Identification of adenylyl cyclase isoforms mediating parathyroid hormone- and calcitonin-stimulated cyclic AMP accumulation in distal tubule cells.

BACKGROUND: The distal convoluted tubule (DCT) is an important nephron site for parathyroid hormone (PTH) and calcitonin regulation of urinary divalent cation excretion. These hormones exert their effects on the DCT in substantial part through activation of adenylyl cyclase (AC); however, it is unknown which AC isoforms are involved. METHODS: To examine this, two mouse DCT cell lines were studied: 209 and D1 cells. AC isoform mRNA expression was analyzed by real-time PCR. Cyclic AMP was measured using enzyme immunoassay. RESULTS: Calcitonin, but not PTH, stimulated cAMP accumulation in 209 cells, while PTH, but not calcitonin, increased cAMP content in D1 cells. Both cell types expressed AC3, AC4, AC6, AC7, and AC9 mRNA; in both cell types, AC6 mRNA was most abundant, followed by AC9, then AC3 and AC7, with relatively very small amounts of AC4 mRNA. Microdissected mouse DCT had a similar pattern of AC isoform mRNA expression although AC5 mRNA was detected. Individual siRNA knockdown of AC6 and AC9 reduced calcitonin-stimulated cAMP accumulation in 209 cells and PTH-induced cAMP levels in D1 cells. Knockdown of AC3 had no effect on hormonal augmentation of cAMP in either cell line. Surprisingly, knockdown of AC7 increased calcitonin-induced cAMP accumulation in 209 cells as well as PTH-stimulated cAMP content in D1 cells. CONCLUSIONS: Taken together, these findings indicate that AC6 and AC9 mediate calcitonin- and PTH-stimulated cAMP accumulation in DCT cells, while activation of AC7 may paradoxically reduce the stimulatory effects of PTH and calcitonin on cultured DCT cAMP levels.

Nephron-specific deletion of the prorenin receptor causes a urine concentration defect.

The prorenin receptor (PRR), a recently discovered component of the renin-angiotensin system, is expressed in the nephron in general and the collecting duct in particular. However, the physiological significance of nephron PRR remains unclear, partly due to developmental abnormalities associated with global or renal-specific PRR gene knockout (KO). Therefore, we developed mice with inducible nephron-wide PRR deletion using Pax8-reverse tetracycline transactivator and LC-1 transgenes and loxP flanked PRR alleles such that ablation of PRR occurs in adulthood, after induction with doxycycline. Nephron-specific PRR KO mice have normal survival to ∼1 yr of age and no renal histological defects. Compared with control mice, PRR KO mice had 65% lower medullary PRR mRNA and protein levels and markedly diminished renal PRR immunofluorescence. During both normal water intake and mild water restriction, PRR KO mice had significantly lower urine osmolality, higher water intake, and higher urine volume compared with control mice. No differences were seen in urine vasopressin excretion, urine Na(+) and K(+) excretion, plasma Na(+), or plasma osmolality between the two groups. However, PRR KO mice had reduced medullary aquaporin-2 levels and arginine vasopressin-stimulated cAMP accumulation in the isolated renal medulla compared with control mice. Taken together, these results suggest nephron PRR can potentially modulate renal water excretion.

Adenylyl cyclase 6 deficiency ameliorates polycystic kidney disease.

cAMP is an important mediator of cystogenesis in polycystic kidney disease (PKD). Several adenylyl cyclase (AC) isoforms could mediate cAMP accumulation in PKD, and identification of a specific pathogenic AC isoform is of therapeutic interest. We investigated the role of AC6 in a mouse model of PKD that is homozygous for the loxP-flanked PKD1 gene and heterozygous for an aquaporin-2-Cre recombinase transgene to achieve collecting duct-specific gene targeting. Collecting duct-specific knockout of polycystin-1 caused massive renal cyst formation, kidney enlargement, and severe kidney failure, with a mean survival time of 2 months. In contrast, coincident collecting duct-specific knockout of polycystin-1 and AC6 (also homozygous for the floxed ADCY6 gene) markedly decreased kidney size and cystogenesis, improved renal function, reduced activation of the B-Raf/ERK/MEK pathway, and greatly increased survival. Absence of collecting duct AC6 did not alter urinary cAMP excretion or kidney cAMP concentration. In conclusion, AC6 is a key mediator of cyst formation and renal injury in a model of PKD.

