Recognition of the urothelium as a new sensory structure has significantly advanced our understanding of bladder physiology[1]. The specific receptor profiles of this tissue layer, distinct from those of the detrusor muscle make it a novel target for therapeutic exploration. Its involvement in pathological conditions has further stimulated intense interest in identifying the pathological regulators in this tissue. Reactive oxygen species (ROS) and oxidative stress is a fundamental mediator underlying many aging and chronic conditions. The ROS-generating NADPH oxidase (Nox) is of particular importance as it produces ROS as its sole function and can be selectively targeted to control excessive ROS without comprising essential biochemical oxidation as seen with all other enzymes in the body [2]. Our recent pilot studies have identified this enzyme in the urothelium and determined the basic mode of actions [3]. We hypothesise that Nox-derived ROS production is upregulated in response to aging and this has specific contribution from different Nox enzyme subtypes. We also hypothesise that Nox proteins are expressed and functional in human urothelium and Nox-derived ROS have physiological relevance and pathological implications in human conditions. This study aimed to explore aging specific changes in bladder urothelium using mouse model,  and to identify the presence and function of major Nox subtypes in human urothelial tissue and examine the physiological and pathological roles of Nox-derived ROS in human samples.

Young adult C57BL/6J mice (3-6 months) and old age mice (1.5-2.5 years) were used in compliance with UK and EU regulations. Aging Nox-1 (B6.129X1-Nox1tm1 Kkr/J), Nox-2 (B6.129S-Cybbtm1Din/J) and Nox4 (B6.129-Nox4tm1Kkr/J) knockout mice on C57BL/6J background and their wild type littermates (The Jackson Laboratory) were also used to dissect molecular mechanisms.  All experimental models were maintained in pathogen-free and standard feeding conditions. Mice were euthanised to obtain bladder tissue in compliance with the regulations. The genotyping was determined for each mouse by PCR with specific primers and gel electrophoresis.  Human bladder biopsy samples were obtained during cystoscopy with informed patient consent and ethical approval. Bladder mucosa and smooth muscle were micro-dissected under microscopic guide. Western blot determined the expression of Nox subtypes with Nox1, Nox2, Nox3, Nox4 and Nox5 primary antibodies and the secondary antibodies conjugated with near-infrared fluorescent dyes. Lucigenin-enhanced chemiluminescence quantified NADPH-stimulated superoxide production in live tissue. 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 tissue preparations. Data are expressed as mean±SEM or median and interquartile range. 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. The association between two variables were determined by correlation analysis.

Aging mouse bladder mucosa generated more superoxide than the young control tissue (RLU/mg tissue: 571±64 vs. 400±41, n=27, 14, p<0.05). This change was not seen in bladder smooth muscle (RLU/mg tissue: 25±3 vs. 34±7, p>0.05). In aging Nox subtype knockout mice, the superoxide production was reduced in Nox1, Nox2 and Nox4 deletion compared with their wildtype littermate controls. Substance P,  an inflammatory factor involved in bladder inflammation and pain, augmented superoxide production from bladder mucosa (170±38, n=11, p<0.05) and smooth muscle (193±41% control, n=11, p<0.05) in aging but not in young age control bladders. In human bladder mucosa biopsies, Western blot showed the expression of Nox 2 (high), Nox1  (low) and Nox5 (low) subtypes (n=5-6 samples).  NADPH stimulated significant superoxide production with a level of 192±65 RLU/mg tissue (n=8, p<0.01 vs. baseline control). The specificity of superoxide detected in the tissue was verified by its sensitivity to superoxide scavenger Tiron (10mM). Angiotensin II, a bladder pathology relevant inflammatory factor and Nox activator, increased the superoxide production to 188±28 % of the control (n=5, p<0.05). In urothelium functional experiments, Angiotensin II also enhanced ATP release from these human mucosa biopsies (control vs. Angiotensin II, 59±16 and 180±62 pmoles/g tissue/min respectively, n=8, p<0.05), with a 266±42% increase. GSK1016790A (1µM), a specific activator for TRPV4 receptor mediating sensory dysfunction and bladder pain, also increased ATP release to 279± 55 % of control (n=12, p<0.01). Control experiments using mouse bladder mucosa showed that GSK1016790A was able to enhance superoxide production (RLU/mg tissue, median with interquartile range: control, 70 [33, 123]; GSK, 136 [44, 219]; n=10, p<0.05, Wilcoxon test). The GSK stimulated peak ATP level and the net increase of ATP release were much higher in samples from the overactive bladders (peak ATP levels, ρmoles/g tissue/min; OAB, 693±239 (n=6) vs. control, 190±98 (n=14), p<0.05; net increase: OAB, 190±98 (n=6) vs. control, 88±49 (n=14), p<0.05). The proportional increase of ATP release triggered by GSK was positively correlated with age in the OAB group (n=6, r=0.88, p<0.05). However, this increase was not correlated with age in the control group.

A significantly higher level of NADPH-dependent superoxide production in aging bladder mucosa shows that Nox-derived ROS production is upregulated in the urothelium during bladder aging.  A lack of such difference in the smooth muscle suggests this change is urothelium-specific. The attenuation of superoxide production in the bladder mucosa in aging Nox1, Nox2 and Nox4 subtype knock out mice compared with their wildtype littermate controls, reveals the contribution of these Nox subtypes to upregulated ROS production in response to bladder aging.  The augmentation of superoxide production from mucosa and smooth muscle by substance P in aging bladder demonstrate an enhanced tissue response to this bladder pathology relevant inflammatory mediator during aging.  Positive western blot results for Nox2, Nox1 and Nox5 in human bladder mucosa identify the existence of these Nox subtypes in human urothelium and stronger Nox2 expression indicates Nox2 dominance. The significant and Tiron-sensitive NADPH dependent superoxide production in human tissue proves that Nox proteins are functional in producing superoxide. The ability of Angiotensin II and TRPV4 activator GSK to enhance superoxide production and also augment ATP release demonstrate that ROS can be upregulated by bladder pathology relevant inflammatory factors and such augmentation further influences urothelial function in human conditions. The higher level of TRPV4 activation augmented urothelial ATP release in biopsies from OAB patients shows that this ROS system is involved in the pathogenesis of bladder overactivity. The positive correlation of TRPV4 activation induced ATP release with age in OAB but not in control samples uncovers aging as a specific driver for OAB progression.

These results demonstrate for the first time the age-dependent ROS upregulation in bladder urothelium, the involvement of different Nox subtypes and increased sensitivity of the Nox-ROS system to pathologically important inflammatory factors during aging. The results also provide the first evidence for molecular and functional expression of Nox proteins and major subtypes in human urothelium. More importantly, the Nox derived ROS in human urothelium can be augmented by key inflammatory factors, have pathological influence on urothelial function, and contribute to overactive bladders and aging pathology.

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