Temporal dynamics of urinary extracellular vesicle excretion and cargo in healthy subjects over 24 h

Urinary extracellular vesicles (uEVs) are promising noninvasive biomarkers of renal physiology and pathology; however, their temporal excretion patterns and optimal normalization strategies remain incompletely defined, particularly because urine concentration varies diurnally. This basic physiology study used a uniquely designed protocol of urine collection to characterize 24-h variation in uEV excretion and cargo in 13 healthy adults, with samples collected at each void over 24 h. Each void and a representative 24-h collection sample were analyzed. uEVs were enriched by low-speed centrifugation with three buffer washes to deplete uromodulin. uEV size, concentration, morphology, and cargo were characterized with nanoparticle tracking analysis, cryo-electron microscopy, immunoblotting, and single EV flow cytometry. uEV concentrations varied up to 100-fold between individuals, ranging from 6.2 × 10⁶ to 4.5 × 10⁹ particles/mL. The relationship between urine flow and uEV excretion varied significantly across the day. uEV concentration was positively associated with urine creatinine, specific gravity, and tumor susceptibility 101 (TSG101) EV cargo signal, allowing normalization of uEVs to urine creatinine and TSG101. Normalized uEV cargo markers exhibited substantial within-individual variation without consistent interindividual trends. When voids were grouped into predefined time periods, sodium chloride cotransporter (NCC) normalized to TSG101 showed a transient dip at noon followed by a significant increase in the next period, indicating a temporal excretion pattern. TSG101 signal did not vary significantly over the day and was notably positively correlated with uEV excretion rate. These findings offer insight into uEV excretion patterns and highlight the importance of normalization, methodological design, and temporal patterns in advancing uEV biomarker research.NEW & NOTEWORTHY Urinary extracellular vesicles (uEVs) are increasingly studied as biomarkers of kidney health, but little is known about the changes in their excretion and cargo over the day. In this 24-h study, fluctuations in uEV concentration and cargo were observed, with sodium chloride cotransporter (NCC) having a temporal pattern with a dip in signal at noon. The results highlight the importance of uEV collection timing and normalization approaches, such as creatinine, which is stably excreted over the day.