Long-term follow-up of TREK-1 KO mice reveals the development of bladder hypertrophy and impaired bladder smooth muscle contractility with age.

Stretch-activated two-pore domain K+ (K2P) channels play important roles in many visceral organs, including the urinary bladder. The TWIK-related K+ channel TREK-1 is the predominantly expressed K2P channel in the urinary bladder of humans and rodents. Downregulation of TREK-1 channels was observed in the urinary bladder of patients with detrusor overactivity, suggesting their involvement in the pathogenesis of voiding dysfunction. This study aimed to characterize the long-term effects of TREK-1 on bladder function with global and smooth muscle-specific TREK-1 knockout (KO) mice. Bladder morphology, bladder smooth muscle (BSM) contractility, and voiding patterns were evaluated up to 12 mo of age. Both sexes were included in this study to probe the potential sex differences. Smooth muscle-specific TREK-1 KO mice were used to distinguish the effects of TREK-1 downregulation in BSM from the neural pathways involved in the control of bladder contraction and relaxation. TREK-1 KO mice developed enlarged urinary bladders (by 60.0% for males and by 45.1% for females at 6 mo; P < 0.001 compared with the age-matched control group) and had a significantly increased bladder capacity (by 137.7% at 12 mo; P < 0.0001) and compliance (by 73.4% at 12 mo; P < 0.0001). Bladder strips isolated from TREK-1 KO mice exhibited decreased contractility (peak force after KCl at 6 mo was 1.6 ± 0.7 N/g compared with 3.4 ± 2.0 N/g in the control group; P = 0.0005). The lack of TREK-1 channels exclusively in BSM did not replicate the bladder phenotype observed in TREK-1 KO mice, suggesting a strong neurogenic origin of TREK-1-related bladder dysfunction.NEW & NOTEWORTHY This study compared voiding function and bladder phenotypes in global and smooth muscle-specific TREK-1 KO mice. We found significant age-related changes in bladder contractility, suggesting that the lack of TREK-1 channel activity might contribute to age-related changes in bladder smooth muscle physiology.

Therapeutic approaches for UCPPS management: research advances, experimental targets, and future directions.

Urologic Chronic Pelvic Pain Syndrome (UCPPS) is a painful chronic condition with persistent pain originating from the pelvis that often leads to detrimental lifestyle changes in the affected patients. The syndrome develops in both sexes, with an estimated prevalence of 5.7% to 26.6% worldwide. This narrative review summarizes currently recommended therapies for UCPPS, followed by the latest animal and clinical research advances in the field. The diagnosis of UCPPS by clinicians has room for improvement despite the changes in the past decade aiming to decrease the time to treatment. Therapeutic approaches targeting growth factors (i.e., NGF, VEGF), amniotic bladder therapy and stem cell treatments gain more attention as experimental treatment options for UCPPS. The development of novel diagnostic tests based on the latest advances in urinary biomarkers would be beneficial to assist with the clinical diagnosis of UCPPS. Future research directions should address the role of chronic psychological stress and the mechanisms of pain refractory to conventional management strategies in UCPPS etiology. Testing the applicability of cognitive behavioral therapy in this cohort of UCPPS patients might be promising to increase their QoL. The search for novel lead compounds and innovative drug delivery systems requires clinically relevant translational animal models. The role of autoimmune responses triggered by environmental factors is another promising research direction to clarify the impact of the immune system in UCPPS pathophysiology. Significance Statement This minireview provides an up-to-date summary of the therapeutic approaches for UCPPS with focus on recent advancements in the clinical diagnosis and treatments of the disease, pathophysiological mechanisms of UCPPS, signaling pathways and molecular targets involved in pelvic nociception.

Association between urinary symptom severity and white matter plaque distribution in women with multiple sclerosis.

