Microtubules are needed for the perinuclear positioning of aquaporin-2 after its endocytic retrieval in renal principal cells

2007 ◽  
Vol 293 (3) ◽  
pp. C1129-C1138 ◽  
Author(s):  
Anna Vossenkämper ◽  
Pavel I. Nedvetsky ◽  
Burkhard Wiesner ◽  
Jens Furkert ◽  
Walter Rosenthal ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Collecting Duct ◽  
Water Channel ◽  
Small Gtpase ◽  
Cell Periphery ◽  
Resting Cells ◽  
Principal Cells ◽  
Aquaporin 2 ◽  
Osmotic Water ◽  
Dependent Transport

Water reabsorption in the renal collecting duct is regulated by arginine vasopressin (AVP). AVP induces the insertion of the water channel aquaporin-2 (AQP2) into the plasma membrane of principal cells, thereby increasing the osmotic water permeability. The redistribution of AQP2 to the plasma membrane is a cAMP-dependent process and thus a paradigm for cAMP-controlled exocytic processes. Using primary cultured rat inner medullary collecting duct cells, we show that the redistribution of AQP2 to the plasma membrane is accompanied by the reorganization of microtubules and the redistribution of the small GTPase Rab11. In resting cells, AQP2 is colocalized with Rab11 perinuclearly. AVP induced the redistribution of AQP2 to the plasma membrane and of Rab11 to the cell periphery. The redistribution of both proteins was increased when microtubules were depolymerized by nocodazole. In addition, the depolymerization of microtubules prevented the perinuclear positioning of AQP2 and Rab11 in resting cells, which was restored if nocodazole was washed out and microtubules repolymerized. After internalization of AQP2, induced by removal of AVP, forskolin triggered the AQP2 redistribution to the plasma membrane even if microtubules were depolymerized and without the previous positioning of AQP2 in the perinuclear recycling compartment. Collectively, the data indicate that microtubule-dependent transport of AQP2 is predominantly responsible for trafficking and localization of AQP2 inside the cell after its internalization but not for the exocytic transport of the water channel. We also demonstrate that cAMP-signaling regulates the localization of Rab11-positive recycling endosomes in renal principal cells.

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 Rab7 involves Vps35 to mediate AQP2 sorting and apical trafficking in collecting duct cells

2020 ◽  
Vol 318 (4) ◽  
pp. F956-F970 ◽  
Author(s):  
Wei-Ling Wang ◽  
Shih-Han Su ◽  
Kit Yee Wong ◽  
Chan-Wei Yang ◽  
Chin-Fu Liu ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Water Channel ◽  
Small Gtpase ◽  
Apical Plasma Membrane ◽  
Water Reabsorption ◽  
Recycling Endosomes ◽  
Early Endosomes ◽  
Vacuolar Protein

Aquaporin-2 (AQP2) is a vasopressin-regulated water channel protein responsible for osmotic water reabsorption by kidney collecting ducts. In response to vasopressin, AQP2 traffics from intracellular vesicles to the apical plasma membrane of collecting duct principal cells, where it increases water permeability and, hence, water reabsorption. Despite continuing efforts, gaps remain in our knowledge of vasopressin-regulated AQP2 trafficking. Here, we studied the functions of two retromer complex proteins, small GTPase Rab7 and vacuolar protein sorting 35 (Vps35), in vasopressin-induced AQP2 trafficking in a collecting duct cell model (mpkCCD cells). We showed that upon vasopressin removal, apical AQP2 returned to Rab5-positive early endosomes before joining Rab11-positive recycling endosomes. In response to vasopressin, Rab11-associated AQP2 trafficked to the apical plasma membrane before Rab5-associated AQP2 did so. Rab7 knockdown resulted in AQP2 accumulation in early endosomes and impaired vasopressin-induced apical AQP2 trafficking. In response to vasopressin, Rab7 transiently colocalized with Rab5, indicative of a role of Rab7 in AQP2 sorting in early endosomes before trafficking to the apical membrane. Rab7-mediated apical AQP2 trafficking in response to vasopressin required GTPase activity. When Vps35 was knocked down, AQP2 accumulated in recycling endosomes under vehicle conditions and did not traffic to the apical plasma membrane in response to vasopressin. We conclude that Rab7 and Vps35 participate in AQP2 sorting in early endosomes under vehicle conditions and apical membrane trafficking in response to vasopressin.

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.

