Tamm-Horsfall protein and urinary exosome isolation

Kidney Int. 2010 Apr;77(8):736-42. doi: 10.1038/ki.2009.550. Epub 2010 Feb 3.

Abstract

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.

Publication types

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

MeSH terms

  • Adult
  • Biomarkers / urine
  • Blotting, Western
  • Exosomes / ultrastructure*
  • Humans
  • Male
  • Microscopy, Electron
  • Mucoproteins / isolation & purification*
  • Mucoproteins / ultrastructure*
  • Urinary Tract / ultrastructure*
  • Uromodulin

Substances

  • Biomarkers
  • Mucoproteins
  • UMOD protein, human
  • Uromodulin