Effect of mineralocorticoid deficiency on ion and urea transporters and aquaporin water channels in the rat

Mineralocorticoid deficiency is associated with impaired urinary concentration and dilution. The present investigation was undertaken to determine the effects of selective mineralocorticoid deficiency on renal sodium and urea transporters and aquaporin water channels and whether these perturbations can be reversed by maintenance of extracellular fluid volume. Mineralocorticoid deficiency was induced by bilateral adrenalectomies with glucocorticoid replacement. Mineralocorticoid deficient rats receiving plain drinking water (MDW) were compared with mineralocorticoid deficient rats receiving saline-drinking water (MDS) in order to maintain extracellular fluid volume, and with controls (CTL). In MDW rats, there was a significant decrease in renal outer medulla Na-K-2Cl co-transporter and outer medulla Na-K-ATPase as well as an increase in inner medulla aquaporins 2 and 3. There were no significant changes in aquaporin-1, aquaporin-4, or urea transporters. These alterations were reversed with maintenance of extracellular fluid volume in MDS rats. Our findings indicate that mineralocorticoid deficiency in the rat is associated with alterations in factors involved in the countercurrent concentrating mechanism (Na-K-2Cl, Na-K-ATPase) and osmotic water equilibration in the collecting duct (AQP2, AQP3). Maintenance of sodium balance and extracellular fluid volume is associated with normalization of these perturbations.     

Altered expression of renal aquaporins and Na(+) transporters in rats treated with L-type calcium blocker

Nifedipine, a calcium antagonist, has diuretic and natriuretic properties. However, the molecular mechanisms by which these effects are produced are poorly understood. We examined kidney abundance of aquaporins (AQP1, AQP2, and AQP3) and major sodium transporters [type 3 Na/H exchanger (NHE-3); type 2 Na-Pi cotransporter (NaPi-2); Na-K-ATPase; type 1 bumetanide-sensitive cotransporter (BSC-1); and thiazide-sensitive Na-Cl cotransporter (TSC)] as well as inner medullary abundance of AQP2, phosphorylated-AQP2 (p-AQP2), AQP3, and calcium-sensing receptor (CaR). Rats treated with nifedipine orally (700 mg/kg) for 19 days had a significant increase in urine output, whereas urinary osmolality and solute-free water reabsorption were markedly reduced. Consistent with this, immunoblotting revealed a significant decrease in the abundance of whole kidney AQP2 (47 +/- 7% of control rats, P < 0.05) and in inner medullary AQP2 (60 +/- 7%) as well as in p-AQP2 abundance (17 +/- 6%) in nifedipine-treated rats. In contrast, whole kidney AQP3 abundance was significantly increased (219 +/- 28%). Of potential importance in modulating AQP2 levels, the abundance of CaR in the inner medulla was significantly increased (295 +/- 25%) in nifedipine-treated rats. Nifedipine treatment was also associated with increased urinary sodium excretion. Consistent with this, semiquantitative immunoblotting revealed significant reductions in the abundance of proximal tubule Na(+) transporters: NHE-3 (3 +/- 1%), NaPi-2 (53 +/- 12%), and Na-K-ATPase (74 +/- 5%). In contrast, the abundance of the distal tubule Na-Cl cotransporter (TSC) was markedly increased (240 +/- 29%), whereas BSC-1 in the thick ascending limb was not altered. In conclusion, 1) increased urine output and reduced urinary concentration in nifedipine-treated-rats may, in part, be due to downregulation of AQP2 and p-AQP2 levels; 2) CaR might be involved in the regulation of water reabsorption in the inner medulla collecting duct; 3) reduced expression of proximal tubule Na(+) transporters (NHE-3, NaPi-2, and Na, K-ATPase) may be involved in the increased urinary sodium excretion; and 4) increase in TSC expression may occur as a compensatory mechanism.     

