Expression and distribution of aquaporin of collecting duct are regulated by vasopressin V2 receptor in rat kidney.

Phosphorylation of Ser256, in a PKA consensus site, in AQP2 (p-AQP2) appears to be critically involved in the vasopressin-induced trafficking of AQP2. In the present study, affinity-purified antibodies that selectively recognize AQP2 phosphorylated at Ser256 were developed. These antibodies were used to determine 1) the subcellular localization of p-AQP2 in rat kidney and 2) changes in distribution and/or levels of p-AQP2 in response to [desamino-Cys1,d-Arg8]vasopressin (DDAVP) treatment or V2-receptor blockade. Immunoelectron microscopy revealed that p-AQP2 was localized in both the apical plasma membrane and in intracellular vesicles of collecting duct principal cells. Treatment of rats with V2-receptor antagonist for 30 min resulted in almost complete disappearance of p-AQP2 labeling of the apical plasma membrane with only marginal labeling of intracellular vesicles remaining. Immunoblotting confirmed a marked decrease in p-AQP2 levels. In control Brattleboro rats (BB), lacking vasopressin secretion, p-AQP2 labeling was almost exclusively present in intracellular vesicles. Treatment of BB rats with DDAVP for 2 h induced a 10-fold increase in p-AQP2 labeling of the apical plasma membrane. The overall abundance of p-AQP2, however, was not increased, as determined both by immunoelectron microscopy and immunoblotting. Consistent with this, 2 h of DDAVP treatment of normal rats also resulted in unchanged p-AQP2 levels. Thus the results demonstrate that AQP2 phosphorylated in Ser256 is present in the apical plasma membrane and in intracellular vesicles and that both the intracellular distribution/trafficking, as well as the abundance of p-AQP2, are regulated via V2 receptors by altering phosphorylation and/or dephosphorylation of Ser256in AQP2.

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Electrolyte handling in the isolated perfused rat kidney: demonstration of vasopressin V2-receptor-dependent calcium reabsorption

Upsala Journal of Medical Sciences ◽

10.1080/03009734.2020.1804496 ◽

2020 ◽

Vol 125 (4) ◽

pp. 274-280

Author(s):

Krister Bamberg ◽

Lena William-Olsson ◽

Ulrika Johansson ◽

Anders Arner ◽

Judith Hartleib-Geschwindner ◽

...

Keyword(s):

Rat Kidney ◽

Isolated Perfused Rat Kidney ◽

Vasopressin V2 Receptor ◽

Perfused Rat Kidney ◽

Calcium Reabsorption ◽

V2 Receptor

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MAP kinases‐Regulated Proteins in Downstream Signaling Pathways of Vasopressin V2 Receptor in Kidney Collecting Duct

The FASEB Journal ◽

10.1096/fasebj.2020.34.s1.03844 ◽

2020 ◽

Vol 34 (S1) ◽

pp. 1-1

Author(s):

Hyo-Ju Jang ◽

Hyun jun Jung ◽

Hyo-Jung Choi ◽

Eui-Jung Park ◽

Hye-Jeong Park ◽

...

Keyword(s):

Signaling Pathways ◽

Map Kinases ◽

Collecting Duct ◽

Downstream Signaling ◽

Kidney Collecting Duct ◽

Vasopressin V2 Receptor ◽

V2 Receptor

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Stability of the vasopressin V2 receptor–adenylyl cyclase system in rat kidney

European Journal of Pharmacology ◽

10.1016/s0014-2999(97)01461-1 ◽

1998 ◽

Vol 341 (1) ◽

pp. 87-94 ◽

Cited By ~ 3

Author(s):

Tiansheng Shen ◽

John Laycock ◽

Yosuke Suzuki ◽

Nicole Defer ◽

Jacques Hanoune

Keyword(s):

Adenylyl Cyclase ◽

Rat Kidney ◽

Adenylyl Cyclase System ◽

Vasopressin V2 Receptor ◽

V2 Receptor

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Tolvaptan as a tool in renal physiology

AJP Renal Physiology ◽

10.1152/ajprenal.00330.2013 ◽

2014 ◽

Vol 306 (3) ◽

pp. F359-F366 ◽

Cited By ~ 21

Author(s):

Carlos A. Miranda ◽

Jae Wook Lee ◽

Chung-Lin Chou ◽

Mark A. Knepper

Keyword(s):

Collecting Duct ◽

Rat Kidney ◽

Water Channel ◽

The United States ◽

Competitive Inhibitor ◽

Renal Physiology ◽

Brattleboro Rat ◽

V2 Receptor

For decades, the Brattleboro rat has been a useful model in kidney physiology. These animals manifest central diabetes insipidus (lack of circulating vasopressin) due to a mutation in the vasopressin-neurophysin gene. V2 receptor-mediated vasopressin actions in the kidney can be assessed in these animals by infusing the V2-selective vasopressin analog 1-desamino-8-d-arginine vasopressin (dDAVP). However, the major commercial supplier in the United States has ceased production, creating the need for another reliable experimental model of V2 receptor-mediated vasopressin action in rodents. We designed an in vivo protocol to investigate vasopressin responses in the rat kidney using osmotic minipumps loaded with tolvaptan, a nonpeptide competitive inhibitor of the vasopressin V2 receptor. Tolvaptan-infused rats had a mean urinary osmolality of <300 vs. >2,000 mosmol/kgH2O in vehicle-infused rats. The tolvaptan infusion produced large decreases in the renal abundance of aquaporin-2 (AQP2), aquaporin-3 (AQP3), the β-subunit of the epithelial sodium channel (β-ENaC), and γ-ENaC that were comparable to the differences seen in vehicle-infused vs. vasopressin-infused Brattleboro rats. Thus we conclude that tolvaptan infusion in rats provides an additional model (besides dDAVP-infusion in the Brattleboro rat) for the assessment of V2 receptor-mediated vasopressin actions in the kidney. We also provide ancillary in vitro data in rat inner-medullary-collecting-duct suspensions showing that tolvaptan can block vasopressin's effects on phosphorylation of the water channel AQP2 in vitro. Specifically, tolvaptan almost completely inhibited the ability of vasopressin to increase AQP2 phosphorylation at Ser256, Ser264, and Ser269, while strongly inhibiting a vasopressin-induced decrease in AQP2 phosphorylation at Ser261.

