Localization of MIWC and GLIP water channel homologs in neuromuscular, epithelial and glandular tissues

1995 ◽  
Vol 108 (9) ◽  
pp. 2993-3002 ◽  
Author(s):  
A. Frigeri ◽  
M.A. Gropper ◽  
F. Umenishi ◽  
M. Kawashima ◽  
D. Brown ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Urinary Bladder ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Water Channel ◽  
Parietal Cells ◽  
Immunogold Electron Microscopy ◽  
Transitional Epithelium ◽  
Principal Cells ◽  
Kidney Collecting Duct

It was shown recently that water channel homologs MIWC (mercurial insensitive water channel) and GLIP (glycerol intrinsic protein) colocalized in basolateral membranes of kidney collecting duct, tracheal and colonic epithelia, and in brain pia mater. We report here an extensive immunolocalization study of MIWC and GLIP in non-epithelial and glandular epithelial tissues in rat. Immunogold electron microscopy confirmed colocalization of MIWC and GLIP in basolateral membrane of principal cells in kidney collecting duct. However, in other epithelia, MIWC but not GLIP was expressed in basolateral membrane of parietal cells in stomach, and in excretory tubules of salivary and lacrimal glands; GLIP but not MIWC was expressed in transitional epithelium of urinary bladder and skin epidermis. In the central nervous system, MIWC was strongly expressed in the ependymal layer lining the aqueductal system, and in astrocytes throughout the spinal cord and in selected regions of brain. MIWC was also expressed in a plasma membrane pattern in skeletal, but not smooth or cardiac muscle. Neither protein was expressed in small intestine, testis, liver, spleen and nerve. The tissue-specific expression of MIWC suggests a role in fluid transport and/or cell volume regulation in stomach and glandular epithelia. The functional role of MIWC expression in the neuromuscular system and of GLIP expression in skin and urinary bladder is uncertain. The specific cellular sites of MIWC expression (astrocytes, trachea, sarcolemma, gastric parietal cells and kidney principal cells) correspond exactly to sites where orthogonal arrays of particles (OAPs) have been visualized by freeze-fracture electron microscopy, suggesting that MIWC may be the OAP protein.

A basolateral CHIP28/MIP26-related protein (BLIP) in kidney principal cells and gastric parietal cells

1994 ◽  
Vol 267 (3) ◽  
pp. C812-C820 ◽  
Author(s):  
G. Valenti ◽  
J. M. Verbavatz ◽  
I. Sabolic ◽  
D. A. Ausiello ◽  
A. S. Verkman ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Basolateral Membrane ◽  
Water Channel ◽  
Protein S ◽  
High Water ◽  
Protein Band ◽  
Parietal Cells ◽  
Toad Urinary Bladder ◽  
Principal Cells ◽  
Gastric Parietal Cells

The water channel CHIP28 accounts for the high water permeability of proximal tubules and thin descending limbs of Henle; a homologous water channel, WCH-CD, in the apical membrane of collecting duct principal cells, may be the vasopressin-sensitive water channel. We show here that one antiserum, raised against CHIP28, immunostains the basolateral membrane of collecting duct principal cells, in addition to staining CHIP28 in other cells. This serum was named anti-basolateral integral protein (anti-BLIP) to distinguish it from other anti-CHIP28 antisera. By Western blotting, BLIP serum recognized both CHIP28 and MIP26, and it stained lens fibers, which contain MIP26 but not CHIP28. BLIP antiserum immunoprecipitated a 28-kDa band, a broad 35- to 50-kDa band, and an approximately 16-kDa band from kidney papilla. It also stained the basolateral membrane of gastric parietal cells, which were not stained with anti-CHIP28 or anti-MIP26 antibodies. BLIP antiserum immunoprecipitated a 28-kDa protein band from stomach; this protein was not precipitated by anti-CHIP28 antibodies. These results suggest that basolateral membranes of principal cells and parietal cells contain a protein(s) that shares common epitopes with CHIP28 and MIP26. Finally, BLIP but not CHIP28 antiserum stained mesothelial (but not epithelial) cells of toad urinary bladder, a further indication that the BLIP antiserum recognizes a protein distinct from CHIP28.