Aquaporins of plasma membranes of epithelial cells

The early 90s have brought us a discovery of a new class of membrane proteins--aquaporins with a function of transmembrane water channels. Being genetically closed proteins aquaporins are members of a large family of channel-forming proteins called MIPs (major intrinsic proteins). All aquaporins, except AQP4, are mercury-sensitive. Many aquaporins have been cloned and identified. Polyclonal antibodies grown against some of them promoted numerous studies of aquaporin localization and distribution in animal and plant tissues. Up to the present, 10 and 2 aquaporins have been described in mammalian and amphibian epithelial tissues, respectively. One of described aquaporins, AQP2, whose localization is confined to kidney collecting duct principal cells, has been found to be a hormone-depending water channel. The insertion of apical vesicles bearing AQP2 was shown to be regulated by vasopressin, meanwhile all other aquaporins are inserted into the plasma membrane constitutively. There is a vast evidence showing that the integrity of microtubules is necessary for both pathways of aquaporin insertion. AQP2 is important for normal kidney functioning and AQP2 mutations cause water-balance disorders. On the contrary, the AQP1 mutations are not accompanied by any evident clinical pathology. This review is focused on a discussion of the data so far available on aquaporin distribution in different animal tissues.     

Water channels in platelet volume regulation

The regulation of platelet volume significantly affects its function. Because water is the major molecule in cells and its active transport via water channels called aquaporins (AQPs) have been implicated in cellular and organelle volume regulation, the presence of water channels in platelets and their potential role in platelet volume regulation was investigated. G-protein-mediated AQP regulation in secretory vesicle swelling has previously been reported in neurons and in pancreatic acinar cells. Mercuric chloride has been demonstrated to inhibit most AQPs except AQP6, which is stimulated by the compound. Exposure of platelets to HgCl(2)-induced swelling in a dose-dependent manner, suggesting the presence of AQP6 in platelets. Immunoblot analysis of platelet protein confirmed the presence of AQP6, and also of G(αo), G(αi-1) and G(αi-3) proteins. Results from this study demonstrate for the first time that in platelets AQP6 is involved in cell volume regulation via a G-protein-mediated pathway.     

Clopidogrel attenuates lithium-induced alterations in renal water and sodium channels/transporters in mice

Lithium (Li) administration causes deranged expression and function of renal aquaporins and sodium channels/transporters resulting in nephrogenic diabetes insipidus (NDI). Extracellular nucleotides (ATP/ADP/UTP), via P2 receptors, regulate these transport functions. We tested whether clopidogrel bisulfate (CLPD), an antagonist of ADP-activated P2Y₁₂ receptor, would affect Li-induced alterations in renal aquaporins and sodium channels/transporters. Adult mice were treated for 14 days with CLPD and/or Li and euthanized. Urine and kidneys were collected for analysis. When administered with Li, CLPD ameliorated polyuria, attenuated the rise in urine prostaglandin E2 (PGE2), and resulted in significantly higher urinary arginine vasopressin (AVP) and aldosterone levels as compared to Li treatment alone. However, urine sodium excretion remained elevated. Semi-quantitative immunoblotting revealed that CLPD alone increased renal aquaporin 2 (AQP2), Na-K-2Cl cotransporter (NKCC2), Na-Cl cotransporter (NCC), and the subunits of the epithelial Na channel (ENaC) in medulla by 25–130 %. When combined with Li, CLPD prevented downregulation of AQP2, Na-K-ATPase, and NKCC2 but was less effective against downregulation of cortical α- or γ-ENaC (70 kDa band). Thus, CLPD primarily attenuated Li-induced downregulation of proteins involved in water conservation (AVP-sensitive), with modest effects on aldosterone-sensitive proteins potentially explaining sustained natriuresis. Confocal immunofluorescence microscopy revealed strong labeling for P2Y₁₂-R in proximal tubule brush border and blood vessels in the cortex and less intense labeling in medullary thick ascending limb and the collecting ducts. Therefore, there is the potential for CLPD to be directly acting at the tubule sites to mediate these effects. In conclusion, P2Y₁₂-R may represent a novel therapeutic target for Li-induced NDI.     

