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Aquaporin‐2 (AQP2) is a member of the aquaporin water channel family. In the kidney, AQP2 is expressed in collecting duct principal cells where it facilitates water reabsorption in response to antidiuretic hormone (arginine vasopressin, AVP). AVP induces the redistribution of AQP2 from intracellular vesicles and its incorporation into the plasma membrane. The plasma membrane insertion of AQP2 represents the crucial step in AVP‐mediated water reabsorption. Dysregulation of the system preventing the AQP2 plasma membrane insertion causes diabetes insipidus (DI), a disease characterised by an impaired urine concentrating ability and polydipsia. There is no satisfactory treatment of DI available. This review discusses kinases that control the localisation of AQP2 and points out potential kinase‐directed targets for the treatment of DI. [ABSTRACT FROM AUTHOR]

The shuttling of renal collecting duct aquaporin-2 (AQP2) between intracellular vesicles and the apical plasma membrane is paramount for regulation of renal water reabsorption. The binding of the circulating antidiuretic hormone arginine vasopressin (AVP) to the basolateral AVP receptor increases intracellular cAMP, which ultimately leads to AQP2 plasma membrane accumulation via a dual effect on AQP2 vesicle fusion with the apical plasma membrane and reduced AQP2 endocytosis. This AQP2 plasma membrane accumulation increases water reabsorption and consequently urine concentration. Conventional fluorescent microscopy provides a lateral resolution of ~250 nm, which is insufficient to resolve the AQP2-containing endosomes/vesicles. Therefore, detailed information regarding the AQP2 vesicular population is still lacking. Newly established 4.5x Expansion Microscopy (ExM) can increase resolution to 60-70 nm. Using 4.5x ExM, we detected AQP2 vesicles/endosomes as small as 79 nm considering an average expansion factor of 4.3 for endosomes. Using different markers of the endosomal system provided detailed information of the cellular AQP2 itinerary upon changes in endogenous cAMP levels. Before cAMP elevation, AQP2 colocalized with early and recycling, but not late endosomes. Forskolininduced cAMP increase was characterized by AQP2 insertion into the plasma membrane and AQP2 withdrawal from large perinuclear endosomes as well as some localization to lysosomal compartments. Forskolin washout promoted AQP2 endocytosis where AQP2 localized to not only early and recycling endosomes but also late endosomes and lysosomes indicating increased AQP2 degradation. Thus, our results show that 4.5 ExM is an attractive approach to obtain detailed information regarding AQP2 shuttling. NEW & NOTEWORTHY: Renal aquaporin-2 (AQP2) imaged by expansion microscopy provides unprecedented 3-D information regarding the AQP2 itinerary in response to changes in cellular cAMP. [ABSTRACT FROM AUTHOR]

Regulated vesicle exocytosis is a key response to extracellular stimuli in diverse physiological processes, including hormone regulated short-term urine concentration. In the renal collecting duct, the water channel aquaporin-2 (AQP2) localizes to the apical plasma membrane as well as to small, subapical vesicles. In response to stimulation with the antidiuretic hormone, arginine vasopressin, aquaporin-2-containing vesicles fuse with the plasma membrane, which increases collecting duct water reabsorption and thus, urine concentration. The nanoscale size of these vesicles has limited analysis of their three-dimensional (3D) organization. Using a cell system combined with 3D superresolution microscopy, we provide the first direct analysis of the 3D network of aquaporin-2-containing exocytic vesicles in a cell culture system. We show that aquaporin-2 vesicles are 43 ± 3 nm in diameter, a size similar to synaptic vesicles, and that one fraction of AQP2 vesicles localized with the subcortical F-actin layer and the other localized in between the F-actin layer and the plasma membrane. Aquaporin-2 vesicles associated with F-actin and this association were enhanced in a serine 256 phospho-mimic of aquaporin-2, whose phosphorylation is a key event in antidiuretic hormone-mediated aquaporin-2 vesicle exocytosis. [ABSTRACT FROM AUTHOR]

