The results of this study demonstrated that water was absorbed from the rat bladder but was not influenced by the administration of any of the three anticholinergics used. In rats that were diuretic with saline, the use of anticholinergics resulted in the renal reabsorption of water in the absence of AVP. This is the first report that anticholinergics increased the cAMP level and that AQP2 was trafficked to the luminal side of the collecting ducts of the kidney.
Several animal studies have shown that water moves from the bladder epithelium to the systemic circulation.8,12−14 To date, among the 11 isoforms of AQP observed in mammals, the expressions of AQP2, 3, 4, 7, 9, and 11 have been observed in the bladder epithelium. However, the function of AQP is not fully elucidated.15 In an earlier investigation, when physiological saline was injected into the bladder, water was absorbed from the bladder epithelium with an enhancement of epithelial AQP2 expression.8 The bladder epithelium has a role in sodium transport, possibly via epithelial sodium channels (ENaCs) or claudin-3,6.16,17 In humans, > 100 ml of bladder urine disappears during sleep at night, when the bladder capacity reaches its functional limit as shown by the time-course monitoring of bladder capacity by transabdominal 3D ultrasound.18
Our present findings in an animal model confirmed that physiological saline and glucose solution injected into the rat bladder were each absorbed through the bladder. The lower the osmotic pressure of the solution administered into the bladder, the higher the concentration effect was, but there was no significant difference. The administration of anticholinergics did not affect the absorption rate. Electrolytes such as Na+ and Cl− were also absorbed with water. Changes in the electrolytes, osmotic pressure, and pH of the solutions were observed when the values obtained before and after the solutions' injection were compared; however, no significant differences were observed in these changes even when the anticholinergics were administered. Therefore, although urine absorption from the urothelium of the bladder is possible, the absorption rate was approx. 10% of the injected amount. The urinary absorption mechanism through the bladder epithelium was not activated by the present administration of anticholinergics. These results suggest that urine production may be suppressed by anticholinergics via an increase in the reabsorption of urine in the kidneys.
With the use of animal diuretic models Watanabe et al. demonstrated that anticholinergics have antidiuretic effects.5 Yamazaki et al. also reported that imidafenacin might enhance the vasopressin-signaling pathway in rats, they did not show that the point of action was the kidney.19 The present study's results demonstrate for the first time that the antidiuretic effect of anticholinergics is to promote renal urine reabsorption. However, the question thus arises: why do anticholinergics promote urine reabsorption in the kidneys?
Cholineacetyltransferase (ChAT) mRNA is localized within the renal cortex collecting ducts, and ChAT-positive cells correspond to the principal cells in the collecting ducts.20 Endogenous ACh has been suggested to be synthesized in renal cortical cells in rabbits.21 An increase in the interstitial sodium concentration stimulated endogenous ACh release in the renal cortex.21,22 It was also reported that the administration of the Na+/K+-ATPase inhibitor ouabain produced a release of ACh and that the ENaC inhibitor benzamil suppressed the release of ACh.22 It thus appears that ACh release is associated with sodium ion transport in the renal cortex.
On the other hand, ACh causes Na+ diuresis in the collecting ducts in accord with the intracellular Na+ concentration.19,23−25 Takeda et al. reported that ACh suppressed the inward current of ENaCs in the collecting ducts of the rabbit renal cortex, inducing natriuresis.23 Garg et al. also demonstrated that the muscarinic receptor agonist carbachol had an inhibitory effect on Na+/K+-ATPase activity in Madin-Darby canine kidney cells via the activation of PKC.24 The acetylcholinesterase inhibitor distigmine activated endogenous ACh and caused an increase in urine output in a study by Yamazaki et al.,19 and Williams et al. observed that exogenous ACh increased sodium excretion in the urine.25
In the present experiment in which saline loading caused Na+ diuresis, the ACh released from the renal cortex may have resulted in a tendency toward natriuresis in an autocrine or paracrine fashion. From this point of view, anticholinergics have the potential to decrease the production of urine through a reabsorption of sodium across the collecting duct cells. In fact, we observed that the high dose of the anticholinergic IM significantly reduced the excretion of urinary sodium, suggesting that sodium reabsorption occurred in the rat kidney. In addition, since the cAMP level was increased by the administration of IM in this study, we speculate that the trafficking of ENaCs to the apical cell membrane occurred, followed by the decrease in the excretion of Na+ in the urine. Increased cAMP in the collecting duct cells resulted in the trafficking of ENaCs as well as AQP2 to the apical cell membrane.26
Under "steady-state" conditions in normally hydrated animals, the majority of AQP2 is located in the apical plasma membrane.27 In microdissected rat terminal inner medullary collecting ducts, carbachol inhibited water permeability by activating the phosphoinositide signaling pathway to increase intracellular calcium and activate PKC, independently of the cAMP-mediated hydro-osmotic effect of AVP.7 It was later clarified that the activation of PKC promoted the internalization of AQP2, and the internalized AQP2 was directed to intracellular vesicles by endocytosis.28,29 Therefore, endogenous ACh works on diuresis by promoting a PKC-mediated internalization of AQP2 regardless of the level of AVP.
