scholarly journals Sorting Nexin 27 Regulates the Lysosomal Degradation of Aquaporin-2 Protein in the Kidney Collecting Duct

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1208 ◽  
Author(s):  
Hyo-Jung Choi ◽  
Hyo-Ju Jang ◽  
Euijung Park ◽  
Stine Julie Tingskov ◽  
Rikke Nørregaard ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Rat Kidney ◽  
Pdz Domain ◽  
Apical Plasma Membrane ◽  
Protein Abundance ◽  
Lysosomal Degradation ◽  
Glutathione S Transferase ◽  
Sorting Nexin ◽  
Aquaporin 2 ◽  
Cell Surface Biotinylation

Sorting nexin 27 (SNX27), a PDZ (Postsynaptic density-95/Discs large/Zonula occludens 1) domain-containing protein, cooperates with a retromer complex, which regulates intracellular trafficking and the abundance of membrane proteins. Since the carboxyl terminus of aquaporin-2 (AQP2c) has a class I PDZ-interacting motif (X-T/S-X-Φ), the role of SNX27 in the regulation of AQP2 was studied. Co-immunoprecipitation assay of the rat kidney demonstrated an interaction of SNX27 with AQP2. Glutathione S-transferase (GST) pull-down assays revealed an interaction of the PDZ domain of SNX27 with AQP2c. Immunocytochemistry of HeLa cells co-transfected with FLAG-SNX27 and hemagglutinin (HA)-AQP2 also revealed co-localization throughout the cytoplasm. When the PDZ domain was deleted, punctate HA-AQP2 labeling was localized in the perinuclear region. The labeling was intensively overlaid by Lysotracker staining but not by GM130 labeling, a cis-Golgi marker. In rat kidneys and primary cultured inner medullary collecting duct cells, the subcellular redistribution of SNX27 was similar to AQP2 under 1-deamino-8-D-arginine vasopressin (dDAVP) stimulation/withdrawal. Cell surface biotinylation assay showed that dDAVP-induced AQP2 translocation to the apical plasma membrane was unaffected after SNX27 knockdown in mpkCCD cells. In contrast, the dDAVP-induced AQP2 protein abundance was significantly attenuated without changes in AQP2 mRNA expression. Moreover, the AQP2 protein abundance was markedly declined during the dDAVP withdrawal period after stimulation under SNX27 knockdown, which was inhibited by lysosome inhibitors. Autophagy was induced after SNX27 knockdown in mpkCCD cells. Lithium-induced nephrogenic diabetes insipidus in rats revealed a significant downregulation of SNX27 in the kidney inner medulla. Taken together, the PDZ domain-containing SNX27 interacts with AQP2 and depletion of SNX27 contributes to the autophagy-lysosomal degradation of AQP2.

scholarly journals Depletion of vacuolar protein sorting-associated protein 35 is associated with increased lysosomal degradation of aquaporin-2

2016 ◽  
Vol 311 (6) ◽  
pp. F1294-F1307 ◽  
Author(s):  
Mi Suk Lee ◽  
Hyo-Jung Choi ◽  
Eui-Jung Park ◽  
Hye-Jeong Park ◽  
Tae-Hwan Kwon
Keyword(s):  
Posttranslational Modifications ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Protein Sorting ◽  
Protein Abundance ◽  
Lysosomal Degradation ◽  
Vacuolar Protein Sorting ◽  
Aquaporin 2 ◽  
Cell Surface Biotinylation ◽  
Vacuolar Protein

The carboxyl terminus of aquaporin-2 (AQP2c) undergoes posttranslational modifications, including phosphorylation and ubiquitination, in the process of regulating aquaporin-2 (AQP2) translocation and protein abundance. We aimed to identify novel proteins interacting with AQP2c. Recombinant AQP2c protein was made in Escherichia coli BL21 (DE3) cells by exploiting the pET32 TrxA fusion system. Lysates of rat kidney inner medullary collecting duct (IMCD) tubule suspensions interacted with rat AQP2c bound to Ni2+-resin were subjected to LC-MS/MS proteomic analysis. Potential interacting proteins were identified, including vacuolar protein sorting-associated protein 35 (Vps35). Coimmunoprecipitation assay demonstrated that Vps35 interacted with AQP2c. Immunohistochemistry of rat kidney revealed that AQP2 and Vps35 were partly colocalized at the intracellular vesicles in collecting duct cells. The role of Vps35 in AQP2 regulation induced by 1-deamino-8D-arginine vasopressin (dDAVP) was examined in mpkCCDc14 cells. Cell surface biotinylation assay demonstrated that dDAVP-induced apical translocation of AQP2 was significantly decreased under siRNA-mediated Vps35 knockdown. dDAVP-induced AQP2 upregulation was less prominent in the cells with Vps35 knockdown. Moreover, AQP2 protein abundance was decreased to a greater extent during the withdrawal period after dDAVP stimulation under Vps35 knockdown, which was significantly inhibited by chloroquine (a blocker of the lysosomal pathway) but not by MG132 (a proteasome inhibitor). Immunocytochemistry demonstrated that internalized AQP2 was more associated with lysosomal-associated membrane protein 1 (LAMP-1) in primary cultured IMCD cells under a Vps35 knockdown situation. Taken together, our results show that Vps35 interacts with AQP2c, and depletion of Vps35 is likely to be associated with decreased AQP2 trafficking and increased lysosomal degradation of AQP2 protein.

