Loss of calcineurin Aα results in altered trafficking of AQP2 and in nephrogenic diabetes insipidus

Abstract

The serine/threonine phosphatase calcineurin is an important signaling molecule involved in kidney development and function. One potential target of calcineurin action is the water channel aquaporin 2 (AQP2). In this study, we examined the effect of loss of calcineurin Aα (CnAα) on AQP2 function in vivo. CnAα null mice were found to have defective post-natal urine-concentrating ability and an impaired urine-concentrating response to vasopressin. Expression of AQP2 is normal but, paradoxically, vasopressin-mediated phosphorylation of the channel is decreased compared with wild-type littermates and there is no accumulation of AQP2 in the apical membrane. Calcineurin protein and activity was found in innermedullary collecting duct vesicles, and loss of calcineurin expression and activity was associated with a loss of AQP2 in the vesicle fraction. As such, the lack of vasopressin-mediated phosphorylation of AQP2 might be the result of a defect in normal trafficking of AQP2 to apical-targeted vesicles. Likewise, treatment of wild-type mice with cyclosporin A to inhibit calcineurin produces a similarly impaired urine-concentrating response to vasopressin and alterations in AQP2 phosphorylation and trafficking. These experiments demonstrate that, CnAα is required for normal intracellular trafficking of AQP2 and loss of calcineurin protein or activity disrupts AQP2 function.

Role of AKAP79/150 Protein in β1-Adrenergic Receptor Trafficking and Signaling in Mammalian Cells

“…In adult cardiac myocytes, calcineurin and PKA bound to muscle AKAP exerted opposite effects on trafficking of myopodin between Z-discs and the nucleus (23). Moreover, a calcineurin-binding AKAP was also involved in the translocation of aquaporin-2 from endosomes to the membrane of inner medullary collecting ducts (40). Trafficking of aquaporin-2 or myopodin was mediated by their phosphorylation by an AKAP-anchored PKA and was inhibited by the phosphatase activity of AKAP-anchored calcineurin (23,40,41).…”

“…Moreover, a calcineurin-binding AKAP was also involved in the translocation of aquaporin-2 from endosomes to the membrane of inner medullary collecting ducts (40). Trafficking of aquaporin-2 or myopodin was mediated by their phosphorylation by an AKAP-anchored PKA and was inhibited by the phosphatase activity of AKAP-anchored calcineurin (23,40,41). Furthermore, translocation of aquaporin-2 from endosomes to the membrane of inner medullary collecting ducts or myopodin from Z-discs to the nucleus was inhibited by manipulations that increased the activity or the amount of calcineurin (23,40).…”

“…Trafficking of aquaporin-2 or myopodin was mediated by their phosphorylation by an AKAP-anchored PKA and was inhibited by the phosphatase activity of AKAP-anchored calcineurin (23,40,41). Furthermore, translocation of aquaporin-2 from endosomes to the membrane of inner medullary collecting ducts or myopodin from Z-discs to the nucleus was inhibited by manipulations that increased the activity or the amount of calcineurin (23,40). Therefore, it is conceivable that loss of the calcineurin-binding site from AKAP5 might have affected optimal SAP97-AKAP5 geometry at the ␤ 1 -AR or interfered with the targeting or efficiency of PKA in phosphorylating Ser 312 or other trafficking proteins.…”

LC-MS/MS analysis of differential centrifugation fractions from native inner medullary collecting duct of rat

“…A second objective of this work was to evaluate the utility of a technique that has been used in many papers to investigate trafficking of the AQP2 water channel (6,7,9,10,12,19). Typically, in such studies, the ratio of the AQP2 in the 17K fraction to that in the 200K has been used to assess the distribution of AQP2 between intracellular vesicles (represented by the 200K fraction) and plasma membrane (represented by the 17K fraction).…”

“…In most papers that followed, the cell fraction obtained from a 17,000-g spin (17K) has been referred to as the "plasma membrane" fraction (for example, see Refs. 6,7,9,10,19), while the fraction obtained from a 200,000-g spin (200K) has been called the "intracellular vesicle" fraction. Marples and colleagues demonstrated in rat kidneys that the ratio of AQP2 in the 17K pellet to that in the 200K pellet increased in response to exposure to the V2 receptor-selective vasopressin analog dDAVP (12).…”