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Atrial natriuretic peptide (ANP)/cGMPs cause diuresis and natriuresis. Their downstream effectors beyond cGMP remain unclear. To elucidate a probable function of cGMP-dependent protein kinase II (cGKII), we investigated renal parameters in different conditions (basal, salt diets, starving, water load) using a genetically modified mouse model (cGKII-KO), but did not detect any striking differences between WT and cGKII-KO. Thus, cGKII is proposed to play only a marginal role in the adjustment of renal concentration ability to varying salt loads without water restriction or starving conditions. When WT mice were subjected to a volume load (performed by application of a 10-mM glucose solution (3 % of BW) via feeding needle), they exhibited a potent diuresis. In contrast, urine volume was decreased significantly in cGKII-KO. We showed that AQP2 plasma membrane (PM) abundance was reduced for about 50 % in WT upon volume load, therefore, this might be a main cause for the enhanced diuresis. In contrast, cGKII-KO mice almost completely failed to decrease AQP2-PM distribution. This significant difference between both genotypes is not induced by an altered p-Ser256-AQP2 phosphorylation, as phosphorylation at this site decreases similarly in WT and KO. Furthermore, sodium excretion was lowered in cGKII-KO mice during volume load. In summary, cGKII is only involved to a minor extent in the regulation of basal renal concentration ability. By contrast, cGKII-KO mice are not able to handle an acute volume load. Our results suggest that membrane insertion of AQP2 is inhibited by cGMP/cGKII. [ABSTRACT FROM AUTHOR]

Nephrogenic diabetes insipidus (NDI) provides an excellent model for the benefits and insights that can be gained from studying rare diseases. The discovery of underlying genes identified key molecules involved in urinary concentration, including the type 2 vasopressin receptor AVPR2 and the water channel AQP2, which constitute obvious pharmacologic targets. Subsequently developed drugs targeting AVPR2 not only provide potential benefit to some patients with NDI, but are now used for much more common clinical applications as diverse as nocturnal enuresis and heart failure. Yet, the story is still evolving: clinical observations and animal experiments continue to discover new ways to affect urinary concentration. These novel pathways can potentially be exploited for therapeutic gain. Here we review the (patho)physiology of water homoeostasis, the current status of clinical management, and potential new treatments. [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]

Background Renal handling of sodium and water is abnormal in chronic kidney disease (CKD). The aim of this study was to test the hypothesis that abnormal activity of the aquaporin-2 water channels (AQP2), the sodium-potassium-2chloride transporter (NKCC2) and/or the epithelial sodium channels (ENaC) contribute to this phenomenon. Methods 23 patients with CKD and 24 healthy controls at baseline and after 3% saline infusion were compared. The following measurements were performed: urinary concentrations of AQP2 (u- AQP2), NKCC2 (u-NKCC2), ENaC (u-ENaCγ), glomerular filtration rate (GFR) estimated by 51Cr-EDTA clearance, free water clearance (CH2O), urinary output (UO), fractional excretion of sodium (FENa), plasma concentrations of AVP, renin (PRC), Angiotensin II (ANG II), Aldosterone (Aldo) and body fluid volumes. Results At baseline, GFR was 34 ml/min in CKD patients and 89 ml/ml in controls. There were no significant differences in u-AQP2, u-NKCC2 or u-ENaCγ, but FENa, p-Aldo and p-AVP were higher in CKD patients than controls. In response to hypertonic saline, patients with CKD had an attenuated decrease in CH2O and UO. A greater increase in U-AQP2 was observed in CKD patients compared to controls. Furthermore, u-NKCC2 increased in CKD patients, whereas u-NKCC2 decreased in controls. Body fluid volumes did not significantly differ. Conclusions In response to hypertonic saline, u-NKCC2 increased, suggesting an increased sodium reabsorption via NKCC2 in patients with CKD. U-AQP2 increased more in CKD patients, despite an attenuated decrease in CH2O. Thus, though high levels of p-AVP and p-Aldo, the kidneys can only partly compensate and counteract acute volume expansion due to a defective tubular response. [ABSTRACT FROM AUTHOR]

Background Tolvaptan is a selective vasopressin receptor antagonist (V2R) that increases free water excretion. We wanted to test the hypotheses that tolvaptan changes both renal handling of water and sodium and systemic hemodynamics during basal conditions and during nitric oxide (NO)-inhibition with L-NG-monomethyl-arginine (L-NMMA). Methods Nineteen healthy subjects were enrolled in a randomized, placebo-controlled, double-blind, crossover study of two examination days. Tolvaptan 15 mg or placebo was given in the morning. L-NMMA was given as a bolus followed by continuous infusion during 60 minutes. We measured urine output(UO), free water clearance (CH2O), fractional excretion of sodium (FENa), urinary aquaporin-2 channels (u-AQP2) and epithelial sodium channels (u-ENaCγ), plasma vasopressin (p-AVP), central and brachial blood pressure(cBP, bBP). Results During baseline conditions, tolvaptan caused a significant increase in UO, CH2O and p-AVP, and FENa was unchanged. During L-NMMA infusion, UO and CH2O decreased more pronounced after tolvaptan than after placebo (-54 vs.-42% and -34 vs.-9% respectively). UAQP2 decreased during both treatments, whereas u-ENaCγ decreased after placebo and increased after tolvaptan. CBP and bBP were unchanged. Conclusion During baseline conditions, tolvaptan increased renal water excretion. During NO-inhibition, the more pronounced reduction in renal water excretion after tolvaptan indicates that NO promotes water excretion in the principal cells, at least partly, via an AVP-dependent mechanism. The lack of decrease in u-AQP2 by tolvaptan could be explained by a counteracting effect of increased plasma vasopressin. The antagonizing effect of NOinhibition on u-ENaC suggests that NO interferes with the transport via ENaC by an AVPdependent mechanism. [ABSTRACT FROM AUTHOR]

