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- Title
Comparison of three methods for isolation of urinary microvesicles to identify biomarkers of nephrotic syndrome.
- Authors
Rood, Ilse M.; Deegens, Jeroen K. J.; Merchant, Michael L.; Tamboer, Wim P. M.; Wilkey, Daniel W.; Wetzels, Jack F. M.; Klein, Jon B.
- Abstract
Urinary microvesicles, such as 40-100 nm exosomes and 100-1000 nm microparticles, contain many proteins that may serve as biomarkers of renal disease. Microvesicles have been isolated by ultracentrifugation or nanomembrane ultrafiltration from normal urine; however, little is known about the efficiency of these methods in isolating microvesicles from patients with nephrotic-range proteinuria. Here we compared three techniques to isolate microvesicles from nephrotic urine: nanomembrane ultrafiltration, ultracentrifugation, and ultracentrifugation followed by size-exclusion chromatography (UC-SEC). Highly abundant urinary proteins were still present in sufficient quantity after ultrafiltration or ultracentrifugation to blunt detection of less abundant microvesicular proteins by MALDI-TOF-TOF mass spectrometry. The microvesicular markers neprilysin, aquaporin-2, and podocalyxin were highly enriched following UC-SEC compared with preparations by ultrafiltration or ultracentrifugation alone. Electron microscopy of the UC-SEC fractions found microvesicles of varying size, compatible with the presence of both exosomes and microparticles. Thus, UC-SEC following ultracentrifugation to further enrich and purify microparticles facilitates the search for prognostic biomarkers that might be used to predict the clinical course of nephrotic syndrome.
- Subjects
NEPHROTIC syndrome treatment; URINALYSIS; BIOMARKERS; BIOMOLECULE separation; ULTRAFILTRATION; ULTRACENTRIFUGATION
- Publication
Kidney International, 2010, Vol 78, Issue 8, p810
- ISSN
0085-2538
- Publication type
Article
- DOI
10.1038/ki.2010.262