MP67-02 THE EFFECT OF ALANINE ON CELL VIABILITY AND AGT ACTIVITY IN TRANSFORMED CHINESE HAMSTER OVARY CELLS

Abstract

You have accessJournal of UrologyStone Disease: Basic Research & Pathophysiology II1 Apr 2016MP67-02 THE EFFECT OF ALANINE ON CELL VIABILITY AND AGT ACTIVITY IN TRANSFORMED CHINESE HAMSTER OVARY CELLS Win Shun Lai, Mary Elaine Killian, Sonia Fargue, John Knight, Ross Holmes, and Dean Assimos Win Shun LaiWin Shun Lai More articles by this author , Mary Elaine KillianMary Elaine Killian More articles by this author , Sonia FargueSonia Fargue More articles by this author , John KnightJohn Knight More articles by this author , Ross HolmesRoss Holmes More articles by this author , and Dean AssimosDean Assimos More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1318AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Primary hyperoxaluria is a spectrum of disorders that causes overproduction of oxalate, leading to kidney stones, nephrocalcinosis, and even end stage renal disease. Primary hyperoxaluria type 1 (PH1) is due to a mutation in the enzyme alanine-glyoxylate aminotransferase (AGT). This leads to a failure in the transamination of glyoxylate to glycine, and causes increased oxalate production. Alanine, a substrate of AGT, is a potential target in improving AGT activity. A previous study showed that alanine supplementation led to decreased production of oxalate in guinea pig peroxisomes. Expanding on this finding, our goal was to determine the effects of alanine supplementation on AGT activity and cell viability in Chinese Hamster Ovary Cells (CHO) expressing various PH1-specific AGT mutants. METHODS CHO cells transformed with glycolate oxidase (GO) and normal or mutant human AGT cDNA were incubated without and with alanine in the setting of a cell-based glycolate toxicity assay. Cell viability and AGT activity in the form of intracellular and extracellular oxalate concentrations were measured using ion chromatography and mass spectrometry. Data were analyzed using Student’s t-test. RESULTS Oxalate concentrations increased and cell viability decreased in the cell lines transformed with GO and an AGT variant after incubation with 750uM glycolate. Upon addition of alanine 5mM, a statistically significant improvement in cell viability was seen for the major (AGT-MA), minor (AGT-mi) alleles, and mutants AGT-170 (G170R), AGT-152 (F152I) and AGT-244 (I244T). A statistically significant reduction in extracellular oxalate was also noted for AGT-MA, AGT-mi, AGT-170 and AGT-244. No significant change in intracellular oxalate was seen. CONCLUSIONS Supplemental alanine reduces oxalate production and improves cell viability in this model for certain AGT variants. This finding warrants further investigation as a potential therapeutic option to reduce oxalate synthesis in patients with PH1. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e881 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Win Shun Lai More articles by this author Mary Elaine Killian More articles by this author Sonia Fargue More articles by this author John Knight More articles by this author Ross Holmes More articles by this author Dean Assimos More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...