Low aquaporin-2 levels in polyuric DI +/+ severe mice with constitutively high cAMP-phosphodiesterase activity

1999 ◽  
Vol 276 (2) ◽  
pp. F179-F190 ◽  
Author(s):  
Jørgen Frøkiaer ◽  
David Marples ◽  
Heinz Valtin ◽  
John F. Morris ◽  
Mark A. Knepper ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Collecting Duct ◽  
Northern Blotting ◽  
Apical Plasma Membrane ◽  
Phosphodiesterase Activity ◽  
Camp Phosphodiesterase ◽  
Aquaporin 2 ◽  
Acute Response ◽  
Camp Levels

In the renal collecting duct, vasopressin acutely activates cAMP production, resulting in trafficking of aquaporin-2 water channels (AQP2) to the apical plasma membrane, thereby increasing water permeability. This acute response is modulated by long-term changes in AQP2 expression. Recently, a cAMP-responsive element has been identified in the AQP2 gene, raising the possibility that changes in cAMP levels may control AQP2 expression. To investigate this possibility, we determined AQP2 protein levels in a strain of mice, DI +/+ severe (DI), which have genetically high levels of cAMP-phosphodiesterase activity, and hence low cellular cAMP levels, and severe polyuria. Semiquantitative immunoblotting of membrane fractions prepared from whole kidneys revealed that AQP2 levels in DI mice were only 26 ± 7% (±SE) of those in control mice ( n = 10, P < 0.01). In addition, semiquantitative Northern blotting revealed a significantly lower AQP2 mRNA expression in kidneys from DI mice compared with control mice (43 ± 6% vs. 100 ± 10%; n = 6 in each group, P < 0.05). AQP3 levels were also reduced. The mice were polyuric and urine osmolalities were accordingly substantially lower in the DI mice than in controls (496 ± 53 vs. 1,696 ± 105 mosmol/kgH2O, respectively). Moreover, there was a linear correlation between urine osmolalities and AQP2 levels ( P < 0.05). Immunoelectron microscopy confirmed the markedly lower expression of AQP2 in collecting duct principal cells in kidneys of DI mice and, furthermore, demonstrated that AQP2 was almost completely absent from the apical plasma membrane. Thus expression of AQP2 and AQP2 trafficking were severely impaired in DI mice. These results are consistent with the view that in vivo regulation of AQP2 expression by vasopressin is mediated by cAMP.

ADH-sensitive cAMP system in papillary collecting duct: effect of osmolality and PGE2

1981 ◽  
Vol 240 (4) ◽  
pp. F311-F318 ◽  
Author(s):  
R. M. Edwards ◽  
B. A. Jackson ◽  
T. P. Dousa
Keyword(s):  
Collecting Duct ◽  
Rat Kidney ◽  
Major Influence ◽  
Phosphodiesterase Activity ◽  
Camp Phosphodiesterase ◽  
Biphasic Effect ◽  
Papillary Collecting Duct ◽  
Camp Metabolism ◽  
Camp System ◽  
Camp Levels

The papillary collecting duct (PCD) is considered to be of major importance in the final elaboration of the urine, but the metabolism of cyclic adenosine 3',5'-monophosphate (cAMP) has not yet been directly studied in the PCD. Therefore, in the present study we examined the basic properties of the cAMP system in isolated PCD microdissected from rat kidney. Vasopressin (VP) caused a marked (5- to 10-fold) stimulation of adenylate cyclase (AdC) but parathyroid hormone, calcitonin, isoproterenol, and bradykinin were without effect. A gradual increase in osmolality from 200 mosM had a biphasic effect on AdC, first enhancing (at 800 mosM) then inhibiting AdC activity at 2,000 mosM. cAMP-phosphodiesterase activity was inhibited as osmolality was increased from 200 to 800 mosM and the inhibition remained constant to 2,000 mosM. Incubation of intact PCD with VP resulted in a threefold increase in cAMP levels. As the osmolality of the incubation medium ws increased from 300 to 2,000 mosM, both basal and VP-stimulated cAMP levels continued to increase. Prostaglandin E2 (PGE2) (10(-5) M) alone (in the absence of vP) caused an increase in AdC activity, but the same dose of PGE2 had no effect on AdC activity stimulated by submaximal or maximal doses of VP. PGE2 (10(-5) M) caused a small increase in cAMP levels in intact PCD. On the other hand, PGE2 inhibited VP-stimulated cAMP levels by 50%. Incubation of PCD with PGE2 had no effect on cAMP-phosphodiesterase activity. The results demonstrate that osmolality in the physiologic range has a major influence on cAMP metabolism in the PCD and document an antagonism between PGE2 and VP at the level of cAMP accumulation in the PCD.