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Phosphoproteomic identification of vasopressin V2 receptor-dependent signaling in the renal collecting duct

AJP Renal Physiology ◽

10.1152/ajprenal.00281.2019 ◽

2019 ◽

Vol 317 (4) ◽

pp. F789-F804 ◽

Cited By ~ 1

Author(s):

Venkatesh Deshpande ◽

Anika Kao ◽

Viswanathan Raghuram ◽

Arnab Datta ◽

Chung-Lin Chou ◽

...

Keyword(s):

Amino Acid ◽

Collecting Duct ◽

Water Channel ◽

Protein Mass ◽

Web Resource ◽

Phosphorylated Amino Acid ◽

Vasopressin V2 Receptor ◽

Duct Cells ◽

V2 Receptor ◽

Collecting Duct Cells

Vasopressin controls water balance largely through PKA-dependent effects to regulate the collecting duct water channel aquaporin-2 (AQP2). Although considerable information has accrued regarding the regulation of water and solute transport in collecting duct cells, information is sparse regarding the signaling connections between PKA and transport responses. Here, we exploited recent advancements in protein mass spectrometry to perform a comprehensive, multiple-replicate analysis of changes in the phosphoproteome of native rat inner medullary collecting duct cells in response to the vasopressin V2 receptor-selective agonist 1-desamino-8D-arginine vasopressin. Of the 10,738 phosphopeptides quantified, only 156 phosphopeptides were significantly increased in abundance, and only 63 phosphopeptides were decreased, indicative of a highly selective response to vasopressin. The list of upregulated phosphosites showed several general characteristics: 1) a preponderance of sites with basic (positively charged) amino acids arginine (R) and lysine (K) in position −2 and −3 relative to the phosphorylated amino acid, consistent with phosphorylation by PKA and/or other basophilic kinases; 2) a greater-than-random likelihood of sites previously demonstrated to be phosphorylated by PKA; 3) a preponderance of sites in membrane proteins, consistent with regulation by membrane association; and 4) a greater-than-random likelihood of sites in proteins with class I COOH-terminal PDZ ligand motifs. The list of downregulated phosphosites showed a preponderance of those with proline in position +1 relative to the phosphorylated amino acid, consistent with either downregulation of proline-directed kinases (e.g., MAPKs or cyclin-dependent kinases) or upregulation of one or more protein phosphatases that selectively dephosphorylate such sites (e.g., protein phosphatase 2A). The phosphoproteomic data were used to create a web resource for the investigation of G protein-coupled receptor signaling and regulation of AQP2-mediated water transport.

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Akt and ERK1/2 pathways are components of the vasopressin signaling network in rat native IMCD

AJP Renal Physiology ◽

10.1152/ajprenal.90339.2008 ◽

2008 ◽

Vol 295 (4) ◽

pp. F1030-F1043 ◽

Cited By ~ 55

Author(s):

Trairak Pisitkun ◽

Vinitha Jacob ◽

Stephen M. Schleicher ◽

Chung-Lin Chou ◽

Ming-Jiun Yu ◽

...

Keyword(s):

Systems Approach ◽

Collecting Duct ◽

Rat Kidney ◽

Signaling Network ◽

The State ◽

Water Excretion ◽

Akt Phosphorylation ◽

Akt Activation ◽

Inhibitory Site ◽

V2 Receptor

Vasopressin regulates water excretion through effects on the renal collecting duct. Vasopressin signaling in the inner medullary collecting duct (IMCD) is mediated by V2 receptor occupation coupled to the generation of cyclic AMP. Here, we employ a “systems” approach to analysis of vasopressin signaling. The objective is to investigate roles of activation of the Akt and ERK1/2 MAP kinase pathways, as well as Ca2+ mobilization, in IMCD cells isolated from rat kidney. The V2 receptor-selective vasopressin analog dDAVP increased the state of Akt activation (increased phosphorylation at T308 and S473) and decreased the state of ERK1/2 activation (decreased phosphorylation at T202 and Y204). Akt activation was blocked by an inhibitor of PI3K, LY294002. In microdissected IMCD segments, nonperiodic spike-like increases in intracellular Ca2+ (FLUO-4) were accelerated by vasopressin. Chelation of Ca2+ or calmodulin inhibition markedly decreased Akt phosphorylation. Decreased ERK1/2 phosphorylation was associated with a decrease in MEK1/2 phosphorylation and an increase in c-Raf phosphorylation at S259 (an inhibitory site). Based on the current findings integrated with previous findings in the IMCD, we now report a 33-node vasopressin signaling network involved in vasopressin regulation of IMCD function.

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