Post-Golgi supramolecular assembly of aquaporin-4 in orthogonal arrays.

The supramolecular assembly of aquaporin-4 (AQP4) in orthogonal arrays of particles (OAPs) involves N-terminus interactions of the M23-AQP4 isoform. We found AQP4 OAPs in cell plasma membranes but not in endoplasmic reticulum (ER) or Golgi, as shown by: (i) native gel electrophoresis of brain and AQP4-transfected cells, (ii) photobleaching recovery of green fluorescent protein-AQP4 chimeras in live cells and (iii) freeze-fracture electron microscopy (FFEM). We found that AQP4 OAP formation in plasma membranes, but not in the Golgi, was not related to AQP4 density, pH, membrane lipid composition, C-terminal PDZ domain interactions or α-syntrophin expression. Remarkably, however, fusion of AQP4-containing Golgi vesicles with (AQP4-free) plasma membrane vesicles produced OAPs, suggesting the involvement of plasma membrane factor(s) in AQP4 OAP formation. In investigating additional possible determinants of OAP assembly we discovered membrane curvature-dependent OAP assembly, in which OAPs were disrupted by extrusion of plasma membrane vesicles to ∼110 nm diameter, but not to ∼220 nm diameter. We conclude that AQP4 supramolecular assembly in OAPs is a post-Golgi phenomenon involving plasma membrane-specific factor(s). Post-Golgi and membrane curvature-dependent OAP assembly may be important for vesicle transport of AQP4 in the secretory pathway and AQP4-facilitated astrocyte migration, and suggests a novel therapeutic approach for neuromyelitis optica.

Lack of an effect of collecting duct-specific deletion of adenylyl cyclase 3 on renal Na+ and water excretion or arterial pressure.

cAMP is a key mediator of connecting tubule and collecting duct (CD) Na(+) and water reabsorption. Studies performed in vitro have suggested that CD adenylyl cyclase (AC)3 partly mediates the actions of vasopressin; however, the physiological role of CD AC3 has not been determined. To assess this, mice were developed with CD-specific disruption of AC3 [CD AC3 knockout (KO)]. Inner medullary CDs from these mice exhibited 100% target gene recombination and had reduced ANG II- but not vasopressin-induced cAMP accumulation. However, there were no differences in urine volume, urinary urea excretion, or urine osmolality between KO and control mice during normal water intake or varying degrees of water restriction in the presence or absence of chronic vasopressin administration. There were no differences between CD AC3 KO and control mice in arterial pressure or urinary Na(+) or K(+) excretion during a normal or high-salt diet, whereas plasma renin and vasopressin concentrations were similar between the two genotypes. Patch-clamp analysis of split-open cortical CDs revealed no difference in epithelial Na(+) channel activity in the presence or absence of vasopressin. Compensatory changes in AC6 were not responsible for the lack of a renal phenotype in CD AC3 KO mice since combined CD AC3/AC6 KO mice had similar arterial pressure and renal Na(+) and water handling compared with CD AC6 KO mice. In summary, these data do not support a significant role for CD AC3 in the regulation of renal Na(+) and water excretion in general or vasopressin regulation of CD function in particular.

Aquaporin 2 promotes cell migration and epithelial morphogenesis.

The aquaporin 2 (AQP2) water channel, expressed in kidney collecting ducts, contributes critically to water homeostasis in mammals. Animals lacking or having significantly reduced levels of AQP2, however, have not only urinary concentrating abnormalities but also renal tubular defects that lead to neonatal mortality from renal failure. Here, we show that AQP2 is not only a water channel but also an integrin-binding membrane protein that promotes cell migration and epithelial morphogenesis. AQP2 expression modulates the trafficking and internalization of integrin ß1, facilitating its turnover at focal adhesions. In vitro, disturbing the interaction between AQP2 and integrin ß1 by mutating the RGD motif led to reduced endocytosis, retention of integrin ß1 at the cell surface, and defective cell migration and tubulogenesis. Similarly, in vivo, AQP2-null mice exhibited significant retention of integrin ß1 at the basolateral membrane and had tubular abnormalities. In summary, these data suggest that the water channel AQP2 interacts with integrins to promote renal epithelial cell migration, contributing to the structural and functional integrity of the mammalian kidney.

Aquaporin 9 expression in the developing rat epididymis is modulated by steroid hormones.

