Youth fountain and aging culprits are usually sought and identified in blood but not urine. Extracellular vesicles (EVs) possess parental cell properties, circulate in blood, CSF and urine, and provide paracrine and remote cell–cell communication messengers. This study investigated whether senescence‐associated secretory phenotype (SASP) and immune defense factors in EVs of urine could serve as biomarkers in elderly individuals with and without a comorbidity. Urine samples from young adults and elderly individuals with and without Parkinson disease (PD) were collected and stored at − 80 °C until studies. Urine EVs were separated from a drop-through solution and confirmed by verifying CD9, CD63, CD81 and syntenin expression. The EVs and drop-through solution were subjected to measurement of SASP cytokines and defense factors by Milliplex array assays. Many SASP cytokines and defense factors could be detected in urinary EVs but not urinary solutions. Elderly individuals (age > 60) had significantly higher levels of the SASP-associated factors IL-8, IP-10, GRO, and MCP-1 in EVs (p < 0.05). In contrast, some defense factors, IL-4, MDC and IFNα2 in EVs had significantly lower levels in elderly adults than in young adults (age < 30). Patients with and without PD exhibited a similar SASP profile in EVs but significantly lower levels of IL-10 in the EVs from patients with PD.
Fig: Isolation and validation of urine EVs. Urine (40 ml) was centrifuged to deplete cell debris, followed by depletion of apoptotic bodies by 1.0 µm and 0.22 µm filters, and finally to isolate EVs from solution by another 0.03 µm filter (A). Urine EVs (UEV) showed a prominent CD9 expression in contrast to plasma EVs (PEV) expressing a higher CD81, and no exosomal markers (CD9, CD63, CD81 or HSP90) was detected in the drop through solution (Soln) in a normal adult. The PEV isolated from the young adult (Y1) by ExoQuick contained some albumin contamination, but UEV, isolated from the young adult (Y1) by a series of membrane filtrations as shown in (A), contained neglectable albumin contamination (B). Further studies showed that similar levels of CD9/CD81/HSP90/syntenin expression were found in the EVs from young adults (n = 4; Y1, Y2, Y3 and Y4) and the EVs from old adults (n = 4; O1, O2, O3 and O4), but some extent of higher CD63/actinin-4 expression in the EVs from the elderly (C). The blots presented here are derived from the images of original western blots which are shown in Supplementary Fig. 1B and C. The flow cytometric analyses of CD63, CD9 and CD47 expression using anti-CD63-beads enriched UEVs showed that CD9 co-expressed with CD63, but not CD47, on UEVs in three replicable experiments (D). We also performed fluorescent nanoparticle tracking analyses (NTA), gating the size between 30 and 300 nm, to visualize the UEVs derived from old (O1) and young (Y1) adults in three replicable experiments (E).
This study used a simple device to separate urinary EVs from solution for comparisons of SASP and defense mediators between young adults and elders with and without PD. Results from this study indicate that aging signature is present in EVs circulating to urine and the signatures include higher inflammatory mediators and lower defense factors in urinary EVs but not solutions, suggesting a simple method to separate urinary EVs from solutions for searching aging mechanistic biomarkers may make prediction of aging and monitoring of anti-senolytic interventions possible.
Yeh, Sh., Lin, CH., Yang, YJ. et al. Higher senescence associated secretory phenotype and lower defense mediator in urinary extracellular vesicles of elders with and without Parkinson disease. Sci Rep 11, 15783 (2021). https://doi.org/10.1038/s41598-021-95062-y