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.
Fernández-Llama
Patricia
P
Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-1603, USA.
Khositseth
Sookkasem
S
Gonzales
Patricia A
PA
Star
Robert A
RA
Pisitkun
Trairak
T
Knepper
Mark A
MA
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