Recognition of the urothelium as a new sensory structure is a major advance in our understanding of bladder physiology[1]. Changes to urothelial structure and function in pathological conditions further emphasize its importance in bladder pathologies. A variety of receptors has been identified and recognised as physiological regulators of the urothelium[2]. A major step forward is the identification of novel pathological regulators in this tissue. Oxidative stress due to excessive reactive oxygen species (ROS) is a fundamental pathological mediator for aging and many chronic diseases. ROS are mainly generated by enzymatic oxidation. However, controlling ROS production by modulating these enzymes is impractical as almost all these enzymes produce ROS as a by-product while carrying essential physiological oxidation and suppressing these enzymes would compromise vital cell function. A unique class of ROS-generating enzyme is NADPH oxidase (Nox) as it is the only enzyme that produces ROS as its sole function and can be targeted without compromising physiological oxidation[3]. Identifying and targeting Nox subtypes in the body has generated intense interest in recent years however this has never been examined in bladder tissue. We hypothesize that major Nox subtypes exist in the urothelium and are a major source for superoxide; this ROS pathway plays a key role in urothelial and bladder function. This study aimed to identify the presence of major Nox subtypes in the urothelium and bladder tissue, to show that these Nox enzymes are a major source of superoxide production and define the importance of urothelial superoxide production in the body, to dissect the contribution of Nox subtypes in superoxide production, and finally to demonstrate its functional importance.

C57BL/6J mice (8-14 weeks) were euthanized in compliance with the current regulations. Bladder and other tissue types were isolated, and mucosa-attached and denuded detrusor muscles and mucosal sheets were micro-dissected. Immunofluorescence and confocal microscopy determined the expression of Nox subtypes with Nox1, Nox2, Nox3 and Nox4 primary antibodies and fluorescent dye-conjugated secondary antibodies. Western blot further verified specific protein bands of Nox subtypes with their primary antibodies and the secondary antibodies conjugated with near-infrared fluorescent dyes. Lucigenin-enhanced chemiluminescence quantified NADPH-stimulated superoxide production in live tissue. Dihydroethidium (DHE) fluorescence measured intracellular superoxide generation. Human bladder mucosal samples were obtained at cystoscopy with ethical approval and informed patient consent. Tissue preparations were incubated in a HEPES-buffered physiological saline. A luciferin-luciferase assay determined tissue ATP release in the superfusate sampled adjacent to the preparations. Data are expressed as mean±SEM. Student’s t-test examined two paired and non-paired normally distributed data sets; non-parametric equivalent tests were used for data sets of unknown distribution. ANOVA with post-hoc pair-wise comparison tested the difference between multiple means.

