Comparison of three methods for isolation of urinary microvesicles to identify biomarkers of nephrotic syndrome

Kidney Int. 2010 Oct;78(8):810-6. doi: 10.1038/ki.2010.262. Epub 2010 Aug 4.


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.

Publication types

  • Comparative Study
  • Evaluation Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Aquaporin 2 / isolation & purification
  • Aquaporin 2 / urine
  • Biomarkers / analysis
  • Cell-Derived Microparticles / chemistry*
  • Exosomes / chemistry
  • Humans
  • Mass Spectrometry
  • Methods
  • Nephrotic Syndrome / diagnosis*
  • Nephrotic Syndrome / urine
  • Neprilysin / isolation & purification
  • Neprilysin / urine
  • Particle Size
  • Proteinuria / diagnosis*
  • Proteomics / methods*
  • Sialoglycoproteins / isolation & purification
  • Sialoglycoproteins / urine


  • Aquaporin 2
  • Biomarkers
  • Sialoglycoproteins
  • podocalyxin
  • Neprilysin