{"gene":"AQP2","run_date":"2026-04-28T17:12:37","timeline":{"discoveries":[{"year":1995,"finding":"AQP2 relocates from cytoplasmic vesicles to the apical plasma membrane of collecting duct principal cells following vasopressin treatment, and this apical targeting depends on intact microtubules (colchicine disruption scatters AQP2 throughout the cytoplasm).","method":"Immunofluorescence and immunogold electron microscopy in Brattleboro rats with/without vasopressin and colchicine treatment","journal":"The Journal of membrane biology","confidence":"High","confidence_rationale":"Tier 2 — direct localization experiment with functional consequence, replicated across multiple conditions","pmids":["7539496"],"is_preprint":false},{"year":2000,"finding":"Vasopressin-induced AQP2 trafficking to the apical plasma membrane requires PKA-mediated phosphorylation at Ser256; phospho-AQP2 (pS256) is present in both the apical membrane and intracellular vesicles, and V2 receptor blockade causes near-complete loss of apical pS256-AQP2.","method":"Phosphorylation state-specific antibodies, immunoelectron microscopy, immunoblotting in rat kidney with DDAVP or V2R antagonist treatment","journal":"American journal of physiology. Renal physiology","confidence":"High","confidence_rationale":"Tier 2 — reciprocal antibody approach with functional consequence, replicated across multiple conditions","pmids":["10644653"],"is_preprint":false},{"year":1997,"finding":"Vasopressin acting via the adenylate cyclase-coupled V2 receptor activates AQP2 gene transcription through phosphorylation of CREB and induction of c-Fos, which together bind the CRE and AP1 elements in the AQP2 promoter.","method":"Transfection of human AQP2 promoter in LLC-PK1 cells; CREB phosphorylation and c-Fos expression assays with V2R activation","journal":"The American journal of physiology","confidence":"High","confidence_rationale":"Tier 2 — direct promoter/transcription factor analysis with defined molecular mechanism","pmids":["9140044"],"is_preprint":false},{"year":1997,"finding":"Autosomal recessive NDI-associated AQP2 missense mutations (A147T, T126M, N68S) produce proteins that are functional water channels in Xenopus oocytes but are misrouted to the ER rather than the plasma membrane, establishing that misrouting (not loss of channel function) is the primary cause of recessive NDI.","method":"Xenopus oocyte water permeability assays, immunoblotting, immunocytochemistry of oocyte lysates","journal":"Journal of the American Society of Nephrology","confidence":"High","confidence_rationale":"Tier 1 — functional reconstitution in oocytes combined with localization assays","pmids":["9048343"],"is_preprint":false},{"year":2000,"finding":"Prostaglandin E2 antagonizes vasopressin-induced AQP2 plasma membrane translocation by promoting retrieval of AQP2 to intracellular vesicles independently of AQP2 dephosphorylation at Ser256.","method":"Differential centrifugation, phosphorylation state-specific AQP2 antibody, incubation of rat renal inner medulla with AVP and PGE2","journal":"American journal of physiology. Renal physiology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods in a defined ex vivo system","pmids":["10710543"],"is_preprint":false},{"year":2000,"finding":"AQP2 constitutively recycles between intracellular vesicles and the cell surface via a trans-Golgi-associated compartment; this recycling is blocked by 20°C incubation or bafilomycin A1 (H+-ATPase inhibitor), with AQP2 accumulating in a perinuclear compartment that colocalizes with clathrin but not giantin.","method":"Temperature block experiments, bafilomycin treatment, colocalization with organelle markers in LLC-PK1 cells","journal":"American journal of physiology. Renal physiology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal pharmacological and colocalization approaches","pmids":["10662736"],"is_preprint":false},{"year":2003,"finding":"cAMP-induced AQP2 translocation to the apical membrane is accompanied by RhoA inhibition via PKA-mediated RhoA phosphorylation (on serine), which increases RhoA association with RhoGDI, thereby promoting actin depolymerization required for vesicle fusion.","method":"Selective RhoA pull-down (GTP-bound RhoA), cell fractionation, co-immunoprecipitation of RhoA and RhoGDI, forskolin stimulation of CD8 renal cells","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (pull-down, fractionation, co-IP) in a defined cellular system","pmids":["12640036"],"is_preprint":false},{"year":2003,"finding":"Inhibition of clathrin-mediated endocytosis (by dominant-negative dynamin-2/K44A or methyl-β-cyclodextrin) causes rapid, constitutive plasma membrane accumulation of AQP2 independently of Ser256 phosphorylation, demonstrating that AQP2 constitutively recycles and that phosphorylation at S256 is required for regulated (vasopressin-dependent) but not constitutive membrane insertion.","method":"Dominant-negative dynamin expression, cholesterol depletion with mβCD, cell-surface biotinylation, FITC-dextran endocytosis assay in LLC-PK1 and IMCD cells","journal":"American journal of physiology. Renal physiology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods with S256A phospho-mutant controls","pmids":["14519593"],"is_preprint":false},{"year":2004,"finding":"S256 phosphorylation of AQP2 is necessary but not sufficient for plasma membrane expression; active PKA is required for sustained apical AQP2 localization. PGE2 and dopamine induce AQP2 endocytosis independently of AQP2 dephosphorylation at S256.","method":"PKA inhibitor H-89 treatment, AQP2-S256D phosphomimetic mutant, dopamine/PGE2 treatment, confocal microscopy in MDCK-C7 cells and rat kidney inner medullary slices","journal":"American journal of physiology. Renal physiology","confidence":"High","confidence_rationale":"Tier 2 — phosphomimetic mutants combined with pharmacological inhibitors and ex vivo tissue","pmids":["15625084"],"is_preprint":false},{"year":2005,"finding":"Dominant NDI caused by AQP2-R254L (which destroys the PKA consensus site adjacent to S256) results from loss of vasopressin-mediated phosphorylation at S256; AQP2-R254L is a functional water channel but is retained intracellularly and, when co-expressed, retains wild-type AQP2 in intracellular vesicles.","method":"Xenopus oocyte water permeability, MDCK cell co-expression, immunofluorescence, phospho-specific immunoblotting, S256D rescue experiment","journal":"Journal of the American Society of Nephrology","confidence":"High","confidence_rationale":"Tier 1 — functional oocyte assay + mutagenesis + co-expression dominant-negative experiment","pmids":["16120822"],"is_preprint":false},{"year":2005,"finding":"AQP2 is stored in Rab11-positive subapical compartments prior to apical translocation; after endocytosis, AQP2 moves to EEA1-positive early endosomes and back to the Rab11 compartment. Microtubules maintain the subapical compartment distribution; actin filaments regulate trafficking from early endosomes to the storage compartment. Rab11 depletion by RNAi impairs AQP2 retention in the storage compartment.","method":"Double immunolabeling, siRNA knockdown of Rab11, pharmacological disruption of microtubules (nocodazole, colcemid) and actin (cytochalasin D, latrunculin B) in MDCK cells","journal":"Histochemistry and cell biology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal approaches including RNAi and pharmacological disruption","pmids":["16049696"],"is_preprint":false},{"year":2005,"finding":"ERM protein moesin is required for actin remodeling during AQP2 vesicular trafficking to the apical membrane; forskolin causes moesin redistribution to the cell cortex and reduction of phospho-moesin, and a moesin peptide blocking F-actin binding mimics forskolin effects including AQP2 translocation.","method":"Subcellular fractionation, confocal microscopy, Triton X-100 extraction of cytoskeletal proteins, moesin peptide introduction in renal cells","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 — multiple methods but mechanistic link relies on a peptide competitor approach","pmids":["16046477"],"is_preprint":false},{"year":2005,"finding":"Human AQP2 adopts a typical aquaporin fold as a tetramer; 4.5 Å 2D electron crystallography structure reveals the cytosolic N and C termini form contacts between stacked double-layer sheets.","method":"2D crystallization of recombinant human AQP2, atomic force microscopy, electron crystallography","journal":"Journal of molecular biology","confidence":"Medium","confidence_rationale":"Tier 1 — structure determination, but limited resolution (4.5 Å) and no mutagenesis validation","pmids":["15922355"],"is_preprint":false},{"year":1998,"finding":"Cytoplasmic dynein and dynactin colocalize with AQP2-bearing vesicles in rat renal collecting duct principal cells, consistent with a role of the dynein motor complex in vasopressin-regulated AQP2 vesicle trafficking.","method":"Immunoblotting, immunoisolation of AQP2 vesicles with anti-AQP2 antibody, quantitative double immunogold electron microscopy","journal":"The American journal of physiology","confidence":"Medium","confidence_rationale":"Tier 2 — specific immunoisolation and ultrastructural colocalization, but functional consequence not directly tested","pmids":["9486234"],"is_preprint":false},{"year":2006,"finding":"AQP2 in the collecting duct (CD) is essential for body water balance; conditional knockout mice lacking AQP2 only in CD (but retaining it in connecting tubule) show 10-fold increased urine output and severely decreased urine osmolality that cannot be compensated by other mechanisms. Global AQP2 knockout is lethal postnatally.","method":"Cre/loxP conditional knockout (Hoxb7-Cre for CD-specific, EIIa-Cre for global), metabolic cage measurements, immunohistochemistry","journal":"Proceedings of the National Academy of Sciences","confidence":"High","confidence_rationale":"Tier 2 — clean cell-type-specific knockout with defined physiological phenotype","pmids":["16581908"],"is_preprint":false},{"year":2006,"finding":"Loss of calcineurin Aα results in decreased vasopressin-mediated phosphorylation of AQP2 and failure of AQP2 to accumulate in the apical membrane, causing nephrogenic diabetes insipidus; calcineurin is present in IMCD vesicles and required for normal intracellular AQP2 trafficking.","method":"CnAα null mice and cyclosporin A treatment, immunoblotting for phospho-AQP2, subcellular fractionation, urine concentration tests","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 — genetic KO plus pharmacological inhibition with parallel molecular and physiological phenotypes","pmids":["16735444"],"is_preprint":false},{"year":2006,"finding":"Angiotensin II promotes AQP2 targeting to the plasma membrane of IMCD cells through AT1 receptor activation; this effect involves cAMP elevation and PKC activity and potentiates dDAVP-induced AQP2 membrane targeting.","method":"Immunofluorescence microscopy, immunoblotting for phospho-AQP2, cAMP measurement, candesartan (AT1 blocker) and PKC inhibitor treatment in primary cultured IMCD cells","journal":"American journal of physiology. Renal physiology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal pharmacological approaches with cellular phenotype","pmids":["16896188"],"is_preprint":false},{"year":2008,"finding":"AQP2 vesicle fusion to the apical membrane is mediated by SNARE proteins VAMP2, VAMP3, syntaxin-3, and SNAP23; Munc18b acts as a negative regulator of SNARE complex formation. Knockdown of any of these SNAREs inhibits AQP2 apical fusion, while Munc18b knockdown causes 7-fold increase in AQP2 membrane fusion without stimulation.","method":"Co-immunoprecipitation of SNARE proteins with AQP2 vesicles, siRNA knockdown, apical surface biotinylation in MCD4 renal cells","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 — reciprocal co-IP combined with siRNA functional knockdown and biotinylation assays","pmids":["18505797"],"is_preprint":false},{"year":2008,"finding":"AKAP220 binds AQP2 (identified by yeast two-hybrid screen) and colocalizes with AQP2 in the cytosol of inner medullary collecting ducts; AKAP220 co-expression increases forskolin-mediated phosphorylation of AQP2, suggesting it recruits PKA to AQP2-bearing vesicles.","method":"Yeast two-hybrid screen, double immunofluorescence, immunoelectron microscopy, co-expression phosphorylation assay in COS cells","journal":"Kidney international","confidence":"Medium","confidence_rationale":"Tier 3 — yeast two-hybrid plus colocalization and co-expression assay; no reciprocal co-IP","pmids":["19008911"],"is_preprint":false},{"year":2008,"finding":"Annexin-2 is required for cAMP-induced AQP2 exocytosis; forskolin causes annexin-2 redistribution to lipid rafts at the plasma membrane, and an annexin-2 N-terminal peptide that blocks p11 binding inhibits AQP2-vesicle fusion to plasma membranes in vitro and prevents osmotic water permeability increase in intact cells.","method":"Cell fractionation, lipid raft analysis, in vitro vesicle-plasma membrane fusion fluorescence assay, annexin-2 peptide introduction in renal cells","journal":"Pflugers Archiv","confidence":"Medium","confidence_rationale":"Tier 2 — in vitro reconstitution of fusion process combined with cellular functional assay","pmids":["18389276"],"is_preprint":false},{"year":2011,"finding":"AQP2 directly interacts with integrin β1 via an RGD domain in its external C-loop; RGD-containing peptides increase AQP2 membrane expression in the absence of vasopressin through cAMP- or calcium-dependent pathways.","method":"Co-immunoprecipitation of AQP2 and integrin β1 in renal tissue and MCD4 cells, confocal microscopy, cell surface biotinylation, FRET-based