Vasopressin increases expression of UT-A1, UT-A3, and ER chaperone GRP78 in the renal medulla of mice with a urinary concentrating defect.
ABSTRACT: Activation of V2 receptors (V2R) during antidiuresis increases the permeability of the inner medullary collecting duct to urea and water. Extracellular osmolality is elevated as the concentrating capacity of the kidney increases. Osmolality is known to contribute to the regulation of collecting duct water (aquaporin-2; AQP2) and urea transporter (UT-A1, UT-A3) regulation. AQP1KO mice are a concentrating mechanism knockout, a defect attributed to the loss of high interstitial osmolality. A V2R-specific agonist, deamino-8-D-arginine vasopressin (dDAVP), was infused into wild-type and AQP1KO mice for 7 days. UT-A1 mRNA and protein abundance were significantly increased in the medullas of wild-type and AQP1KO mice following dDAVP infusion. The mRNA and protein abundance of UT-A3, the basolateral urea transporter, was significantly increased by dDAVP in both wild-type and AQP1KO mice. Semiquantitative immunoblots revealed that dDAVP infusion induced a significant increase in the medullary expression of the endoplasmic reticulum (ER) chaperone GRP78. Immunofluorescence studies demonstrated that GRP78 expression colocalized with AQP2 in principal cells of the papillary tip of the renal medulla. Using immunohistochemistry and immunogold electron microscopy, we demonstrate that vasopressin induced a marked apical targeting of GRP78 in medullary principal cells. Urea-sensitive genes, GADD153 and ATF4 (components of the ER stress pathway), were significantly increased in AQP1KO mice by dDAVP infusion. These findings strongly support an important role of vasopressin in the activation of an ER stress response in renal collecting duct cells, in addition to its role in activating an increase in UT-A1 and UT-A3 abundance.
Project description:To investigate the role of inner medullary collecting duct (IMCD) urea transporters in the renal concentrating mechanism, we deleted 3 kb of the UT-A urea transporter gene containing a single 140-bp exon (exon 10). Deletion of this segment selectively disrupted expression of the two known IMCD isoforms of UT-A, namely UT-A1 and UT-A3, producing UT-A1/3(-/-) mice. In isolated perfused IMCDs from UT-A1/3(-/-) mice, there was a complete absence of phloretin-sensitive or vasopressin-stimulated urea transport. On a normal protein intake (20% protein diet), UT-A1/3(-/-) mice had significantly greater fluid consumption and urine flow and a reduced maximal urinary osmolality relative to wild-type controls. These differences in urinary concentrating capacity were nearly eliminated when urea excretion was decreased by dietary protein restriction (4% by weight), consistent with the 1958 Berliner hypothesis stating that the chief role of IMCD urea transport in the concentrating mechanism is the prevention of urea-induced osmotic diuresis. Analysis of inner medullary tissue after water restriction revealed marked depletion of urea in UT-A1/3(-/-) mice, confirming the concept that phloretin-sensitive IMCD urea transporters play a central role in medullary urea accumulation. However, there were no significant differences in mean inner medullary Na(+) or Cl(-) concentrations between UT-A1/3(-/-) mice and wild-type controls, indicating that the processes that concentrate NaCl were intact. Thus, these results do not corroborate the predictions of passive medullary concentrating models stating that NaCl accumulation in the inner medulla depends on rapid vasopressin-regulated urea transport across the IMCD epithelium.
Project description:The urea channel Slc14a2 (or UT-A1) mediates vasopressin-regulated urea transport across the inner medullary collecting duct (IMCD). Previously, UT-A1 was found to present in a high molecular weight complex, suggesting UT-A1 is involved in certain protein-protein interactions. The present study sought to identify the proteins that interact with UT-A1 in this complex for a better understanding of how UT-A1 is regulated. Rat IMCD suspensions were treated with or without V2 receptor agonist, dDAVP, followed by in-cell crosslinking using BSOCOES and detergent solubilization. Immunoprecipitation using Dynabeads coated with UT-A1 specific antibody successfully pulled down the UT-A1 proteins. In-gel digestion protocol was carried out to prepare samples for liquid chromatographic mass spectrometry analysis of tryptic peptides using a Velos-Orbitrap mass spectrometer. The peptides passing stringent spectral quality thresholds were quantified (label-free) to identify those with (UTA-1 antibody/preimmune IgG) >4. A total of 128 UT-A1 interacting proteins were identified. Gene Ontology analysis maps the distribution of these proteins throughout major cell compartments: endoplasmic reticulum, Golgi, endosomes, cytosol and plasma membrane. Among them are four protein kinases (Cdc42bpb, Phkb, Camk2d, Mtor) that play roles in vasopressin-regulated phosphorylation of UT-A1. Non-label quantification was also performed to determine the stoichiometry of UT-A3 with UT-A1, the result does not support an oligomeric complex formation of UT-A1/A3. In conclusion, we have provided a refined list of UT-A1 binding proteins which can be useful for further analysis of the vasopressin signaling pathway in regulation of UT-A1 in IMCD.
