Circadian variation in the release of small extracellular vesicles can be normalized by vesicle number or TSG101

Abstract

Numerous candidate biomarkers in urine extracellular vesicles (EVs) have been described for kidney diseases, but none are yet in clinical use, possibly due to a lack of proper normalization. Proper normalization corrects for normal biological variation in urine flow rate or concentration, which can vary by over one order of magnitude. Here, we observed inter- and intra-animal variation in urine excretion rates of small EVs (<200 nm in diameter) in healthy rats as a series of six 4-h fractions. To visualize intra-animal variation, we normalized a small EV excretion rate to a peak excretion rate, revealing a circadian pattern for each rat. This circadian pattern was distinct from urine volume, urine albumin, urine creatinine, and urine albumin-to-creatinine ratio. Furthermore, urine small EV excretion was not significantly altered by sex, food/water deprivation, or ischemic acute kidney injury. Urine excretion of the exosomal/small EV marker protein tumor susceptibility gene 101 (TSG101) displayed a similar circadian pattern to urine small EV excretion; both measurements were highly correlated ( R2 = 0.85), with an average stoichiometry of 10.0 molecules of TSG101/vesicle in healthy rats. The observed stoichiometry of TSG101/vesicle in rat urine translated to human spot urine samples (10.2 molecules/vesicle) and cultured kidney-derived cell lines (human embryonic kidney-293 and normal rat kidney 52E cells). Small EV number and its surrogate, TSG101 protein, can normalize for circadian variation when testing candidate biomarkers in small EVs. Just as creatinine has emerged as the customary normalization factor for liquid-phase urine biomarkers, vesicle number and its surrogate, molecules of exosome/small EV-associated TSG101, should be considered as viable, normalizing factors for small EV biomarkers.

Decreased Excretion of Urinary Exosomal Aquaporin-2 in a Puromycin Aminonucleoside-Induced Nephrotic Syndrome Model

“…TSG101 and ALIX have been reported to play a pivotal role in the biogenesis of exosomes and to be marker proteins for exosomes [8,20]. Representative immunoblots of urinary exosomal TSG101 These data indicated that the decreased expression of renal AQP2 was accompanied by evident proteinuria in the present experimental model of nephrotic syndrome, in good agreement with a previous report [18].…”

“…TSG101 is a component of ESCRT-I, and ALIX is associated with ESCRT proteins [1]. As TSG101 and Alix play important roles in the biogenesis of exosomes, these proteins have been used as markers of urinary exosomes [8,20]. In the present study, we observed that the excretion of UE-TSG101 and UE-ALIX was decreased on day 5 and increased on day 8, suggesting that the number of exosomes released into urine was increased on day 5 and decreased on day 8 [8,20].…”

“…These findings strongly suggest that the excretion of UE-AQP2 is altered under the conditions of nephrotic syndrome, but this issue has not yet been investigated. In the present study, we investigated the excretion of UE-AQP2 in nephrotic syndrome using rats treated with puromycin aminonucleoside (PAN), as well as the release of exosomal marker proteins, including tumor susceptibility gene 101 protein (TSG101) and ALG-2 interacting protein X (ALIX) [8,20].…”

Plasma extracellular vesicles detected by Single Molecule array technology as a liquid biopsy for colorectal cancer

“…EVs were purified from approximately 50 mL of cell culture medium collected from U87, A549, MIA PaCa-2 and HT-29 cell lines via differential ultracentrifugation, followed by lysis in a lysis buffer as previously described [18]. Protein lysates were separated by 10% sodium lauryl sulphate polyacrylamide (SDS-PAGE) gel, and then transferred onto apolyvinylidene difluoride (PVDF) membrane (Sigma-Aldrich).…”

The proteome of small urinary extracellular vesicles after kidney transplantation as an indicator of renal cellular biology and a source for markers predicting outcome

“…This also holds true when broadening the filter to the top 50 proteins, indicating a decrease in ALIX-positive vesicles with potential implications for vesicle formation and late endosome maturation throughout renal transplantation. Further indication for an alteration in the release of vesicles is given by the reduction in TSG101 right after transplantation as TSG was proposed as a surrogate marker for urinary vesicle number in a recent study (29).…”