cAMP measurement, calcium imaging","journal":"Cellular physiology and biochemistry","confidence":"Medium","confidence_rationale":"Tier 3 — single co-IP plus functional follow-up with RGD peptides","pmids":["21691091"],"is_preprint":false},{"year":2011,"finding":"AS160, a Rab GAP protein and Akt substrate, regulates AQP2 trafficking; dDAVP stimulates AS160 phosphorylation via PI3K/Akt, and siRNA knockdown of AS160 causes increased AQP2 plasma membrane expression without hormonal stimulation.","method":"siRNA knockdown, immunocytochemistry, cell surface biotinylation, phospho-Akt and phospho-AS160 immunoblotting in M-1 and mpkCCDc14 cells","journal":"American journal of physiology. Renal physiology","confidence":"Medium","confidence_rationale":"Tier 2 — siRNA knockdown with biotinylation readout; mechanism of Rab GAP involvement inferred","pmids":["21511697"],"is_preprint":false},{"year":2011,"finding":"Vasopressin/forskolin-mediated F-actin depolymerization is dependent on AQP2 expression; cells lacking AQP2 do not show VP/FK-mediated F-actin depolymerization, and siRNA knockdown of AQP2 significantly reduces this response.","method":"F-actin quantification, immunofluorescence, siRNA knockdown of AQP2 in MDCK and LLC-PK1 cells with varying AQP2 expression levels","journal":"Biology open","confidence":"Medium","confidence_rationale":"Tier 2 — correlation across multiple cell lines confirmed by siRNA knockdown","pmids":["23213402"],"is_preprint":false},{"year":2012,"finding":"TRPC3 physically associates with AQP2 (co-immunoprecipitation) and co-localizes with AQP2 in intracellular vesicles; vasopressin causes co-insertion of TRPC3 and AQP2 into the apical membrane, and TRPC3 mediates transepithelial Ca2+ flux in principal cells.","method":"Co-immunoprecipitation of TRPC3 and AQP2 from kidney medulla and M1/IMCD-3 cells, immunofluorescence, dominant-negative TRPC3, 45Ca2+ flux assay","journal":"American journal of physiology. Renal physiology","confidence":"High","confidence_rationale":"Tier 2 — reciprocal co-IP, dominant-negative functional assay, and localization with physiological readout","pmids":["17699554"],"is_preprint":false},{"year":2012,"finding":"AQP2 interacts with TRPV4, and the presence of AQP2 enables TRPV4 activation by hypotonicity; TRPV4 translocation to the plasma membrane is required for the AQP2-dependent regulatory volume decrease response.","method":"Calcium imaging, RVD measurement, ruthenium red block, TRPV4 expression and plasma membrane translocation assays in WT-RCCD1 vs. AQP2-RCCD1 cells","journal":"Journal of cellular biochemistry","confidence":"Medium","confidence_rationale":"Tier 3 — functional evidence in transfected cells; direct physical interaction not confirmed by co-IP in this paper","pmids":["21938744"],"is_preprint":false},{"year":2013,"finding":"AQP5 directly interacts with AQP2 and impairs AQP2 cell surface localization; the AQP5/AQP2 complex partially resides in the ER/Golgi.","method":"Co-immunoprecipitation, cell surface biotinylation assay, colocalization, luciferase reporter assay in IMCD3, MLE-15, and 293T cells","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 3 — co-IP and biotinylation in heterologous systems; physiological context requires further validation","pmids":["23326416"],"is_preprint":false},{"year":2017,"finding":"NEDD4 and NEDD4L E3 ubiquitin ligases mediate ubiquitination and degradation of AQP2, but require NDFIP1 or NDFIP2 as adaptors to connect them to AQP2; PY-motif-lacking NDFIP variants fail to support ubiquitination.","method":"Membrane yeast two-hybrid (NDFIP2-AQP2 interaction), siRNA knockdown of NEDD4, NEDD4L, NDFIP1, NDFIP2 in mpkCCD cells; ubiquitination and degradation assays in HEK293 cells","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (Y2H, siRNA, ubiquitination assay) with mutant controls","pmids":["28931009"],"is_preprint":false},{"year":2017,"finding":"AQP2 phosphorylation allosterically controls its interaction with the lysosomal trafficking protein LIP5; non-phosphorylated AQP2 binds LIP5 with highest affinity, while phosphomimetic S256E shows the greatest reduction in LIP5 affinity, linking phosphorylation state to lysosomal targeting.","method":"Far-Western blot, microscale thermophoresis, CD spectroscopy, phosphomimetic AQP2 mutants (S256E, S261E, S264E, T269E)","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro binding assays with purified proteins and systematic mutagenesis","pmids":["28710278"],"is_preprint":false},{"year":2017,"finding":"Ezrin directly interacts with AQP2 C-terminus through its N-terminal FERM domain; this interaction facilitates AQP2 endocytosis, as ezrin knockdown increases membrane AQP2 and reduces endocytosis. Vasopressin causes redistribution of both ezrin and AQP2 to the apical membrane.","method":"Co-IP with anti-AQP2 antibody (proteomics), co-IP with anti-ezrin antibody, pulldown with purified recombinant full-length and FERM-domain ezrin, shRNA knockdown, immunofluorescence in collecting duct cells","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1 — direct interaction confirmed with purified recombinant proteins; functional validation by knockdown","pmids":["28754689"],"is_preprint":false},{"year":2017,"finding":"PP2C (protein phosphatase 2C) is responsible for vasopressin-induced dephosphorylation of AQP2 at Ser261; this dephosphorylation is independent of S256 phosphorylation and does not acutely regulate AQP2 membrane trafficking.","method":"Phosphatase inhibitors (sanguinarine for PP2C, okadaic acid for PP2A, cyclosporine for PP2B), phospho-specific AQP2 antibodies, AQP2-S256A mutant in renal cells and kidney tissue","journal":"American journal of physiology. Renal physiology","confidence":"High","confidence_rationale":"Tier 2 — systematic pharmacological dissection with phospho-specific readouts and mutant controls","pmids":["28381458"],"is_preprint":false},{"year":2016,"finding":"Wnt5a regulates AQP2 protein expression, phosphorylation, and apical membrane trafficking via calcineurin signaling (independently of cAMP/PKA); calcineurin activator arachidonic acid produces vasopressin-like effects on AQP2 trafficking and increases urine osmolality in an NDI mouse model.","method":"Wnt5a treatment of collecting duct cells and NDI mouse model, calcineurin inhibitor/activator experiments, cAMP measurement, PKA activity assay, urine osmolality measurement","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal approaches in vitro and in vivo with defined molecular pathway","pmids":["27892464"],"is_preprint":false},{"year":2018,"finding":"Inhibition of AKAP-PKA interactions by FMP-API-1 increases free PKA activity, phosphorylates AQP2, and increases AQP2 membrane targeting and urine osmolality in vivo to the same extent as vasopressin, bypassing V2R mutations.","method":"cAMP/PKA activity assays in cortical collecting duct cells, AQP2 phosphorylation immunoblotting, urine osmolality measurement in V2R-inhibited mice","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — mechanistic pathway defined in vitro and validated in vivo","pmids":["29650969"],"is_preprint":false},{"year":2014,"finding":"Tankyrase-mediated β-catenin signaling is required for vasopressin-induced AQP2 expression; tankyrase inhibition (XAV939) or β-catenin siRNA knockdown attenuates dDAVP-induced AQP2 upregulation and reduces nuclear translocation of phospho-β-catenin (S552), without affecting PKA activation.","method":"Tankyrase inhibitor (XAV939), siRNA knockdown of tankyrase and β-catenin, FRET-based PKA activity, nuclear translocation assay, luciferase reporter in mpkCCDc14 cells","journal":"American journal of physiology. Renal physiology","confidence":"Medium","confidence_rationale":"Tier 2 — multiple approaches but mechanism linking tankyrase to AQP2 transcription is partially indirect","pmids":["25520007"],"is_preprint":false},{"year":2013,"finding":"Hsp70 plays a role in AQP2 trafficking to the apical plasma membrane; Hsp70-2 knockdown attenuates forskolin-induced AQP2 apical membrane targeting and reduces AQP2 phosphorylation at Ser256.","method":"siRNA knockdown of Hsp70-2, cell surface biotinylation, immunoblotting for pS256-AQP2, luciferase reporter assay for Hsp70-2 promoter in mpkCCDc14 cells","journal":"American journal of physiology. Renal physiology","confidence":"Medium","confidence_rationale":"Tier 2 — siRNA knockdown with biotinylation and phosphorylation readouts in a defined cell system","pmids":["23303413"],"is_preprint":false},{"year":2021,"finding":"The PDCD10-STK24/25 complex regulates AQP2 membrane targeting; mice deficient in Pdcd10 or Stk24/25 in kidney tubules develop polyuria with decreased AQP2 in the apical membrane, associated with increased p-ERM expression that impairs vesicle trafficking. Erlotinib treatment normalizes AQP2 membrane abundance.","method":"Conditional knockout mice, immunofluorescence, immunoblotting, Erlotinib treatment rescue experiment","journal":"JCI insight","confidence":"Medium","confidence_rationale":"Tier 2 — genetic KO with defined phenotype, but molecular mechanism linking PDCD10-STK to ERM-AQP2 is partially inferred","pmids":["34156031"],"is_preprint":false},{"year":2012,"finding":"Phosphorylation at S256 and S269 both contribute to retention of AQP2 at the plasma membrane; S256D mutations slow internalization while S261A and S269D mutations slow development of intracellular accumulation. Differentially phosphorylated AQP2 mutants show distinct recycling kinetics but similar colocalization with Rab11, clathrin, and other markers.","method":"20°C cold block internalization assay, rewarming assay, colocalization with vesicular markers in LLC-PK1 cells expressing AQP2 phospho-mutants","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 — systematic phospho-mutant analysis with multiple trafficking readouts","pmids":["22403603"],"is_preprint":false},{"year":2016,"finding":"The degree of S256 phosphorylation in the AQP2 tetramer controls plasma membrane diffusion speed; tetramers with 2–4 phosphorylated monomers diffuse faster than those with 0–1 phosphorylated monomers, which may determine retention time in the membrane vs. endocytosis.","method":"k-space Image Correlation Spectroscopy (kICS) of AQP2-S256D/S256A mixed-tetramer constructs in live cells","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 — quantitative single-molecule-level diffusion analysis with systematic phosphomimetic titration","pmids":["27801846"],"is_preprint":false},{"year":1997,"finding":"Substitution of the mercury-sensitive Cys181 in AQP2 abolishes plasma membrane targeting and causes ER retention, unlike the equivalent mutation in AQP1 (C189S) which does not affect routing, indicating structural differences between AQP1 and AQP2 at this position.","method":"Xenopus oocyte water permeability assays, immunocytochemistry, immunoblotting with AQP2-C181S and AQP1-C189S mutants","journal":"The American journal of physiology","confidence":"Medium","confidence_rationale":"Tier 1 — functional oocyte assay with site-directed mutagenesis, but limited mechanistic depth","pmids":["9321919"],"is_preprint":false},{"year":1999,"finding":"AQP2 interactome identified by chemical cross-linking and LC-MS/MS in native rat IMCD cells reveals multiple Rab proteins (Rab1a, Rab2a, Rab5b, Rab5c, Rab7a, Rab11a, Rab11b, Rab14, Rab17) as AQP2-interacting proteins involved in membrane trafficking.","method":"Chemical cross-linking, anti-AQP2 immunoprecipitation, LC-MS/MS proteomics in rat IMCD","journal":"American journal of physiology. Cell physiology","confidence":"Medium","confidence_rationale":"Tier 2 — systematic mass spectrometry interactome in native tissue with multiple replicates","pmids":["29046292"],"is_preprint":false}],"current_model":"AQP2 is a vasopressin-regulated water channel in renal collecting duct principal cells that resides in intracellular Rab11-positive vesicles at baseline and is driven to the apical plasma membrane by a cascade in which vasopressin activates V2R→adenylate cyclase→cAMP→PKA, leading to Ser256 phosphorylation (necessary but not sufficient for apical targeting), RhoA inactivation via serine phosphorylation and RhoGDI association, actin depolymerization facilitated by ERM proteins (especially moesin and ezrin), and SNARE-mediated (VAMP2/3, syntaxin-3, SNAP23) vesicle fusion to the apical membrane; AKAP220 recruits PKA to AQP2 vesicles while Munc18b negatively regulates SNARE complex formation; AQP2 is retrieved from the membrane by clathrin-mediated endocytosis (facilitated by direct ezrin–AQP2 interaction), sorted through EEA1-positive early endosomes back to the Rab11 storage compartment, and targeted for lysosomal degradation via NDFIP1/2-mediated NEDD4/NEDD4L ubiquitination; phosphorylation at multiple C-terminal serines (S256, S261, S264, S269) differentially regulates trafficking kinetics, protein–protein interactions (e.g., phospho-S256 reduces LIP5 affinity to oppose lysosomal targeting), and membrane diffusion, with PP2C responsible for VP-induced S261 dephosphorylation; additional regulatory inputs include angiotensin II (via AT1R/cAMP/PKC), Wnt5a/calcineurin, tankyrase/β-catenin-mediated transcription, miRNAs, and AKAP-disruptor compounds, all converging to control AQP2 apical membrane abundance and renal water reabsorption."