Hypertonicity Is Involved in Redirecting the Aquaporin-2 Water Channel into the Basolateral, Instead of the Apical, Plasma Membrane of Renal Epithelial Cells

We previously showed that in polarized Madin–Darby canine kidney (MDCK) cells, aquaporin-2 (AQP2) is continuously targeted to the basolateral plasma membrane from which it is rapidly retrieved by clathrin-mediated endocytosis. It then undertakes microtubule-dependent transcytosis toward the apical plasma membrane. In this study, we found that treatment with chlorpromazine (CPZ, an inhibitor of clathrin-mediated endocytosis) results in AQP2 accumulation in the basolateral, but not the apical plasma membrane of epithelial cells. In MDCK cells, both AQP2 and clathrin were concentrated in the basolateral plasma membrane after CPZ treatment (100 µM for 15 min), and endocytosis was reduced. Then, using rhodamine phalloidin staining, we found that basolateral, but not apical, F-actin was selectively reduced by CPZ treatment. After incubation of rat kidney slices in situ with CPZ (200 µM for 15 min), basolateral AQP2 and clathrin were increased in principal cells, which simultaneously showed a significant decrease of basolateral compared to apical F-actin staining. These results indicate that clathrin-dependent transcytosis of AQP2 is an essential part of its trafficking pathway in renal epithelial cells and that this process can be inhibited by selectively depolymerizing the basolateral actin pool using CPZ.

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Purinergic control of apical plasma membrane PI(4,5)P2 levels sets ENaC activity in principal cells

AJP Renal Physiology ◽

10.1152/ajprenal.00403.2007 ◽

2008 ◽

Vol 294 (1) ◽

pp. F38-F46 ◽

Cited By ~ 60

Author(s):

Oleh Pochynyuk ◽

Vladislav Bugaj ◽

Alain Vandewalle ◽

James D. Stockand

Keyword(s):

Plasma Membrane ◽

Epithelial Cells ◽

Purinergic Receptors ◽

Apical Membrane ◽

Purinergic Signaling ◽

Apical Plasma Membrane ◽

Resting Cells ◽

Open Probability ◽

Renal Epithelial Cells ◽

Principal Cells

Activity of the epithelial sodium channel (ENaC) is limiting for Na+ reabsorption at the distal nephron. Phosphoinositides, such as phosphatidylinositol 4,5-biphosphate [PI(4,5)P2] modulate the activity of this channel. Activation of purinergic receptors triggers multiple events, including activation of PKC and PLC, with the latter depleting plasma membrane PI(4,5)P2. Here, we investigate regulation of ENaC in renal principal cells by purinergic receptors via PLC and PI(4,5)P2. Purinergic signaling rapidly decreases ENaC open probability and apical membrane PI(4,5)P2 levels with similar time courses. Moreover, inhibiting purinergic signaling with suramin rescues ENaC activity. The PLC inhibitor U73122, but not U73343, its inactive analog, recapitulates the action of suramin. In contrast, modulating PKC signaling failed to affect purinergic regulation of ENaC. Unexpectedly, inhibiting either purinergic receptors or PLC in resting cells dramatically increased ENaC activity above basal levels, indicating tonic activation of purinergic signaling in these polarized renal epithelial cells. Increased ENaC activity was associated with elevation of apical membrane PI(4,5)P2 levels. Subsequent treatment with ATP in the presence of inhibited purinergic signaling failed to decrease ENaC activity and apical membrane PI(4,5)P2 levels. Dwell-time analysis reveals that depletion of PI(4,5)P2 forces ENaC toward a closed state. In contrast, increasing PI(4,5)P2 levels above basal values locks the channel in an open state interrupted by brief closings. Thus our results suggest that purinergic control of apical membrane PI(4,5)P2 levels is a major regulator of ENaC activity in renal epithelial cells.

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

AJP Cell Physiology ◽

10.1152/ajpcell.00255.2021 ◽

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.

