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 ClockΔ19/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, ClockΔ19/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.


Results
The circadian rhythm of [Ca 2+ ] i in MPCUCs. We examined the time-dependent changes in stretchevoked [Ca 2+ ] i in wild-type (WT) and Clock Δ19/Δ19 mice.
In both groups, the cell in the post-stretch conditions after Fura2 loading were different compared with cells in the pre-stretch conditions. A few cells in the field of view were observed to be removed from the silicon chamber after stretch stimulation, and the Fura2 ratios showed large changes around these removed cells (Fig. 1A,B). In WT mice, the largest increase in the Fura2 ratio occurred around 12 h at post-stretch (Fig. 1A, right panels) and this was confirmed by the traces of the Fura2 ratio changes shown in Fig. 2A. However, in the Clock Δ19/Δ19 mice, the time-dependent changes at post-stretch were lower or absent from 0 h to 20 h compared with WT mice (Fig. 1B, right panels). In the traces of the Fura2 ratio changes, the tendency of no change was observed and the peak values were almost constant from 0 h to 20 h, at around 2.5 (Fig. 2B). There was also a difference in the decreasing pattern after stretch stimulation. The increasing Fura2 ratio returned to the baseline in some cells, and other cells maintained higher level after stretch stimulation during measurement for 5 min (Fig. 2).
The time-dependent change in the average stretch-evoked [Ca 2+ ] i from 0 h to 20 h showed a circadian rhythm in the WT mice. The peak time was observed at 12 h, which corresponded to the beginning of the active phase, and the nadir was at 0 h, which corresponded to the beginning of the sleep phase (Fig. 3, left). Clock Δ19/Δ19 mice also showed significant time-dependent changes in the average stretch-evoked [Ca 2+ ] i (Fig. 3, right). However, this pattern was not the circadian rhythm observed in WT mice. There were no conspicuous differences in the situations of cultured cells such as size and detachment after stretching in conditions (a), (b), and (c) (upper left panels in Fig. 4A-C), compared with condition (N). However, the Fura2 ratio images and the trace charts in conditions (a), (b), and (c) showed that the prominent [Ca 2+ ] i at post-stretch at 12 h, which was observed in condition (N), had disappeared and the levels remained constant between 0 h and 12 h (upper right and lower panels in Fig. 4A-C).

