To investigate the effect of detrusor overactivity (DO) on the urethral expression of caveolin (CAV)-1, −2, and −3 of urethra in an animal model of cyclophosphamide (CYP)-induced cystitis rat.
Female Sprague-Dawley rats were divided into the control group (n=20) and the cystitis group (n=20). Cystitis was induced by intraperitoneal injection of CYP (200 mg/kg). An urodynamic study was done 3 days after the CYP injection to measure functional change of the urinary bladder and urethra. Cellular localization and expression of CAV-1, −2, and −3 in the rat urethra were determined by immunohistochemistry (IHC) and Western blot.
Urodynamic experiments demonstrated a decreased contraction interval in the cystitis group compared to the control (3.9±1.0 minutes vs. 6.6±1.2 minutes, P<0.05). Conversely, contraction pressure increased significantly in the cystitis group compared to the control (22.4±0.7 mmHg vs. 11.5±0.4 mmHg, P<0.05). The urethral pressure was decreased in the cystitis group compared to the control (4.05±2.5 mmHg vs. 5.8±2.8 mmHg, P<0.05). The IHC and Western blot data showed that CAV-1, −2, and −3 expression decreased significantly in the cystitis group compared control group (P<0.05).
The decreased urethral CAV-1, −2, and −3 in the DO rats suggests that CAVs might be related with the functional change of urethra in association with DO of urinay bladder.
- The current study was designed to investigate the functional change and the expression of caveolin (CAV)-1, −2, and −3 in the urethra and to suggest the potential implications of CAVs on urethral function in a detrusor overactivity (DO) animal model of cyclophosphamide (CYP)-induced cystitis rat. No study, to date, has yet evaluated the expression of CAVs in the urethra when the pathological changes of the urinary bladder occur.
- Our study indicates that the functional change of urethra and the decreased expression of urethral CAV-1, −2, and −3 was observed in the animal model of DO by CYP-induced cystitis rat.
- These data suggest that urethral CAVs may, in part, be involved with the functional change of urethra in association with DO of urinary bladder.
Caveolins (CAVs) are flask-shaped small invaginations of the plasma membrane that are thought to play an important role in cell surface signaling, endocytosis, and intracellular cholesterol transport [
It has been reported that the CAVs is associated with physiological function of the urinary bladder. We suggested previously that CAV-1 might be involved in urinary bladder signaling activity and play a role in bladder dysfunction in a cyclophosphamide (CYP)-induced rat cystitis model showing detrusor overactivity (DO) [
However, it seems like that the urethral function is tightly associated with the physiologic alteration of the urinary bladder. Wise et al. [
Until now, studies on the association between lower urinary tract and CAV have been largely confined to the bladder. However, a study was conducted on the relationship between urethralfunction and CAV using CAV knock out (KO) mice [
The current study was designed to investigate the functional change and the expression of CAV-1, −2, and −3 in the urethra and to suggest the potential implications of CAVs on urethral function in a DO animal model of CYP-induced cystitis rat. No study, to date, has yet evaluated the expression of CAVs in the urethra when the pathological changes of the urinary bladder occur.
Female Sprague-Dawley rats (age: 14 weeks, weight: 250–300 g, n=40) were divided into control (n=20) and experimental cystitis group (n=20). The cystitis group was induced by intraperitoneal injection of CYP (200 mg/kg) (Sigma Chemical Co., St. Louis, MO, USA) [
Urodynamic studies were conducted 3 days later to measure contraction interval and contraction pressure. The rats (n=5/group) were anesthetized with a subcutaneous injection of urethane (1.2 g/kg) 3 days after the CYP injection. To monitor the vesical and urethral pressure, the rats were prepared using the method of Bae et al [
Midurethral tissue (n=5/group, 20 sections/sample) was placed in 4% paraformaldehyde fixative for 16 hours, washed, and dehydrated. The tissue was embedded in paraffin, and 6-μm sections were prepared. The urethral sections were deparaffinized by incubation in Histochoice clearing agent (Amresco LLC, Solana, OH, USA) for 90 minutes, rehydrated in a graded ethanol series, washed in Tris-buffered saline (TBS; 10mM Tris-HCl, pH 7.6, 150mM NaCl), and stained with hematoxylin and eosin. IHC was performed using a labeled streptavidin biotin immunoperoxidase procedure (Universal Dako LSAB+ Kit; Dako North America Inc., Carpentaria, CA, USA). To quench endogenous peroxidase activity, the sections were treated in 3% hydrogen peroxide (Dako) for 5 minutes, washed with TBS, and incubated for 30 minutes at room temperature with monoclonal mouse anti-CAV-1, −2, and −3 antibodies (1:100; BD Biosciences, San Jose, CA, USA) diluted in TBS, followed by sequential incubations with biotinylated link (Dako) and streptavidin peroxidase (Dako) for 15 minutes each. The sections were incubated for 1 minute with substrate-chromogen solution (Dako) and counterstained for 2 minutes with hematoxylin (1:2 dilution) diluted in distilled water. The sections were then treated with 3% ammonia water (Junsei Chemical Co., Ltd., Tokyo, Japan) and mounted with Dako fluorescent mounting medium (Dako). The sections were examined and photographed using a model BX53 microscope (Olympus, Tokyo, Japan).