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 Decreased aquaporin-2 expression and apical plasma membrane delivery in kidney collecting ducts of polyuric hypercalcemic rats.

1998 ◽  
Vol 9 (12) ◽  
pp. 2181-2193 ◽  
Author(s):  
J H Earm ◽  
B M Christensen ◽  
J Frøkiaer ◽  
D Marples ◽  
J S Han ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Molecular Mechanisms ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Water Channel ◽  
Urine Osmolality ◽  
Apical Plasma Membrane ◽  
Similar Reduction ◽  
Aquaporin 2 ◽  
Urine Production

Hypercalcemia is frequently associated with a urinary concentrating defect and overt polyuria. The molecular mechanisms underlying this defect are poorly understood. Dysregulation of aquaporin-2 (AQP2), the predominant vasopressin-regulated water channel, is known to be associated with a range of congenital and acquired water balance disorders including nephrogenic diabetes insipidus and states of water retention. This study examines the effect of hypercalcemia on the expression of AQP2 in rat kidney. Rats were treated orally for 7 d with dihydrotachysterol, which produced significant hypercalcemia with a 15 +/- 2% increase in plasma calcium concentration. Immunoblotting and densitometry of membrane fractions revealed a significant decrease in AQP2 expression in kidney inner medulla of hypercalcemic rats to 45.7 +/- 6.8% (n = 11) of control levels (100 +/- 12%, n = 9). A similar reduction in AQP2 expression was seen in cortex (36.9 +/- 4.2% of control levels, n = 6). Urine production increased in parallel, from 11.3 +/- 1.4 to a maximum of 25.3 +/- 1.9 ml/d (P < 0.01), whereas urine osmolality decreased from 2007 +/- 186 mosmol/kg x H2O to 925 +/- 103 mosmol/kg x H2O (P < 0.01). Immunocytochemistry confirmed a decrease in total AQP2 labeling of collecting duct principal cells from kidneys of hypercalcemic rats, and reduced apical labeling. Immunoelectron microscopy demonstrated a significant reduction in AQP2 labeling of the apical plasma membrane, consistent with the development of polyuria. In summary, the results strongly suggest that AQP2 downregulation and reduced apical plasma membrane delivery of AQP2 play important roles in the development of polyuria in association with hypercalcemia.

scholarly journals α-Actinin 4 Links Vasopressin Short-Term and Long-Term Regulation of Aquaporin-2 in Kidney Collecting Duct Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Cheng-Hsuan Ho ◽  
Hsiu-Hui Yang ◽  
Shih-Han Su ◽  
Ai-Hsin Yeh ◽  
Ming-Jiun Yu
Keyword(s):  
Gene Expression ◽  
Glucocorticoid Receptor ◽  
Nuclear Translocation ◽  
Collecting Duct ◽  
Apical Plasma Membrane ◽  
Membrane Insertion ◽  
Short Term ◽  
Aquaporin 2 ◽  
Mrna And Protein Expression

