Exploring urinary extracellular vesicles in the post-COVID-19 condition: a pilot study

Introduction: Recent studies have emphasized the importance of monitoring kidney function in the post-COVID-19 phase due to the persistence of inflammation and vascular alterations that could be associated with the development of the post-COVID condition (PCC). Objectives: To analyze urinary extracellular vesicles (uEVs) as possible biomarkers of renal dysfunction in patients presenting PCC. Methods: This is a cross-sectional study performed with patients diagnosed with COVID-19 during 2020-2022 (IRB#59213722.5.0000.5243). Laboratory and clinical data were obtained from medical records and PCC was diagnosed using the WHO definition Information on the need for hospitalization during the acute phase of the most severe episode of COVID-19 was also recorded. For uEV detection, the first urine of the morning was collected, centrifuged at x 600xg for 5 minutes to remove debris and other contaminants and stored at -80°C. uEVs were isolated by differential centrifugation (20,000xg for 20 minutes at 4ºC) and identified by nanoscale flow cytometry (Cytoflex S, Beckman Coulter). We identified total (annexin V⁺) and podocyte-derived (annexin V⁺ podoplanin) uEVs considering size calibration (100-900 nm). For negative controls during acquisition, solutions of 0.1% Triton-X, Milli Q water, Annexin V buffer and antibody mixture were used. Results: We included 42 patients (57±13 years old, 81% female), 17 (40.5%) with PCC. We observed that the PCC patients that were hospitalized were significantly older (P=0.001). When evaluating total and podocyte-derived uEVs, we found no difference between PCC and non-PCC groups. However, we observed significantly higher total uEVs in PCC individuals who were hospitalized during the acute infection (30,132±15,296 vs 1,660±7,669 counts/mL, P=0.01). Podocyte uEVs were also elevated in the same group, but without statistical significance (812±1,714 vs 459±732 counts/mL, p=0.83). Importantly, a multivariate analysis showed that the higher levels of total uEVs in PCC patients were significantly associated with hospitalization (OR 1.50; P=0.04), but not with age (OR 0.06, P=0.07). Regarding the usual renal function parameters, no difference was found between groups. Conclusions: Our results suggest that patients who required hospitalization during SARS-CoV-2 infection present elevated uEVs after the acute phase, regardless of PCC diagnosis. These alterations might be considered as early markers of renal dysfunction in the post-COVID period.