Fluid and solute transport across the epithelium of the male excurrent duct is important for sperm maturation and storage. Aquaporin 9 (AQP9), which allows permeation of water and neutral solutes, is abundant throughout the male reproductive tract, where it is expressed at the apical membrane of rat epididymal principal cells as early as at 1 week of age. We evaluated the effect of neonatal exposure to: 1) a GNRH antagonist (GNRHa); 2) diethylstilbestrol (DES); 3) ethinyl estradiol (EE); 4) DES plus testosterone (DES+TE); and 5) the anti-androgen flutamide on AQP9 expression in the epididymis of peripubertal rats. Control groups received the vehicle alone. In 25-day-old rats, quantification of the mean pixel intensity of immunofluorescence-stained sections showed a significant decrease in AQP9 staining in the apical membrane of epididymal principal cells after treatments with GNRHa, DES, or flutamide, compared to controls. These results were confirmed by western blotting. While EE induced a marked decrease in AQP9 levels by western blotting, the decrease in AQP9-associated fluorescence was not significant compared to controls. DES+TE-treated rats showed levels of AQP9 protein similar to controls, indicating maintenance of AQP9 expression by testosterone treatment in the presence of DES. Our data show that expression of AQP9 in the developing rat epididymis is downregulated by neonatal DES, GNRHa, EE, and flutamide, and that the effects mediated by estrogens can be prevented by testosterone administration.

Osmolar regulation of endothelin-1 production by the inner medullary collecting duct.

AIMS: Endothelin-1 (ET-1) is an autocrine inhibitor of collecting duct (CD) Na(+) and water reabsorption. CD ET-1 production is increased by a high salt diet and is important in promoting a natriuretic response. The mechanisms by which a high salt diet enhances CD ET-1 are being uncovered. In particular, elevated tubule fluid flow, as occurs in salt loading, enhances CD ET-1 synthesis. Tubule fluid solute content and interstitial osmolality can also be altered by a high salt diet, however their effect on CD ET-1 alone, or in combination with flow, is poorly understood. MAIN METHODS: ET-1 mRNA production by a mouse inner medullary CD cell line (mIMCD3) in response to changing flow and/or osmolality was assessed. KEY FINDINGS: Flow or hyperosmolality (using NaCl, mannitol or urea) individually caused an ~2-fold increase in ET-1 mRNA, while flow and hyperosmolality together increased ET-1 mRNA by ~14 fold. The hyperosmolality effect alone and the synergistic effect of flow + hyperosmolality was inhibited by chelation of intracellular Ca(2+), however were not altered by blockade of downstream Ca(2+)-signaling pathways (calcineurin or NFATc), inhibition of cellular Ca(2+) entry channels (purinergic receptors or polycystin-2), or blockade of the epithelial Na(+) channel. Inhibition of NFAT5 with rottlerin or NFAT5 siRNA greatly reduced the stimulatory effect of osmolality alone and osmolality + flow on mIMCD3 ET-1 mRNA levels. SIGNIFICANCE: Both flow and osmolality individually and synergistically stimulate mIMCD3 ET-1 mRNA content. These findings may be relevant to explaining high salt diet induction of CD ET-1 production.

Rapid Identification of Novel Inhibitors of the Human Aquaporin-1 Water Channel.

Aquaporins (AQPs) are a family of membrane proteins that function as channels facilitating water transport in response to osmotic gradients. These play critical roles in several normal physiological and pathological states and are targets for drug discovery. Selective inhibition of the AQP1 water channel may provide a new approach for the treatment of several disorders including ocular hypertension/glaucoma, congestive heart failure, brain swelling associated with a stroke, corneal and macular edema, pulmonary edema, and otic disorders such as hearing loss and vertigo. We developed a high-throughput assay to screen a library of compounds as potential AQP1 modulators by monitoring the fluorescence dequenching of entrapped calcein in a confluent layer of AQP1-overexpressing CHO cells that were exposed to a hypotonic shock. Promising candidates were tested in a Xenopus oocyte-swelling assay, which confirmed the identification of two lead classes of compounds belonging to aromatic sulfonamides and dihydrobenzofurans with IC50 s in the low micromolar range. These selected compounds directly inhibited water transport in AQP1-enriched stripped erythrocyte ghosts and in proteoliposomes reconstituted with purified AQP1. Validation of these lead compounds, by the three independent assays, establishes a set of attractive AQP1 blockers for developing novel, small-molecule functional modulators of human AQP1.