AIMS: Multiple sclerosis (MS) is characterized by demyelinated white matter plaque throughout the central nervous system. Plaque involvement in regions that regulate micturition may be associated with urinary symptom severity in patients with MS. The aim of this prospective study is to investigate the relationship between cerebral plaque volume (PV), location, and urinary symptoms in women with MS. METHODS: We conducted a case-control pilot study of women with MS undergoing routine yearly brain MRI. Women were administered the American Urologic Association-Symptom Index (AUA-SI) and divided into two groups: severe urinary symptoms (AUA-SI ≥20) and mild symptoms (AUA-SI ≤7). PV and location in the brain were determined using a validated automated white matter lesion segmentation algorithm. RESULTS: This study of 36 women found that the median total PV did not differ between groups. Women with severe urinary symptoms had larger median PV in the left frontal lobe (LFL) and right limbic lobe (RLL) compared with women with mild urinary symptoms. Within the RLL, women with severe symptoms had a larger median PV in the right cingulate gyrus (RCG). There was a moderate correlation between LFL lesion volume and RLL lesion volume with the AUA emptying subscore; however, these regions did not correlate with the storage subscore. CONCLUSIONS: This preliminary study found urinary symptom severity in women with MS is associated with PV in the RCG and LFL, and not total cerebral PV. These findings may explain why disease burden alone is not a predictor of severity or type of voiding dysfunction in patients with MS.

Doxorubicin induces detrusor smooth muscle impairments through myosin dysregulation, leading to a risk of lower urinary tract dysfunction.

Cytotoxic chemotherapy is the foundation for the treatment of the wide variety of childhood malignancies; however, these therapies are known to have a variety of deleterious side effects. One common chemotherapy used in children, doxorubicin (DOX), is well known to cause cardiotoxicity and cardiomyopathy. Recent studies have revealed that DOX impairs skeletal and smooth muscle function and contributes to fatigue and abnormal intestinal motility in patients. In this study, we tested the hypothesis that systemic DOX administration also affects detrusor smooth muscle (DSM) function in the urinary bladder, especially when administered at a young age. The effects on the DSM and bladder function were assessed in BALB/cJ mice that received six weekly intravenous injections of DOX (3 mg·kg-1·wk-1) or saline for the control group. Systemic DOX administration resulted in DSM hypertrophy, increased voiding frequency, and a significant attenuation of DSM contractility, followed by a slower relaxation compared with the control group. Gene expression analyses revealed that unlike DOX-induced cardiotoxicity, the bladders from DOX-administered animals showed no changes in oxidative stress markers; instead, downregulation of large-conductance Ca2+-activated K+ channels and altered expression of myosin light-chain kinase coincided with reduced myosin light-chain phosphorylation. These results indicate that in vivo DOX exposure caused DSM dysfunction by dysregulation of molecules involved in the detrusor contractile-relaxation mechanisms. Collectively, our findings suggest that survivors of childhood cancer treated with DOX may be at increased risk of bladder dysfunction and benefit from followup surveillance of bladder function.

Association of genetic polymorphisms in the pore domains of mechano-gated TREK-1 channel with overactive lower urinary tract symptoms in humans.

AIMS: Mechanosensitivity of the urinary bladder is regulated by many factors including mechano-gated two-pore domain (K2 P, KCNK) potassium channels. TWIK-related K+ channel, TREK-1, is a predominantly expressed member of K2 P channel family in the human detrusor, and its expression and function are diminished in patients with overactive lower urinary tract symptoms (LUTS). The changes in channel activity may result from spontaneously occurring gene mutations. The aim of this study was to compare single nucleotide polymorphisms (SNPs) in TREK-1 channel between patients with LUTS and healthy donors. METHODS: Six SNPs (rs370266806, rs373919966, rs758937019, rs769301539, rs772497750, and rs775158737) in two pore domains of human TREK-1 gene were analyzed using TaqMan SNP genotyping assay with manufacturer-designed primers and allele-specific probes. The screening was done in control bladders and detrusor specimens from patients with overactive LUTS. Statistical analyses were performed using R, Fisher's exact test and Hardy-Weinberg Equilibrium. RESULTS: Six SNPs in two pore domains of the human TREK-1 gene were analyzed in human bladder specimens. The frequencies of rs758937019-CT genotype (P = 0.0016) and rs758937019-T allele (P = 0.0022) were significantly higher in the group with overactive LUTS. There was no significant association of rs775158737-GA genotype and rs775158737-A allele with the overactive LUTS, though they were present only in the overactive LUTS group. CONCLUSIONS: Our results provide evidence that altered expression and function of TREK-1 channel in patients with overactive LUTS could be due to genetic polymorphisms in the pore domains of TREK-1 channel (rs758937019).

Altered detrusor contractility and voiding patterns in mice lacking the mechanosensitive TREK-1 channel.

