Hypothesis / aims of study
Advanced age is associated with increased incidence of nocturia, the nocturnal need to void that is preceded and followed by sleep. The pathophysiology of nocturia is multi-factorial, but dysregulation of arginine vasopressin (AVP, anti-diuretic hormone) signaling is believed to be a contributing factor. Nocturia is believed to be a consequence of nocturnal polyuria, marked by poor water reabsorption by the collecting ducts dew to deficient endocrine AVP signaling during sleep. However, we have evidence that the bladder may also be involved in altering urine/blood osmolarity and decreasing stored urine volumes. The functional role of AVP receptors in the urinary bladder was recently demonstrated as intraluminal desmopressin (dAVP, vasopressin receptor 2 agonist) promotes bladder smooth muscle relaxation in a mucosa-dependent manner [1]. However, if AVP signaling in the bladder impacts nocturia remains an open question. In this study, we hypothesized that the age-associated rise in the prevalence of nocturia is linked to a decline in water and salt reabsorption by aging bladders and tested this hypothesis by measuring the effect of aging and AVP/dAVP on water and urea reabsorption in the mouse bladders.
Study design, materials and methods
Adult (6-8 months old) and aged (20-24 months old) female and male mice were anesthetized with isoflurane (5% induction, 1-1.5% maintenance) and catheterized through the urethras with PE10 /30 G needle catheters. Bladders were emptied and instilled with 150 microl saline containing 5 microCi/ml 3H water and 5 microCi/ml 14C urea. Additional animal groups were instilled with the same solution with the addition of 100 nM AVP, 100 nM dAVP or vehicle (0.1% DMSO). The tails were snipped 1 mm from the tip and 10 microl of blood was withdrawn before, immediately after and every 15 minutes then (total volume not to exceed 100 microl). Blood was mixed with 2 ml of scintillation fluid immediately upon withdrawal and disintegrations per minute (DPM) were measured using a liquid scintillation counter.
Results
Our preliminary experiments demonstrated that higher bladder volume/distention led to increased bladder reabsorption regardless of gender, presumably due to distention-evoked opening of tight junctions and higher aquaporin 2 expression in the urothelium. Given the rapid systemic uptake in mice due to their high heart rates and small blood volumes (< 2 ml), in these experiments we used a lower volume determined from the mean voided volumes recorded in metabolic cage experiments (underdistended bladder). Figure 1A-D shows the effect of AVP and dAVP on the systemic reuptake of water in adult female, adult male, aged female and aged male mice, respectively, during first 60 minutes following instillation. The rise in DPM stabilizes with the reabsorption of instilled radiolabeled as well as not radiolabeled water excreted into urine at later time points as ureters were not ligated. Figure 2 demonstrates the % dose of 3H water reabsorbed at 15 minutes post instillation (=(dpm⁄(ml ) of blood ÷ Vd)/(dpm⁄(ml ) of instilled dose) × 100).
Interpretation of results
In the absence of AVP/dAVP, female mice exhibited more than two-fold age-related decline in water reabsorption. Both AVP and dAVP significantly increased the rate of water reabsorption in adult female and aged male mice during the first 30 minutes. In adult male mice the effect was more prominent with dAVP than AVP. At 15 minutes post instillation, both AVP and dAVP more than doubled the % dose absorbed in aged male mice compared to just a 60% increase noted only with AVP in aged females (**p < 0.0001). While dAVP was more efficacious than AVP in both adult and aged female mice at 15 min timepoint, none of the agonists had a significant effect in adult males at the same time. Overall, both AVP and dAVP raised the rate of water reabsorption 5 fold to > 10 mL/min from < 2 mL/min with just saline in aged animals.