The objective of this study was to solve the problem of inconsistent isolation of urinary exosomes. We proposed that exosomes were being variably entrapped by polymeric networks of Tamm-Horsfall protein and showed such entrapment by electron microscopy. Our experiments showed that dissolution of the THP polymeric network through the use of the reducing agent DTT markedly reduced the amount of exosomal marker proteins (TSG101 and Alix) in the low-speed fraction from centrifugation of urine samples. Therefore, the exosomes were more completely retrieved in the subsequent high-speed fraction. We recommend therefore that the standard protocol for exosome isolation by differential centrifugation be modified to use DTT treatment of the resuspended low-speed pellet with a repeat of the low-speed (17,000 × g
) spin. represents our current protocol based on the findings of this study. In addition we include a written protocol in the Supplementary Materials
Current procedure for urinary exosome isolation
Tamm-Horsfall protein is related to the ZP family of proteins. ZP proteins form a polymeric meshwork that creates a barrier around a fertilized egg to prevent polyspermy. The polymerization is through disulfide linkages between ZP protein monomers resulting in a linear polymer. The pattern of distribution of cysteines in THP proteins is similar to that seen in ZP proteins. The success of DTT treatment in disrupting THP polymerization strongly supports the notion that disulfide linkages are also responsible for THP polymerization. THP was found to form ropelike structures by lateral aggregation of the THP monomeric chains (). The physical basis for the lateral association is presently unknown.
In addition, we looked in additional detail about urine storage for exosome isolation. Our previous study concluded that freezing samples at −20 °C markedly reduced the retrieval of exosomes from urine samples14
but did not explain the loss. Here we compared storage at 4 and −80 °C with the yield using fresh urine samples. The immunoblotting did not show a substantial loss of exosomal marker proteins at either temperature. However, there was a small amount of retention of exosomal marker proteins in the 17,000 × g
pellet even with DTT, suggesting that the mechanism of this small retention may depend on a process other than entrapment by THP networks (). Thus, analysis of fresh samples seems to be superior. However, freezing at −80 °C appears to be a reasonable choice if long-term storage is necessary. Other than the temperature, the ionic strength and pH of urine can also contribute to THP aggregation.15
However, this study did not investigate the role of these factors on THP aggregation and exosome isolation. Thus, future studies are needed to address this issue experimentally.
Biomarker discovery and exploitation depend on development of methods for quantifying urinary excretion rates of proteins. However, the timed collections needed for the calculation of such rates are not always practical in a clinic setting. Thus, a normalization variable is needed to substitute for time in calculations of relative excretion rate of exosomal proteins. (Because water excretion is highly variable, comparison of absolute protein concentrations in urine is never a reasonable alternative.) One alternative for a normalizing variable would be exosomal markers such as Alix or TSG101. Such measurements, however, add to the complexity of the overall determination. Here we show that the level of THP in the 200,000 × g pellet correlates very highly with the levels of Alix or TSG101 (). Hence, THP may be a suitable normalizing variable, especially as its level is easily assessed by densitometry of Coomassie-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS—PAGE) and does not require an immunoassay. THP is secreted by the thick ascending limb of Henle’s loop and, although its secretion is unlikely to be invariant among subjects, it is more likely to be relatively constant in the same subject. The advantage of THP over measurement of creatinine or some other substance filtered at the glomerulus is that it can be measured in the same high-speed differential centrifugation fraction that is used to assay potential biomarkers. Hence, factors that would cause THP to vary either physiologically or technically (in the isolation process) would be common to THP determination and the determination of any other protein in that fraction. Thus, we would expect that THP normalization would give a smaller systematic error than normalization by creatinine. In this study, we investigated the potential use of THP as a normalizing variable for estimation of relative excretion rates of exosomal proteins only in normal human subjects. Further studies are required to address the validity of THP as a normalizing variable in subjects with renal disease.