Tissue homogenates (n=10/group) were prepared from urethral tissue. Protein concentration in each sample was assayed using the BCA protein assay kit (Thermo Scientific, Rockford, IL, USA) using bovine serum albumin as the standard (Thermo Scientific), according to the manufacturer’s protocol. Equal amounts of protein (50 μg) were resuspended in sample buffer and separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were transferred to 0.2 μm pore polyvinylidene difluoride membranes (Amersham Pharmacia Biotech, Cambridge, UK). Nonspecific binding was inhibited by 60 minutes incubation with 5% skimmed milk in TBS containing 0.05% Tween-20 (TBS-T). The membranes were incubated overnight at 4°C with monoclonal mouse anti-CAV-1, −2, or −3 antibody (1:1,000; BD Biosciences). Monoclonal mouse antibodies for CAVs (1:2,000; BD Biosciences Immunocytometry Systems, San Jose, CA, USA) and a polyclonal rabbit antibody against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:4,000; Cell Signaling Technology Inc., Danvers, MA, USA) were used as an internal control. The membranes were rinsed extensively with TBS-T solution and incubated for 60 minutes at room temperature with goat anti-mouse-immunoglobulin G (IgG) and goat anti-rabbit-IgG conjugated to horseradish peroxidase (1:2,500; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) secondary antibody. The bands were visualized using the WEST-ZOL plus Western Blot Detection System (iNtRON Biotechnology Inc., Seongnam, Korea) and were imaged with the LAS-3000 system (Life-science Fujifilm Global, Tokyo, Japan). Densitometry was performed using the Multi gauge V3.0 (Fujifilm Global) chemiluminescence system and analysis software.
Results are expressed as means±standard deviation. The urodynamic quantitative data are expressed as mean±standard error. The Mann-Whitney test was used to test the null hypothesis of no differences in mean expression levels between the groups. A P-value of <0.05 was considered significant.
All animals survived the 3 days after the CYP injection. Body weight between the groups was not different.
Cystometrograms were performed 3 days after the CYP injection. Nonvoiding spontaneous contractions were detected in the cystitis group. The contraction interval decreased in the cystitis group (3.9±1.0 minutes) compared to that in the control group (6.6±1.2 minutes) (P<0.05). Conversely, contraction pressure was significantly higher in the cystitis group (22.4± 0.7 mmHg) than that in the control group (11.5±0.4 mmHg) (P<0.05) (
The urethral walls from inside to outside consist of urothelium, suburothelial lamina propria, inner longitudinal smooth muscle, outer circular smooth muscle, and an adventitia of loose connective tissue. The suburothelial layer is rich in microvasculature. All microvascular structures were surrounded by scattered smooth muscle bundles and connective tissue. The urethral tissue from the cystitis group showed a thickened submucosa, infiltration of inflammatory cells into the interstitium, and swelling, and irregular arrangement of muscular structure compared to those in the control group (
In the urothelium and suburothelial tissue, CAV-1 and CAV-2 were expressed in the capillaries, venules, and arterioles of the suburothelial layer just beneath the urothelium, whereas CAV-3 was expressed only in urothelium. CAVs were stained brown via immunolabeling throughout the urethral tissue (
Western blot analysis revealed CAV-1, −2, and −3 protein band at 21, 20, and 18 kDa, respectively (
In the present study, the expression of CAV-1, −2, and −3 in the urethra decreased significantly in the CYP-induced cystitis rat showing obvious DO of the urinary bladder. And the urethral pressure was decreased in the cystitis group compared the control. These results suggest that DO in the urinary bladder would have an impact on the urethral function and that CAV may play a role in the decreased urethral function in association with dysfunctional change of urinary bladder.