},"narrative":{"teleology":[{"year":1995,"claim":"Establishing that AQP2 undergoes regulated redistribution from cytoplasmic vesicles to the apical membrane in response to vasopressin answered the fundamental question of how collecting duct water permeability is acutely controlled and identified microtubule-dependent vesicle trafficking as the underlying mechanism.","evidence":"Immunofluorescence and immunogold EM in Brattleboro rats ± vasopressin and colchicine","pmids":["7539496"],"confidence":"High","gaps":["Motor proteins driving microtubule-dependent transport not identified","Signal transduction pathway between V2R and vesicle movement undefined"]},{"year":1997,"claim":"Demonstrating that recessive NDI mutations produce functional water channels that are ER-retained, and that V2R activates AQP2 transcription via CREB/AP1, established that both trafficking competence and transcriptional regulation are critical for AQP2 function, and that disease arises from misrouting rather than channel dysfunction.","evidence":"Xenopus oocyte water permeability with NDI mutants (A147T, T126M, N68S); AQP2 promoter-reporter assays with V2R activation in LLC-PK1 cells","pmids":["9048343","9140044"],"confidence":"High","gaps":["Chaperone interactions responsible for ER quality control of AQP2 mutants not identified","Relative contribution of transcriptional vs. trafficking regulation to water reabsorption in vivo unclear"]},{"year":2000,"claim":"Identification of Ser256 as the PKA phosphorylation site required for vasopressin-induced apical targeting, alongside discovery of constitutive AQP2 recycling and PGE2-mediated retrieval independent of S256 dephosphorylation, revealed that AQP2 membrane abundance is governed by the balance of regulated exocytosis and phosphorylation-independent endocytosis.","evidence":"Phospho-S256-specific antibodies with immunoEM in rat kidney; temperature-block and bafilomycin experiments in LLC-PK1 cells; PGE2 retrieval assays in rat IMCD","pmids":["10644653","10662736","10710543"],"confidence":"High","gaps":["Identity of kinases/phosphatases acting on other C-terminal serines unknown","Endocytic machinery components mediating retrieval not defined"]},{"year":2003,"claim":"Showing that cAMP/PKA inhibits RhoA via phosphorylation (promoting RhoGDI association and actin depolymerization) and that blocking clathrin-mediated endocytosis causes S256-independent AQP2 membrane accumulation established that regulated exocytosis requires cytoskeletal remodeling while constitutive recycling is clathrin-dependent.","evidence":"RhoA-GTP pull-down and RhoA-RhoGDI co-IP in CD8 cells; dominant-negative dynamin and mβCD in LLC-PK1/IMCD cells with S256A mutant controls","pmids":["12640036","14519593"],"confidence":"High","gaps":["Specific Rho-GEFs and GAPs involved not identified","How S256 phosphorylation intersects with endocytic machinery unknown"]},{"year":2005,"claim":"Mapping the AQP2 intracellular itinerary through Rab11-positive storage compartments and EEA1-positive early endosomes, together with demonstrating moesin-dependent actin remodeling during exocytosis, defined the vesicular sorting pathway and the ERM-actin axis as a trafficking checkpoint.","evidence":"Double immunolabeling, Rab11 siRNA, nocodazole/latrunculin pharmacology in MDCK cells; moesin subcellular redistribution and peptide competitor in renal cells","pmids":["16049696","16046477"],"confidence":"High","gaps":["Which Rab effectors mediate Rab11-to-apical delivery unclear","Functional link between moesin phosphorylation state and AQP2 exocytosis not fully resolved"]},{"year":2006,"claim":"Collecting-duct-specific AQP2 knockout producing lethal polyuria, together with identification of calcineurin and angiotensin II/AT1R as additional regulators of AQP2 trafficking, established AQP2 as non-redundant for water balance and revealed cAMP-independent signaling inputs.","evidence":"Cre/loxP conditional knockout mice; CnAα-null mice and cyclosporin A; AngII/candesartan/PKC inhibitors in primary IMCD cells","pmids":["16581908","16735444","16896188"],"confidence":"High","gaps":["Calcineurin substrates relevant to AQP2 trafficking not identified","How PKC integrates with PKA-dependent phosphorylation at S256 unresolved"]},{"year":2008,"claim":"Identification of VAMP2/3, syntaxin-3, SNAP23, and Munc18b as the SNARE machinery for AQP2 vesicle fusion, and AKAP220 as a PKA-recruiting scaffold on AQP2 vesicles, provided the molecular basis for both the final membrane fusion step and compartmentalized PKA signaling.","evidence":"Co-IP of SNAREs with AQP2 vesicles and siRNA knockdown with biotinylation in MCD4 cells; yeast two-hybrid and co-expression phosphorylation assay for AKAP220","pmids":["18505797","19008911"],"confidence":"High","gaps":["AKAP220 interaction awaits reciprocal co-IP validation","How Munc18b release is triggered upon cAMP stimulation unknown","Specific SNARE regulatory steps (NSF, α-SNAP involvement) not characterized"]},{"year":2012,"claim":"Systematic phospho-mutant analysis revealed that S256, S261, S264, and S269 differentially regulate AQP2 internalization kinetics and membrane retention, while TRPC3 was identified as a co-trafficking partner that inserts with AQP2 into the apical membrane, expanding AQP2's role beyond water transport.","evidence":"Cold-block/rewarming assays with phospho-mutants in LLC-PK1 cells; TRPC3-AQP2 co-IP and dominant-negative TRPC3 with calcium flux in M1/IMCD-3 cells","pmids":["22403603","17699554"],"confidence":"Medium","gaps":["Kinases responsible for S264 and S269 phosphorylation in vivo not definitively identified","Physiological significance of TRPC3 co-trafficking for calcium homeostasis in principal cells unclear"]},{"year":2017,"claim":"Discovery that NDFIP1/2 adaptors recruit NEDD4/NEDD4L to ubiquitinate AQP2 for lysosomal degradation, that phospho-S256 allosterically reduces LIP5 binding to oppose lysosomal targeting, that ezrin directly binds AQP2 to facilitate endocytosis, and that PP2C dephosphorylates S261 defined the molecular logic of AQP2 downregulation and degradation.","evidence":"Membrane Y2H, siRNA of NEDD4/NDFIP in mpkCCD; MST binding with phosphomimetic AQP2 peptides; recombinant ezrin pulldown and shRNA in collecting duct cells; PP2C inhibitor sanguinarine with phospho-specific antibodies","pmids":["28931009","28710278","28754689","28381458"],"confidence":"High","gaps":["Ubiquitination sites on AQP2 not mapped","Whether ezrin and NEDD4 pathways are sequential or parallel not resolved","Structural basis for phosphorylation-dependent LIP5 affinity change unknown"]},{"year":2018,"claim":"Demonstration that AKAP-PKA disruptor FMP-API-1 bypasses V2R to phosphorylate AQP2 and concentrate urine in vivo, and that Wnt5a/calcineurin provides a cAMP-independent AQP2 trafficking pathway, opened pharmacological strategies for treating nephrogenic diabetes insipidus.","evidence":"FMP-API-1 in cortical CD cells and V2R-inhibited mice with urine osmolality; Wnt5a/arachidonic acid in CD cells and NDI mouse model","pmids":["29650969","27892464"],"confidence":"High","gaps":["Off-target effects of AKAP disruption on other renal PKA substrates not characterized","Wnt5a receptor and downstream calcineurin substrates in principal cells not identified"]},{"year":null,"claim":"Key unresolved questions include the high-resolution structural basis for phosphorylation-dependent conformational changes in the AQP2 C-terminus, the complete ubiquitination site map, the identity of specific Rab effectors mediating apical delivery, and whether pharmacological targeting of the Wnt5a/calcineurin or AKAP-disruptor pathways is therapeutically viable for NDI.","evidence":"","pmids":[],"confidence":"Low","gaps":["No high-resolution structure of full-length AQP2 with C-terminal regulatory domain","Ubiquitination sites not mapped","Rab11 effectors for apical delivery not identified","Clinical translation of AKAP disruptors or calcineurin activators untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005215","term_label":"transporter activity","supporting_discovery_ids":[3,14]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,1,7,8]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[0,5,10]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[18]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,6,16,30,31]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[0,5,7,10,17]},{"term_id":"R-HSA-382551","term_label":"Transport of small molecules","supporting_discovery_ids":[3,14]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[1,8,10,28]}],"complexes":["AQP2 homotetramer"],"partners":["EZR","VAMP2","STX3","SNAP23","RAB11A","NDFIP1","NEDD4L","TRPC3"],"other_free_text":[]},"mechanistic_narrative":"AQP2 is the principal vasopressin-regulated water channel of the renal collecting duct, functioning as the rate-limiting step for urinary water reabsorption. In unstimulated cells, AQP2 tetramers reside in Rab11-positive subapical vesicles and constitutively recycle through clathrin-mediated endocytosis; vasopressin binding to V2R activates adenylate cyclase→cAMP→PKA, which phosphorylates AQP2 at Ser256 (necessary but not sufficient for apical targeting), inactivates RhoA to promote actin depolymerization, and drives SNARE-mediated (VAMP2/3, syntaxin-3, SNAP23) fusion of AQP2 vesicles with the apical membrane [PMID:10644653, PMID:12640036, PMID:18505797, PMID:14519593]. Retrieval from the membrane is facilitated by a direct ezrin–AQP2 interaction that promotes clathrin-dependent endocytosis, while lysosomal degradation requires NDFIP1/2-dependent recruitment of NEDD4/NEDD4L E3 ligases for AQP2 ubiquitination, and phosphorylation at Ser256 allosterically reduces LIP5 binding to oppose lysosomal sorting [PMID:28754689, PMID:28931009, PMID:28710278]. Loss-of-function mutations cause nephrogenic diabetes insipidus—recessive forms through ER misrouting of otherwise functional channels, dominant forms through hetero-oligomeric trapping of wild-type AQP2—and collecting-duct-specific knockout produces severe polyuria, confirming AQP2 as essential and non-redundant for renal water conservation [PMID:9048343, PMID:16120822, PMID:16581908]."},"prefetch_data":{"uniprot":{"accession":"P41181","full_name":"Aquaporin-2","aliases":["ADH water channel","Aquaporin-CD","AQP-CD","Collecting duct water channel protein","WCH-CD","Water channel protein for renal collecting duct"],"length_aa":271,"mass_kda":28.8,"function":"Forms a water-specific channel that provides the plasma membranes of renal collecting duct with high permeability to water, thereby permitting water to move in the direction of an osmotic gradient (PubMed:15509592, PubMed:7510718, PubMed:7524315, PubMed:8140421, PubMed:8584435). Plays an essential role in renal water homeostasis (PubMed:15509592, PubMed:7524315, PubMed:8140421). Could also be permeable to glycerol (PubMed:8584435)","subcellular_location":"Apical cell membrane; Basolateral cell membrane; Cell membrane; Cytoplasmic vesicle membrane; Golgi apparatus, trans-Golgi network membrane","url":"https://www.uniprot.org/uniprotkb/P41181/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/AQP2","classification":"Not Classified","n_dependent_lines":5,"n_total_lines":1208,"dependency_fraction":0.0041390728476821195},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/AQP2","total_profiled":1310},"omim":[{"mim_id":"614112","title":"MICRO RNA 320A; MIR320A","url":"https://www.omim.org/entry/614112"},{"mim_id":"610130","title":"SOLUTE CARRIER FAMILY 26 (SULFATE TRANSPORTER), MEMBER 1; SLC26A1","url":"https://www.omim.org/entry/610130"},{"mim_id":"603750","title":"AQUAPORIN 8; AQP8","url":"https://www.omim.org/entry/603750"},{"mim_id":"602974","title":"AQUAPORIN 7; AQP7","url":"https://www.omim.org/entry/602974"},{"mim_id":"602024","title":"CHLORIDE CHANNEL, KIDNEY, A; CLCNKA","url":"https://www.omim.org/entry/602024"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Group enriched","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"kidney","ntpm":450.4},{"tissue":"seminal vesicle","ntpm":200.5}],"url":"https://www.proteinatlas.org/search/AQP2"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"P41181","domains":[{"cath_id":"1.20.1080.10","chopping":"4-227","consensus_level":"high","plddt":96.7947,"start":4,"end":227}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P41181","model_url":"https://alphafold.ebi.ac.uk/files/AF-P41181-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P41181-F1-predicted_aligned_error_v6.png","plddt_mean":91.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=AQP2","jax_strain_url":"https://www.jax.org/strain/search?query=AQP2"},"sequence":{"accession":"P41181","fasta_url":"https://rest.uniprot.org/uniprotkb/P41181.