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BACKGROUND: The NaCl cotransporter NCC in the kidney distal convoluted tubule (DCT) regulates urinary NaCl excretion and BP. Aldosterone increases NaCl reabsorption via NCC over the long-term by altering gene expression. But the acute effects of aldosterone in the DCT are less well understood. METHODS: Proteomics, bioinformatics, and cell biology approaches were combined with animal models and gene-targeted mice. RESULTS: Aldosterone significantly increases NCC activity within minutes in vivo or ex vivo. These effects were independent of transcription and translation, but were absent in the presence of high potassium. In vitro, aldosterone rapidly increased intracellular cAMP and inositol phosphate accumulation, and altered phosphorylation of various kinases/kinase substrates within the MAPK/ERK, PI3K/AKT, and cAMP/PKA pathways. Inhibiting GPR30, a membrane-associated receptor, limited aldosterone's effects on NCC activity ex vivo, and NCC phosphorylation was reduced in GPR30 knockout mice. Phosphoproteomics, network analysis, and in vitro studies determined that aldosterone activates EGFR-dependent signaling. The EGFR immunolocalized to the DCT and EGFR tyrosine kinase inhibition decreased NCC activity ex vivo and in vivo. CONCLUSIONS: Aldosterone acutely activates NCC to modulate renal NaCl excretion.
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Aldosterona/farmacología , Túbulos Renales Distales/metabolismo , Transducción de Señal , Tiazidas/farmacología , Aldosterona/metabolismo , Animales , Presión Sanguínea , Calcio/metabolismo , Línea Celular , Membrana Celular/metabolismo , Biología Computacional , AMP Cíclico/metabolismo , Receptores ErbB/metabolismo , Síndrome de Gitelman/metabolismo , Riñón/metabolismo , Masculino , Ratones , Mineralocorticoides/metabolismo , Fosforilación , Proteómica , Receptores de Estrógenos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Cloruro de Sodio/metabolismo , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismoRESUMEN
The targeting of the water channel aquaporin-2 (AQP2) to the apical plasma membrane of kidney collecting duct principal cells is regulated mainly by the antidiuretic peptide hormone arginine vasopressin (AVP). This process is of crucial importance for the maintenance of body water homeostasis. In this brief review we assess the role of cyclic adenosine monophosphate (cAMP) and discuss the emerging concept that type 2 AVP receptor (V2R)-mediated AQP2 trafficking is cAMP-independent.
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Acuaporina 2/metabolismo , Agua Corporal/metabolismo , Membrana Celular/metabolismo , Células Epiteliales/metabolismo , Túbulos Renales Colectores/metabolismo , Equilibrio Hidroelectrolítico , Animales , Arginina Vasopresina/metabolismo , AMP Cíclico/metabolismo , Humanos , Transporte de Proteínas , Receptores de Vasopresinas/metabolismo , Sistemas de Mensajero SecundarioRESUMEN
Apical membrane targeting of the collecting duct water channel aquaporin-2 (AQP2) is essential for body water balance. As this event is regulated by Gs coupled 7-transmembrane receptors such as the vasopressin type 2 receptor (V2R) and the prostanoid receptors EP2 and EP4, it is believed to be cAMP dependent. However, on the basis of recent reports, it was hypothesized in the current study that increased cAMP levels are not necessary for AQP2 membrane targeting. The role and dynamics of cAMP signaling in AQP2 membrane targeting in Madin-Darby canine kidney and mouse cortical collecting duct (mpkCCD14) cells was examined using selective agonists against the V2R (dDAVP), EP2 (butaprost), and EP4 (CAY10580). During EP2 stimulation, AQP2 membrane targeting continually increased during 80 min of stimulation; whereas cAMP levels reached a plateau after 10 min. EP4 stimulation caused a rapid and transient increase in AQP2 membrane targeting, but did not significantly increase cAMP levels. After washout of the EP2 agonist or dDAVP, AQP2 membrane abundance remained elevated for at least 80 min, whereas cAMP levels rapidly decreased. Similar effects of the EP2 agonist were also observed for AQP2 constitutively nonphosphorylated at ser-269. The adenylyl cyclase inhibitor SQ22536 did not prevent AQP2 targeting during stimulation of each receptor, nor after dDAVP washout. In conclusion, this study demonstrates that although direct stimulation with cAMP causes AQP2 membrane targeting, cAMP is not necessary for receptor-mediated AQP2 membrane targeting and Gs-coupled receptors can also signal through an alternative pathway that increases AQP2 membrane targeting.
