Intravital imaging of mouse urothelium reveals activation of extracellular signal-regulated kinase by stretch-induced intravesical release of ATP

To better understand the roles played by signaling molecules in the bladder, we established a protocol of intravital imaging of the bladder of mice expressing a Förster/fluorescence resonance energy transfer (FRET) biosensor for extracellular signal-regulated kinase (ERK), which plays critical roles not only in cell growth but also stress responses. With an upright two-photon excitation microscope and a vacuum-stabilized imaging window, cellular ERK activity was visualized in the whole bladder wall, from adventitia to urothelium. We found that bladder distention caused by elevated intravesical pressure (IVP) activated ERK in the urothelium, but not in the detrusor smooth muscle. When bladder distension was prevented, high IVP failed to activate ERK, suggesting that mechanical stretch, but not the high IVP, caused ERK activation. To delineate its molecular mechanism, the stretch-induced ERK activation was reproduced in an hTERT-immortalized human urothelial cell line (TRT-HU1) in vitro. We found that uniaxial stretch raised the ATP concentration in the culture medium and that inhibition of ATP signaling by apyrase or suramin suppressed the stretch-induced ERK activation in TRT-HU1 cells. In agreement with this in vitro observation, pretreatment with apyrase or suramin suppressed the high IVP-induced urothelial ERK activation in vivo. Thus, we propose that mechanical stretch induces intravesical secretion of ATP and thereby activates ERK in the urothelium. Our method of intravital imaging of the bladder of FRET biosensor-expressing mice should open a pathway for the future association of physiological stimuli with the activities of intracellular signaling networks

Development and validation of prediction model for incident overactive bladder: The Nagahama study.

OBJECTIVES We aimed to develop models to predict new-onset overactive bladder in 5 years using a large prospective cohort of the general population. METHODS This is a secondary analysis of a longitudinal cohort study in Japan. The baseline characteristics were measured between 2008 and 2010, with follow-ups every 5 years. We included subjects without overactive bladder at baseline and with follow-up data 5 years later. Overactive bladder was assessed using the overactive bladder symptom score. Baseline characteristics (demographics, health behaviors, comorbidities, and overactive bladder symptom scores) and blood test data were included as predictors. We developed two competing prediction models for each sex based on logistic regression with penalized likelihood (LASSO). We chose the best model separately for men and women after evaluating models' performance in terms of discrimination and calibration using an internal validation via 200 bootstrap resamples and a temporal validation. RESULTS We analyzed 7218 participants (male: 2238, female: 4980). The median age was 60 and 55 years, and the number of new-onset overactive bladder was 223 (10.0%) and 288 (5.8%) per 5 years in males and females, respectively. The in-sample estimates for C-statistic, calibration intercept, and slope for the best performing models were 0.77 (95% confidence interval 0.74-0.80), 0.28 and 1.15 for males, and 0.77 (95% confidence interval 0.74-0.80), 0.20 and 1.08 for females. Internal and temporal validation gave broadly similar estimates of performance, indicating low optimism. CONCLUSION We developed risk prediction models for new-onset overactive bladder among men and women with good predictive ability

Authentic role of ATP signaling in micturition reflex

Adenosine triphosphate (ATP) is a signaling molecule that regulates cellular processes. Based on previous studies of bladder function over the past decade, bladder ATP signaling was thought to have an essential role in the normal micturition reflex. In this study, we performed detailed analyses of bladder function in purinergic receptor-deficient mice using the automated voided stain on paper method and video-urodynamics. Unexpectedly, a lack of P2X2 or P2X3 receptors did not affect bladder function under normal physiological conditions, indicating that bladder ATP signaling is not essential for normal micturition reflex. In contrast, we found that lipopolysaccharide (LPS) induced markedly high levels of ATP release from the urothelium. In addition, LPS-induced rapid bladder hyperactivity was attenuated in P2X2 -/- and P2X3 -/- mice. Contrary to the previous interpretation, our present findings indicate that bladder ATP signaling has a fundamental role in the micturition reflex, especially in bladder dysfunction, under pathological conditions. Therefore, the bladder ATP signaling pathway might be a highly promising therapeutic target for functional bladder disorders. This study newly defines an authentic role for bladder ATP signaling in the micturition reflex

膀胱のコネキシン43と概日時計は日内排尿リズムに関与している

京都大学0048新制・課程博士博士(医学)甲第17826号医博第3824号新制||医||999(附属図書館)30641京都大学大学院医学研究科医学専攻(主査)教授 渡邉 大, 教授 村井 俊哉, 教授 柳田 素子学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDFA

Chronoradiation Therapy for Prostate Cancer: Morning Proton Beam Therapy Ameliorates Worsening Lower Urinary Tract Symptoms