Aquaporin-3 functions as a glycerol transporter in mammalian skin

The AQPs (aquaporins) are a family of homologous water transporting proteins expressed in many mammalian epithelial, endothelial and other cell types. Phenotype analysis of mice lacking individual AQPs has been informative in elucidating their role in mammalian physiology. For example, phenotype analysis has indicated an important role of AQPs in the renal urinary concentrating mechanism (AQP1-AQP4), brain water balance and neural signal transduction (AQP4), exocrine gland secretion (AQP5) and ocular fluid balance (AQP1, AQP5). In skin, the aquaglyceroporin AQP3 is expressed in the basal layer of epidermal keratinocytes. Mice deficient in AQP3 have dry skin with reduced SC (stratum corneum) hydration, decreased elasticity and impaired biosynthesis. Mechanistic analysis of the altered skin phenotype in AQP3 deficiency suggested that the glycerol rather than the water transporting function of AQP3 is important in skin physiology. The glycerol content of SC and epidermis of AQP3 deficient mice is reduced, whereas that of dermis and serum is normal. The dry, relatively inelastic skin in AQP3 null mice is probably related to the humectant properties of glycerol, and the impaired SC repair to impaired epidermal biosynthetic function. The key role of AQP3 in epidermal physiology might be exploited in the development of improved cosmetics and new therapies for skin diseases associated with altered skin water content.     

Impaired hearing in mice lacking aquaporin-4 water channels.

A role for aquaporins (AQPs) in hearing has been suggested from the specific expression of aquaporins in inner ear and the need for precise volume regulation in epithelial cells involved in acoustic signal transduction. Using mice deficient in selected aquaporins as controls, we localized AQP1 in fibrocytes in the spiral ligament and AQP4 in supporting epithelial cells (Hensen's, Claudius, and inner sulcus cells) in the organ of Corti. To determine whether aquaporins play a role in hearing, auditory brain stem response (ABR) thresholds were compared in wild-type mice and transgenic null mice lacking (individually) AQP1, AQP3, AQP4, and AQP5. In 4-5-week-old mice in a CD1 genetic background, ABR thresholds in response to a click stimulus were remarkably increased by >12 db in AQP4 null mice compared with wild-type mice (p < 0.001), whereas ABR thresholds were not affected by AQP1, AQP3, or AQP5 deletion. In a C57/bl6 background, nearly all AQP4 null mice were deaf, whereas ABRs could be elicited in wild-type controls. ABRs in AQP4 null CD1 mice measured in response to tone bursts (4-20 kHz) indicated a frequency-independent hearing deficit. Light microscopy showed no differences in cochlear morphology of wild-type versus AQP4 null mice. These results provide the first direct evidence that an aquaporin water channel plays a role in hearing. AQP4 may facilitate rapid osmotic equilibration in epithelial cells in the organ of Corti, which are subject to large K(+) fluxes during mechano-electric signal transduction.     

Aquaporin-mediated fluid regulation in the inner ear

1. The sensory functions of the inner ear (hearing and balance) critically depend on the precise regulation of two fluid compartments of highly desparate ion composition, i.e., the endolymph and the perilymph.2. The parameters volume, ion composition, and pH need to be held athomeostasis irrespective of the hydration status of the total organism.3. Specific cellular water channels, aquaporins, have been shown to be essential for the fluid regulation of several organs, e.g., kidney, lung, and brain.4. Because of functional similarities of water regulation in the kidney and inner ear this review initially summarizes some aquaporin functions in the kidney and then focuses on 6 out of 11 mammalian aquaporins that are present in the inner ear (AQP1-6).5. Their potential role in the inner ear fluid control will be discussed on the basis of the respective expression patterns and individual pore properties.6. Further, a working model is presented of how the endolymphatic sac may contribute to inner ear fluid regulation.     

Saccharomyces cerivisiae as a model system for kidney disease: what can yeast tell us about renal function?

