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Polarized Trafficking of AQP2 Revealed in Three Dimensional Epithelial Culture.

Rice WL, Li W, Mamuya F, McKee M, Păunescu TG, Lu HA - PLoS ONE (2015)

Bottom Line: Here we report the successful application of a 3-dimensional Madin-Darby canine kidney (MDCK) epithelial model to study polarized AQP2 trafficking.Therefore we have established a 3D culture model for the study of trafficking and regulation of both the apical and basolaterally targeted AQP2.The new model will enable further characterization of the complex mechanism regulating bi-polarized trafficking of AQP2 in vitro.

View Article: PubMed Central - PubMed

Affiliation: Center for Systems Biology, Program in Membrane Biology, Division of Nephrology, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, United States of America.

ABSTRACT
In renal collecting duct (CD) principal cells (PCs), vasopressin (VP) acts through its receptor, V2R, to increase intracellular cAMP leading to phosphorylation and apical membrane accumulation of the water channel aquaporin 2 (AQP2). The trafficking and function of basolaterally located AQP2 is, however, poorly understood. Here we report the successful application of a 3-dimensional Madin-Darby canine kidney (MDCK) epithelial model to study polarized AQP2 trafficking. This model recapitulates the luminal architecture of the CD and bi-polarized distribution of AQP2 as seen in kidney. Without stimulation, AQP2 is located in the subapical and basolateral regions. Treatment with VP, forskolin (FK), or 8-(4-Chlorophenylthio)-2'-O-methyladenosine 3',5'-cyclic monophosphate monosodium hydrate (CPT-cAMP) leads to translocation of cytosolic AQP2 to the apical membrane, but not to the basolateral membrane. Treating cells with methyl-β-cyclodextrin (mβCD) to acutely block endocytosis causes accumulation of AQP2 on the basolateral membrane, but not on the apical membrane. Our data suggest that AQP2 may traffic differently at the apical and basolateral domains in this 3D epithelial model. In addition, application of a panel of phosphorylation specific AQP2 antibodies reveals the polarized, subcellular localization of differentially phosphorylated AQP2 at S256, S261, S264 and S269 in the 3D culture model, which is consistent with observations made in the CDs of VP treated animals, suggesting the preservation of phosphorylation dependent regulatory mechanism of AQP2 trafficking in this model. Therefore we have established a 3D culture model for the study of trafficking and regulation of both the apical and basolaterally targeted AQP2. The new model will enable further characterization of the complex mechanism regulating bi-polarized trafficking of AQP2 in vitro.

No MeSH data available.


Related in: MedlinePlus

Formation of well-polarized epithelial cyst by AQP2 expressing MDCK cells.Single MDCK cells expressing rat AQP2 plated on matrigel form well-polarized cyst. The apical membrane domain is ringed by the tight junction protein (A) Zona Occludens-1 (green) and highlighted by (F) podocalyxin (green). (B) Integrin β1, (C) Na+/K+-ATPase and (D) β-catenin (green) are located on the basolateral membrane. (E, F), AQP2 (red) is distributed diffusely in the cytosol and in the basolateral region under baseline, non-stimulated conditions. DAPI (blue) stains nuclei. Images are single confocal planes taken through the middle of a spherical cyst. Bars = 10 μm.
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pone.0131719.g001: Formation of well-polarized epithelial cyst by AQP2 expressing MDCK cells.Single MDCK cells expressing rat AQP2 plated on matrigel form well-polarized cyst. The apical membrane domain is ringed by the tight junction protein (A) Zona Occludens-1 (green) and highlighted by (F) podocalyxin (green). (B) Integrin β1, (C) Na+/K+-ATPase and (D) β-catenin (green) are located on the basolateral membrane. (E, F), AQP2 (red) is distributed diffusely in the cytosol and in the basolateral region under baseline, non-stimulated conditions. DAPI (blue) stains nuclei. Images are single confocal planes taken through the middle of a spherical cyst. Bars = 10 μm.

Mentions: AQP2 expressing MDCK cells form polarized spherical cyst structures after 5–7 days of culture on matrigel (Fig 1 and S1 Fig). The tissue architecture of the”cyst” closely resembles the mature collecting duct epithelium observed in vivo: it is polarized with an outward facing basal membrane in contact with the extracellular matrix (ECM), lateral membranes that connect neighboring cells and a free apical membrane facing a fluid filled lumen. This polarized epithelium mimic expresses well-defined epithelial polarity markers: the classic apical markers GP-135 (podocalyxin) and zona occludens -1 are clearly detected on the apical membrane (Fig 1A and 1F respectively), while basolateral proteins such as integrin β1, Na+/K+-ATPase and β-catenin are restricted to the basolateral domain of the cyst (Fig 1B, 1C and 1D respectively). These results are consistent with other reports using similar models and observations of epithelium in vivo [30–33]. In the cyst, AQP2 locates mainly in vesicles throughout the cytosol and in the basolateral membrane region under baseline, non-stimulated conditions (Fig 1E and 1F), similar to what is observed in the connecting tubule and inner medullary collecting duct in normal rats [34].


