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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

Subcellular distribution of phosphorylated AQP2 in MDCK cells grown in 3D culture and in Brattleboro rat kidney.Antibodies recognizing AQP2 phosphorylated at S256, S261, S264 or S269 highlighted the subcellular localization of phosphorylated AQP2 in MDCK-AQP2 cysts (A-H). The staining pattern of differentially phosphorylated AQP2 in the cyst is reminiscent of that observed in Brattleboro rat kidneys (I-P). Prior to stimulation, cysts were pre-incubated in serum free medium for 120 min. Comparing the distribution of phosphorylated AQP2 in cyst versus kidney tissue, pS256 is found in the cytosol and apical membrane in both the cyst and kidney tissue without VP treatment (A, I) Total AQP2 staining of the tissue in panel I can be seen in Fig 3B. After stimulation with VP (E,M), the redistribution of pS256 AQP2 to the apical membrane is pronounced in the kidney tissue, but is not significantly different in the cyst tissue (quantification in panel R). VP stimulation resulted in a reduction of both apical and cytosolic pS261 fluorescence intensity in cyst and kidney tissue (B vs. F, J vs. N). Under stimulated conditions, some non-specific labeling of the nuclei by the p261 antibody can be observed (F). pS264 staining signal translocated from the cytosolic compartment (C,K) to the apical membrane (G, O) after VP stimulation in both cyst and kidney tissue (H, P). In both the cyst, and the rat tissue pS269 staining was only observed following stimulation, and solely located on the apical membrane (H,P). Bar = 10 μm. (Q) Differential phosphorylation of AQP2 at various serine residues after FK stimulation was detected by western blot in AQP2 expressing MDCK cells grown as a monolayer culture. The bar graph represents quantification of western blots (means ± SE, N = 3 experiments): total AQP2 and phosphorylation antibody results are presented as relative to loading control. No significant difference was observed between non-stimulated and VP/FK simulated conditions for AQP2 pS256 or pS264. On the other hand, stimulation resulted in a significant (asterisk) decrease in pS261 (p = .002) and a significant increase in pS269 (p = .04). (R) Quantitative assessment of cyst immunofluorescence images reveal significant (P < = 0.05) increases in apical, relative to intracellular, pS264 (N = 4 cysts) and pS269 (N = 8 cysts) AQP2 staining following AVP stimulation. No significant difference in apical, relative to intracellular pS261 (N = 5 cysts) fluorescence is observed due to concomitant decreases (relative to treatment) in both apical and intracellular pS261 staining.
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pone.0131719.g005: Subcellular distribution of phosphorylated AQP2 in MDCK cells grown in 3D culture and in Brattleboro rat kidney.Antibodies recognizing AQP2 phosphorylated at S256, S261, S264 or S269 highlighted the subcellular localization of phosphorylated AQP2 in MDCK-AQP2 cysts (A-H). The staining pattern of differentially phosphorylated AQP2 in the cyst is reminiscent of that observed in Brattleboro rat kidneys (I-P). Prior to stimulation, cysts were pre-incubated in serum free medium for 120 min. Comparing the distribution of phosphorylated AQP2 in cyst versus kidney tissue, pS256 is found in the cytosol and apical membrane in both the cyst and kidney tissue without VP treatment (A, I) Total AQP2 staining of the tissue in panel I can be seen in Fig 3B. After stimulation with VP (E,M), the redistribution of pS256 AQP2 to the apical membrane is pronounced in the kidney tissue, but is not significantly different in the cyst tissue (quantification in panel R). VP stimulation resulted in a reduction of both apical and cytosolic pS261 fluorescence intensity in cyst and kidney tissue (B vs. F, J vs. N). Under stimulated conditions, some non-specific labeling of the nuclei by the p261 antibody can be observed (F). pS264 staining signal translocated from the cytosolic compartment (C,K) to the apical membrane (G, O) after VP stimulation in both cyst and kidney tissue (H, P). In both the cyst, and the rat tissue pS269 staining was only observed following stimulation, and solely located on the apical membrane (H,P). Bar = 10 μm. (Q) Differential phosphorylation of AQP2 at various serine residues after FK stimulation was detected by western blot in AQP2 expressing MDCK cells grown as a monolayer culture. The bar graph represents quantification of western blots (means ± SE, N = 3 experiments): total AQP2 and phosphorylation antibody results are presented as relative to loading control. No significant difference was observed between non-stimulated and VP/FK simulated conditions for AQP2 pS256 or pS264. On the other hand, stimulation resulted in a significant (asterisk) decrease in pS261 (p = .002) and a significant increase in pS269 (p = .04). (R) Quantitative assessment of cyst immunofluorescence images reveal significant (P < = 0.05) increases in apical, relative to intracellular, pS264 (N = 4 cysts) and pS269 (N = 8 cysts) AQP2 staining following AVP stimulation. No significant difference in apical, relative to intracellular pS261 (N = 5 cysts) fluorescence is observed due to concomitant decreases (relative to treatment) in both apical and intracellular pS261 staining.

Mentions: We next examined the phosphorylation of AQP2 and its trafficking in the polarized epithelial cyst model. Immunostaining using antibodies specific for AQP2 phosphorylation at S256, S261, S264, or S269 in the absence (Fig 5A–5D) or presence (Fig 5E–5H) of VP treatment reveals that all four phosphorylation specific antibodies are able to recognize AQP2 in the 3D epithelial culture and that the staining patterns of individually phosphorylated AQP2 are reminiscent of what was observed in the dDAVP treated Brattleboro rat kidneys (Fig 5I–5L no treatment, and Fig 5M–5P dDAVP treated).


