). AQP2 is an integral membrane protein, and investigators thus far have been puzzled with regard to the mechanism of its secretion into the urine. Biochemical studies (5) and immunoelectron microscopy (6) have demonstrated that AQP2 is present in small, low-density membrane vesicles but have not provided an explanation for the appearance of these vesicles in the urine. Immunoblotting of urinary membrane fractions has revealed that, in addition to AQP2, the kidneys excrete membranes containing apical plasma-membrane transporter proteins from each renal tubule segment (5), suggesting that analysis of urinary membrane fractions could provide noninvasive information about the pathophysiological state of the entire renal tubule.

We hypothesize that AQP2 and other apical plasma-membrane proteins are excreted through the process of exosome formation, i.e., delivery of the internal vesicles of multivesicular bodies (MVBs) to the urinary space by fusion of the outer membrane of MVBs with the apical plasma membrane of renal tubule epithelial cells. This hypothesis leads to explicit predictions that can be tested directly. First, the urinary vesicles should be oriented “cytoplasmic-side inward,” a unique characteristic of the internal vesicles of MVBs (7). Second, the urinary vesicles should be small (<100 nm in diameter) and relatively uniform in size, consistent with the internal vesicles of MVBs (8) and exosomes secreted by other tissues (7). Third, the urinary vesicles should contain proteins typical of MVBs and of exosomes formed by other cell types (7).

Here we use immunoelectron microscopy and nanospray liquid chromatography-tandem MS (LC-MS/MS) analysis of urinary membrane proteins to show that AQP2 and other apical plasma-membrane proteins are excreted through the process of exosome formation in agreement with the predictions above. The proteomic analysis revealed the presence of 21 proteins known to be associated with specific renal diseases or blood pressure regulation. These studies demonstrate the potential for using the urinary exosomes as a starting material in studies aimed at disease biomarker discovery.

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Acknowledgments

We thank Drs. Roger Wiggins (University of Michigan, Ann Arbor), Gregory Germino (The Johns Hopkins University, Baltimore), John Edwards (Saint Louis University), William Sly (Saint Louis University), John Hoyer (University of Pennsylvania, Philadelphia), and Kenneth Bernstein (Emory University, Atlanta) for kindly providing antibodies; Dr. Xiaoyan Wang for preparing human kidney samples; Dr. Zu-Xi Yu for assistance with electron microscopy; Angel Aponte for help with 2D electrophoresis; Dr. Guanghui Wang for bioinformatics assistance; and Dr. Kriang Tungsanga for career development advice and support. This work was supported by the intramural budget of the National Heart, Lung, and Blood Institute (Z01-HL-01282-KE). T.P. was supported by an International Society of Nephrology Fellowship Award.

Acknowledgments

Notes

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: APN, aminopeptidase N; AQP1 and AQP2, aquaporin-1 and -2; LC-MS/MS, liquid chromatography-tandem MS; MVBs, multivesicular bodies; NCC, thiazide-sensitive Na-Cl cotransporter; THP, Tamm-Horsfall protein; VPS, vacuolar protein-sorting.

Notes

This paper was submitted directly (Track II) to the PNAS office.