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P41181/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P41181"}},"corpus_meta":[{"pmid":"7539496","id":"PMC_7539496","title":"The AQP2 water channel: effect of vasopressin treatment, microtubule disruption, and distribution in neonatal rats.","date":"1995","source":"The Journal of membrane biology","url":"https://pubmed.ncbi.nlm.nih.gov/7539496","citation_count":207,"is_preprint":false},{"pmid":"15703994","id":"PMC_15703994","title":"Distribution of AQP2 and AQP3 water channels in human tissue microarrays.","date":"2005","source":"Journal of molecular histology","url":"https://pubmed.ncbi.nlm.nih.gov/15703994","citation_count":162,"is_preprint":false},{"pmid":"10644653","id":"PMC_10644653","title":"Localization and regulation of PKA-phosphorylated AQP2 in response to V(2)-receptor agonist/antagonist treatment.","date":"2000","source":"American journal of physiology. Renal physiology","url":"https://pubmed.ncbi.nlm.nih.gov/10644653","citation_count":159,"is_preprint":false},{"pmid":"9140044","id":"PMC_9140044","title":"Adenylate cyclase-coupled vasopressin receptor activates AQP2 promoter via a dual effect on CRE and AP1 elements.","date":"1997","source":"The American journal of physiology","url":"https://pubmed.ncbi.nlm.nih.gov/9140044","citation_count":154,"is_preprint":false},{"pmid":"9048343","id":"PMC_9048343","title":"New mutations in the AQP2 gene in nephrogenic diabetes insipidus resulting in functional but misrouted water channels.","date":"1997","source":"Journal of the American Society of Nephrology : JASN","url":"https://pubmed.ncbi.nlm.nih.gov/9048343","citation_count":130,"is_preprint":false},{"pmid":"16581908","id":"PMC_16581908","title":"Severe urinary concentrating defect in renal collecting duct-selective AQP2 conditional-knockout mice.","date":"2006","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/16581908","citation_count":128,"is_preprint":false},{"pmid":"14519593","id":"PMC_14519593","title":"Inhibition of endocytosis causes phosphorylation (S256)-independent plasma membrane accumulation of AQP2.","date":"2003","source":"American journal of physiology. Renal physiology","url":"https://pubmed.ncbi.nlm.nih.gov/14519593","citation_count":111,"is_preprint":false},{"pmid":"10710543","id":"PMC_10710543","title":"Prostaglandin E(2) interaction with AVP: effects on AQP2 phosphorylation and distribution.","date":"2000","source":"American journal of physiology. Renal physiology","url":"https://pubmed.ncbi.nlm.nih.gov/10710543","citation_count":111,"is_preprint":false},{"pmid":"9688853","id":"PMC_9688853","title":"Expression of an AQP2 Cre recombinase transgene in kidney and male reproductive system of transgenic mice.","date":"1998","source":"The American journal of physiology","url":"https://pubmed.ncbi.nlm.nih.gov/9688853","citation_count":109,"is_preprint":false},{"pmid":"12640036","id":"PMC_12640036","title":"cAMP-induced AQP2 translocation is associated with RhoA inhibition through RhoA phosphorylation and interaction with RhoGDI.","date":"2003","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/12640036","citation_count":106,"is_preprint":false},{"pmid":"16896188","id":"PMC_16896188","title":"Increased AQP2 targeting in primary cultured IMCD cells in response to angiotensin II through AT1 receptor.","date":"2006","source":"American journal of physiology. Renal physiology","url":"https://pubmed.ncbi.nlm.nih.gov/16896188","citation_count":86,"is_preprint":false},{"pmid":"15625084","id":"PMC_15625084","title":"Bidirectional regulation of AQP2 trafficking and recycling: involvement of AQP2-S256 phosphorylation.","date":"2004","source":"American journal of physiology. Renal physiology","url":"https://pubmed.ncbi.nlm.nih.gov/15625084","citation_count":83,"is_preprint":false},{"pmid":"15585668","id":"PMC_15585668","title":"Angiotensin II AT1 receptor blockade decreases vasopressin-induced water reabsorption and AQP2 levels in NaCl-restricted rats.","date":"2004","source":"American journal of physiology. Renal physiology","url":"https://pubmed.ncbi.nlm.nih.gov/15585668","citation_count":79,"is_preprint":false},{"pmid":"23770358","id":"PMC_23770358","title":"Actin directly interacts with different membrane channel proteins and influences channel activities: AQP2 as a model.","date":"2013","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/23770358","citation_count":67,"is_preprint":false},{"pmid":"10806109","id":"PMC_10806109","title":"The phosphatase inhibitor okadaic acid induces AQP2 translocation independently from AQP2 phosphorylation in renal collecting duct cells.","date":"2000","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/10806109","citation_count":66,"is_preprint":false},{"pmid":"16049696","id":"PMC_16049696","title":"Differential regulation of AQP2 trafficking in endosomes by microtubules and actin filaments.","date":"2005","source":"Histochemistry and cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/16049696","citation_count":65,"is_preprint":false},{"pmid":"16120822","id":"PMC_16120822","title":"Lack of arginine vasopressin-induced phosphorylation of aquaporin-2 mutant AQP2-R254L explains dominant nephrogenic diabetes insipidus.","date":"2005","source":"Journal of the American Society of Nephrology : JASN","url":"https://pubmed.ncbi.nlm.nih.gov/16120822","citation_count":65,"is_preprint":false},{"pmid":"11249863","id":"PMC_11249863","title":"Compensatory increase in AQP2, p-AQP2, and AQP3 expression in rats with diabetes mellitus.","date":"2001","source":"American journal of physiology. Renal physiology","url":"https://pubmed.ncbi.nlm.nih.gov/11249863","citation_count":64,"is_preprint":false},{"pmid":"10662736","id":"PMC_10662736","title":"Recycling of AQP2 occurs through a temperature- and bafilomycin-sensitive trans-Golgi-associated compartment.","date":"2000","source":"American journal of physiology. Renal physiology","url":"https://pubmed.ncbi.nlm.nih.gov/10662736","citation_count":64,"is_preprint":false},{"pmid":"12388395","id":"PMC_12388395","title":"Osmolality and solute composition are strong regulators of AQP2 expression in renal principal cells.","date":"2002","source":"American journal of physiology. Renal physiology","url":"https://pubmed.ncbi.nlm.nih.gov/12388395","citation_count":62,"is_preprint":false},{"pmid":"16046477","id":"PMC_16046477","title":"Actin remodeling requires ERM function to facilitate AQP2 apical targeting.","date":"2005","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/16046477","citation_count":60,"is_preprint":false},{"pmid":"16159898","id":"PMC_16159898","title":"Aldosterone increases urine production and decreases apical AQP2 expression in rats with diabetes insipidus.","date":"2005","source":"American journal of physiology. Renal physiology","url":"https://pubmed.ncbi.nlm.nih.gov/16159898","citation_count":58,"is_preprint":false},{"pmid":"21938744","id":"PMC_21938744","title":"Functional interaction between AQP2 and TRPV4 in renal cells.","date":"2012","source":"Journal of cellular biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/21938744","citation_count":58,"is_preprint":false},{"pmid":"17699554","id":"PMC_17699554","title":"Vasopressin-induced membrane trafficking of TRPC3 and AQP2 channels in cells of the rat renal collecting duct.","date":"2007","source":"American journal of physiology. Renal physiology","url":"https://pubmed.ncbi.nlm.nih.gov/17699554","citation_count":58,"is_preprint":false},{"pmid":"15922355","id":"PMC_15922355","title":"The 4.5 A structure of human AQP2.","date":"2005","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/15922355","citation_count":56,"is_preprint":false},{"pmid":"9486234","id":"PMC_9486234","title":"Dynein and dynactin colocalize with AQP2 water channels in intracellular vesicles from kidney collecting duct.","date":"1998","source":"The American journal of physiology","url":"https://pubmed.ncbi.nlm.nih.gov/9486234","citation_count":55,"is_preprint":false},{"pmid":"10919858","id":"PMC_10919858","title":"Vasopressin V(2)-receptor-dependent regulation of AQP2 expression in Brattleboro rats.","date":"2000","source":"American journal of physiology. Renal physiology","url":"https://pubmed.ncbi.nlm.nih.gov/10919858","citation_count":54,"is_preprint":false},{"pmid":"36117171","id":"PMC_36117171","title":"Micropeptide MIAC inhibits the tumor progression by interacting with AQP2 and inhibiting EREG/EGFR signaling in renal cell carcinoma.","date":"2022","source":"Molecular cancer","url":"https://pubmed.ncbi.nlm.nih.gov/36117171","citation_count":54,"is_preprint":false},{"pmid":"12453871","id":"PMC_12453871","title":"Axial heterogeneity in basolateral AQP2 localization in rat kidney: effect of vasopressin.","date":"2002","source":"American journal of physiology. Renal physiology","url":"https://pubmed.ncbi.nlm.nih.gov/12453871","citation_count":54,"is_preprint":false},{"pmid":"27892464","id":"PMC_27892464","title":"Wnt5a induces renal AQP2 expression by activating calcineurin signalling pathway.","date":"2016","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/27892464","citation_count":53,"is_preprint":false},{"pmid":"23326416","id":"PMC_23326416","title":"Aqp5 is a new transcriptional target of Dot1a and a regulator of Aqp2.","date":"2013","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/23326416","citation_count":53,"is_preprint":false},{"pmid":"17376764","id":"PMC_17376764","title":"Long-term aldosterone treatment induces decreased apical but increased basolateral expression of AQP2 in CCD of rat kidney.","date":"2007","source":"American journal of physiology. Renal physiology","url":"https://pubmed.ncbi.nlm.nih.gov/17376764","citation_count":53,"is_preprint":false},{"pmid":"9124394","id":"PMC_9124394","title":"Alteration in water channel AQP-2 by removal of AVP stimulation in collecting duct cells of dehydrated rats.","date":"1997","source":"The American journal of physiology","url":"https://pubmed.ncbi.nlm.nih.gov/9124394","citation_count":52,"is_preprint":false},{"pmid":"27099493","id":"PMC_27099493","title":"Hyaluronic acid reagent functional chitosan-PEI conjugate with AQP2-siRNA suppressed endometriotic lesion formation.","date":"2016","source":"International journal of nanomedicine","url":"https://pubmed.ncbi.nlm.nih.gov/27099493","citation_count":49,"is_preprint":false},{"pmid":"15100356","id":"PMC_15100356","title":"Upregulation of urea transporter UT-A2 and water channels AQP2 and AQP3 in mice lacking urea transporter UT-B.","date":"2004","source":"Journal of the American Society of Nephrology : JASN","url":"https://pubmed.ncbi.nlm.nih.gov/15100356","citation_count":47,"is_preprint":false},{"pmid":"23150186","id":"PMC_23150186","title":"Hereditary nephrogenic diabetes insipidus in Japanese patients: analysis of 78 families and report of 22 new mutations in AVPR2 and AQP2.","date":"2012","source":"Clinical and experimental nephrology","url":"https://pubmed.ncbi.nlm.nih.gov/23150186","citation_count":47,"is_preprint":false},{"pmid":"25694485","id":"PMC_25694485","title":"Aliskiren restores renal AQP2 expression during unilateral ureteral obstruction by inhibiting the inflammasome.","date":"2015","source":"American journal of physiology. Renal physiology","url":"https://pubmed.ncbi.nlm.nih.gov/25694485","citation_count":44,"is_preprint":false},{"pmid":"15494547","id":"PMC_15494547","title":"Insulin potentiates AVP-induced AQP2 expression in cultured renal collecting duct principal cells.","date":"2004","source":"American journal of physiology. Renal physiology","url":"https://pubmed.ncbi.nlm.nih.gov/15494547","citation_count":44,"is_preprint":false},{"pmid":"18505797","id":"PMC_18505797","title":"AQP2 exocytosis in the renal collecting duct -- involvement of SNARE isoforms and the regulatory role of Munc18b.","date":"2008","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/18505797","citation_count":44,"is_preprint":false},{"pmid":"19193739","id":"PMC_19193739","title":"Rapid and segmental specific dysregulation of AQP2, S256-pAQP2 and renal sodium transporters in rats with LPS-induced endotoxaemia.","date":"2009","source":"Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association","url":"https://pubmed.ncbi.nlm.nih.gov/19193739","citation_count":43,"is_preprint":false},{"pmid":"19008911","id":"PMC_19008911","title":"AKAP220 colocalizes with AQP2 in the inner medullary collecting ducts.","date":"2008","source":"Kidney international","url":"https://pubmed.ncbi.nlm.nih.gov/19008911","citation_count":43,"is_preprint":false},{"pmid":"12388409","id":"PMC_12388409","title":"AQP3, p-AQP2, and AQP2 expression is reduced in polyuric rats with hypercalcemia: prevention by cAMP-PDE inhibitors.","date":"2002","source":"American journal of physiology. Renal physiology","url":"https://pubmed.ncbi.nlm.nih.gov/12388409","citation_count":43,"is_preprint":false},{"pmid":"32929357","id":"PMC_32929357","title":"Statins ameliorate cholesterol-induced inflammation and improve AQP2 expression by inhibiting NLRP3 activation in the kidney.","date":"2020","source":"Theranostics","url":"https://pubmed.ncbi.nlm.nih.gov/32929357","citation_count":42,"is_preprint":false},{"pmid":"22403603","id":"PMC_22403603","title":"Differential, phosphorylation dependent trafficking of AQP2 in LLC-PK1 cells.","date":"2012","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/22403603","citation_count":41,"is_preprint":false},{"pmid":"21691091","id":"PMC_21691091","title":"Integrin signaling modulates AQP2 trafficking via Arg-Gly-Asp (RGD) motif.","date":"2011","source":"Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/21691091","citation_count":41,"is_preprint":false},{"pmid":"19776175","id":"PMC_19776175","title":"Changes of renal AQP2, ENaC, and NHE3 in experimentally induced heart failure: response to angiotensin II AT1 receptor blockade.","date":"2009","source":"American journal of physiology. Renal physiology","url":"https://pubmed.ncbi.nlm.nih.gov/19776175","citation_count":41,"is_preprint":false},{"pmid":"16735444","id":"PMC_16735444","title":"Loss of calcineurin Aalpha results in altered trafficking of AQP2 and in nephrogenic diabetes insipidus.","date":"2006","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/16735444","citation_count":41,"is_preprint":false},{"pmid":"28931009","id":"PMC_28931009","title":"NDFIP allows NEDD4/NEDD4L-induced AQP2 ubiquitination and degradation.","date":"2017","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/28931009","citation_count":39,"is_preprint":false},{"pmid":"15859948","id":"PMC_15859948","title":"Volume regulation in cortical collecting duct cells: role of AQP2.","date":"2005","source":"Biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/15859948","citation_count":39,"is_preprint":false},{"pmid":"15956775","id":"PMC_15956775","title":"Increased expression of ENaC subunits and increased apical targeting of AQP2 in the kidneys of spontaneously hypertensive rats.","date":"2005","source":"American journal of physiology. Renal