The circadian rhythm system regulates various physiological processes of internal organs through clock genes (positive regulators Clock/Bmal1 and negative regulators Per2/Cry2)，including urinary storage and voiding functions. Mammals urinate less frequently during sleep than during wakefulness, which is closely associated with increased in functional bladder capacity during sleep. However, the mechanism underlying the circadian rhythm regulation of bladder function remains unclear. The BDNF-TrkB-MAPK signaling pathway has been shown to play an important role in bladder function regulation, but its interaction with clock genes and its role in the circadian rhythm are not fully understood. The aim of this study was to reveal the role of BDNF-TrkB-MAPK pathway in the circadian clock gene-guarded rhythmic bladder functions, and to explore its potential association with the pathogenesis of nocturia.

Twelve-week C57 and age-matched CRY1-/-CRY2-/- female mice were reared in 12 hours of light /12 hours of darkness environment, and were switched to constant darkness environment as needed. Voiding behavior was continuously recorded for 72 hours using a non-invasive automatic quantitative voiding monitor. Urine spot distribution and volume scatter plots were generated. Bladder detrusor muscle tissues were collected every 4 hours, and qRT-PCR and Western blot techniques were used to detect the rhythmic expression of biological clock genes (Bmal1, Clock, Per2, Cry2) and BDNF-TrkB-MAPK signaling pathway molecules in the bladder. Cosine fitting analysis was performed to assess their phase and amplitude characteristics.

In wild type mice,  urinary frequency showed a periodic variation of lower daytime and higher nighttime activity.  This rhythm remained stable even under constant darkness. The clock genes Bmal1 and Clock mRNA and protein expression peaking during the active nighttime phase.  Conversely, Cry2 and Per2 expression was higher during the inactive phase.  In addition, the mRNA and protein level of BDNF, TrkB and MAPK showed  a decrease during the day and an increase at night.  However, in CRY1-/-CRY2-/- mice, the circadian rhythm of clock genes was completely abolished, and the bladder urination circadian rhythm was significantly disrupted, with loss of circadian expression of the BDNF-TrkB-MAPK pathway molecules.

(1) In wild-type mice, bladder function follows a circadian rhythm with higher urinary frequency during the night and lower during the day. This pattern persists an endogenous circadian regulation rather than environmental influences.
(2) The clock genes Bmal1 and Clock mRNA and protein expression peaking during the active nighttime phase, synchronized with increased bladder contraction excitability at night. Conversely, Cry2 and Per2 expression was higher during the inactive phase, correlating with increased bladder capacity.
(3) The expression of BDNF, TrkB, and MAPK also follows a rhythmic fluctuation, which aligns with the rhythmic excitability of the bladder, suggesting these molecules are involved in the circadian regulation of bladder function.
(4) In CRY1-/-CRY2-/- mice, which lack functional CRY1 and CRY2 proteins,  the circadian rhythm of bladder urination is disrupted, and the rhythmic expression of the BDNF-TrkB-MAPK pathway is lost. This suggests that clock gene and  BDNF-TrkB-MAPK pathway are essential for maintaining the circadian regulation of bladder function.

The rhythmic voiding behavioral pattern is regulated by a set of peripheral clock genes. The BDNF-TrkB-MAPK signaling pathway plays a critical role in regulating bladder excitability which is closely associated with the oscillatory expression of clock genes. Nocturia may occur, due to the disruption of circadian clock genes and BDNF-TrkB-MAPK pathway molecules, potentially contributing to increased bladder excitability and deceased functional capacity during inactive sleep period. Restoration of circadian rhythm disturbances could be a potential target for nocturia, but further research is needed to define the regulatory mechanisms between clock genes and BDNF-TrkB-MAPK pathway.

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