Nephrogenic syndrome of inappropriate antidiuresis (NSIAD) is a recently identified chromosome X-linked disease associated with gain-of-function mutations of the V2 vasopressin receptor (V2R), a G-protein-coupled receptor. It is characterized by inability to excrete a free water load, hyponatremia, and undetectable vasopressin-circulating levels. Hyponatremia can be quite severe in affected male children. To gain a deeper insight into the functional properties of the V2R active mutants and how they might translate into the pathological outcome of NSIAD, in this study, we have expressed the wild-type V2R and three constitutively active V2R mutants associated with NSIAD (R137L, R137C, and the F229V) in MCD4 cells, a cell line derived from renal mouse collecting duct, stably expressing the vasopressin-sensitive water channel aquaporin-2 (AQP2). Our findings indicate that in cells expressing each active mutant, AQP2 was constitutively localized to the apical plasma membrane in the absence of vasopressin stimulation. In line with these observations, under basal conditions, osmotic water permeability in cells expressing the constitutively active mutants was significantly higher compared to that of cells expressing the wild-type V2R. Our findings demonstrate a direct link between activating mutations of the V2R and the perturbation of water balance in NSIAD. In addition, this study provides a useful cell-based assay system to assess the functional consequences of newly discovered activating mutations of the V2R on water permeability in kidney cells and to screen the effect of drugs on the mutated receptors. [ABSTRACT FROM AUTHOR]

Fine tuning of urine concentration occurs in the renal collecting duct in response to circulating levels of arginine vasopressin (AVP). AVP stimulates intracellular cAMP production, which mediates exocytosis of sub-apical vesicles containing the water channel aquaporin-2 (AQP2). Protein Kinase A (PKA) phosphorylates AQP2 on serine-256 (S256), which triggers plasma membrane accumulation of AQP2. This mediates insertion of AQP2 into the apical plasma membrane, increasing water permeability of the collecting duct. AQP2 is a homo-tetramer. When S256 on all four monomers is changed to the phosphomimic aspartic acid (S256D), AQP2-S256D localizes to the plasma membrane and internalization is decreased. In contrast, when S256 is mutated to alanine (S256A) to mimic non-phosphorylated AQP2, AQP2-S256A localizes to intracellular vesicles as well as the plasma membrane, with increased internalization from the plasma membrane. S256 phosphorylation is not necessary for exocytosis and dephosphorylation is not necessary for endocytosis, however, the degree of S256 phosphorylation is hypothesized to regulate the kinetics of AQP2 endocytosis and thus, retention time in the plasma membrane. Using k-space Image Correlation Spectroscopy (kICS), we determined how the number of phosphorylated to non-phosphorylated S256 monomers in the AQP2 tetramer affects diffusion speed of AQP2 in the plasma membrane. When all four monomers mimicked constitutive phosphorylation (AQP2-S256D), diffusion was faster than when all four were non-phosphorylated (AQP2-S256A). AQP2-WT diffused at a speed similar to that of AQP2-S256D. When an average of two or three monomers in the tetramer were constitutively phosphorylated, the average diffusion coefficients were not significantly different to that of AQP2-S256D. However, when only one monomer was phosphorylated, diffusion was slower and similar to AQP2-S256A. Thus, AQP2 with two to four phosphorylated monomers has faster plasma membrane kinetics, than the tetramer which contains just one or no phosphorylated monomers. This difference in diffusion rate may reflect behavior of AQP2 tetramers destined for either plasma membrane retention or endocytosis. [ABSTRACT FROM AUTHOR]

The post-translational modifications (PTMs) phosphorylation and ubiquitylation regulate plasma membrane protein function. Here, we examine the interplay between phosphorylation and ubiquitylation of the membrane protein aquaporin-2 (AQP2) and demonstrate that phosphorylation can override the previously suggested dominant endocytic signal of K63-linked polyubiquitylation. In polarized epithelial cells, although S256 is an important phosphorylation site for AQP2 membrane localization, the rate of AQP2 endocytosis was reduced by prolonging phosphorylation specifically at S269. Despite their close proximity, AQP2 phosphorylation at S269 and ubiquitylation at K270 can occur in parallel, with increased S269 phosphorylation and decreased AQP2 endocytosis occurring when K270 polyubiquitylation levels are maximal. In vivo studies support this data, with maximal levels of AQP2 ubiquitylation occurring in parallel to maximal S269 phosphorylation and enhanced AQP2 plasma membrane localization. In conclusion, we demonstrate for the first time that although K63-linked polyubiquitylation marks AQP2 for endocytosis, site-specific phosphorylation can counteract polyubiquitylation to determine its final localization. Similar mechanisms might exist for other plasma membrane proteins. [ABSTRACT FROM AUTHOR]