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

AJP Renal Physiology ◽

10.1152/ajprenal.00359.2009 ◽

2010 ◽

Vol 298 (2) ◽

pp. F266-F278 ◽

Cited By ~ 27

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.

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

AJP Cell Physiology ◽

10.1152/ajpcell.00109.2012 ◽

2013 ◽

Vol 304 (1) ◽

pp. C38-C48 ◽

Cited By ~ 45

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.

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Differential localization of human nongastric H+-K+-ATPase ATP1AL1 in polarized renal epithelial cells

AJP Renal Physiology ◽

10.1152/ajprenal.2000.279.3.f417 ◽

2000 ◽

Vol 279 (3) ◽

pp. F417-F425 ◽

Cited By ~ 14

Author(s):

Jürgen Reinhardt ◽

Alexander V. Grishin ◽

Hans Oberleithner ◽

Michael J. Caplan

Keyword(s):

Plasma Membrane ◽

Epithelial Cells ◽

Plasma Membranes ◽

Mdck Cells ◽

Apical Plasma Membrane ◽

Ion Pump ◽

Β Subunit ◽

Ion Pumps ◽

Α Subunit ◽

Renal Epithelial Cells

The human H+-K+-ATPase, ATP1AL1, belongs to the subgroup of nongastric, K+-transporting ATPases. In concert with the structurally related gastric H+-K+-ATPase, it plays a major role in K+ reabsorption in various tissues, including colon and kidney. Physiological and immunocytochemical data suggest that the functional heteromeric ion pumps are usually found in the apical plasma membranes of renal epithelial cells. However, the low expression levels of characteristic nongastric ion pumps makes it difficult to verify their spatial distribution in vivo. To investigate the sorting behavior of ATP1AL1, we expressed this pump by stable transfection in MDCK and LLC-PK1 renal epithelial cell lines. Stable interaction of ATP1AL1 with either the endogenous Na+-K+-ATPase β-subunit or the gastric H+-K+-ATPase β-subunit was tested by confocal immunofluorescence microscopy and surface biotinylation. In cells transfected with ATP1AL1 alone, the α-subunit accumulated intracellularly, consistent with its inability to assemble and travel to the plasma membrane with the endogenous Na+-K+-ATPase β-subunit. Cotransfection of ATP1AL1 with the gastric H+-K+-ATPase β-subunit resulted in plasma membrane localization of both pump subunits. In cotransfected MDCK cells the heteromeric ion pump was predominantly polarized to the apical plasma membrane. Functional expression of ATP1AL1 was confirmed by 86Rb+uptake measurements. In contrast, cotransfected LLC-PK1cells accumulate ATP1AL1 at the lateral membrane. The distinct polarization of ATP1AL1 indicates that the α-subunit encodes sorting information that is differently interpreted by cell type-specific sorting mechanisms.

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Quantitative analysis of aquaporin-2 phosphorylation

AJP Renal Physiology ◽

10.1152/ajprenal.00580.2009 ◽

2010 ◽

Vol 298 (4) ◽

pp. F1018-F1023 ◽

Cited By ~ 34

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.

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Effect on stability, degradation, expression, and targeting of aquaporin-2 water channel by hyperosmolality in renal epithelial cells

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2005.10.127 ◽

2005 ◽

Vol 338 (3) ◽

pp. 1593-1599 ◽

Cited By ~ 36

Author(s):

Fuminori Umenishi ◽

Takefumi Narikiyo ◽

Robert W. Schrier

Keyword(s):

Epithelial Cells ◽

Water Channel ◽

Renal Epithelial Cells ◽

Aquaporin 2

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

Journal of the American Society of Nephrology ◽

10.1681/asn.v9122181 ◽

1998 ◽

Vol 9 (12) ◽

pp. 2181-2193 ◽

Cited By ~ 1

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

AJP Cell Physiology ◽

10.1152/ajpcell.1995.269.3.c655 ◽

1995 ◽

Vol 269 (3) ◽

pp. C655-C664 ◽

Cited By ~ 212

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.

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