Mouse Urinary Bladder Research Articles

84 - Effects of aging and vasopressin receptor agonists on water reabsorption in the mouse urinary bladder

84 - Effects of aging and vasopressin receptor agonists on water reabsorption in the mouse urinary bladder

26 - Endogenous production of arginine vasopressin in the mouse urinary bladder

26 - Endogenous production of arginine vasopressin in the mouse urinary bladder

Neurogenic-derived 6-nitrodopamine is the most potent endogenous modulator of the mouse urinary bladder relaxation

6-Nitrodopamine (6-ND) modulates vas deferens, seminal vesicles, and corpus cavernosum contractility; however, its role on the lower urinary tract organs has not been evaluated. Investigations of isolated urinary bladders from wild-type (WT) mice revealed 6-ND release was comparable to that of dopamine and adrenaline, whereas noradrenaline was hardly detected, as assessed by liquid chromatography coupled to tandem mass spectrometry. In vitro, 6-ND induced concentration-dependent relaxations in carbachol pre-contracted bladders with high potency (pEC50: 8.04 ± 0.86), independently of eNOS/sGC activity. Co-incubation of 6-ND (1–10 μM) antagonizes the contractile effects of acetylcholine (p < 0.05). Experiments using nitric oxide synthase (NOS) knockout mice demonstrated that 6-ND release from isolated urinary bladder was significantly reduced by neuronal NOS (nNOS−/−) deletion and abolished by triple NOSs deletion (n/i/eNOS−/−), while no significant changes were observed in endothelial (eNOS−/−) or inducible (iNOS−/−) knockout mice. Incubation with tetrodotoxin resulted in a significant decrease in 6-ND release in bladders obtained from WT, but not in nNOS−/− mice. The bladders from nNOS−/− and n/i/eNOS−/− mice exhibited significantly higher contractile responses to electric field stimulation (EFS), compared to eNOS−/−, iNOS−/−, or WT bladders. The hyperreactivity observed in triple NOS knockouts was reversed by the incubation with bladder mucosal layer obtained from a donor WT mice, but not with the muscular layer. These findings clearly demonstrate 6-ND is the most potent endogenous relaxing agent of urinary bladder, and inhibition of its release is associated with bladder hyperreactivity.

Competitive fitness of asymptomatic bacteriuria E. coli strain 83972 against uropathogens in human urine.

Urinary tract infection (UTI) is one of the most common bacterial infections worldwide. The main causative agent of UTI is uropathogenic Escherichia coli (UPEC). There is an immediate need for novel prophylactic and treatment strategies against UTI because of the increasing incidence of antimicrobial resistance among uropathogens. ABU 83972, an asymptomatic bacteriuria-causing E. coli strain, prevents UTI by suppressing the colonization of UPEC. However, the nature of competition and growth repression of UPEC by ABU 83972 is unclear and is the subject of our investigation. Here, we characterized the growth kinetics of ABU 83972 and uropathogens in human urine and laboratory media. Next, we performed a series of competitive co-culture experiments where ABU 83972 and uropathogens were inoculated at a 1:1 ratio in human urine and in various media, and their relative abundance was determined. In human urine, ABU 83972 outcompeted UPEC and additional uropathogens, reaching up to 90% of the total population after 24 hours of incubation. In contrast, UPEC outcompeted ABU 83972 in LB and M9 minimal media and exhibited superior colonization than ABU 83972 in the mouse urinary bladder. Since engineered living materials (ELMs) can be used to retain an organism of interest in a particular location, we developed ABU 83972-containing ELMs that effectively outcompeted UPEC in human urine. In summary, our work establishes that ABU 83972 outcompetes UPEC in a milieu- and cell-density-dependent manner, highlighting the importance of the metabolites and nutrients found in the human urine as determinants of the competitive fitness of ABU 83972.

Urothelium-derived prostanoids enhance contractility of urinary bladder smooth muscle and stimulate bladder afferent nerve activity in the mouse.