Project description:Urea transport (UT) proteins of the UT-A class are expressed in epithelial cells in kidney tubules, where they are required for the formation of a concentrated urine by countercurrent multiplication. Here, using a recently developed high-throughput assay to identify UT-A inhibitors, a screen of 50,000 synthetic small molecules identified UT-A inhibitors of aryl-thiazole, ?-sultambenzosulfonamide, aminocarbonitrile butene, and 4-isoxazolamide chemical classes. Structure-activity analysis identified compounds that inhibited UT-A selectively by a noncompetitive mechanism with IC50 down to ?1 ?M. Molecular modeling identified putative inhibitor binding sites on rat UT-A. To test compound efficacy in rats, formulations and administration procedures were established to give therapeutic inhibitor concentrations in blood and urine. We found that intravenous administration of an indole thiazole or a ?-sultambenzosulfonamide at 20 mg/kg increased urine output by 3-5-fold and reduced urine osmolality by ?2-fold compared to vehicle control rats, even under conditions of maximum antidiuresis produced by 1-deamino-8-D-arginine vasopressin (DDAVP). The diuresis was reversible and showed urea > salt excretion. The results provide proof of concept for the diuretic action of UT-A-selective inhibitors. UT-A inhibitors are first in their class salt-sparing diuretics with potential clinical indications in volume-overload edemas and high-vasopressin-associated hyponatremias.
Project description:Protein phosphorylation is an important component of vasopressin signaling in the renal collecting duct, but the database of known phosphoproteins is incomplete. We used tandem mass spectrometry to identify vasopressin-regulated phosphorylation events in isolated rat inner medullary collecting duct (IMCD) suspensions. Using multiple search algorithms to identify the phosphopeptides from spectral data, we expanded the size of the existing collecting duct phosphoproteome database from 367 to 1187 entries. Label-free quantification in vasopressin- and vehicle-treated samples detected a significant change in the phosphorylation of 29 of 530 quantified phosphopeptides. The targets include important structural, regulatory, and transporter proteins. The vasopressin-regulated sites included two known sites (Ser-486 and Ser-499) present in the urea channel UT-A1 and one previously unknown site (Ser-84) on vasopressin-sensitive urea channels UT-A1 and UT-A3. In vitro assays using synthetic peptides showed that purified protein kinase A (PKA) could phosphorylate all three sites, and immunoblotting confirmed the PKA dependence of Ser-84 and Ser-486 phosphorylation. These results expand the known list of collecting duct phosphoproteins and highlight the utility of targeted phosphoproteomic approaches.
Project description:Urea transport (UT) proteins facilitate the concentration of urine by the kidney, suggesting that inhibition of these proteins could have therapeutic use as a diuretic strategy. We screened 100,000 compounds for UT-B inhibition using an optical assay based on the hypotonic lysis of acetamide-loaded mouse erythrocytes. We identified a class of triazolothienopyrimidine UT-B inhibitors; the most potent compound, UTB(inh)-14, fully and reversibly inhibited urea transport with IC(50) values of 10 nM and 25 nM for human and mouse UT-B, respectively. UTB(inh)-14 competed with urea binding at an intracellular site on the UT-B protein. UTB(inh)-14 exhibited low toxicity and high selectivity for UT-B over UT-A isoforms. After intraperitoneal administration of UTB(inh)-14 in mice to achieve predicted therapeutic concentrations in the kidney, urine osmolality after administration of 1-deamino-8-D-arginine-vasopressin was approximately 700 mosm/kg H(2)O lower in UTB(inh)-14-treated mice than vehicle-treated mice. UTB(inh)-14 also increased urine output and reduced urine osmolality in mice given free access to water. UTB(inh)-14 did not reduce urine osmolality in UT-B knockout mice. In summary, these data provide proof of concept for the potential utility of UT inhibitors to reduce urinary concentration in high-vasopressin, fluid-retaining conditions. The diuretic mechanism of UT inhibitors may complement the action of conventional diuretics, which target sodium transport.
