Effects of fatty acid metabolites on nocturia.

Dysregulation of circadian rhythm can cause nocturia. Levels of fatty acid metabolites, such as palmitoylethanolamide (PEA), 9-hydroxy-10E,12Z-octadecadienoic acid (9-HODE), and 4-hydroxy-5E,7Z,10Z,13Z,16Z,19Z-docosahexaenoic acid (4-HDoHE), are higher in the serum of patients with nocturia; however, the reason remains unknown. Here, we investigated the circadian rhythm of fatty acid metabolites and their effect on voiding in mice. WT and Clock mutant (ClockΔ19/Δ19) mice, a model for nocturia with circadian rhythm disorder, were used. Levels of serum PEA, 9-HODE, and 4-HDoHEl were measured every 8 h using LC/MS. Voiding pattern was recorded using metabolic cages after administration of PEA, 9-HODE, and 4-HDoHE to WT mice. Levels of serum PEA and 9-HODE fluctuated with circadian rhythm in WT mice, which were lower during the light phase. In contrast, circadian PEA and 9-HODE level deteriorated or retreated in ClockΔ19/Δ19 mice. Levels of serum PEA, 9-HODE, and 4-HDoHE were higher in ClockΔ19/Δ19 than in WT mice. Voiding frequency increased in PEA- and 4-HDoHE-administered mice. Bladder capacity decreased in PEA-administered mice. The changes of these bladder functions in mice were similar to those in elderly humans with nocturia. These findings highlighted the novel effect of lipids on the pathology of nocturia. These may be used for development of biomarkers and better therapies for nocturia.

Urinary metabolites identified using metabolomic analysis as potential biomarkers of nocturia in elderly men.

PURPOSE: To investigate the association between nocturia and urinary metabolites in elderly men using metabolomic analysis. METHODS: We recruited 66 men aged 65-80 years. The 3-day frequency volume chart (FCV), International Prostate Symptom Score (IPSS), and quality of life score were used to assess micturition behavior. Participants with the total IPSS > 0 and ≥ 1.5 micturition on an average for three nights were included in the nocturia group. Participants with the total IPSS < 8 and < 1.5 micturition at night were included in the control group. We conducted a comprehensive metabolomic analysis of urine samples. Metabolites were compared between the groups using an unpaired t test. A multivariable logistic regression analysis was used to determine the relationship between nocturia and these metabolites. RESULTS: The nocturia and control groups consisted of 45 and 21 men, respectively. There were no differences in the background factors between the groups except for receiving anticholinergic drug and having life style-related diseases. The FVC revealed that nocturnal urine volume, 24 h micturition frequency, and nocturnal micturition frequency were significantly higher in the nocturia group than in the control group. The metabolomic analysis revealed 16 metabolites, which were differentially expressed between the groups. The multivariate analysis showed that increased serotonin level and decreased 3-hydroxypropionic acid and 3-indoleacetonitrile levels were associated with nocturia. CONCLUSIONS: These findings suggest that abnormal urinary metabolites including serotonin, 3-hydroxypropionic acid, and 3-indoleacetonitrile are involved in the pathogenesis of nocturia in elderly men.

Intermittent restraint stress induces circadian misalignment in the mouse bladder, leading to nocturia.

