Lixivaptan, a New Generation Diuretic, Counteracts Vasopressin-Induced Aquaporin-2 Trafficking and Function in Renal Collecting Duct Cells

Abstract

Vasopressin V2 receptor (V2R) antagonists (vaptans) are a new generation of diuretics. Compared with classical diuretics, vaptans promote the excretion of retained body water in disorders in which plasma vasopressin concentrations are inappropriately high for any given plasma osmolality. Under these conditions, an aquaretic drug would be preferable over a conventional diuretic. The clinical efficacy of vaptans is in principle due to impaired vasopressin-regulated water reabsorption via the water channel aquaporin-2 (AQP2). Here, the effect of lixivaptan-a novel selective V2R antagonist-on the vasopressin-cAMP/PKA signaling cascade was investigated in mouse renal collecting duct cells expressing AQP2 (MCD4) and the human V2R. Compared to tolvaptan-a selective V2R antagonist indicated for the treatment of clinically significant hypervolemic and euvolemic hyponatremia-lixivaptan has been predicted to be less likely to cause liver injury. In MCD4 cells, clinically relevant concentrations of lixivaptan (100 nM for 1 h) prevented dDAVP-induced increase of cytosolic cAMP levels and AQP2 phosphorylation at ser-256. Consistent with this finding, real-time fluorescence kinetic measurements demonstrated that lixivaptan prevented dDAVP-induced increase in osmotic water permeability. These data represent the first detailed demonstration of the central role of AQP2 blockade in the aquaretic effect of lixivaptan and suggest that lixivaptan has the potential to become a safe and effective therapy for the treatment of disorders characterized by high plasma vasopressin concentrations and water retention.

Keywords: Aquaporin-2; V2R; lixivaptan; vaptans; vasopressin.

Figure 1

Measurements of intracellular cAMP by FRET. (A) Schematic model showing a FRET probe containing the cAMP-binding sequence of Epac1 sandwiched between ECFP (donor) and EYFP (acceptor). Binding of cAMP to the Epac1 results in an intermolecular steric conformation change causing an increase in the distance between the fluorescent donor and the acceptor, thereby decreasing the FRET process. (B) dDAVP stimulation significantly increased cAMP levels with respect to cells left untreated or in presence of lixivaptan (LXV), with or without dDAVP (*** p < 0.0001, n = 160 cells). Co-treatment with LXV prevented cAMP increase induced by dDAVP. All data were analyzed by one-way ANOVA followed by Newman–Keuls multiple comparisons test and are expressed as means ± SEM. (C) Representative transfected cells with H96 probe showing the FRET signal (ratio 535/30 nm) depicted in false color.

Figure 2

Effect of lixivaptan on pS256-AQP2 levels. (A) Equal amount of proteins from MCD4 cells were immunoblotted for evaluation of pS256-AQP2 and total AQP2 levels. (B) Statistical analysis revealed that lixivaptan prevented the increase of AQP2 phosphorylated at S256 induced by dDAVP. No alterations in pS256-AQP2 levels were observed by the sole treatment with lixivaptan. dDAVP alone induced a significant increase in pS256-AQP2 compared to cells under basal condition. Data are expressed as means ± SEM and were analyzed by one-way ANOVA followed by Newman–Keuls multiple comparisons test ($ p < 0.0001 dDAVP vs. dDAVP+LXV, # p < 0.001 dDAVP vs. CTR, £ p < 0.01 LXV vs. dDAVP, * p < 0.05 LXV vs. dDAVP+LXV, n.s. CTR vs. dDAVP+LXV or LXV; n = 5).

Figure 3

Effect of lixivaptan on osmotic water permeability. (A) Representative normalized traces of the time course of alterations in the osmotic water permeability in response to exposure to a hyperosmotic solution, followed as modulation of normalized calcein fluorescence intensity. Osmotic permeability measurements studies in MCD4 cells expressing human V2R and AQP2 were performed as described in Materials and Methods. (B) The temporal osmotic response is indicated as 1/τ (s⁻¹). Lixivaptan prevented dDAVP-induced cell swelling (*** p < 0.0001). Data were analyzed by one-way ANOVA followed by Newman–Keuls multiple comparisons test and are expressed as means ± SEM.