Water permeability of the kidney collecting ducts is regulated by the peptide hormone vasopressin. Between minutes and hours (short-term), vasopressin induces trafficking of the water channel protein aquaporin-2 to the apical plasma membrane of the collecting duct principal cells to increase water permeability. Between hours and days (long-term), vasopressin induces aquaporin-2 gene expression. Here, we investigated the mechanisms that bridge the short-term and long-term vasopressin-mediated aquaporin-2 regulation by α-actinin 4, an F-actin crosslinking protein and a transcription co-activator of the glucocorticoid receptor. Vasopressin induced F-actin depolymerization and α-actinin 4 nuclear translocation in the mpkCCD collecting duct cell model. Co-immunoprecipitation followed by immunoblotting showed increased interaction between α-actinin 4 and glucocorticoid receptor in response to vasopressin. ChIP-PCR showed results consistent with α-actinin 4 and glucocorticoid receptor binding to the aquaporin-2 promoter. α-actinin 4 knockdown reduced vasopressin-induced increases in aquaporin-2 mRNA and protein expression. α-actinin 4 knockdown did not affect vasopressin-induced glucocorticoid receptor nuclear translocation, suggesting independent mechanisms of vasopressin-induced nuclear translocation of α-actinin 4 and glucocorticoid receptor. Glucocorticoid receptor knockdown profoundly reduced vasopressin-induced increases in aquaporin-2 mRNA and protein expression. In the absence of glucocorticoid analog dexamethasone, vasopressin-induced increases in glucocorticoid receptor nuclear translocation and aquaporin-2 mRNA were greatly reduced. α-actinin 4 knockdown further reduced vasopressin-induced increase in aquaporin-2 mRNA in the absence of dexamethasone. We conclude that glucocorticoid receptor plays a major role in vasopressin-induced aquaporin-2 gene expression that can be enhanced by α-actinin 4. In the absence of vasopressin, α-actinin 4 crosslinks F-actin underneath the apical plasma membrane, impeding aquaporin-2 membrane insertion. Vasopressin-induced F-actin depolymerization in one hand facilitates aquaporin-2 apical membrane insertion and in the other hand frees α-actinin 4 to enter the nucleus where it binds glucocorticoid receptor to enhance aquaporin-2 gene expression.

Localization and regulation of PKA-phosphorylated AQP2 in response to V2-receptor agonist/antagonist treatment

2000 ◽  
Vol 278 (1) ◽  
pp. F29-F42 ◽  
Author(s):  
Birgitte Mønster Christensen ◽  
Marina Zelenina ◽  
Anita Aperia ◽  
Søren Nielsen
Keyword(s):  
Plasma Membrane ◽  
Immunoelectron Microscopy ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Fold Increase ◽  
Apical Plasma Membrane ◽  
Complete Disappearance ◽  
Vasopressin Secretion ◽  
V2 Receptor ◽  
Intracellular Vesicles

Phosphorylation of Ser256, in a PKA consensus site, in AQP2 (p-AQP2) appears to be critically involved in the vasopressin-induced trafficking of AQP2. In the present study, affinity-purified antibodies that selectively recognize AQP2 phosphorylated at Ser256 were developed. These antibodies were used to determine 1) the subcellular localization of p-AQP2 in rat kidney and 2) changes in distribution and/or levels of p-AQP2 in response to [desamino-Cys1,d-Arg8]vasopressin (DDAVP) treatment or V2-receptor blockade. Immunoelectron microscopy revealed that p-AQP2 was localized in both the apical plasma membrane and in intracellular vesicles of collecting duct principal cells. Treatment of rats with V2-receptor antagonist for 30 min resulted in almost complete disappearance of p-AQP2 labeling of the apical plasma membrane with only marginal labeling of intracellular vesicles remaining. Immunoblotting confirmed a marked decrease in p-AQP2 levels. In control Brattleboro rats (BB), lacking vasopressin secretion, p-AQP2 labeling was almost exclusively present in intracellular vesicles. Treatment of BB rats with DDAVP for 2 h induced a 10-fold increase in p-AQP2 labeling of the apical plasma membrane. The overall abundance of p-AQP2, however, was not increased, as determined both by immunoelectron microscopy and immunoblotting. Consistent with this, 2 h of DDAVP treatment of normal rats also resulted in unchanged p-AQP2 levels. Thus the results demonstrate that AQP2 phosphorylated in Ser256 is present in the apical plasma membrane and in intracellular vesicles and that both the intracellular distribution/trafficking, as well as the abundance of p-AQP2, are regulated via V2 receptors by altering phosphorylation and/or dephosphorylation of Ser256in AQP2.

scholarly journals Chlorpromazine Induces Basolateral Aquaporin-2 Accumulation via F-Actin Depolymerization and Blockade of Endocytosis in Renal Epithelial Cells

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 1057
Author(s):  
Richard Bouley ◽  
Naofumi Yui ◽  
Abby Terlouw ◽  
Pui W. Cheung ◽  
Dennis Brown
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Mdck Cells ◽  
Rat Kidney ◽  
Apical Plasma Membrane ◽  
Rhodamine Phalloidin ◽  
Renal Epithelial Cells ◽  
Aquaporin 2 ◽  
Basolateral Plasma Membrane