BACKGROUND: Previously published results from our laboratory identified a mechano-gated two-pore domain potassium channel, TREK-1, as a main mechanosensor in the smooth muscle of the human urinary bladder. One of the limitations of in vitro experiments on isolated human detrusor included inability to evaluate in vivo effects of TREK-1 on voiding function, as the channel is also expressed in the nervous system, and may modulate micturition via neural pathways. Therefore, in the present study, we aimed to assess the role of TREK-1 channel in bladder function and voiding patterns in vivo by using TREK-1 knockout (KO) mice. METHODS: Adult C57BL/6 J wild-type (WT, N = 32) and TREK-1 KO (N = 33) mice were used in this study. The overall phenotype and bladder function were evaluated by gene and protein expression of TREK-1 channel, in vitro contractile experiments using detrusor strips in response to stretch and pharmacological stimuli, and cystometry in unanesthetized animals. RESULTS: TREK-1 KO animals had an elevated basal muscle tone and enhanced spontaneous activity in the detrusor without detectable changes in bladder morphology/histology. Stretch applied to isolated detrusor strips increased the amplitude of spontaneous contractions by 109% in the TREK-1 KO group in contrast to a 61% increase in WT mice (p ≤ 0.05 to respective baseline for each group). The detrusor strips from TREK-1 KO mice also generated more contractile force in response to electric field stimulation and high potassium concentration in comparison to WT group (p ≤ 0.05 for both tests). However, cystometric recordings from TREK-1 KO mice revealed a significant increase in the duration of the intermicturition interval, enhanced bladder capacity and increased number of non-voiding contractions in comparison to WT mice. CONCLUSIONS: Our results provide evidence that global down-regulation of TREK-1 channels has dual effects on detrusor contractility and micturition patterns in vivo. The observed differences are likely due to expression of TREK-1 channel not only in detrusor myocytes but also in afferent and efferent neural pathways involved in regulation of micturition which may underly the "mixed" voiding phenotype in TREK-1 KO mice.

Early life voiding dysfunction leads to lower urinary tract dysfunction through alteration of muscarinic and purinergic signaling in the bladder.

Lower urinary tract dysfunction (LUTD) is a common problem in children and constitutes up to 40% of pediatric urology clinic visits. Improved diagnosis and interventions have been leading to better outcomes in many patients, whereas some children are left untreated or do not respond to the treatment successfully. In addition, many of these patients are lost by the pediatric urologists during their teenage years, and the outcome in later life largely remains unidentified. Studies suggest childhood LUTD is associated with subsequent adult urinary tract symptoms. However, whether and how early life LUTD attributes to urinary symptoms in those patients later in life remains to be elucidated. In the current study, we investigated the effects of early life voiding perturbation on bladder function using a neonatal maternal separation (NMS) protocol in mice. The NMS group displayed a delayed development of voluntary voiding behavior, a significant reduction of functional bladder capacity, and bladder overactivity compared with control mice later in life. In vitro evaluation of detrusor smooth muscle and molecular study showed a decrease in muscarinic contribution alongside an increase in purinergic contribution in detrusor contractility in NMS mice compared with control group. These results suggest that early life bladder dysfunction interfered with the normal maturation of the voluntary micturition control and facilitated LUTD in a later stage, which is at least partly attributed to an alteration of muscarinic and purinergic signaling in the urinary bladder.

Preventative effects of a HIF inhibitor, 17-DMAG, on partial bladder outlet obstruction-induced bladder dysfunction.

Posterior urethral valves are the most common cause of partial bladder outlet obstruction (PBOO) in the pediatric population. Pathological changes in the bladder developed during PBOO are responsible for long-lasting voiding dysfunction in this population despite early surgical interventions. Increasing evidence showed PBOO induces an upregulation of hypoxia-inducible factors (HIFs) and their transcriptional target genes, and they play a role in pathophysiological changes in the obstructed bladders. We hypothesized that blocking HIF pathways can prevent PBOO-induced bladder dysfunction. PBOO was surgically created by ligation of the bladder neck in male C57BL/6J mice for 2 wk. PBOO mice received intraperitoneal injection of either saline or 17-DMAG (alvespimycin, 3 mg/kg) every 48 h starting from day 1 postsurgery. Sham-operated animals received injection of saline on the same schedule as PBOO mice and served as controls. The bladders were harvested after 2 wk, and basal activity and evoked contractility of the detrusor smooth muscle (DSM) were evaluated in vitro. Bladder function was assessed in vivo by void spot assay and cystometry in conscious, unrestrained mice. Results indicated the 17-DMAG treatment preserved DSM contractility and partially prevented the development of detrusor over activity in obstructed bladders. In addition, PBOO caused a significant increase in the frequency of micturition, which was significantly reduced by 17-DMAG treatment. The 17-DMAG treatment improved urodynamic parameters, including increases in the bladder pressure at micturition and nonvoid contractions observed in PBOO mice. These results demonstrate that treatment with 17-DMAG, a HIF inhibitor, significantly alleviated PBOO-induced bladder pathology in vivo.

