Redistribution of aquaporin-2 water channels induced by vasopressin in rat kidney inner medullary collecting duct

1995 ◽  
Vol 269 (3) ◽  
pp. C655-C664 ◽  
Author(s):  
D. Marples ◽  
M. A. Knepper ◽  
E. I. Christensen ◽  
S. Nielsen
Keyword(s):  
Plasma Membrane ◽  
Immunoelectron Microscopy ◽  
Collecting Duct ◽  
Water Channel ◽  
Fold Increase ◽  
Apical Plasma Membrane ◽  
Water Loading ◽  
Aquaporin 2 ◽  
Intracellular Vesicle ◽  
Intracellular Vesicles

Aquaporin-2 (AQP2) is the predominant vasopressin-regulated water channel of the renal collecting duct. We tested whether vasopressin induces translocation of AQP2 from intracellular vesicles into the apical plasma membrane. AQP2 was quantitated in plasma membrane and intracellular vesicle fractions prepared from the inner medulla of one kidney from each rat before or 20 min after intravenous 1-desamino-8-D-arginine vasopressin (DDAVP) treatment, using immunoblotting and densitometry. Contralateral kidneys were prepared for immunofluorescence and immunoelectron microscopy. Immunoblotting revealed that, compared with untreated controls, DDAVP treatment significantly increased the fraction of AQP2 protein associated with the plasma membrane fraction relative to intracellular vesicles. This increase averaged 2.0-fold in untreated rats and 2.9-fold in rats water loaded for 12 h. Water loading, presumably by suppressing circulating vasopressin levels, decreased the fraction of AQP2 associated with the plasma membrane by 55%, suggesting retrieval of AQP2 from the plasma membrane. In rats sequentially thirsted for 48 h to increase expression and then water loaded for 72 h to minimize plasma membrane labeling, DDAVP caused a 12-fold increase in the plasma membrane to intracellular vesicle labeling ratio. The accentuation of the DDAVP response seen after water loading is consistent with the observed increase in the fraction of AQP2 in the intracellular pool available for insertion. Immunofluorescence confirmed a marked DDAVP-induced redistribution of AQP2 from intracellular to plasma membrane domains. Furthermore, quantitative immunoelectron microscopy demonstrated a 3.4-fold increase in apical plasma membrane to intracellular vesicle labeling ratio. These results provide a direct in vivo demonstration of vasopressin-induced translocation of AQP2 into the apical plasma membrane.

Localization and regulation of PKA-phosphorylated AQP2 in response to V2-receptor agonist/antagonist treatment

2000 ◽  
Vol 278 (1) ◽  
pp. F29-F42 ◽  
Author(s):  
Birgitte Mønster Christensen ◽  
Marina Zelenina ◽  
Anita Aperia ◽  
Søren Nielsen
Keyword(s):  
Plasma Membrane ◽  
Immunoelectron Microscopy ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Fold Increase ◽  
Apical Plasma Membrane ◽  
Complete Disappearance ◽  
Vasopressin Secretion ◽  
V2 Receptor ◽  
Intracellular Vesicles

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.

scholarly journals A Role for VAMP8/Endobrevin in Surface Deployment of the Water Channel Aquaporin 2

2009 ◽  
Vol 30 (1) ◽  
pp. 333-343 ◽  
Author(s):  
Cheng-Chun Wang ◽  
Chee Peng Ng ◽  
Hong Shi ◽  
Hwee Chien Liew ◽  
Ke Guo ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Collecting Duct ◽  
Water Channel ◽  
Snare Proteins ◽  
Snare Protein ◽  
Aquaporin 2 ◽  
Duct Cells ◽  
Collecting Duct Cells ◽  
Intracellular Vesicles

ABSTRACT Vesicle-associated-membrane protein 8 (VAMP8) is highly expressed in the kidney, but the exact physiological and molecular functions executed by this v-SNARE protein in nephrons remain elusive. Here, we show that the depletion of VAMP8 in mice resulted in hydronephrosis. Furthermore, the level of the vasopressin-responsive water channel aquaporin 2 (AQP2) was increased by three- to fivefold in VAMP8-null mice. Forskolin and [desamino-Cys1, D-Arg8]-vasopressin (DDAVP)-induced AQP2 exocytosis was impaired in VAMP8-null collecting duct cells. VAMP8 was revealed to colocalize with AQP2 on intracellular vesicles and to interact with the plasma membrane t-SNARE proteins syntaxin4 and syntaxin3, suggesting that VAMP8 mediates the regulated fusion of AQP2-positive vesicles with the plasma membrane.

