MP20-14 UNCOUPLED ENDOTHELIAL NITRIC OXIDE SYNTHASE CONTRIBUTES TO OXALATE-INDUCED OXIDATIVE CELL INJURY IN RENAL EPITHELIAL CELLS: EFFECT OF ANTIOXIDANTS

Abstract

You have accessJournal of UrologyStone Disease: Basic Research I1 Apr 2014MP20-14 UNCOUPLED ENDOTHELIAL NITRIC OXIDE SYNTHASE CONTRIBUTES TO OXALATE-INDUCED OXIDATIVE CELL INJURY IN RENAL EPITHELIAL CELLS: EFFECT OF ANTIOXIDANTS Vijayalakshmi Thamilselvan, Mani Menon, and Sivagnanam Thamilselvan Vijayalakshmi ThamilselvanVijayalakshmi Thamilselvan More articles by this author , Mani MenonMani Menon More articles by this author , and Sivagnanam ThamilselvanSivagnanam Thamilselvan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.736AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Hyperoxaluria is one of the major risk factors for calcium oxalate urolithiasis and studies support the concept that cellular injury is a critical factor in calcium oxalate crystal nucleation and development of kidney stones. We have previously shown that oxalate-induced reactive oxygen species (ROS) mediates oxidative renal injury via NADPH oxidase (NOX) activation and mitochondrial dysfunction in renal epithelial cells. Here we demonstrate that Uncoupling of Endothelial Nitric Oxide Synthase (eNOS) due to increased NOX and mitochondrial dependent ROS generation also mediates oxalate-induced oxidative cell injury in renal epithelial cells. METHODS LLC-PK1 cells were exposed to oxalate (0.5 mM-1.0 mM) for different time periods in the presence or absence of inhibitors to NOX (Diphenyleneiodonium), mitochondrial electron transport chain (ETC) (rotenone and antimycin A), Nitric oxide synthase (L-NAME) and antioxidants (vitamin E and ascorbic acid). The ROS production, LDH release, NOX activity (lucigenin chemiluminescence), and nitric oxide (NO) production were determined. Mitochondrial ROS was determined by MitoSox oxidation. Protein expression levels of eNOS and nitrotyrosine were determined by western analysis. RESULTS Treatment of LLC-PK1 cells with oxalate increased superoxide, H2O2 generation, LDH release, NOX activity and mitochondrial superoxide generation in a dose and time dependent manner. Pretreatment of LLC-PK1 cells with inhibitors of NOX, mitochondria, and nitric oxide synthase significantly decreased oxalate-induced ROS and LDH release. Oxalate-induced uncoupling of eNOS in LLC-PK1 cells was confirmed by decreased levels of nitric oxide production, eNOS protein expression and increased levels of nitrotyrosine. NOX and mitochondrial inhibitors significantly attenuated oxalate-induced eNOS uncoupling and NO-dependent damage. Vitamin E and ascorbic acid treatment effectively prevented oxalate-induced eNOS uncoupling mediated renal cell injury. CONCLUSIONS We demonstrate that oxalate-induced eNOS uncoupling contributes to additional formation of ROS and subsequent induction of oxidative renal cell injury. Combination of vitamin E and ascorbic acid is more efficient in preventing NOX/mitochondria/eNOS uncoupling mediated renal cell injury. These novel findings suggest that a combination of vitamin E and ascorbic acid should be considered when treating patients with recurrent calcium oxalate kidney stones. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e201 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Vijayalakshmi Thamilselvan More articles by this author Mani Menon More articles by this author Sivagnanam Thamilselvan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...