The transitional epithelial cells (urothelium) that line the lumen of the urinary bladder form a barrier between potentially harmful pathogens, toxins, and other bladder contents and the inner layers of the bladder wall. The urothelium, however, is not simply a passive barrier, as it can produce signaling factors, such as ATP, nitric oxide, prostaglandins, and other prostanoids, that can modulate bladder function. We investigated whether substances produced by the urothelium could directly modulate the contractility of the underlying urinary bladder smooth muscle. Force was measured in isolated strips of mouse urinary bladder with the urothelium intact or denuded. Bladder strips developed spontaneous tone and phasic contractions. In urothelium-intact strips, basal tone, as well as the frequency and amplitude of phasic contractions, were 25%, 32%, and 338% higher than in urothelium-denuded strips, respectively. Basal tone and phasic contractility in urothelium-intact bladder strips were abolished by the cyclooxygenase (COX) inhibitor indomethacin (10 µM) or the voltage-dependent Ca2+ channel blocker diltiazem (50 µM), whereas blocking neuronal sodium channels with tetrodotoxin (1 µM) had no effect. These results suggest that prostanoids produced in the urothelium enhance smooth muscle tone and phasic contractions by activating voltage-dependent Ca2+ channels in the underlying bladder smooth muscle. We went on to demonstrate that blocking COX inhibits the generation of transient pressure events in isolated pressurized bladders and greatly attenuates the afferent nerve activity during bladder filling, suggesting that urothelial prostanoids may also play a role in sensory nerve signaling.NEW & NOTEWORTHY This paper provides evidence for the role of urothelial-derived prostanoids in maintaining tone in the urinary bladder during bladder filling, not only underscoring the role of the urothelium as more than a barrier but also contributing to active regulation of the urinary bladder. Furthermore, cyclooxygenase products greatly augment sensory nerve activity generated by bladder afferents during bladder filling and thus may play a role in perception of bladder fullness.

Application of clustering strategy for automatic segmentation of tissue regions in mass spectrometry imaging.

Mass spectrometry imaging (MSI) has been widely used in biomedical research fields. Each pixel in MSI consists of a mass spectrum that reflects the molecule feature of the tissue spot. Because MSI contains high-dimensional datasets, it is highly desired to develop computational methods for data mining and constructing tissue segmentation maps. To visualize different tissue regions based on mass spectrum features and improve the efficiency in processing enormous data, we proposed a computational strategy that consists of four procedures including preprocessing, data reduction, clustering, and quantitative validation. In this study, we examined the combination of t-distributed stochastic neighbor embedding (t-SNE) and hierarchical clustering (HC) for MSI data analysis. Using publicly available MSI datasets, one dataset of mouse urinary bladder, and one dataset of human colorectal cancer, we demonstrated that the generated tissue segmentation maps from this combination were superior to other data reduction and clustering algorithms. Using the staining image as a reference, we assessed the performance of clustering algorithms with external and internal clustering validation measures, including purity, adjusted Rand index (ARI), Davies-Bouldin index (DBI), and spatial aggregation index (SAI). The result indicated that SAI delivered excellent performance for automatic segmentation of tissue regions in MSI. We used a clustering algorithm to construct tissue automatic segmentation in MSI datasets. The performance was evaluated by comparing it with the stained image and calculating clustering validation indexes. The results indicated that SAI is important for automatic tissue segmentation in MSI, different from traditional clustering validation measures. Compared to the reports that used internal clustering validation measures such as DBI, our method offers more effective evaluation of clustering results for MSI segmentation. We envision that the proposed automatic image segmentation strategy can facilitate deep learning in molecular feature extraction and biomarker discovery for the biomedical applications of MSI.

Pharmacological Studies on the Ca2+ Influx Pathways in Platelet-Activating Factor (PAF)-Induced Mouse Urinary Bladder Smooth Muscle Contraction.