Immunofluorescence confocal microscopy demonstrated presence of Nox1, Nox2 and Nox4 subtypes in mouse bladder urothelial layer and smooth muscle, with higher intensity in the urothelium (n=6 bladders). Western blot further demonstrated specific protein bands for Nox1, Nox2 and Nox4 in bladder mucosa and smooth muscle (n=7). Lucigenin assay showed significant NADPH-dependent superoxide production in bladder tissues, sensitive to superoxide scavenger Tiron (10mM), with the main source from the mucosa. Superoxide production in bladder mucosa (RLU/mg tissue: 530±8, mean±SEM, n=16) was many fold higher than that in detrusor muscle (21±4, n=16, p<0.01), aorta (70±23, n=8, p<0.01), brain (12±3, n=7, p<0.01), kidney (95±22, n=7, p<0.01), ventricle (7±1, n=7, p<0.01) and liver (81±13, n=7, p<0.01). DHE imaging revealed positive staining in bladder tissue, with stronger intensity in the urothelium (n=8). Superoxide scavenger Tiron abolished the DHE fluorescence. The broad spectrum Nox inhibitor diphenyleneiodonium (DPI, 20μM) reduced superoxide production to 26±3% of control (n=7, p<0.01) in bladder mucosa. Mitochondria de-coupler FCCP (1μM) suppressed superoxide production to 75±11 % of control (n=7; p<0.01) in bladder mucosa. Xanthine oxidase inhibitor oxypurinol (100μM) produced no significant effect (85±13 % of control, n=8, p>0.05). Nox1 selective inhibitor NoxA1ds (5μM) inhibited superoxide production to 88±6 % of control (n=25, p<0.01). Nox2 specific inhibitor GSK2759039 (1μM) reduced superoxide production to 76±6 % of control (n=15, p<0.01). In a further set of experiments with combined inhibitors, a combination of Nox1 inhibitor NoxA1ds and Nox2 inhibitor GSK2759039 reduced the superoxide production to 71±15% of control (n=11, p<0.05), while addition of Nox1/Nox4 dual selective inhibitor GSK137831 (2μM) in the presence of the above two inhibitors reduced the superoxide production further to 53± 7% of control (p<0.01), revealing additional Nox4-selective inhibition. Application of exogenous ROS H2O2 (100µM) increased the ATP release from mucosa-attached bladder strips to 239±43% of control (n=5, p<0.01). Angiotensin II (1µM), an inflammatory mediator and Nox activator as shown in vascular tissue, increased the superoxide production (RLU/unit tissue: 275±76 to 317±112, n=16, p<0.05) and also augmented ATP release from bladder mucosa (222±20% of control, n=24, p<0.01). Angiotensin II also increased ATP release from human bladder mucosa samples (n=5).

Positive confocal immunofluorescence images identify the existence of Nox1, 2 and 4 subtypes in the bladder with urothelial dominance. Western blot specific protein bands provide further proof for expression of Nox1, 2 and 4 subtype molecules in bladder tissue and the urothelial importance. Significant NADPH-dependent and Tiron-sensitive lucigenin intensity suggests Nox-driven superoxide production from the bladder and presents direct evidence that Nox enzymes are functional; a stronger signal from the mucosa proves its main source from the urothelium. The strong DHE signal and consistently greater intensity in the mucosa further demonstrate the intracellular source of superoxide. The multi-fold higher levels of superoxide in the bladder mucosa, compared to detrusor muscle and several other major types of tissue known to generate significant ROS, demonstrate that the urothelium is the most predominant tissue in the body for superoxide production. The exceptionally high capacity of the urothelium to produce superoxide explains why bladder is so sensitive to inflammation, pain and overactivity. The differential sensitivities of the bladder tissue to Nox and mitochondrial inhibitors identifies that urothelial superoxide production is mainly from Nox enzymes and, to a lesser extent, from mitochondria. The inhibitory effects of Nox1 specific inhibitor NoxA1ds and Nox2 specific inhibitor GSK2759039 on superoxide production suggest that some Nox activity are from Nox1 and Nox2 subtypes. The additional inhibition of superoxide production by Nox1/Nox4 dual inhibitor GSK137831 in the presence of Nox1 and Nox2 inhibitors to suppress Nox1 and Nox2 enzymes, uncovers additional Nox4 activity. The ability of exogenous ROS H2O2 to increase ATP release emphasizes that ROS can influence the key urothelial and bladder function. The stimulatory effect of angiotensin II, an inflammatory factor, on superoxide production and ATP release from the urothelial tissues underscores the importance of Nox-derived superoxide in bladder function and further suggests its pathological implications and human relevance.

These results demonstrate for the first time that the main Nox subtypes Nox1, Nox2 and Nox4 are expressed in the bladder wall, predominantly located in the urothelium; these Nox enzymes are functional in producing superoxide, with contribution from each subtype. More importantly the study has discovered that the urothelium is the most active tissue in the body for superoxide production. The main enzymatic source for superoxide production in the bladder is Nox enzymes as opposed to mitochondrial electron transport. Furthermore Nox-derived superoxide has functional importance in the bladder and also has pathological significance and human relevance. Exceptionally high levels of Nox-driven superoxide explain why bladder urothelium is susceptible to oxidative stress, inflammation and sensory dysfunction.

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