Purpose . The circadian clock is a molecular oscillator system consisting of a group of genes formulating transcription-translation feedback loops, and exists in most body cells and organs. Involvement of the circadian clock is reported in the normal and pathological physiology of various organs, including brain, heart, liver, adrenal grand, and others. However, it is poorly investigated in the urinary bladder. We aimed to investigate the presence of functional circadian clock in the bladder, and to demonstrate its contribution to the diurnal control of micturition rhythm, associated with genetic oscillation of Connexin43 (Cx43), a gap junction protein in the bladder.
Methods . 1. Using a novel method for automatic recording of mouse micturition named automated Voided Stain On Paper (aVSOP) method, micturition of Cx43 heterozygote knock-out mice was compared with that of littermates. 2. Diurnal micturition rhythm in wild-type mice and Cry-null mice, having a dysfunctional biological clock, was measured in 12-hour light/dark and constant dark conditions by aVSOP method, and circadian expressions of Cx43 and clock genes in the bladder were assessed in mRNA and protein levels. 3. Using ex-vivo bladder slice culture of Per2-luciferase knock-in mice, an internal oscillation of the expression of Per2, a clock gene, was recorded. An internal oscillation of Cx43 expression and gap junction function were assessed in cultured rat bladder smooth muscle cells (BSMC), whose circadian clock was synchronized by serum-shock, a method for evaluating clock gene oscillations in-vitro. 5. A transcriptional regulatory link between the circadian clock and Cx43 was investigated by promoter-reporter assay and chromatin-immunoprecipitation assay.
Results . Urine volume voided per micturition in heterozygote Cx43 mice was significantly larger than that in littermates (p<0.05 by two-way ANOVA), indicating that Cx43 is a negative regulator of functional bladder capacity. Bladder Cx43 levels and functional bladder capacity showed circadian oscillations in wild-type mice, but such rhythms were completely lost in Cry-null mice. Both bladder smooth muscle ex-vivo and BSMC in-vitro had internal genetic oscillation rhythm, associated with oscillations of Cx43 and gap junction function represented by dye-transfer experiments. A clock regulator, Rev-erba, upregulated Cx43 transcription as a co-factor of a transcription factor Sp1, using Sp1 cis-elements of the promoter.
Conclusion . Circadian genetic oscillation of Cx43 is associated with the biological clock and generates circadian rhythm of gap junction function. This may contribute to diurnal changes in functional bladder capacity peaking at resting phase, i.e. light phase for rodents, avoiding disturbance of sleep by micturition. These findings highlight a new insight into pathophysiology of nocturnal enuresis and nocturia, characterized by impairment of the diurnal micturition rhythm.