Abstract: The interplay between actin and 10 membrane channel proteins that have been shown to directly bind to actin are reviewed. The 10 membrane channel proteins covered in this review are aquaporin 2 (AQP2), cystic fibrosis transmembrane conductance regulator (CFTR), ClC2, short form of ClC3 (sClC3), chloride intracellular channel 1 (CLIC1), chloride intracellular channel 5 (CLIC5), epithelial sodium channel (ENaC), large-conductance calcium-activated potassium channel (Maxi-K), transient receptor potential vanilloid 4 (TRPV4), and voltage-dependent anion channel (VDAC), with particular attention to AQP2. In regard to AQP2, most reciprocal interactions between actin and AQP2 occur during intracellular trafficking, which are largely mediated through indirect binding. Actin and the actin cytoskeleton work as cables, barriers, stabilizers, and force generators for motility. However, as with ENaC, the effects of actin cytoskeleton on channel gating should be investigated further. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé. [Copyright &y& Elsevier]

Introduction: Phosphatase and tensin (PTEN) is a multifunctional gene associated with the normal development and physiological function of various tissues including the kidney. However, its role in renal tubular reabsorption function has not been well elucidated., Methods: We generated a renal tubule-specific Pten knockout mouse model by crossing Pten fl/fl mice with Ksp-Cre transgenic mice, evaluated the effect of Pten loss on renal tubular function, and investigated the underlying mechanisms., Results: Pten loss resulted in abnormal renal structure and function and water retention in multiple organs. Our results also demonstrated that aquaporin-2 (AQP2), an important water channel protein, was upregulated and concentrated on the apical plasma membrane of collecting duct cells, which could be responsible for the impaired water balance in Pten loss mice. The regulation of Pten loss on AQP2 was mediated by protein kinase B (AKT) activation., Conclusions: Our results reveal a connection between PTEN gene inactivation and water retention, suggesting the importance of PTEN in normal kidney development and function., Competing Interests: The authors have no conflicts of interest to declare., (Copyright © 2022 by The Author(s). Published by S. Karger AG, Basel.)

Recent discoveries have shed new light on the understanding of water metabolism: (1.) in addition to hypothalamic osmoreceptor cells expressing a TRPV1 variant, there are peripheral TRPV4 receptors sensing tonicity in the portal vein and changing central vasopressin secretion and peripheral autonomic activity; (2.) the central osmoregulatory gain of angiotensin action participates in the non-osmotic release of vasopressin induced by hypovolaemia; (3.) prostaglandins EP2 receptors on principal cells of the collecting ducts positively regulate urine concentration mechanisms. These new developments are important clinically for the understanding of hereditary polyuric states. We recommend sequencing of the nephrogenic diabetes insipidus genes in all affected patients. This genomic information is key to the routine care of patients with congenital polyuria and, as in other genetic diseases, reduces health costs and confers psychological benefits on patients and families.

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]

Regulation of water permeability of collecting duct principal cells by neurohypophysial hormone vasopressin underlies the functioning of kidneys as the main effector organs providing the maintenance of the water-electrolyte balance of the organism. Clathrin-mediated endocytosis of water channels and V2 vasopressin receptors is an important step of the antidiuretic mechanism of vasopressin. Internalization of the receptors does not only reduce the number of receptors in plasma membrane, but also activates alternative intracellular signaling pathways. This review outlines the current concept of the mechanism of clathrinmediated endocytosis of V2 receptors and AQP2 water channels, which is necessary for better understanding the molecular mechanism of vasopressin action. [ABSTRACT FROM AUTHOR]

Trepiccione F, Capasso G, Nielsen S, Christensen BM. Evaluation of cellular plasticity in the collecting duct during recovery from lithium-induced nephrogenic diabetes insipidus. Am J Physiol Renal Physiol 305: F919 -F929, 2013. First published July 3, 2013; doi:10.1152/ajprenal.00152.2012.--The cellular morphology of the collecting duct is altered by chronic lithium treatment. We have previously shown that lithium increases the fraction of type-A intercalated cells and lowers the fraction of principal cells along the collecting duct. Moreover, type-A intercalated cells acquire a longrow distribution pattern along the tubules. In the present study, we show that these morphological changes reverse progressively after discontinuation of lithium and finally disappear after 19 days from lithium suspension. In this time frame we have identified for the first time, in vivo, a novel cellular type positive for both intercalated and principal cells functional markers, as recognized by colabeling with H+-ATPase/aquaporin-4 (AQP4) and anion exchanger-1 (AE-1)/ AQP2 and Foxi1/AQP4. This cell type is mainly present after 6 days of lithium washout, and it disappears in parallel with the long-row pattern of the type-A intercalated cells. It usually localizes either in the middle or at the edge of the long-row pattern. Its ultrastructure resembles the intercalated cells as shown both by differential interference contrast and by electron microscopy. The time course of appearance, the localization along the collecting duct, and the ultrastructure suggest that the cells double labeled for principal and intercalated cells markers could represent a transition element driving the conversion of intercalated cells into principal cells. [ABSTRACT FROM AUTHOR]