Ion channels, solute transporters, aquaporins, and factors required for signal transduction are vital for kidney function. Because mutations in these proteins or in associated regulatory factors can lead to disease, an investigation into their biogenesis, activities, and interplay with other proteins is essential. To this end, the yeast, Saccharomyces cerevisiae, represents a powerful experimental system. Proteins expressed in yeast include the following: 1) ion channels, including the epithelial sodium channel, members of the inward rectifying potassium channel family, and cystic fibrosis transmembrane conductance regulator; 2) plasma membrane transporters, such as the Na(+)-K(+)-ATPase, the Na(+)-phosphate cotransporter, and the Na(+)-H(+) ATPase; 3) aquaporins 1-4; and 4) proteins such as serum/glucocorticoid-induced kinase 1, phosphoinositide-dependent kinase 1, Rh glycoprotein kidney, and trehalase. The variety of proteins expressed and studied emphasizes the versatility of yeast, and, because of the many available tools in this organism, results can be obtained rapidly and economically. In most cases, data gathered using yeast have been substantiated in higher cell types. These attributes validate yeast as a model system to explore renal physiology and suggest that research initiated using this system may lead to novel therapeutics.     

Rapid aquaporin translocation regulates cellular water flow: mechanism of hypotonicity-induced subcellular localization of aquaporin 1 water channel

The control of cellular water flow is mediated by the aquaporin (AQP) family of membrane proteins. The structural features of the family and the mechanism of selective water passage through the AQP pore are established, but there remains a gap in our knowledge of how water transport is regulated. Two broad possibilities exist. One is controlling the passage of water through the AQP pore, but this only has been observed as a phenomenon in some plant and microbial AQPs. An alternative is controlling the number of AQPs in the cell membrane. Here, we describe a novel pathway in mammalian cells whereby a hypotonic stimulus directly induces intracellular calcium elevations through transient receptor potential channels, which trigger AQP1 translocation. This translocation, which has a direct role in cell volume regulation, occurs within 30 s and is dependent on calmodulin activation and phosphorylation of AQP1 at two threonine residues by protein kinase C. This direct mechanism provides a rationale for the changes in water transport that are required in response to constantly changing local cellular water availability. Moreover, because calcium is a pluripotent and ubiquitous second messenger in biological systems, the discovery of its role in the regulation of AQP translocation has ramifications for diverse physiological and pathophysiological processes, as well as providing an explanation for the rapid regulation of water flow that is necessary for cell homeostasis.     

Aquaporin water channels: molecular mechanisms for human diseases

Although water is the major component of all biological fluids, the molecular pathways for water transport across cell membranes eluded identification until the discovery of the aquaporin family of water channels. The atomic structure of mammalian AQP1 illustrates how this family of proteins is freely permeated by water but not protons (hydronium ions, H3O+). Definition of the subcellular sites of expression predicted their physiological functions and potential clinical disorders. Analysis of several human disease states has confirmed that aquaporins are involved in multiple different illnesses including abnormalities of kidney function, loss of vision, onset of brain edema, starvation, and arsenic toxicity.     

Comparative functional analysis of aquaporins/glyceroporins in mammals and anurans

Maintenance of fluid homeostasis is critical to establishing and maintaining normal physiology. The landmark discovery of membrane water channels (aquaporins; AQPs) ushered in a new area in osmoregulatory biology that has drawn from and contributed to diverse branches of biology, from molecular biology and genomics to systems biology and evolution, and from microbial and plant biology to animal and translational physiology. As a result, the study of AQPs provides a unique and integrated backdrop for exploring the relationships between genes and genome systems, the regulation of gene expression, and the physiologic consequences of genetic variation. The wide species distribution of AQP family members and the evolutionary conservation of the family indicate that the control of membrane water flux is a critical biological process. AQP function and regulation is proving to be central to many of the pathways involved in individual physiologic systems in both mammals and anurans. In mammals, AQPs are essential to normal secretory and absorptive functions of the eye, lung, salivary gland, sweat glands, gastrointestinal tract, and kidney. In urinary, respiratory, and gastrointestinal systems, AQPs are required for proper urine concentration, fluid reabsorption, and glandular secretions. In anurans, AQPs are important in mediating physiologic responses to changes in the external environment, including those that occur during metamorphosis and adaptation from an aquatic to terrestrial environment and thermal acclimation in anticipation of freezing. Therefore, an understanding of AQP function and regulation is an important aspect of an integrated approach to basic biological research.     