We previously showed that in polarized Madin–Darby canine kidney (MDCK) cells, aquaporin-2 (AQP2) is continuously targeted to the basolateral plasma membrane from which it is rapidly retrieved by clathrin-mediated endocytosis. It then undertakes microtubule-dependent transcytosis toward the apical plasma membrane. In this study, we found that treatment with chlorpromazine (CPZ, an inhibitor of clathrin-mediated endocytosis) results in AQP2 accumulation in the basolateral, but not the apical plasma membrane of epithelial cells. In MDCK cells, both AQP2 and clathrin were concentrated in the basolateral plasma membrane after CPZ treatment (100 µM for 15 min), and endocytosis was reduced. Then, using rhodamine phalloidin staining, we found that basolateral, but not apical, F-actin was selectively reduced by CPZ treatment. After incubation of rat kidney slices in situ with CPZ (200 µM for 15 min), basolateral AQP2 and clathrin were increased in principal cells, which simultaneously showed a significant decrease of basolateral compared to apical F-actin staining. These results indicate that clathrin-dependent transcytosis of AQP2 is an essential part of its trafficking pathway in renal epithelial cells and that this process can be inhibited by selectively depolymerizing the basolateral actin pool using CPZ.

scholarly journals Aliskiren increases aquaporin-2 expression and attenuates lithium-induced nephrogenic diabetes insipidus

2017 ◽  
Vol 313 (4) ◽  
pp. F914-F925 ◽  
Author(s):  
Yu Lin ◽  
Tiezheng Zhang ◽  
Pinning Feng ◽  
Miaojuan Qiu ◽  
Qiaojuan Liu ◽  
...  
Keyword(s):  
Diabetes Insipidus ◽  
Nephrogenic Diabetes Insipidus ◽  
Collecting Duct ◽  
Renin Inhibitor ◽  
Protein Abundance ◽  
Aquaporin 2 ◽  
Direct Renin Inhibitor ◽  
Collecting Ducts ◽  
Dry Food ◽  
Concentrating Defect

The direct renin inhibitor aliskiren has been shown to be retained and persist in medullary collecting ducts even after treatment is discontinued, suggesting a new mechanism of action for this drug. The purpose of the present study was to investigate whether aliskiren regulates renal aquaporin expression in the collecting ducts and improves urinary concentrating defect induced by lithium in mice. The mice were fed with either normal chow or LiCl diet (40 mmol·kg dry food−1·day−1 for 4 days and 20 mmol·kg dry food−1·day−1 for the last 3 days) for 7 days. Some mice were intraperitoneally injected with aliskiren (50 mg·kg body wt−1·day−1 in saline). Aliskiren significantly increased protein abundance of aquaporin-2 (AQP2) in the kidney inner medulla in mice. In inner medulla collecting duct cell suspension, aliskiren markedly increased AQP2 and phosphorylated AQP2 at serine 256 (pS256-AQP2) protein abundance, which was significantly inhibited both by adenylyl cyclase inhibitor MDL-12330A and by PKA inhibitor H89, indicating an involvement of the cAMP-PKA signaling pathway in aliskiren-induced increased AQP2 expression. Aliskiren treatment improved urinary concentrating defect in lithium-treated mice and partially prevented the decrease of AQP2 and pS256-AQP2 protein abundance in the inner medulla of the kidney. In conclusion, the direct renin inhibitor aliskiren upregulates AQP2 protein expression in inner medullary collecting duct principal cells and prevents lithium-induced nephrogenic diabetes insipidus likely via cAMP-PKA pathways.

Advances in aquaporin-2 trafficking mechanisms and their implications for treatment of water balance disorders

2020 ◽  
Vol 319 (1) ◽  
pp. C1-C10 ◽  
Author(s):  
Robert A. Fenton ◽  
Sathish K. Murali ◽  
Hanne B. Moeller
Keyword(s):  
Protein Interactions ◽  
Collecting Duct ◽  
Water Channel ◽  
Plasma Osmolality ◽  
Carboxyl Terminus ◽  
Apical Plasma Membrane ◽  
Tail Domain ◽  
Water Reabsorption ◽  
Aquaporin 2