Platelet-activating factor (PAF) not only acts as a mediator of platelet aggregation, inflammation, and allergy responses but also as a constrictor of various smooth muscle (SM) tissues, including gastrointestinal, tracheal/bronchial, and pregnancy uterine SMs. Previously, we reported that PAF induces basal tension increase (BTI) and oscillatory contraction (OC) in mouse urinary bladder SM (UBSM). In this study, we examined the Ca2+ influx pathways involved in PAF-induced BTI and OC in the mouse UBSM. PAF (10-6 M) induced BTI and OC in mouse UBSM. However, the PAF-induced BTI and OC were completely suppressed by extracellular Ca2+ removal. PAF-induced BTI and OC frequencies were markedly suppressed by voltage-dependent Ca2+ channel (VDCC) inhibitors (verapamil (10-5 M), diltiazem (10-5 M), and nifedipine (10-7 M)). However, these VDCC inhibitors had a minor effect on the PAF-induced OC amplitude. The PAF-induced OC amplitude in the presence of verapamil (10-5 M) was strongly suppressed by SKF-96365 (3 × 10-5 M), an inhibitor of receptor-operated Ca2+ channel (ROCC) and store-operated Ca2+ channel (SOCC), but not by LOE-908 (3 × 10-5 M) (an inhibitor of ROCC). Overall, PAF-induced BTI and OC in mouse UBSM depend on Ca2+ influx and the main Ca2+ influx pathways in PAF-induced BTI and OC may be VDCC and SOCC. Of note, VDCC may be involved in PAF-induced BTI and OC frequency, and SOCC might be involved in PAF-induced OC amplitude.

Muscarinic receptor-induced contractility of mouse urinary bladder strips is not influenced by the urothelium

The urothelium is thought to exert a direct influence on urinary bladder smooth muscle (UBSM) contractility by releasing active agents such as acetylcholine, ATP, and nitric oxide. Previous studies suggest that urothelial cells express muscarinic receptors, and upon stimulation, factors released from the urothelium may modulate UBSM contractility in response to muscarinic receptor activation on UBSM. This study aimed to determine whether the urinary bladder urothelium affects the contractility of UBSM in response to muscarinic stimulation. We hypothesized that the muscarinic receptor-induced contractile response of UBSM would be attenuated in the presence of the urothelium.Urinary bladders were isolated from male C57Bl6/J mice post-euthanasia. After dissection, bladders were placed in ice-cold nominally Ca2+-free dissection solution. Urinary bladders were opened and cut into strips with and without the urothelium. Strips were placed in myograph tissue baths (DMT) with physiological saline solution for contractility analysis. Each strip was stretched to 1g and was left to equilibrate for 45 minutes (min). Strips were washed 2-3x before applying carbachol (CCh). CCh was applied at concentrations of 0.001 μM, 0.01 μM, 0.1 μM,1 μM, and 10 μM with at least a 5-min waiting period between doses. [GH1] Contractile responses were analyzed using LabChart software and GraphPad Prism. Data were analyzed using a 2-way ANOVA with Tukey’s test for multiple comparisons. P&lt;0.05 was considered statistically significant. CCh induced a biphasic contraction in UBSM strips, which consisted of a rapid initial phasic contraction followed by a smaller stable elevation in tone. In strips without the urothelium (n=16 strips from 8 mice), peak contractions were 0.069 ± 0.104, 0.087 ± 0.102, 0.335 ± 0.302, 0.992 ± 0.606, and 1.197 ± 0.636 g in concentrations 0.001 μM, 0.01 μM, 0.1 μM, 1 μM, and 10 μM of CCh, respectively. In urothelium-denuded strips (n=15 strips from 8 mice), peak contractions were 0.054 ± 0.132, 0.077 ± 0.160, 0.365 ± 0.323, 1.222 ± 0.791, 1.416 ± 1.075 g across the same CCh concentration range. There were no significant differences in peak contraction responses to CCh at any concentration between urothelium-intact and -denuded UBSM strips (P = 0.2809). Similar results were obtained when quantifying steady-state contraction amplitudes. The contractile response to the muscarinic agent CCh in UBSM strips is not altered by the presence of the urothelium. This finding is in contrast with previous studies, and it suggests that muscarinic receptor stimulation on urothelial cells does not alter the contractile response induced by the activation of muscarinic receptors in the UBSM. This work was supported by a grant from the National Institute of Diabetes and Digestive and Kidney Diseases (R01DK125543). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Benzbromarone inhibition of phasic smooth muscle contractions of mouse urinary bladder