Altered expression and modulation of the two-pore-domain (K2P) mechanogated potassium channel TREK-1 in overactive human detrusor.

Detrusor overactivity (DO) is the abnormal response of the urinary bladder to physiological stretch during the filling phase of the micturition cycle. The mechanisms of bladder smooth muscle compliance upon the wall stretch are poorly understood. We previously reported that the function of normal detrusor is regulated by TREK-1, a member of the mechanogated subfamily of two-pore-domain potassium (K2P) channels. In the present study, we aimed to identify the changes in expression and function of TREK-1 channels under pathological conditions associated with DO, evaluate the potential relationship between TREK-1 channels and cytoskeletal proteins in the human bladder, and test the possibility of modulation of TREK-1 channel expression by small RNAs. Expression of TREK-1 channels in DO specimens was 2.7-fold decreased compared with control bladders and was associated with a significant reduction of the recorded TREK-1 currents. Isolated DO muscle strips failed to relax when exposed to a TREK-1 channel opener. Immunocytochemical labeling revealed close association of TREK-1 channels with cell cytoskeletal proteins and caveolins, with caveolae microdomains being severely disrupted in DO specimens. Small activating RNA (saRNA) tested in vitro provided evidence that expression of TREK-1 protein could be partially upregulated. Our data confirmed a significant downregulation of TREK-1 expression in human DO specimens and provided evidence of close association between the channel, cell cytoskeleton, and caveolins. Upregulation of TREK-1 expression by saRNA could be a future step for the development of in vivo pharmacological and genetic approaches to treat DO in humans.

Urinary Biomarkers in Women with Refractory Urgency Urinary Incontinence Randomized to Sacral Neuromodulation versus OnabotulinumtoxinA Compared to Controls.

PURPOSE: We measured urinary biomarker levels in women with refractory urgency urinary incontinence and controls at baseline and 6 months after treatment with sacral neuromodulation or intradetrusor injection of onabotulinumtoxinA. We also assessed the association of baseline biomarkers with posttreatment urgency urinary incontinence episodes and overactive bladder symptom bother outcomes. MATERIALS AND METHODS: First morning urine samples were collected from consented trial participants and age matched women without urgency urinary incontinence. Biomarkers reflecting general inflammation, neuroinflammation, afferent neurotransmitters and tissue remodeling were measured using standardized enzyme-linked immunosorbent assay and activity assays as appropriate. Symptom bother was assessed by the overactive bladder questionnaire and urgency urinary incontinence episodes were determined by bladder diary. Linear models were used to examine differences in mean biomarker levels and the change in urgency urinary incontinence episodes and symptom bother between baseline and 6 months. Modest evidence of a potential association was represented by p ≤0.01 and p ≤0.004 represented moderate evidence of an association with outcomes. RESULTS: Baseline biomarker levels differed little between cases and controls except tropoelastin (p = 0.001) and N-terminal telopeptide collagen type 1 (p <0.001). Changes in biomarker levels 6 months after intervention included decreases in collagenase (p <0.001) in both treatment groups and increases in interleukin-8 (p = 0.002) and matrix metalloprotease-9 (p <0.001) in the onabotulinumtoxinA group. Higher baseline calcitonin gene-related peptide across both treatments (p = 0.007) and nerve growth factor in the onabotulinumtoxinA arm (p = 0.007) were associated with less reduction in overactive bladder symptom bother. CONCLUSIONS: Refractory urgency urinary incontinence is a complex condition. These data suggest that matrix remodeling and neuropeptide mediation may be involved in its pathophysiological mechanisms and response to treatment.