Water channel-carrying vesicles in the rat IMCD contain cellubrevin

1995 ◽  
Vol 269 (3) ◽  
pp. C797-C801 ◽  
Author(s):  
N. Franki ◽  
F. Macaluso ◽  
W. Schubert ◽  
L. Gunther ◽  
R. M. Hays
Keyword(s):  
Electron Microscopy ◽  
Membrane Protein ◽  
Arginine Vasopressin ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Water Channel ◽  
Immunogold Electron Microscopy ◽  
Cyclic Process ◽  
Docking Proteins ◽  
Medullary Collecting Duct

Antidiuretic hormone (arginine vasopressin) induces a cyclic process of docking, fusion, and endocytosis of water channel-containing vesicles in the collecting duct. There is now evidence that docking and endocytosis are mediated by an array of proteins associated with vesicles and target membranes. In recent studies, we have shown that cellubrevin, a member of the vesicle-associated membrane protein family, as well as other docking proteins, are expressed in the rat inner medullary collecting duct. We now show by immunogold electron microscopy that cellubrevin is present on vesicles containing water channels, that it is associated with both coated and uncoated vesicles, and that it is present on the apical membrane. Cellubrevin, therefore, is in a position to mediate one or more steps in arginine vasopressin-induced water channel cycling.

Immunolocalization and effect of dehydration on AQP3, a basolateral water channel of kidney collecting ducts

1997 ◽  
Vol 272 (2) ◽  
pp. F235-F241 ◽  
Author(s):  
K. Ishibashi ◽  
S. Sasaki ◽  
K. Fushimi ◽  
T. Yamamoto ◽  
M. Kuwahara ◽  
...  
Keyword(s):  
Xenopus Oocytes ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Broad Band ◽  
Water Channel ◽  
In Vitro Translation ◽  
Mercury Chloride ◽  
Channel Activity ◽  
Kidney Medulla ◽  
Principal Cells

Aquaporin-3 (AQP3) is unique in its structure (lowest homology with other aquaporins) and in its function (significantly conductive to both small nonelectrolytes and water). However, there is a controversy among researchers on its water transport and induction by dehydration. We examined its localization and the effect of dehydration on its expression in the kidney, as well as its water channel activity when expressed in Xenopus oocytes. In vitro translation using reticulocyte lysate revealed that the size of rat AQP3 was 26 kDa, and the band shifted to around 31 kDa with microsomal fraction, which was sensitive to the digestion with N-glycosidase F. In Western blot analysis of rat kidney medulla, AQP3 appeared as a sharp band at 27 kDa and a broad band at 34-40 kDa. In immunohistochemistry, AQP3 was localized to principal cells and absent in intercalated cells in outer medulla. In inner medulla, AQP3 was restricted to inner medullary collecting duct (IMCD) cells. AQP3 was confined to the basolateral membrane of these cells. Although dehydration of rats for 2 days did not change the distribution pattern of AQP3 in IMCD cells, the dehydration increased AQP3 mRNA by twofold with slight increase of its protein level in kidney medulla. Finally, we confirmed its water channel activity when expressed in Xenopus oocytes. The human AQP3 stimulated osmotic water permeability by eightfold, which was inhibited by 0.3 mM mercury chloride by 34% and reversed by beta-mercaptoethanol. Our results indicate that AQP3 is a glycosylated protein and a mercury-sensitive water channel localized at the basolateral membrane of principal cells and IMCD cells, and its expression is induced by dehydration at both protein and mRNA level.

Functional characterization and cell immunolocalization of AQP-CD water channel in kidney collecting duct

1994 ◽  
Vol 267 (4) ◽  
pp. F573-F582 ◽  
Author(s):  
K. Fushimi ◽  
S. Sasaki ◽  
T. Yamamoto ◽  
M. Hayashi ◽  
T. Furukawa ◽  
...  
Keyword(s):  
Water Permeability ◽  
Expression Vector ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Functional Characterization ◽  
Protein A ◽  
Water Channel ◽  
Kidney Medulla ◽  
Principal Cells ◽  
Kidney Collecting Duct

