Hypothesis / aims of study
Normal urinary tract function is characterized by decreased urine production and increased stored volumes during sleep versus awake hours. However, this alters with aging potentially leading to nocturia, the nocturnal need to void, that is preceded and followed by sleep which is a consequence of circadian desynchronization. The homeostasis of plasma osmolality is maintained despite fluctuations in urine osmolality through endocrine signalling of the hypothalamic pituitary axis triggering the release of arginine vasopressin (AVP or antidiuretic hormone) from the pituitary. AVP targets vasopressin receptors (VR) located on the apical epithelial membrane of the collecting ducts in the kidney to induce transport of water and solutes. It has been previously reported that AVP enhances spontaneous contractions in rat bladders which is contrasted by the relaxing effect of desmopressin, a VR2 selective agonist [1], indicating a complex interaction of AVP-VR signalling in the urinary bladder. Therefore, we investigated the localisation and differential activities of VR in adult and aged mouse urinary bladder to better define its actions.
Study design, materials and methods
Subjects. The study utilized adult (12-24 weeks, N=5) and aged (20-28 months, N=8) female C57Bl/6 mice purchased from Jackson Laboratories. Mice were socially housed in a centralized husbandry facility and maintained on a 12-hour light/dark cycle (7am-7pm).
Metabolic cage assessments of adult and aged mice. Voiding behaviour analysis was performed using customized metabolic cages (Columbus Instruments Inc.) where the mice were maintained in a climate-controlled cabinet with the same 12-hour light/dark cycle as their normal housing facility. Food and water were provided ad libitum and their consumption recorded. Data were acquired through associated OxyMax (Columbus Instruments Inc.) and LabChart (AD Instruments) software for up to 48 hours.
Immunofluorescence. Paraffin embedded bladder sections from adult and aged female mice were processed for immunofluorescent detection of pre-proAVP (phosphoSolutions LLC), VR1b (Alomone Labs) and VR2 (Abcam) using standard methods. Fluorescent labelling was imaged and recorded using both BX63 widefield and FV3000 confocal microscopes (Olympus).
Western blot. Protein lysates from isolated bladder mucosa and detrusor layers of adult female mice were analysed by standard western blot technique for expression of pre-proAVP and VR2. Protein bands were normalized to mouse beta-actin before relative expression of mucosa versus detrusor was performed.
RT-PCR. Female adult mice were humanely sacrifice and bladder and pituitary gland tissue samples were collected. Tissues were processed to obtain mRNA that were analysed for AVP expression using probe-based RT-PCR protocol (Bio-Rad PrimePCR® probe assays) normalised to 18s ribosome expression.
Data and statistical analysis. Data are expressed as mean ± standard error of mean. Pairwise comparisons were performed using Student’s t-test where the null hypothesis was rejected at p<0.05.
Interpretation of results
The molecular data support the existence of a non-neuronal source of AVP in the bladder urothelium which are localised adjacent to cells expressing their cognate receptors. The expression of AVP and VR appear to alter with advanced age and correlate with changes in voiding behaviour, namely increased voided volumes and greater urine output. How the bladder contributes to this alteration still requires further investigation.