A new ‘tail’ of aquaporin‐2

Abstract

The kidney collecting duct principal cell is pivotal in the maintenance of body water balance. In conditions where plasma osmolality is increased and preservation of body water is required, the antidiuretic hormone vasopressin (VP) is released from the pituitary gland. VP regulates the water permeability of the collecting duct by leading to apical plasma membrane accumulation of the water channel aquaporin-2 (AQP2). The increase in water permeability allows transcellular water reabsorption as a function of the osmotic differences between the tubular lumen and the interstitium. In the presence of VP this reabsorption occurs along the entire connecting tubule and collecting duct, resulting in concentration of urine and body water conservation. VP causes apical plasma membrane accumulation of AQP2 via its binding to the basolateral V2-receptor (V2R), a Gsα protein-coupled receptor. Activation of adenylate cyclase downstream of V2R causes an increase in cAMP followed by protein kinase A (PKA)-dependent modulations in the poly-phosphorylated C-terminal region of AQP2. Thus, VP causes phosphorylation to increase at residues Ser256, Ser264 and Ser269 of AQP2, whereas Ser261 phosphorylation decreases (Hoffert et al. 2006). In particular, phosphorylation of Ser256 is generally considered the first phosphorylation site and critical for regulated membrane targeting. More recent studies also point to an important role for Ser269 in this event (Moeller et al. 2014). Thus, the general paradigm for understanding VP-mediated membrane accumulation of AQP2 involves both an increase in cAMP and hierarchical phosphorylation events. However, as highlighted in the paper by Cheung et al. (2019) in this issue of The Journal of Physiology this may not be the whole truth concerning VP-induced AQP2 membrane targeting in the collecting duct principal cell. Cheung et al. (2019) have examined the role of inhibition of the non-receptor tyrosine kinase Src in AQP2 membrane accumulation. Using immunohistochemistry, Src was shown to localize to both the basolateral and the apical plasma membrane in principal cells in cortex, outer and inner medulla of rat kidney. Thus, Src was present in AQP2 expressing cells in the relevant intracellular compartment, the apical membrane, for potential regulation of AQP2. In LLC-PK1 cells expressing AQP2, the authors found that, just like VP, 30 min of Src inhibition via dasatinib (Das) caused membrane accumulation of AQP2. Das also caused AQP2 membrane accumulation in rat kidney slices. In the cell model, both Das and VP reduced Src phosphorylation at Tyr416, an activating phosphorylation site. siRNA-mediated knockdown of Src also resulted in AQP2 membrane accumulation. This finding was independent of Ser256 phosphorylation as demonstrated in 256A-AQP2 expressing cells, a phosphorylation-deficient cell line. Furthermore, Src inhibition resulted in increased levels of Ser269 phosphorylation whereas Ser256 was unaffected. The ability of Src inhibition to mediate increases in Ser269 phosphorylation was further supported by the observation of this phosphorylation event in S256A-AQP2 expressing cells treated with Das, but also when treated with VP. However, only in Das-treated 256A-AQP2 cells, Ser269 phosphorylation coincided with increased membrane abundance of AQP2. Confirming these observations, the Das-induced AQP2 membrane targeting was prevented in 269A-AQP2 expressing cells. Src inhibition by Das did not change the intracellular cAMP levels. The balance of exocytic and endocytic events was studied using cell model assays, which revealed that Src inhibition affects AQP2 membrane targeting both by enhancing exocytosis and by decreasing endocytosis. Several important perspectives concerning our understanding of AQP2 regulation are revealed by these findings – both at the level of basic research and at a clinical level. First, as has also been emphasized by other research groups recently (Olesen & Fenton, 2017), and even though cAMP must still be considered a major factor for AQP2 membrane accumulation, this signalling pathway may not be the only important mechanism of VP-mediated AQP2 membrane accumulation. There may easily be other downstream signalling events yet to be discovered. The results of Cheung et al. (2019) are in line with this notion, and Src inhibition may turn out to be a significant new player in the regulation of AQP2 trafficking. Second, posttranslational modifications of the C-terminus of AQP2 have often been considered a regulatory event affecting the whole polyphosphorylated region. However, using mutant cell lines, phosphospecific antibodies and Src inhibition, Cheung et al. elegantly succeed in targeting just one phosphorylation site in this region and thereby allow us to understand the specific relevance of this particular site for AQP2 regulation. The finding of a significant role for regulated Ser269 phosphorylation in AQP2 membrane targeting corroborates previous findings in this field (Moeller et al. 2009). Pinpointing regulation of phosphorylation of single residues in this important region may indeed be the road to gaining more insight into the specific roles of other phosphorylation sites currently less well understood, e.g. Ser261 and Ser264. Finally, a number of studies over the years have highlighted that AQP2 membrane targeting can occur independently of VP – either dependent on or independent of cAMP/PKA. In this respect, Cheung et al. (2019) demonstrate that Src inhibition in their model systems can, independently of VP, cause AQP2 membrane accumulation. In conditions of a defective intracellular response to VP, such as is the case with X-linked nephrogenic diabetes insipidus (NDI), which results from loss of function mutations of the V2R, this insight could pave the way for new potential treatments for this condition.