Background Desmopressin (dDAVP) induces a decrease in immunolabelling of aquaporin (AQP) 4 protein in the terminal inner medulla (IM) of the Brattleboro (BB) rat kidney. It is disputed, however, whether the decreased labelling reflects real down-regulation of protein abundance, or whether it is a result of epitope shielding in the AQP4 protein, preventing binding of the antibody as previously suggested. Furthermore, it is unknown if vasopressin regulates AQP4 in the connecting tubule (CNT) and in the cortical collecting duct (CCD). Using BB rats, we aimed to determine (i) whether the dDAVP-induced decrease in AQP4 labelling in the terminal IM reflects down-regulation in protein abundance and (ii) whether dDAVP increases the AQP4 protein abundance in the CNT and the CCD. Methods BB rats received dDAVP or saline (control) via osmotic minipumps pumps for 6 days. Results Immunolight microscopy revealed strong AQP4 labelling in the initial inner medullary collecting duct (IMCD1), weak labelling in the middle IMCD (IMCD2) and weak/absent labelling in the terminal IMCD (IMCD3) after 6 days of dDAVP administration. AQP3 labelling was similar to that of AQP4. Two-hour administration with dDAVP (previously described BB rats) did not change the labelling pattern of AQP4, suggesting that potential acute phosphorylation did not induce epitope shielding, thereby preventing the binding of the antibody. Conclusions In BB rats, long-term administration with dDAVP (i) increased the AQP4 protein abundance in the IMCD1 and decreased the abundance in the IMCD2 and the IMCD3, and (ii) increased the AQP4 protein abundance in the CNT and the CCD. [ABSTRACT FROM AUTHOR]

Melarsoprol and pentamidine represent the two main classes of drugs, the arsenicals and diamidines, historically used to treat the diseases caused by African trypanosomes: sleeping sickness in humans and Nagana in livestock. Cross-resistance to these drugs was first observed over 60 years ago and remains the only example of cross-resistance among sleeping sickness therapies. A Trypanosoma brucei adenosine transporter is well known for its role in the uptake of both drugs. More recently, aquaglyceroporin 2 (AQP2) loss of function was linked to melarsoprol–pentamidine cross-resistance. AQP2, a channel that appears to facilitate drug accumulation, may also be linked to clinical cases of resistance. Here, we review these findings and consider some new questions as well as future prospects for tackling the devastating diseases caused by these parasites. [ABSTRACT FROM AUTHOR]

Protein kinase A (PKA) directly phosphorylates aquaporin-2 (AQP2) water channels in renal collecting ducts to reabsorb water from urine for the maintenance of systemic water homeostasis. More than 50 functionally distinct PKA-anchoring proteins (AKAPs) respectively create compartmentalized PKA signaling to determine the substrate specificity of PKA. Identification of an AKAP responsible for AQP2 phosphorylation is an essential step toward elucidating the molecular mechanisms of urinary concentration. PKA activation by several compounds is a novel screening strategy to uncover PKA substrates whose phosphorylation levels were nearly perfectly correlated with that of AQP2. The leading candidate in this assay proved to be an AKAP termed lipopolysaccharide-responsive and beige-like anchor protein (LRBA). We found that LRBA colocalized with AQP2 in vivo, and Lrba knockout mice displayed a polyuric phenotype with severely impaired AQP2 phosphorylation. Most of the PKA substrates other than AQP2 were adequately phosphorylated by PKA in the absence of LRBA, demonstrating that LRBA-anchored PKA preferentially phosphorylated AQP2 in renal collecting ducts. Furthermore, the LRBA-PKA interaction, rather than other AKAP-PKA interactions, was robustly dissociated by PKA activation. AKAP-PKA interaction inhibitors have attracted attention for their ability to directly phosphorylate AQP2. Therefore, the LRBA-PKA interaction is a promising drug target for the development of anti-aquaretics.

The aim of this study was to identify mutations in three different genes, the arginine-vasopressin-neurophysin II ( AVP- NPII) gene, the arginine-vasopressin receptor 2 ( AVPR2) gene, and the vasopressin-sensitive water channel aquaporin-2 ( AQP2) gene in Turkish patients affected by central diabetes insipidus or nephrogenic diabetes insipidus. This study included 15 patients from unrelated families. Prospective clinical data were collected for all patients including the patients underwent a water deprivation-desmopressin test. The coding regions of the AVPR2, AQP2, and AVP- NPII genes were amplified by polymerase chain reaction and submitted to direct sequence analysis. Of the 15 patients with diabetes insipidus referred to Gulhane Military Medical Academy, Department of Endocrinology and Metabolism, eight patients have AVPR2 mutations, five patients have AQP2 mutations and two patients have AVP-NPII mutations. Of the patients, which have AVPR2 mutations, one is compound heterozygous for AVPR2 gene. Seven of these mutations are novel. Comparison of the clinical outcomes of these mutations may facilitate in understanding the functions of AVP- NPII, AQP2, and AVPR2 genes in future studies. [ABSTRACT FROM AUTHOR]

Abstract: This review describes the discovery of rat aquaporin 2 AQP2 as a vasopressin-regulated water channel, and subsequent isolation of human AQP2. Regarding the structure and function of AQP2, further structural analysis is necessary to understand the basic properties of individual channel function, for examples, such as possible regulation by gating. The critical importance of AQP2 in the urine concentrating ability is demonstrated by a human disease, nephrogenic diabetes insipidus (NDI), and by gene targeting of AQP2 in mice. AQP2 is regulated by many mechanisms from gene transcription to final protein degradation, and vasopressin-stimulated recycling of AQP2 is important for accumulation of AQP2 at the apical membrane. In AQP2-affected NDI, comparison of genotype (types of mutations and mutated residues) and phenotype (clinical characteristics) provides better understanding of both clinical entity of the disease and molecular mechanisms regulating AQP2. Finally, it has become increasingly clear that AQP2 is greatly involved in many human abnormal water balance disorders that await new therapies and clinical markers. [Copyright &y& Elsevier]