Based on the results of our present experiments, we speculate that the three anticholinergics exerted an antidiuretic effect by inhibiting the internalization of AQP2 in the renal cortex collecting ducts. In fact, we observed the immunofluorescent localization of AQP2 on the luminal side of the collecting ducts. It is likely that anticholinergics will be effective for the reabsorption of urine when endogenous ACh causes diuresis. This study is the first to reveal that anticholinergics showed trafficking of AQP2 on the luminal side. However, it is not yet known why this trafficking is accompanied by an increase in cAMP.
mAChRs are expressed in the kidney glomeruli, proximal tubules, and collecting ducts.6 The presence of mAChRs in the kidney was confirmed in rabbits, and both principal cells and intercalated cells of the collecting ducts showed M1mAChR, with stronger reactivity in medullary cells than in cortical cells.30 M4- and M5mAChRs were also present in intercalated-like cells.6 The M2- and M4mAChRs are coupled to G protein (Gi/o) to inhibit stimulated adenylate cyclase and reduce intracellular cAMP levels, while M1- and M3mAChRs are coupled to the phosphoinositide pathway through Gq/11.31 In addition, M1- and M3mAChRs have been suggested to modulate cAMP production in reconstituted systems in transfected cell lines.32,33
In the present study, the level of cAMP before the saline administration (the non-diuretic state) was the same as that in the diuretic state, suggesting that endogenous ACh does not affect the production of cAMP. We speculate that cAMP will be produced when the M2- or M4mACh-mediated inhibitory influences on adenylate cyclase are released by the administration of an anticholinergic. In fact, IM has a high affinity for M1 and M3 receptors, but it also has an affinity for M4 receptors.34 AT and TO have no muscarinic receptor selectivity. We observed a significant increase in cAMP production following the IM administration, inducing the enhancement of AQP2 trafficking to the apical cell membrane. The underlying mechanism of AQP2 trafficking will be elucidated in more detail in future experiments.
The urinary release of AQP2 is influenced by the action of AVP in the kidney collecting ducts.35 An increase in the urinary release of AQP2 has been reported in patients with SIADH (syndrome of inappropriate secretion of anti-diuretic hormone), cognitive heart failure, or hepatic cirrhosis, and in pregnant women, while a decrease in the urinary release of AQP2 was reported in patients with chronic kidney diseases (including polycystic kidney) and patients treated with a V2 receptor antagonist.35–37 The urinary release of AQP2 is related to the apical trafficking of AQP2, which indicates that AQP2 in urine is a useful biomarker that reflects the potency of AVP in the renal collecting ducts.37 In the present study, dDAVP significantly increased the urinary AQP2, while IM administration also increased AQP2 but not significantly. We speculate that the reason why the increase in cAMP/AQP2 by the administration of an anticholinergic is smaller than that produced by treatment with dDAVP is not the direct action of the anticholinergic on cAMP production; rather, it is the effect of blocking the inhibitory system via the ACh receptors.
Study limitations
An important limitation of the present study is that we did not measure the endogenous ACh release from the renal cortex during saline loading. A microdialysis technique is necessary to measure the ACh release from the renal cortex.22 We also did not evaluate the trafficking of ENaCs in order to determine the reason for the decrease in urinary Na+ with the high dose of anticholinergics. Further studies may reveal the precise effects of anticholinergics on sodium channels.