Hypothesis / aims of study
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.
Study design, materials and methods
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.
Interpretation of results
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.