Abstract
Introduction: Age-related dysfunctions are multifactorial and are associated with reactive
oxygen species (ROS) induced damage to cells and tissues. Overactive bladder (OAB) syndrome presents a major health and social challenge to our ageing society, but the underlying mechanisms are poorly understood. NADPH oxidase (Nox) enzymes generate ROS, particularly superoxide (O2•-), as a sole product, offering a specific pharmaceutical target for managing bladder pathologies. Recognition of bladder urothelium as a novel sensory structure has stimulated research interest. The role of Nox-derived O2•- in bladder urothelium function and ageing has never been studied. Role of inflammatory mediators in O2•- generation and urothelial function was explored.
Methods: Normal young vs aged C57BL/6 J mice and wildtype (WT) vs Nox knockout
(Nox-/-) mice were used as experimental models. Tissue samples were prepared by microdissection. Lucigenin-enhanced chemiluminescence measured NADPH-dependent O2•- production. Luciferin-luciferase assay quantified urothelial ATP release, a main urothelial sensory transducer. Immunohistochemistry and Western blotting determined the expression of Nox enzymes and various DNA damage response (DDR) components.
Results: Nox2 and Nox4 subtypes were expressed within the mice bladder wall where Nox2
being the main subtype. Urothelium is the main origin of Nox-derived O2•- and ATP release. Inflammatory mediators can mediate Nox-derived O2•- and urothelial ATP release through specific pathways. Key specific pathways identified include protein kinase C (PKC), Rac and p47phox. Members of DDR pathway (p53, p16 and γH2AX) were expressed in the mice bladder wall. Tissue type and age specific differences were identified for each study.
Conclusion: These results provide the first evidence for role of inflammatory mediators in urothelial function and identify mechanisms of action for O2•- generation and ATP release associated with bladder inflammation and ageing.