Calcium-activated chloride (ClCa) channels play a key role in generating pacemaker currents responsible for maintaining motility in the phasic smooth muscle of the gastrointestinal tract. These channels may play a similar role in the urinary bladder, resulting in the bladder exhibiting spontaneous phasic contractions. These phasic contractions are known to underlie non-voiding contractions and transient pressure events. The purpose of this study was to determine whether the putative ClCa channel inhibitor benzbromarone has an inhibitory effect on transient pressure events in the isolated perfused mouse urinary bladder. We hypothesized benzbromarone would abolish bladder transient pressure events. Urinary bladders were isolated from male C57Bl6/J mice (7-8 wks old, 23.2-24.5 g body weight) post-euthanasia. Isolated bladders were placed in ice-cold nominally Ca-free dissection solution. Ureters were tied off, the bladder was cannulated through the urethra, and submerged in an organ bath containing physiological saline solution (37 °C, pH 7.4). The bladder was then attached to a syringe pump and inline pressure transducer to infuse at a constant rate of 1.8 mL/hr while recording intraluminal pressure. Urinary bladders were filled until they reached an intravesical pressure of 25 mmHg, which was defined as bladder capacity. Upon reaching bladder capacity, the bladder was emptied through the urethral cannula. Three fills were performed under baseline conditions to establish a consistent pressure-volume relationship. Benzbromarone (10 μM) was applied for 20 minutes. Bladders were subsequently filled to 80 % capacity and transient pressure events allowed to stabilize for at least 5 minutes. Transient pressure events were recorded in the absence and presence of benzbromarone. Transient pressure event frequency, amplitude, and duration were analyzed using a custom MatLab algorithm. Under baseline conditions, transient pressure events occurred at a frequency of 5.2 ± 0.8 events per minute (n = 3). Inhibition of ClCa with benzbromarone decreased transient pressure event frequency to 2.1±1.1 events per minute (n = 3, p = 0.08, paired t-test). Similarly, benzbromarone decreased transient pressure event amplitude from a baseline value of 0.22 ± 0.02 mmHg (n = 3) to 0.06 ± 0.04 mmHg (n = 3, p = 0.17, paired t-test). Transient pressure event duration was unaffected by benzbromarone (baseline duration 5.3 ± 0.3 sec, benzbromarone duration 5.2 ± 2.7 sec, n = 3, p= 0.98, paired t-test). In summary, we found that the putativeClCa channel inhibitor benzbromarone disrupted the rhythmicity and amplitude of transient pressure events, while leaving the duration of these contractile events unaltered. This suggests that ClCa channels play a role in generating the rhythm of phasic smooth muscle contractions in the bladder. The cellualr and molecular basis underlying the involvement of ClCa channels in the urinary bladder remains to be explored. This work was supported by a grant from the National Institute of Diabetes and Digestive and Kidney Diseases (R01DK125543). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Glutamate Transporters GltS, GltP and GltI Are Involved in Escherichia coli Tolerance In Vitro and Pathogenicity in Mouse Urinary Tract Infections.