Regulated exocytosis: Renal Aquaporin-2 3D Vesicular Network Organization and Association with F-actin

2021 ◽  
Author(s):  
Mikkel R. Holst ◽  
Louis Gammelgaard ◽  
Jesse Aaron ◽  
Frédéric H. Login ◽  
Sampavi Rajkumar ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Antidiuretic Hormone ◽  
Collecting Duct ◽  
Water Channel ◽  
Urine Concentration ◽  
Apical Plasma Membrane ◽  
Aquaporin 2 ◽  
3D Network ◽  
Vesicle Exocytosis ◽  
A Cell

Regulated vesicle exocytosis is a key response to extracellular stimuli in diverse physiological processes; including hormone regulated short-term urine concentration. In the renal collecting duct, the water channel aquaporin-2 localizes to the apical plasma membrane as well as small, sub-apical vesicles. In response to stimulation with the antidiuretic hormone, arginine vasopressin, aquaporin-2 containing vesicles fuse with the plasma membrane, which increases collecting duct water reabsorption and thus, urine concentration. The nano-scale size of these vesicles has limited analysis of their 3D organization. Using a cell system combined with 3D super resolution microscopy, we provide the first direct analysis of the 3D network of aquaporin-2 containing exocytic vesicles in a cell culture system. We show that aquaporin-2 vesicles are 43 ± 3nm in diameter, a size similar to synaptic vesicles, and that one fraction of AQP2 vesicles localized with the sub-cortical F-actin layer and the other localized in between the F-actin layer and the plasma membrane. Aquaporin-2 vesicles associated with F-actin and this association was enhanced in a serine 256 phospho-mimic of aquaporin-2, whose phosphorylation is a key event in antidiuretic hormone-mediated aquaporin-2 vesicle exocytosis.

scholarly journals Lovastatin-induced cholesterol depletion affects both apical sorting and endocytosis of aquaporin-2 in renal cells

2010 ◽  
Vol 298 (2) ◽  
pp. F266-F278 ◽  
Author(s):  
G. Procino ◽  
C. Barbieri ◽  
M. Carmosino ◽  
F. Rizzo ◽  
G. Valenti ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Molecular Mechanisms ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Water Channel ◽  
Membrane Rafts ◽  
Apical Plasma Membrane ◽  
Cholesterol Depletion ◽  
Aquaporin 2 ◽  
Apical Sorting

Vasopressin causes the redistribution of the water channel aquaporin-2 (AQP2) from cytoplasmic storage vesicles to the apical plasma membrane of collecting duct principal cells, leading to urine concentration. The molecular mechanisms regulating the selective apical sorting of AQP2 are only partially uncovered. In this work, we investigate whether AQP2 sorting/trafficking is regulated by its association with membrane rafts. In both MCD4 cells and rat kidney, AQP2 preferentially associated with Lubrol WX-insoluble membranes regardless of its presence in the storage compartment or at the apical membrane. Block-and-release experiments indicate that 1) AQP2 associates with detergent-resistant membranes early in the biosynthetic pathway; 2) strong cholesterol depletion delays the exit of AQP2 from the trans-Golgi network. Interestingly, mild cholesterol depletion promoted a dramatic accumulation of AQP2 at the apical plasma membrane in MCD4 cells in the absence of forskolin stimulation. An internalization assay showed that AQP2 endocytosis was clearly reduced under this experimental condition. Taken together, these data suggest that association with membrane rafts may regulate both AQP2 apical sorting and endocytosis.