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)

Subcellular distribution of phosphorylated AQP2 in MDCK cells grown in 3D culture and in Brattleboro rat kidney.Antibodies recognizing AQP2 phosphorylated at S256, S261, S264 or S269 highlighted the subcellular localization of phosphorylated AQP2 in MDCK-AQP2 cysts (A-H). The staining pattern of differentially phosphorylated AQP2 in the cyst is reminiscent of that observed in Brattleboro rat kidneys (I-P). Prior to stimulation, cysts were pre-incubated in serum free medium for 120 min. Comparing the distribution of phosphorylated AQP2 in cyst versus kidney tissue, pS256 is found in the cytosol and apical membrane in both the cyst and kidney tissue without VP treatment (A, I) Total AQP2 staining of the tissue in panel I can be seen in Fig 3B. After stimulation with VP (E,M), the redistribution of pS256 AQP2 to the apical membrane is pronounced in the kidney tissue, but is not significantly different in the cyst tissue (quantification in panel R). VP stimulation resulted in a reduction of both apical and cytosolic pS261 fluorescence intensity in cyst and kidney tissue (B vs. F, J vs. N). Under stimulated conditions, some non-specific labeling of the nuclei by the p261 antibody can be observed (F). pS264 staining signal translocated from the cytosolic compartment (C,K) to the apical membrane (G, O) after VP stimulation in both cyst and kidney tissue (H, P). In both the cyst, and the rat tissue pS269 staining was only observed following stimulation, and solely located on the apical membrane (H,P). Bar = 10 μm. (Q) Differential phosphorylation of AQP2 at various serine residues after FK stimulation was detected by western blot in AQP2 expressing MDCK cells grown as a monolayer culture. The bar graph represents quantification of western blots (means ± SE, N = 3 experiments): total AQP2 and phosphorylation antibody results are presented as relative to loading control. No significant difference was observed between non-stimulated and VP/FK simulated conditions for AQP2 pS256 or pS264. On the other hand, stimulation resulted in a significant (asterisk) decrease in pS261 (p = .002) and a significant increase in pS269 (p = .04). (R) Quantitative assessment of cyst immunofluorescence images reveal significant (P < = 0.05) increases in apical, relative to intracellular, pS264 (N = 4 cysts) and pS269 (N = 8 cysts) AQP2 staining following AVP stimulation. No significant difference in apical, relative to intracellular pS261 (N = 5 cysts) fluorescence is observed due to concomitant decreases (relative to treatment) in both apical and intracellular pS261 staining.
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Related In: Results  -  Collection

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pone.0131719.g005: Subcellular distribution of phosphorylated AQP2 in MDCK cells grown in 3D culture and in Brattleboro rat kidney.Antibodies recognizing AQP2 phosphorylated at S256, S261, S264 or S269 highlighted the subcellular localization of phosphorylated AQP2 in MDCK-AQP2 cysts (A-H). The staining pattern of differentially phosphorylated AQP2 in the cyst is reminiscent of that observed in Brattleboro rat kidneys (I-P). Prior to stimulation, cysts were pre-incubated in serum free medium for 120 min. Comparing the distribution of phosphorylated AQP2 in cyst versus kidney tissue, pS256 is found in the cytosol and apical membrane in both the cyst and kidney tissue without VP treatment (A, I) Total AQP2 staining of the tissue in panel I can be seen in Fig 3B. After stimulation with VP (E,M), the redistribution of pS256 AQP2 to the apical membrane is pronounced in the kidney tissue, but is not significantly different in the cyst tissue (quantification in panel R). VP stimulation resulted in a reduction of both apical and cytosolic pS261 fluorescence intensity in cyst and kidney tissue (B vs. F, J vs. N). Under stimulated conditions, some non-specific labeling of the nuclei by the p261 antibody can be observed (F). pS264 staining signal translocated from the cytosolic compartment (C,K) to the apical membrane (G, O) after VP stimulation in both cyst and kidney tissue (H, P). In both the cyst, and the rat tissue pS269 staining was only observed following stimulation, and solely located on the apical membrane (H,P). Bar = 10 μm. (Q) Differential phosphorylation of AQP2 at various serine residues after FK stimulation was detected by western blot in AQP2 expressing MDCK cells grown as a monolayer culture. The bar graph represents quantification of western blots (means ± SE, N = 3 experiments): total AQP2 and phosphorylation antibody results are presented as relative to loading control. No significant difference was observed between non-stimulated and VP/FK simulated conditions for AQP2 pS256 or pS264. On the other hand, stimulation resulted in a significant (asterisk) decrease in pS261 (p = .002) and a significant increase in pS269 (p = .04). (R) Quantitative assessment of cyst immunofluorescence images reveal significant (P < = 0.05) increases in apical, relative to intracellular, pS264 (N = 4 cysts) and pS269 (N = 8 cysts) AQP2 staining following AVP stimulation. No significant difference in apical, relative to intracellular pS261 (N = 5 cysts) fluorescence is observed due to concomitant decreases (relative to treatment) in both apical and intracellular pS261 staining.
Mentions: We next examined the phosphorylation of AQP2 and its trafficking in the polarized epithelial cyst model. Immunostaining using antibodies specific for AQP2 phosphorylation at S256, S261, S264, or S269 in the absence (Fig 5A–5D) or presence (Fig 5E–5H) of VP treatment reveals that all four phosphorylation specific antibodies are able to recognize AQP2 in the 3D epithelial culture and that the staining patterns of individually phosphorylated AQP2 are reminiscent of what was observed in the dDAVP treated Brattleboro rat kidneys (Fig 5I–5L no treatment, and Fig 5M–5P dDAVP treated).

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