Roflumilast and aquaporin-2 regulation in rat renal inner medullary collecting duct

Abstract

Roflumilast is a cyclic nucleotide phosphodiesterase inhibitor that is FDA-approved for treatment of chronic obstructive pulmonary disease. With a view toward possible use for treatment of patients with X-linked nephrogenic diabetes insipidus (NDI) due to hemizygous mutations in the V2 vasopressin receptor, this study sought to determine the effect of roflumilast on aquaporin-2 (AQP2) phosphorylation, AQP2 trafficking, and water permeability in the rat inner medullary collecting duct (IMCD). In the presence of the vasopressin analog dDAVP (0.1 nmol/L), both roflumilast and its active metabolite roflumilast N-oxide (RNO) significantly increased phosphorylation at S256, S264, and S269, and decreased phosphorylation at S261 (immunoblotting) in IMCD suspensions in a dose-dependent manner (3-3000 nmol/L). Another commonly used phosphodiesterase inhibitor, IBMX, affected phosphorylation only at the highest concentration in this range. However, neither roflumilast nor RNO had an effect on AQP2 phosphorylation in the absence of vasopressin. Furthermore, roflumilast alone did not increase AQP2 trafficking to the plasma membrane (immunofluorescence) or increase water permeability in freshly microdissected perfused IMCD segments. We conclude that roflumilast can be used to enhance vasopressin's action on AQP2 activity in the renal collecting duct, but has no detectable effect in the absence of vasopressin. These findings suggest that roflumilast may not have a beneficial effect in X-linked NDI, but could find useful application in acquired NDI.

Keywords: X chromosome; cAMP phosphodiesterase; kidney; nephrogenic diabetes insipidus.

Figure 1

Effect of roflumilast on AQP2 phosphorylation changes in rat IMCD suspensions. IMCD suspensions were incubated with vehicle or roflumilast (30 nmol/L) in the absence or presence of 0.1 nmol/L dDAVP for 30 min. (A–E) immunoblots for the four vasopressin‐regulated serine sites (S256, S261, S264, and S269) of AQP2 and total AQP2. (F–O) band density analysis depicted as log₂ value of the effect of vehicle or roflumilast normalized by vehicle in the absence (F–J) or presence (K–O) of dDAVP. *Statistically significant (P < 0.05). IMCD, inner medullary collecting duct.

Figure 2

Effect of roflumilast N‐oxide on AQP2 phosphorylation changes in rat IMCD suspensions. IMCD suspensions were incubated with vehicle or RNO (30 nmol/L) in the absence or presence of 0.1 nmol/L dDAVP for 30 min. (A–E) immunoblots for the four vasopressin‐regulated serine sites (S256, S261, S264, and S269) of AQP2 and total AQP2. (F–O) band density analysis depicted as log₂ value of the effect of vehicle or RNO normalized by vehicle in the absence (F–J) or presence (K–O) of dDAVP. *Statistically significant (P < 0.05). IMCD, inner medullary collecting duct; RNO, roflumilast N‐oxide.

Figure 3

Dose‐dependent effect of roflumilast and IBMX on AQP2 phosphorylation changes in IMCD suspensions in the presence of 0.1 nmol/L AVP. The dose range (3 nmol/L, 30 nmol/L, 300 nmol/L, and 3000 nmol/L) and the incubation time (30 min) were the same for both agents. (A&B) immunoblots for total AQP2 and the four vasopressin‐regulated serine sites (S256, S261, S264, and S269) of AQP2. (C&D) line graphs summarizing the changes in normalized band densities of the total and four phosphorylation sites of AQP2 elicited by roflumilast (C) or IBMX (D). n = 6 for both groups. *Statistically significant (P < 0.05). IBMX, isobutyl‐1‐methylxanthine; IMCD, inner medullary collecting duct.

Figure 4

Effect of roflumilast on AQP2 phosphorylation changes in rat IMCD suspensions in the presence of a selective EP4 agonist ONO‐AE1‐329 (30 min incubation period). (A) immunoblots for the four vasopressin‐regulated serine sites (S256, S261, S264, and S269) of AQP2 and total AQP2. (B–F) bar graphs of the normalized band density analysis. IMCD, inner medullary collecting duct.

Figure 5

Immunofluorescence labeling of AQP2 in microdissected IMCD segments. Freshly microdissected IMCDs were incubated with vehicle (A) 30 nmol/L roflumilast (C) or 0.1 nmol/L dDAVP (D) for 30 min, followed by fixation and immunofluorescence staining for AQP2. B, using an AQP1 antibody at a concentration similar to that used for AQP2 antibody in A, C and D, AQP1 identified only the thin structures attached to an IMCD segment (pretreated with 0.1 nmol/L dDAVP) but not IMCD. Green: AQP2 or AQP1, blue: DAPI. (n = 3). IMCD, inner medullary collecting duct.

Figure 6

Osmotic water permeability (P _f) measurements in microdissected IMCD segments. IMCD tubules were isolated and perfused for osmotic water permeability measurements in three 30‐min experimental periods: basal, roflumilast and roflumilast+dDAVP (upper panel) or basal, RNO and RNO+dDAVP (lower panel). Data from the same experimental period were pooled for statistical analysis. Water permeability measured at 30 nmol/L roflumilast or RNO alone was not statistically different from the basal condition, whereas in the presence of 0.1 nmol/L dDAVP, water permeability was significantly increased (P < 0.001, n = 6). IMCD, inner medullary collecting duct; RNO, roflumilast N‐oxide.