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High concentrations of urinary calcium counteract vasopressin action via the activation of the Calcium‐Sensing Receptor (CaSR) expressed in the luminal membrane of the collecting duct cells, which impairs the trafficking of aquaporin‐2 (AQP2). In line with these findings, we provide evidence that, with respect to wild‐type mice, CaSR knock‐in (KI) mice mimicking autosomal dominant hypocalcaemia, display a significant decrease in the total content of AQP2 associated with significantly higher levels of AQP2 phosphorylation at Ser261, a phosphorylation site involved in AQP2 degradation. Interestingly, KI mice also had significantly higher levels of phosphorylated p38MAPK, a downstream effector of CaSR and known to phosphorylate AQP2 at Ser261. Moreover, ATF1 phosphorylated at Ser63, a transcription factor downstream of p38MAPK, was significantly higher in KI. In addition, KI mice had significantly higher levels of AQP2‐targeting miRNA137 consistent with a post‐transcriptional downregulation of AQP2. In vivo treatment of KI mice with the calcilytic JTT‐305, a CaSR antagonist, increased AQP2 expression and reduced AQP2‐targeting miRNA137 levels in KI mice. Together, these results provide direct evidence for a critical role of CaSR in impairing both short‐term vasopressin response by increasing AQP2‐pS261, as well as AQP2 abundance, via the p38MAPK‐ATF1‐miR137 pathway. Key points: Calcium‐Sensing Receptor (CaSR) activating mutations are the main cause of autosomal dominant hypocalcaemia (ADH) characterized by inappropriate renal calcium excretion leading to hypocalcaemia and hypercalciuria. Current treatments of ADH patients with parathyroid hormone, although improving hypocalcaemia, do not improve hypercalciuria or nephrocalcinosis.In vivo treatment with calcilytic JTT‐305/MK‐5442 ameliorates most of the ADH phenotypes of the CaSR knock‐in mice including hypercalciuria or nephrocalcinosis and reverses the downregulation of the vasopressin‐sensitive aquaporin‐2 (AQP2) expression, providing direct evidence for a critical role of CaSR in impairing vasopressin response.The beneficial effect of calcilytic in reducing the risk of renal calcification may occur in a parathyroid hormone‐independent action through vasopressin‐dependent inhibition of cAMP synthesis in the thick ascending limb and in the collecting duct.The amelioration of most of the abnormalities in calcium metabolism including hypercalciuria, renal calcification, and AQP2‐mediated osmotic water reabsorption makes calcilytic a good candidate as a novel therapeutic agent for ADH. [ABSTRACT FROM AUTHOR]

Aquaporin‐2 (AQP2) is a member of the aquaporin water channel family. In the kidney, AQP2 is expressed in collecting duct principal cells where it facilitates water reabsorption in response to antidiuretic hormone (arginine vasopressin, AVP). AVP induces the redistribution of AQP2 from intracellular vesicles and its incorporation into the plasma membrane. The plasma membrane insertion of AQP2 represents the crucial step in AVP‐mediated water reabsorption. Dysregulation of the system preventing the AQP2 plasma membrane insertion causes diabetes insipidus (DI), a disease characterised by an impaired urine concentrating ability and polydipsia. There is no satisfactory treatment of DI available. This review discusses kinases that control the localisation of AQP2 and points out potential kinase‐directed targets for the treatment of DI. [ABSTRACT FROM AUTHOR]

Nephrogenic syndrome of inappropriate antidiuresis (NSIAD) is a rare X‐linked disease caused by gain‐of‐function mutations of arginine vasopressin receptor 2 (V2R). Patients with NSIAD are characterized by the inability to excrete a free water load and by inappropriately increased urinary osmolality despite very low levels of plasma vasopressin, resulting in euvolaemic hyponatraemia. To dissect the signalling downstream V2R constitutively active variants, Flp‐In T‐REx Madin‐Darby canine kidney (FTM) cells, stably transfected with V2R mutants (R137L, R137C and F229V) and AQP2‐wt or non‐phosphorylatable AQP2‐S269A/AQP2‐S256A, were used as cellular models. All three activating V2R mutations presented constitutive plasma membrane expression of AQP2‐wt and significantly higher basal water permeability. In addition, V2R‐R137L/C showed significantly higher activity of Rho‐associated kinase (ROCK), a serine/threonine kinase previously suggested to be involved in S269‐AQP2 phosphorylation downstream of these V2R mutants. Interestingly, FTM cells expressing V2R‐R137L/C mutants and AQP2‐S269A showed a significant reduction in AQP2 membrane abundance and a significant reduction in ROCK activity, indicating the crucial importance of S269‐AQP2 phosphorylation in the gain‐of‐function phenotype. Conversely, V2R‐R137L/C mutants retained the gain‐of‐function phenotype when AQP2‐S256A was co‐expressed. In contrast, cells expressing the F229V mutant and the non‐phosphorylatable AQP2‐S256A had a significant reduction in AQP2 membrane abundance along with a significant reduction in basal osmotic water permeability, indicating a crucial role of Ser256 for this mutant. These data indicate that the constitutive AQP2 trafficking associated with the gain‐of‐function V2R‐R137L/C mutants causing NSIAD is protein kinase A independent and requires an intact Ser269 in AQP2 under the control of ROCK phosphorylation. Key points: Nephrogenic syndrome of inappropriate antidiuresis is caused by two constitutively active variant phenotypes of AVPR2, one sensitive to vaptans (V2R‐F229V) and the other vaptan resistant (V2R‐R137C/L). In renal cells, all three activating arginine vasopressin receptor 2 (V2R) variants display constitutive AQP2 plasma membrane expression and high basal water permeability.In cells expressing V2R‐R137L/C mutants, disruption of the AQP2‐S269 phosphorylation site caused the loss of the gain‐of‐function phenotype, which, in contrast, was retained in V2R‐F229V‐expressing cells.Cells expressing the V2R‐F229V mutant were instead sensitive to disruption of the AQP2‐S256 phosphorylation site.The serine/threonine kinase Rho‐associated kinase (ROCK) was found to be involved in AQP2‐S269 phosphorylation downstream of the V2R‐R137L/C mutants.These findings might have clinical relevance for patients with nephrogenic syndrome of inappropriate antidiuresis. [ABSTRACT FROM AUTHOR]