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.

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 SNAP-25-associated Hrs-2 protein colocalizes with AQP2 in rat kidney collecting duct principal cells

2001 ◽  
Vol 281 (3) ◽  
pp. F546-F556 ◽  
Author(s):  
Alok Shukla ◽  
Henrik Hager ◽  
Thomas Juhl Corydon ◽  
Andrew J. Bean ◽  
Ronald Dahl ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Laser Scanning ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Northern Blotting ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Apical Plasma Membrane ◽  
Membrane Domains ◽  
Rt Pcr

The vasopressin-induced trafficking of aquaporin-2 (AQP2) water channels in kidney collecting duct is likely mediated by vesicle-targeting proteins ( N-ethylmaleimide-sensitive factor attachment protein receptors). Hrs-2 is an ATPase believed to have a modulatory role in regulated exocytosis. To examine whether Hrs-2 is expressed in rat kidney, we carried out RT-PCR combined with DNA sequence analysis and Northern blotting using a digoxigenin-labeled Hrs-2 RNA probe. RT-PCR and Northern blotting revealed that Hrs-2 mRNA is localized in all zones of rat kidney. The presence of Hrs-2 protein in rat kidney was confirmed by immunoblotting, revealing a 115-kDa protein in kidney and brain membrane fractions corresponding to the expected molecular size of Hrs-2. Immunostaining and confocal laser scanning microscopy of LLC-PK1 cells (a porcine proximal tubule cell line) transfected with Hrs-2 DNA confirmed the specificity of the antibody and revealed that Hrs-2 is mainly localized in intracellular compartments, including cathepsin D-containing lysosomal/endosomal compartments. The cellular and subcellular localization of Hrs-2 in rat kidney was examined by immunocytochemistry and confocal laser scanning microscopy. Hrs-2 immunoreactivity was observed in collecting duct principal cells, and weaker labeling was detected in other nephron segments. The labeling was predominantly present in intracellular vesicles, but labeling was also observed in the apical plasma membrane domains of some cells. Colabeling with AQP2 revealed colocalization in vesicles and apical plasma membrane domains, suggesting a role for Hrs-2 in regulated AQP2 trafficking.

Differential subcellular localization of ENaC subunits in mouse kidney in response to high- and low-Na diets

2000 ◽  
Vol 279 (2) ◽  
pp. F252-F258 ◽  
Author(s):  
Johannes Loffing ◽  
Laurence Pietri ◽  
Fintan Aregger ◽  
May Bloch-Faure ◽  
Urs Ziegler ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Sodium Intake ◽  
Collecting Duct ◽  
Mouse Kidney ◽  
Plasma Aldosterone ◽  
Dietary Sodium ◽  
Apical Plasma Membrane ◽  
Cytoplasmic Staining ◽  
Α Subunit

Previous electrophysiological experiments on renal cortical collecting ducts indicated that dietary sodium intake and variations in aldosterone plasma levels regulate the abundance of functional epithelial Na channels (ENaC) in the apical plasma membrane. In mouse kidney we investigated by immunohistochemistry whether feeding for 3 wk a diet with high (3% Na) and low (0.05% Na) Na content influences the distribution pattern of ENaC. In mice of all experimental groups, ENaC was apparent in cells from the late portion of the distal convoluted tubule (DCT2) down to the medullary collecting duct (CD). In mice on a high-Na diet (plasma aldosterone: 40.8 ± 2.0 ng/dl), the α-subunit was undetectable, and the β- and γ-ENaC were detected in the cytoplasm, but not in the apical plasma membrane of the cells. In contrast, in mice on a low-Na diet (plasma aldosterone: 93.6 ± 9.3 ng/dl) all three ENaC subunits were displayed in the subapical cytoplasm and in the apical membrane of DCT2, connecting tubule (CNT), and, although less prominent, in cortical CD cells. Apical plasma membrane immunostaining progressively decreased along the cortical CD, simultaneously with increasing cytoplasmic staining for β- and γ-ENaC. Thus our data on mice adapted to moderately low and high Na intake suggest that regulation of ENaC function in vivo involves shifts of β- and γ-subunits from the cytoplasm to the apical plasma membrane and vice versa, respectively. The insertion of these subunits into the apical plasma membrane coincides with upregulation of the α-subunit and its insertion into the apical plasma membrane.

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.

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.

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