Tamm-Horsfall protein and urinary exosome isolation.
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.
Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-1603, USA.
Patricia
P
Fernández-Llama
Sookkasem
S
Khositseth
Patricia A
PA
Gonzales
Robert A
RA
Star
Trairak
T
Pisitkun
Mark A
MA
Knepper
D000328:Adult; D015415:Biomarkers / Q000652:urine; D015153:Blotting, Western; D055354:Exosomes / Q000648:ultrastructure*; D006801:Humans; D008297:Male; D008854:Microscopy, Electron; D009088:Mucoproteins / Q000302:isolation & purification* / Q000648:ultrastructure*; D014551:Urinary Tract / Q000648:ultrastructure*; D058949:Uromodulin
D015415:Biomarkers; D009088:Mucoproteins; C548612:UMOD protein, human; D058949:Uromodulin