To verify the roles of GltS, GltP, and GltI in E. coli tolerance and pathogenicity, we quantified and compared the relative abundance of gltS, gltP, and gltI in log-phase and stationary-phase E. coli and constructed their knockout mutant strains in E. coli BW25113 and uropathogenic E. coli (UPEC) separately, followed by analysis of their abilities to tolerate antibiotics and stressors, their capacity for adhesion to and invasion of human bladder epithelial cells, and their survival ability in mouse urinary tracts. Our results showed that gltS, gltP, and gltI transcripts were higher in stationary phase E. coli than in log-phase incubation. Furthermore, deletion of gltS, gltP, and gltI genes in E. coli BW25113 results in decreased tolerance to antibiotics (levofloxacin and ofloxacin) and stressors (acid pH, hyperosmosis, and heat), and loss of gltS, gltP, and gltI in uropathogenic E. coli UTI89 caused attenuated adhesion and invasion in human bladder epithelial cells and markedly reduced survival in mice. The results showed the important roles of the glutamate transporter genes gltI, gltP, and gltS in E. coli tolerance to antibiotics (levofloxacin and ofloxacin) and stressors (acid pH, hyperosmosis, and heat) in vitro and in pathogenicity in mouse urinary tracts and human bladder epithelial cells, as shown by reduced survival and colonization, which improves our understanding of the molecular mechanisms of bacterial tolerance and pathogenicity.

Phosphodiesterase1 inhibitor "Vinpocetine" ameliorates the inflammation, apoptosis and oxidative stress induced by cyclophosphamide in urinary bladder: an experimental study.

Hemorrhagic cystitis often develops in patients treated with cyclophosphamide (CP). Vincamine (vinca alkaloid) is the source of the synthetic derivative vinpocetine (Vinpo). Worldwide, Vinpo is used as a cerebroprotective drug. As it has anti-oxidant, anti-thrombotic and anti-inflammatory effects but the power of Vinpo to prevent CP induced cystitis has not been studied. This research was planned to explore the effect of Vinpo (10-30mg/kg, orally) administered 1 or 4h before inducing cystitis by CP injection (300mg/kg, i.p.) on the urinary bladder of mice. Administration of Vinpo 30mg/kg, 4h before CP injection ameliorated inflammatory markers. It reduced inducible nitric oxide synthase (iNOS), tumor necrosis factor- α (TNF-α), and BCL2 Associated X (Bax) expression in the bladder and increased the total antioxidant capacity level. Histological examination of the bladder has further supported these results. The present study suggests a protective effect of Vinpo (30mg/kg, 4h before CP injection) against CP-induced bladder inflammation. This proposes that Vinpo 30mg/kg may become a promising pharmacological drug to prevent urinary adverse effects in patients treated with chemotherapy using CP.

Platelet-activating factor (PAF) strongly enhances contractile mechanical activities in guinea pig and mouse urinary bladder

In this study, we investigated the effects of platelet-activating factor (PAF) on the basal tone and spontaneous contractile activities of guinea pig (GP) and mouse urinary bladder (UB) smooth muscle (UBSM) tissues to determine whether PAF could induce UBSM tissue contraction. In addition, we examined the mRNA expression of the PAF receptor, PAF-synthesizing enzyme (lysophosphatidylcholine acyltransferase, LPCAT), and PAF-degrading enzyme (PAF acetylhydrolase, PAF-AH) in GP and mouse UB tissues using RT-qPCR. PAF (10−9–10−6 M) strongly enhanced the basal tone and spontaneous contractile activities (amplitude and frequency) of GP and mouse UBSM tissues in a concentration-dependent manner. The enhancing effects of PAF (10−6 M) on both GP and mouse UBSM contractile activities were strongly suppressed by pretreatment with apafant (a PAF receptor antagonist, GP: 10−5 M; mouse: 3 × 10−5 M). The PAF receptor (Ptafr), LPCAT (Lpcat1, Lpcat2), and PAF-AH (Pafah1b3, Pafah2) mRNAs were detected in GP and mouse UB tissues. These findings reveal that PAF strongly enhances the contractile mechanical activities of UBSM tissues through its receptor and suggest that the PAF-synthesizing and -degrading system exists in UBSM tissues. PAF may serve as both an endogenous UBSM constrictor and an endogenous mediator leading to detrusor overactivity.

