Abstract P252: Differential Expression Of ACE2, Neprilysin And ACE In Extracellular Vesicles Isolated From Human Urine

Abstract

The Ang II convertase ACE2 is present in urine and may correlate with various renal pathologies. We reported functional ACE2 in urinary exosomes and now extend our studies to distinguish exosomes (EXOs) and microvesicles (MVs) containing ACE2, as well as neprilysin (NEP) and ACE. EXO purification was modified by an intermediate centrifugation step (20k xg, 60 mins) to pellet MVs followed by 100k xg to pellet EXOs. The MV, EXO, and 100k supernatant (soluble, SOL) fractions were assessed for ACE2 activity with MCA-APK-DNP, and NEP and ACE activities with MCA-RPPGFSAFK-Dnp with selective inhibitors. Peptidase activities were expressed as relative fluorescent units (RFU x10 6 /min/mL), and data are means ± SEM (*P<0.05; **P<0.01, N=4). Morning collections from male subjects [51 to 65 years of age, non-smokers] were collected, treated with Cibacron blue to bind albumin, 0.2 μM filtration, and 100 kDa Amicon concentration prior to high speed and ultracentrifugation steps. ACE2 activity was >3-fold higher in the EXO versus the MV fractions with minor activity in the SOL fraction. In contrast, NEP activity was highest in the MV fraction [~2-fold > EXO] while endopeptidase activity was barely detectable in the SOL fraction. ACE activity was similar in MVs and EXOs, but NEP activity was 40-fold higher than ACE in both the MV and EXO fractions. Differential centrifugation of the 100-kDa urine concentrate reveals a unique profile of ACE2, NEP and ACE expression in MVs and EXOs. The functional significance of these peptidase activities in different vesicular populations is currently unknown; however, their origin may reflect the distinct regulation and release of ACE2 and NEP/ACE in urinary vesicles.