The advent of modern quantitative protein mass spectrometry techniques around the turn of the 21st century has contributed to a revolution in biology referred to as 'systems biology'. These methods allow identification and quantification of thousands of proteins in a biological specimen, as well as detection and quantification of post‐translational protein modifications including phosphorylation. Here, we discuss these methodologies and show how they can be applied to understand the effects of the peptide hormone vasopressin to regulate the molecular water channel aquaporin‐2. The emerging picture provides a detailed framework for understanding the molecular mechanisms involved in water balance disorders. [ABSTRACT FROM AUTHOR]

Aquaporin‐2 (AQP2) water channels are proteins that are recycled between intracellular vesicles and the apical plasma membrane in renal collecting ducts. Lipopolysaccharide‐responsive beige‐like anchor protein (LRBA) is a protein kinase A (PKA) anchoring protein that creates compartmentalized PKA signalling responsible for AQP2 phosphorylation. In response to increased plasma osmolality, vasopressin/cyclic adenosine monophosphate (cAMP)/PKA signalling phosphorylates AQP2, promoting AQP2 trafficking into the apical plasma membrane and increasing water reabsorption from urine. However, the molecular mechanisms by which LRBA mediates vasopressin‐induced AQP2 phosphorylation remain unknown. To investigate AQP2 intracellular localization and phosphorylation status in vivo, a density gradient ultracentrifugation technique was combined with an in situ proximity ligation assay, super‐resolution structured illumination microscopy and immunoelectron microscopy. Most of the AQP2 was localized on the recycling endosome in the presence of tolvaptan, a vasopressin type 2 receptor (V2R) antagonist. Desmopressin, a V2R agonist, phosphorylated AQP2, translocating it from the recycling endosome to the apical plasma membrane. In contrast, LRBA was constitutively localized at the recycling endosome. Therefore, LRBA and AQP2 were well colocalized in the absence of vasopressin stimulation. The loss of LRBA/PKA signalling by Lrba knockout impaired vasopressin‐induced AQP2 phosphorylation, resulting in AQP2 retention at the recycling endosome. Defective AQP2 trafficking caused low urinary concentrating ability in Lrba−/− mice. The LRBA‐PKA complex created compartmentalized PKA signalling at the recycling endosome, which facilitated AQP2 phosphorylation in response to vasopressin. Key points: Membrane proteins are continuously internalized into the endosomal system via endocytosis, after which they are either recycled back to the plasma membrane or degraded at the lysosome.In T cells, lipopolysaccharide‐responsive beige‐like anchor protein (LRBA) binds directly to the cytotoxic T lymphocyte antigen 4 (CTLA‐4), a checkpoint immune molecule, to prevent CTLA‐4 lysosomal degradation and promote its vesicle recycling.LRBA has different physiological functions in renal collecting ducts. LRBA and aquaporin‐2 (AQP2) water channels were colocalized on the recycling endosome in vivo in the absence of the anti‐diuretic hormone vasopressin.LRBA promoted vasopressin‐induced AQP2 trafficking, increasing water reabsorption from urine via AQP2. LRBA determined renal responsiveness to vasopressin at recycling endosomes.LRBA is a ubiquitously expressed anchor protein. LRBA signalosomes might regulate membrane trafficking of several constitutively recycled proteins at recycling endosomes. [ABSTRACT FROM AUTHOR]

The article discusses the regulation of water reabsorption in the collecting ducts of the kidney, focusing on the role of aquaporin 2 (AQP2) and the lipopolysaccharide-responsive beige-like anchor protein (LRBA). LRBA acts as an anchoring protein for vasopressin/PKA-mediated phosphorylation of AQP2, influencing its localization from recycling endosomes to the apical membrane. The study sheds light on a new layer of regulation in water reabsorption and provides a basis for further research on LRBA's role in protein trafficking and water balance in the kidney. [Extracted from the article]