HIF-1 mediated activation of antimicrobial peptide LL-37 in type 2 diabetic patients

Infections are common in patients with diabetes, but increasing antibiotic resistance hampers successful bacterial clearance and calls for alternative treatment strategies. Hypoxia-inducible factor 1 (HIF-1) is known to influence the innate immune defense and could therefore serve as a possible target. However, the impact of high glucose on HIF-1 has received little attention and merits closer investigation. Here, we show that higher levels of proinflammatory cytokines and CAMP, encoding for the antimicrobial peptide cathelicidin, LL-37, correlate with HIF-1 in type 2 diabetic patients. Chemical activation of HIF-1 further enhanced LL-37, IL-1β, and IL-8 in human uroepithelial cells exposed to high glucose. Moreover, HIF-1 activation of transurethrally infected diabetic mice resulted in lower bacterial load. Drugs activating HIF-1 could therefore in the future potentially have a therapeutic role in clearing bacteria in diabetic patients with infections where antibiotic treatment failed.Key messages• Mohanty et al. “HIF-1 mediated activation of antimicrobial peptide LL-37 in type 2 diabetic patients.”• Our study highlights induction of the antimicrobial peptide, LL-37, and strengthening of the innate immunity through hypoxia-inducible factor 1 (HIF-1) in diabetes.• Our key observations are:1. HIF-1 activation increased LL-37 expression in human urothelial cells treated with high glucose. In line with that, we demonstrated that patients with type 2 diabetes living at high altitude had increased levels of the LL-37.2. HIF-1 activation increased IL-1β and IL-8 in human uroepithelial cells treated with high glucose concentration.3. Pharmacological activation of HIF-1 decreased bacterial load in the urinary bladder of mice with hereditary diabetes.• We conclude that enhancing HIF-1 may along with antibiotics in the future contribute to the treatment in selected patient groups where traditional therapy is not possible.

Does and duration of exposure time dependent regulation of proteins in the urinary bladder of hamsters exposed to sodium arsenite

Does and duration of exposure time dependent regulation of proteins in the urinary bladder of hamsters exposed to sodium arsenite

A temporal Ca2+ desensitization of myosin light chain kinase in phasic smooth muscles induced by CaMKKβ/PP2A pathways.

Cell signaling pathways regulating myosin regulatory light chain (LC20) phosphorylation contribute to determining contractile responses in smooth muscles. Following excitation and contraction, phasic smooth muscles, such as the digestive tract and urinary bladder, undergo relaxation due to a decline of cellular Ca2+ concentration and decreased Ca2+ sensitivity of LC20 phosphorylation, named Ca2+ desensitization. Here, we determined the mechanisms underlying the temporal Ca2+ desensitization of LC20 phosphorylation in phasic smooth muscles using permeabilized strips of the mouse ileum and urinary bladder. Upon stimulation with pCa6.0 at 20°C, contraction and LC20 phosphorylation peaked within 30 s and then declined to about 50% of the peak force at 2 min after stimulation. During the relaxation phase after the contraction, LC20 kinase [myosin light chain kinase (MLCK)] was inactivated, but no fluctuation in LC20 phosphatase activity occurred, suggesting that MLCK inactivation is a cause of the Ca2+-induced Ca2+ desensitization of LC20 phosphorylation. MLCK inactivation was associated with phosphorylation at the calmodulin-binding domain of the kinase. Treatment with STO-609 and TIM-063 antagonists for Ca2+/calmodulin (CaM)-dependent protein kinase kinase-β (CaMKKβ) attenuated both the phasic response of the contraction and MLCK phosphorylation, whereas neither CaM kinase II, AMP-activated protein kinase, nor p21-activated kinase induced MLCK inactivation in phasic smooth muscles. Conversely, protein phosphatase 2A inhibition amplified the phasic response. Signaling pathways through CaMKKβ and protein phosphatase 2A may contribute to regulating the phasic response of smooth muscle contraction.

