THE ADAPTED-ULTRACENTRIFUGATION METHOD OF ISOLATION OF URINARY EXTRACELLULAR VESICLES (EVS) IN PATIENTS WITH AUTOSOMAL DOMINANT POLYCYSTIC KIDNEY DISEASE (ADPKD)

YANEFF A; VLACHOVSKY S; PERONI RN; ROSEMBERG ML; RIERA N; GOETTE NP ; AZURMENDI PJ; BRANCA BE; DAVIO CA; ODDO EM

Mar del Plata

Congreso; 1. LXVII Reunión Científica Anual de la Sociedad Argentina de Investigación Clínica; 2022

Sociedad Argentina de Investigación Clínica

ADPKD is characterized by an intracellular calcium depletion that belongs to deficient activity of the polycystin 1 or 2 complex which, in turn, stimulates cAMP production. Urine EVs are extracellular microvesicles, which play an essential role in cell-to-cell communication, possibly by interactions between the primary cilium. The gold standard for EVs isolation is a 200,000xg ultracentrifugation,a method that is not currently accessible to clinical laboratories. Previous results (Rosenberg, et al., abstract #0689, SAIC 2019) obtained with EVs extracted by a commercial kit based on precipitation showed low protein and RNA recovery and aggregated vesicles that jeopardize subsequent studies. Therefore, our objective was to isolate EVs from morning urine by centrifugation method, equalizing time and speed (50,000xg for 4h at 4ºC) compared to the ultracentrifugation, and the enrichment step using membrane filters (0.22 um) and 200 mg/ml of dithiothreitol from urine of 4 patients and 2 control subjects. EVs were characterized as spherical, 30-150 nm particles using electron microscopy and dynamic light scattering. Exosomal marker CD63, evaluated by flow cytometry, showed 60 ± 16 % of positive particles. Protein content, measured by Bradford, ranged from 0.9-1.3 ug/ul, 9±1 times more than the precipitation method. Preliminary cAMP quantification in the EVs was 2.0 ± 0.2 nmol/ml, showing a 22 ±2 fold more recovery than precipitation method. The adapted-ultracentrifugation method here recovers particles that fulfill conditions of EVs, and enriches it to improve future studies for biomarkers of disease progression.