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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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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
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
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