In kidney collecting duct cells, filamentous actin (F-actin) depolymerization is a critical step in vasopressin-induced trafficking of aquaporin-2 to the apical plasma membrane. However, the molecular components of this response are largely unknown. Using stable isotope-based quantitative protein mass spectrometry and surface biotinylation, we identified 100 proteins that showed significant abundance changes in the apical plasma membrane of mouse cortical collecting duct cells in response to vasopressin. Fourteen of these proteins are involved in actin cytoskeleton regulation, including actin itself, 10 actin-associated proteins, and 3 regulatory proteins. Identified were two integral membrane proteins (Clmn, Nckap1) and one actin-binding protein (Mpp5) that link F-actin to the plasma membrane, five F-actin end-binding proteins (Arpc2, Arpc4, Gsn, Scin, and Capzb) involved in F-actin reorganization, and two actin adaptor proteins (Dbn1, Lasp1) that regulate actin cytoskeleton organization. There were also protease (Capn1), protein kinase (Cdc42bpb), and Rho guanine nucleotide exchange factor 2 (Arhgef2) that mediate signal-induced F-actin changes. Based on these findings, we devised a live-cell imaging method to observe vasopressininduced F-actin dynamics in polarized mouse cortical collecting duct cells. In response to vasopressin, F-actin gradually disappeared near the center of the apical plasma membrane while consolidating laterally near the tight junction. This F-actin peripheralization was blocked by calcium ion chelation. Vasopressin-induced apical aquaporin- 2 trafficking and forskolin-induced water permeability increase were blocked by F-actin disruption. In conclusion, we identified a vasopressin-regulated actin network potentially responsible for vasopressin-induced apical F-actin dynamics that could explain regulation of apical aquaporin-2 trafficking and water permeability increase. [ABSTRACT FROM AUTHOR]

Myo-inositol (MI; hexahydroxycyclohexane, C(6)H(6)O(12)) is a small neutral molecule used as a compatible osmolyte in the kidney medulla. At high concentrations, MI appears to act as a chemical chaperone and was shown to promote plasma membrane expression of the impaired cystic fibrosis chloride channel (Delta508-CFTR). In the present study, we measured whether MI could increase expression of two human aquaporin 2 (AQP2) mutants which were recently identified as causing nephrogenic diabetes insipidus (NDI). Both proteins (D150E and G196D) were expressed in Xenopus laevis oocytes, but only D150E displayed an increase in oocyte water permeability (P (f)). Adding 5 mM MI to the bathing solution for 24 h produced a 50% increase in the D150E-associated P (f), while it had no effect on noninjected oocytes or on oocytes expressing wt-AQP2 or G196D. Western blots performed on purified plasma membrane preparations confirmed that MI increased the amount of D150E present at the plasma membrane, while G196D was always undetectable. X. laevis oocytes are remarkably impermeable to MI, and the effect of MI on D150E expression does not require the presence of intracellular MI. The effect of external MI was dose-dependent (K (0.5) was 130 microM) and specific with respect to other forms of inositols. Further studies on a second group of AQP2 mutants causing NDI showed that K228E activity was similarly stimulated by MI, while V71M, A70D and S256L were not. It is concluded that physiological concentrations of extracellular MI can stimulate the expression of a specific subgroup of AQP2 mutants. [ABSTRACT FROM AUTHOR]

AVP and atrial natriuretic peptide (ANP) have opposite effects in the kidney. AVP induces antidiuresis by insertion of aquaporin-2 (AQP2) water channels into the plasma membrane of collecting duct principal cells. ANP acts as a diuretic factor. An ANPand nitric oxide (NO)/soluble guanylate cyclase (sGC)-induced insertion of AQP2 into the plasma membrane is reported from different models. However, functional data on the insertion of AQP2 is missing. We used primary cultured inner medullary collecting duct (IMCD) cells and digital holographic microscopy, calcein-quenching measurements, and immunofluorescence and Western blotting to analyze the effects of ANP and NO donors on AQP2 phosphorylation, membrane expression, and water permeability. While AVP led to acceleration in osmotically induced swelling, ANP had no effect. However, in AVP-pretreated cells ANP significantly decreased the kinetics of cell swelling. This effect was mimicked by 8-bromo-cGMP and blunted by PKG inhibition. Stimulation of the NO/sOC pathway or direct activation of sGC with BAY 58-2667 had similar effects to ANP. In cells treated with AVP, AQP2 was predominantly localized in the plasma membrane, and after additional incubation with ANP AQP2 was mostly localized in the cytosol, indicating an increased retrieval of AQP2 from the plasma membrane by ANP. Western blot analysis showed that ANP was able to reduce AVP-induced phosphorylation of AQP2 at position S256. In conclusion, we show that the diuretic action of ANP or NO in the IMCD involves a decreased localization of AQP2 in the plasma membrane which is mediated by cGMP and PKG. [ABSTRACT FROM AUTHOR]