Background and Purpose: Worsening lower urinary tract symptoms (LUTS) are a frequent adverse event following proton beam therapy (PBT) for localized prostate cancer. We investigated the differences in worsening LUTS among patients who received PBT at different times of day. Participants and Methods: Among 173 patients who underwent PBT for prostate cancer, 168 patients (median age 68.5 years) completed international prostate symptom score (IPSS) questionnaires and were included. Changes in the IPSS from baseline to the end of PBT were assessed by multiple linear regression analysis for age, National Comprehensive Cancer Network risk classification, androgen deprivation therapy, fractional PBT dose, clinical target volume, severity of IPSS, diabetes, LUTS medication use before PBT, anti-coagulant therapy and radiation time of day (morning (08:30–10:30), around noon (10:31–14:30), and late afternoon (14:31–16:30)). Results: IPSS total score and IPSS-Quality of Life (QoL) score (12 patients were excluded due to missing IPSS-QoL score) increased from eight to 14.9 (p < 0.0001) and from two to four (p < 0.0001), respectively. Time of day (morning) was the only determinant for worsening LUTS (β = −0.24, p < 0.01), voiding subscore (β = −0.22, p < 0.05) and IPSS-QoL (β = −0.27, p < 0.005), and was a determinant in item four (urgency) (β = −0.28, p < 0.005) with age (β = 0.19, p < 0.05). Conclusions: Morning PBT for localized prostate cancer significantly ameliorated worsening LUTS and improved QoL compared with treatment around noon or late afternoon. Chronoradiation therapy for localized prostate cancer may be effective and further research to elucidate the underlying mechanism is warranted

Chronobiology of micturition: putative role of the circadian clock.

[Purpose]Mammals urinate less frequently during the sleep period than the awake period. This is modulated by a triad of factors, including decreased arousal in the brain, a decreased urine production rate in the kidneys and increased functional bladder capacity during sleep. The circadian clock is genetic transcription-translation feedback machinery. It exists in most organs and cells, termed the peripheral clock, which is orchestrated by the central clock in the suprachiasmatic nucleus of the brain. We discuss the linkage between the day and night change in micturition frequency and the genetic rhythm maintained by the circadian clock system, focusing on the brain, kidney and bladder. [Material and Methods]We performed an inclusive review of the literature on the diurnal change in micturition frequency, urine volume, functional bladder capacity and urodynamics in humans and rodents, relating this to recent basic biological findings about the circadian clock. [Results]In humans various behavioral studies demonstrated a diurnal functional change in the kidney and bladder. Conversely, patients with nocturnal enuresis and nocturia showed impairment in this triad of factors. Rats and mice, which are nocturnal animals, also have a micturition frequency rhythm that is decreased during the day, which is the sleep phase for them. Mice with a genetically defective circadian clock system show impaired physiological rhythms in the triad of factors. The existence of the circadian clock has been proven in the brain, kidney and bladder, in which thousands of circadian oscillating genes exist. In the kidney they include genes involved in the regulation of water and major electrolytes. In the bladder they include connexin 43, a gene associated with the regulation of bladder capacity. [Conclusions]Recent progress in molecular biology about the circadian clock provides an opportunity to investigate the genetic basis of the micturition rhythm or impairment of the rhythm in nocturnal enuresis and nocturia. If this approach is to be translated clinically, a strategy is to analyze and treat the triad of micturition factors as separate parts of 1 problem. The other way could be to cope with this triad of problems simultaneously, if possible, by treating the circadian physiological rhythm itself. The discoveries reviewed point toward further investigation of the micturition rhythm by basic and translational chronobiology

Regulation of connexin 43 by basic fibroblast growth factor in the bladder: transcriptional and behavioral implications.

[Purpose]: Basic fibroblast growth factor is a candidate causative factor of detrusor overactivity in bladder outlet obstruction cases through up-regulation of the gap junction protein connexin 43. We addressed the transcriptional and behavioral implications of this axis. [Materials and Methods]: Cx43 and Cx45 mRNA expression was assessed by real-time reverse transcriptase-polymerase chain reaction in the bladder of a rat bladder outlet obstruction model and in cultured rat bladder smooth muscle cells with and without basic fibroblast growth factor treatment. Involvement of the extracellular signal regulated kinase 1/2-activator protein-1 pathway was evaluated by immunofluorescence study and a promoter-reporter assay in bladder smooth muscle cells. The effect of basic fibroblast growth factor on micturition behavior was measured in unrestrained rats under a 12-hour light/dark cycle using a controlled release system from gelatin hydrogels fixed on the bladder. The expression of extracellular signal regulated kinase 1/2 and connexin 43 protein was assessed by Western blotting of rat bladder protein. [Results]: Cx43 but not Cx45 mRNA expression was increased in the bladder of the obstruction model and in bladder smooth muscle cells treated with basic fibroblast growth factor. The mitogen-activated and extracellular signal-regulated kinase kinase inhibitor PD98059 blocked the stimulatory effect of basic fibroblast growth factor on connexin 43 protein expression and promoter activity, which was also decreased by mutation or deletion of an activator protein-1 cis-element of the connexin 43 promoter. In vivo application of basic fibroblast growth factor on the bladder increased urinary frequency during the latter half of the dark phase, ie the late active phase of rats (F=5.1, 2-way ANOVA p<0.05). The expression of phospho-extracellular signal regulated kinase 1/2 and connexin 43 protein was increased in the bladder. [Conclusions]: The extracellular signal regulated kinase 1/2-activator protein-1-connexin 43 axis could be a potential therapeutic target for increased urinary frequency