Hypothesis / aims of study
Recognition of the urothelium as a new sensory structure and increasing evidence for its potential role in mediating bladder dysfunction has led to great interest in identifying its pathological mediators (1). Oxidative stress due to reactive oxygen species (ROS) is a fundamental pathological mediator. Biological ROS are mainly produced from oxidizing enzymes. ROS-generating enzyme NADPH-oxidase (Nox) has particular importance as it is the only enzyme in the body that produces ROS as its sole function and can thus serve as specific ROS-controlling target without compromising normal biochemical oxidation. Identifying and targeting Nox subtypes in the body has been a subject of intense interest in recent years (2). The role of Nox-driven ROS has been demonstrated in aging, inflammation and aging-related chronic diseases in many organ systems and tissue types. Our recent study has identified the existence of such ROS-generating system in bladder urothelium and its physiological importance (3). To gain further molecular insight and understand its pathological significance, this study tested the hypothesis that Nox subtype-specific molecules Nox 1 and Nox 2 contribute to endogenous Nox activity for NADPH-dependent superoxide production in bladder urothelium and Nox-derived ROS production can be modulated by the inflammatory mediators encountered in bladder pathologies. The objectives of this study were to examine the effect of Nox-subtype deletion and several pathologically important inflammatory factors on Nox-derived superoxide production in bladder urothelial tissue.
Study design, materials and methods
Wild type C57BL/6J mice (aged 8-16 weeks), Nox-1 (B6.129X1-Nox1tm1Kkr/J) and Nox-2 (B6.129S-Cybbtm1Din/J) knockout mice of the same age on C57BL/6J background and their wild type littermates (The Jackson Laboratory) were used as experimental models maintained in standard feeding conditions and 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. Bladder mucosa and full-thickness bladder tissue were isolated under microscopic guidance. Tissue preparations were incubated in a HEPES-buffered physiological saline. The effect of inflammatory mediators was evaluated in the presence and absence of these substances in the same physiological saline. NADPH-dependent superoxide production in live tissue was determined by lucigenin-enhanced chemiluminescence and measured on a luminescence plate reader (LUMIstar Omega BMG LABTECH). The superoxide specificity was verified by its scavenger Tiron. A luciferin-luciferase assay determined tissue ATP release in the superfusate sampled adjacent to the preparations using a luminometer (LKB). 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.
In Nox1 knockout mice, the level of NADPH-dependent superoxide production in bladder mucosa was 65±8% of the value in wild type littermate controls (mean±SEM; n=6, p<0.01). In Nox2 knockout mice, the level of the superoxide production was 78± 6 % of the wildtype littermate control (n=9, p<0.01). Application of angiotensin II (1µM), a trophic factor and vessel constrictor which is endogenously released and involved in bladder outflow obstruction and hypertrophy, increased the superoxide production in bladder mucosa from 86±6 to 138±12 RLU/mg tissue (n=16, p<0.01). GSK1016790A (1µM), a specific activator for TRPV4 receptor, which participates in sensory dysfunction and bladder pain, enhanced the superoxide production in bladder mucosa from 90±6 to 119 ±6 RLU/mg tissue (n=10, p<0.01). Endothelin-1 (1µM), an inflammatory factor endogenously released in tissue ischemia, also augmented the superoxide production in bladder mucosa from 45±8 to 90±18 RLU/mg tissue (n=10, p<0.05). Further experiments on urothelial ATP release using angiotensin II and GSK 1016790A showed that these inflammatory mediators also enhanced ATP release from the urothelium (angiotensin II: 239±43 % of control, n=5, p<0.01; GSK1016790A: 180±35% of control, n=8, p<0.05).
Interpretation of results
The lower level of NADPH-dependent superoxide production from the urothelium in Nox1 knockout mice compared to that from their littermate controls shows that Nox-1 subtype deletion reduces the superoxide production and hence proves the contribution of Nox1 subtype to endogenous superoxide production in the urothelium. A similar change to the NADPH-dependent superoxide production from the urothelium in Nox2 knockout mice demonstrates that Nox2 deletion also attenuates NADPH-dependent superoxide generation and supports for Nox2 subtype contribution to endogenous superoxide production in the urothelium. These results uncover Nox1 and Nox2 subtypes as molecular basis for Nox-derived superoxide production in bladder urothelium and provide a rationale for targeting Nox1 and Nox2 to selectively reduce excessive ROS production and oxidative damage to the bladder. The ability to augment NADPH-dependent superoxide generation by the three pathologically important inflammatory mediators, which are encountered in bladder pathologies and involve bladder outflow obstruction, bladder hypertrophy, tissue ischemia, bladder sensation and bladder pain, suggests the wide pathological implications of Nox-derived superoxide production in the urothelium and bladder. The consistent stimulatory effect of these mediators on urothelial ATP release consolidates the functional relevance of these findings. Thus Nox-derived ROS generating machinery in the bladder serves as a downstream cellular pathway whereby inflammatory mediators amplify inflammatory actions through further ROS generation and oxidative damage.