TAMM-HORSFALL PROTEIN AND URINARY EXOSOME ISOLATION: PP.9.380

Abstract

Urinary exosomes have been proposed as a starting material for discovery of protein biomarkers of disease. Currently, standard protocols for urinary exosome isolation involve a two-step differential centrifugation process. Due to their low density, exosomes are expected to remain in the 17,000 Xg supernatant and to sediment only when the sample is spun at 200,000 Xg. Here we show, using immunoblotting and electron microscopy, that urinary exosomes are also present in the 17,000 Xg pellet as a result of entrainment by polymeric Tamm-Horsfall protein (THP). This diminishes the reproducibility of isolation. We show, by electron microscopy, that the addition of DTT to the 17,000 Xg pellet results in disruption of the THP polymeric network presumably by reduction of disulfide bonds that link the monomers. This procedure was shown to deplete the exosomal proteins Alix, TSG101, and CD9 from the 17,000 Xg pellet. Also, by shifting the THP to the 200,000 Xg pellet, the use of DTT makes it theoretically feasible to use THP in this fraction to normalize excretion rates of other proteins in spot urines. Immunoblotting to test this idea showed a high degree of correlation between exosomal proteins and THP. We conclude that 1) THP polymeric networks entrain urinary exosomes; 2) the yield of exosomes by differential centrifugation can be increased by chemical reduction of the sample; and 3) THP may be a suitable normalizing variable for urinary exosome studies when quantitative urine collections are not practical. Optimize urinary exosome isolation is a key point in the field of biomarker discovery