Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, C029

Oral Communications

The bladder mucosa produces the highest level of NADPH oxidase-derived superoxide in the body: pathological significance and therapeutic potential

M. Roberts1, J. Amosah1, M. R. Ruggieri2, D. Wu1

1. School of Biosciences and Medicine, University of Surrey, Guildford, SURREY, United Kingdom. 2. Department of Anatomy and Cell Biology, Temple University, Philadelphia, Pennsylvania, United States.

Lower urinary tract diseases affect 1/3 of world population, with 546 million having overactive bladder (OAB) in 20181. Despite the exceptionally high prevalence, specific treatments are lacking. The urothelium, the bladder epithelial lining, has generated intense interest for new targets following recent identification of its novel sensory role2. The tissue is also highly susceptible to age-related dysfunction3,4. The role of oxidative stress and reactive oxygen species (ROS) in several bladder pathologies has been demonstrated, including OAB5. However, the sources of ROS are unknown, which may provide therapeutic targets. NADPH oxidases (Nox) have gained increasing interest, as they produce ROS as a sole function and have the advantage over other ROS-generating enzymes with little side effects. We hypothesise that the urothelium is capable of producing high levels of ROS, through Nox enzymes, contributing to age-related bladder pathologies. Tissues from bladder and other organs were isolated from mice (12wk, male C57BL/6) by micro- dissection in accordance with UK/EU regulations. Immunofluorescence and Western blot determined Nox subtype expression. Lucigenin-enhanced chemiluminescence quantified superoxide (SO) release from live tissue. Data are expressed as medians±interquartiles, tested by Wilcoxon's (non-Gaussian data). Western blotting (n=10 bladders) and immunohistochemistry (n=5) demonstrated Nox1, 2 and 4 subtype expression throughout the bladder, with no Nox3 or 5. The mucosa had significantly higher expression than smooth muscle (SM) of NOX2 (mucosa: 4.5[2.9, 6.7] Vs SM: 1.7[0.8, 2.0] p<0.05) and Nox4 (mucosa: 1.02[0.7, 1.9] Vs SM: 0.6[0.4, 0.8] p<0.05), but similar Nox1 expression. Chemiluminescence identified significantly higher SO release from mucosa compared to other bladder tissues and major organs (mean light units (MLU): mucosa[n=102]: 340[182, 539] SM[n=102]: 16[12, 24] Aorta[n=38]: 33[21, 86]Cerebrum[n=34]: 17[10, 29] Kidney[n=39]: 109[63, 146] Liver[n=34]: 97[62, 116] Ventricle[n=34]:7.4[5.0, 13] Large int.[n=16]: 106[60, 228] Small int.[n=14]:15 [11, 27] p<0.05 for all mucosa Vs. other tissue). The contributions to mucosal SO from mitochondria were 35% with uncoupling agent (con: 90[58, 106] 1µM FCCP: 56[52, 63] n=9, p<0.05) and 15% with complex1 inhibitor (con: 92[82, 128] 10µM rotenone: 74[56, 86] n=13, p<0.05). Nox contributed 75% of SO production (con: 82[47, 97] 20µM DPI: 7[5, 17] n=7, p<0.05) with Nox1 accounting for 20% (con: 86[72,104] 5µM NoxA1ds: 66[57, 91] p<0.05). This study identified for the first time an exceptionally high level of ROS from the urothelium, compared to that in other major organs known to produce high levels of ROS. These findings explain the highly inflammatory nature of bladder urothelium and demonstrate that Nox enzymes are a major contributor to age-dependent bladder pathologies and serve as a novel group of drug targets.

Where applicable, experiments conform with Society ethical requirements