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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

Regulated trafficking of AQP2 is intact in MDCK cysts.AQP2 trafficking in MDCK-AQP2 cysts is intact and staining patterns for total AQP2 are comparable to those observed in Brattleboro, and normal rat kidney. (A) MDCK-AQP2 cysts were incubated in serum free medium for 120 minutes. Addition of AVP, FK, or CPT-cAMP to the medium for 40 minutes resulting in apical membrane accumulation of AQP2 (arrows). Bar = 10 μm. (D) Asterisk denotes that significantly (P < = 0.05) more apical, but not basolateral staining of AQP2, relative to intracellular AQP2, was observed following stimulation with AVP, FK or CPT-cAMP. N = 5 cysts (NT, non-treated), N = 12 cysts (AVP/FK/CPT-cAMP stimulated). The data for AVP, FK and cAMP-treated cysts were pooled together because we saw no statistically significant difference in Apical/Internal total AQP2 or Basolateral/Internal total AQP2 between the treatment modalities. (B) In the Brattleboro rat kidney AQP2 was located mainly in the subapical region while apical membrane accumulation of AQP2 was seen after treatment with dDAVP for 3 days. Bar = 10 μm. (C) Similarly, in a tissue slice culture from normal rat kidney, incubation in medium without VP resulted in AQP2 in the cytosol and subapical region. dDAVP treatment for 20 minutes resulted in AQP2 translocation to the apical membrane, with AQP2 still detectable in the cytosol. (E) In transmission electron micrographs, AQP2 in the MDCK-AQP2 cyst is labeled with 15nm gold particles. AQP2 gold particles distributed diffusely throughout the cytosol under baseline, non-stimulated conditions (left panel) while AQP2 accumulated on the apical membrane after VP stimulation (right panel), but not on the basolateral membrane (S2 Fig). Bars = 500 nm
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pone.0131719.g003: Regulated trafficking of AQP2 is intact in MDCK cysts.AQP2 trafficking in MDCK-AQP2 cysts is intact and staining patterns for total AQP2 are comparable to those observed in Brattleboro, and normal rat kidney. (A) MDCK-AQP2 cysts were incubated in serum free medium for 120 minutes. Addition of AVP, FK, or CPT-cAMP to the medium for 40 minutes resulting in apical membrane accumulation of AQP2 (arrows). Bar = 10 μm. (D) Asterisk denotes that significantly (P < = 0.05) more apical, but not basolateral staining of AQP2, relative to intracellular AQP2, was observed following stimulation with AVP, FK or CPT-cAMP. N = 5 cysts (NT, non-treated), N = 12 cysts (AVP/FK/CPT-cAMP stimulated). The data for AVP, FK and cAMP-treated cysts were pooled together because we saw no statistically significant difference in Apical/Internal total AQP2 or Basolateral/Internal total AQP2 between the treatment modalities. (B) In the Brattleboro rat kidney AQP2 was located mainly in the subapical region while apical membrane accumulation of AQP2 was seen after treatment with dDAVP for 3 days. Bar = 10 μm. (C) Similarly, in a tissue slice culture from normal rat kidney, incubation in medium without VP resulted in AQP2 in the cytosol and subapical region. dDAVP treatment for 20 minutes resulted in AQP2 translocation to the apical membrane, with AQP2 still detectable in the cytosol. (E) In transmission electron micrographs, AQP2 in the MDCK-AQP2 cyst is labeled with 15nm gold particles. AQP2 gold particles distributed diffusely throughout the cytosol under baseline, non-stimulated conditions (left panel) while AQP2 accumulated on the apical membrane after VP stimulation (right panel), but not on the basolateral membrane (S2 Fig). Bars = 500 nm

Mentions: To demonstrate the utility of this model to study polarized trafficking of AQP2, the epithelial “cysts” were treated with arginine vasopressin (AVP), forskolin (FK) CPT-cAMP, or m0βCD. Application of AVP, FK or CPT-cAMP, leads to apical accumulation of AQP2 (Fig 3A) suggesting an intact, regulated trafficking pathway of AQP2 at the apical and subapical domain in cells that form the cyst. While clearly weaker than the apical accumulation of total AQP2 seen in vivo, this data is consistent with our observations in the Brattleboro (Fig 3B) [40] and normal (Fig 3C) rat kidney, in which a similar apical redistribution of AQP2 is detected after 1-desamino-8-D-arginine vasopressin (dDAVP) treatment. The apical trafficking of AQP2 in the cysts was further confirmed by immunogold electron microscopy (Fig 3E). Under non-stimulated conditions, AQP2 is found in the cytosol and subapical domain while a clear apical accumulation of AQP2 occurs after VP treatment. Despite the clear apical membrane redistribution of total AQP2 in response to AVP, FK or CPT-cAMP, there was no significant enrichment of AQP2 signal on the basolateral membrane in response to these treatments (Fig 3D, S2 Fig), suggesting that AQP2 located near/at the basolateral region may not be readily subject to VP/cAMP regulation as is the apically located protein.