Intermittent stress disrupts the circadian rhythm in clock genes such as Per2 only in peripheral organs without any effect on the central circadian clock in the suprachiasmatic nucleus. Here, the effect of restraint stress (RS) on circadian bladder function was investigated based on urination behavior and gene expression rhythms. Furthermore, PF670462 (PF), a Per2 phosphorylation enzyme inhibitor, was administered to investigate the effects on circadian bladder re-alignment after RS. Two-hour RS during the light (sleep) phase was applied to mice (RS mice) for 5 days. The following parameters were then examined: urination behaviors; clock gene expression rhythms and urinary sensory-related molecules such as piezo type mechanosensitive ion channel component 1 (Piezo1), transient receptor potential cation channel subfamily V member 4 (TRPV4), and Connexin26 (Cx26) in the bladder mucosa; Per2 expression in the excised bladder of Per2luciferase knock-in mice (Per2::luc); in vivo Per2 expression rhythms in the bladder of Per2::luc mice. Control mice did not show altered urination behavior in the light phase, whereas RS mice exhibited a higher voiding frequency and lower bladder capacity. In the bladder mucosa, RS mice also showed abrogated or misaligned Piezo1, TRPV4, Connexin26, and clock gene expression. The rhythmic expression of Per2 was also altered in RS mice both in excised- and in vivo bladder, compared with control mice. After PF administration, voiding frequency was reduced and bladder capacity was increased during the light phase in RS mice; the in vivo Per2 expression rhythm was also fully restored. Therefore, RS can alter circadian gene expression in the bladder during the light phase and might cause nocturia via changes in circadian bladder function due the dysregulation of clock genes. Amending the circadian rhythm therapeutically could be applied for nocturia.

The oscillation of intracellular Ca2+ influx associated with the circadian expression of Piezo1 and TRPV4 in the bladder urothelium.

We previously showed that bladder functions are controlled by clock genes with circadian rhythm. The sensation of bladder fullness (SBF) is sensed by mechano-sensor such as Piezo1 and TRPV4 in the mouse bladder urothelium. However, functional circadian rhythms of such mechano-sensors remain unknown. To investigate functional circadian changes of these mechano-sensors, we measured circadian changes in stretch-evoked intracellular Ca2+ influx ([Ca2+] i ) using mouse primary cultured urothelial cells (MPCUCs). Using Ca2+ imaging, stretch-evoked [Ca2+] i was quantified every 4 h in MPCUCs derived from wild-type (WT) and Clock Δ19/Δ19 mice, which showed a nocturia phenotype. Furthermore, a Piezo1 inhibitor GsMTx4 and a TRPV4 inhibitor Ruthenium Red were applied and stretch-evoked [Ca2+] i in MPCUCs was measured to investigate their contribution to SBF. Stretch-evoked [Ca2+] i showed a circadian rhythm in the WT mice. In contrast, Clock Δ19/Δ19 mice showed disrupted circadian rhythm. The administration of both GsMTx4 and Ruthenium Red eliminated the circadian rhythm of stretch-evoked [Ca2+] i in WT mice. We conclude that SBF may have a circadian rhythm, which is created by functional circadian changes of Piezo1 and TRPV4 being controlled by clock genes to be active during wakefulness and inactive during sleep. Abnormalities of clock genes disrupt SBF, and induce nocturia.

Clock Genes Regulate the Circadian Expression of Piezo1, TRPV4, Connexin26, and VNUT in an Ex Vivo Mouse Bladder Mucosa.

OBJECTIVES: ClockΔ19/Δ19 mice is an experimental model mouse for nocturia (NOC). Using the bladder mucosa obtained from ClockΔ19/Δ19 mice, we investigated the gene expression rhythms of mechanosensory cation channels such as transient receptor potential cation channel subfamily V member 4 (TRPV4) and Piezo1, and main ATP release pathways including vesicular nucleotide transporter (VNUT) and Connexin26(Cx26), in addition to clock genes. MATERIALS AND METHODS: Eight- to twelve-week-old male C57BL/6 mice (WT) and age- and sex-matched C57BL/6 ClockΔ19/Δ19 mice, which were bred under 12-h light/dark conditions for 2 weeks, were used. Gene expression rhythms and transcriptional regulation mechanisms in clock genes, mechanosensor, Cx26 and VNUT were measured in the mouse bladder mucosa, collected every 4 hours from WT and ClockΔ19/Δ19 mice using quantitative RT-PCR, a Western blot analysis, and ChIP assays. RESULTS: WT mice showed circadian rhythms in clock genes as well as mechanosensor, Cx26 and VNUT. Their expression was low during the sleep phase. The results of ChIP assays showed Clock protein binding to the promotor regions and the transcriptional regulation of mechanosensor, Cx26 and VNUT. In contrast, all of these circadian expressions were disrupted in ClockΔ19/Δ19 mice. The gene expression of mechanosensor, Cx26 and VNUT was maintained at a higher level in spite of the sleep phase. CONCLUSIONS: Mechanosensor, Cx26 and VNUT expressed with circadian rhythm in the mouse bladder mucosa. The disruption of circadian rhythms in these genes, induced by the abnormalities in clock genes, may be factors contributing to NOC because of hypersensitivity to bladder wall extension.