Previous studies have demonstrated that CAVs are involved in the regulation of different signaling pathways during bladder smooth muscle contraction [
Urethral function is affected by abnormal detrusor activities [
CYP-induced chemical cystitis in animals and human studies show a mucosal erosion or edema, hemorrhagic changes, and infiltration of leukocytes in the urinary bladder [
In the histopathologic study, CAV-1 and CAV-2 were expressed in the capillaries, venules, and arterioles of the suburothelial layer just beneath the urothelium in the urethra, whereas CAV-3 was expressed only in the urothelium. In the smooth muscle layers, CAV-1 and CAV-2 were predominantly expressed in the inner longitudinal smooth muscle cells, whereas CAV-3 was predominantly expressed in outer circular smooth muscle cells. The distinct expression feature suggests that CAV would have specific functional roles by each CAV subtype in the different urethral tissue layers and smooth muscle layer.
Significant advances in the understanding of bladder sensory function in terms of crosstalk between the urothelium and afferent nerves have been made in recent years. The urothelium has been thought to be a passive barrier between the urinary tract and urine. However, the urothelium is now understood to be an interactive organ that senses a variety of signals from the urinary bladder [
This is the first study showing the possible occurrence of altered signaling process via revealing the change of the expression of CAVs in the urethra in association with bladder dysfunction. There are several potential implications of altered CAV expression on the urethra when the urinary bladder shows abnormal detrusor function. First, decreased CAV expression in the urethra may be associated with impaired urethral function induced by disruption of signal transduction pathways because CAVs have been known for the key regulators of signaling process involved in cellular reactions and smooth muscle contractions. The urothelium can sense chemical and mechanical stimuli that may relay the status of the urothelial environment to the underlying nervous and muscular systems [
A limitation of our study is that the precise pathophysiological mechanism or functional activity of CAVs on the urethra was not fully unveiled. Further studies are needed to understand the exact roles of CAVs in the urethra by revealing their functional responses assessed by
In conclusion, our study indicates that the functional change of urethra and the decreased expression of urethral CAV-1, −2, and −3 was observed in the animal model of DO by CYP-induced cystitis rat. These data suggest that urethral CAVs may, in part, be involved with the functional change of urethra in association with DO of urinary bladder.
This study was supported by a grant of the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2014-1825); by a grant CRI 17015-1, Chonnam National University Hospital Research Institute of Clinical Medicine.
All experiments were approved by the Ethics Committee of Chonnam National University Medical School (CNU IACUC-H-2015-10).
No potential conflict of interest relevant to this article was reported.
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Representative urodynamic profiles: control group (A, C) and cystitis group (B, D). Nonvoiding spontaneous contractions were found in the cystitis group. The contraction interval in the cystitis group was shorter than that in the control (P<0.05). However, contraction pressure during voiding increased significantly in the cystitis compared the control group (P<0.05). The lower panels (E, F) denote the means±standard deviations of 5 experiments under each condition, as determined by cystometrograms. Panels C and D: the magnification image of each contraction of panels A and B. *P<0.05 vs. control.
Hematoxylin and eosin staining. The urethral walls from inside to outside consist of urothelium, suburothelial lamina propria, inner longitudinal smooth muscle, outer circular smooth muscle, and an adventitia of loose connective tissue. The urethras in the cystitis group showed thickened submucosa, infiltration of inflammatory cells into the interstitium, swelling, and irregularly arranged muscle structure (B, D, and F), compared to those in the control (A, C, and E).
Immunohistochemistry of caveolin (CAV)-1 (A–H), CAV-2 (I–P), and CAV-3 (Q–X). CAVs were stained brown via immunolabeling (arrows). CAV-1 and CAV-2 were expressed in capillaries, venules, and arterioles in the suburothelial layer beneath the urothelium (B, F, J, and N), whereas CAV-3 was expressed in the urothelium (R and V). CAV-1, −2, and −3 were all expressed in urethral smooth muscle cells. CAV-1 and CAV-2 were mainly expressed in inner longitudinal smooth muscle cells (C, G, K, and O), whereas CAV-3 was predominantly expressed in outer circular smooth muscle cells (T, X). CAV-1, −2, and −3 expression was decreased in the cystitis group compared the control group. The horizontal scale bar at the bottom indicates magnification.
Western blot analysis of caveolin (CAV)-1, −2, and −3. The expression of CAV-1, −2, and −3 was decreased markedly in the cystitis group compared the control. The anti-CAV −1, −2, −3 antibody recognized a 21, 20, and 18 kDa band, respectively. The anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody recognized a 37 kDa band. Lower panels denote mean± standard deviation of 10 experiments for each condition, as determined by densitometry relative to GAPDH. *P<0.05.