Whole-body water balance is predominantly controlled by the kidneys, which have the ability to concentrate or dilute the urine in the face of altered fluid and solute intake. Regulated water excretion is controlled by various hormones and signaling molecules, with the antidiuretic hormone arginine vasopressin (AVP) playing an essential role, predominantly via its modulatory effects on the function of the water channel aquaporin-2 (AQP2). The clinical conditions, central and nephrogenic diabetes insipidus, emphasize the importance of the AVP-AQP2 axis. In this article, we summarize the most important and recent studies on AVP-regulated trafficking of AQP2, with focus on the cellular components mediating (1) AQP2 vesicle targeting to the principal cell apical plasma membrane, (2) docking and fusion of AQP2-containing vesicles, (3) regulated removal of AQP2 from the plasma membrane, and (4) posttranslational modifications of AQP2 that control several of these processes. Insight into the molecular mechanisms responsible for regulated AQP2 trafficking is proving to be fundamental for development of novel therapies for water balance disorders. [ABSTRACT FROM AUTHOR]

Background: Urinary excretion of aquaporin 2 (AQP2) is a useful marker of kidney collecting duct function. A specific and convenient method to measure AQP2 in human urine would help to treat water balance disorders. It is unknown whether urinary excretion of AQP2 shows any daily variance. Methods: A sandwich enzyme-linked immunosorbent assay (ELISA) method for AQP2 was established using two different kinds of antibodies, and its sensitivity and specificity were examined. Daily variance of urinary excretion of AQP2 and responses to acute water load were examined. Results: The established ELISA specifically detected urine AQP2 with high sensitivity (detected as low as 0.34 pmol/mL). Urinary excretion of AQP2 did not show daily variance between 9 a.m. and 9 p.m. in healthy subjects. Conclusions: The developed ELISA method using two different antibodies is convenient and highly sensitive, and could be useful in clinical practice. Urinary excretion of AQP2 is relatively constant from morning to night, and spot urine sampling at any time during this time period does not affect the results. [ABSTRACT FROM AUTHOR]

Abstract: Ethnopharmacological relevance: A major physiological role of the kidney is to regulate body water and urine concentration. Aquaporin-2 (AQP2), a family of water channels, plays an important role in the urinary concentrating process and regulation of water balance in the kidney. The dried sclerotia of Poria cocos Wolf has been known to have a diuretic effect and used for the treatment of chronic edema and nephrosis. Aim of the study: This study was conducted to evaluate the inhibitory effect of the sclerotia of Poria cocos (WPC) on hypertonic stress-induced AQP2 expression and apoptosis in inner medullary collecting duct cell lines (IMCD-3). Materials and methods: Hypertonic stress was induced by 175mM NaCl. Inhibitory effect of WPC on hypertonic stress-induced AQP2 expression and apoptosis were determined by western blot, RT-PCR, and immunofluorescence. Results: Hypertonic stress (175mM NaCl) increased in the levels of AQP2 expression by hypertonicity in IMCD-3 cells. WPC attenuated the hypertonicity-induced increase in protein and mRNA levels of AQP2 in a concentration-dependent manner. Pretreatment with WPC attenuated hypertonicity-induced cell death. Hypertonicity increased serum- and glucocorticoid-inducible protein kinase (Sgk1) phosphorylation, however, WPC attenuated the hypertonicity-induced Sgk1 activation. Tonicity-responsive enhancer binding protein (TonEBP) mRNA was also recovered by WPC under hypertonic stress. Pretreatment with WPC presented the similar effect of PKA inhibitor which decreased hypertonic stress-induced AQP2 expression. Hypertonicity increased cAMP levels and the changes were blocked by WPC. On the other hand, hypertonic stress-induced Bax or caspase-3 expression was decreased by WPC, resulting in anti-apoptotic effect. Conclusions: These results provided evidence that the beneficial effect of WPC in water balance against in vitro hypertonic stress of renal collecting ducts. In addition, WPC exhibits anti-apoptotic property response to hypertonic stress. Thus, these data suggests that WPC has benefit for the therapeutic approach to the inhibition of renal disorder. [Copyright &y& Elsevier]

Previously we demonstrated that neonatally induced partial unilateral ureteral obstruction (PUUO) in rats is associated with changes in the abundance of renal acid-base transporters that were paralleled by reduction in renal functions dependent on the severity and duration of obstruction. The aim of the present study was to identify whether changes in renal aquaporin abundance are age-dependent. Semiquantitative immunoblotting and immunohistochemistry were used to examine the changes in abundance of AQP1, AQP2, p-S256AQP2 (AQP2 phosphorylated at consensus site Ser) and AQP3 in the kidneys of rats with neonatally induced PUUO within the first 48 h of life, and then monitored for 7 or 14 weeks. Protein abundance of AQP2 and AQP3 increased in both obstructed and non-obstructed kidneys 7 weeks after induction of neonatal PUUO (PUUO-7W). In contrast, AQP1 and AQP2 protein abundance in the obstructed kidney were reduced after 14 weeks of PUUO (PUUO-14W). Importantly, pS256-AQP2 protein abundance was reduced in obstructed kidneys of both PUUO-7W and PUUO-14W. Immunohistochemistry confirmed the persistent pS256-AQP2 downregulation in both PUUO-7W and PUUO-14W rats. The study shows that the protein abundance of AQP1, AQP2, and AQP3 in the obstructed kidney is increased in PUUO-7W, which may be a compensatory phenomenon and reduced in PUUO-14W rats suggesting a time-/age-dependent dysregulation in response to PUUO. pS256-AQP2 protein abundance is reduced consistent with obstruction-induced direct effects in the apical part of the collecting duct principal cells in response to PUUO. [ABSTRACT FROM AUTHOR]