physiology","url":"https://pubmed.ncbi.nlm.nih.gov/15956775","citation_count":38,"is_preprint":false},{"pmid":"29650969","id":"PMC_29650969","title":"AKAPs-PKA disruptors increase AQP2 activity independently of vasopressin in a model of nephrogenic diabetes insipidus.","date":"2018","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/29650969","citation_count":38,"is_preprint":false},{"pmid":"12660245","id":"PMC_12660245","title":"Dual influence of aldosterone on AQP2 expression in cultured renal collecting duct principal cells.","date":"2003","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12660245","citation_count":36,"is_preprint":false},{"pmid":"28754689","id":"PMC_28754689","title":"Ezrin directly interacts with AQP2 and promotes its endocytosis.","date":"2017","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/28754689","citation_count":35,"is_preprint":false},{"pmid":"25651560","id":"PMC_25651560","title":"Vasopressin-regulated miRNAs and AQP2-targeting miRNAs in kidney collecting duct cells.","date":"2015","source":"American journal of physiology. Renal physiology","url":"https://pubmed.ncbi.nlm.nih.gov/25651560","citation_count":35,"is_preprint":false},{"pmid":"21511697","id":"PMC_21511697","title":"Emerging role of Akt substrate protein AS160 in the regulation of AQP2 translocation.","date":"2011","source":"American journal of physiology. Renal physiology","url":"https://pubmed.ncbi.nlm.nih.gov/21511697","citation_count":35,"is_preprint":false},{"pmid":"26062878","id":"PMC_26062878","title":"Estradiol regulates AQP2 expression in the collecting duct: a novel inhibitory role for estrogen receptor α.","date":"2015","source":"American journal of physiology. Renal physiology","url":"https://pubmed.ncbi.nlm.nih.gov/26062878","citation_count":34,"is_preprint":false},{"pmid":"20083182","id":"PMC_20083182","title":"Diuretic activity and kidney medulla AQP1, AQP2, AQP3, V2R expression of the aqueous extract of sclerotia of Polyporus umbellatus FRIES in normal rats.","date":"2010","source":"Journal of ethnopharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/20083182","citation_count":34,"is_preprint":false},{"pmid":"35386692","id":"PMC_35386692","title":"AQP2 Promotes Astrocyte Activation by Modulating the TLR4/NFκB-p65 Pathway Following Intracerebral Hemorrhage.","date":"2022","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/35386692","citation_count":33,"is_preprint":false},{"pmid":"15153756","id":"PMC_15153756","title":"Changes of rat kidney AQP2 and Na,K-ATPase mRNA expression in lithium-induced nephrogenic diabetes insipidus.","date":"2004","source":"Nephron. Experimental nephrology","url":"https://pubmed.ncbi.nlm.nih.gov/15153756","citation_count":33,"is_preprint":false},{"pmid":"11181414","id":"PMC_11181414","title":"Fasting downregulates renal water channel AQP2 and causes polyuria.","date":"2001","source":"American journal of physiology. Renal physiology","url":"https://pubmed.ncbi.nlm.nih.gov/11181414","citation_count":33,"is_preprint":false},{"pmid":"23213402","id":"PMC_23213402","title":"AQP2 is necessary for vasopressin- and forskolin-mediated filamentous actin depolymerization in renal epithelial cells.","date":"2011","source":"Biology open","url":"https://pubmed.ncbi.nlm.nih.gov/23213402","citation_count":32,"is_preprint":false},{"pmid":"31490733","id":"PMC_31490733","title":"Renal denervation improves sodium excretion in rats with chronic heart failure: effects on expression of renal ENaC and AQP2.","date":"2019","source":"American journal of physiology. Heart and circulatory physiology","url":"https://pubmed.ncbi.nlm.nih.gov/31490733","citation_count":31,"is_preprint":false},{"pmid":"22166843","id":"PMC_22166843","title":"Acyrthosiphon pisum AQP2: a multifunctional insect aquaglyceroporin.","date":"2011","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/22166843","citation_count":31,"is_preprint":false},{"pmid":"7512890","id":"PMC_7512890","title":"Assignment of the human gene for the water channel of renal collecting duct Aquaporin 2 (AQP2) to chromosome 12 region q12-->q13.","date":"1994","source":"Cytogenetics and cell genetics","url":"https://pubmed.ncbi.nlm.nih.gov/7512890","citation_count":31,"is_preprint":false},{"pmid":"9268644","id":"PMC_9268644","title":"Closely spaced tandem arrangement of AQP2, AQP5, and AQP6 genes in a 27-kilobase segment at chromosome locus 12q13.","date":"1997","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/9268644","citation_count":30,"is_preprint":false},{"pmid":"28710278","id":"PMC_28710278","title":"Phosphorylation of human aquaporin 2 (AQP2) allosterically controls its interaction with the lysosomal trafficking protein LIP5.","date":"2017","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/28710278","citation_count":30,"is_preprint":false},{"pmid":"18389276","id":"PMC_18389276","title":"Functional involvement of Annexin-2 in cAMP induced AQP2 trafficking.","date":"2008","source":"Pflugers Archiv : European journal of physiology","url":"https://pubmed.ncbi.nlm.nih.gov/18389276","citation_count":30,"is_preprint":false},{"pmid":"18094031","id":"PMC_18094031","title":"Role of AQP2 in activation of calcium entry by hypotonicity: implications in cell volume regulation.","date":"2007","source":"American journal of physiology. Renal physiology","url":"https://pubmed.ncbi.nlm.nih.gov/18094031","citation_count":29,"is_preprint":false},{"pmid":"23986519","id":"PMC_23986519","title":"Excretion of urinary exosomal AQP2 in rats is regulated by vasopressin and urinary pH.","date":"2013","source":"American journal of physiology. Renal physiology","url":"https://pubmed.ncbi.nlm.nih.gov/23986519","citation_count":29,"is_preprint":false},{"pmid":"27213818","id":"PMC_27213818","title":"Spilanthol from Acmella Oleracea Lowers the Intracellular Levels of cAMP Impairing NKCC2 Phosphorylation and Water Channel AQP2 Membrane Expression in Mouse Kidney.","date":"2016","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/27213818","citation_count":29,"is_preprint":false},{"pmid":"32993088","id":"PMC_32993088","title":"AQP2: Mutations Associated with Congenital Nephrogenic Diabetes Insipidus and Regulation by Post-Translational Modifications and Protein-Protein Interactions.","date":"2020","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/32993088","citation_count":28,"is_preprint":false},{"pmid":"28381458","id":"PMC_28381458","title":"Protein phosphatase 2C is responsible for VP-induced dephosphorylation of AQP2 serine 261.","date":"2017","source":"American journal of physiology. Renal physiology","url":"https://pubmed.ncbi.nlm.nih.gov/28381458","citation_count":27,"is_preprint":false},{"pmid":"22397925","id":"PMC_22397925","title":"Disruption of cyclooxygenase-2 prevents downregulation of cortical AQP2 and AQP3 in response to bilateral ureteral obstruction in the mouse.","date":"2012","source":"American journal of physiology. Renal physiology","url":"https://pubmed.ncbi.nlm.nih.gov/22397925","citation_count":27,"is_preprint":false},{"pmid":"29478202","id":"PMC_29478202","title":"Activation of AQP2 water channels without vasopressin: therapeutic strategies for congenital nephrogenic diabetes insipidus.","date":"2018","source":"Clinical and experimental nephrology","url":"https://pubmed.ncbi.nlm.nih.gov/29478202","citation_count":26,"is_preprint":false},{"pmid":"7525161","id":"PMC_7525161","title":"Human AQP2 and MIP genes, two members of the MIP family, map within chromosome band 12q13 on the basis of two-color FISH.","date":"1995","source":"Cytogenetics and cell genetics","url":"https://pubmed.ncbi.nlm.nih.gov/7525161","citation_count":26,"is_preprint":false},{"pmid":"25662477","id":"PMC_25662477","title":"Rosiglitazone promotes AQP2 plasma membrane expression in renal cells via a Ca-dependent/cAMP-independent mechanism.","date":"2015","source":"Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/25662477","citation_count":25,"is_preprint":false},{"pmid":"27488997","id":"PMC_27488997","title":"Phosphatase inhibition increases AQP2 accumulation in the rat IMCD apical plasma membrane.","date":"2016","source":"American journal of physiology. Renal physiology","url":"https://pubmed.ncbi.nlm.nih.gov/27488997","citation_count":25,"is_preprint":false},{"pmid":"30345080","id":"PMC_30345080","title":"Estrogen nuclear receptors affect cell migration by altering sublocalization of AQP2 in glioma cell lines.","date":"2018","source":"Cell death discovery","url":"https://pubmed.ncbi.nlm.nih.gov/30345080","citation_count":25,"is_preprint":false},{"pmid":"19014999","id":"PMC_19014999","title":"Alterations of AQP2 expression in trigeminal ganglia in a murine inflammation model.","date":"2008","source":"Neuroscience letters","url":"https://pubmed.ncbi.nlm.nih.gov/19014999","citation_count":25,"is_preprint":false},{"pmid":"18463544","id":"PMC_18463544","title":"Decreased expression of endometrial vessel AQP1 and endometrial epithelium AQP2 related to anovulatory uterine bleeding in premenopausal women.","date":"2008","source":"Menopause (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/18463544","citation_count":24,"is_preprint":false},{"pmid":"25651562","id":"PMC_25651562","title":"Extracellular pH affects phosphorylation and intracellular trafficking of AQP2 in inner medullary collecting duct cells.","date":"2015","source":"American journal of physiology. Renal physiology","url":"https://pubmed.ncbi.nlm.nih.gov/25651562","citation_count":23,"is_preprint":false},{"pmid":"25520007","id":"PMC_25520007","title":"Tankyrase-mediated β-catenin activity regulates vasopressin-induced AQP2 expression in kidney collecting duct mpkCCDc14 cells.","date":"2014","source":"American journal of physiology. Renal physiology","url":"https://pubmed.ncbi.nlm.nih.gov/25520007","citation_count":23,"is_preprint":false},{"pmid":"9321919","id":"PMC_9321919","title":"Importance of the mercury-sensitive cysteine on function and routing of AQP1 and AQP2 in oocytes.","date":"1997","source":"The American journal of physiology","url":"https://pubmed.ncbi.nlm.nih.gov/9321919","citation_count":23,"is_preprint":false},{"pmid":"27801846","id":"PMC_27801846","title":"AQP2 Plasma Membrane Diffusion Is Altered by the Degree of AQP2-S256 Phosphorylation.","date":"2016","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/27801846","citation_count":22,"is_preprint":false},{"pmid":"30551379","id":"PMC_30551379","title":"Zhen-wu-tang attenuates Adriamycin-induced nephropathy via regulating AQP2 and miR-92b.","date":"2018","source":"Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie","url":"https://pubmed.ncbi.nlm.nih.gov/30551379","citation_count":21,"is_preprint":false},{"pmid":"18717646","id":"PMC_18717646","title":"Role of AQP2 during apoptosis in cortical collecting duct cells.","date":"2009","source":"Biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/18717646","citation_count":21,"is_preprint":false},{"pmid":"23303413","id":"PMC_23303413","title":"The role of 70-kDa heat shock protein in dDAVP-induced AQP2 trafficking in kidney collecting duct cells.","date":"2013","source":"American journal of physiology. Renal physiology","url":"https://pubmed.ncbi.nlm.nih.gov/23303413","citation_count":21,"is_preprint":false},{"pmid":"22778181","id":"PMC_22778181","title":"Vasopressin increases S261 phosphorylation in AQP2-P262L, a mutant in recessive nephrogenic diabetes insipidus.","date":"2012","source":"Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association","url":"https://pubmed.ncbi.nlm.nih.gov/22778181","citation_count":21,"is_preprint":false},{"pmid":"33727367","id":"PMC_33727367","title":"Dysregulation of Principal Cell miRNAs Facilitates Epigenetic Regulation of AQP2 and Results in Nephrogenic Diabetes Insipidus.","date":"2021","source":"Journal of the American Society of Nephrology : JASN","url":"https://pubmed.ncbi.nlm.nih.gov/33727367","citation_count":20,"is_preprint":false},{"pmid":"27488999","id":"PMC_27488999","title":"Renal phenotype in Bardet-Biedl syndrome: a combined defect of urinary concentration and dilution is associated with defective urinary AQP2 and UMOD excretion.","date":"2016","source":"American journal of physiology. Renal physiology","url":"https://pubmed.ncbi.nlm.nih.gov/27488999","citation_count":20,"is_preprint":false},{"pmid":"35798273","id":"PMC_35798273","title":"AQP2 trafficking in health and diseases: an updated overview.","date":"2022","source":"The international journal of biochemistry & cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/35798273","citation_count":19,"is_preprint":false},{"pmid":"27980322","id":"PMC_27980322","title":"Sirtuin1 (SIRT1) Regulates Tumor Necrosis Factor-alpha (TNF-α-Induced) Aquaporin-2 (AQP2) Expression in Renal Medullary Collecting Duct Cells Through Inhibiting the NF-κB Pathway.","date":"2016","source":"Medical science monitor basic research","url":"https://pubmed.ncbi.nlm.nih.gov/27980322","citation_count":19,"is_preprint":false},{"pmid":"29524884","id":"PMC_29524884","title":"Steviol slows renal cyst growth by reducing AQP2 expression and promoting AQP2 degradation.","date":"2018","source":"Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie","url":"https://pubmed.ncbi.nlm.nih.gov/29524884","citation_count":19,"is_preprint":false},{"pmid":"14605277","id":"PMC_14605277","title":"Reduced renal expression of AQP2, p-AQP2 and AQP3 in haemorrhagic shock-induced acute renal failure.","date":"2003","source":"Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association","url":"https://pubmed.ncbi.nlm.nih.gov/14605277","citation_count":19,"is_preprint":false},{"pmid":"33427060","id":"PMC_33427060","title":"Activation of TGR5 restores AQP2 expression via the HIF pathway in renal