Direct demonstration of aquaporin-2 water channel recycling in stably transfected LLC-PK1 epithelial cells

1996 ◽  
Vol 270 (3) ◽  
pp. F548-F553 ◽  
Author(s):  
T. Katsura ◽  
D. A. Ausiello ◽  
D. Brown
Keyword(s):  
Protein Synthesis ◽  
Plasma Membrane ◽  
De Novo ◽  
Collecting Duct ◽  
Water Channel ◽  
Hormonal Stimulation ◽  
Aquaporin 2 ◽  
Direct Demonstration ◽  
Intracellular Vesicles ◽  
De Novo Protein Synthesis

Vasopressin-dependent membrane insertion of aquaporin-2 (AQP-2) in collecting duct principal cells has been demonstrated in vivo and in vitro. However, the hypothesis that the AQP-2 molecule recycles between intracellular vesicles and the plasma membrane in response to hormonal stimulation and withdrawal remains to be demonstrated directly. In the present study, we examined AQP-2 recycling between intracellular vesicles and the plasma membrane in the absence of de novo protein synthesis using LLC-PK1 cells transfected with an AQP-2-c-myc construct. Cells were treated with cycloheximide for 30 min prior to vasopressin stimulation, and all subsequent treatments were performed in the continued presence of cycloheximide. Complete inhibition of AQP-2 biosynthesis by cycloheximide was verified by immuno-precipitation. Immunofluorescence revealed that AQP-2 was located on intracellular vesicles in nonstimulated cells but was relocated to the plasma membrane after vasopressin treatment, even in the presence of cycloheximide. After vasopressin washout, AQP-2 was retrieved to intracellular vesicles and was relocated to the plasma membrane after restimulation with forskolin. Subsequent forskolin washout resulted in AQP-2 endocytosis, and a second stimulation with forskolin resulted in relocation to the plasma membrane. These data, obtained in the absence of de novo protein synthesis, clearly indicate that AQP-2 can be recycled multiple times between intracellular vesicles and the plasma membrane.

scholarly journals Basolateral targeting and microtubule-dependent transcytosis of the aquaporin-2 water channel

2013 ◽  
Vol 304 (1) ◽  
pp. C38-C48 ◽  
Author(s):  
Naofumi Yui ◽  
Hua A. J. Lu ◽  
Ying Chen ◽  
Naohiro Nomura ◽  
Richard Bouley ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cold Shock ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Water Channel ◽  
Apical Plasma Membrane ◽  
Indirect Pathway ◽  
Basolateral Membranes ◽  
Principal Cells ◽  
Aquaporin 2

The aquaporin-2 (AQP2) water channel relocates mainly to the apical plasma membrane of collecting duct principal cells after vasopressin (VP) stimulation. AQP2 transport to this membrane domain is assumed to be a direct route involving recycling of intracellular vesicles. However, basolateral plasma membrane expression of AQP2 is observed in vivo in principal cells. Here, we asked whether there is a transcytotic pathway of AQP2 trafficking between apical and basolateral membranes. We used MDCK cells in which AQP2 normally accumulates apically after VP exposure. In contrast, both site-specific biotinylation and immunofluorescence showed that AQP2 is strongly accumulated in the basolateral membrane, along with the endocytic protein clathrin, after a brief cold shock (4°C). This suggests that AQP2 may be constitutively targeted to basolateral membranes and then retrieved by clathrin-mediated endocytosis at physiological temperatures. Rab11 does not accumulate in basolateral membranes after cold shock, suggesting that the AQP2 in this location is not associated with Rab11-positive vesicles. After rewarming (37°C), basolateral AQP2 staining is diminished and it subsequently accumulates at the apical membrane in the presence of VP/forskolin, suggesting that transcytosis can be followed by apical insertion of AQP2. This process is inhibited by treatment with colchicine. Our data suggest that the cold shock procedure reveals the presence of microtubule-dependent AQP2 transcytosis, which represents an indirect pathway of apical AQP2 delivery in these cells. Furthermore, our data indicate that protein polarity data obtained from biotinylation assays, which require cells to be cooled to 4°C during the labeling procedure, should be interpreted with caution.

scholarly journals Quantitative analysis of aquaporin-2 phosphorylation

2010 ◽  
Vol 298 (4) ◽  
pp. F1018-F1023 ◽  
Author(s):  
Luke Xie ◽  
Jason D. Hoffert ◽  
Chung-Lin Chou ◽  
Ming-Jiun Yu ◽  
Trairak Pisitkun ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Collecting Duct ◽  
Water Channel ◽  
Apical Plasma Membrane ◽  
Specific Antibodies ◽  
Protein Mass ◽  
Aquaporin 2 ◽  
Protein Mass Spectrometry ◽  
V2 Receptor ◽  
A Site

