Hypothesis / aims of study
Cytochrome B5 reductase type-3 (CYB5R3) is a flavoprotein involved in multiple metabolic and signaling pathways within the mitochondria, endoplasmic reticulum and cytosol. CYB5R3 activity can decrease due to inflammation /oxidative stress or natural aging leading to significant impediments on cellular function [1]. However, its contribution to urinary bladder function has yet to be defined. We hypothesized that selective knockdown of CYB5R3 in the urinary bladder could be used to model pathological situations where the enzyme activity may be downregulated. To address this hypothesis, we generated a conditional CYB5R3 knockout mouse and performed intravesical instillations of 4-hydroxytamoxifen (4-OHT, active metabolite of tamoxifen) to selectively target the urothelium. The voiding activity of mice was monitored over 12 weeks with morphological changes to the bladder wall assessed by histology. The goal was to determine whether bladder CYB5R3 deficiency could mimic established pathological models including inflammatory insults or natural aging.
Study design, materials and methods
Generation of the 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 a β-actin promoter (CAG-Cre, Jackson laboratories, stock#:004682) to generate the conditional CYB5R3flox/flox+CAG-Cre (CYB5R3 KO) mouse (Figure 1A). Both mouse strains were based on a C57Bl/6 background. At 8-12 weeks of age, female CYB5R3 KO and age matched CYB5R3 wildtype (CAG-Cre expressing CYB5R3flox/wt or wt/wt, henceforth, WT) mice were anesthetized with isoflurane and using sterile surgical conditions, the urinary bladder was instilled with 1 mg/ml 4-OHT (200 µl total, dissolved in ethanol/CremophorEL/saline) using a PE-10 catheter for 30 minutes which was repeated two more times with 48-hour intervals. The mice were treated with the analgesic, ketoprofen (3 mg/kg, IM) 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 for up to 12 weeks. Filter papers lining the metabolic cages were imaged using a ChemiDocMP (Bio-Rad) and analyzed for urine spotting with FIJI ImageJ software (National Institutes of Health). Voiding frequency and volume 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 (7 am - 7 pm).
Histology. Mice were humanely sacrificed for tissue collection at 12 weeks after 4-OHT treatment. Urinary bladders were fixed in 10% neutral buffered formalin and processed for paraffin embedding. Tissues were sectioned 3 to 4 µm thick and processed for chemical staining (hematoxylin-eosin for morphology or Van Gieson for collagen) or immunofluorescence to detect CYB5R3 (Proteintech) and cytokeratin-20 (Abcam) localization. Slides were imaged using a widefield microscope (Olympus BX63) under brightfield and fluorescent illumination. Images were analyzed using FIJI ImageJ.
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.
Results
CYB5R3 was found to be robustly expressed throughout the mouse urothelium, blood vessels and red blood cells (Figure 1B). CYB5R3 KO mice showed significant downregulation of expression throughout the bladder wall at 12 weeks after 4-OHT treatment (Figure 1C). CYB5R3 KO and WT mice did not exhibit significant differences in voiding patterns at up to 6 weeks after 4-OHT instillation when examined by urine spot tests (not shown). However metabolic cage assessments between 8 to 12 week following instillation showed that KO mice had a reduction in mean voided volumes and increased voiding frequency over a 24-hour period (Figure 1D and 1E, N=4 per group, averaged from three separate recording sessions, **p=0.006, *p=0.01). There were no significant differences in body weight (22 ± 1 grams versus 24 ± 1 grams) or bladder weights (25 ± 6 mg versus 28 ± 6 mg) between KO and WT mice as a result of 4-OHT treatment at the 12-week endpoint.
Histological analysis of bladder walls showed indications of disruption to the apical cells of the urothelium of KO mice (Figure 2A, H&E staining, disruption indicated in black arrows) compared to WT (Figure 2B). This correlated with patchy, low intensity immunolabeling of cytokeratin-20, a marker for apical urothelial cells, in the KO (Figure 2C) versus WT (Figure 2D). CYB5R3 KO also showed evidence of chronic inflammation and fibrosis seen by increased collagen content in the lamina propria and its infiltration into the detrusor layer (Figure 2E, Van Gieson staining) compared to WT mice which underwent the same 4-OHT treatment (Figure 2F). Furthermore, there was evidence of lipofuscin-like vesicle accumulation in KO urothelium which were identified as autofluorescent, circular structures visible under multiple excitation wavelengths (Figure 2G, representative image under 488 nm excitation). The vesicles in KO mouse bladders were larger (Figure 2H) and covered a greater area of the urothelial surface (Figure 2I) compared to WT mice.
Interpretation of results
CYB5R3 KO in the bladder was associated with changes in voiding behavior at 12 weeks after 4-OHT treatment. This correlated with morphological changes to the urothelium, disruption of the apical cell layer and accumulation of large vesicles. Accumulation of lipofuscin has been associated with aging mouse bladders and correlate with urothelial metabolic dysfunction [3]. Therefore, the CYB5R3 KO recapitulate pathophysiological features of the aging mouse urothelium