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Acuaporina 2/metabolismo , Membrana Celular/metabolismo , Túbulos Renales Colectores/metabolismo , Subtipo EP2 de Receptores de Prostaglandina E/metabolismo , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Receptores de Vasopresinas/metabolismo , Transducción de Señal/fisiología , Alprostadil/análogos & derivados , Alprostadil/farmacología , Animales , Línea Celular , Membrana Celular/efectos de los fármacos , AMP Cíclico/metabolismo , Desamino Arginina Vasopresina/farmacología , Dinoprostona/análogos & derivados , Dinoprostona/farmacología , Perros , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/efectos de los fármacos , Ratones , Pirrolidinonas/farmacología , Subtipo EP2 de Receptores de Prostaglandina E/agonistas , Subtipo EP4 de Receptores de Prostaglandina E/agonistas , Receptores de Vasopresinas/agonistas , Transducción de Señal/efectos de los fármacosRESUMEN
Aquaporin 4 (AQP4) is the predominant water channel in the mammalian brain and is mainly expressed in the perivascular glial endfeet at the brain-blood interface. AQP4 serves as a water entry site during brain edema formation, and regulation of AQP4 may therefore be of therapeutic interest. Phosphorylation of aquaporins can regulate plasma membrane localization and, possibly, the unit water permeability via gating of the AQP channel itself. In vivo phosphorylation of six serine residues in the COOH terminus of AQP4 has been detected by mass spectrometry: Ser(276), Ser(285), Ser(315), Ser(316), Ser(321), and Ser(322). To address the role of these phosphorylation sites for AQP4 function, serine-to-alanine mutants were created to abolish the phosphorylation sites. All mutants were detected at the plasma membrane of transfected C6 cells, with the fraction of the total cellular AQP4 expressed at the plasma membrane of transfected C6 cells being similar between the wild-type (WT) and mutant forms of AQP4. Activation of protein kinases A, C, and G in primary astrocytic cultures did not affect the plasma membrane abundance of AQP4. The unit water permeability was determined for the mutant AQP4s upon heterologous expression in Xenopus laevis oocytes (along with serine-to-aspartate mutants of the same residues to mimic a phosphorylation). None of the mutant AQP4 constructs displayed alterations in the unit water permeability. Thus phosphorylation of six different serine residues in the COOH terminus of AQP4 appears not to be required for proper plasma membrane localization of AQP4 or to act as a molecular switch to gate the water channel.