Urinary neurotrophic peptides in postmenopausal women with and without overactive bladder.

AIMS: The aim of this study was to compare the expression of urinary nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), substance P (SP), and calcitonin-gene related peptide (CGRP) in women with and without overactive bladder (OAB). We sought to determine factors associated with higher expression of these neuropeptides. METHODS: Participants with OAB and age-matched controls were enrolled. Symptom severity was assessed with validated questionnaires. Urinary neurotrophin levels, symptom scores, and clinical data were compared between the groups. Multivariate analysis determined independent factors associated with urinary neurotrophin levels. RESULTS: Sixty-seven women (38 OAB, 29 controls) were included. Women with OAB and controls were similar in age, race, body mass index, parity, and smoking status. Women with OAB were more likely to report a history of pelvic pain and pelvic surgery. Neurotrophic factor levels normalized to urinary creatinine did not differ between the groups. Increasing age was associated with greater urinary levels of BDNF and NGF (ß = 0.23, 95%CI 0.11-0.34 and 0.75, 95%CI 0.17-1.33, respectively, P < 0.02). Higher urinary NGF was associated with increasing BMI (ß = 0.81, 95%CI 0.05-1.57, P = 0.04) while pain was associated with elevated urinary SP (ß = 0.21, 95%CI 0.09-0.33, P = 0.001). CONCLUSIONS: Our data does not support a relationship between urinary neurotrophin levels and OAB in age-matched postmenopausal women. Further research is necessary to elucidate the role of urinary neurotrophins in the diagnosis and management of OAB. Neurourol. Urodynam. 36:740-744, 2017. © 2016 Wiley Periodicals, Inc.

Inhibition of HIF Reduces Bladder Hypertrophy and Improves Bladder Function in Murine Model of Partial Bladder Outlet Obstruction.

PURPOSE: Posterior urethral valves are the most common cause of partial bladder outlet obstruction in the pediatric population. However, to our knowledge the etiology and the detailed mechanisms underlying pathological changes in the bladder following partial bladder outlet obstruction remain to be elucidated. Recent findings suggest that hypoxia and associated up-regulation of HIFs (hypoxia-inducible factors) have a key role in partial bladder outlet obstruction induced pathology in the bladder. We examined the effects of pharmacological inhibition of HIF pathways by 17-DMAG (17-(dimethylaminoethylamino)-17-demethoxygeldanamycin) in pathophysiological phenotypes after partial bladder outlet obstruction. MATERIALS AND METHODS: Partial bladder outlet obstruction was surgically created in male C57BL/6J mice. The animals received oral administration of 17-DMAG or vehicle daily starting from the initiation of obstruction up to 5 days. Sham operated mice served as controls. Bladders were harvested from each group 2, 4 and 7 days postoperatively, and analyzed for histological and biochemical changes. Bladder function was assessed by in vitro muscle contractility recordings. RESULTS: Partial bladder outlet obstruction caused a significant increase in the bladder mass accompanying enhanced collagen deposition in the bladder wall while 17-DMAG treatment suppressed those increases. Treatment with 17-DMAG attenuated the degree of up-regulation of HIFs and their target genes involving the development of tissue fibrosis in obstructed bladders. Treatment with 17-DMAG improved the decreased responses of obstructed bladder strips to electrical field stimulation and KCl. CONCLUSIONS: In vivo 17-DMAG treatment decreased partial bladder outlet obstruction induced pathophysiological changes in the bladder. HIF pathway inhibition has a potential clinical implication for the development of novel pharmacological therapies to treat bladder pathology associated with partial bladder outlet obstruction.

Basal and stress-activated hypothalamic pituitary adrenal axis function in postmenopausal women with overactive bladder.

