A Novel PKA-Independent Signaling Pathway Controlling AQP2 Trafficking as a Possible Cause for the Syndrome of Inappropriate Antidiuresis

Background: Renal water reabsorption is controlled by vasopressin (AVP) which activates phosphorylation of AQP2 at serine 256 (pS256) and translocation to the plasma membrane. Besides S256, AVP causes dephosphoryation of S261. Recent studies showed that cyclin-dependent kinases can phosphorylate S261 AQP2 peptides in vitro. In an attempt to investigate the possible role of cdks on AQP2 phosphorylation, we identified a PKA-independent pathway regulating AQP2 trafficking. Methods: MDCK cells or kidney slices were left untreated or forskolin stimulated with or without roscovitine (10μM), a specific cdks inhibitor. Results: In ex-vivo kidney slices and MDCK cells roscovitine increased pS256 and decreased pS261.The changes in AQP2 phosphorylation were paralleled by an increase in cell surface AQP2 expression and osmotic water permeability in the absence of forskolin stimulation. Of note, roscovitine did alter neither cAMP intracellular level nor PKA activity. Because phosphorylation results from the balance between kinase and phosphatase activity we evaluated the possible contribution of protein phosphatases PP1, PP2A and PP2B. Of these, roscovitine treatment specifically reduced PP2A activity in MDCK cells. Interestingly, in PKD1+/- mice displaying a syndrome of inappropriate antidiuresis with high level of pS256 despite unchanged AVP and cAMP (Alharabi et. al 2007) we found a reduced PP2A expression and activity. Indeed similarly to what previously found in PKD1+/- mice, roscovitine significantly decreased intracellular calcium in MDCK. Conclusions: Our data indicate that a reduced activity of PP2A, secondary to reduced intracellular Ca2+ levels, promotes AQP2 trafficking independently from the AVP-PKA axis. This pathway may be relevant for explaining pathological states characterized by inappropriate AVP secretion and positive water balance.