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Acuaporina 4/metabolismo , Membrana Celular/metabolismo , Activación del Canal Iónico/fisiología , Serina/metabolismo , Secuencia de Aminoácidos , Animales , Acuaporina 4/genética , Membrana Celular/genética , Células Cultivadas , Femenino , Datos de Secuencia Molecular , Fosforilación/fisiología , Transporte de Proteínas/fisiología , Ratas , Ratas Sprague-Dawley , Serina/genética , Xenopus laevisRESUMEN
In the kidney, the actions of vasopressin on its type-2 receptor (V2R) induce increased water reabsorption alongside polyphosphorylation and membrane targeting of the water channel aquaporin-2 (AQP2). Loss-of-function mutations in the V2R cause X-linked nephrogenic diabetes insipidus. Treatment of this condition would require bypassing the V2R to increase AQP2 membrane targeting, but currently no specific pharmacological therapy is available. The present study examined specific E-prostanoid receptors for this purpose. In vitro, prostaglandin E2 (PGE2) and selective agonists for the E-prostanoid receptors EP2 (butaprost) or EP4 (CAY10580) all increased trafficking and ser-264 phosphorylation of AQP2 in Madin-Darby canine kidney cells. Only PGE2 and butaprost increased cAMP and ser-269 phosphorylation of AQP2. Ex vivo, PGE2, butaprost, or CAY10580 increased AQP2 phosphorylation in isolated cortical tubules, whereas PGE2 and butaprost selectively increased AQP2 membrane accumulation in kidney slices. In vivo, a V2R antagonist caused a severe urinary concentrating defect in rats, which was greatly alleviated by treatment with butaprost. In conclusion, EP2 and EP4 agonists increase AQP2 phosphorylation and trafficking, likely through different signaling pathways. Furthermore, EP2 selective agonists can partially compensate for a nonfunctional V2R, providing a rationale for new treatment strategies for hereditary nephrogenic diabetes insipidus.
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Acuaporina 2/metabolismo , Diabetes Insípida Nefrogénica/metabolismo , Subtipo EP2 de Receptores de Prostaglandina E/metabolismo , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Alprostadil/análogos & derivados , Alprostadil/farmacología , Animales , Antagonistas de los Receptores de Hormonas Antidiuréticas , Acuaporina 2/genética , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Diabetes Insípida Nefrogénica/genética , Diabetes Insípida Nefrogénica/prevención & control , Dinoprostona/análogos & derivados , Dinoprostona/farmacología , Perros , Relación Dosis-Respuesta a Droga , Immunoblotting , Riñón/efectos de los fármacos , Riñón/metabolismo , Masculino , Microscopía Confocal , Fosforilación/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Pirrolidinonas/farmacología , Ratas , Ratas Wistar , Subtipo EP2 de Receptores de Prostaglandina E/agonistas , Subtipo EP2 de Receptores de Prostaglandina E/genética , Subtipo EP4 de Receptores de Prostaglandina E/agonistas , Subtipo EP4 de Receptores de Prostaglandina E/genética , Receptores de Vasopresinas/genética , Receptores de Vasopresinas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vasopresinas/metabolismo , Vasopresinas/farmacologíaRESUMEN
Prostanoids are prominent, yet complex, components in the maintenance of body water homeostasis. Recent functional and molecular studies have revealed that the local lipid mediator PGE2 is involved both in water excretion and absorption. The biologic actions of PGE2 are exerted through four different G-protein-coupled receptors; designated EP1-4, which couple to separate intracellular signaling pathways. Here, we discuss new developments in our understanding of the actions of PGE2 that have been uncovered utilizing receptor specific agonists and antagonists, EP receptor and PG synthase knockout mice, polyuric animal models, and the new understanding of the molecular regulation of collecting duct water permeability. The role of PGE2 in urinary concentration comprises a variety of mechanisms, which are not fully understood and likely depend on which receptor is activated under a particular physiologic condition. EP3 and microsomal PG synthase type 1 play a role in decreasing collecting duct water permeability and increasing water excretion, whereas EP2 and EP4 can bypass vasopressin signaling and increase water reabsorption through two different intracellular signaling pathways. PGE2 has an intricate role in urinary concentration, and we now suggest how targeting specific prostanoid receptor signaling pathways could be exploited for the treatment of disorders in water balance.
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Dinoprostona/orina , Capacidad de Concentración Renal/fisiología , Enfermedades Renales/orina , Equilibrio Hidroelectrolítico/fisiología , Desequilibrio Hidroelectrolítico/orina , Animales , Humanos , Enfermedades Renales/fisiopatología , Desequilibrio Hidroelectrolítico/fisiopatologíaRESUMEN
The cellular functions of many eukaryotic membrane proteins, including the vasopressin-regulated water channel aquaporin-2 (AQP2), are regulated by posttranslational modifications. In this article, we discuss the experimental discoveries that have advanced our understanding of how posttranslational modifications affect AQP2 function, especially as they relate to the role of AQP2 in the kidney. We review the most recent data demonstrating that glycosylation and, in particular, phosphorylation and ubiquitination are mechanisms that regulate AQP2 activity, subcellular sorting and distribution, degradation, and protein interactions. From a clinical perspective, posttranslational modification resulting in protein misrouting or degradation may explain certain forms of nephrogenic diabetes insipidus. In addition to providing major insight into the function and dynamics of renal AQP2 regulation, the analysis of AQP2 posttranslational modification may provide general clues as to the role of posttranslational modification for regulation of other membrane proteins.
