Background: The vasopressin receptor antagonist tolvaptan has emerged as tool in the management of hyponatremia. However, no direct evidence that the aquaretic effect of tolvaptan is based on impairment of vasopressin stimulated AQP2 phosphorylation and targeting to the plasma membrane has been provided. Methods: MDCK stably expressing hAQP2 or rat kidney slides were exposed to DDAVP or forskolin (FK) stimulation in the presence or in the absence of tolvaptan (10nM). The effect of these treatments on cAMP levels, AQP2 phosphorylation, intracellular calcium concentration and osmotic water permeability was analyzed. Results: In MDCK cells, DDAVP treatment significantly increased cAMP levels paralleled by an increase in p256AQP2. Pretreatment with tolvaptan significantly reduced both effects. Surprisingly, tolvaptan pretreatment strongly reduced the increase in p256AQP2 elicited by FK, a direct activator of adenylyl cyclase. Similar results were obtained in rat kidney slides. In line, tolvaptan prevented the increase in the osmotic water permeability promoted by either DDAVP or FK in MDCK. We therefore analyzed whether tolvaptan had, per se, a cellular effect. Calibration of cellular calcium in MCDK cells revealed that tolvaptan caused a significant increase in intracellular calcium (tolvaptan 64.0±2 nM; ctr 32±1.7 nM). Since p256AQP2 can be de-phosphorylated by PP2A, a calcium depended serine/threonine phosphatase, rat kidney slides were pretreated with tolvaptan and exposed to FK in the presence or absence of caliculyn (5pM) a specific inhibitor of PP2A. Under these conditions tolvaptan failed to prevent FK-induced increase in p256AQP2 suggesting that tolvaptan, activates PP2A. Conclusions: Tolvaptan prevents vasopressin induced increase in p256AQP2, AQP2 trafficking and increase in osmotic water permeability. Moreover tolvaptan increases basal intracellular calcium, which might have relevant consequences in modulating p256AQP2 levels and therefore the clinical response to the drug.

Modulation of cAMP Signaling, AQP2 Phosphorylation and Osmotic Water Permeability in Response to DDAVP or FK under Tolvaptan Treatment in Renal Cells

TAMMA, GRAZIA;Di Mise A;RANIERI, MARIANNA;SVELTO, Maria;VALENTI, Giovanna
2013

Abstract

Background: The vasopressin receptor antagonist tolvaptan has emerged as tool in the management of hyponatremia. However, no direct evidence that the aquaretic effect of tolvaptan is based on impairment of vasopressin stimulated AQP2 phosphorylation and targeting to the plasma membrane has been provided. Methods: MDCK stably expressing hAQP2 or rat kidney slides were exposed to DDAVP or forskolin (FK) stimulation in the presence or in the absence of tolvaptan (10nM). The effect of these treatments on cAMP levels, AQP2 phosphorylation, intracellular calcium concentration and osmotic water permeability was analyzed. Results: In MDCK cells, DDAVP treatment significantly increased cAMP levels paralleled by an increase in p256AQP2. Pretreatment with tolvaptan significantly reduced both effects. Surprisingly, tolvaptan pretreatment strongly reduced the increase in p256AQP2 elicited by FK, a direct activator of adenylyl cyclase. Similar results were obtained in rat kidney slides. In line, tolvaptan prevented the increase in the osmotic water permeability promoted by either DDAVP or FK in MDCK. We therefore analyzed whether tolvaptan had, per se, a cellular effect. Calibration of cellular calcium in MCDK cells revealed that tolvaptan caused a significant increase in intracellular calcium (tolvaptan 64.0±2 nM; ctr 32±1.7 nM). Since p256AQP2 can be de-phosphorylated by PP2A, a calcium depended serine/threonine phosphatase, rat kidney slides were pretreated with tolvaptan and exposed to FK in the presence or absence of caliculyn (5pM) a specific inhibitor of PP2A. Under these conditions tolvaptan failed to prevent FK-induced increase in p256AQP2 suggesting that tolvaptan, activates PP2A. Conclusions: Tolvaptan prevents vasopressin induced increase in p256AQP2, AQP2 trafficking and increase in osmotic water permeability. Moreover tolvaptan increases basal intracellular calcium, which might have relevant consequences in modulating p256AQP2 levels and therefore the clinical response to the drug.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11586/95031
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