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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

Ultrastructure of MDCK-AQP2 cysts.The ultrastructure of the cyst closely resembles the polarized architecture of renal tubular epithelium in animal kidney. (A) A differential interference contrast image of a whole cyst. The lumen is clearly visible despite some remnants of cells that were shed in the lumen during lumen formation. The basal membrane is in contact with the Matrigel substrate. Scale = 10 μm. (B) In a TEM cross section of a single cell from a MDCK-AQP2 cyst, microvilli can be seen on the apical membrane facing the lumen (L) while the nucleus (N) is oriented toward the basal membrane. Scale = 2 μm. (C) At the apical domain, neighboring cells interact through tight junctions (TJ) and desmosomes (D) and the presence of clathrin-coated pits (arrow) indicate active endocytosis in the apical and subapical region. Scale = 500 nm. (D) Similarly, clathrin coated vesicles are observed at the basal and lateral membranes of the cyst cells (arrows). Scale = 500 nm. In all images (M) indicates mitochondria.
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pone.0131719.g002: Ultrastructure of MDCK-AQP2 cysts.The ultrastructure of the cyst closely resembles the polarized architecture of renal tubular epithelium in animal kidney. (A) A differential interference contrast image of a whole cyst. The lumen is clearly visible despite some remnants of cells that were shed in the lumen during lumen formation. The basal membrane is in contact with the Matrigel substrate. Scale = 10 μm. (B) In a TEM cross section of a single cell from a MDCK-AQP2 cyst, microvilli can be seen on the apical membrane facing the lumen (L) while the nucleus (N) is oriented toward the basal membrane. Scale = 2 μm. (C) At the apical domain, neighboring cells interact through tight junctions (TJ) and desmosomes (D) and the presence of clathrin-coated pits (arrow) indicate active endocytosis in the apical and subapical region. Scale = 500 nm. (D) Similarly, clathrin coated vesicles are observed at the basal and lateral membranes of the cyst cells (arrows). Scale = 500 nm. In all images (M) indicates mitochondria.

Mentions: The ultrastructure of the MDCK cell cysts was examined by transmission electron microscopy (TEM), as shown in Fig 2. Multiple microvilli are observed on the apical membrane facing the lumen while the nuclei are oriented toward the basal membrane (Fig 2B). The presence of tight junctions (TJ) on the apical pole of the lateral membranes between two adjacent cells is clearly revealed by the EM (TJ in Fig 2C). Multiple desmosomes (D in Fig 2C) are clearly visualized along the lateral membrane below the apical pole/TJ. Clathrin coated vesicles (arrows) are visible in both the apical and basolateral regions of the cells, suggesting the presence of an active endocytic trafficking interface on both the apical and basolateral membranes.


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)

Ultrastructure of MDCK-AQP2 cysts.The ultrastructure of the cyst closely resembles the polarized architecture of renal tubular epithelium in animal kidney. (A) A differential interference contrast image of a whole cyst. The lumen is clearly visible despite some remnants of cells that were shed in the lumen during lumen formation. The basal membrane is in contact with the Matrigel substrate. Scale = 10 μm. (B) In a TEM cross section of a single cell from a MDCK-AQP2 cyst, microvilli can be seen on the apical membrane facing the lumen (L) while the nucleus (N) is oriented toward the basal membrane. Scale = 2 μm. (C) At the apical domain, neighboring cells interact through tight junctions (TJ) and desmosomes (D) and the presence of clathrin-coated pits (arrow) indicate active endocytosis in the apical and subapical region. Scale = 500 nm. (D) Similarly, clathrin coated vesicles are observed at the basal and lateral membranes of the cyst cells (arrows). Scale = 500 nm. In all images (M) indicates mitochondria.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131719.g002: Ultrastructure of MDCK-AQP2 cysts.The ultrastructure of the cyst closely resembles the polarized architecture of renal tubular epithelium in animal kidney. (A) A differential interference contrast image of a whole cyst. The lumen is clearly visible despite some remnants of cells that were shed in the lumen during lumen formation. The basal membrane is in contact with the Matrigel substrate. Scale = 10 μm. (B) In a TEM cross section of a single cell from a MDCK-AQP2 cyst, microvilli can be seen on the apical membrane facing the lumen (L) while the nucleus (N) is oriented toward the basal membrane. Scale = 2 μm. (C) At the apical domain, neighboring cells interact through tight junctions (TJ) and desmosomes (D) and the presence of clathrin-coated pits (arrow) indicate active endocytosis in the apical and subapical region. Scale = 500 nm. (D) Similarly, clathrin coated vesicles are observed at the basal and lateral membranes of the cyst cells (arrows). Scale = 500 nm. In all images (M) indicates mitochondria.
Mentions: The ultrastructure of the MDCK cell cysts was examined by transmission electron microscopy (TEM), as shown in Fig 2. Multiple microvilli are observed on the apical membrane facing the lumen while the nuclei are oriented toward the basal membrane (Fig 2B). The presence of tight junctions (TJ) on the apical pole of the lateral membranes between two adjacent cells is clearly revealed by the EM (TJ in Fig 2C). Multiple desmosomes (D in Fig 2C) are clearly visualized along the lateral membrane below the apical pole/TJ. Clathrin coated vesicles (arrows) are visible in both the apical and basolateral regions of the cells, suggesting the presence of an active endocytic trafficking interface on both the apical and basolateral membranes.

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