Lack of an effect of nephron-specific deletion of adenylyl cyclase 3 on renal sodium and water excretion or arterial pressure.

Adenylyl cyclase (AC)-stimulated cAMP plays a key role in modulating transport and channel activity along the nephron. However, the role of individual adenylyl cyclase isoforms in such regulation is largely unknown. Since adenylyl cyclase 3 (AC3) is expressed throughout nephron, we investigated its role in the physiologic regulation of renal Na(+) and water transport. Mice were generated with inducible nephron knockout of AC3 (AC3 KO) by breeding mice with loxP-flanked critical exons in the Adcy3 gene with mice expressing Pax8-rtTA and LC-1 transgenes. After doxycycline treatment at 1 month of age, nephron AC3 KO mice had 100% Adcy3 gene recombination in all renal tubule segments, but not in glomeruli. Sodium intake, urinary Na(+) excretion, glomerular filtration rate, and blood pressure were similar between nephron KO mice and the controls during normal, high, and low Na(+) diets. Plasma renin concentration was not different between the two groups during varied Na(+) intake. Moreover, there were no differences in urine volume and urine osmolality between the two genotypes during normal or restricted water intake. In conclusion, these data suggested that AC3 is not involved in the physiological regulation of nephron Na(+) and water handling.

Luminal regulation of Na(+)/H(+) exchanger gene expression in rat ileal mucosa.

It is well recognized that ileostomy patients suffer from chronic depletion of Na(+) through the stoma effluent. In this study we evaluated the effects of ileostomy on messenger RNA levels that encode different Na(+)/H(+) exchanger isoforms (NHE-2 and NHE-3). Loop ileostomies were created in Sprague-Dawley rats. Segments of diverted ileum were harvested for quantitation of mRNA levels encoding these isoforms and the Na(+)/K(+) ATPase in mucosal scrapings and for immunofluorescence microscopy, specifically of the NHE-3 protein. Our studies indicate that as early as 8 days after diversion, NHE-3 gene expression is selectively attenuated in poststomal ileal mucosa. Mucosal morphology remains undisturbed, and the distribution of protein expression along the crypt/villus axis is not altered. Infusion of Na(+) or the enterocyte nutrient, glutamine, into the lumen of the diverted segment restores or even augments mRNA levels for NHE-3, again without altering the histologic appearance or distribution of the protein along the crypt/villus axis. These effects are specific because nonpolar osmolytes (mannitol) and related organic nutrients not specific for the enterocyte (i.e., butyrate) have no effect on mRNA levels of NHE-3. Further work is required to understand how the early changes in mRNA contribute to mucosal function and response to luminal diversion.

High resolution helium ion scanning microscopy of the rat kidney.

Helium ion scanning microscopy is a novel imaging technology with the potential to provide sub-nanometer resolution images of uncoated biological tissues. So far, however, it has been used mainly in materials science applications. Here, we took advantage of helium ion microscopy to explore the epithelium of the rat kidney with unsurpassed image quality and detail. In addition, we evaluated different tissue preparation methods for their ability to preserve tissue architecture. We found that high contrast, high resolution imaging of the renal tubule surface is possible with a relatively simple processing procedure that consists of transcardial perfusion with aldehyde fixatives, vibratome tissue sectioning, tissue dehydration with graded methanol solutions and careful critical point drying. Coupled with the helium ion system, fine details such as membrane texture and membranous nanoprojections on the glomerular podocytes were visualized, and pores within the filtration slit diaphragm could be seen in much greater detail than in previous scanning EM studies. In the collecting duct, the extensive and striking apical microplicae of the intercalated cells were imaged without the shrunken or distorted appearance that is typical with conventional sample processing and scanning electron microscopy. Membrane depressions visible on principal cells suggest possible endo- or exocytotic events, and central cilia on these cells were imaged with remarkable preservation and clarity. We also demonstrate the use of colloidal gold probes for highlighting specific cell-surface proteins and find that 15 nm gold labels are practical and easily distinguishable, indicating that external labels of various sizes can be used to detect multiple targets in the same tissue. We conclude that this technology represents a technical breakthrough in imaging the topographical ultrastructure of animal tissues. Its use in future studies should allow the study of fine cellular details and provide significant advances in our understanding of cell surface structures and membrane organization.

