The DOI matches its Crossref record; 17 of the 17 references it cites were found in a registry or an index.
Tamm-Horsfall protein and urinary exosome isolation
Open accessAbstract
Urinary exosomes have been proposed as starting material for discovery of protein biomarkers of kidney disease. Current protocols for their isolation use a two-step differential centrifugation process. Due to their low density, exosomes are expected to remain in the low-speed (17,000 x g) supernatant and to sediment only when the sample is spun at high speed (200,000 x g). Analysis using western blot and electron microscopy found that urinary exosomes are also present in the low-speed pellet entrapped by polymeric Tamm-Horsfall protein, thus diminishing the procedure's reproducibility. Here we show that addition of dithiothreitol to the low-speed pellet disrupted the polymeric network, presumably by reduction of disulfide bonds linking the monomers. This modification shifted the exosomal proteins from the low- to the high-speed pellet. Also, by shifting the Tamm-Horsfall protein to the high-speed pellet, the use of dithiothreitol makes it feasible to use Tamm-Horsfall protein to normalize excretion rates of exosomal proteins in spot urines. We tested this by western blot, and found that there was a high degree of correlation between exosomal proteins and Tamm-Horsfall protein in the high-speed pellet. Since the yield of exosomes by differential centrifugation can be increased by chemical reduction, Tamm-Horsfall protein may be a suitable normalizing variable for urinary exosome studies when quantitative urine collections are not practical.
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“TSG101 and Alix are two other proteins sometimes used as exosome markers [38, 46, 47].”
From Biogenesis of extracellular vesicles (EV): exosomes, microvesicles, retrovirus-like vesicles, and apoptotic bodies (2013) · cites this paper as [47]
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Cites
Identification and proteomic profiling of exosomes in human urine
Pisitkun, T., Shen, R., & Knepper, M. A. (2004) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.0403453101 · Cited by 2,162
Self-citationProteomic profiling of exosomes: Current perspectives
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Prostate cancer-derived urine exosomes: a novel approach to biomarkers for prostate cancer
Nilsson, J. A., Skog, J., Nordstrand, A., Baranov, V. I., Mincheva‐Nilsson, L., Breakefield, X. O., & Widmark, A. (2009) · British Journal of Cancer · doi:10.1038/sj.bjc.6605058 · Cited by 786
Wassarman, P. M. (1988) · Annual Review of Biochemistry · doi:10.1146/annurev.bi.57.070188.002215 · Cited by 733
Large-scale proteomics and phosphoproteomics of urinary exosomes
Gonzales, P. A., Pisitkun, T., Hoffert, J. D., Tchapyjnikov, D., Star, R. A., Kleta, R., Wang, N. S., & Knepper, M. A. (2009) · Journal of the American Society of Nephrology · doi:10.1681/asn.2008040406 · Cited by 727
Self-citationZhou, H., Yuen, P. S. T., Pisitkun, T., Gonzales, P. A., Yasuda, H., Dear, J. W., Gross, P., Knepper, M. A., & Star, R. A. (2006) · Kidney International · doi:10.1038/sj.ki.5000273 · Cited by 586
Self-citationTamm, I., & Horsfall, F. L. (1952) · The Journal of Experimental Medicine · doi:10.1084/jem.95.1.71 · Cited by 450
Discovery of Urinary Biomarkers
Pisitkun, T., Johnstone, R. M., & Knepper, M. A. (2006) · Molecular & Cellular Proteomics · doi:10.1074/mcp.r600004-mcp200 · Cited by 403
Self-citationCD24 is a marker of exosomes secreted into urine and amniotic fluid
Keller, S., Rupp, C., Stoeck, A., Runz, S., Fogel, M., Lugert, S., Hager, H. D., Abdel‐Bakky, M. S., Gutwein, P., & Altevogt, P. (2007) · Kidney International · doi:10.1038/sj.ki.5002486 · Cited by 384
Characterization of PKD Protein-Positive Exosome-Like Vesicles
Hogan, M. C., Manganelli, L., Woollard, J. R., Masyuk, A. I., Masyuk, T. V., Tammachote, R., Huang, B. Q., Leontovich, A. A., Beito, T. G., Madden, B. J., et al. (2009) · Journal of the American Society of Nephrology · doi:10.1681/asn.2008060564 · Cited by 338
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“TSG101 and Alix are two other proteins sometimes used as exosome markers [38, 46, 47].”
Isolation of Extracellular Vesicles: General Methodologies and Latest Trends
Konoshenko, M. Y., Lekchnov, E. A., Vlassov, A. V., & Laktionov, P. P. (2018) · BioMed Research International · doi:10.1155/2018/8545347 · Cited by 1,302
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Metadata
- Authors
- Fernández-Llama, P., Khositseth, S., Gonzales, P. A., Star, R. A., Pisitkun, T., & Knepper, M. A.
- Affiliations
- National Institutes of Health (US)
- Corresponding author
- Knepper, Mark A.
- Venue
- Kidney International
- Publisher
- Elsevier BV
- Published
- 2010-04
- Volume
- 77
- Issue
- 8
- Pages
- 736-742
- ISSN
- 0085-2538 (ISSN-L)
- PMC
- PMC3398710
- OpenAlex
- W2038509755
- Cited by
- 290
- References
- 17
- Citations by year
- 2026: 7 · 2025: 10 · 2024: 12 · 2023: 12 · 2022: 23 · 2021: 27 · 2020: 37 · 2019: 18 · 2018: 22 · 2017: 20 · 2016: 18 · 2015: 18 · 2014: 22 · 2013: 18 · 2012: 13
- FWCI
- 5.74
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- Locations
- Kidney International (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Funders
- Sociedad Española de Nefrología
- National Institutes of Health (Z99 HL999999)
- National Heart, Lung, and Blood Institute (Z01HL001285, ZIAHL001285, Z01-HL001285)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Extracellular vesicles in disease
- Health Sciences › Medicine › Nephrology › Renal Diseases and Glomerulopathies
- Health Sciences › Medicine › Urology › Kidney Stones and Urolithiasis Treatments
- Keywords
- uromodulin, urinary exosomes, exosome isolation, differential centrifugation, dithiothreitol, disulfide bond reduction, kidney disease biomarkers, spot urine, urinary protein excretion
- MeSH terms
- Adult, Humans, Male, Microscopy, Electron, Mucoproteins, Urinary Tract, Blotting, Western, Biomarkers, Exosomes, Uromodulin
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