467 QUANTITATIVE PROTEOMIC ANALYSIS OF EXTRACELLULAR MICROVESICLES IN AN IN VITRO MODEL OF URINARY TRACT OBSTRUCTION

Abstract

You have accessJournal of UrologyPediatrics: Congenital Anomalies - Kidney & Ureter1 Apr 2012467 QUANTITATIVE PROTEOMIC ANALYSIS OF EXTRACELLULAR MICROVESICLES IN AN IN VITRO MODEL OF URINARY TRACT OBSTRUCTION Dennis Orton, Alan Doucette, Geoffrey Maksym, and Dawn MacLellan Dennis OrtonDennis Orton Halifax, Canada More articles by this author , Alan DoucetteAlan Doucette Halifax, Canada More articles by this author , Geoffrey MaksymGeoffrey Maksym Halifax, Canada More articles by this author , and Dawn MacLellanDawn MacLellan Halifax, Canada More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.535AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Congenital hydronephrosis affects up to 1% of children. An in vitro model of urinary traction obstruction (UTO) using renal proximal tubule cells (NRK-52E) subjected to mechanical stretch has helped to elucidate the molecular pathology of UTO (Nguyen, et al., Nephron. Exp. Nephrol., 2006). Renal proximal tubule cells have been suggested to release protein-containing extracellular microvesicles (exosomes) into the urinary space that reflect the proteomic profile of these cells in vivo. The goal of this study was to confirm that proximal tubule cells generate extracellular microvesicles in vitro, and characterize specific changes in protein abundance in these microvesicles that result from mechanical stretch using quantitative proteomics. METHODS NRK-52E cells were grown on BioFlex plates and subjected to 24 hours of stretch-relaxation, with a maximum of 20% maximal biaxial stretch on a FX-4000 Flexercell Strain Unit (Flexcell, Hillsborough, NC, USA). The efficacy of UTO simulation is measured by cell death ELISA (Roche, Indianapolis, IN, USA). Microvesicles are isolated by ultracentrifugation from the cell culture media. Relative protein abundance is then quantitatively measured by spectral counting following mass spectrometry using a ThermoFisher LTQ linear ion trap mass spectrometer with 5 replicates. RESULTS Microvesicle isolation was confirmed by electron microscopy and immunoblotting for a known exosomal protein, Annexin 1. On average, stretched cells showed a 3 fold increase in cell death. Quantitative analysis reveals 25 proteins with altered abundance as a result of stretch. Histone proteins H4, H2B-291, H2b-61, H3 and H1 had the most significant changes in abundance, while other proteins, gelsolin, nebulin, haptoglobin, an integral membrane serine 2 protease, and polyubiquitin were also noted to have significantly altered abundances in stretch cell extracellular microvesicles (p-value <0.05 by student’s t-test). Fibronectin 1 (FN1) abundance was investigated by peak area integration, and found to be slightly increased in stretched cell microvesicles. CONCLUSIONS Renal proximal tubule cells generate extracellular microvesicles whose protein profile is altered in an in vitro model of UTO. Quantitative proteomic analysis of these extracellular microvesicles demonstrates proteins important in apoptosis, the cytoskeleton, and cellular response to oxidative stress are altered in abundance after mechanical stretch, providing insight into the pathophysiology of UTO. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e191 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Dennis Orton Halifax, Canada More articles by this author Alan Doucette Halifax, Canada More articles by this author Geoffrey Maksym Halifax, Canada More articles by this author Dawn MacLellan Halifax, Canada More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...