References

1. Anderson, N. L. & Anderson, N. G. (2002) Mol. Cell. Proteomics1, 845-867. [[PubMed]
2. Thongboonkerd, V., Klein, J. B., Jevans, A. W. & McLeish, K. R. (2004) Contrib. Nephrol.141, 292-307. [[PubMed]
3. Kanno, K., Sasaki, S., Hirata, Y., Ishikawa, S., Fushimi, K., Nakanishi, S., Bichet, D. G. & Marumo, F. (1995) N. Engl. J. Med.332, 1540-1545. [[PubMed]
4. Ishikawa, S. E. & Schrier, R. W. (2003) Clin. Endocrinol. (Oxford)58, 1-17. [[PubMed]
5. McKee, J. A., Kumar, S., Ecelbarger, C. A., Fernández-Llama, P., Terris, J. & Knepper, M. A. (2000) J. Am. Soc. Nephrol.11, 2128-2132. [[PubMed]
6. Wen, H., Frokiaer, J., Kwon, T. H. & Nielsen, S. (1999) J. Am. Soc. Nephrol.10, 1416-1429. [[PubMed]
7. Thery, C., Zitvogel, L. & Amigorena, S. (2002) Nat. Rev. Immunol.2, 569-579. [[PubMed]
8. Nielsen, S., Muller, J. & Knepper, M. A. (1993) Am. J. Physiol.265, F225-F238. [[PubMed]
9. DiGiovanni, S. R., Nielsen, S., Christensen, E. I. & Knepper, M. A. (1994) Proc. Natl. Acad. Sci. USA91, 8984-8988.
10. Hellman, U., Wernstedt, C., Gonez, J. & Heldin, C. H. (1995) Anal. Biochem.224, 451-455. [[PubMed]
11. Stange, T., Kettmann, U. & Holzhausen, H. J. (2000) Eur. J. Histochem.44, 157-164. [[PubMed]
12. Sheikh-Hamad, D., Youker, K., Truong, L. D., Nielsen, S. & Entman, M. L. (2000) Am. J. Physiol.279, C136-C146. [[PubMed]
13. Obermuller, N., Bernstein, P., Velazquez, H., Reilly, R. F., Moser, D., Ellison, D. H. & Bachmann, S. (1995) Am. J. Physiol.269, F900-F910. [[PubMed]
14. Nielsen, S., Chou, C. L., Marples, D., Christensen, E. I., Kishore, B. K. & Knepper, M. A. (1995) Proc. Natl. Acad. Sci. USA92, 1013-1017.
15. Katzmann, D. J., Babst, M. & Emr, S. D. (2001) Cell106, 145-155. [[PubMed]
16. Bishop, N. & Woodman, P. (2001) J. Biol. Chem.276, 11735-11742. [[PubMed]
17. Babst, M., Odorizzi, G., Estepa, E. J. & Emr, S. D. (2000) Traffic1, 248-258. [[PubMed]
18. Kloor, M., Bork, P., Duwe, A., Klaes, R., von Knebel, D. M. & Ridder, R. (2002) Biochim. Biophys. Acta1579, 219-224. [[PubMed]
19. Babst, M., Katzmann, D. J., Estepa-Sabal, E. J., Meerloo, T. & Emr, S. D. (2002) Dev. Cell3, 271-282. [[PubMed]
20. Howard, T. L., Stauffer, D. R., Degnin, C. R. & Hollenberg, S. M. (2001) J. Cell Sci.114, 2395-2404. [[PubMed]
21. Kranz, A., Kinner, A. & Kolling, R. (2001) Mol. Biol. Cell12, 711-723.
22. Katoh, K., Shibata, H., Suzuki, H., Nara, A., Ishidoh, K., Kominami, E., Yoshimori, T. & Maki, M. (2003) J. Biol. Chem.278, 39104-39113. [[PubMed]
23. Peck, J. W., Bowden, E. T. & Burbelo, P. D. (2004) Biochem. J.377, 693-700.
24. Odorizzi, G., Katzmann, D. J., Babst, M., Audhya, A. & Emr, S. D. (2003) J. Cell Sci.116, 1893-1903. [[PubMed]
25. Bateman, A., Coin, L., Durbin, R., Finn, R. D., Hollich, V., Griffiths-Jones, S., Khanna, A., Marshall, M., Moxon, S., Sonnhammer, E. L., et al. (2004) Nucleic Acids Res.32, Database issue, D138-D141.
26. Babst, M., Wendland, B., Estepa, E. J. & Emr, S. D. (1998) EMBO J.17, 2982-2993.
27. Healy, D. P. & Song, L. (1999) Hypertension33, 740-745. [[PubMed]
28. Farjah, M., Washington, T. L., Roxas, B. P., Geenen, D. L. & Danziger, R. S. (2004) Hypertension43, 282-285. [[PubMed]
29. Kitamura, S., Carbini, L. A., Simmons, W. H. & Scicli, A. G. (1999) Am. J. Physiol.276, H1664-H1671. [[PubMed]
30. Inguimbert, N., Coric, P., Poras, H., Meudal, H., Teffot, F., Fournie-Zaluski, M. C. & Roques, B. P. (2002) J. Med. Chem.45, 1477-1486. [[PubMed]
31. Nakanishi, T., Shiigai, T. & Endou, H. (1986) Clin. Exp. Hypertens. A8, 91-112. [[PubMed]
32. Achan, V., Broadhead, M., Malaki, M., Whitley, G., Leiper, J., MacAllister, R. & Vallance, P. (2003) Arterioscler. Thromb. Vasc. Biol.23, 1455-1459. [[PubMed]
33. Mitra, K., Ubarretxena-Belandia, I., Taguchi, T., Warren, G. & Engelman, D. M. (2004) Proc. Natl. Acad. Sci. USA101, 4083-4088.
34. Thery, C., Boussac, M., Veron, P., Ricciardi-Castagnoli, P., Raposo, G., Garin, J. & Amigorena, S. (2001) J. Immunol.166, 7309-7318. [[PubMed]
35. Bonifacino, J. S. & Traub, L. M. (2003) Annu. Rev. Biochem.72, 395-447. [[PubMed]
36. Sato, S., Zhu, X. L. & Sly, W. S. (1990) Proc. Natl. Acad. Sci. USA87, 6073-6076.
37. Hawari, F. I., Rouhani, F. N., Cui, X., Yu, Z. X., Buckley, C., Kaler, M. & Levine, S. J. (2004) Proc. Natl. Acad. Sci. USA101, 1297-1302.