Vasopressin-induced differential stimulation of AQP4 splice variants regulates the in-membrane assembly of orthogonal arrays.

Aquaporin-4 (AQP4) is a basolateral water channel in collecting duct principal cells and assembles into orthogonal array particles (OAPs), the size of which appears to depend on relative expression levels of AQP4 splice variants. Because the higher-order organization of AQP4 was perturbed by vasopressin in Brattleboro rats and phosphorylation sites have been identified on AQP4, we investigated whether vasopressin and forskolin (Fk) affect AQP4 assembly and/or expression in LLC-PK(1) cells stably transfected with the AQP4 splice variant M23, which is responsible for formation of OAPs, and/or the splice variant M1, which does not form OAPs. Our data show that [lys(8)]-vasopressin (LVP) and Fk treatment led to differential increases in expression levels of M23-AQP4 and M1-AQP4 that varied as a function of incubation time. At early time points (day 1) expression of M1 was significantly stimulated (4.5-fold), over that of M23 (1.6-fold), but after 3 days the expression of M23 became predominant (4.1-fold) over that of M1 (1.9-fold). This pattern of stimulation was dependent on an intact AQP4 residue serine 111 and required protein synthesis. In cells expressing both M1 and M23 (M1/M23 approximately 1), with small sized OAPs at the membrane, the LVP/Fk-induced stimulation of M23 was modified and mimicked that of M1 when expressed alone, suggesting a dominant role for M1. In Brattleboro kidney inner medulla, an 8-day chronic exposure to the vasopressin agonist (dDAVP) led to reduction in M1 and a significant increase in M23 immunoblot staining (M1/M23 = 2/3 --> 1/4). These results indicate that AQP4 organization and expression are regulated by vasopressin in vivo and in vitro and demonstrate that the dominant role for M1 is restricted to a one-to-one interaction between AQP4 splice variants that regulates the membrane expression of OAPs.

Aquaporin-4 dynamics in orthogonal arrays in live cells visualized by quantum dot single particle tracking.

Freeze-fracture electron microscopy (FFEM) indicates that aquaporin-4 (AQP4) water channels can assemble in cell plasma membranes in orthogonal arrays of particles (OAPs). We investigated the determinants and dynamics of AQP4 assembly in OAPs by tracking single AQP4 molecules labeled with quantum dots at an engineered external epitope. In several transfected cell types, including primary astrocyte cultures, the long N-terminal "M1" form of AQP4 diffused freely, with diffusion coefficient approximately 5 x 10(-10) cm(2)/s, covering approximately 5 microm in 5 min. The short N-terminal "M23" form of AQP4, which by FFEM was found to form OAPs, was relatively immobile, moving only approximately 0.4 microm in 5 min. Actin modulation by latrunculin or jasplakinolide did not affect AQP4-M23 diffusion, but deletion of its C-terminal postsynaptic density 95/disc-large/zona occludens (PDZ) binding domain increased its range by approximately twofold over minutes. Biophysical analysis of short-range AQP4-M23 diffusion within OAPs indicated a spring-like potential, with a restoring force of approximately 6.5 pN/microm. These and additional experiments indicated that 1) AQP4-M1 and AQP4-M23 isoforms do not coassociate in OAPs; 2) OAPs can be imaged directly by total internal reflection fluorescence microscopy; and 3) OAPs are relatively fixed, noninterconvertible assemblies that do not require cytoskeletal or PDZ-mediated interactions for formation. Our measurements are the first to visualize OAPs in live cells.

Role of NHERF1, cystic fibrosis transmembrane conductance regulator, and cAMP in the regulation of aquaporin 9.