Bladder oxidative stress in sleep apnea contributes to detrusor instability and nocturia.

PURPOSE: Obstructive sleep apnea is associated with voiding symptoms in humans and animals, and yet its effects on the urinary tract are poorly understood. We examined bladder structure and function, markers of oxidative damage and the redox survival pathway in a rat model of obstructive sleep apnea to identify changes. MATERIALS AND METHODS: To model obstructive sleep apnea we used a rat oxycycler system to create cyclical interruption in breathing oxygen, thereby producing intermittent hypoxemia. Male Sprague Dawley® rats were divided into an obstructive sleep apnea, a sham treated and a control group of 8 each. After 8-week exposure to obstructive sleep apnea conditions we assessed daytime and nighttime rat voiding behavior in metabolic cages. Cystometrograms were done and bladder tissue was processed for biochemical assays, enzyme-linked immunosorbent assay and transmission electron microscopy. RESULTS: Increased urinary frequency and total urine output developed in rats exposed to obstructive sleep apnea conditions. Cystometric changes included detrusor instability, bladder noncompliance and increased spontaneous contractions. These changes were associated with bladder oxidative stress characterized by significant increases in tissue levels of malondialdehyde and advanced oxidation protein products. Obstructive sleep apnea activated cell survival signaling manifested by increased expression of PI3K and phosphorylated Akt1. Transmission electron microscopy revealed marked ultrastructural damage to subcellular elements. CONCLUSIONS: Intermittent hypoxia in obstructive sleep apnea causes oxidative stress with ultrastructural and functional changes in the bladder. Sleep apnea related nocturia/voiding symptoms could be the result of these direct changes. Untreated sleep apnea has significant health consequences. Identifying urinary oxidative stress products in patients with nocturia may be useful as an economical noninvasive biomarker to identify undiagnosed obstructive sleep apnea.

Prediction of nocturia severity in men: nocturnal urine overproduction vs race or metabolic risk factors.

IMPORTANCE: Nocturia is one of the most common and bothersome of lower urinary tract symptoms. OBJECTIVE: To examine the effect of race and metabolic risk factors on nocturia severity in men as measured by the number of nightly voids. DESIGN, SETTING, AND PARTICIPANTS: A retrospective review from 2011 to 2013 was performed at a Veterans Affairs-based urology clinic in Brooklyn, New York, among 104 adult men 18 years or older who completed a 24-hour frequency and volume chart. Metabolic risk factors included race and a history of diabetes mellitus, hypertension, and obstructive sleep apnea. The 24-hour frequency and volume chart data included the nocturia index (nocturnal urine volume divided by maximal voided volume), the nocturnal polyuria index (nocturnal urine volume divided by 24-hour volume), and nocturnal urine production (nocturnal urine volume per hours slept). A nocturia index of less than 2 vs 2 or higher, a nocturnal polyuria index of less than 33% vs 33% or higher, and nocturnal urine production of less than 90 vs 90 mL/h or higher were chosen as clinically relevant cutoff points for nocturia severity. Nocturia severity was compared by race, the aforementioned variables, and the presence or absence of diabetes mellitus, hypertension, and obstructive sleep apnea. MAIN OUTCOMES AND MEASURES: The number of nightly voids. RESULTS: One hundred four adult men (mean age, 64 years; age range, 24-92 years) completed a 24-hour frequency and volume chart (mean number of nightly voids, 2.93; range, 0-15). The number of nightly voids was not statistically different for white vs black race (3.00 vs 2.93, P = .86) or for the presence vs the absence of diabetes mellitus (3.00 vs 2.88, P = .85), hypertension (2.94 vs 2.80, P = .75), and obstructive sleep apnea (3.29 vs 2.83, P = .50). However, nocturia severity was significantly different based on a nocturia index of less than 2 vs 2 or higher (1.39 vs 3.60), a nocturnal polyuria index of less than 33% vs 33% or higher (1.83 vs 3.65), and nocturnal urine production of less than 90 vs 90 mL/h or higher (2.27 vs 3.77) (P < .001 for all). CONCLUSIONS AND RELEVANCE: Neither race nor metabolic risk factors affect nocturia severity. In contrast, variables that denote nocturnal urine overproduction sharply discriminate the risk of nocturia severity and suggest that variable data may provide useful clinical correlation.