Vasopressin-regulated water permeability of the kidney collecting duct is a key component of the urine concentration machinery. Recently, a cDNA for AQP-CD, the vasopressin-regulated water channel, initially reported as WCH-CD, has been isolated (K. Fushimi, S. Uchida, Y. Hara, Y. Hirata, F. Marumo, and S. Sasaki. Nature Lond. 361: 549-552, 1993). AQP-CD was expressed in oocyte membrane using a Xenopus expression vector, and functional characteristics of AQP-CD were examined. Osmotic water permeability (Pf) of oocytes expressing AQP-CD was 138 +/- 19 microns/s (mean +/- SE), 12 times greater than the control (11 +/- 3 microns/s), 90% inhibited by 0.3 mM HgCl2, and weakly temperature dependent (energy of activation for Pf was 4.0 kcal/mol). Urea influx measured from 15-min [14C]urea uptake by oocytes injected with AQP-CD/expression vector 1 cRNA was 86 +/- 17% of the control. Two-electrode voltage-clamp experiments revealed insignificant ion conductance of AQP-CD. Immunoblots of membranes from rat kidney medulla and oocytes expressing AQP-CD using anti-AQP-CD COOH-terminal antibody showed a 29-kDa protein and 35- to 50-kDa high-molecular-mass forms. Immunohistochemistry showed apical and subapical localization of AQP-CD in the collecting duct principal cells. Our results indicated that AQP-CD is a 29-kDa protein, a selective water channel, distinct from a urea channel, and localized to the membranes of vasopressin-sensitive components in kidney collecting duct principal cells.

scholarly journals Endocytic vesicles from renal papilla which retrieve the vasopressin-sensitive water channel do not contain a functional H+ ATPase.

1990 ◽  
Vol 111 (2) ◽  
pp. 379-389 ◽  
Author(s):  
W I Lencer ◽  
A S Verkman ◽  
M A Arnaout ◽  
D A Ausiello ◽  
D Brown
Keyword(s):  
Plasma Membrane ◽  
Water Permeability ◽  
Collecting Duct ◽  
Water Channel ◽  
Water Channels ◽  
Proton Pumps ◽  
Principal Cells ◽  
Kidney Collecting Duct ◽  
Fluorescence Imaging Microscopy ◽  
Membrane Components

The water permeability of the kidney collecting duct epithelium is regulated by vasopressin (VP)-induced recycling of water channels between an intracellular vesicular compartment and the plasma membrane of principal cells. To test whether the water channels pass through an acidic endosomal compartment during the endocytic portion of this pathway, we measured ATP-dependent acidification of FITC-dextran-labeled endosomes in isolated microsomal fractions from different regions of Brattleboro rat kidneys. Both VP-deficient controls and rat treated with exogenous VP were examined. ATP-dependent acidification was not detectable in endosomes containing water channels from distal papilla (osmotic water permeability Pf = 0.038 +/- 0.004 cm/s). In contrast, the addition of ATP resulted in a strong acidification of renal cortical endosomes (pHmin = 5.8, initial rate = 0.18-0.25 pH U/s). Acidification of cortical endosomes was reversed with nigericin and strongly inhibited by N-ethyl-maleimide. Passive proton permeability was similar and low in both cortical and papillary endosomes from rats treated or not treated with VP. The fraction of labeled endosomes present in microsomal preparations was determined by fluorescence imaging microscopy of microsomes nonspecifically bound to poly-l-lysine-coated coverslips and was 25% in cortical preparations compared to 14% (+VP) and 9% (-VP) in papillary preparations. The fraction of cortical endosomes was enriched 1.5-fold by immunoabsorption to coverslips coated with mAbs against the bovine vacuolar proton pump. In contrast, the fraction of papillary endosomes was depleted more than twofold by immunoabsorption to identical coverslips. Finally, sections of distal papilla stained with antibodies against the lysosomal glycoprotein LGP120 showed that most of the entrapped FITC-dextran did not colocalize with this lysosomal protein. These results demonstrate that vesicles which internalize water channels in kidney collecting duct principal cells lack functional proton pumps, and do not deliver the bulk of their FITC-dextran content to lysosomes. The data suggest that the principal cell contains a specialized nonacidic apical endocytic compartment which functions primarily to recycle membrane components, including water channels, to the plasma membrane.

scholarly journals Basolateral targeting and microtubule-dependent transcytosis of the aquaporin-2 water channel

2013 ◽  
Vol 304 (1) ◽  
pp. C38-C48 ◽  
Author(s):  
Naofumi Yui ◽  
Hua A. J. Lu ◽  
Ying Chen ◽  
Naohiro Nomura ◽  
Richard Bouley ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cold Shock ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Water Channel ◽  
Apical Plasma Membrane ◽  
Indirect Pathway ◽  
Basolateral Membranes ◽  
Principal Cells ◽  
Aquaporin 2