Altered regulation of renal sodium transporters and natriuretic peptide system in DOCA–salt hypertensive rats

Although deoxycorticosterone acetate (DOCA)–salt hypertension is a volume dependent model of hypertension, it shows polyuria and natriuresis. It is expected that dysregulation of aquaporin water channels (AQPs) and sodium transporters associated with natriuretic peptide (NP) system may play an escape role in sodium retaining state. One week after left unilateral nephrectomy, rats were subcutaneously implanted with silastic DOCA (200 mg/kg) strips. Physiologic saline was supplied as a drinking water to all animals. 4 weeks after operation, the protein expression of AQPs, sodium transporters, and endopeptidase (NEP) was determined in the kidneys by semiquantitative immunoblotting and immunohistochemistry. The mRNA expression of NP system was determined by real-time polymerase chain reaction. The amount of urinary ANP excretion was measured by radioimmunoassay. In DOCA–salt rats, urine osmolality was decreased while urinary excretion of sodium was increased. The expression of AQP1-3 as well as that of α-1 subunit of Na,K–ATPase, NHE3, NKCC2 and NCC was decreased in the kidney. The mRNA expression of ANP, brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP) was increased in the kidney. The expression of NEP was decreased, and urinary ANP excretion was increased. Downregulation of AQPs and sodium transporters may contribute to mineralocorticoid escape in DOCA–salt hypertension. Increased expression of natriuretic peptides associated with downregulation of NEP may play a role in natriuresis.     

Expression and localization of aquaporins in rat gastrointestinal tract

A family of water-selective channels, aquaporins (AQP), has beendemonstrated in various organs and tissues. However, the localizationand expression of the AQP family members in the gastrointestinal tracthave not been entirely elucidated. This study aimed to demonstrate theexpression and distribution of several types of the AQP family and tospeculate on their role in water transport in the rat gastrointestinal tract. By RNase protection assay, expression of AQP1-5 and AQP8 was examined in various portions through the gastrointestinal tract.AQP1 and AQP3 mRNAs were diffusely expressed from esophagus to colon,and their expression was relatively intense in the small intestine andcolon. In contrast, AQP4 mRNA was selectively expressed in the stomachand small intestine and AQP8 mRNA in the jejunum and colon.Immunohistochemistry and in situ hybridization demonstrated cellularlocalization of these AQP in these portions. AQP1 was localized onendothelial cells of lymphatic vessels in the submucosa and laminapropria throughout the gastrointestinal tract. AQP3 was detected on thecircumferential plasma membranes of stratified squamous epithelialcells in the esophagus and basolateral membranes of cardiac glandepithelia in the lower stomach and of surface columnar epithelia in thecolon. However, AQP3 was not apparently detected in the smallintestine. AQP4 was present on the basolateral membrane of the parietalcells in the lower stomach and selectively in the basolateral membranesof deep intestinal gland cells in the small intestine. AQP8 mRNAexpression was demonstrated in the absorptive columnar epithelial cellsof the jejunum and colon by in situ hybridization. These findings mayindicate that water crosses the epithelial layer through these waterchannels, suggesting a possible role of the transcellular route forwater intake or outlet in the gastrointestinal tract.     

