Regulation of urothelial and bladder function by oxidative stress – the role of NOX enzymes

BackgroundThe urothelial layer of the bladder has recently been highlighted as a major sensory hub, sending, and receiving signals in response to bladder stretch. Its pathological significance has generated intense interest as changes in the urothelium have been discovered in bladder pathologies and ageing. “Inflamm-aging” is a new viewpoint for aging-associated disorders, contributing low-grade chronic inflammation to their pathogenesis. Oxidative damage has also been recognised as a cause of aging. Of all ROS generating enzymes, NADPH Oxidase (NOX) is of particular interest as it is the body’s only enzyme whose sole purpose is to create superoxide. This study aimed to explore the pathophysiological role of NOX and inflammatory mediators and determine the relevant changes in the aging bladder.MethodsExperimental models of male C57BL/6J mice and DH Guinea-pigs, and human samples were used for this study. The effect of NOX deletion was also evaluated. Stimulated release of reactive oxygen species, ATP and muscle force was measured in response to inflammatory mediators (Angiotensin-II, Endothelin-1 and Bradykinin) in young and aging samples. Immunohistochemistry and Western blotting determined the presence and localisation of NOX subtypes.ResultsNOX1, NOX2, and NOX4 subtypes were present throughout the urothelium, suburothelium and smooth muscle. The urothelium expressed 437% more ROS than the smooth muscle. ROS mimetics stimulated, while ROS scavengers attenuated urothelial ATP release. Angiotensin-II, Endothelin-1 and bradykinin significantly increased the release of ROS and ATP. A calcium-dependent mechanism for ROS production was identified. NOX2 deletion significantly reduced Angiotensin-II evoked ATP release by 48.73%. Specific changes in aging tissue were identified. Conclusion These results reveal new evidence for the role of NOX in urothelial and bladder function and identifies ROS upregulation by inflammatory mediators as a pathogenic mechanism. This demonstrates the pathological significance of inflammation and oxidative stress via the NOX pathway in bladder aging and dysfunction