Cisplatin (CP) induced acute renal failure (ARF) has previously been associated with decreased urinary prostaglandin E2 (PGE2) excretion and reduced aquaporin 2 (AQP2) expression in kidney collecting duct. In this study we examined the expression of cyclooxygenase (COX)-1 and -2 as well as AQP2 and the Na-K-2Cl cotransporter in kidneys from rats with CP induced ARF. Rats were treated with either CP or saline and followed for 5 days. Kidneys were dissected into three zones and prepared for immunoblotting, quantitative polymerase chain reaction (QPCR) and immunohistochemistry. Renal content and urinary PGE2 excretion was measured. Cisplatin treatment was associated with polyuria and a significant decreased creatinine clearance. Inner medullary PGE2 content and urinary PGE2 excretion was decreased in CP-treated rats. QPCR and semiquatitative immunoblotting demonstrated that CP treatment reduced COX-2, AQP2 and Na-K-2Cl cotransporter abundance in the different kidney zones, whereas no change in COX-1 was observed. Results were confirmed by immunohistochemistry. Cyclooxygenase-2 expression is decreased in inner medulla and cortex. Consistent with this urinary PGE2 levels were reduced. These data suggest that downregulation of COX-2 is responsible for impaired de novo generation of vasodilatory prostaglandins which may play an important role for the CP induced renal vasoconstriction and development of nephropathy. [ABSTRACT FROM AUTHOR]

To investigate the role of the mineralocorticoid receptor (MR) in renal ENaC-mediated sodium reabsorption, we have previously used the Cre-loxP system to generate mice with principal-cell specific MR ablation (MRAQP2Cre mice). To restrict Cre expression to principal cells, we have used the regulatory elements of the mouse aquaporin-2 (AQP2) gene to drive Cre expression. Since AQP2 is already expressed during renal development, MR ablation took place long before the analysis performed at the adult stage. To investigate whether the early onset of MR ablation affected the adult renal sodium handling, we developed a transgene expressing the CreERT2 fusion protein under control of the regulatory elements of the AQP2 gene (AQP2CreERT2). Immunofluorescence revealed MR loss in the collecting duct (CD) and late connecting tubule after induction of MR ablation by tamoxifen in MRAQP2CreERT2 mice that equals the MR loss in MRAQP2Cre mice. Surprisingly, tamoxifen-independent MR loss is observed in CDs of noninduced mutants without affecting circulating aldosterone levels. Under a low-salt diet, the induced ablation of MR at the adult stage recapitulates the renal sodium wasting observed in mice with constitutive early-onset MR ablation. The AQP2CreERT2 transgene is a new tool for investigating in vivo the function of genes downstream of MR in renal ENaC-mediated sodium reabsorption by inducible somatic gene inactivation. [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]

Cyclooxygenase-2 activity is required for the development of lithium-induced polyuria. However, the involvement of a specific, terminal prostaglandin (PG) isomerase has not been evaluated. The present study was undertaken to assess lithium-induced polyuria in mice deficient in microsomal prostaglandin E synthase-l (mPGES-1). A 2-wk administration of LiCI (4 mmol·kg·day-1 ip) in mPGES-1 +/+ mice led to a marked polyuria with hyposmotic urine. This was associated with elevated renal mPGES-1 protein expression and increased urine POE2 excretion. In contrast, mPGES-1 -/- mice were largely resistant to lithium-induced polyuria and a urine concentrating defect, accompanied by nearly complete blockade of high urine PGE2 and cAMP output. Immunoblotting, immunohistochemistry, and quantitative (q) RT-PCR consistently detected a significant decrease in aquaporin-2 (AQP2) protein expression in both the renal cortex and medulla of lithium-treated +/+ mice. This decrease was significantly attenuated in the -/- mice. qRT-PCR detected similar patterns of changes in AQP2 mRNA in the medulla but not in the cortex. Similarly, the total protein abundance of the Na-K-2Cl cotransporter (NKCC2) in the medulla but not in the cortex of the +/+ mice was significantly reduced by lithium treatment. In contrast, the dowregulation of renal medullary NKCC2 expression was significantly attenuated in the -/- mice. We conclude that mPGES-I-derived PGE2 mediates lithium-induced polyuria likely via inhibition of AQP2 and NKCC2 expression. [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]

