Hypothesis / aims of study
Molecular activity effected by circadian clock genes is responsible for a majority of diurnal variations observed in living organisms. Circadian rhythms are regulated by clock gene products, and in the case of mammals, these clock gene products are present in most cells and organs. The suprachiasmatic nucleus (SCN) of the brain works as a master pacemaker, and is synchronized with a peripheral clock that exists in multiple tissues throughout the whole body, including the lungs, liver, kidneys, bladder, etc. It is reported that transient receptor potential cation channel subfamily V member 4 (TRPV4), vesicular nucleotide transporter (VNUT), and Piezo1 show circadian rhythms in the bladder mucosa and that these circadian rhythms are hindered by clock gene abnormality1). However, the effect of bladder clock genes on bladder dysfunction remains unclear. In this study, we investigate the expression and circadian rhythm of TRPV1, TRPV4, VUNT, Piezo1, and clock genes in the bladders of spontaneously hypertensive rats (SHR).
Study design, materials and methods
Male Wistar rats (control group) and male SHRs (SHR group) were used in this study. The experimental animals were placed 12 h of alternating light and dark conditions. The light period began at zeitgeber time (ZT) 0 followed by the dark period at ZT 12. Upon completing 18 weeks of age, urination was evaluated using a metabolism gauge (MG). The parameters evaluated included urine volume, urination frequency, and urine volume per void, which defines bladder capacity, for 24 h, during the light and dark periods, and these parameters were compared between the two groups. After collecting MG data, bladders were harvested every 4 h at six time points (ZT 3, 7, 11, 15, 19, and 23) from the control and the SHR group (n = 6 for each time), and the gene expression of Per2, Cry2, Bmal1, Clock, Rev-erbα, TRPV1, TRPV4, VUNT, and Piezo1 was examined using qRT-PCR.
Results
Rat total body and bladder weight were significantly lower in the SHR group than in the control group. The urination frequency for 24 h, both during the light and dark periods, was significantly higher in the SHR group than in the control group. In both the groups, urinary frequency was significantly higher during the dark period than during the light period. Urine volume for 24 h and during the dark period alone was significantly lower in the SHR group than in the control group. In both the groups, urine volume during the dark period was significantly higher than that during the light period. Urine volume per void was significantly lower in the SHR group than the control group during both light and dark periods. In the control group urine volume per void was significantly lower during the dark than during the light period. However, there was no significant difference in urine volume per void between the light and dark periods in the SHR groups. In the SHR bladders, we observed significant increase in the expression of Per2 and Rev-erbα at all time points and increase in Cry2, Bmal1, and Clock expression in some but not all time points compared to the control group. In both the groups, Per2, Bmal1, and Rev-erbα expression had the circadian rhythms with peaks and nadirs, respectively, at ZT 15 and 3 for Per2, 23 and 11 for Bmal1, and 7 and 19 for Rev-erbα. Expression of TRPV1, TRPV4, VUNT, and Piezo1 were significantly higher at point ZT19 in the SHR group than in the control group.
Interpretation of results
The mRNA expression of each clock gene in the SHRs was higher than that of the control group. Circadian rhythms were observed in Per2, Bmal1, Rev-erbα, and the variation showed similarities between the control group and the SHRgroup. At time point ZT19, the expression of TRPV1, TRPV4, VUNT, and Piezo1 in Wistar rats decreased; however, the expression of these genes in SHRs remained consistent or mildly decreased. These results may correlate with the decreasing urine volume per void and increasing urination frequency during the dark period in SHR.