Mutations in aquaporin-2 (AQP2) that interfere with its cellular processing can produce autosomal recessive nephrogenic diabetes insipidus (NDI). Prior gene knock-in of the human NDI-causing AQP2 mutation T126M produced mutant mice that died by age 7 days. Here, we used a novel "conditional gene knock-in" strategy to generate adult, AQP2-T126M mutant mice. Mice separately heterozygous for floxed wild-type AQP2 and AQP2-T126M were bred to produce hemizygous mice, which following excision of the wild-type AQP2 gene by tamoxifen-induced Cre-recombinase gave AQP2T126M/- mice. AQP2T126M/- mice were polyuric (9-14 ml urine/ day) compared to AQP2+/+ mice (1.6 ml/day) and had reduced urine osmolality (400 vs. 1800 mosmol). Kidneys of AQP2T126M/- mice expressed core-glycosylated AQP2- T126M protein in an endoplasmic reticulum pattern. Screening of candidate protein folding "correctors" in AQP2-T126M-transfected kidney cells showed increased AQP2-T126M plasma membrane expression with the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). 17-AAG increased urine osmolality in AQP2T126M/- mice by >300 mosmol but had no effect in AQP2-/- mice. Kidneys of 17-AAG-treated AQP2T126M/- mice showed partial rescue of defective AQP2-T126M cellular processing. Our results establish an adult mouse model of NDI and demonstrate partial restoration urinary concentration function by a compound currently in clinical trials for other indications. [ABSTRACT FROM AUTHOR]

The cAMP-dependent aquaporin-2 (AQP2) redistribution from intracellular vesicles into the plasma membrane of renal collecting duct principal cells induces water reabsorption and fine-tunes body water homeostasis. However, the mechanisms controlling the localization of AQP2 are not understood in detail. Using immortalized mouse medullary collecting duct (MCD4) and primary rat inner medullary collecting duct (IMCD) cells as model systems, we here discovered a key regulatory role of Aurora kinase A (AURKA) in the control of AQP2. The AURKA-selective inhibitor Aurora-A inhibitor I and novel derivatives as well as a structurally different inhibitor, Alisertib, prevented the cAMP-induced redistribution of AQP2. Aurora-A inhibitor I led to a depolymerization of actin stress fibers, which serve as tracks for the translocation of AQP2-bearing vesicles to the plasma membrane. The phosphorylation of cofilin-1 (CFL1) inactivates the actin-depolymerizing function of CFL1. Aurora-A inhibitor I decreased the CFL1 phosphorylation, accounting for the removal of the actin stress fibers and the inhibition of the redistribution of AQP2. Surprisingly, Alisertib caused an increase in actin stress fibers and did not affect CFL1 phosphorylation, indicating that AURKA exerts its control over AQP2 through different mechanisms. An involvement of AURKA and CFL1 in the control of the localization of AQP2 was hitherto unknown. [ABSTRACT FROM AUTHOR]

Aquaporins (AQPs) are membrane proteins serving in the transfer of water and small solutes across cellular membranes. AQPs play a variety of roles in the body such as urine formation, prevention from dehydration in covering epithelia, water handling in the blood–brain barrier, secretion, conditioning of the sensory system, cell motility and metastasis, formation of cell junctions, and fat metabolism. The kidney plays a central role in water homeostasis in the body. At least seven isoforms, namely AQP1, AQP2, AQP3, AQP4, AQP6, AQP7, and AQP11, are expressed. Among them, AQP2, the anti-diuretic hormone (ADH)-regulated water channel, plays a critical role in water reabsorption. AQP2 is expressed in principal cells of connecting tubules and collecting ducts, where it is stored in Rab11-positive storage vesicles in the basal state. Upon ADH stimulation, AQP2 is translocated to the apical plasma membrane, where it serves in the influx of water. The translocation process is regulated through the phosphorylation of AQP2 by protein kinase A. As soon as the stimulation is terminated, AQP2 is retrieved to early endosomes, and then transferred back to the Rab 11-positive storage compartment. Some AQP2 is secreted via multivesicular bodies into the urine as exosomes. Actin plays an important role in the intracellular trafficking of AQP2. Recent findings have shed light on the molecular basis that controls the trafficking of AQP2. [ABSTRACT FROM AUTHOR]