The aquaporin-2 (AQP2) water channel relocates mainly to the apical plasma membrane of collecting duct principal cells after vasopressin (VP) stimulation. AQP2 transport to this membrane domain is assumed to be a direct route involving recycling of intracellular vesicles. However, basolateral plasma membrane expression of AQP2 is observed in vivo in principal cells. Here, we asked whether there is a transcytotic pathway of AQP2 trafficking between apical and basolateral membranes. We used MDCK cells in which AQP2 normally accumulates apically after VP exposure. In contrast, both site-specific biotinylation and immunofluorescence showed that AQP2 is strongly accumulated in the basolateral membrane, along with the endocytic protein clathrin, after a brief cold shock (4°C). This suggests that AQP2 may be constitutively targeted to basolateral membranes and then retrieved by clathrin-mediated endocytosis at physiological temperatures. Rab11 does not accumulate in basolateral membranes after cold shock, suggesting that the AQP2 in this location is not associated with Rab11-positive vesicles. After rewarming (37°C), basolateral AQP2 staining is diminished and it subsequently accumulates at the apical membrane in the presence of VP/forskolin, suggesting that transcytosis can be followed by apical insertion of AQP2. This process is inhibited by treatment with colchicine. Our data suggest that the cold shock procedure reveals the presence of microtubule-dependent AQP2 transcytosis, which represents an indirect pathway of apical AQP2 delivery in these cells. Furthermore, our data indicate that protein polarity data obtained from biotinylation assays, which require cells to be cooled to 4°C during the labeling procedure, should be interpreted with caution.

scholarly journals Vasopressin induces apical expression of caveolin in rat kidney collecting duct principal cells

2013 ◽  
Vol 305 (12) ◽  
pp. F1783-F1795 ◽  
Author(s):  
Teodor G. Păunescu ◽  
Hua A. J. Lu ◽  
Leileata M. Russo ◽  
Núria M. Pastor-Soler ◽  
Mary McKee ◽  
...  
Keyword(s):  
Apical Membrane ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Immunogold Electron Microscopy ◽  
Bb Rat ◽  
Membrane Domain ◽  
Sprague Dawley ◽  
Principal Cells ◽  
Bb Rats ◽  
Long Evans

Caveolin (Cav)1 is expressed in the basolateral membrane domain of renal collecting duct (CD) principal cells (PCs), where it is associated with caveolae. To reveal any potential involvement of Cav1 in vasopressin signaling, we used specific monoclonal and polyclonal antibodies to examine its localization in CD PCs of Brattleboro (BB) rats treated with vasopressin (DDAVP). Compared with controls, immunofluorescence revealed a time-dependent increase in Cav1 expression in the apical membrane domain of PCs, where it overlapped with aquaporin-2 (AQP2). After 24 h of DDAVP treatment, Cav1 was visible as an increased number of small apical spots. The staining gradually became more extensive, and, after 2 wk of DDAVP, it occupied the majority of the apical membrane domain of many PCs. Cav1 also assumed an apical localization in PCs of DDAVP-treated Sprague-Dawley and Long-Evans rats. Similarly, Cav2 appeared at the apical pole of PCs after DDAVP treatment of BB, Sprague-Dawley, and Long-Evans rats. Immunogold electron microscopy confirmed bipolar Cav1 membrane expression in DDAVP-treated BB rats, whereas caveolae were only detected on the basolateral membrane. Immunoblot analysis of BB rat whole kidney homogenates revealed no significant increase in Cav1 levels in DDAVP-treated rats, suggesting that DDAVP induces Cav1 relocalization or modifies its targeting. We conclude that Cav1 and Cav2 trafficking and membrane localization are dramatically altered by the action of DDAVP. Importantly, the absence of apical caveolae indicates that while Cavs may have an as yet undetermined role in vasopressin-regulated signaling processes, this is probably unrelated to AQP2 internalization by caveolae.

scholarly journals Bilateral ureteral obstruction is rapidly accompanied by ER stress and activation of autophagic degradation of IMCD proteins, including AQP2

2020 ◽  
Vol 318 (1) ◽  
pp. F135-F147
Author(s):  
Poorichaya Somparn ◽  
Chatikorn Boonkrai ◽  
Komgrid Charngkaew ◽  
Nusara Chomanee ◽  
Kenneth G. Hodge ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Mass Spectrometry ◽  
Endoplasmic Reticulum ◽  
Ureteral Obstruction ◽  
Collecting Duct ◽  
Immunoblot Analysis ◽  
Immunogold Electron Microscopy ◽  
Sham Operation ◽  
Autophagic Degradation ◽  
Cell Cell