INTRODUCTION AND HYPOTHESIS: The aim of this study was to measure physiologic and psychologic stress reactivity in women with overactive bladder (OAB). There is growing evidence in preclinical models that central nervous system dysregulation, particularly in response to psychological stress, may contribute to lower urinary tract symptoms in women with OAB. METHODS: Postmenopausal women with OAB and healthy controls underwent Structured Clinical Interview for DSM-IV Axis I disorders (SCID) to identify those without identifiable psychiatric disease. Eligible participants underwent physiologic measures including basal (cortisol-awakening response; CAR) and stress-activated salivary cortisol levels, heart rate (HR), urinary metanephrines and neurotrophins, as well as validated symptom assessment for stress, anxiety, depression, and bladder dysfunction at baseline and during, and following an acute laboratory stressor, the Trier Social Stress Test (TSST). RESULTS: Baseline measures of cortisol reactivity measured by CAR showed blunted response among women with OAB (p = 0.015), while cortisol response to the TSST was greater in the OAB group (p = 0.019). Among OAB patients, bladder urgency as measured by visual analog scale (VAS) increased from pre- to post-TSST (p = 0.04). There was a main effect of TSST on HR (p < 0.001), but no group interaction. CONCLUSIONS: Preliminary findings suggest that women with OAB have greater physiologic and psychologic stress reactivity than healthy controls. Importantly for women with OAB, acute stress appears to exacerbate bladder urgency. Evaluation of the markers of stress response may suggest targets for potential diagnostic and therapeutic interventions.

Regulation of urinary bladder function by protein kinase C in physiology and pathophysiology.

BACKGROUND: Protein kinase C (PKC) is expressed in many tissues and organs including the urinary bladder, however, its role in bladder physiology and pathophysiology is still evolving. The aim of this review was to evaluate available evidence on the involvement of PKC in regulation of detrusor contractility, muscle tone of the bladder wall, spontaneous contractile activity and bladder function under physiological and pathophysiological conditions. METHODS: This is a non-systematic review of the published literature which summarizes the available animal and human data on the role of PKC signaling in the urinary bladder under different physiological and pathophysiological conditions. A wide PubMed search was performed including the combination of the following keywords: "urinary bladder", "PKC", "detrusor contractility", "bladder smooth muscle", "detrusor relaxation", "peak force", "detrusor underactivity", "partial bladder outlet obstruction", "voltage-gated channels", "bladder nerves", "PKC inhibitors", "PKC activators". Retrieved articles were individually screened for the relevance to the topic of this review with 91 citations being selected and included in the data analysis. DISCUSSION: Urinary bladder function includes the ability to store urine at low intravesical pressure followed by a subsequent release of bladder contents due to a rapid phasic contraction that is maintained long enough to ensure complete emptying. This review summarizes the current concepts regarding the potential contribution of PKC to contractility, physiological voiding, and related signaling mechanisms involved in the control of both the storage and emptying phases of the micturition cycle, and in dysfunctional voiding. Previous studies linked PKC activation exclusively with an increase in generation of the peak force of smooth muscle contraction, and maximum force generation in the lower urinary tract. More recent data suggests that PKC presents a broader range of effects on urinary bladder function including regulation of storage, emptying, excitability of the detrusor, and bladder innervation. In this review, we evaluated the mechanisms of peripheral and local regulation of PKC signaling in the urinary bladder, and their impact on different phases of the micturition cycle under physiological and pathophysiological conditions.

Protein kinase C modulates frequency of micturition and non-voiding contractions in the urinary bladder via neuronal and myogenic mechanisms.

BACKGROUND: Protein Kinase C (PKC) dysfunction is implicated in a variety of smooth muscle disorders including detrusor overactivity associated with frequency and urgency of micturition. In this study, we aimed to evaluate the modulatory effects of endogenous PKC-dependent pathways on bladder storage and emptying function. METHODS: We utilized in vivo cystometry and in vitro organ bath studies using isolated bladder muscle strips (BMS) from rats to measure contractility, intravesical pressure, and voided volume. Both in vitro and in vivo results were statistically analyzed using one-way repeated measures ANOVA between the groups followed by Bonferroni's post-test, as appropriate (Systat Software Inc., San Jose, CA). RESULTS: Effects of PKC activators, phorbol-12,13-dibutyrate (PDBu), and phorbol-12,13-myristate (PMA), were concentration-dependent, with high concentrations increasing frequency of micturition, and sensitivity of intramural nerves to electrical field stimulation (EFS), in vitro, while lower concentrations had no effect on BMS sensitivity to EFS. The PKC inhibitors, bisindolylmaleimide1 (Bim-1), (28 nM), and Ro318220 (50 µM) triggered an increase in the number of non-voiding contractions (NVC), and a decrease in the voided volume associated with reduced ability to maintain contractile force upon EFS, but did not affect peak force in vitro. Both low (50 nM) and high PDBu 1 micromolar (1 uM) decreased the sensitivity of BMS to carbachol. Application of a low concentration of PDBu inhibited spontaneous contractions, in vitro, and Bim-1-induced NVC, and restored normal voiding frequency during urodynamic recordings in vivo. CONCLUSIONS: In summary, the effects of low PKC stimulation include inhibition of smooth muscle contractile responses, whereas high levels of PKC stimulation increased nerve-mediated contractions in vitro, and micturition contractions in vivo. These results indicate that endogenous PKC signaling displays a concentration-dependent contraction profile in the urinary bladder via both smooth muscle and nerve-mediated pathways.

