MP02-05 CDKN2A REGULATES OXIDATIVE STRESS AND AUTOPHAGY IN UROTHELIAL DEFENSE AGAINST URINARY TRACT INFECTION

Abstract

You have accessJournal of UrologyCME1 May 2022MP02-05 CDKN2A REGULATES OXIDATIVE STRESS AND AUTOPHAGY IN UROTHELIAL DEFENSE AGAINST URINARY TRACT INFECTION Yan Liu, Hongying Huang, Herbert Lepor, Xue-Ru Wu, and Ellen Shapiro Yan LiuYan Liu More articles by this author , Hongying HuangHongying Huang More articles by this author , Herbert LeporHerbert Lepor More articles by this author , Xue-Ru WuXue-Ru Wu More articles by this author , and Ellen ShapiroEllen Shapiro More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002514.05AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The Cyclin-Dependent Kinase Inhibitor 2A gene (CDKN2A) plays a critical role in innate urothelial defenses against uropathogenic E.coli (UPEC). In response to urinary tract infection (UTI), the CDKN2A gene is hypermethylated, and gene expression is downregulated; loss of CDKN2A led to increased intracellular bacterial communities (IBCs) and worsened bacterial burden. This study aims to uncover the molecular mechanism underlying the CDKN2A mediated defensive role in UTI. METHODS: Human bladder cancer cells 5637 harboring wildtype CDKN2A were transfected with CDKN2A-siRNA or control scrambled siRNA and then infected by a cystitis strain UIT89 at a multiplicity of infection of 1:100. Seven-week-old female CDKN2A knockout (KO) and wildtype (WT) mice were infected through transurethral inoculation of 107 CFU of UTI89. Mice were euthanized, and bladders were lysed and plated to assess the UPEC colonization. IBCs were evaluated by staining tissue sections with anti-E.coli and UPIa antibodies. Whole-mount urothelium and cultured 5637 cells were incubated with 10 μM DCFDA to examine the reactive oxygen species (ROS) level. Autophagy was assessed by LC3 western blot. Expelled UPEC were analyzed by bacterial expulsion assay. Neutrophil infiltration was examined by neutrophil marker Ly6G immuno-staining and by myeloperoxidase activity assay. RESULTS: UPEC inoculation induced a rapid increase of ROS in both infected 5637 bladder cancer cells and mice bladders. At 8 hours post-infection (hpi), the ROS returned to baseline level in 5637 cells but remained high in 5637 cells with CDKN2A knockdown. The CDKN2A knockdown cells also showed a 50% reduction in autophagic flux and expelled 3-time less UPEC than the control cells. Consistently, ROS level in CDKN2A KO mice was 2-fold higher than that in the WT mice at 3 hpi and 3.2-fold higher at 24 hpi. Accompanying the severe and prolonged oxidative stress in CDKN2A KO mice were the significantly less accumulation of the autophagosomal marker LC3-II and two times more intracellular bacterial communities. Furthermore, the UPEC colonization in CDKN2A KO mice was 5-fold more at 24 hpi, and the neutrophil infiltration was significantly lower than the control mice. CONCLUSIONS: CDKN2A plays a critical role in regulating redox homeostasis and autophagy in response to UTI. Loss of CDKN2A is detrimental to the expulsion of UPEC and neutrophil infiltration. CDKN2A may have significant clinical implications for understanding and treating acute and recurrent UTIs. Source of Funding: None © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e11 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yan Liu More articles by this author Hongying Huang More articles by this author Herbert Lepor More articles by this author Xue-Ru Wu More articles by this author Ellen Shapiro More articles by this author Expand All Advertisement PDF DownloadLoading ...