Cytochrome B5 reductase type-3 (CYB5R3) is a ubiquitous flavoprotein involved in many important cellular metabolic processes and signalling pathways. One of the more unique processes it has been linked to is nitric oxide (NO) signalling where it is involved in redox cycling of the haem moiety of soluble guanylate cyclase (sGC) and maintaining its ability to generate cGMP [1].  It is hypothesized that CYB5R3 deficiency due to inflammation/aging is a contributing factor to reduce NO mediated signalling [2]. In the urinary bladder, it has been reported that NO-induced cGMP generation occurs in the urothelium and platelet derived growth factor receptor alpha (PDGFRα) positive cells [3] and are believed to modulate contractile and afferent nerve activities. Therefore, our aim was to interrogate the effect of decreasing CYB5R3 activity in these cell types to determine their effect on voiding behaviour in a mouse model.

Generation of uroplakin-2 (UPK2) and PDGFRα linked CYB5R3 deficient mouse.  A mouse with loxP sites flanking exon3 of the CYB5R3 gene [1] was crossed with a mouse expressing Cre recombinase linked to the UPK2 (Jackson labs, stock #029281) or PDGFRα (Jackson labs, stock #013148) promoter.  From the cross with each Cre strain, CYB5R3 homozygous (flox/flox) were used as the deficient mouse and compared against Cre expressing CYB5R3 wildtype (wt/wt) littermates.  All mouse strains were based on a C57Bl/6 background and maintained in a centralized husbandry facility on a 12-hour light/dark cycle (7 am – 7pm). 

Metabolic cage assessments of adult and aged mice.  Voiding behaviour analysis was performed in adult male (10-12 week) mice using customized metabolic cages (Columbus Instruments Inc.) where the mice were maintained in a climate-controlled cabinet with the same 12-hour light/dark cycle as their normal housing facility. Food and water were provided ad libitum and their consumption recorded. Data were acquired through associated OxyMax (Columbus Instruments Inc.) and LabChart (AD Instruments) software for up to 48 hours.  

Data and statistical analysis. Data are expressed as mean ± standard error of mean. Pairwise comparisons were performed using Student’s t-test where the null hypothesis was rejected at p<0.05.

Both UPK2 and PDGFRα-CYB5R3 deficient and wildtype mice were found to be viable and did not show significant differences in body weight or show overt phenotypes. Metabolic cage analysis showed UPK2-CYB5R3 deficient mice showed decreases in voided volume and voiding frequency but not in total urine output or water consumption compared to their wildtype littermates (Figure 1A). In contrast, PDGFRα-CYB5R3 deficient mice showed a smaller but significant increase in voiding frequency when compared to wildtype littermates (Figure 1B).

Deficiency of CYB5R3 in the urothelium (UPK2-CYB5R3) showed indications of altered voiding behaviour compared to wildtype littermates. In contrast, reducing CYB5R3 in PDGFRα-expressing cells appeared to only cause a modest but significant increase in urinary frequency in mice.  These data indicate reduction of urothelial or fibroblast CYB5R3 can alter bladder activity.

Understanding of the physiological role of NO signalling in the urinary bladder has been elusive. It has been reported that PDE5 inhibitors show efficacy in reducing lower urinary tract symptoms secondary to benign prostatic hyperplasia without amelioration of outlet resistance. Thus, the bladder could potentially be a site of action for NO-cGMP modulating drugs. We chose the UPK2 and PDGFRα selective knockdown model as it is reported that the urothelium can release NO through mechanical distention and bladder PDGFRα positive fibroblasts generate cGMP in response to NO donors and form close appositions to nitrergic nerves.  We propose that the CYB5R3 deficient mouse better recapitulates the pathological downregulation of NO signalling compared to sGC knockdown and may be a useful model for future investigations.

Durgin et al.  Loss of smooth muscle CYB5R3 amplifies angiotensin II-induced hypertension by increasing sGC heme oxidation.  JCI Insight, 4(19):e129183, 2019.
Fan et al.  Cyb5r3 links FoxO1-dependent mitochondrial dysfunction with ß-cell failure.  Molecular Metabolism, 34:97-111, 2020.
Winder et al. Signalling Molecules in the Urothelium. Biomed Res Int, 2014: 297295, 2014.

Continence 12S (2024) 101376
DOI: 10.1016/j.cont.2024.101376