Water and solute transport across the plasma membrane of cells is a crucial biological function that is mediated mainly by aquaporins and aquaglyceroporins. The regulation of these membrane proteins is still incompletely understood. Using the male reproductive tract as a model system in which water and glycerol transport are critical for the establishment of fertility, we now report a novel pathway for the regulation of aquaporin 9 (AQP9) permeability. AQP9 is the major aquaglyceroporin of the epididymis, liver, and peripheral leukocytes, and its COOH-terminal portion contains a putative PDZ binding motif (SVIM). Here we show that NHERF1, cystic fibrosis transmembrane conductance regulator (CFTR), and AQP9 co-localize in the apical membrane of principal cells of the epididymis and the vas deferens, and that both NHERF1 and CFTR co-immunoprecipitate with AQP9. Overlay assays revealed that AQP9 binds to both the PDZ1 and PDZ2 domains of NHERF1, with an apparently higher affinity for PDZ1 versus PDZ2. Pull-down assays showed that the AQP9 COOH-terminal SVIM motif is essential for interaction with NHERF1. Functional assays on isolated tubules perfused in vitro showed a high permeability of the apical membrane to glycerol, which is inhibited by the AQP9 inhibitor, phloretin, and is markedly activated by cAMP. The CFTR inhibitors DPC, GlyH-101 and CFTRinh-172 all significantly reduced the cAMP-activated glycerol-induced cell swelling. We propose that CFTR is an important regulator of AQP9 and that the interaction between AQP9, NHERF1, and CFTR may facilitate the activation of AQP9 by cAMP.

Compensatory membrane expression of the V-ATPase B2 subunit isoform in renal medullary intercalated cells of B1-deficient mice.

Mice deficient in the ATP6V1B1 ("B1") subunit of the vacuolar proton-pumping ATPase (V-ATPase) maintain body acid-base homeostasis under normal conditions, but not when exposed to an acid load. Here, compensatory mechanisms involving the alternate ATP6V1B2 ("B2") isoform were examined to explain the persistence of baseline pH regulation in these animals. By immunocytochemistry, the mean pixel intensity of apical B2 immunostaining in medullary A intercalated cells (A-ICs) was twofold greater in B1-/- mice than in B1+/+ animals, and B2 was colocalized with other V-ATPase subunits. No significant upregulation of B2 mRNA or protein expression was detected in B1-/- mice compared with wild-type controls. We conclude that increased apical B2 staining is due to relocalization of B2-containing V-ATPase complexes from the cytosol to the plasma membrane. Recycling of B2-containing holoenzymes between these domains was confirmed by the intracellular accumulation of B1-deficient V-ATPases in response to the microtubule-disrupting drug colchicine. V-ATPase membrane expression is further supported by the presence of "rod-shaped" intramembranous particles seen by freeze fracture microscopy in apical membranes of normal and B1-deficient A-ICs. Intracellular pH recovery assays show that significant (28-40% of normal) V-ATPase function is preserved in medullary ICs from B1-/- mice. We conclude that the activity of apical B2-containing V-ATPase holoenzymes in A-ICs is sufficient to maintain baseline acid-base homeostasis in B1-deficient mice. However, our results show no increase in cell surface V-ATPase activity in response to metabolic acidosis in ICs from these animals, consistent with their inability to appropriately acidify their urine under these conditions.

Postnatal expression of aquaporins in epithelial cells of the rat epididymis.

The mammalian aquaporins (AQPs) are a family of 13 transmembrane channel proteins that are involved in the transport of water in numerous organs. In the male excurrent duct, the movement of fluid and solutes across the epithelium is essential for establishing the proper luminal environment in which sperm mature and are stored. AQP9 is abundantly expressed in the efferent ducts, the epididymis, and the vas deferens, where it could represent an important apical pathway for transmembrane water and solute movement. However, other organs in which water transport is critical, including the kidney, the lung, or the eye, express several different AQPs with a cell-specific pattern. To undertake a systematic analysis of the expression of known AQPs in the postnatal and adult rat epididymis, we examined the expression of their respective mRNAs in epithelial cells isolated by laser capture microdissection (LCM), and we determined their corresponding protein expression pattern by immunofluorescence and Western blotting. Our data show that, whereas AQP9 is the main AQP of the epididymis, the mRNA specific for Aqp2, 5, 7, and 11 are also expressed in epididymal epithelial cells. AQP5 protein colocalizes with AQP9 in the apical membrane of a subpopulation of principal cells in the corpus and cauda regions. Aqp2 mRNA was detected in epithelial cells after the second postnatal week and the amount significantly increased up to adulthood. However, AQP2 protein was detected only in the distal cauda of young rats (between the second and fourth postnatal week). No AQP2 protein was detected in the adult epididymis, indicating that posttranscriptional mechanisms are involved in the regulation of AQP2 expression. In addition, epididymal epithelial cells express significant amounts of the mRNAs coding for AQP7 and 11. No mRNA or protein for AQPs 0, 4, 6, and 8 were detectable in epithelial cells, and Aqp1 was detected in whole epididymal samples, but not in epithelial cells. Thanks to the recent development of microdissection technologies, our observations suggest that epididymal epithelial cells express several members of the AQP family with a region-specific pattern. AQPs may be involved not only in the transepithelial transport of water in the epididymis but also in the postnatal development of this organ, as suggested by the differential expression of AQP2.