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Acuaporina 2/metabolismo , Activación del Canal Iónico , Riñón/metabolismo , Procesamiento Proteico-Postraduccional , Agua/metabolismo , Secuencia de Aminoácidos , Animales , Endocitosis , Exocitosis , Glicosilación , Humanos , Datos de Secuencia Molecular , Fosforilación , Proteínas Quinasas/metabolismo , Transporte de Proteínas , UbiquitinaciónRESUMEN
Targeting the collecting duct water channel aquaporin 2 (AQP2) to the plasma membrane is essential for the maintenance of mammalian water homeostasis. The vasopressin V2 receptor (V2R), which is a GS protein-coupled receptor that increases intracellular cAMP levels, has a major role in this targeting process. Although a rise in cAMP levels and activation of protein kinase A are involved in facilitating the actions of V2R, studies in knockout mice and cell models have suggested that cAMP signalling pathways are not an absolute requirement for V2R-mediated AQP2 trafficking to the plasma membrane. In addition, although AQP2 phosphorylation is a known prerequisite for V2R-mediated plasma membrane targeting, none of the known AQP2 phosphorylation events appears to be rate-limiting in this process, which suggests the involvement of other factors; cytoskeletal remodelling has also been implicated. Notably, several regulatory processes and signalling pathways involved in AQP2 trafficking also have a role in the pathophysiology of autosomal dominant polycystic kidney disease, although the role of AQP2 in cyst progression is unknown. Here, we highlight advances in the field of AQP2 regulation that might be exploited for the treatment of water balance disorders and provide a rationale for targeting these pathways in autosomal dominant polycystic kidney disease.
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Acuaporina 2/metabolismo , Túbulos Renales Colectores/metabolismo , Riñón Poliquístico Autosómico Dominante/metabolismo , Equilibrio Hidroelectrolítico/fisiología , Agua/metabolismo , Proteínas de Anclaje a la Quinasa A/metabolismo , Antagonistas de los Receptores de Hormonas Antidiuréticas/uso terapéutico , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Fluconazol , Humanos , Riñón Poliquístico Autosómico Dominante/tratamiento farmacológico , Transporte de Proteínas , Proteínas Proto-Oncogénicas pp60(c-src)/antagonistas & inhibidores , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Receptores de Vasopresinas/metabolismo , Transducción de Señal , Tolvaptán/uso terapéutico , Proteína Wnt-5a/metabolismoRESUMEN
Arginine vasopressin (AVP) stimulates the concentration of renal urine by increasing the principal cell expression of aquaporin-2 (AQP2) water channels. Prostaglandin E2 (PGE2) and prostaglandin2α (PGF2α) increase the water absorption of the principal cell without AVP, but PGE2 decreases it in the presence of AVP. The underlying mechanism of this paradoxical response was investigated here. Mouse cortical collecting duct (mkpCCDc14) cells mimic principal cells as they endogenously express AQP2 in response to AVP. PGE2 increased AQP2 abundance without desmopressin (dDAVP), while in the presence of dDAVP, PGE2, and PGF2α reduced AQP2 abundance. dDAVP increased the cellular PGD2 and PGE2 release and decreased the PGF2α release. MpkCCD cells expressed mRNAs for the receptors of PGE2 (EP1/EP4), PGF2 (FP), and TxB2 (TP). Incubation with dDAVP increased the expression of EP1 and FP but decreased the expression of EP4. In the absence of dDAVP, incubation of mpkCCD cells with an EP4, but not EP1/3, agonist increased AQP2 abundance, and the PGE2-induced increase in AQP2 was blocked with an EP4 antagonist. Moreover, in the presence of dDAVP, an EP1/3, but not EP4, agonist decreased the AQP2 abundance, and the addition of EP1 antagonists prevented the PGE2-mediated downregulation of AQP2. Our study shows that in mpkCCDc14 cells, reduced EP4 receptor and increased EP1/FP receptor expression by dDAVP explains the differential effects of PGE2 and PGF2α on AQP2 abundance with or without dDAVP. As the V2R and EP4 receptor, but not the EP1 and FP receptor, can couple to Gs and stimulate the cyclic adenosine monophosphate (cAMP) pathway, our data support a view that cells can desensitize themselves for receptors activating the same pathway and sensitize themselves for receptors of alternative pathways.