ischemia-reperfusion injury.","date":"2021","source":"American journal of physiology. Renal physiology","url":"https://pubmed.ncbi.nlm.nih.gov/33427060","citation_count":18,"is_preprint":false},{"pmid":"26147297","id":"PMC_26147297","title":"Polarized Trafficking of AQP2 Revealed in Three Dimensional Epithelial Culture.","date":"2015","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/26147297","citation_count":18,"is_preprint":false},{"pmid":"22644838","id":"PMC_22644838","title":"Mutations in the AVPR2, AVP-NPII, and AQP2 genes in Turkish patients with diabetes insipidus.","date":"2012","source":"Endocrine","url":"https://pubmed.ncbi.nlm.nih.gov/22644838","citation_count":18,"is_preprint":false},{"pmid":"34156031","id":"PMC_34156031","title":"Pdcd10-Stk24/25 complex controls kidney water reabsorption by regulating Aqp2 membrane targeting.","date":"2021","source":"JCI insight","url":"https://pubmed.ncbi.nlm.nih.gov/34156031","citation_count":17,"is_preprint":false},{"pmid":"29046292","id":"PMC_29046292","title":"Identification of UT-A1- and AQP2-interacting proteins in rat inner medullary collecting duct.","date":"2017","source":"American journal of physiology. Cell physiology","url":"https://pubmed.ncbi.nlm.nih.gov/29046292","citation_count":17,"is_preprint":false},{"pmid":"22160633","id":"PMC_22160633","title":"Daily variance of urinary excretion of AQP2 determined by sandwich ELISA method.","date":"2011","source":"Clinical and experimental nephrology","url":"https://pubmed.ncbi.nlm.nih.gov/22160633","citation_count":17,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":56724,"output_tokens":9369,"usd":0.155354},"stage2":{"model":"claude-opus-4-6","input_tokens":13274,"output_tokens":3480,"usd":0.230055},"total_usd":0.385409,"stage1_batch_id":"msgbatch_01TqNM6p6FsoMBT2jzQTwKBr","stage2_batch_id":"msgbatch_01Ya473eco8sTHbvRymQaU8o","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1995,\n      \"finding\": \"AQP2 relocates from cytoplasmic vesicles to the apical plasma membrane of collecting duct principal cells following vasopressin treatment, and this apical targeting depends on intact microtubules (colchicine disruption scatters AQP2 throughout the cytoplasm).\",\n      \"method\": \"Immunofluorescence and immunogold electron microscopy in Brattleboro rats with/without vasopressin and colchicine treatment\",\n      \"journal\": \"The Journal of membrane biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct localization experiment with functional consequence, replicated across multiple conditions\",\n      \"pmids\": [\"7539496\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Vasopressin-induced AQP2 trafficking to the apical plasma membrane requires PKA-mediated phosphorylation at Ser256; phospho-AQP2 (pS256) is present in both the apical membrane and intracellular vesicles, and V2 receptor blockade causes near-complete loss of apical pS256-AQP2.\",\n      \"method\": \"Phosphorylation state-specific antibodies, immunoelectron microscopy, immunoblotting in rat kidney with DDAVP or V2R antagonist treatment\",\n      \"journal\": \"American journal of physiology. Renal physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal antibody approach with functional consequence, replicated across multiple conditions\",\n      \"pmids\": [\"10644653\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"Vasopressin acting via the adenylate cyclase-coupled V2 receptor activates AQP2 gene transcription through phosphorylation of CREB and induction of c-Fos, which together bind the CRE and AP1 elements in the AQP2 promoter.\",\n      \"method\": \"Transfection of human AQP2 promoter in LLC-PK1 cells; CREB phosphorylation and c-Fos expression assays with V2R activation\",\n      \"journal\": \"The American journal of physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct promoter/transcription factor analysis with defined molecular mechanism\",\n      \"pmids\": [\"9140044\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"Autosomal recessive NDI-associated AQP2 missense mutations (A147T, T126M, N68S) produce proteins that are functional water channels in Xenopus oocytes but are misrouted to the ER rather than the plasma membrane, establishing that misrouting (not loss of channel function) is the primary cause of recessive NDI.\",\n      \"method\": \"Xenopus oocyte water permeability assays, immunoblotting, immunocytochemistry of oocyte lysates\",\n      \"journal\": \"Journal of the American Society of Nephrology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — functional reconstitution in oocytes combined with localization assays\",\n      \"pmids\": [\"9048343\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Prostaglandin E2 antagonizes vasopressin-induced AQP2 plasma membrane translocation by promoting retrieval of AQP2 to intracellular vesicles independently of AQP2 dephosphorylation at Ser256.\",\n      \"method\": \"Differential centrifugation, phosphorylation state-specific AQP2 antibody, incubation of rat renal inner medulla with AVP and PGE2\",\n      \"journal\": \"American journal of physiology. Renal physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods in a defined ex vivo system\",\n      \"pmids\": [\"10710543\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"AQP2 constitutively recycles between intracellular vesicles and the cell surface via a trans-Golgi-associated compartment; this recycling is blocked by 20°C incubation or bafilomycin A1 (H+-ATPase inhibitor), with AQP2 accumulating in a perinuclear compartment that colocalizes with clathrin but not giantin.\",\n      \"method\": \"Temperature block experiments, bafilomycin treatment, colocalization with organelle markers in LLC-PK1 cells\",\n      \"journal\": \"American journal of physiology. Renal physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal pharmacological and colocalization approaches\",\n      \"pmids\": [\"10662736\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"cAMP-induced AQP2 translocation to the apical membrane is accompanied by RhoA inhibition via PKA-mediated RhoA phosphorylation (on serine), which increases RhoA association with RhoGDI, thereby promoting actin depolymerization required for vesicle fusion.\",\n      \"method\": \"Selective RhoA pull-down (GTP-bound RhoA), cell fractionation, co-immunoprecipitation of RhoA and RhoGDI, forskolin stimulation of CD8 renal cells\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (pull-down, fractionation, co-IP) in a defined cellular system\",\n      \"pmids\": [\"12640036\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Inhibition of clathrin-mediated endocytosis (by dominant-negative dynamin-2/K44A or methyl-β-cyclodextrin) causes rapid, constitutive plasma membrane accumulation of AQP2 independently of Ser256 phosphorylation, demonstrating that AQP2 constitutively recycles and that phosphorylation at S256 is required for regulated (vasopressin-dependent) but not constitutive membrane insertion.\",\n      \"method\": \"Dominant-negative dynamin expression, cholesterol depletion with mβCD, cell-surface biotinylation, FITC-dextran endocytosis assay in LLC-PK1 and IMCD cells\",\n      \"journal\": \"American journal of physiology. Renal physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods with S256A phospho-mutant controls\",\n      \"pmids\": [\"14519593\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"S256 phosphorylation of AQP2 is necessary but not sufficient for plasma membrane expression; active PKA is required for sustained apical AQP2 localization. PGE2 and dopamine induce AQP2 endocytosis independently of AQP2 dephosphorylation at S256.\",\n      \"method\": \"PKA inhibitor H-89 treatment, AQP2-S256D phosphomimetic mutant, dopamine/PGE2 treatment, confocal microscopy in MDCK-C7 cells and rat kidney inner medullary slices\",\n      \"journal\": \"American journal of physiology. Renal physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — phosphomimetic mutants combined with pharmacological inhibitors and ex vivo tissue\",\n      \"pmids\": [\"15625084\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Dominant NDI caused by AQP2-R254L (which destroys the PKA consensus site adjacent to S256) results from loss of vasopressin-mediated phosphorylation at S256; AQP2-R254L is a functional water channel but is retained intracellularly and, when co-expressed, retains wild-type AQP2 in intracellular vesicles.\",\n      \"method\": \"Xenopus oocyte water permeability, MDCK cell co-expression, immunofluorescence, phospho-specific immunoblotting, S256D rescue experiment\",\n      \"journal\": \"Journal of the American Society of Nephrology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — functional oocyte assay + mutagenesis + co-expression dominant-negative experiment\",\n      \"pmids\": [\"16120822\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"AQP2 is stored in Rab11-positive subapical compartments prior to apical translocation; after endocytosis, AQP2 moves to EEA1-positive early endosomes and back to the Rab11 compartment. Microtubules maintain the subapical compartment distribution; actin filaments regulate trafficking from early endosomes to the storage compartment. Rab11 depletion by RNAi impairs AQP2 retention in the storage compartment.\",\n      \"method\": \"Double immunolabeling, siRNA knockdown of Rab11, pharmacological disruption of microtubules (nocodazole, colcemid) and actin (cytochalasin D, latrunculin B) in MDCK cells\",\n      \"journal\": \"Histochemistry and cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal approaches including RNAi and pharmacological disruption\",\n      \"pmids\": [\"16049696\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"ERM protein moesin is required for actin remodeling during AQP2 vesicular trafficking to the apical membrane; forskolin causes moesin redistribution to the cell cortex and reduction of phospho-moesin, and a moesin peptide blocking F-actin binding mimics forskolin effects including AQP2 translocation.\",\n      \"method\": \"Subcellular fractionation, confocal microscopy, Triton X-100 extraction of cytoskeletal proteins, moesin peptide introduction in renal cells\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple methods but mechanistic link relies on a peptide competitor approach\",\n      \"pmids\": [\"16046477\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Human AQP2 adopts a typical aquaporin fold as a tetramer; 4.5 Å 2D electron crystallography structure reveals the cytosolic N and C termini form contacts between stacked double-layer sheets.\",\n      \"method\": \"2D crystallization of recombinant human AQP2, atomic force microscopy, electron crystallography\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — structure determination, but limited resolution (4.5 Å) and no mutagenesis validation\",\n      \"pmids\": [\"15922355\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Cytoplasmic dynein and dynactin colocalize with AQP2-bearing vesicles in rat renal collecting duct principal cells, consistent with a role of the dynein motor complex in vasopressin-regulated AQP2 vesicle trafficking.\",\n      \"method\": \"Immunoblotting, immunoisolation of AQP2 vesicles with anti-AQP2 antibody, quantitative double immunogold electron microscopy\",\n      \"journal\": \"The American journal of physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — specific immunoisolation and ultrastructural colocalization, but functional consequence not directly tested\",\n      \"pmids\": [\"9486234\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"AQP2 in the collecting duct (CD) is essential for body water balance; conditional knockout mice lacking AQP2 only in CD (but retaining it in connecting tubule) show 10-fold increased urine output and severely decreased urine osmolality that cannot be compensated by other mechanisms. Global AQP2 knockout is lethal postnatally.\",\n      \"method\": \"Cre/loxP conditional knockout (Hoxb7-Cre for CD-specific, EIIa-Cre for global), metabolic cage measurements, immunohistochemistry\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean cell-type-specific knockout with defined physiological phenotype\",\n      \"pmids\": [\"16581908\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Loss of calcineurin Aα results in decreased vasopressin-mediated phosphorylation of AQP2 and failure of AQP2 to accumulate in the apical membrane, causing nephrogenic diabetes insipidus; calcineurin is present in IMCD vesicles and required for normal intracellular AQP2 trafficking.\",\n      \"method\": \"CnAα null mice and cyclosporin A treatment, immunoblotting for phospho-AQP2, subcellular fractionation, urine concentration tests\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO plus pharmacological inhibition with parallel molecular and physiological phenotypes\",\n      \"pmids\": [\"16735444\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Angiotensin II promotes AQP2 targeting to the plasma membrane of IMCD cells through AT1 receptor activation; this effect involves cAMP elevation and PKC activity and potentiates dDAVP-induced AQP2 membrane targeting.\",\n      \"method\": \"Immunofluorescence microscopy, immunoblotting for phospho-AQP2, cAMP measurement, candesartan (AT1 blocker) and PKC inhibitor treatment in primary cultured IMCD cells\",\n      \"journal\": \"American journal of physiology. Renal physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal pharmacological approaches with cellular phenotype\",\n      \"pmids\": [\"16896188\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"AQP2 vesicle fusion to the apical membrane is mediated by SNARE proteins VAMP2, VAMP3, syntaxin-3, and SNAP23; Munc18b acts as a negative regulator of SNARE complex formation. Knockdown of any of these SNAREs inhibits AQP2 apical fusion, while Munc18b knockdown causes 7-fold increase in AQP2 membrane fusion without stimulation.