Aquaporin 2 is a vasopressin-independent, constitutive apical membrane protein in rat vas deferens

2000 ◽  
Vol 278 (4) ◽  
pp. C791-C802 ◽  
Author(s):  
Anna L. Stevens ◽  
Sylvie Breton ◽  
Corinne E. Gustafson ◽  
Richard Bouley ◽  
Raoul D. Nelson ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Cellular Localization ◽  
Vas Deferens ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Water Channel ◽  
Sperm Concentration ◽  
Apical Plasma Membrane ◽  
Principal Cells ◽  
Aquaporin 2

Aquaporin 2 (AQP2), the vasopressin-regulated water channel, was originally identified in renal collecting duct principal cells. However, our recent description of AQP2 in the vas deferens indicated that this water channel may have extra-renal functions, possibly related to sperm concentration in the male reproductive tract. In this study, we have examined the regulation and membrane insertion pathway of AQP2 in the vas deferens. The amino acid sequence of vas deferens AQP2 showed 100% identity to the renal protein. AQP2 was highly expressed in the distal portion (ampulla) of the vas deferens, but not in the proximal portion nearest the epididymis. It was concentrated on the apical plasma membrane of vas deferens principal cells, and very little was detected on intracellular vesicles. Protein expression levels and cellular localization patterns were similar in normal rats and vasopressin-deficient Brattleboro homozygous rats, and were not changed after 36 h of dehydration, or after 3 days of vasopressin infusion into Brattleboro rats. AQP2 was not found in apical endosomes (labeled with Texas Red-dextran) in vas deferens principal cells, indicating that it is not rapidly recycling in this tissue. Finally, vasopressin receptors were not detectable on vas deferens epithelial cell membranes using a [3H]vasopressin binding assay. These data indicate that AQP2 is a constitutive apical membrane protein in the vas deferens, and that it is not vasopressin-regulated in this tissue. Thus AQP2 contains targeting information that can be interpreted in a cell-type-specific fashion in vivo.

Escape from vasopressin-induced antidiuresis: role of vasopressin resistance of the collecting duct

1998 ◽  
Vol 274 (6) ◽  
pp. F1161-F1166 ◽  
Author(s):  
Carolyn A. Ecelbarger ◽  
Chung-Lin Chou ◽  
Alanna J. Lee ◽  
Susan R. DiGiovanni ◽  
Joseph G. Verbalis ◽  
...  
Keyword(s):  
Water Permeability ◽  
Collecting Duct ◽  
Water Channel ◽  
Phosphodiesterase Inhibitor ◽  
Intracellular Camp ◽  
Protein Subunit ◽  
Aquaporin 2 ◽  
Camp Generation ◽  
Osmotic Water ◽  
Camp Levels

Previously, we demonstrated that escape from vasopressin-induced antidiuresis (“vasopressin escape”) in rats is associated with a large, selective decrease in whole kidney expression of aquaporin-2, the vasopressin-regulated water channel. Here, we show that isolated perfused inner medullary collecting ducts (IMCDs) from vasopressin-escape rats {desamino-[d-arginine]vasopressin (DDAVP)/water-loaded} have dramatically reduced vasopressin-dependent osmotic water permeabilities [46% of control rats (DDAVP alone)], which coincides with a fall in inner medullary aquaporin-2 protein abundance as measured by immunoblotting in the opposite kidney. Furthermore, we demonstrate in IMCD suspensions that cAMP accumulation in response to DDAVP is substantially reduced in the vasopressin-escape rats both in the presence and absence of the phosphodiesterase inhibitor IBMX. By immunoblotting, we show that the abundance of two proteins important in cAMP generation: the stimulatory heterotrimeric G protein subunit Gsα and adenylyl cyclase type VI, do not change. We conclude that vasopressin escape is associated with relative vasopressin resistance of the collecting duct cells manifested by decreased intracellular cAMP levels. The decreased cAMP levels can contribute to the demonstrated decrease in collecting duct water permeability in two ways: 1) by causing a decrease in aquaporin-2 expression and 2) by limiting the acute action of vasopressin to increase collecting duct water permeability.

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 Endocytic vesicles from renal papilla which retrieve the vasopressin-sensitive water channel do not contain a functional H+ ATPase.

1990 ◽  
Vol 111 (2) ◽  
pp. 379-389 ◽  
Author(s):  
W I Lencer ◽  
A S Verkman ◽  
M A Arnaout ◽  
D A Ausiello ◽  
D Brown
Keyword(s):  
Plasma Membrane ◽  
Water Permeability ◽  
Collecting Duct ◽  
Water Channel ◽  
Water Channels ◽  
Proton Pumps ◽  
Principal Cells ◽  
Kidney Collecting Duct ◽  
Fluorescence Imaging Microscopy ◽  
Membrane Components

