title: Comparison of three methods for isolation of urinary microvesicles to identify biomarkers of nephrotic syndrome.
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.
authors.affiliation: Department of Nephrology, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands.
authors.forename: Ilse M
authors.initials: IM
authors.lastname: Rood
authors.forename: Jeroen K J
authors.initials: JK
authors.lastname: Deegens
authors.forename: Michael L
authors.initials: ML
authors.lastname: Merchant
authors.forename: Wim P M
authors.initials: WP
authors.lastname: Tamboer
authors.forename: Daniel W
authors.initials: DW
authors.lastname: Wilkey
authors.forename: Jack F M
authors.initials: JF
authors.lastname: Wetzels
authors.forename: Jon B
authors.initials: JB
authors.lastname: Klein
mesh_terms: D051399:Aquaporin 2 / Q000302:isolation & purification / Q000652:urine; D015415:Biomarkers / Q000032:analysis; D055252:Cell-Derived Microparticles / Q000737:chemistry*; D055354:Exosomes / Q000737:chemistry; D006801:Humans; D013058:Mass Spectrometry; D008722:Methods; D009404:Nephrotic Syndrome / Q000175:diagnosis* / Q000652:urine; D015260:Neprilysin / Q000302:isolation & purification / Q000652:urine; D010316:Particle Size; D011507:Proteinuria / Q000175:diagnosis*; D040901:Proteomics / Q000379:methods*; D012795:Sialoglycoproteins / Q000302:isolation & purification / Q000652:urine
chemical_list: D051399:Aquaporin 2; D015415:Biomarkers; D012795:Sialoglycoproteins; C001643:podocalyxin; D015260:Neprilysin