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)

Regulated trafficking of AQP2 is intact in MDCK cysts.AQP2 trafficking in MDCK-AQP2 cysts is intact and staining patterns for total AQP2 are comparable to those observed in Brattleboro, and normal rat kidney. (A) MDCK-AQP2 cysts were incubated in serum free medium for 120 minutes. Addition of AVP, FK, or CPT-cAMP to the medium for 40 minutes resulting in apical membrane accumulation of AQP2 (arrows). Bar = 10 μm. (D) Asterisk denotes that significantly (P < = 0.05) more apical, but not basolateral staining of AQP2, relative to intracellular AQP2, was observed following stimulation with AVP, FK or CPT-cAMP. N = 5 cysts (NT, non-treated), N = 12 cysts (AVP/FK/CPT-cAMP stimulated). The data for AVP, FK and cAMP-treated cysts were pooled together because we saw no statistically significant difference in Apical/Internal total AQP2 or Basolateral/Internal total AQP2 between the treatment modalities. (B) In the Brattleboro rat kidney AQP2 was located mainly in the subapical region while apical membrane accumulation of AQP2 was seen after treatment with dDAVP for 3 days. Bar = 10 μm. (C) Similarly, in a tissue slice culture from normal rat kidney, incubation in medium without VP resulted in AQP2 in the cytosol and subapical region. dDAVP treatment for 20 minutes resulted in AQP2 translocation to the apical membrane, with AQP2 still detectable in the cytosol. (E) In transmission electron micrographs, AQP2 in the MDCK-AQP2 cyst is labeled with 15nm gold particles. AQP2 gold particles distributed diffusely throughout the cytosol under baseline, non-stimulated conditions (left panel) while AQP2 accumulated on the apical membrane after VP stimulation (right panel), but not on the basolateral membrane (S2 Fig). Bars = 500 nm
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493001&req=5

pone.0131719.g003: Regulated trafficking of AQP2 is intact in MDCK cysts.AQP2 trafficking in MDCK-AQP2 cysts is intact and staining patterns for total AQP2 are comparable to those observed in Brattleboro, and normal rat kidney. (A) MDCK-AQP2 cysts were incubated in serum free medium for 120 minutes. Addition of AVP, FK, or CPT-cAMP to the medium for 40 minutes resulting in apical membrane accumulation of AQP2 (arrows). Bar = 10 μm. (D) Asterisk denotes that significantly (P < = 0.05) more apical, but not basolateral staining of AQP2, relative to intracellular AQP2, was observed following stimulation with AVP, FK or CPT-cAMP. N = 5 cysts (NT, non-treated), N = 12 cysts (AVP/FK/CPT-cAMP stimulated). The data for AVP, FK and cAMP-treated cysts were pooled together because we saw no statistically significant difference in Apical/Internal total AQP2 or Basolateral/Internal total AQP2 between the treatment modalities. (B) In the Brattleboro rat kidney AQP2 was located mainly in the subapical region while apical membrane accumulation of AQP2 was seen after treatment with dDAVP for 3 days. Bar = 10 μm. (C) Similarly, in a tissue slice culture from normal rat kidney, incubation in medium without VP resulted in AQP2 in the cytosol and subapical region. dDAVP treatment for 20 minutes resulted in AQP2 translocation to the apical membrane, with AQP2 still detectable in the cytosol. (E) In transmission electron micrographs, AQP2 in the MDCK-AQP2 cyst is labeled with 15nm gold particles. AQP2 gold particles distributed diffusely throughout the cytosol under baseline, non-stimulated conditions (left panel) while AQP2 accumulated on the apical membrane after VP stimulation (right panel), but not on the basolateral membrane (S2 Fig). Bars = 500 nm
Mentions: To demonstrate the utility of this model to study polarized trafficking of AQP2, the epithelial “cysts” were treated with arginine vasopressin (AVP), forskolin (FK) CPT-cAMP, or m0βCD. Application of AVP, FK or CPT-cAMP, leads to apical accumulation of AQP2 (Fig 3A) suggesting an intact, regulated trafficking pathway of AQP2 at the apical and subapical domain in cells that form the cyst. While clearly weaker than the apical accumulation of total AQP2 seen in vivo, this data is consistent with our observations in the Brattleboro (Fig 3B) [40] and normal (Fig 3C) rat kidney, in which a similar apical redistribution of AQP2 is detected after 1-desamino-8-D-arginine vasopressin (dDAVP) treatment. The apical trafficking of AQP2 in the cysts was further confirmed by immunogold electron microscopy (Fig 3E). Under non-stimulated conditions, AQP2 is found in the cytosol and subapical domain while a clear apical accumulation of AQP2 occurs after VP treatment. Despite the clear apical membrane redistribution of total AQP2 in response to AVP, FK or CPT-cAMP, there was no significant enrichment of AQP2 signal on the basolateral membrane in response to these treatments (Fig 3D, S2 Fig), suggesting that AQP2 located near/at the basolateral region may not be readily subject to VP/cAMP regulation as is the apically located protein.

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