\",\n      \"method\": \"Co-immunoprecipitation of SNARE proteins with AQP2 vesicles, siRNA knockdown, apical surface biotinylation in MCD4 renal cells\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP combined with siRNA functional knockdown and biotinylation assays\",\n      \"pmids\": [\"18505797\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"AKAP220 binds AQP2 (identified by yeast two-hybrid screen) and colocalizes with AQP2 in the cytosol of inner medullary collecting ducts; AKAP220 co-expression increases forskolin-mediated phosphorylation of AQP2, suggesting it recruits PKA to AQP2-bearing vesicles.\",\n      \"method\": \"Yeast two-hybrid screen, double immunofluorescence, immunoelectron microscopy, co-expression phosphorylation assay in COS cells\",\n      \"journal\": \"Kidney international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — yeast two-hybrid plus colocalization and co-expression assay; no reciprocal co-IP\",\n      \"pmids\": [\"19008911\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Annexin-2 is required for cAMP-induced AQP2 exocytosis; forskolin causes annexin-2 redistribution to lipid rafts at the plasma membrane, and an annexin-2 N-terminal peptide that blocks p11 binding inhibits AQP2-vesicle fusion to plasma membranes in vitro and prevents osmotic water permeability increase in intact cells.\",\n      \"method\": \"Cell fractionation, lipid raft analysis, in vitro vesicle-plasma membrane fusion fluorescence assay, annexin-2 peptide introduction in renal cells\",\n      \"journal\": \"Pflugers Archiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vitro reconstitution of fusion process combined with cellular functional assay\",\n      \"pmids\": [\"18389276\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"AQP2 directly interacts with integrin β1 via an RGD domain in its external C-loop; RGD-containing peptides increase AQP2 membrane expression in the absence of vasopressin through cAMP- or calcium-dependent pathways.\",\n      \"method\": \"Co-immunoprecipitation of AQP2 and integrin β1 in renal tissue and MCD4 cells, confocal microscopy, cell surface biotinylation, FRET-based cAMP measurement, calcium imaging\",\n      \"journal\": \"Cellular physiology and biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single co-IP plus functional follow-up with RGD peptides\",\n      \"pmids\": [\"21691091\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"AS160, a Rab GAP protein and Akt substrate, regulates AQP2 trafficking; dDAVP stimulates AS160 phosphorylation via PI3K/Akt, and siRNA knockdown of AS160 causes increased AQP2 plasma membrane expression without hormonal stimulation.\",\n      \"method\": \"siRNA knockdown, immunocytochemistry, cell surface biotinylation, phospho-Akt and phospho-AS160 immunoblotting in M-1 and mpkCCDc14 cells\",\n      \"journal\": \"American journal of physiology. Renal physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — siRNA knockdown with biotinylation readout; mechanism of Rab GAP involvement inferred\",\n      \"pmids\": [\"21511697\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Vasopressin/forskolin-mediated F-actin depolymerization is dependent on AQP2 expression; cells lacking AQP2 do not show VP/FK-mediated F-actin depolymerization, and siRNA knockdown of AQP2 significantly reduces this response.\",\n      \"method\": \"F-actin quantification, immunofluorescence, siRNA knockdown of AQP2 in MDCK and LLC-PK1 cells with varying AQP2 expression levels\",\n      \"journal\": \"Biology open\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — correlation across multiple cell lines confirmed by siRNA knockdown\",\n      \"pmids\": [\"23213402\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"TRPC3 physically associates with AQP2 (co-immunoprecipitation) and co-localizes with AQP2 in intracellular vesicles; vasopressin causes co-insertion of TRPC3 and AQP2 into the apical membrane, and TRPC3 mediates transepithelial Ca2+ flux in principal cells.\",\n      \"method\": \"Co-immunoprecipitation of TRPC3 and AQP2 from kidney medulla and M1/IMCD-3 cells, immunofluorescence, dominant-negative TRPC3, 45Ca2+ flux assay\",\n      \"journal\": \"American journal of physiology. Renal physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP, dominant-negative functional assay, and localization with physiological readout\",\n      \"pmids\": [\"17699554\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"AQP2 interacts with TRPV4, and the presence of AQP2 enables TRPV4 activation by hypotonicity; TRPV4 translocation to the plasma membrane is required for the AQP2-dependent regulatory volume decrease response.\",\n      \"method\": \"Calcium imaging, RVD measurement, ruthenium red block, TRPV4 expression and plasma membrane translocation assays in WT-RCCD1 vs. AQP2-RCCD1 cells\",\n      \"journal\": \"Journal of cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — functional evidence in transfected cells; direct physical interaction not confirmed by co-IP in this paper\",\n      \"pmids\": [\"21938744\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"AQP5 directly interacts with AQP2 and impairs AQP2 cell surface localization; the AQP5/AQP2 complex partially resides in the ER/Golgi.\",\n      \"method\": \"Co-immunoprecipitation, cell surface biotinylation assay, colocalization, luciferase reporter assay in IMCD3, MLE-15, and 293T cells\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — co-IP and biotinylation in heterologous systems; physiological context requires further validation\",\n      \"pmids\": [\"23326416\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"NEDD4 and NEDD4L E3 ubiquitin ligases mediate ubiquitination and degradation of AQP2, but require NDFIP1 or NDFIP2 as adaptors to connect them to AQP2; PY-motif-lacking NDFIP variants fail to support ubiquitination.\",\n      \"method\": \"Membrane yeast two-hybrid (NDFIP2-AQP2 interaction), siRNA knockdown of NEDD4, NEDD4L, NDFIP1, NDFIP2 in mpkCCD cells; ubiquitination and degradation assays in HEK293 cells\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (Y2H, siRNA, ubiquitination assay) with mutant controls\",\n      \"pmids\": [\"28931009\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"AQP2 phosphorylation allosterically controls its interaction with the lysosomal trafficking protein LIP5; non-phosphorylated AQP2 binds LIP5 with highest affinity, while phosphomimetic S256E shows the greatest reduction in LIP5 affinity, linking phosphorylation state to lysosomal targeting.\",\n      \"method\": \"Far-Western blot, microscale thermophoresis, CD spectroscopy, phosphomimetic AQP2 mutants (S256E, S261E, S264E, T269E)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro binding assays with purified proteins and systematic mutagenesis\",\n      \"pmids\": [\"28710278\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Ezrin directly interacts with AQP2 C-terminus through its N-terminal FERM domain; this interaction facilitates AQP2 endocytosis, as ezrin knockdown increases membrane AQP2 and reduces endocytosis. Vasopressin causes redistribution of both ezrin and AQP2 to the apical membrane.\",\n      \"method\": \"Co-IP with anti-AQP2 antibody (proteomics), co-IP with anti-ezrin antibody, pulldown with purified recombinant full-length and FERM-domain ezrin, shRNA knockdown, immunofluorescence in collecting duct cells\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct interaction confirmed with purified recombinant proteins; functional validation by knockdown\",\n      \"pmids\": [\"28754689\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"PP2C (protein phosphatase 2C) is responsible for vasopressin-induced dephosphorylation of AQP2 at Ser261; this dephosphorylation is independent of S256 phosphorylation and does not acutely regulate AQP2 membrane trafficking.\",\n      \"method\": \"Phosphatase inhibitors (sanguinarine for PP2C, okadaic acid for PP2A, cyclosporine for PP2B), phospho-specific AQP2 antibodies, AQP2-S256A mutant in renal cells and kidney tissue\",\n      \"journal\": \"American journal of physiology. Renal physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — systematic pharmacological dissection with phospho-specific readouts and mutant controls\",\n      \"pmids\": [\"28381458\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Wnt5a regulates AQP2 protein expression, phosphorylation, and apical membrane trafficking via calcineurin signaling (independently of cAMP/PKA); calcineurin activator arachidonic acid produces vasopressin-like effects on AQP2 trafficking and increases urine osmolality in an NDI mouse model.\",\n      \"method\": \"Wnt5a treatment of collecting duct cells and NDI mouse model, calcineurin inhibitor/activator experiments, cAMP measurement, PKA activity assay, urine osmolality measurement\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal approaches in vitro and in vivo with defined molecular pathway\",\n      \"pmids\": [\"27892464\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Inhibition of AKAP-PKA interactions by FMP-API-1 increases free PKA activity, phosphorylates AQP2, and increases AQP2 membrane targeting and urine osmolality in vivo to the same extent as vasopressin, bypassing V2R mutations.\",\n      \"method\": \"cAMP/PKA activity assays in cortical collecting duct cells, AQP2 phosphorylation immunoblotting, urine osmolality measurement in V2R-inhibited mice\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic pathway defined in vitro and validated in vivo\",\n      \"pmids\": [\"29650969\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Tankyrase-mediated β-catenin signaling is required for vasopressin-induced AQP2 expression; tankyrase inhibition (XAV939) or β-catenin siRNA knockdown attenuates dDAVP-induced AQP2 upregulation and reduces nuclear translocation of phospho-β-catenin (S552), without affecting PKA activation.\",\n      \"method\": \"Tankyrase inhibitor (XAV939), siRNA knockdown of tankyrase and β-catenin, FRET-based PKA activity, nuclear translocation assay, luciferase reporter in mpkCCDc14 cells\",\n      \"journal\": \"American journal of physiology. Renal physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple approaches but mechanism linking tankyrase to AQP2 transcription is partially indirect\",\n      \"pmids\": [\"25520007\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Hsp70 plays a role in AQP2 trafficking to the apical plasma membrane; Hsp70-2 knockdown attenuates forskolin-induced AQP2 apical membrane targeting and reduces AQP2 phosphorylation at Ser256.\",\n      \"method\": \"siRNA knockdown of Hsp70-2, cell surface biotinylation, immunoblotting for pS256-AQP2, luciferase reporter assay for Hsp70-2 promoter in mpkCCDc14 cells\",\n      \"journal\": \"American journal of physiology. Renal physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — siRNA knockdown with biotinylation and phosphorylation readouts in a defined cell system\",\n      \"pmids\": [\"23303413\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The PDCD10-STK24/25 complex regulates AQP2 membrane targeting; mice deficient in Pdcd10 or Stk24/25 in kidney tubules develop polyuria with decreased AQP2 in the apical membrane, associated with increased p-ERM expression that impairs vesicle trafficking. Erlotinib treatment normalizes AQP2 membrane abundance.\",\n      \"method\": \"Conditional knockout mice, immunofluorescence, immunoblotting, Erlotinib treatment rescue experiment\",\n      \"journal\": \"JCI insight\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with defined phenotype, but molecular mechanism linking PDCD10-STK to ERM-AQP2 is partially inferred\",\n      \"pmids\": [\"34156031\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Phosphorylation at S256 and S269 both contribute to retention of AQP2 at the plasma membrane; S256D mutations slow internalization while S261A and S269D mutations slow development of intracellular accumulation. Differentially phosphorylated AQP2 mutants show distinct recycling kinetics but similar colocalization with Rab11, clathrin, and other markers.\",\n      \"method\": \"20°C cold block internalization assay, rewarming assay, colocalization with vesicular markers in LLC-PK1 cells expressing AQP2 phospho-mutants\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — systematic phospho-mutant analysis with multiple trafficking readouts\",\n      \"pmids\": [\"22403603\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The degree of S256 phosphorylation in the AQP2 tetramer controls plasma membrane diffusion speed; tetramers with 2–4 phosphorylated monomers diffuse faster than those with 0–1 phosphorylated monomers, which may determine retention time in the membrane vs. endocytosis.\",\n      \"method\": \"k-space Image Correlation Spectroscopy (kICS) of AQP2-S256D/S256A mixed-tetramer constructs in live cells\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — quantitative single-molecule-level diffusion analysis with systematic phosphomimetic titration\",\n      \"pmids\": [\"27801846\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"Substitution of the mercury-sensitive Cys181 in AQP2 abolishes plasma membrane targeting and causes ER retention, unlike the equivalent mutation in AQP1 (C189S) which does not affect routing, indicating structural differences between AQP1 and AQP2 at this position.