Spatiotemporal mapping of sensory and motor innervation of the embryonic and postnatal mouse urinary bladder

The primary function of the urinary bladder is to store urine (continence) until a suitable time for voiding (micturition). These distinct processes are determined by the coordinated activation of sensory and motor components of the nervous system, which matures to enable voluntary control at the time of weaning. Our aim was to define the development and maturation of the nerve-organ interface of the mouse urinary bladder by mapping the organ and tissue distribution of major classes of autonomic (motor) and sensory axons. Innervation of the bladder was evident from E13 and progressed dorsoventrally. Increasing defasciculation of axon bundles to single axons within the muscle occurred through the prenatal period, and in several classes of axons underwent further maturation until P7. Urothelial innervation occurred more slowly than muscle innervation and showed a clear regional difference, from E18 the bladder neck having the highest density of urothelial nerves. These features of innervation were similar in males and females but varied in timing and tissue density between different axon classes. We also analysed the pelvic ganglion, the major source of motor axons that innervate the lower urinary tract and other pelvic organs. Cholinergic, nitrergic (subset of cholinergic) and noradrenergic neuronal cell bodies were present prior to visualization of these axon classes within the bladder. Examination of cholinergic structures within the pelvic ganglion indicated that connections from spinal preganglionic neurons to pelvic ganglion neurons were already present by E12, a time at which these autonomic ganglion neurons had not yet innervated the bladder. These putative preganglionic inputs increased in density prior to birth as axon terminal fields continued to expand within the bladder tissues. Our studies also revealed in numerous pelvic ganglion neurons an unexpected transient expression of calcitonin gene-related peptide, a peptide commonly used to visualise the peptidergic class of visceral sensory axons. Together, our outcomes enhance our understanding of neural regulatory elements in the lower urinary tract during development and provide a foundation for studies of plasticity and regenerative capacity in the adult system.

Urothelium-Specific Deletion of Connexin43 in the Mouse Urinary Bladder Alters Distension-Induced ATP Release and Voiding Behavior.

Connexin43 (Cx43), the main gap junction and hemichannel forming protein in the urinary bladder, participates in the regulation of bladder motor and sensory functions and has been reported as an important modulator of day–night variations in functional bladder capacity. However, because Cx43 is expressed throughout the bladder, the actual role played by the detrusor and the urothelial Cx43 is still unknown. For this purpose, we generated urothelium-specific Cx43 knockout (uCx43KO) mice using Cre-LoxP system. We evaluated the day–night micturition pattern and the urothelial Cx43 hemichannel function of the uCx43KO mice by measuring luminal ATP release after bladder distention. In wild-type (WT) mice, distention-induced ATP release was elevated, and functional bladder capacity was decreased in the animals’ active phase (nighttime) when Cx43 expression was also high compared to levels measured in the sleep phase (daytime). These day–night differences in urothelial ATP release and functional bladder capacity were attenuated in uCx43KO mice that, in the active phase, displayed lower ATP release and higher functional bladder capacity than WT mice. These findings indicate that urothelial Cx43 mediated ATP signaling and coordination of urothelial activity are essential for proper perception and regulation of responses to bladder distension in the animals’ awake, active phase.

Prevention of urinary infection through the incorporation of silver-ricinoleic acid-polystyrene nanoparticles on the catheter surface.