In mammals, conservation of body water is critical for survival and is dependent on the kidneys’ ability to minimize water loss in the urine during periods of water deprivation. The collecting duct water channel aquaporin-2 (AQP2) plays an essential role in this homeostatic response by facilitating water reabsorption along osmotic gradients. The ability to increase the levels of AQP2 in the apical plasma membrane following an increase in plasma osmolality is a rate-limiting step in water reabsorption, a process that is tightly regulated by the antidiuretic hormone arginine vasopressin (AVP). In this review, the focus is on the role of the carboxyl-terminus of AQP2 as a key regulatory point for AQP2 trafficking. We provide an overview of AQP2 structure, disease-causing mutations in the AQP2 carboxyl-terminus, the role of posttranslational modifications such as phosphorylation and ubiquitylation in the tail domain, and their implications for balanced trafficking of AQP2. Finally, we discuss how various modifications of the AQP2 tail facilitate selective protein-protein interactions that modulate the AQP2 trafficking mechanism.

Immunocytochemical localization of Na+ channels in rat kidney medulla

1989 ◽  
Vol 256 (2) ◽  
pp. F366-F369 ◽  
Author(s):  
D. Brown ◽  
E. J. Sorscher ◽  
D. A. Ausiello ◽  
D. J. Benos
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Apical Plasma Membrane ◽  
Na Channel ◽  
Thick Ascending Limb ◽  
Na Channels ◽  
Kidney Medulla ◽  
Principal Cells

Amiloride-sensitive Na+ channels were localized in semithin frozen sections of rat renal medullary collecting ducts, using polyclonal antibodies directed against purified bovine kidney Na+ channel protein. The apical plasma membrane of collecting duct principal cells was heavily stained by indirect immunofluorescence, whereas intercalated cells were negative. Basolateral plasma membranes of both cell types were unstained, as were subapical vesicles in the cytoplasm of these cells. In the thick ascending limb of Henle, some scattered granular fluorescence was seen in the cytoplasm and close to the apical pole of epithelial cells, suggesting the presence of antigenic sites associated with some membrane domains in these cells. No staining was detected in thin limbs of Henle, or in proximal tubules in the outer medulla. These results show that amiloride-sensitive sodium channels are located predominantly on the apical plasma membrane of medullary collecting duct principal cells, the cells that are involved in Na+ homeostasis in this region of the kidney.

scholarly journals Maintained ENaC trafficking in aldosterone-infused rats during mineralocorticoid and glucocorticoid receptor blockade

2007 ◽  
Vol 292 (1) ◽  
pp. F382-F394 ◽  
Author(s):  
Jakob Nielsen ◽  
Tae-Hwan Kwon ◽  
Jørgen Frøkiær ◽  
Mark A. Knepper ◽  
Søren Nielsen
Keyword(s):  
Plasma Membrane ◽  
Glucocorticoid Receptor ◽  
Collecting Duct ◽  
Kidney Cortex ◽  
Apical Plasma Membrane ◽  
Protein Abundance ◽  
Cortical Collecting Duct ◽  
Plasma Aldosterone Concentration ◽  
Mediated Effects ◽  
Plasma Membrane Domain

Aldosterone induces redistribution of epithelial sodium channel (ENaC) to the apical plasma membrane from intracellular vesicles in renal connecting tubule (CNT) and cortical collecting duct (CCD). The role of the classical mineralocorticoid receptor (MR) in ENaC trafficking is still debated. We examined whether the MR antagonist spironolactone affects ENaC regulation in the kidney cortex of aldosterone-infused rats. Aldosterone infusion for 7 days resulted in a plasma aldosterone concentration in the high physiological range (3 to 4 nM). Aldosterone infusion decreased plasma K+ concentration compared with untreated control rats. Cotreatment with spironolactone completely blocked the aldosterone-induced decrease in plasma K+. Immunoblotting and immunohistochemistry showed increased protein abundance of Na-K-ATPase α1-subunit and NCC in the kidney cortex, in response to aldosterone infusion that was blocked by spironolactone. In contrast, aldosterone-induced redistribution of ENaC subunits from the cytoplasm to the apical plasma membrane domain in CNT and CCD was unaffected by spironolactone. Immunoblotting of αENaC showed increased protein abundance in aldosterone-infused rats that was not blocked by spironolactone treatment. To exclude possible glucocorticoid receptor (GR)-mediated effects of aldosterone, we treated aldosterone-infused rats with both spironolactone and the GR antagonist RU486. Combined MR and GR blockade prevented neither ENaC trafficking nor the upregulation of αENaC protein abundance in aldosterone-infused rats. We provide new evidence for ENaC trafficking occurring independent of MR and GR activation in aldosterone-infused rats.

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