[Nocturia and the circadian rhythm].

Nocturia, undesired micturition at night, is present in 60% to 90% of individuals older than 60 years and causes significant deterioration in quality of life. Prevention of micturition at night is regulated by a triad of factors including decreased arousal in the brain, decreased urine production rate in the kidneys and increased functional bladder capacity. There is increasing evidence that the triad of micturition is modulated by the circadian clock system. Risk factors of nocturia such as hypertension, cardiovascular diseases, diabetes mellitus, cerebrovascular accidents and depression have also been associated with dysfunction of the circadian clock system. The etiology of nocturia might be elucidated by investigating it as dysfunction of peripheral or central circadian clock.

Involvement of urinary bladder Connexin43 and the circadian clock in coordination of diurnal micturition rhythm.

Nocturnal enuresis in children and nocturia in the elderly are two highly prevalent clinical conditions characterized by a mismatch between urine production rate in the kidneys and storage in the urinary bladder during the sleep phase. Here we demonstrate, using a novel method for automated recording of mouse micturition, that connexin43, a bladder gap junction protein, is a negative regulator of functional bladder capacity. Bladder connexin43 levels and functional capacity show circadian oscillations in wild-type mice, but such rhythms are completely lost in Cry-null mice having a dysfunctional biological clock. Bladder muscle cells have an internal clock, and show oscillations of connexin43 and gap junction function. A clock regulator, Rev-erbα, upregulates connexin43 transcription as a cofactor of Sp1, using Sp1 cis-elements of the promoter. Therefore, circadian oscillation of connexin43 is associated with the biological clock and contributes to diurnal changes in bladder capacity, which avoids disturbance of sleep by micturition.

The effect of genital and lower urinary tract symptoms on steroid receptor expression in women with genital prolapse.

INTRODUCTION AND HYPOTHESIS: This study evaluates the expression of estrogen receptor (ER) isoforms alpha (α) and beta (ß) and progesterone receptor (PR) in vaginal and periurethral tissue in women with genital prolapse in relation to genital and lower urinary tract symptoms (LUTS). METHODS: Forty-seven postmenopausal women without systemic estrogen therapy underwent pelvic organ prolapse quantification and urodynamic assessment. LUTS were evaluated by CATI questionnaire. Biopsies from vaginal and periurethral tissue were obtained during prolapse surgery. The steroid receptor gene expression was measured by RT-PCR. RESULTS: The expression of PR in periurethral and ER ß in vaginal tissue varied with prolapse extent. Nulliparous women showed a significantly higher expression of PR in periurethral tissue. Women with a positive stress test and those with overactive bladder symptoms showed a significantly lower amount of PR in vaginal tissue. CONCLUSION: Changes in PR expression in vaginal or periurethral tissue might be a marker of structural and endocrine changes.

Biochemical and body composition analysis of nocturia in the elderly.

AIMS: Nocturia is a common symptom in the elderly, and various contributing factors have been suggested. Therefore, in order to investigate which factors are strongly related to occurrence of nocturia, we performed a suite of examinations. METHODS: One hundred eighty volunteers were divided into three groups: a young adult control group (60 healthy persons without nocturia), an elderly control group (60 healthy persons with a low mean frequency of nocturnal urination, i.e., twice per night) in the elderly.