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BACKGROUND: Acute renal failure (ARF) is a frequent complication of sepsis. Characteristics of ARF in sepsis are impaired urinary concentration, increased natriuresis and decreased glomerular filtration rate (GFR), in which inducible nitric oxide synthase (iNOS) has been revealed to play a role. Aims. We aimed to investigate renal water and sodium excretion and in parallel the segmental regulation of renal AQP2 and major sodium transporters in rats with acute LPS-induced endotoxaemia. Next, we aimed to examine the changes of iNOS expression and activated macrophage infiltration in the kidney and the effects of iNOS inhibition on AQP2 and NKCC2 expression in LPS rats. METHODS: Rats were treated with LPS (i.p.) or with LPS + iNOS inhibitor L-NIL, and 6 h later kidneys were subjected to semiquantitative immunoblotting and immunohistochemistry. RESULTS: Polyuria and increased natriuresis were seen 6 h after LPS injection alongside downregulation of both AQP2 and S256-phosphorylated AQP2 in CTX/OSOM and ISOM but not in inner medulla (IM). Thick ascending limb sodium transporters NHE3 and NKCC2 were downregulated in ISOM and NaPi2 was decreased in CTX/OSOM, whereas NCC and ENaC were not consistently downregulated. Immunolabelling intensity of iNOS was increased in vascular structures and transitional epithelium, and an infiltration of activated macrophages was seen in CTX and ISOM. L-NIL co-treatment prevented the downregulation of NKCC2 but not AQP2 in LPS rats. CONCLUSIONS: Early downregulation of AQP2 and sodium transporters takes place segmentally in the kidney after LPS administration. In addition, an infiltration of activated macrophages and increased iNOS expression may play a role in the urinary concentrating defect in acute LPS-induced entotoxaemia.
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Acuaporina 2/metabolismo , Endotoxemia/inducido químicamente , Endotoxemia/metabolismo , Riñón/metabolismo , Lipopolisacáridos/farmacología , Anestesia , Animales , Western Blotting , Regulación hacia Abajo , Endotoxemia/patología , Inhibidores Enzimáticos/farmacología , Immunoblotting , Técnicas para Inmunoenzimas , Riñón/citología , Riñón/efectos de los fármacos , Lisina/análogos & derivados , Lisina/farmacología , Macrófagos/metabolismo , Masculino , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II/metabolismo , Fosforilación/efectos de los fármacos , Ratas , Ratas Wistar , Sodio/orina , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Miembro 1 de la Familia de Transportadores de Soluto 12RESUMEN
The Epstein-Barr virus (EBV) BILF1 gene encodes a constitutively active G protein-coupled receptor (GPCR) that downregulates major histocompatibility complex (MHC) class I and induces signaling-dependent tumorigenesis. Different BILF1 homologs display highly conserved extracellular loops (ECLs) including the conserved cysteine residues involved in disulfide bridges present in class A GPCRs (GPCR bridge between transmembrane helix 3 [TM-3] and ECL-2) and in chemokine receptors (CKR bridge between the N terminus and ECL-3). In order to investigate the roles of the conserved residues in the receptor functions, 25 mutations were created in the extracellular domains. Luciferase reporter assays and flow cytometry were used to investigate the G protein signaling and MHC class I downregulation in HEK293 cells. We find that the cysteine residues involved in the GPCR bridge are important for both signaling and MHC class I downregulation, whereas the cysteine residues in the N terminus and ECL-3 are dispensable for signaling but important for MHC class I downregulation. Multiple conserved residues in the extracellular regions are important for