Functional interaction between AQP2 and TRPV4 in renal cells

We have previously demonstrated that renal cortical collecting duct cells (RCCD(1)), responded to hypotonic stress with a rapid activation of regulatory volume decrease (RVD) mechanisms. This process requires the presence of the water channel AQP2 and calcium influx, opening the question about the molecular identity of this calcium entry path. Since the calcium permeable nonselective cation channel TRPV4 plays a crucial role in the response to mechanical and osmotic perturbations in a wide range of cell types, the aim of this work was to test the hypothesis that the increase in intracellular calcium concentration and the subsequent rapid RVD, only observed in the presence of AQP2, could be due to a specific activation of TRPV4. We evaluated the expression and function of TRPV4 channels and their contribution to RVD in WT-RCCD(1) (not expressing aquaporins) and in AQP2-RCCD(1) (transfected with AQP2) cells. Our results demonstrated that both cell lines endogenously express functional TRPV4, however, a large activation of the channel by hypotonicity only occurs in cells that express AQP2. Blocking of TRPV4 by ruthenium red abolished calcium influx as well as RVD, identifying TRPV4 as a necessary component in volume regulation. Even more, this process is dependent on the translocation of TRPV4 to the plasma membrane. Our data provide evidence of a novel association between TRPV4 and AQP2 that is involved in the activation of TRPV4 by hypotonicity and regulation of cellular response to the osmotic stress, suggesting that both proteins are assembled in a signaling complex that responds to anisosmotic conditions.     

Clinical update on renal aquaporins

Following the discovery of the aquaporin-1 water channel over a decade ago, molecular techniques have been developed to examine the role of renal aquaporin water channels under numerous physiological and pathological conditions. The present article reviews current knowledge regarding the function and dysfunction of renal aquaporins in disorders of water metabolism.     

Active water selective channels in the stomach: investigation of aquaporins after ethanol and capsaicin treatment in rats

Recent studies discovered the existence of aquaporins (AQP), suggesting their roles in the active, ATP dependent water secretion or absorption. Our recent development of the monoclonal antibody family against aquaporins (Type 1 and 4) allowed us a good opportunity to investigate the mechanism of the gastric mucosal edema in a rat model. THE AIM OF OUR STUDY was to evaluate the changes in the tissue level of aquaporins (AQP1 and AQP4) after ethanol and capsaicin treatment in rat stomach. MATERIALS AND METHODS: the experiments were carried out on Sprague-Dawley rats weighing 150-200 g. The animals were fasted for 24 h, after the 1 ml of ethanol (50% v/v) or capsaicin (2 mg/ml) was given intragastrically. Rats were sacrificed after 5, 30, 60, 120 and 240 min, the tissue level of AQP1 and AQP4 was investigated immunoserologically by ELISA and dot-blot methods using our monoclonal antibodies. The location of these aquaporins in the gastric tissue was demonstrated by immunohistochemistry. RESULTS: (1) in ethanol-treated stomach, both AQP1 and AQP4 increased after 5 min simultaneously with gastritis, then decreased dramatically depending on time. (2) In the capsaicin-treated group there were no changes in the tissue level of aquaporins in the first hour. After 60 min both AQP1 and AQP4 increased in the stomach without any macroscopically detectable changes, then decreased depending on time. (3) The immunohistochemical investigations using our monoclonal antibodies seem to support our present quantitative results. CONCLUSION: chemically induced gastric mucosal lesions are started by an extended edema. In the induction of the edema and the subsequent gastric injury, aquaporins (both AQP1 and AQP4) play an important role in the maintenance of mucosal integrity.     

Potential role of the glial water channel aquaporin-4 in epilepsy

Recent studies have implicated glial cells in novel physiological roles in the CNS, such as modulation of synaptic transmission, so it is possible that glial cells might have a functional role in the hyperexcitability that is characteristic of epilepsy. Indeed, alterations in distinct astrocyte membrane channels, receptors and transporters have all been associated with the epileptic state. This paper focuses on the potential roles of the glial water channel aquaporin-4 (AQP4) in modulating brain excitability and in epilepsy. We review studies of seizure phenotypes, K(+) homeostasis and extracellular space physiology of mice that lack AQP4 (AQP4(-/-) mice) and discuss the human studies demonstrating alterations of AQP4 in specimens of human epilepsy tissue. We conclude with new studies of AQP4 regulation by seizures and discuss its potential role in the development of epilepsy (epileptogenesis). Although many questions remain unanswered, the available data indicate that AQP4 and its molecular partners might represent important new therapeutic targets.