The pathophysiology of nocturia is multifactorial. Its etiology remains uncertain in many patients who are refractory to treatments. Further researches are necessary to determine the causes of nocturia and effective treatments for it. Previously, we reported that urination behavior in mice showed circadian rhythm, which was regulated by clock genes, and the abnormalities of clock genes might cause nocturia because of the loss of circadian bladder function. Furthermore, we also reported that the sensation of bladder fullness showed circadian rhythm through gene expression rhythm of mechanosensors such as Piezo1 and TRPV4 involving the regulation by clock genes in the mouse bladder urothelium (1).
 On the other hand, it has been reported that anxiety or post-traumatic-stress-disorder could increase nocturnal voiding. Particularly, some types of intermittent stress such as restraint stress (RS) caused disruption of the circadian rhythm only in peripheral organs without any effect on the central nervous system in mice (2). At ICS2017, we have already reported that intermittent RS induced nocturia in mice and hypothesized that abnormal circadian clock induced by RS could occur in the mice bladder and caused to nocturia.
 To reveal whether RS affects the alternation of gene expression rhythm and causes to nocturia in mice, we investigated the expression rhythm of clock genes and mechanosensors in the mice bladder mucosa after RS.

Male C57BL/6 (WT) mice and Period2luciferase knock-in mice (Per2::luc) aged 8 -12 weeks were used. Per2 is one of the clock genes and act as a negative transcriptional factor for clock-controlled-genes. Mice were bred under 12 h light/dark conditions. The light period started from 6 am [Zeitgeber time (ZT) 0)]. Mice were subjected to RS for 2 hrs from ZT4 to ZT6, the rest phase in mice, by enfolding using metal mesh sized 12cm×12cm. Five-time RS was applied to mice until the experiments begun.
 Mice bladder was excised every 4 h from ZT0 both control- and RS mice after RS. The bladder mucosa were isolated and then mRNA was extracted from them. The gene expression rhythm in clock genes and mechanosensor was measured using RT-PCR. Per2 expression in the bladder for Per2::luc mice was measured using in vivo imaging. These data were compared between control and RS mice. Data were processed using an one-way ANOVA with Bonferroni’s test. Data are presented as means ± SE. ** p < 0.01; n.s., not significant.

WT mice showed the time-dependent gene expression in clock genes and mechanosensors. However, these rhythms were disrupted or the oscillation pattern was altered in RS mice. In in vivo imaging of the mice bladder, WT mice also showed the circadian Per2 expression rhythm. However, this rhythm was altered in RS mice, although circadian Per2 expression was maintained in RS mice (Fig. 1).

We demonstrated that the circadian gene expressions in clock genes and mechanosensors were altered by intermittent RS during the rest phase, sleeping phase, in mice, which might induce nocturia through the abnormal circadian bladder function. These results indicated that exposure to stress could be one of the causes of nocturia. Further studies may provide the new therapeutic strategy for nocturia.

Abnormalities of circadian clock in the bladder induced by physical stress are one of important factors in changes of urination pattern, particularly nocturia.

Ihara, T., Mitsui, T., Nakamura, Y. et al.: The oscillation of intracellular Ca2+ influx associated with the circadian expression of Piezo1 and TRPV4 in the bladder urothelium. Sci Rep, 2017, In press.
Tahara, Y., Shiraishi, T., Kikuchi, Y. et al.: Entrainment of the mouse circadian clock by sub-acute physical and psychological stress. Sci Rep, 5: 11417, 2015