Interstitial cystitis/bladder pain syndrome (IC/BPS) is characterized by bladder and/or pelvic pain and increased urinary urgency, frequency, and nocturia, resulting in a severe impact on the patient’s quality of life. The pathophysiology of IC/BPS is poorly understood, and the key mechanisms implicated in causing or maintaining IC/BPS include chronic inflammation, autoimmune dysregulation, urothelial dysfunction, and oxidative stress [1]. The nitric oxide (NO)/soluble guanylate cyclase (sGC)/cyclic‐GMP (cGMP) signaling pathway is ubiquitous and regulates several functions in physiological systems, including the lower urinary tract, under many pathophysiological conditions. [2]. 
A previous study showed increased intravesical NO levels in IC/BPS patients [3]. However, the major source NO production and impact on the NO/sGC/cGMP pathway have not yet been determined. We investigated the expression of genes of the NO/sGC/cGMP pathway and NO and superoxide anion (O2-) levels in urothelial samples obtained from control and IC/BPS patients.

All aspects of the study protocol received full board review and approval from the Institutional Committee. Informed consent was obtained from all participants prior to sample acquisition. Samples were obtained from 5 patients with diagnosed IC/BPS (46.9 ± 11.9 years old) and 7 control patients (49.8 ± 10.3 years old) without morphologic or clinical features of IC/BPS. Biopsies of the urothelium were obtained through cystoscopy using cold cup forceps  and immediately submerged in cold Krebs-Henseleit solution (118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3 and 5.6 mM glucose, pH 7.4). Tissues were processed within one hour of removal, segmented to different analyses, and stored accordingly at -80 ºC. RT-PCR of NOS2 (iNOS), NOS3 (eNOS), GUCY1B1 (sGC subunit β1), PRKG (protein kinase cGMP dependent 1, PKG1), PDE5A (phosphodiesterase type 5A), and SOD1 (superoxide dismutase type 1) was determined in urothelium samples. Nitric oxide (NO) and O2- generation were determined by histochemistry of frozen urothelium sections using DAF-2A and DHE fluorescent probes, respectively. Additionally, DHE was carried out in the presence or absence of the NOS inhibitor L-NAME (1 mM) and the iNOS selective inhibitor 1400 W (10 µM). Data are presented as the mean ± SEM. The Shapiro-Wilk test was performed to confirm normality. A two-tailed unpaired Student’s t-test was performed, p < 0.05 was reported as significant.

In relation to the control samples, urothelial mRNA expression (Table 1) of NOS2 was reduced by 56% in samples from IC/BPS patients, although statistical significance was not yet reached, while mRNA expression of SOD1 was significantly reduced by approximately 39%. On the other hand, PDE5A mRNA was significantly increased by approximately 66% in relation to the controls. No statistically significant differences were observed in NOS3 or PRKG1 mRNA expression levels. 
Nitric oxide production, as evaluated by the DAF-2A assay, showed no differences between the control (6.07 ± 0.62 fluorescence/µm2) and IC/BPS (6.39 ± 1.20 fluorescence/µm2) samples (Fig.2A). The basal levels of O2- were also not different between the control (23.73 ± 3.37 fluorescence/µm2) and IC/BPS (19.34 ± 3.88 fluorescence/µm2) samples (Fig.2B). Pre-incubation (30 min) with L-NAME reduced O2- generation in both control (~66% reduction, p < 0.05) and IC/BPS samples (Fig.2B), although L-NAME inhibition was higher in IC/BPS (~85% reduction, p < 0.05). Pre-incubation (30 min) with iNOS inhibitor 1400W did not produce any significant changes in O2- generation in control, but increased significantly by >3 times O2- generation in IC/BPS samples (Fig.2B).

The observed alterations in mRNA expression levels of GUCY1B1 and SOD1 and, remarkably, PDE5A between the control and IC/BPS samples suggest potential dysregulation in the urothelial NO/sGC/cGMP pathway. Despite no significant difference in NO or O2- basal levels between the control and IC/BPS samples, the inhibitory response of L-NAME was greater in the IC/BPS samples than in the controls, suggesting that the contribution of NOS to O2- generation in IC/BPS was higher. Interestingly, inhibition of iNOS by 1400W had no effect on control samples, whereas in IC/BPS samples it markedly increased O2- levels. It can be suggested that selective iNOS inhibition favors greater O2- generation, not by directly inhibiting its formation, but by reducing NO synthesis, which will rapidly react with O2- , possibly derived from dysfunctional uncoupled eNOS, to form peroxynitrite (ONOO-; not detectable by DHE). Thus, the level of ROS in IC/BPS may depend on different sources for NO and O2- generation, iNOS and eNOS, respectively, suggesting that ONOO- is the main effector molecule. Future analyses will further explore this point.

Despite the small experimental sample size, these results are interesting and may contribute not only to understanding the role of the NO/sGC/cGMP pathway in the urothelium but also of the contribution of ROS generation to IC/BPS and the potential therapeutic drugs, such as PDE5 inhibitors.

Continence 12S (2024) 101471
DOI: 10.1016/j.cont.2024.101471