The action of vasopressin in rodent collecting ducts to regulate water permeability depends in part on increases in phosphorylation of the water channel aquaporin-2 (AQP2) at three sites: Ser256, Ser264, and Ser269. Previous studies of AQP2 phosphorylation have depended largely on qualitative data using protein mass spectrometry and phospho-specific antibodies. Here, we use a new method employing phospho-specific antibodies to determine the percentage of total AQP2 phosphorylated at each site in the presence and absence of the V2-receptor-selective vasopressin analog dDAVP in rat renal inner medullary collecting duct (IMCD) and cultured mpkCCD cells. Phosphorylation of Ser269, a site previously implicated in plasma membrane retention, was found to increase from 3 to 26% of total AQP2 in rat IMCD cells following dDAVP. Quantification of immunogold labeling of the opposite kidneys from the same rats estimated that 11% of total AQP2 is present in the apical plasma membrane (APM) without injection of dDAVP and 25% is present in the APM after dDAVP. Surprisingly, the baseline level of Ser256 phosphorylation was constitutively high, and there was no increase with dDAVP (confirmed in 2 more sets of rats). In general, Ser264 phosphorylation remained below 5% of total. The pattern of response was similar in cultured mpkCCD cells (large increase in Ser269 phosphorylation following dDAVP, but constitutively high levels of Ser256 phosphorylation). We suggest from these studies that Ser269 phosphorylation may be a more consistent indicator of vasopressin action and AQP2 membrane abundance than is Ser256 phosphorylation.

Immunocytochemical localization of pendrin in intercalated cell subtypes in rat and mouse kidney

2002 ◽  
Vol 283 (4) ◽  
pp. F744-F754 ◽  
Author(s):  
Young-Hee Kim ◽  
Tae-Hwan Kwon ◽  
Sebastian Frische ◽  
Jin Kim ◽  
C. Craig Tisher ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Immunoelectron Microscopy ◽  
Collecting Duct ◽  
Mouse Kidney ◽  
Laser Scanning Microscopy ◽  
Apical Plasma Membrane ◽  
Intercalated Cells ◽  
Type B ◽  
Connecting Tubule ◽  
Intracellular Vesicles

Recent studies have demonstrated that a novel anion exchanger, pendrin, is expressed in the apical domain of type B intercalated cells in the mammalian collecting duct. The purpose of this study was 1) to determine the expression and distribution of pendrin along the collecting duct and connecting tubule of mouse and rat kidney and establish whether pendrin is expressed in the non-A-non-B intercalated cells and 2) to determine the intracellular localization of pendrin in the different populations of intercalated cells by immunoelectron microscopy. A peptide-derived affinity-purified antibody was generated that specifically recognized pendrin in immunoblots of rat and mouse kidney. Immunohistochemistry and confocal laser scanning microscopy demonstrated the presence of pendrin in apical domains of all type B intercalated cells in mouse and rat connecting tubule and collecting duct. In addition, strong pendrin immunostaining was observed in non-A-non-B intercalated cells. There was no labeling of type A intercalated cells. Immunoelectron microscopy demonstrated that pendrin was located in the apical plasma membrane and intracellular vesicles of both type B intercalated cells and non-A-non-B cells; the latter was identified by the presence of H+-ATPase in the apical plasma membrane. The results of this study demonstrate that both pendrin and H+-ATPase are expressed in the apical plasma membrane of non-A-non-B intercalated cells, suggesting that these cells are capable of both HCO[Formula: see text] and proton secretion. Furthermore, the presence of pendrin in both the apical plasma membrane and the apical intracellular vesicles of type B and non-A-non-B intercalated cells suggests that HCO[Formula: see text] secretion may be regulated by trafficking of pendrin between the two membrane compartments.

scholarly journals Decreased aquaporin-2 expression and apical plasma membrane delivery in kidney collecting ducts of polyuric hypercalcemic rats.