Membrane water channel aquaporin-2 (AQP2) and glucose transporter 4 (GLUT4) exhibit a common feature in that they are stored in intracellular storage compartments and undergo translocation to the plasma membrane upon hormonal stimulation. We compared the intracellular localization and trafficking of AQP2 and GLUT4 in polarized Madin-Darby canine kidney cells stably transfected with human AQP2 (MDCK-hAQP2) by immunofluorescence microscopy. When expressed in MDCK-hAQP2 cells, GLUT4 and GLUT4—EGFP were predominantly localized in the perinuclear region close to and within the Golgi apparatus, similar to endogenous GLUT4 in adipocytes and myocytes. In addition, GLUT4 was occasionally seen in EEA1-positive early endosomes. AQP2, on the other hand, was sequestered in subapical Rab11-positive vesicles. In the basal state, the intracellular storage site of GLUT4 was distinct from that of AQP2. Forskolin induced translocation of AQP2 from the subapical storage vesicles to the apical plasma membrane, which did not affect GLUT4 localization. When forskolin was washed out, AQP2 was first retrieved to early endosomes from the apical plasma membrane, where it was partly colocalized with GLUT4. AQP2 was then transferred to Rab11-positive storage vesicles. These results show that AQP2 and GLUT4 share a common compartment after retrieval from the plasma membrane, but their storage compartments are distinct from each other in polarized MDCK-hAQP2 cells. [ABSTRACT FROM AUTHOR]

TonEBP stimulates genes whose products drive cellular accumulation of organic osmolytes and HSP70, which protect cells from the deleterious effects of hypertonicity and urea, respectively. Mice deficient in the TonEBP gene display severe atrophy of the renal medulla because cells failed to adapt to the hyperosmolality. Emerging data suggest that TonEBP plays a key role in the urinary concentrating mechanism by stimulating the UT-A urea transporters and possibly AQP2 water channel. Thus, TonEBP is an essential regulator in the urinary concentrating mechanism. Studies on structural basis of TonEBP function have revealed the structure of the DNA binding domain, and defined the transactivation domains. Molecular mechanisms underlying the nucleocytoplasmic trafficking, transactivation, and phosphorylation in response to changes in tonicity need to be understood in molecular detail. Such knowledge is needed for the identification of the sensor that detects changes in ambient tonicity and signals to TonEBP. [ABSTRACT FROM AUTHOR]

Urinary exosomes containing apical membrane and intracellular fluid are normally secreted into the urine from all nephron segments, and may carry protein markers of renal dysfunction and structural injury. We studied methods for collection, storage, and preservation of urinary exosomal proteins. We collected urine from healthy volunteers, added protease inhibitors, and stored urine samples at 4, −20, and −80°C for 1 week or 7 months. Samples were thawed with and without extensive vortexing, and three fractions were isolated: urinary sediment, supernatant, and exosome fraction. Protein concentration, electrophoresis patterns, and abundance of seven exosome-associated proteins were measured. Exosome-associated proteins were not detected in sediment or supernatant fractions. Protease inhibitors prevented degradation of exosome-associated proteins. Freezing at −20°C caused a major loss in exosomes compared to fresh urine. In contrast, recovery after freezing at −80°C was almost complete. Extensive vortexing after thawing markedly increased exosome recovery in urine frozen at −20 or −80°C, even if frozen for 7 months. The recovery from first and second morning urine was similar. The abundance of cytosolic exosome-associated proteins did not decrease during long-term storage. We concluded: (1) protease inhibitors are essential for preservation; (2) storage at −80°C with extensive vortexing after thawing maximizes the recovery of urinary exosomes; (3) the difference between first and second morning urine exosome-associated protein was small, suggesting minimal protein degradation in the urinary tract/bladder; (4) urinary exosomes remain intact during long-term storage. These urine collection, storage, and processing conditions may be useful for future biomarker discovery efforts.Kidney International (2006) 69, 1471–1476. doi:10.1038/sj.ki.5000273; published online 22 February 2006 [ABSTRACT FROM AUTHOR]