Nosocominal infections associated with biofilm formation on urinary catheters cause serious complications. The aim of this study was to investigate the feasibility of the polyurethane (PU) catheter modified with tetracycline hydrochloride (TCH) attached Ag nanoparticles embedded PolyRicinoleic acid-Polystyrene Nanoparticles (PU-TCH-AgNPs-PRici-PS NPs) and the influence on antimicrobial and antibiofilm activity of urinary catheters infected by Escherichia coli and Staphylococcus aureus. For this purpose, AgNPs embedded PRici graft PS graft copolymers (AgNPs-PRici-g-PS) were synthesized via free radical polymerization and characterized by FTIR, HNMR and DSC. AgNPs-PRici-PS NPs were prepared and optimized by the different parameters and the optimized size of nanoparticle was found as about 150 ± 1 nm. The characterization of the nanoparticles and the morphological evaluation were carried out by FTIR and SEM. Short term stability of nanoparticles was realised at 4°C for 30 days. In vitro release profiles of TCH and Ag NPs were also investigated. The formation of biofilm on PU modified TCH-Ag NPs-PRici-PS NPs, was evaluated and the biocompatibility test of the nanoparticles was realized via the mouse fibroblast (L929) and mouse urinary bladder cells (G/G An1). This is the first time that TCH-AgNPs-PRici-PS NPs used in the modification of PU catheter demonstrated high antimicrobial and antibiofilm activities against the urinary tract infection.

A Method to Isolate Pericytes From the Mouse Urinary Bladder for the Study of Diabetic Bladder Dysfunction.

PurposePericytes surround the endothelial cells in microvessels and play a distinct role in controlling vascular permeability and maturation. The loss of pericyte function is known to be associated with diabetic retinopathy and erectile dysfunction. This study aimed to establish a technique for the isolation of pericytes from the mouse urinary bladder and an in vitro model that mimics in vivo diabetic bladder dysfunction.MethodsTo avoid contamination with epithelial cells, the urothelial layer was meticulously removed from the underlying submucosa and detrusor muscle layer. The tissues were cut into multiple pieces, and the fragmented tissues were settled by gravity into collagen I-coated culture plates. The cells were cultured under normal-glucose (5 mmol/L) or high-glucose (30 mmol/L) conditions, and tube formation, cell proliferation, and TUNEL assays were performed. We also performed hydroethidine staining to measure superoxide anion production.ResultsWe successfully isolated high-purity pericytes from the mouse urinary bladder. The cells were positively stained for platelet-derived growth factor receptor-β and NG2 and negatively stained for smooth muscle cell markers (desmin and myosin) and an endothelial cell marker (CD31). The number of tubes formed and the number of proliferating cells were significantly lower when the pericytes were exposed to high-glucose conditions compared with normal-glucose conditions. In addition, there were significant increases in superoxide anion production and the number of apoptotic cells when the pericytes were cultured under high-glucose conditions.ConclusionsTo the best of our knowledge, this is the first study to isolate and culture pericytes from the mouse urinary bladder. Our model would be a useful tool for screening the efficacy of therapeutic candidates targeting pericyte function in diabetic bladder dysfunction and exploring the functional role of specific targets at the cellular level.

A new method for purifying Brazilin from lignum sappan – Cytotoxic and anti-proliferative effects in cell lines and improved survival in mice bearing urinary bladder carcinoma

Background: Urinary bladder carcinoma is characterized by high post-operative recurrence rates. Sufu'ning lotion, prepared with Lignum Sappan, can reduce the recurrence of urinary bladder carcinoma. Objectives: The present study describes a methodology for extracting and obtaining high purity brazilin from Lignum sappan and evaluates its effects on urinary bladder carcinoma, in vitro and in vivo . Materials and Methods: The brazilin was extracted from Lignum sappan, purified and identified. The purified brazilin was assessed in vitro for its effects on tumor cell viability and growth using human (T24) and mouse urinary bladder (BTT) cancer cell lines. The role of brazilin in the inhibition of the cell cycle was determined by flow cytometry. The in vivo activity of brazilin was assessed based on the survival of tumor-bearing mice. Results: Brazilin was purified of 99.10% purity and was bioactive as it significantly (P