\",\n      \"method\": \"Xenopus oocyte water permeability assays, immunocytochemistry, immunoblotting with AQP2-C181S and AQP1-C189S mutants\",\n      \"journal\": \"The American journal of physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — functional oocyte assay with site-directed mutagenesis, but limited mechanistic depth\",\n      \"pmids\": [\"9321919\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"AQP2 interactome identified by chemical cross-linking and LC-MS/MS in native rat IMCD cells reveals multiple Rab proteins (Rab1a, Rab2a, Rab5b, Rab5c, Rab7a, Rab11a, Rab11b, Rab14, Rab17) as AQP2-interacting proteins involved in membrane trafficking.\",\n      \"method\": \"Chemical cross-linking, anti-AQP2 immunoprecipitation, LC-MS/MS proteomics in rat IMCD\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — systematic mass spectrometry interactome in native tissue with multiple replicates\",\n      \"pmids\": [\"29046292\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"AQP2 is a vasopressin-regulated water channel in renal collecting duct principal cells that resides in intracellular Rab11-positive vesicles at baseline and is driven to the apical plasma membrane by a cascade in which vasopressin activates V2R→adenylate cyclase→cAMP→PKA, leading to Ser256 phosphorylation (necessary but not sufficient for apical targeting), RhoA inactivation via serine phosphorylation and RhoGDI association, actin depolymerization facilitated by ERM proteins (especially moesin and ezrin), and SNARE-mediated (VAMP2/3, syntaxin-3, SNAP23) vesicle fusion to the apical membrane; AKAP220 recruits PKA to AQP2 vesicles while Munc18b negatively regulates SNARE complex formation; AQP2 is retrieved from the membrane by clathrin-mediated endocytosis (facilitated by direct ezrin–AQP2 interaction), sorted through EEA1-positive early endosomes back to the Rab11 storage compartment, and targeted for lysosomal degradation via NDFIP1/2-mediated NEDD4/NEDD4L ubiquitination; phosphorylation at multiple C-terminal serines (S256, S261, S264, S269) differentially regulates trafficking kinetics, protein–protein interactions (e.g., phospho-S256 reduces LIP5 affinity to oppose lysosomal targeting), and membrane diffusion, with PP2C responsible for VP-induced S261 dephosphorylation; additional regulatory inputs include angiotensin II (via AT1R/cAMP/PKC), Wnt5a/calcineurin, tankyrase/β-catenin-mediated transcription, miRNAs, and AKAP-disruptor compounds, all converging to control AQP2 apical membrane abundance and renal water reabsorption.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"AQP2 is the principal vasopressin-regulated water channel of the renal collecting duct, functioning as the rate-limiting step for urinary water reabsorption. In unstimulated cells, AQP2 tetramers reside in Rab11-positive subapical vesicles and constitutively recycle through clathrin-mediated endocytosis; vasopressin binding to V2R activates adenylate cyclase→cAMP→PKA, which phosphorylates AQP2 at Ser256 (necessary but not sufficient for apical targeting), inactivates RhoA to promote actin depolymerization, and drives SNARE-mediated (VAMP2/3, syntaxin-3, SNAP23) fusion of AQP2 vesicles with the apical membrane [PMID:10644653, PMID:12640036, PMID:18505797, PMID:14519593]. Retrieval from the membrane is facilitated by a direct ezrin–AQP2 interaction that promotes clathrin-dependent endocytosis, while lysosomal degradation requires NDFIP1/2-dependent recruitment of NEDD4/NEDD4L E3 ligases for AQP2 ubiquitination, and phosphorylation at Ser256 allosterically reduces LIP5 binding to oppose lysosomal sorting [PMID:28754689, PMID:28931009, PMID:28710278]. Loss-of-function mutations cause nephrogenic diabetes insipidus—recessive forms through ER misrouting of otherwise functional channels, dominant forms through hetero-oligomeric trapping of wild-type AQP2—and collecting-duct-specific knockout produces severe polyuria, confirming AQP2 as essential and non-redundant for renal water conservation [PMID:9048343, PMID:16120822, PMID:16581908].\",\n  \"teleology\": [\n    {\n      \"year\": 1995,\n      \"claim\": \"Establishing that AQP2 undergoes regulated redistribution from cytoplasmic vesicles to the apical membrane in response to vasopressin answered the fundamental question of how collecting duct water permeability is acutely controlled and identified microtubule-dependent vesicle trafficking as the underlying mechanism.\",\n      \"evidence\": \"Immunofluorescence and immunogold EM in Brattleboro rats ± vasopressin and colchicine\",\n      \"pmids\": [\"7539496\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Motor proteins driving microtubule-dependent transport not identified\", \"Signal transduction pathway between V2R and vesicle movement undefined\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Demonstrating that recessive NDI mutations produce functional water channels that are ER-retained, and that V2R activates AQP2 transcription via CREB/AP1, established that both trafficking competence and transcriptional regulation are critical for AQP2 function, and that disease arises from misrouting rather than channel dysfunction.\",\n      \"evidence\": \"Xenopus oocyte water permeability with NDI mutants (A147T, T126M, N68S); AQP2 promoter-reporter assays with V2R activation in LLC-PK1 cells\",\n      \"pmids\": [\"9048343\", \"9140044\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Chaperone interactions responsible for ER quality control of AQP2 mutants not identified\", \"Relative contribution of transcriptional vs. trafficking regulation to water reabsorption in vivo unclear\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Identification of Ser256 as the PKA phosphorylation site required for vasopressin-induced apical targeting, alongside discovery of constitutive AQP2 recycling and PGE2-mediated retrieval independent of S256 dephosphorylation, revealed that AQP2 membrane abundance is governed by the balance of regulated exocytosis and phosphorylation-independent endocytosis.\",\n      \"evidence\": \"Phospho-S256-specific antibodies with immunoEM in rat kidney; temperature-block and bafilomycin experiments in LLC-PK1 cells; PGE2 retrieval assays in rat IMCD\",\n      \"pmids\": [\"10644653\", \"10662736\", \"10710543\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of kinases/phosphatases acting on other C-terminal serines unknown\", \"Endocytic machinery components mediating retrieval not defined\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Showing that cAMP/PKA inhibits RhoA via phosphorylation (promoting RhoGDI association and actin depolymerization) and that blocking clathrin-mediated endocytosis causes S256-independent AQP2 membrane accumulation established that regulated exocytosis requires cytoskeletal remodeling while constitutive recycling is clathrin-dependent.\",\n      \"evidence\": \"RhoA-GTP pull-down and RhoA-RhoGDI co-IP in CD8 cells; dominant-negative dynamin and mβCD in LLC-PK1/IMCD cells with S256A mutant controls\",\n      \"pmids\": [\"12640036\", \"14519593\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific Rho-GEFs and GAPs involved not identified\", \"How S256 phosphorylation intersects with endocytic machinery unknown\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Mapping the AQP2 intracellular itinerary through Rab11-positive storage compartments and EEA1-positive early endosomes, together with demonstrating moesin-dependent actin remodeling during exocytosis, defined the vesicular sorting pathway and the ERM-actin axis as a trafficking checkpoint.\",\n      \"evidence\": \"Double immunolabeling, Rab11 siRNA, nocodazole/latrunculin pharmacology in MDCK cells; moesin subcellular redistribution and peptide competitor in renal cells\",\n      \"pmids\": [\"16049696\", \"16046477\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which Rab effectors mediate Rab11-to-apical delivery unclear\", \"Functional link between moesin phosphorylation state and AQP2 exocytosis not fully resolved\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Collecting-duct-specific AQP2 knockout producing lethal polyuria, together with identification of calcineurin and angiotensin II/AT1R as additional regulators of AQP2 trafficking, established AQP2 as non-redundant for water balance and revealed cAMP-independent signaling inputs.\",\n      \"evidence\": \"Cre/loxP conditional knockout mice; CnAα-null mice and cyclosporin A; AngII/candesartan/PKC inhibitors in primary IMCD cells\",\n      \"pmids\": [\"16581908\", \"16735444\", \"16896188\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Calcineurin substrates relevant to AQP2 trafficking not identified\", \"How PKC integrates with PKA-dependent phosphorylation at S256 unresolved\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identification of VAMP2/3, syntaxin-3, SNAP23, and Munc18b as the SNARE machinery for AQP2 vesicle fusion, and AKAP220 as a PKA-recruiting scaffold on AQP2 vesicles, provided the molecular basis for both the final membrane fusion step and compartmentalized PKA signaling.\",\n      \"evidence\": \"Co-IP of SNAREs with AQP2 vesicles and siRNA knockdown with biotinylation in MCD4 cells; yeast two-hybrid and co-expression phosphorylation assay for AKAP220\",\n      \"pmids\": [\"18505797\", \"19008911\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"AKAP220 interaction awaits reciprocal co-IP validation\", \"How Munc18b release is triggered upon cAMP stimulation unknown\", \"Specific SNARE regulatory steps (NSF, α-SNAP involvement) not characterized\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Systematic phospho-mutant analysis revealed that S256, S261, S264, and S269 differentially regulate AQP2 internalization kinetics and membrane retention, while TRPC3 was identified as a co-trafficking partner that inserts with AQP2 into the apical membrane, expanding AQP2's role beyond water transport.\",\n      \"evidence\": \"Cold-block/rewarming assays with phospho-mutants in LLC-PK1 cells; TRPC3-AQP2 co-IP and dominant-negative TRPC3 with calcium flux in M1/IMCD-3 cells\",\n      \"pmids\": [\"22403603\", \"17699554\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Kinases responsible for S264 and S269 phosphorylation in vivo not definitively identified\", \"Physiological significance of TRPC3 co-trafficking for calcium homeostasis in principal cells unclear\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Discovery that NDFIP1/2 adaptors recruit NEDD4/NEDD4L to ubiquitinate AQP2 for lysosomal degradation, that phospho-S256 allosterically reduces LIP5 binding to oppose lysosomal targeting, that ezrin directly binds AQP2 to facilitate endocytosis, and that PP2C dephosphorylates S261 defined the molecular logic of AQP2 downregulation and degradation.\",\n      \"evidence\": \"Membrane Y2H, siRNA of NEDD4/NDFIP in mpkCCD; MST binding with phosphomimetic AQP2 peptides; recombinant ezrin pulldown and shRNA in collecting duct cells; PP2C inhibitor sanguinarine with phospho-specific antibodies\",\n      \"pmids\": [\"28931009\", \"28710278\", \"28754689\", \"28381458\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ubiquitination sites on AQP2 not mapped\", \"Whether ezrin and NEDD4 pathways are sequential or parallel not resolved\", \"Structural basis for phosphorylation-dependent LIP5 affinity change unknown\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Demonstration that AKAP-PKA disruptor FMP-API-1 bypasses V2R to phosphorylate AQP2 and concentrate urine in vivo, and that Wnt5a/calcineurin provides a cAMP-independent AQP2 trafficking pathway, opened pharmacological strategies for treating nephrogenic diabetes insipidus.\",\n      \"evidence\": \"FMP-API-1 in cortical CD cells and V2R-inhibited mice with urine osmolality; Wnt5a/arachidonic acid in CD cells and NDI mouse model\",\n      \"pmids\": [\"29650969\", \"27892464\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Off-target effects of AKAP disruption on other renal PKA substrates not characterized\", \"Wnt5a receptor and downstream calcineurin substrates in principal cells not identified\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the high-resolution structural basis for phosphorylation-dependent conformational changes in the AQP2 C-terminus, the complete ubiquitination site map, the identity of specific Rab effectors mediating apical delivery, and whether pharmacological targeting of the Wnt5a/calcineurin or AKAP-disruptor pathways is therapeutically viable for NDI.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No high-resolution structure of full-length AQP2 with C-terminal regulatory domain\", \"Ubiquitination sites not mapped\", \"Rab11 effectors for apical delivery not identified\", \"Clinical translation of AKAP disruptors or calcineurin activators untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005215\", \"supporting_discovery_ids\": [3, 14]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 1, 7, 8]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [0, 5, 10]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [18]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 6, 16, 30, 31]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [0, 5, 7, 10, 17]},\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [3, 14]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [1, 8, 10, 28]}\n    ],\n    \"complexes\": [\n      \"AQP2 homotetramer\"\n    ],\n    \"partners\": [\n      \"EZR\",\n      \"VAMP2\",\n      \"STX3\",\n      \"SNAP23\",\n      \"RAB11A\",\n      \"NDFIP1\",\n      \"NEDD4L\",\n      \"TRPC3\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}