Vasopressin-induced trafficking of aquaporin-2 (AQP2) water channels in kidney collecting duct cells is critical to regulate the urine concentration. To better understand the mechanism of subcellular trafficking of AQP2, we examined MDCK cells expressing AQP2 as a model. We first performed double-immunolabeling of AQP2 with endosomal marker proteins, and showed that AQP2 is stored at a Rab11-positive subapical compartment. After the translocation to the plasma membrane, AQP2 was endocytosed to EEA1-positive early endosomes, and then transferred back to the original Rab11-positive compartment. When Rab11 was depleted by RNA interference, retention of AQP2 at the subapical storage compartment was impaired. We next examined the role of cytoskeleton in the AQP2 trafficking and localization. By the treatment with microtubule-disrupting agent such as nocodazole or colcemid, the distribution of AQP2 storage compartment was altered. The disruption of actin filaments with cytochalasin D or latrunculin B induced the accumulation of AQP2 in EEA1-positive early endosomes. Altogether, our data suggest that Rab11 and microtubules maintain the proper distribution of the subapical AQP2 storage compartment, and actin filaments regulate the trafficking of AQP2 from early endosomes to the storage compartment. [ABSTRACT FROM AUTHOR]

NSIAD is a rare X-linked condition, caused by activating mutations in the AVPR2 gene coding for the vasopressin V2 receptor (V2R) associated with hyponatremia, despite undetectable plasma vasopressin levels. We have recently provided in vitro evidence that, compared to V2R-wt, expression of activating V2R mutations R137L, R137C and F229V cause a constitutive redistribution of the AQP2 water channel to the plasma membrane, higher basal water permeability and significantly higher basal levels of p256-AQP2 in the F229V mutant but not in R137L or R137C. In this study, V2R mutations were expressed in collecting duct principal cells and the associated signalling was dissected. V2R-R137L and R137C mutants had significantly higher basal pT269-AQP2 levels -independently of S256 and PKA-which were reduced to control by treatment with Rho kinase (ROCK) inhibitor. Interestingly, ROCK activity was found significantly higher in V2R-R137L along with activation of the Gα12/13–Rho–ROCK pathway. Of note, inhibition of ROCK reduced the basal elevated osmotic water permeability to control. To conclude, our data demonstrate for the first time that the gain-of-function mutation of the V2R, R137L causing NSIAD, signals through an alternative PKA-independent pathway that increases AQP2 membrane targeting through ROCK-induced phosphorylation at S/T269 independently of S256 of AQP2. [ABSTRACT FROM AUTHOR]

Aldosterone indirectly regulates water reabsorption in the distal tubule by regulating sodium reabsorption. However, the direct effect of aldosterone on vasopressin-regulated water and urea permeability in the rat inner medullary collecting duct (IMCD) has not been tested. We investigated whether aldosterone regulates osmotic water permeability in isolated perfused rat IMCDs. Adding aldosterone (500 nM) to the bath significantly decreased osmotic water permeability in the presence of vasopressin (50 pM) in both male and female rat IMCDs. Aldosterone significantly decreased aquaporin-2 (AQP2) phosphorylation at S256 but did not change it at S261. Previous studies show that aldosterone can act both genomically and non-genomically. We tested the mechanism by which aldosterone attenuates osmotic water permeability. Blockade of gene transcription with actinomycin D did not reverse aldosterone-attenuated osmotic water permeability. In addition to AQP2, the urea transporter UT-A1 contributes to vasopressin-regulated urine concentrating ability. We tested aldosterone-regulated urea permeability in vasopressin-treated IMCDs. Blockade of gene transcription did not reverse aldosterone-attenuated urea permeability. In conclusion, aldosterone directly regulates water reabsorption through a non-genomic mechanism. Aldosterone-attenuated water reabsorption may be related to decreased trafficking of AQP2 to the plasma membrane. There may be a sex difference apparent in the inhibitory effect of aldosterone on water reabsorption in the inner medullary collecting duct. This study is the first to show a direct effect of aldosterone to inhibit vasopressin-stimulated osmotic water permeability and urea permeability in perfused rat IMCDs. [ABSTRACT FROM AUTHOR]