Abstract: Birds and mammals are the only vertebrates that can concentrate urine. Avian kidneys contain structurally primitive loopless nephrons and also more advanced looped nephrons, in the cortical and medullary regions, respectively. We have identified the gene sequence of an aquaporin 2 (AQP2)-homologue water channel in collecting ducts of kidneys from adult quail, Coturnix japonica. Although immunoreactive quail AQP2 (qAQP2) was found in both types of nephrons, the expression is enhanced more clearly in the medullary regions after water deprivation. We therefore hypothesized that regulation of qAQP2 expression in quail kidneys via antidiuretic hormone (ADH) may require more advanced nephron structure. In this study, we determined the expression of qAQP2 mRNA in tissues isolated from the cortical and medullary regions before and after water deprivation, by conventional reverse transcriptase-polymerase chain reaction (RT-PCR) and quantitative real-time PCR. In both normally hydrated and water-deprived groups, qAQP2 mRNA levels in the medullary regions were significantly higher (P <0.01) than in the cortical regions. In medullary areas, qAQP2 mRNA levels (real-time PCR normalized with 18S) were significantly higher (P <0.01, ANOVA) after water deprivation (1.09±0.10) than in normally hydrated controls (0.46±0.08). In cortical areas, qAQP2 mRNA levels were also higher after water deprivation (0.37±0.05) than in controls (0.11±0.02). qAQP2 mRNA signals determined by in situ hybridization of digoxigenin-labeled riboprobe were also enhanced after water deprivation in both cortical and medullary collecting ducts. The results suggest that, contrary to our hypothesis, the endogenous production of ADH by water deprivation stimulates qAQP2 mRNA in both loopless and looped nephrons. [Copyright &y& Elsevier]

Hypertonicity in the renal medulla stimulates local cyclooxygenase 2 expression, leading to abundant PGE2 production. Here we found that mRNA expression by the PGE2-activated G-protein-coupled receptors, EP3 and EP4 in the renal medulla was decreased by furosemide treatment, a procedure that reduces medullary hypertonicity. When HepG2 cells were cultured in hypertonic conditions by addition of salt or sorbitol, EP3 expression was induced. A specific EP3 agonist inhibited cAMP production, indicating receptor functionality, and this led to a substantial increase in cell survival in hypertonic media. Survival was independent of the SLC5A3 inositol transporter and aldose reductase expression, suggesting that EP3 promoted cell survival under hypertonic conditions independent of cellular organic osmolyte accumulation. Reduced cAMP production did not contribute to increased survival. EP4 expression was stimulated by hypertonicity in MDCK and HepG2 cells, which was associated with increased cAMP production in response to an EP4 agonist. Our study shows that local hypertonicity promotes PGE2 signaling in the renal medulla by stimulating cognate receptor and cyclooxygenase 2 expression that likely regulates local hemodynamics and tubular transport.Kidney International (2009) 75, 278–284. doi:10.1038/ki.2008.498 [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]

Volume depletion due to persistent glucosuria-induced osmotic diuresis is a significant problem in uncontrolled diabetes mellitus (DM). Angiotensin II receptor blockers (ARBs), such as candesartan, slow the progression of chronic kidney disease in patients with DM. However, mice with genetic knockout of components of the renin-angiotensin system have urine concentrating defects, suggesting that ARBs may exacerbate the volume depletion. Therefore, the effect of candesartan on UT-A1, UT-A3, NKCC2, and aquaporin-2 (AQP2) protein abundances was determined in control and 3-wk DM rats. Aldosterone levels in control rats (0.36 ± 0.06 nM) and candesartan-treated rats (0.34 ± 0.14 nM) were the same. DM rats had higher aldosterone levels (1.48 ± 0.37 nM) that were decreased by candesartan (0.97 ± 0.26 nM). Western analysis showed that UT-A1 expression was increased in DM rats compared with controls in inner medullary (IM) tip (158 ± 13%) and base (120 ± 25%). UT-A3 abundance was increased in IM tip (123 ± 11%) and base (146 ± 17%) of DM rats vs. controls. UT-A3 was unchanged in candesartan-treated control rats. In candesartan-treated DM rats, UT-A3 increased in IM tip (160 ± 14%) and base (210 ± 19%). Candesartan-treated DM rats had slightly higher AQP2 in IM (46%, P < 0.05) vs. control rats. NKCC2/BSC1 was increased 145 ± 10% in outer medulla of DM vs. control rats. We conclude that candesartan augments compensatory changes in medullary transport proteins, reducing the losses of solute and water during uncontrolled DM. These changes may represent a previously unrecognized beneficial effect of type 1 ARBs in DM. [ABSTRACT FROM AUTHOR]

Summary: Water reabsorption in the kidney represents a critical physiological event in the maintenance of body water homeostasis. This highly regulated process relies largely on vasopressin (VP) action and on the VP-sensitive water channel (AQP2) that is expressed in principal cells of the kidney collecting duct. Defects in the VP signaling pathway and/or in AQP2 cell surface expression can lead to an inappropriate reduction in renal water reabsorption and the development of nephrogenic diabetes insipidus, a disease characterized by polyuria and polydipsia. This review focuses on the major regulatory steps that are involved in AQP2 trafficking and function. Specifically, we begin with a discussion on VP-receptor–independent mechanisms of AQP2 trafficking, with special emphasis on the nitric oxide–cyclic guanosine monophosphate signaling pathway, followed by a review of the mechanisms that govern AQP2 endocytosis and exocytosis. We then discuss emerging data illustrating roles played by the actin cytoskeleton on AQP2 trafficking, and lastly we consider elements that affect AQP2 protein expression in cells. Recent advances in each topic are summarized and are presented in the context of their potential to serve as a basis for the development of novel therapies that may ultimately improve life quality of nephrogenic diabetes insipidus patients. [Copyright &y& Elsevier]

