Glucocorticoids coordinate the bladder peripheral clock and diurnal micturition pattern in mice

Abstract

Peripheral clocks function to regulate each organ and are synchronized though various molecular and behavioral signals. However, signals that entrain the bladder clock remain elusive. Here, we show that glucocorticoids are a key cue for the bladder clock in vitro and in vivo. A pBmal1-dLuc human urothelial cell-line showed significant shifts in gene expression after cortisol treatment. In vivo, rhythmic bladder clock gene expression was unchanged by bilateral adrenalectomy but shifted 4 h forward by corticosterone administration at the inactive phase. Moreover, the bladder clock shifted 8-12 h in mice that underwent both bilateral adrenalectomy and corticosterone administration at the inactive phase. These mice showed decreases in the diurnal rhythm of volume voided per micturition, while maintaining diurnal activity rhythms. These results indicate that the diurnal rhythm of glucocorticoid signaling is a zeitgeber that overcomes other bladder clock entrainment factors and coordinates the diurnal rhythm of volume voided per micturition.

Fig. 1. TRT-HU1 Bmal1-Luciferase cell luminescence and effect of reagents.

a schematic diagram of the mean quantified Bmal1-Luciferase (Bmal1-Luc) relative luminescence intensities in 35 mm dishes after serum shock. b Successive changes of relative luminescence intensities of Bmal1-Luc cells by adding DDW, NA 10 μM, Carb 10 μM, ATP 10 μM, PGE2 10 μM, and DEX 0.1 μM to the medium, compared with 0.2% DMSO. One representative of two experiments with similar results is shown (DDW: distilled water, NA: noradrenaline, Carb: carbachol, ATP: adenosine triphosphate, PGE2: prostaglandin E2, DEX: dexamethasone).

Fig. 2. Bmal1-Luc cell luminescence intensities with cortisol addition.

a Successive changes of luminescence intensities of Bmal1-Luc cells by adding 1 nM, 10 nM, 25 nM, 75 nM, and 100 nM cortisol to the medium. One representative of two experiments with similar results is shown. b Successive changes of relative luminescence intensities of Bmal1-Luc cells by administrating 25 nM cortisol and glucocorticoid receptor inhibitor (GRi) at 40 h and 52 h after serum shock (Blue arrows). One representative of two experiments with similar results is shown.

Fig. 3. Effect of corticosterone administration at the non-physiological timing.

a Diurnal change in serum corticosterone levels in normal mice at our facility. Each data series represents the mean ± SEM and 4 mice per time point. b A schematic diagram of the experiment that mice were orally administered corticosterone (CORT) for 7 consecutive days at ZT 1 and sacrificed every 4 h on day 8 (ZT 4,8,12,16,20,24). c Relative mRNA accumulation of Bmal1, Per2, and Rev-erbα in the liver and bladder from mice vehicle (solid line) and CORT (dash line) at ZT 1. Each data series represents the mean ± SEM and 5 mice per time point. Differences were determined by two-way ANOVA and Sidak’s multiple comparisons test. *p < 0.01 v.s. vehicle (ZT: Zeitgeber time).

Fig. 4. Effect of bilateral adrenalectomy.

a A schematic diagram of the experiment that bilateral adrenalectomy (ADx) performed on Day 1 and sacrifice death on Day 8. b Relative mRNA accumulation of Bmal1, Per2, and Rev-erbα in the liver and bladder from sham (solid line) and ADx (dash line). Each data series represents the mean ± SEM and 5 mice per time point. Differences were determined by two-way ANOVA and Sidak’s multiple comparisons test. *p < 0.01 v.s. sham.

Fig. 5. Effect of bilateral adrenalectomy and corticosterone administration at the non-physiological timing.

a A schematic diagram of the bilateral adrenalectomy (ADx) performed on Day 1 and corticosterone administered orally for 7 days at ZT 1 from Day 8 after ADx and sacrifice on Day 15 (ADx + CORT). b Relative mRNA accumulation of Bmal1, Per2, and Rev-erbα in the liver and bladder from ADx + vehicle (solid line) and ADx + CORT (dash line). Each data series represents the mean ± SEM and 5 mice per time point. Differences were determined by two-way ANOVA and Sidak’s multiple comparisons test. *p < 0.01 v.s. ADx + vehicle. c Temporal mRNA accumulation of clock genes in the bladder from ADx + vehicle (solid line) and ADx + CORT (dash line) by analyzing RNA-seq. Each data series represents the 3 mice per time point.

Fig. 6. The diurnal rhythm of micturition.

a Volume voided per micturition per 8-h averages measured on 3 consecutive days in ADx + vehicle (solid black line) and ADx + CORT (dash red line). Each data series represents the mean ± SEM and 5 mice per time point. b Average of 3 days results of measured volume voided per micturition average per 8-h in ADx + vehicle (solid black line) and ADx + CORT (dash red line). Each data series represents the ratio of ZT14, 22 to ZT6 as 1 in the same and the mean ± SEM and 5 mice per time point. Differences were determined by two-way ANOVA and Sidak’s multiple comparisons test. ^#p < 0.01 v.s. ADx + vehicle and Dunnett’s multiple comparisons test. *p < 0.01 v.s. ZT 14 in ADx + vehicle and ADx + CORT. c Total voided volume per h per 8-h averages measured on 3 consecutive days in ADx + vehicle (solid line) and ADx + CORT (dash red line). Each data series represents the mean ± SEM and 5 mice per time point.

Fig. 7. Global transcriptome analysis using RNA-seq in ADx + vehicle and ADx + CORT mice.

a PCA plots with N = 3 at ZT 4, 8, 12, 16, 20, and 24 in the ADx + vehicle and ADx + CORT groups, respectively. b Pathway analysis was performed using Metascape for the diurnal rhythmicity genes (defined as greater than the MaxCorr of 0.85 from the cosine curve with a 1.5-fold amplitude of expression level) in the ADx + vehicle group.

Fig. 8. A schematic diagram of this study.

The present study suggests that glucocorticoid secreted by the adrenal cortex acts on the glucocorticoid receptor in the bladder to be an entrainer of the bladder clock and that a mismatch between the central and the peripheral bladder clocks decrease the diurnal rhythm of volume voided per micturition. (GC: glucocorticoid, GR: glucocorticoid receptor).