Hypothesis / aims of study
A variety of organisms have many physiological and behavioral processes with rhythmicity of approximately 24 hours, and this is called circadian rhythm. The central master clock located at the suprachiasmatic nucleus in mammals generates this rhythm through mechanisms of transcription-translation feedback loops. In addition to the circadian rhythm of whole body controlled by the central clock, each organ has its own functional circadian rhythm modulated by the peripheral clock. Several reports have shown that peripheral clocks are important for the circadian rhythm of each organ function (ref. 1), and the bladder is considered as well (ref. 2). However, the physiological cues to control the peripheral clock in the bladder has not been revealed yet.
Study design, materials and methods
1) In vitro experiments. For the screening of physiological signals to modulate the peripheral clock in the urothelium, we created the hTERT-immortalized human urothelial cells stably expressing pBmal1-dLuc (Bmal1-Luc) by transfecting the lentiviral vector carrying it. After synchronization of the clock by serum shock, bioluminescence was continuously monitored for 3-5 days with administration of noradrenaline (10 μM), carbachol (10 μM), ATP (10 μM), and prostaglandin E2 (10 μM) and dexamethasone (0.1μM).
2) In vivo experiments. This study used 8-week-old male C57BL/6 mice. All mice were housed under a 14-hour light/10-hour dark (L/D) cycle condition [light-on at 0500 a.m. and Zeitgeber time (ZT) at 0]. To investigate the influence of corticosterone on the bladder clock and micturition behavior in mice, we created three models and their controls: a) bilateral adrenalectomy (ADx) or the sham operation b) corticosterone (0.2 mg/body) or the vehicle (methylcellulose) administration orally at a non-physiological timing of ZT1 (CORT), and c) ADx with oral CORT administration at ZT1 (ADx+CORT) or the vehicle. Both ADx and sham-operated mice were allowed to recover for 1 week postoperatively. All mice had free access to 1% NaCl solution after the operation. Bladders were extracted and genes expressions were evaluated by real-time qPCR. RNA sequences for the bladder samples were performed in the experiment c). The micturition behavior was measured using aVSOP method using a filter paper. The averages of 8 hours (4 hours before and after) were graphed starting from the beginning of the dark period. The amount of volume voided per micturition was averaged the amount of volume voided per micturition in the elapsed time. The amount of urine volume per hour was calculated by dividing the amount of total voided volume by the elapsed time.
Results
1) In vitro experiments. After administration of noradrenaline, carbachol, ATP, and prostaglandin E2, the rhythm and amplitude of luminescence did not change, but only dexamethasone significantly decreased the amplitude, shifted the peak forward 10 hrs, and reduced the period from 24 hrs to 22 hrs. The amplitude of luminescence decreased after the administration of above 25 nM of cortisol, a physiological glucocorticoid (GC) in humans. The cortisol administration at the physiological timing after serum shock, a compatible to the time that the blood cortisol level in humans elevated, increased the amplitude but did not influence on the phase of the luminescence rhythm. However, the cortisol administration at non-physiological timing reversed the phase of the luminescence rhythm. These changes in phase and amplitude induced by the cortisol administration were inhibited when a mifepristone 10 nM, a glucocorticoid receptor inhibitor, was administered simultaneously with cortisol.
2) In vivo experiments. The ADx mice had little change on clock genes’ expressions in the bladder compared with the sham mice, but the CORT mice and the ADX+CORT mice had significant alterations on the diurnal rhythm of clock genes’ expressions compared with the controls. The peaks of Bmal1 and Rev-erbα shifted forward 4 hrs in the CORT mice and, of note, 8-12 hrs in the ADx+CORT mice (Fig. 1). Moreover, the RNA-seq revealed that the peaks of most of the clock genes shifted forward 8 to 12 hrs in the ADx+CORT mice. As for micturition behavior analysis, all model mice except the ADx+CORT maintained the diurnal rhythm of volume voided per micturition with decreased during the active period and increased during the inactive period; however, the ADx+CORT disrupted the diurnal rhythm of volume voided per micturition (Fig. 2).
Interpretation of results
From the in vitro experiments, the GCs could be selected as a candidate physiological signal for the control of the peripheral bladder clock. From the in vivo experiments, the findings of the inversed rhythm of clock genes in the bladder of the ADx+CORT mice suggests that the peripheral bladder clock can be mainly controlled by the diurnal rhythm of cortisol level. The clock genes in the bladder of CORT mice, a model of non-physiological timing administration of corticosterone, shifted several hours forward only and kept their rhythms suggests that the maintained physiological peak of the corticosterone could attenuate the shift. The disrupted the diurnal rhythm of volume voided per micturition accompanied with the inversed rhythm of the bladder peripheral clock in ADx+CORT mice indicates that the synchronized rhythm of the central clock and the bladder peripheral clock is important for creating the diurnal rhythm of micturition.