Response of the human detrusor to stretch is regulated by TREK-1, a two-pore-domain (K2P) mechano-gated potassium channel.

The mechanisms of mechanosensitivity underlying the response of the human bladder to stretch are poorly understood. Animal data suggest that stretch-activated two-pore-domain (K2P) K(+) channels play a critical role in bladder relaxation during the filling phase. The objective of this study was to characterize the expression and function of stretch-activated K2P channels in the human bladder and to clarify their physiological role in bladder mechanosensitivity. Gene and protein analysis of the K2P channels TREK-1, TREK-2 and TRAAK in the human bladder revealed that TREK-1 is the predominantly expressed member of the mechano-gated subfamily of K2P channels. Immunohistochemical labelling of bladder wall identified higher levels of expression of TREK-1 in detrusor smooth muscle cells in comparison to bladder mucosa. Functional characterization and biophysical properties of the predominantly expressed member of the K2P family, the TREK-1 channel, were evaluated by in vitro organ bath studies and the patch-clamp technique. Electrophysiological recordings from single smooth muscle cells confirmed direct activation of TREK-1 channels by mechanical stretch and negative pressure applied to the cell membrane. Inhibition of TREK-1 channels in the human detrusor significantly delayed relaxation of the stretched bladder smooth muscle strips and triggered small-amplitude spontaneous contractions. Application of negative pressure to cell-attached patches (-20 mmHg) caused a 19-fold increase in the open probability (NPo) of human TREK-1 channels. l-Methionine (1 mm), a specific TREK-1 inhibitor, dramatically decreased the NPo of TREK-1 channels from 0.045 ± 0.003 to 0.008 ± 0.001 (n = 8, P ≤ 0.01). Subsequent addition of arachidonic acid (10 µm), a channel opener, increased the open probability of methionine-inhibited unitary currents up to 0.43 ± 0.05 at 0 mV (n = 9, P ≤ 0.05). The results of our study provide direct evidence that the response of the human detrusor to mechanical stretch is regulated by activation of mechano-gated TREK-1 channels. Impaired mechanosensation and mechanotransduction associated with the changes in stretch-activated K2P channels may underlie myogenic bladder dysfunction in humans.

Coronavirus-induced demyelination of neural pathways triggers neurogenic bladder overactivity in a mouse model of multiple sclerosis.

In the present study, we aimed to determine whether mice with coronavirus-induced encephalomyelitis (CIE) develop neurogenic bladder dysfunction that is comparable with the neurogenic detrusor overactivity observed in patients with multiple sclerosis. Adult mice (C57BL/6J, 8 wk of age, n = 146) were inoculated with a neurotropic strain of mouse hepatitis virus (A59 strain) and followed for 4 wk. Inoculation with the virus caused a significant neural deficit in mice with an average clinical symptom score of 2.6 ± 0.5 at 2 wk. These changes were accompanied by 25 ± 5% weight loss at 1 and 2 wk postinoculation (P ≤ 0.001 vs. baseline) followed by a recovery phase. Histological analysis of spinal cord sections revealed multifocal sites of demyelinated lesions. Assessment of micturition patterns by filter paper assay determined an increase in the number of small and large urine spots in CIE mice starting from the second week after inoculation. Cystometric recordings in unrestrained awake animals confirmed neurogenic bladder overactivity at 4 wk postinoculation. One week after inoculation with the A59 strain of mouse hepatitis virus, mice became increasingly sensitive to von Frey filament testing with responses enhanced by 45% (n = 8, P ≤ 0.05 vs. baseline at 4 g); however, this initial increase in sensitivity was followed by gradual and significant diminution of abdominal sensitivity to mechanical stimulation by 4 wk postinoculation. Our results provide direct evidence showing that coronavirus-induced demyelination of the central nervous system causes the development of a neurogenic bladder that is comparable with neurogenic detrusor overactivity observed in patients with multiple sclerosis.

