Cytochrome B5 reductase type-3 (CYB5R3) is a flavoprotein that regulates redox pathways in the mitochondria, endoplasmic reticulum and nitric oxide signalling by reduction of the soluble guanylate cyclase haem group.  Inflammatory conditions can downregulate CYB5R3 activity with detrimental consequences to critical cellular processes [1].  We hypothesized that selective knockdown of CYB5R3 in the urinary bladder could be used to model pathological situations that result in chronic inflammation including exposure to ionising radiation, urinary tract infections or natural aging.  To address this hypothesis, we generated a conditional CYB5R3 knockout mouse and performed 4-hydroxytamoxifen (4-OHT, active metabolite of tamoxifen) injections into the urinary bladder wall.  The voiding activity of mice was monitored over 6 weeks and bladder contractile function assessed by isometric tension recordings.  This study aimed to generate a new model for evaluating the consequences of metabolic/mitochondrial dysfunction and resultant inflammation localized to the urinary bladder.

Generation of CYB5R3 conditional knockout mouse.  A mouse with loxP sites flanking exon3 of the CYB5R3 gene [2] (CYB5R3flox/flox) was crossed with a mouse expressing the tamoxifen inducible Cre recombinase under the β-actin promoter (CAG-Cre, Jackson laboratories, stock#:004682) to generate the conditional CYB5R3flox/flox+CAG-Cre (CYB5R3 KO) mouse.  Both mouse strains were based on a C57Bl/6 background.  At 8-12 weeks of age, female CYB5R3 KO and age matched CYB5R3 wildtype (WT) mice were used for 4-OHT bladder wall injections.  Mice were anesthetized with isoflurane and a lower midline incision made to expose the urinary bladder using sterile surgical conditions.  The bladder wall was injected from the serosal surface at 4 locations with 0.5 mg/ml 4-OHT (10 µl total, dissolved in ethanol/CremophorEL/saline) using a 32-gauge insulin syringe.  The incisions were sutured, and mice were given prophylactic ampicillin (100 mg/kg, SQ, 7 days) and ketoprofen (3 mg/kg, IM, 3 days) during the recovery period.

Urine void spot and metabolic cage assessments.  Voiding activity was assessed by two-hour urine spot tests starting one week before 4-OHT injections and performed weekly thereafter up to 6 weeks.  Filter papers lining the metabolic cages were imaged using a ChemiDocMP (Bio-Rad) and analysed with FIJI ImageJ software.  The area of urine spots was measured and analysed as previously described [3] where primary void spots (PVS) were determined as those ≥80 mm2.  24-hour voiding analysis was also performed using metabolic cages (Columbus Instruments Inc.) where the mice were maintained in a climate-controlled cabinet with a 12-hour light/dark cycle (7am-7pm).

Isolated bladder strip contractility. Mice were humanely sacrificed for tissue collection at 6 weeks after surgery.  Urinary bladders were dissected into strips in the dome to base orientation and examined by isometric tension recordings.  Strips were stimulated by electrical field stimulation and stretched incrementally to determine the baseline tension for optimal alignment of contractile fibres, i.e., optimal length.  Responses were measured to purinergic (10 µM α,β-methylene ATP) and muscarinic (0.1-10 µM oxotremorine-M) agonists and direct depolarization (120 mM KCl).  Tension was normalised to force per cross-sectional area of tissue (mN/mm2).

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.

CYB5R3 KO and WT mice exhibited differences in voiding behaviour at 28 days following 4-OHT treatment (Fig 1A-C).  The KO mice showed increased number of PVS which were corroborated by metabolic cage studies that indicated smaller voided volumes and increased number of voiding events (Fig 1D and 1E).  There was no significant difference in total voided volumes or total water intake (not shown).  Gross examination of isolated bladder showed no significant difference in wet weights or obvious signs of irritation in KO or WT mice.  Lastly, muscle strip experiments demonstrated no significant differences in responses to agonist stimulation, force generation or tissue elasticity between KO and WT animals (Fig 1F).

Decreased CYB5R3 levels in the bladder induced urinary frequency and decreased voided volumes.  Altered voiding behaviour was not associated with changes to the contractile or viscoelastic properties of the detrusor.  Thus, observed bladder overactivity is likely neurogenic in origin and mediated by changes in cellular metabolism and localised inflammation.

CYB5R3 is an important redox regulator in many cellular processes and its dysregulation is associated with multiple inflammatory pathologies.  The CYB5R3 KO mouse represents a unique animal model for studying the consequences of metabolic/mitochondrial dysfunction in the urinary bladder.

Fan et al.  Cyb5r3 links FoxO1-dependent mitochondrial dysfunction with ß-cell failure.  Molecular Metabolism, 34:97-111, 2020.
Durgin et al.  Loss of smooth muscle CYB5R3 amplifies angiotensin II-induced hypertension by increasing sGC heme oxidation.  JCI Insight, 4(19):e129183, 2019.
Chen et al.  Evaluating the voiding spot assay in mice: a simple method with complex environmental interactions.  AJP Renal, 313(6): F1274–F1280, 2017.

Continence 2S2 (2022) 100459
DOI: 10.1016/j.cont.2022.100459