Abstract: Cyclic adenosine monophosphate (cAMP) is a central second messenger controlling a plethora of vital functions. Studies of cAMP dynamics in living cells have revealed markedly inhomogeneous concentrations of the second messenger in different compartments. Moreover, cAMP effectors such as cAMP-dependent protein kinase (PKA) and cAMP-activated GTP-exchange factors (Epacs) are tethered to specific cellular sites. Both the tailoring of cAMP concentrations, and the activities of cAMP-dependent signalling systems at specific cellular locations are prerequisites for most, if not all, cAMP-dependent processes. This review focuses on the role of compartmentalized cAMP signalling in exocytic processes in non-neuronal cells. Particularly, the insertion of aquaporin-2 into the plasma membrane of renal principal cells as an example for a cAMP-dependent exocytic process in a non-secretory cell type, renin secretion from juxtaglomerular cells as a cAMP-triggered exocytosis from an endocrine cell, insulin release from pancreatic β-cells as a Ca2+-mediated and cAMP-potentiated exocytic processes in an endocrine cell, and cAMP- or Ca2+-triggered H+ secretion from gastric parietal cells as an exocytic process in an exocrine cell are discussed. The selected examples of cAMP-regulated exocytic pathways are reviewed with regard to key proteins involved: adenylyl cyclases, phosphodiesterases, PKA, A kinase anchoring proteins (AKAPs) and Epacs. [Copyright &y& Elsevier]

To stimulate renal water reabsorption, vasopressin induces phosphorylation of Aquaporin-2 (AQP2) water channels at S256 and their redistribution from vesicles to the apical membrane, whereas vasopressin removal results in AQP2 ubiquitination at K270 and its internalization to multivesicular bodies (MVB). AQP2-E258K causes dominant nephrogenic diabetes insipidus (NDI), but its subcellular location is unclear, and the molecular reason for its involvement in dominant NDI is unknown. To unravel these, AQP2-E258K was studied in transfected polarized Madin–Darby canine kidney (MDCK) cells. In MDCK cells, AQP2-E258K mainly localized to MVB/lysosomes (Lys). Upon coexpression, wild-type (wt) AQP2 and AQP2-E258K formed multimers, which also localized to MVB/Lys, independent of forskolin stimulation. Orthophosphate labeling revealed that forskolin increased phosphorylation of wt-AQP2 and AQP2-E258K but not AQP2-S256A, indicating that the E258K mutation does not interfere with the AQP2 phosphorylation at S256. In contrast to wt-AQP2 but consistent with the introduced protein kinase C (PKC) consensus site, AQP2-E258K was phosphorylated by phorbol esters. Besides the 29-kDa band, however, an additional band of about 35 kDa was observed for AQP2-E258K only, which represented AQP2-E258K uniquely monoubiquitinated at K228 only. Analysis of several mutants interfering with AQP2-E258K phosphorylation, and/or ubiquitination, however, revealed that the MVB/lysosomal sorting of AQP2-E258K occurred independent of its monoubiquitination or phosphorylation by PKC. Instead, our data reveal that the loss of the E258 in AQP2-E258K is fundamental to its missorting to MVB/Lys and indicate that this amino acid has an important role in the proper structure formation of the C-terminal tail of AQP2. [ABSTRACT FROM AUTHOR]

Abstract: For decades, growth hormone (GH) has been known to influence electrolyte and water handling in humans and animals. However, the molecular mechanisms underlying the GH-induced anti-natriuretic and anti-diuretic effects have remained elusive. This review will examine the existing literature on renal electrolyte and water handling following acute and chronic GH-exposure. Renal responses to GH differ in acute and chronic models. Acute application of GH results in a reduced urinary electrolyte and water excretion, whereas the chronic effects of GH are more diverse, as this state likely represents a complex mixture of primary and secondary actions of GH as well as compensatory mechanisms. During chronic GH-exposure an initial sodium retaining state often occurs, followed by a normalization of the urinary sodium excretion, although extracellular volume expansion still persists. We recently described a possible mechanism by which GH acutely increases renal electrolyte and water reabsorption, by modulation of the kidney specific Na+, K+, 2Cl− co-transporter (NKCC2). The primary aim of this review is to investigate how GH-induced regulation of NKCC2 may be involved in the complex renal changes previously described during acute and chronic GH. We propose, that the GH-induced increase in NKCC2 activity may explain the initial water and sodium retention seen in a number of studies. Moreover, renal changes seen during prolonged GH-exposure may now be seen on the background of the acute stimulation of NKCC2. Additionally, GH also promotes renal acidification, thus influencing renal acid/base handling. The GH-induced renal acidification is partly compatible with changes in NKCC2 activity. Finally, we review the available data on changes in hormonal systems affecting tubular transport during acute and chronic GH-exposure. [Copyright &y& Elsevier]

Hypokalemia causes a significant decrease in the tonicity of the renal medullary interstitium in association with reduced expression of sodium transporters in the distal tubule. We asked whether hypokalemia caused downregulation of the tonicity-responsive enhancer binding protein (TonEBP) transcriptional activator in the renal medulla due to the reduced tonicity. We found that the abundance of TonEBP decreased significantly in the outer and inner medullas of hypokalemic rats. Underlying mechanisms appeared different in the two regions because the abundance of TonEBP mRNA was lower in the outer medulla but unchanged in the inner medulla. Immunohistochemical examination of TonEBP revealed cell type-specific differences. TonEBP expression decreased dramatically in the outer and inner medullary collecting ducts, thick ascending limbs, and interstitial cells. In the descending and ascending thin limbs, TonEBP abundance decreased modestly. In the outer medulla, TonEBP shifted to the cytoplasm in the descending thin limbs. As expected, transcription of aldose reductase, a target of TonEBP, was decreased since the abundance of mRNA and protein was reduced. Downregulation of TonEBP appeared to have also contributed to reduced expression of aquaporin-2 and UT-A urea transporters in the renal medulla. In cultured cells, expression and activity of TonEBP were not affected by reduced potassium concentrations in the medium. These data support the view that medullary tonicity regulates expression and nuclear distribution of TonEBP in the renal medulla in cell type-specific manners. [ABSTRACT FROM AUTHOR]