Project description:Inhibitors of kidney urea transporter (UT) proteins have potential use as salt-sparing diuretics ('urearetics') with a different mechanism of action than diuretics that target salt transporters. To study UT inhibition in rats, we screened about 10,000 drugs, natural products and urea analogs for inhibition of rat UT-A1. Drug and natural product screening found nicotine, sanguinarine and an indolcarbonylchromenone with IC50 of 10-20??M. Urea analog screening found methylacetamide and dimethylthiourea (DMTU). DMTU fully and reversibly inhibited rat UT-A1 and UT-B by a noncompetitive mechanism with IC50 of 2-3?mM. Homology modeling and docking computations suggested DMTU binding sites on rat UT-A1. Following a single intraperitoneal injection of 500?mg/kg DMTU, peak plasma concentration was 9?mM with t1/2 of about 10?h, and a urine concentration of 20-40?mM. Rats chronically treated with DMTU had a sustained, reversible reduction in urine osmolality from 1800 to 600 mOsm, a 3-fold increase in urine output, and mild hypokalemia. DMTU did not impair urinary concentrating function in rats on a low protein diet. Compared to furosemide-treated rats, the DMTU-treated rats had greater diuresis and reduced urinary salt loss. In a model of syndrome of inappropriate antidiuretic hormone secretion, DMTU treatment prevented hyponatremia and water retention produced by water-loading in dDAVP-treated rats. Thus, our results establish a rat model of UT inhibition and demonstrate the diuretic efficacy of UT inhibition.
Project description:Arginine vasopressin (AVP) enhances water reabsorption in the renal collecting duct by vasopressin V? receptor (V?R)-mediated activation of adenylyl cyclase (AC), cAMP-promoted phosphorylation of aquaporin-2 (AQP2), and increased abundance of AQP2 on the apical membrane. Multiple isoforms of adenylate cyclase exist, and the roles of individual AC isoforms in water homeostasis are not well understood. Here, we found that levels of AC6 mRNA, the most highly expressed AC isoform in the inner medulla, inversely correlate with fluid intake. Moreover, mice lacking AC6 had lower levels of inner medullary cAMP, reduced abundance of phosphorylated AQP2 (at both serine-256 and serine-269), and lower urine osmolality than wild-type mice. Water deprivation or administration of the V?R agonist dDAVP did not increase urine osmolality of AC6-deficient mice to the levels of wild-type mice. Furthermore, AC6-deficient mice lacked dDAVP-promoted inner medullary cAMP formation and phosphorylation of serine-269 and had attenuated increases in both phosphorylation of serine-256 and apical membrane AQP2 trafficking. In summary, AC6 expression determines inner medullary cAMP formation and AQP2 phosphorylation and trafficking, the absence of which causes nephrogenic diabetes insipidus.
Project description:Satavaptan (SR121463) is a vasopressin V2 receptor antagonist that has been shown to improve hyponatremia in patients with cirrhosis, congestive heart failure, and syndrome of inappropriate antidiuresis. While known to inhibit adenylyl cyclase-mediated accumulation of intracellular cyclic AMP and potentially recruit ?-arrestin in kidney cell lines, very little is known regarding the signaling pathways that are affected by this drug. To this end, we carried out a global quantitative phosphoproteomic analysis of native rat inner medullary collecting duct cells pretreated with satavaptan or vehicle control followed by the V2 receptor agonist desmopressin (dDAVP) for 0.5, 2, 5, or 15 min. A total of 2,449 unique phosphopeptides from 1,160 proteins were identified. Phosphopeptides significantly changed by satavaptan included many of the same kinases [protein kinase A, phosphoinositide 3-kinase, mitogen-activated protein kinase kinase kinase 7 (TAK1), and calcium/calmodulin-dependent kinase kinase 2] and channels (aquaporin-2 and urea transporter UT-A1) regulated by vasopressin. Time course clustering and kinase motif analysis suggest that satavaptan blocks dDAVP-mediated activation of basophilic kinases, while also blocking dDAVP-mediated inhibition of proline-directed kinases. Satavaptan affects a variety of dDAVP-mediated processes including regulation of cell-cell junctions, actin cytoskeleton dynamics, and signaling through Rho GTPases. These results demonstrate that, overall, satavaptan acts as a selective V2 receptor antagonist and affects many of the same signaling pathways regulated by vasopressin. This study represents the first "systems-wide" analysis of a "vaptan"-class drug and provides a wealth of new data regarding the effects of satavaptan on vasopressin-mediated phosphorylation events.