Aquaporin-2 localization in clathrin-coated pits: inhibition of endocytosis by dominant-negative dynamin.

Before the identification of aquaporin (AQP) proteins, vasopressin-regulated "water channels" were identified by freeze-fracture electron microscopy as aggregates or clusters of intramembraneous particles (IMPs) on hormonally stimulated target cell membranes. In the kidney collecting duct, these IMP clusters were subsequently identified as possible sites of clathrin-coated pit formation on the plasma membrane, and a clathrin-mediated mechanism for internalization of vasopressin-sensitive water channels was suggested. Using an antibody raised against the extracellular C loop of AQP2, we now provide direct evidence that AQP2 is concentrated in clathrin-coated pits on the apical surface of collecting duct principal cells. Furthermore, by using a fracture-label technique applied to LLC-PK(1) cells expressing an AQP2-c-myc construct, we show that AQP2 is located in IMP aggregates and is concentrated in shallow membrane invaginations on the surface of forskolin-stimulated cells. We also studied the functional role of clathrin-coated pits in AQP2 trafficking by using a GTPase-deficient dynamin mutation (K44A) to inhibit clathrin-mediated endocytosis. Immunofluorescence labeling and freeze-fracture electron microscopy showed that dominant-negative dynamin 1 and dynamin 2 mutants prevent the release of clathrin-coated pits from the plasma membrane and induce an accumulation of AQP2 on the plasma membrane of AQP2-transfected cells. These data provide the first direct evidence that AQP2 is located in clathrin-coated pits and show that AQP2 recycles between the plasma membrane and intracellular vesicles via a dynamin-dependent endocytotic pathway. We propose that the IMP clusters previously associated with vasopressin action represent sites of dynamin-dependent, clathrin-mediated endocytosis in which AQP2 is concentrated before internalization.

Partitioning of aquaporin-4 water channel mRNA and protein in gastric glands.

Immunolocalization studies in proximal, middle, and distal stomach indicated that aquaporin-4 (AQP4) protein is localized only in parietal cells located in the middle or deep regions of the gastric glands. In studies using in situ hybridization, AQP4 mRNA failed to localize in parietal cells but was identified in neighboring mucosal cells that were triangular in shape and smaller than parietal cells in size, and in columnar cells at the base of the gastric gland. This spatial separation of mRNA and protein was also observed in other species and with other kind of mRNA/protein. In neonatal and adolescent rats, the appearance of morphologically mature parietal cells was preceded by identification of mRNA-bearing triangular cells. Cells harboring both protein and mRNA were observed in postnatal rats and in the pyloric region of the glandular stomach, during induced hypergastrinaemia. The results suggest that such cells represent a transition between those that bear only mRNA and those that are terminally differentiated, expressing proteins that are related to acid secretion.

Expression of aquaporin 9 in the adult rat epididymal epithelium is modulated by androgens.

Reabsorption of fluid and solutes across the epithelium lining the male excurrent duct is important for adequate sperm maturation, concentration, and storage. Water channels contribute to water movement across epithelia in many tissues. Aquaporin 9 (AQP9) is abundantly expressed in the apical membrane of principal cells that line the epididymis, and in reabsorptive and secretory epithelial cells of the male reproductive tract. In this study we show that the nonsteroidal antiandrogen flutamide, given to adult rats at a dose of 50 mg x kg(-1) x day(-1) for 2 wk via osmotic minipumps significantly decreased the amount of AQP9 in the epididymis. This down-regulation was observed by immunofluorescence of cryostat tissue sections and by Western blotting of epididymal brush border membrane preparations. In addition, castrated adult rats showed lower levels of epididymal AQP9 compared with adult controls, whereas systemic testosterone treatment of castrated adult rats induced a recovery of the expression of AQP9 to control levels. These data indicate that the expression of AQP9, a likely candidate for apical transepithelial fluid and solute transport in several regions of the male reproductive tract, is modulated by androgens in the adult rat epididymis.