Discussion
We previously confirmed the circadian rhythm of clock genes, Piezo1 and TRPV4, which was regulated by clock genes in MPCUCs 9 . We suggested that one of the contributing factors of nocturia in Clock Δ19/Δ19 mice was loss of circadian expression pattern of mechano-sensors, which send signals of bladder fullness 5,7,8 . The present study demonstrated that the sensation of bladder fullness (SBF), which is caused by [Ca 2+ ] i , might have a circadian rhythm in MPCUCs. In WT mice, SBF becomes sensitive in the active phase and dose not in the sleep phase. Meanwhile, abnormalities in clock genes cause loss of the circadian SBF. Thus, abnormality of the clock genes causes loss of circadian SBF, which induces nocturia 3,9 . In the Ca 2+ imaging experiments, a few cells were observed to be removed from the silicon chamber at post-stretch (live images in Figs 1 and 4). These removed cells caused an increased Fura2 ratio change in the cells close to the removed cells. This was possibly because the removed cells stimulate neighboring cells directly, or emitted a substance from that stimulated them indirectly. Epithelial cells, including those in the gut, trachea, and urinary tract, undergo prompt turnover, and are removed from tissues by means of apoptosis and necrosis 12,13 . We hypothesized that intracellular substances were released into extracellular region after cell death induced by removal, which might activate neighboring cells 14 . Indeed, the bladder urothelium under the condition of bacterial cystitis is activated directly by the inflammation, and indirectly by apoptosis-mediated-inflammation, resulting in irritative bladder symptoms 15,16 .
Three criteria in the analysis were applied to exclude the variants and obtain even conditions among the cells. Furthermore, the stretch distance and speed were fixed as 100 μm and 100 μm/s, respectively, in all experiments. Because the change in the field of view of the microscope during the experiments varied too greatly to detect candidate cells for analysis when the stretch distance was longer than 100 μm. A stretch speed faster than 100 μm/s also caused increased variation in circadian change detection by the other stimulations, such as removing cultured cells and unevenly processing in stretch transmission. Using these criteria, the stretch-evoked [Ca 2+ ] i showed a clear circadian rhythm in WT mice. The time-dependent changes observed in Clock Δ19/Δ19 mice also showed statistical differences. However, this was not the typical circadian rhythm as observed in WT mice (Fig. 3). Furthermore, these rhythms were consistent with the mRNA and protein expression patterns of mechano-sensors in MPCUCs, as reported previously 10 . The mRNA expression rhythm of Piezo1 in Clock Δ19/Δ19 mice was apparently different from the circadian mRNA expression pattern in WT mice. However, a statistical analysis showed significant differences in time-dependent changes of Piezo1 mRNA expression pattern in both WT and Clock Δ19/Δ19 mice. Interestingly, this tendency was confirmed by the stretch response results (Fig. 3). In this study, we applied a single stretch stimulation. However, the response of MPCUCs seemed to vary according to the stretch distance. Miyamoto et al. mentioned that a weak stretch, such as 100 μm, was sensed by Piezo1, whereas a stronger stretch over 100 μm was sensed by TRPV4 7 . In addition, this difference might be involved in the sensitivity of their mechano-sensor functions. Piezo1 can sense the stretch stimulation directly 17 , whereas TRPV4 needs to form a molecular complex by binding to the actin cytoskeletal structure and a cell junction to be activated as a mechano-sensor 18 . Thus, it is possible that that circadian rhythm of stretch-evoked [Ca 2+ ] i associated with a 100 μm-stretch might be driven mainly by the circadian expression of Piezo1. In contrast, because the voiding behavior is altered in different conditions, such as cold stress 19 , and osmotic stimuli showed different reactions for stretch in rodents 7 , the circadian rhythm of TRPV4 might contribute to another circadian function in the bladder, such as diurnal change of urine sensation accompanying temperature variation 20 , blood flow to regulate vasodilation 21 , and barrier homeostasis from external stimuli, urine osmotic, or pH change in the dark and light cycle 22,23 .
The way of stretch stimulation may influence the result. Some cells were recovered to the initial level after increasing, which were considered to be normal, however some remained high (Fig. 2). We analyzed the duration of Fura2 ratio increasing to see whether these factors could show circadian rhythm since rhythmic Ca 2+ dynamics was reported to play an important role that determined not only daily activity rhythm but also the regular circadian clock function in mice 24 . However, we could not see any differences in time-dependent change. For another reason of difference in Fura2 ratio recovery pattern, the normalizing in each cell culture might be insufficient. We believed that cell condition was as close to a certain state as possible, however, there were many factors that were difficult to normalize such as cell overlap and degree of adhesion. These might influence the reaction to the stretch stimulation. By advanced experiment, it may be possible to prove the circadian rhythm of the retaining period in intracellular Ca 2+ concentration, which determine the duration of urine sensation.
The circadian stretch-evoked [Ca 2+ ] i in WT mice was abolished in Ca 2+ free BSS, and 10 μM GsMTx4, and 10 μM RR treated condition (Figs 4 and 5). The average Fura2 ratio change at 0 h and 12 h were lower than those under normal conditions (Fig. 5). These results indicated that the circadian rhythm of stretch-evoked [Ca 2+ ] i was associated with the function of Pizeo1 and TRPV4. GsMTx4 can inhibit not only Pizeo1, but also various types of stretch-activated-channels (SACs). However, TRPV4 is not inhibited by GsMTx4 7,25 . By contrast, RR is a more non-specific SAC inhibitor that can block both Piezo1 and TRPV4 7,8,26 . If RR was a specific inhibitor of TRPV4, the suggestion that TRPV4 is involved in the regulating the circadian rhythm of SBF would be more reliable. Considering the result showing the abrogation of the circadian rhythm of stretch-evoked [Ca 2+ ] i after RR administration, SACs other than TRPV4 are more likely to participate in creating the circadian rhythm of SBF. However, these results suggest that the loss of circadian rhythm in SBF is one of the factors that contribute to nocturia, and Clock Δ19/Δ19 mice might sense a constant level of SBF throughout the day. This would explain why the Clock Δ19/Δ19 mice voided with the same frequency in the dark and light and why the circadian change of bladder capacity between dark and light was not observed in Clock Δ19/Δ19 mice 3 .
It is controversial to discuss the phenomenon observed in an in vitro experiment as an in vivo phenotype. However, the diurnal change in urination pattern is affected by many factors such as functional bladder capacity and bladder smooth muscle contraction 2,27 . Urine production in the kidney is also one of the factors that (c); P < 0.01, P < 0.01, and P < 0.01, by the Mann-Whitney U-test, respectively. The numbers of analyzed cells are indicated in parenthesis below of the chart. The numbers above error-bar indicate the average of the measured values. Time 0 means 12 hours after horse serum shock (HSS). Statistical analyses were performed using Student's t-test in A and D, Welch's t-test in B, and the Mann-Whitney u-test in C. Data are presented as the means ± standard error (SE). * or # P < 0.05, **P < 0.01, n.s., not significant. determine urination behavior 4,28 . Moreover, in addition to activation in local organs, the sensitivity of the central nervous system or the external environment, such as temperature and irregular life style, also promote urination 19,29,30 . Interestingly, the influences of these factors on the circadian clock in local organs often lead to various body symptoms 1,31 . Although SBF signal that is integrated in central nervous system resulting in voiding behavior and the mechanisms between each oscillator are left undetermined, we suggest that Ca 2+ dynamics associated with gene expression rhythm is one of important contributor to determine SBF. Thus, other factors than the circadian rhythm of SBF, which may also contribute to nocturia, should be identified. Further investigations are needed to reveal the underlying mechanism of the relationship between the circadian clock in the bladder and nocturia.
For another limitation in the present study, the silicon chamber coatings were different between WT and Clock Δ19/Δ19 mice. We used Cell Tak (CORNING, Corning, NY) only in Clock Δ19/Δ19 mice because of the difficulty to maintain adhesion during stretching. This difference might alter the cellular property in stretched reaction. However, we did not compare the differences of stretch-evoked [Ca 2+ ] i level between WT and Clock Δ19/Δ19 mice directly, then we concluded circadian rhythm of stretch-evoked [Ca 2+ ] i in the WT and their loss in Clock Δ19/Δ19 mice (Fig. 3). It is not contradictory to state that SBF may have the circadian rhythm associated with the expression rhythm of Piezo1 and TRPV4 under the regulation of clock genes.
In conclusion, the circadian SBF is created by a circadian rhythm of [Ca 2+ ] i through mechano-sensors in MPCUCs, which are regulated by clock genes, are associated with sensitivity in the active phase and insensitivity in the sleep phase. In contrast, the change of circadian rhythm of [Ca 2+ ] i leads constant urination, which might be one of the factors contributing to nocturia. Thus, remodeling of abnormalities in clock genes, which regulates [Ca 2+ ] i through mechano-sensors, which might have a great potential as a new target in nocturia.

