Presentation Authors: Maxwell W Roberts, Josephine Amosah, Guildford, United Kingdom, Guiping Sui, London, United Kingdom, Rui Wu, Coventry, United Kingdom, Simon Archer, Guildford, United Kingdom, Michael Ruggieri, Philadelphia, PA, Changhao Wu*, Guildford, United Kingdom
Introduction: Recognition of the urothelium as a new sensory structure and its potential role in mediating bladder dysfunction has led to great interest in identifying its pathological mediators. Oxidative stress is a fundamental pathological mediator and ROS-generating enzyme NADPH-oxidase (Nox) has particular importance as it is the only enzyme in the body that produces ROS as its sole function and can serve as specific ROS-controlling target without compromising normal biochemical oxidation. Our recent study has identified such system in bladder urothelium (1). To explore molecular mechanisms of superoxide production and the pathological significance of Nox-associated oxidative stress, we examined the effect of Nox-subtype deletion and several pathologically important inflammatory factors on Nox-derived superoxide production in bladder urothelial tissue.
Methods: Wild type C57BL/6J mice, Nox-1 and Nox-2 knockout mice and their wild type littermates were used in compliance with the regulations. Bladder mucosa and full-thickness bladder tissue were isolated under microscopic guidance. NADPH-dependent superoxide production in live tissue was determined by lucigenin-enhanced chemiluminescence.
Results: Nox-1 deletion reduced the superoxide production to 64.7Â±7.5% of wild type littermate control (meanÂ±SEM; n=6, p < 0.01), showing Nox-1 contribution to endogenous superoxide production. Nox-2 deletion reduced the superoxide level to 78.0Â± 5.8 % of the control (n=9, p < 0.01), demonstrating Nox-2 contribution. Angiotensin II (1ÂµM), a trophic factor and vessel constrictor which is endogenously released and involved in bladder outflow obstruction and hypertrophy, increased the superoxide production from 85.6Â±6.3 to 138Â±11.7 RLU/mg tissue (n=16, p < 0.01). TRPV4-specific activator GSK1016790A (1ÂµM), which participates in sensory dysfunction and bladder pain, enhanced the superoxide production in mucosa-attached bladder strips from 89.7Â±5.5 to 118.8 Â±5.6 RLU/mg tissue (n=10, p < 0.01). Endothelin-1 (1ÂµM), an inflammatory factor endogenously released in tissue ischemia, also augmented the superoxide production in bladder mucosa from 44.5Â±7.6 to 90.2Â±17.9 RLU/mg tissue (n=10, p < 0.05).
Conclusions: These new data show that Nox-1 and Nox-2 subtypes contribute to endogenous superoxide production in bladder urothelium. The results also provide the first evidence that inflammatory factors angiotensin II, GSK1016790A and endothelin-1 can upregulate the Nox-derived superoxide production in bladder mucosa and demonstrate the pathological significance of Nox-associated oxidative stress.
Source of Funding: BBSRC BB/P004695/1; NIA 1R01AG049321-01A1_x000D_
ReferenceRoberts M, Amosah J, Sui G, Wu R, Archer S, Ruggieri, M and Wu C (2018). Identifying the functional subtypes of NADPH oxidases (Nox) from the urothelium: implication for ROS-controlling targets. J Ur