What is the role of covert infection in detrusor overactivity, and other LUTD? ICI-RS 2013.

A think tank was convened at the fourth ICI-RS meeting, which took place June 5-7, 2013 in Bristol UK, to consider current evidence and controversies surrounding the possible role of 'covert infection' in the pathophysiology of refractory detrusor overactivity (DO) and other lower urinary tract disorders (LUTD). The topic was chosen because several authors from different centers worldwide have recently published evidence which supports this concept. However, to date there is inconsistency regarding terminology and microbiological definitions, which were discussed by the participants. The mechanisms whereby infection/inflammation could actually promote aberrant detrusor contractions in the human remain controversial, and are more fully described in this report. Future requirements for research into this topic were outlined.

Lower urinary dysfunction as a long-term effect of childhood vincristine treatment, with potential influences by sex and dose.

Vincristine (VCR) is one of the most widely used chemotherapy agents in treating pediatric cancer. Nonetheless, it is known to cause dose-dependent neurotoxicity which can impact virtually every organ system. Despite its widespread use, the precise impact of VCR on the lower urinary tract (LUT) remains inadequately elucidated. Our initial clinical and translational investigations suggest a sex-specific influence of childhood VCR exposure on LUT function. Thus, the current study aimed to investigate the late effects of systemic VCR exposure on LUT physiology and the underlying mechanisms, focusing on dosage and male-sex, employing juvenile CD-1 mice as a model. Male mice subjected to VCR exhibited augmented functional bladder capacity accompanied by frequent non-void contractions during awake cystometry, alongside mast cell accumulation within the bladder, compared to the saline-treated control group. Noteworthy functional changes were observed in bladder strips from the VCR group, including decreased nerve-mediated contraction, heightened contractile responses to cholinergic and purinergic agonists, enhanced responsiveness to histamine-primarily via histamine receptor 1 (Hrh1)-and an augmented relaxation effect with compound 48/80 (a mast cell degranulator), relative to the control group. Significant changes in gene expression levels associated with neuroinflammation and nociception were observed in both the bladder and lumbosacral dorsal root ganglia (Ls-DRG) of the VCR group. These findings suggest that VCR exposure during childhood, particularly in males, triggers neuroimmune responses in the bladder and Ls-DRG, amplifying responsiveness to neurotransmitters in the bladder, thereby contributing to LUT dysfunction characterized by a mixed bladder phenotype as a late effect during survivorship.

Spontaneous and evoked contractions are regulated by PKC-mediated signaling in detrusor smooth muscle: involvement of BK channels.

Protein kinase C (PKC) and large conductance Ca(2+)-activated potassium channels (BK) are downregulated in the detrusor smooth muscle (DSM) in partial bladder outlet obstruction (PBOO). DSM from these bladders display increased spontaneous activity. This study examines the involvement of PKC in the regulation of spontaneous and evoked DSM contractions and whether pharmacologic inhibition of PKC in normal DSM contributes to increased detrusor excitability. Results indicate the PKC inhibitor bisindolylmaleimide 1 (Bim-1) prevented a decline in the amplitude of spontaneous DSM contractions over time in vitro, and these contractions persist in the presence of tetrodotoxin. Bim-1 also reduced the basal DSM tone, and the ability to maintain force in response to electrical field stimulation, but did not affect maximum contraction. The PKC activator phorbol-12,13-dibutyrate (PDBu) significantly reduced the amplitude and increased the frequency of spontaneous contractions at low concentrations (10 nM), while causing an increase in force at higher concentrations (1 µM). Preincubation of DSM strips with iberiotoxin prevented the inhibition of spontaneous contractions by PDBu. The BK channel openers isopimaric acid and NS1619 reduced the Bim-1-induced enhancement of spontaneous contractions in DSM strips. Our data suggest that PKC has a biphasic activation profile in the DSM and that it may play an important role in maintaining the quiescent state of the normal bladder during storage through the effects on BK channel, while helping to maintain force required for bladder emptying. The data also suggest that PKC dysfunction, as seen in PBOO, contributes to detrusor overactivity.