The water permeability of the kidney collecting duct epithelium is regulated by vasopressin (VP)-induced recycling of water channels between an intracellular vesicular compartment and the plasma membrane of principal cells. To test whether the water channels pass through an acidic endosomal compartment during the endocytic portion of this pathway, we measured ATP-dependent acidification of FITC-dextran-labeled endosomes in isolated microsomal fractions from different regions of Brattleboro rat kidneys. Both VP-deficient controls and rat treated with exogenous VP were examined. ATP-dependent acidification was not detectable in endosomes containing water channels from distal papilla (osmotic water permeability Pf = 0.038 +/- 0.004 cm/s). In contrast, the addition of ATP resulted in a strong acidification of renal cortical endosomes (pHmin = 5.8, initial rate = 0.18-0.25 pH U/s). Acidification of cortical endosomes was reversed with nigericin and strongly inhibited by N-ethyl-maleimide. Passive proton permeability was similar and low in both cortical and papillary endosomes from rats treated or not treated with VP. The fraction of labeled endosomes present in microsomal preparations was determined by fluorescence imaging microscopy of microsomes nonspecifically bound to poly-l-lysine-coated coverslips and was 25% in cortical preparations compared to 14% (+VP) and 9% (-VP) in papillary preparations. The fraction of cortical endosomes was enriched 1.5-fold by immunoabsorption to coverslips coated with mAbs against the bovine vacuolar proton pump. In contrast, the fraction of papillary endosomes was depleted more than twofold by immunoabsorption to identical coverslips. Finally, sections of distal papilla stained with antibodies against the lysosomal glycoprotein LGP120 showed that most of the entrapped FITC-dextran did not colocalize with this lysosomal protein. These results demonstrate that vesicles which internalize water channels in kidney collecting duct principal cells lack functional proton pumps, and do not deliver the bulk of their FITC-dextran content to lysosomes. The data suggest that the principal cell contains a specialized nonacidic apical endocytic compartment which functions primarily to recycle membrane components, including water channels, to the plasma membrane.

A rat kidney tubule suspension for the study of vasopressin-induced shuttling of AQP2 water channels

2002 ◽  
Vol 283 (5) ◽  
pp. F1160-F1166 ◽  
Author(s):  
Stephen Shaw ◽  
David Marples
Keyword(s):  
Plasma Membrane ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Primary Cultures ◽  
Water Channels ◽  
Apical Plasma Membrane ◽  
Cellular Mechanisms ◽  
Principal Cells ◽  
Microtubule Disruption ◽  
Osmotic Water

AVP increases the osmotic water permeability of renal collecting ducts by inducing the translocation of specific aquaporin-2 (AQP2) water channels from cytoplasmic vesicles to the apical plasma membrane of the principal cells. Here, we report a novel inner medullary tubule suspension for the study of this phenomenon that overcomes some of the drawbacks faced by present techniques; both primary cultures of inner medullary collecting duct cells and cell lines expressing AQP2 show aberrant trafficking and/or signaling pathways. The tubule suspensions were prepared by proteolytic digestion of inner medullas dissected from freshly isolated rat kidneys. After drug treatment, cellular distribution of AQP2 was determined by membrane fractionation and Western blotting or by immunocytochemistry. Treatment of suspensions with 1 nM AVP caused redistribution of AQP2 to the apical plasma membrane of the principal cells, a process inhibited by microtubule disruption or PKA inhibition. We conclude that this method provides a valuable new approach to the study of the cellular mechanisms involved in the response of the collecting duct to AVP.

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 Cyclin-Dependent Kinase 18 Controls Trafficking of Aquaporin-2 and Its Abundance through Ubiquitin Ligase STUB1, Which Functions as an AKAP

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 673 ◽  
Author(s):  
Alessandro Dema ◽  
Dörte Faust ◽  
Katina Lazarow ◽  
Marc Wippich ◽  
Martin Neuenschwander ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Ubiquitin Ligase ◽  
Protein Complex ◽  
Collecting Duct ◽  
Water Channel ◽  
Fine Tuning ◽  
Cyclin Dependent Kinase ◽  
Water Reabsorption ◽  
Aquaporin 2 ◽  
Anchoring Protein

Arginine-vasopressin (AVP) facilitates water reabsorption in renal collecting duct principal cells through regulation of the water channel aquaporin-2 (AQP2). The hormone binds to vasopressin V2 receptors (V2R) on the surface of the cells and stimulates cAMP synthesis. The cAMP activates protein kinase A (PKA), which initiates signaling that causes an accumulation of AQP2 in the plasma membrane of the cells facilitating water reabsorption from primary urine and fine-tuning of body water homeostasis. AVP-mediated PKA activation also causes an increase in the AQP2 protein abundance through a mechanism that involves dephosphorylation of AQP2 at serine 261 and a decrease in its poly-ubiquitination. However, the signaling downstream of PKA that controls the localization and abundance of AQP2 is incompletely understood. We carried out an siRNA screen targeting 719 kinase-related genes, representing the majority of the kinases of the human genome and analyzed the effect of the knockdown on AQP2 by high-content imaging and biochemical approaches. The screening identified 13 hits whose knockdown inhibited the AQP2 accumulation in the plasma membrane. Amongst the candidates was the so far hardly characterized cyclin-dependent kinase 18 (CDK18). Our further analysis revealed a hitherto unrecognized signalosome comprising CDK18, an E3 ubiquitin ligase, STUB1 (CHIP), PKA and AQP2 that controls the localization and abundance of AQP2. CDK18 controls AQP2 through phosphorylation at serine 261 and STUB1-mediated ubiquitination. STUB1 functions as an A-kinase anchoring protein (AKAP) tethering PKA to the protein complex and bridging AQP2 and CDK18. The modulation of the protein complex may lead to novel concepts for the treatment of disorders which are caused or are associated with dysregulated AQP2 and for which a satisfactory treatment is not available, e.g., hyponatremia, liver cirrhosis, diabetes insipidus, ADPKD or heart failure.

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