Project description:Of the three major protein variants produced by the UT-A gene (UT-A1, UT-A2, and UT-A3) UT-A1 is the largest. It contains UT-A3 as its NH(2)-terminal half and UT-A2 as its COOH-terminal half. When being part of UT-A1, UT-A3 and UT-A2 are joined by a segment, Lp, whose central part, Lc, is not part of UT-A3 or UT-A2 but is present only in UT-A1. Lc contains the phosphorylation sites S486 and S499 that are involved in protein kinase A-dependent activation, as well as the binding site for snapin, a protein involved in soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE)-mediated vesicle trafficking and fusion to the plasma membrane. We attached Lc to UT-A2 and UT-A3 to test how these phosphorylation sites influenced their urea transport activity. Adding Lc to UT-A2 conferred stimulation by cAMP to the cAMP-unresponsive UT-A2, and adding Lc to UT-A3 did not further enhance its already existing cAMP response. These findings suggest that the responsiveness to vasopressin that is observed with UT-A1 can be introduced into the unresponsive UT-A2 variant through the Lc segment that is unique to UT-A1. In UT-A3, however, the Lc segment plays no significant role in its activation by cAMP. In addition, the Lc segment also gave UT-A2 the ability to bind snapin and, in Xenopus oocytes, to be stimulated in its urea transport activity by snapin and syntaxins 3 and 4, in the same way as UT-A1.
Project description:AIM: Aquaporin-2 (AQP2) is a vasopressin-regulated water channel located in the collecting tubule and collecting duct cells of mammalian kidney. The aim of this study is to investigate whether PKC? plays a role in vasopressin-induced AQP2 trafficking in mouse inner medullary collecting duct 3 (mIMCD3) cells. METHODS: AQP2-mIMCD3 stable cell line was constructed by transfection of mouse inner medullary collecting duct 3 (mIMCD3) cells with AQP2-GFP construct. Then the cells were transfected with PKC? shRNA, PKC? A/25E, or PKC? scrambled shRNA. The expression levels of PKC?, AQP2, and phospho-S256-AQP2 were analyzed using Western blot. The interaction between AQP2 and PKC? was examined using immunoprecipitation. The distribution of AQP2 and microtubules was studied using immunocytochemistry. The AQP2 trafficking was examined using the biotinylation of surface membranes. RESULTS: Treatment of AQP2-mIMCD3 cells with 100 ?mol/L of 1-desamino-8-D-arginine vasopressin (DdAVP) for 30 min stimulated the translocation of AQP2 from the cytoplasm to plasma membrane through influencing the microtubule assembly. Upregulation of active PKC? by transfection with PKC? A/25E plasmids resulted in de-polymerization of ?-tubulin and redistributed AQP2 in the cytoplasm. Down-regulation of PKC? by PKC? shRNA partially inhibited DdAVP-stimulated AQP2 trafficking without altering ?-tubulin distribution. Although 100 ?mol/L of DdAVP increased AQP2 phosphorylation at serine 256, down-regulation of PKC? by PKC? shRNA did not influence DdAVP-induced AQP2 phosphorylation, suggesting that AQP2 phosphorylation at serine 256 was independent of PKC?. Moreover, PKC? did not physically interact with AQP2 in the presence or absence of DdAVP. CONCLUSION: Our results suggested that PKC? regulates AQP2 trafficking induced by DdAVP via microtubule assembly.