the receptor-induced MHC class I downregulation, but not for signaling, indicating distinct structural requirements for these two functions. In an engineered receptor containing a binding site for Zn+2 ions in a complex with an aromatic chelator (phenanthroline or bipyridine), a ligand-driven inhibition of both the receptor signaling and MHC class I downregulation was observed. Taken together, this suggests that distinct regions in EBV-BILF1 can be pharmacologically targeted to inhibit the signaling-mediated tumorigenesis and interfere with the MHC class I downregulation.IMPORTANCE G protein-coupled receptors constitute the largest family of membrane proteins. As targets of >30% of the FDA-approved drugs, they are valuable for drug discovery. The receptor is composed of seven membrane-spanning helices and intracellular and extracellular domains. BILF1 is a receptor encoded by Epstein-Barr virus (EBV), which evades the host immune system by various strategies. BILF1 facilitates the virus immune evasion by downregulating MHC class I and is capable of inducing signaling-mediated tumorigenesis. BILF1 homologs from primate viruses show highly conserved extracellular domains. Here, we show that conserved residues in the extracellular domains of EBV-BILF1 are important for downregulating MHC class I and that the receptor signaling and immune evasion can be inhibited by drug-like small molecules. This suggests that BILF1 could be a target to inhibit the signaling-mediated tumorigenesis and interfere with the MHC class I downregulation, thereby facilitating virus recognition by the immune system.
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Regulación hacia Abajo , Herpesvirus Humano 4/fisiología , Antígenos de Histocompatibilidad Clase I/biosíntesis , Interacciones Huésped-Patógeno , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Proteínas Virales/metabolismo , Análisis Mutacional de ADN , Citometría de Flujo , Genes Reporteros , Células HEK293 , Herpesvirus Humano 4/genética , Humanos , Luciferasas/análisis , Luciferasas/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Receptores Acoplados a Proteínas G/genética , Proteínas Virales/genéticaRESUMEN
The purpose of the present studies was to determine the effects of high-dose aldosterone and dDAVP treatment on renal aquaporin-2 (AQP2) regulation and urinary concentration. Rats were treated for 6 days with either vehicle (CON; n = 8), dDAVP (0.5 ng/h, dDAVP, n = 10), aldosterone (Aldo, 150 microg/day, n = 10) or combined dDAVP and aldosterone treatment (dDAVP+Aldo, n = 10) and had free access to water with a fixed food intake. Aldosterone treatment induced hypokalemia, decreased urine osmolality, and increased the urine volume and water intake in ALDO compared with CON and dDAVP+Aldo compared with dDAVP. Immunohistochemistry and semiquantitative laser confocal microscopy revealed a distinct increase in basolateral domain AQP2 labeling in cortical collecting duct (CCD) principal cells and a reduction in apical domain labeling in Aldo compared with CON rats. Given the presence of hypokalemia in aldosterone-treated rats, we studied dietary-induced hypokalemia in rats, which also reduced apical AQP2 expression in the CCD but did not induce any increase in basolateral AQP2 expression in the CCD as observed with aldosterone treatment. The aldosterone-induced basolateral AQP2 expression in the CCD was thus independent of hypokalemia but was dependent on the presence of sodium and aldosterone. This redistribution was clearly blocked by mineralocorticoid receptor blockade. The increased basolateral expression of AQP2 induced by aldosterone may play a significant role in water metabolism in conditions with increased sodium reabsorption in the CCD.