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MP42-15: The urothelium is the most predominant tissue in the body for superoxide production: key role of NADPH oxidases

Saturday, May 13
3:30 PM - 5:30 PM
Location: BCEC: Room 160

Presentation Authors: Max Roberts, Josephine Amosah, Lisa Adjei, Guildford, United Kingdom, Guiping Sui, London, United Kingdom, Rui Wu, Coventry, United Kingdom, Simon Archer, Jonathan Johnston, Guildford, United Kingdom, Michael Ruggieri, Philadelphia, PA, Changhao Wu*, Guildford, United Kingdom

Introduction: Recognition of the urothelium as a new sensory structure has significantly advanced our understanding of bladder function. Key to further progress is the identification of novel pathological regulators in this tissue. Oxidative stress is a fundamental pathological mediator; ROS generating enzyme NADPH oxidase (Nox enzyme) has attracted intense interest recently as it is the only enzyme that produces ROS as its sole function and can be targeted without compromising normal biochemical oxidation. Our recent pilot study provided initial evidence for the presence of such system in bladder urothelium and its potential functional significance. This study aimed firstly to define the importance of urothelial superoxide production in the body and secondly to dissect the enzymatic sources of superoxide production in the bladder.

Methods: C57BL/6J mice were euthanized. Bladder and other types of tissue were isolated. Lucigenin-enhanced chemiluminescence quantified superoxide production in live tissue. Western blot determined Nox subtype expressions.

Results: Superoxide production in bladder mucosa (RLU/mg tissue: 536.8±104.8, mean±SEM) was many folds as high as those in detrusor muscle (21.8±3.8, n=15, p<0.01), aorta (67.2±26.6, n=7, p<0.05), brain (9.4±1.7, n=6, p<0.01), kidney (84.3±23.0, n=6, p<0.05), ventricle (21.8±3.8, n=6, p<0.01) and liver (80.8±12.9, n=7, p<0.05). NADPH oxidase inhibitor diphenyleneiodonium (DPI, 20µM)reduced superoxide production to 10.8±3.4 % of control (n=6, p<0.01) in bladder mucosa and to 30.8±8.4% of control (n=6, p<0.01) in detrusor. Mitochondria de-coupler FCCP (10µM) suppressed superoxide production to 51.8±10.5 % of control (n=6; p<0.01) in bladder mucosa and to 59.8±10.4 % of control (n=6, p<0.05) in detrusor. Xanthine oxidase inhibitor oxypurinol (100µM) produced no significant effect in bladder mucosa (87.9±17.4 % of control, n=6, p>0.05) but a small inhibition in detrusor (78.0±6.6% of control). Western blot showed specific bands for Nox1, Nox2 and Nox4 but no Nox3 expression in bladder mucosa and detrusor with significantly higher expression in bladder mucosa (p<0.05, n=4-6).

Conclusions: These data demonstrate for the first time that the urothelium is the most active tissue for superoxide production in the body. Nox enzymes are the main enzymatic source for superoxide in bladder. The main Nox subtypes are Nox1, Nox2 and Nox4, mainly located in the urothelium. Exceptionally high levels of Nox-driven superoxide explain why bladder urothelium is prone to oxidative stress, inflammation and sensory dysfunction.

Source Of Funding: BBSRC BB/P004695/1; NIA 1R01AG049321-01A1

Changhao Wu, PhD

University of Surrey

Dr Changhao Wu has obtained both MD and PhD degrees and is currently appointed as a reader (Professor) at University of Surrey, UK. Dr Wu has wide interest in biomedical research and extensive experience in basic science and translational research in urinary track, cardiaovascular and liver diseases. His main focus is on urinary track cell physiology, pathophysiology and aging, in particualr the role of oxidative stress, inflammatory factors and urotheliual signalling in these processes with both molecular and functional approaches using both experimental models and human samples.


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MP42-15: The urothelium is the most predominant tissue in the body for superoxide production: key role of NADPH oxidases

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