Polarized Trafficking of AQP2 Revealed in Three Dimensional Epithelial Culture.

Rice WL, Li W, Mamuya F, McKee M, Păunescu TG, Lu HA - PLoS ONE (2015)

Formation of well-polarized epithelial cyst by AQP2 expressing MDCK cells.Single MDCK cells expressing rat AQP2 plated on matrigel form well-polarized cyst. The apical membrane domain is ringed by the tight junction protein (A) Zona Occludens-1 (green) and highlighted by (F) podocalyxin (green). (B) Integrin β1, (C) Na+/K+-ATPase and (D) β-catenin (green) are located on the basolateral membrane. (E, F), AQP2 (red) is distributed diffusely in the cytosol and in the basolateral region under baseline, non-stimulated conditions. DAPI (blue) stains nuclei. Images are single confocal planes taken through the middle of a spherical cyst. Bars = 10 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493001&req=5

pone.0131719.g001: Formation of well-polarized epithelial cyst by AQP2 expressing MDCK cells.Single MDCK cells expressing rat AQP2 plated on matrigel form well-polarized cyst. The apical membrane domain is ringed by the tight junction protein (A) Zona Occludens-1 (green) and highlighted by (F) podocalyxin (green). (B) Integrin β1, (C) Na+/K+-ATPase and (D) β-catenin (green) are located on the basolateral membrane. (E, F), AQP2 (red) is distributed diffusely in the cytosol and in the basolateral region under baseline, non-stimulated conditions. DAPI (blue) stains nuclei. Images are single confocal planes taken through the middle of a spherical cyst. Bars = 10 μm.
Mentions: AQP2 expressing MDCK cells form polarized spherical cyst structures after 5–7 days of culture on matrigel (Fig 1 and S1 Fig). The tissue architecture of the”cyst” closely resembles the mature collecting duct epithelium observed in vivo: it is polarized with an outward facing basal membrane in contact with the extracellular matrix (ECM), lateral membranes that connect neighboring cells and a free apical membrane facing a fluid filled lumen. This polarized epithelium mimic expresses well-defined epithelial polarity markers: the classic apical markers GP-135 (podocalyxin) and zona occludens -1 are clearly detected on the apical membrane (Fig 1A and 1F respectively), while basolateral proteins such as integrin β1, Na+/K+-ATPase and β-catenin are restricted to the basolateral domain of the cyst (Fig 1B, 1C and 1D respectively). These results are consistent with other reports using similar models and observations of epithelium in vivo [30–33]. In the cyst, AQP2 locates mainly in vesicles throughout the cytosol and in the basolateral membrane region under baseline, non-stimulated conditions (Fig 1E and 1F), similar to what is observed in the connecting tubule and inner medullary collecting duct in normal rats [34].

Bottom Line: Here we report the successful application of a 3-dimensional Madin-Darby canine kidney (MDCK) epithelial model to study polarized AQP2 trafficking.Therefore we have established a 3D culture model for the study of trafficking and regulation of both the apical and basolaterally targeted AQP2.The new model will enable further characterization of the complex mechanism regulating bi-polarized trafficking of AQP2 in vitro.

View Article: PubMed Central - PubMed

Affiliation: Center for Systems Biology, Program in Membrane Biology, Division of Nephrology, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, United States of America.

ABSTRACT
In renal collecting duct (CD) principal cells (PCs), vasopressin (VP) acts through its receptor, V2R, to increase intracellular cAMP leading to phosphorylation and apical membrane accumulation of the water channel aquaporin 2 (AQP2). The trafficking and function of basolaterally located AQP2 is, however, poorly understood. Here we report the successful application of a 3-dimensional Madin-Darby canine kidney (MDCK) epithelial model to study polarized AQP2 trafficking. This model recapitulates the luminal architecture of the CD and bi-polarized distribution of AQP2 as seen in kidney. Without stimulation, AQP2 is located in the subapical and basolateral regions. Treatment with VP, forskolin (FK), or 8-(4-Chlorophenylthio)-2'-O-methyladenosine 3',5'-cyclic monophosphate monosodium hydrate (CPT-cAMP) leads to translocation of cytosolic AQP2 to the apical membrane, but not to the basolateral membrane. Treating cells with methyl-β-cyclodextrin (mβCD) to acutely block endocytosis causes accumulation of AQP2 on the basolateral membrane, but not on the apical membrane. Our data suggest that AQP2 may traffic differently at the apical and basolateral domains in this 3D epithelial model. In addition, application of a panel of phosphorylation specific AQP2 antibodies reveals the polarized, subcellular localization of differentially phosphorylated AQP2 at S256, S261, S264 and S269 in the 3D culture model, which is consistent with observations made in the CDs of VP treated animals, suggesting the preservation of phosphorylation dependent regulatory mechanism of AQP2 trafficking in this model. Therefore we have established a 3D culture model for the study of trafficking and regulation of both the apical and basolaterally targeted AQP2. The new model will enable further characterization of the complex mechanism regulating bi-polarized trafficking of AQP2 in vitro.

No MeSH data available.


Related in: MedlinePlus