1998 ◽  
Vol 9 (12) ◽  
pp. 2181-2193 ◽  
Author(s):  
J H Earm ◽  
B M Christensen ◽  
J Frøkiaer ◽  
D Marples ◽  
J S Han ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Molecular Mechanisms ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Water Channel ◽  
Urine Osmolality ◽  
Apical Plasma Membrane ◽  
Similar Reduction ◽  
Aquaporin 2 ◽  
Urine Production

Hypercalcemia is frequently associated with a urinary concentrating defect and overt polyuria. The molecular mechanisms underlying this defect are poorly understood. Dysregulation of aquaporin-2 (AQP2), the predominant vasopressin-regulated water channel, is known to be associated with a range of congenital and acquired water balance disorders including nephrogenic diabetes insipidus and states of water retention. This study examines the effect of hypercalcemia on the expression of AQP2 in rat kidney. Rats were treated orally for 7 d with dihydrotachysterol, which produced significant hypercalcemia with a 15 +/- 2% increase in plasma calcium concentration. Immunoblotting and densitometry of membrane fractions revealed a significant decrease in AQP2 expression in kidney inner medulla of hypercalcemic rats to 45.7 +/- 6.8% (n = 11) of control levels (100 +/- 12%, n = 9). A similar reduction in AQP2 expression was seen in cortex (36.9 +/- 4.2% of control levels, n = 6). Urine production increased in parallel, from 11.3 +/- 1.4 to a maximum of 25.3 +/- 1.9 ml/d (P < 0.01), whereas urine osmolality decreased from 2007 +/- 186 mosmol/kg x H2O to 925 +/- 103 mosmol/kg x H2O (P < 0.01). Immunocytochemistry confirmed a decrease in total AQP2 labeling of collecting duct principal cells from kidneys of hypercalcemic rats, and reduced apical labeling. Immunoelectron microscopy demonstrated a significant reduction in AQP2 labeling of the apical plasma membrane, consistent with the development of polyuria. In summary, the results strongly suggest that AQP2 downregulation and reduced apical plasma membrane delivery of AQP2 play important roles in the development of polyuria in association with hypercalcemia.

H(+)V-ATPase-dependent luminal acidification in the kidney collecting duct and the epididymis/vas deferens: vesicle recycling and transcytotic pathways

2000 ◽  
Vol 203 (1) ◽  
pp. 137-145 ◽  
Author(s):  
D. Brown ◽  
S. Breton
Keyword(s):  
Plasma Membrane ◽  
B Cells ◽  
Vas Deferens ◽  
Collecting Duct ◽  
Kidney Cortex ◽  
Apical Plasma Membrane ◽  
Intercalated Cells ◽  
A Cells ◽  
Basolateral Plasma Membrane ◽  
Intracellular Vesicles

Many vertebrate transporting epithelia contain characteristic ‘mitochondria-rich’ cells that express high levels of a vacuolar proton-pumping ATPase (H(+)V-ATPase) on their plasma membrane and on intracellular vesicles. In the kidney cortex, A-cells and B-cells are involved in proton secretion and bicarbonate secretion, respectively, in the distal nephron and collecting duct. A-cells have an H(+)V-ATPase on their apical plasma membrane and on intracellular vesicles, whereas the cellular location of the H(+)V-ATPase can be apical, basolateral, bipolar or diffuse in B-cells. The rat epididymis and vas deferens also contain a distinct population of H(+)V-ATPase-rich epithelial cells. These cells are involved in generating a low luminal pH, which is involved in sperm maturation and in maintaining sperm in an immotile state during their passage through the epididymis and vas deferens. In both kidney and reproductive tract, H(+)V-ATPase-rich cells have a high rate of apical membrane recycling. H(+)V-ATPase molecules are transported between the cell surface and the cytoplasm in vesicles that have a well-defined ‘coat’ structure formed of the peripheral V(1) subunits of the H(+)V-ATPase. In addition, we propose that B-type intercalated cells have a transcytotic pathway that enables them to shuttle H(+)V-ATPase molecules from apical to basolateral plasma membrane domains. This hypothesis is supported by data showing that A-cells and B-cells have different intracellular trafficking pathways for LGP120, a lysosomal glycoprotein. LGP120 was found both on the basolateral plasma membrane and in lysosomes in B-cells, whereas no LGP120 was detectable in the plasma membrane of A-cells. We propose that the ‘polarity reversal’ of the H(+)V-ATPase in B-intercalated cells is mediated by a physiologically regulated transcytotic pathway that may be similar to that existing in some other cell types.

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