Cytochrome B5 reductase type-3 (CYB5R3) is a ubiquitous flavoprotein enzyme involved in many important metabolic and signaling pathways, particularly, nitric oxide (NO•) – soluble Guanylate Cyclase (sGC) – cyclic GMP (cGMP) [1]. Its deletion in the urothelium has previously been reported to induce oxidative stress, inflammation and fibrosis and mimic age-related pathophysiology in the urinary bladder [2]. In this study, we have demonstrated increased pelvic pain in mice with conditional selective knockout of CYB5R3 in bladder urothelium that is alleviated by restoration of sGC-cGMP pathway with sGC activator, cinaciguat.

The conditional CYB3R5flox/flox+CAG-Cre mouse (CYB5R3 KO) was generated by crossing mice with loxP sites flanking exon3 of the CYB5R3 gene (CYB5R3flox/flox) with mice with tamoxifen-inducible Cre recombinase under a β-actin promoter (CAG-Cre). 2-3 month old CYB5R3 KO and wildtype littermates were treated with three 30 minutes long 4-OH-tamoxifen (4-OHT, 1 mg/ml, 200 µl, in ethanol/cremophorEL/saline) bladder instillations with 48 hour intervals.

Starting 1 week before 4-OHT instillations and at least weekly thereafter mice were placed in the metabolic cages (Columbus Instruments Inc.) for voiding frequency and volume analysis followed by pelvic pain evaluation with an automatic testing system (Topcat methodology, 5-6 repeats per mouse every 1-2 minutes). Some of the mice were gavaged with cinaciguat (10 mg/kg/day/14 days in 0.5% methylcellulose and 10% DMSO) starting approximately 8 weeks following instillations.

Metabolic cage assessments as soon as 8 weeks following 4-OHT instillation demonstrated a reduction in voided volumes and increased voiding frequency over a 24-hour period (Figure 1A and B). There were no changes in water consumption or total urine output. Sensitivity tests showed a significant decrease in withdrawal thresholds in the CYB5R3 KO animals in comparison to the WT littermates (Figure 1C).

Cinaciguat gavage did not affect WT animals. CYB5R3 KO started showing signs of improvement as soon as after 4 days of treatment. By one week of cinaciguat treatment, the withdrawal thresholds in the CYB5R3 KO animals were equivalent to WT littermate values. Voiding function assessments also demonstrated lower frequency and higher voided volumes compared to untreated CYB5R3 KO mice.

The CYB5R3 KO animals have lower withdrawal thresholds in the pelvic area suggestive of a pelvic pain sensation. Their voiding function was also altered with higher frequency and lower voided volumes. Cinaciguat gavage did not only restore proper bladder function, but also increased withdrawal threshold decreasing abnormal sensation as soon as after a week of treatment.

The physiological role of NO• signaling in the urinary bladder is not completely understood. It has been reported that the urothelium can release NO• in response to mechanical distention and pathological downregulation of NO• signaling due to the loss of nitrergic nerves or inflammation in aging causes bladder dysfunction. We have demonstrated that the bladder dysfunction in mice with conditional selective knockout of CYB5R3 in urothelium is accompanied by pelvic pain alleviated by cinaciguat treatment.

Durgin BG et al. JCI Insight. 2019. 4(19):e129183. PMID: 31487266.
Ikeda Y et al. Continence. 2023. 7(1):100858.