Release of bilateral ureteral obstruction (BUO) is associated with reduced expression of renal aquaporins (AQPs), polyuria, and impairment of urine-concentrating capacity. Recently, we demonstrated that 24 h of BUO is associated with increased cyclooxygenase (COX)-2 expression in the inner medulla (IM) and that selective COX-2 inhibition prevents downregulation of AQP2. In the present study, we tested the hypothesis that COX-2 activity increases in the postobstructive phase and that this increase in COX-2 activity contributes to polyuria and impaired urine-concentrating capacity. We examined the effect of the selective COX-2 inhibitor parecoxib (5 mg·kg-1·day-1 via osmotic minipumps) on renal functions and protein abundance of AQP2, AQP3, Na-K-2Cl cotransporter type 2 (NKCC2), and Na-K-ATPase 3 days after release of BUO. At 3 days after release of BUO, rats exhibited polyuria, dehydration and urine and IM tissue osmolality were decreased. There were inverse changes of COX-1 and COX-2 in the IM: COX-2 mRNA, protein, and activity increased, while COX-1 mRNA and protein decreased. Parecoxib reduced urine output 1 day after release of BUO, but sodium excretion and glomerular filtration rate were unchanged. Parecoxib normalized urinary PGE2 and PGI2 excretion and attenuated downregulation of AQP2 and AQP3, while phosphorylated AQP2 and NKCC2 remained suppressed. Parecoxib did not improve urine-concentrating capacity in response to 24 h of water deprivation. We conclude that decreased NKCC2 and collapse of the IM osmotic gradient, together with suppressed phosphorylated AQP2, are likely causes for the impaired urine-concentrating capacity and that COX-2 activity is not likely to mediate these changes in the chronic postobstructive phase after ureteral obstruction. [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]

Objective: To study the expression of aquaporin-2 (AQP2) in human endometrium. Design: Prospective clinical study. Setting: Hospital-based unit for gynecology and obstetrics and research laboratories. Patient(s): Healthy women with proven fertility who were divided into four groups according to LH peak. Intervention(s): Endometrial biopsies were obtained from 34 women on cycle days LH+4 to LH+14. Main Outcome Measure(s): Localization of AQP2 in human endometrium during normal cycle using immunohistochemistry, verification of AQP2 expression through detection of AQP2 mRNA in reverse transcriptase–polymerase chain reaction (RT-PCR), detection of pinopodes using scanning electron microscopy, and confirmation of AQP2 on pinopodes in confocal microscopy. Result(s): Immunostaining of AQP2 is present in the luminal and glandular epithelium but not in the stroma. Some vessels stained positive for AQP2. When present, pinopodes stained positive, which was confirmed using confocal microscopy. A significant increase in staining intensity was seen in the glandular and luminal epithelium during the mid and late luteal phases of the cycle. The presence of AQP2 in human endometrium was also confirmed by RT-PCR. Conclusion(s): AQP2 is present in the human endometrium. The expression of AQP2 appears to be cycle dependent and suggests a role for AQP2 in implantation, edema, and/or menstruation. [Copyright &y& Elsevier]

Sodium retention is a hallmark of nephrotic syndrome (NS). Puromycin aminonucleoside (PAN)-induced NS is associated with high aldosterone levels and increased ENaC expression and apical targeting. However, the mechanisms associated with increased apical targeting of ENaC in NS remain undefined, and it is unclear whether this is secondary to high aldosterone levels and whether aldosterone and/or apical ENaC targeting are important for the development of sodium retention. This study aimed at uncovering I) whether aldosterone is essential for sodium retention in PAN-induced NS, 2) whether ENaC expression or apical targeting is secondary to high aldosterone levels, and 3) the role of aldosterone in the dysregulation of sodium trans- porters in NS. Puromycin treatment of adrenalectomized (ADX) rats supplemented with dexamethasone induced sodium retention despite the absence of aldosterone. Immunocytochemical analyses revealed an absence of enhanced apical targeting of ENaC subunits in PAN- treated ADX (ADX-PAN) rats, with distribution of labeling similar to adrenalectomized dexamethasone-treated control rats (ADX). More- over, ENaC subunit abundance was increased in ADX-PAN rats. The abundance of aquaporin-2 was unchanged, whereas apical targeting was enhanced. Key sodium transporters were downregulated as previously observed in nonadrenalectomized puromycin-treated rats (Kim SW, Wang W, Nielsen J, Praetorius J, Kwon TH, Knepper MA, Frøkiær J, and Nielsen S. Am J Physiol Renal Physiol 286: F922- F935, 2004), whereas the global expression of the α1-subunit of the Na-K-ATPase was unchanged. In conclusion, PAN treatment in the absence of aldosterone induced sodium retention, increased ENaC expression, but did not change the subcellular distribution of ENaC. This indicates that the previously observed enhanced apical targeting of ENaC in PAN-induced NS (Kim SW, Wang W, Nielsen J, Praetorius J, Kwon TH, Knepper MA, Frøkiær J, and Nielsen S. Am J Physiol Renal Physiol 286: F922-F935, 2004) is caused by aldosterone and that development of sodium retention can occur in the absence of aldosterone in NS. [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]