Materials and Methods
Animals. Eight to twelve-week-old male C57BL/6 WT mice and age and sex-matched C57BL/6 Clock Δ19/Δ19 were used in the following experiments. Clock Δ19/Δ19 mice have an A to T mutation in the 5′ splice site of intron 19 of the Clock gene, and an in-frame deletion of entire exon 19 (Clock Δ19/Δ19 ), which results in loss of normal transcriptional activity and show a nocturia phenotype 3 . All experiments were performed using these mice. All procedures were conducted in accordance with the "Guiding Principles in the Care and Use of Animals in the Field of Physiologic Sciences" published by the Physiologic Society of Japan. In addition, all experimental protocols were approved by the Animal Care Committee of the University of Yamanashi (Chuo, Yamanashi, Japan).
To reset and synchronize the gene expression rhythms in each cell, 50% heat inactivated horse serum (Gibco TM , Thermo Fisher Scientific, Waltham, MA) was added for 2 h (Horse Serum Shock; HSS). The cells were then maintained in Dulbecco's modified Eagle medium (DMEM) without Phenol Red (WAKO) with 1% Penicillin/Streptomycin (P/S; Gibco TM ). Experiments were performed 12 h after HSS. The time of the first experiment was defined as 0 h (12 hours after HSS). For the in vitro time course, the light period was set from 0 h to 12 h, and the dark period was set from 12 h to 20 h, according to gene expression rhythm in the mouse bladder mucosa, in which peak time was observed at zeitgeber time 12 9,10 . Mechanical stretch experiment. The mechanical stretch experiment was conducted as previously described using elastic silicon chambers and an extension device (STREX) (Supplementary Figure. 1) 7,8 . After the chambers were attached to the extension device and detection reagents or solutions were added, they were left for 15 min to avoid stimulation by the moving or adding of the reagents or solutions. All experiments were done at room temperature and the cell extension system was fixed at a stretch distance of 100 μm, and a stretch speed of 100 μm/s in all experiments.

Measurement of intracellular Ca 2+ influx ([Ca 2+ ] i ). MPCUCs were loaded with the fluorescent Ca 2+
indicator Fura-2 AM (10 μM; Life Technologies, Carlsbad, CA) with 0.04% Pluronic F-127 (Sigma-Aldrich, St. Louis, MO) for 60 min at room temperature. The cells were then washed with balanced salt solution (BSS). 10 μM GsMTx4 (Peptide Institute, INC., Osaka, Japan) and 10 μM RR (WAKO) were applied directly into the chamber. Measurement of [Ca 2+ ] i was performed for 5 min at pre-and post-stretch stimulation, using previously described methods with slight modifications 7,8 . The ratio-changes were calculated by subtracting basal values from the peak values. The analysis conditions were required to satisfy the following three criteria. 1; The basal values of the Fura2 ratio must be less than 1.5. 2; The cell-cultures must be larger than the field view of the microscope. 3; When the cells were not fixed on the silicon chamber bottom at post-stretch, the cells around them were excluded from the analysis.

Statistical analyses.
Experimental values were expressed as means ± standard error (SE). The significances of the differences between two groups were analyzed using Student's t-test, Welch's t-test, or Mann-Whitney's U-test, depending on the distribution of the samples. A one-way analysis of variation (ANOVA) was used to compare differences among the time points in each group. A P value of less than 0.05 was considered significant.