After the release of bilateral ureteral obstruction (BUO), postobstructive diuresis from an impaired urine concentration mechanism is associated with reduced aquaporin 2 (AQP2) abundance in the inner medullary collecting duct (IMCD). However, the underlying molecular mechanism of this AQP2 reduction is incompletely understood. To elucidate the mechanisms responsible for this phenomenon, we studied molecular changes in IMCDs isolated from rats with 4-h BUO or sham operation at the early onset of AQP2 downregulation using mass spectrometry-based proteomic analysis. Two-hundred fifteen proteins had significant changes in abundances, with 65% of them downregulated in the IMCD of 4-h BUO rats compared with sham rats. Bioinformatic analysis revealed that significantly changed proteins were associated with functional Gene Ontology terms, including “cell-cell adhesion,” “cell-cell adherens junction,” “mitochondrial inner membrane,” “endoplasmic reticulum chaperone complex,” and the KEGG pathway of glycolysis/gluconeogenesis. Targeted liquid chromatography-tandem mass spectrometry or immunoblot analysis confirmed the changes in 19 proteins representative of each predominant cluster, including AQP2. Electron microscopy demonstrated disrupted tight junctions, disorganized adherens junctions, swollen mitochondria, enlargement of the endoplasmic reticulum lumen, and numerous autophagosomes/lysosomes in the IMCD of rats with 4-h BUO. AQP2 and seven proteins chosen as representative of the significantly altered clusters had a significant increase in immunofluorescence-based colocalization with autophagosomes/lysosomes. Immunogold electron microscopy confirmed colocalization of AQP2 with the autophagosome marker microtubule-associated protein 1A/1B-light chain 3 and the lysosomal marker cathepsin D in IMCD cells of rats with 4-h BUO. We conclude that enhanced autophagic degradation of AQP2 and other critical proteins, as well as endoplasmic reticulum stress in the IMCD, are initiated shortly after BUO.

Expression, functional analysis, and in situ hybridization of a cloned rat kidney collecting duct water channel

1994 ◽  
Vol 266 (1) ◽  
pp. C189-C197 ◽  
Author(s):  
T. Ma ◽  
H. Hasegawa ◽  
W. R. Skach ◽  
A. Frigeri ◽  
A. S. Verkman
Keyword(s):  
Base Pair ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Water Channel ◽  
Cloning And Expression ◽  
Base Pairs ◽  
Kidney Medulla ◽  
Coding Sequence ◽  
Kidney Collecting Duct ◽  
Protein Size

The cloning and expression of an apical membrane water channel from rat kidney collecting duct (WCH-CD) homologous to a 28-kDa integral membrane protein (CHIP28) was reported recently (K. Fushimi, S. Uchida, Y. Hara, Y. Hirata, F. Marumo, and S. Sasaki. Nature Lond. 361: 549-552, 1993). We obtained an approximately 1.8-kilobase clone from a rat kidney lambda gt10 cDNA library by a polymerase chain reaction cloning method; whereas the coding sequence (814 base pairs, predicted protein size 29 kDa) was identical to that reported, we identified an in-frame ATG codon at base pair -123 predicting a protein size of 33 kDa. On Northern blots probed by cDNAs corresponding to the WCH-CD coding sequence (base pairs +1 to +814) or 5'-untranslated sequence (-403 to -16), a single band at 1.9 kilobases was observed in kidney medulla greater than in cortex but not in other tissues; mRNA expression was increased strongly by dehydration. Translation and oocyte expression studies were performed to identify the translation start site. The short (base pairs +1 to +814) and long (base pairs -123 to +814) cDNAs were subcloned in vector pSP64 containing the 5'-untranslated Xenopus globin sequence upstream to the ATGs; a 30-base pair c-myc sequence was engineered at the COOH- terminal for antibody recognition.(ABSTRACT TRUNCATED AT 250 WORDS)

Depletion of intercalated cells from collecting ducts of carbonic anhydrase II-deficient (CAR2 null) mice

1995 ◽  
Vol 269 (6) ◽  
pp. F761-F774 ◽  
Author(s):  
S. Breton ◽  
S. L. Alper ◽  
S. L. Gluck ◽  
W. S. Sly ◽  
J. E. Barker ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Collecting Duct ◽  
Water Channel ◽  
Immunofluorescent Staining ◽  
Specific Marker ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Principal Cells ◽  
Collecting Tubules ◽  
Deficient Mice

The kidneys of mice (CAR2-null mice) that are genetically devoid of carbonic anhydrase type II (CAII) were screened by immunocytochemistry with antibodies that distinguish intercalated and principal cells. Immunofluorescent localization of the anion exchanger AE1 and of the 56-kDa subunit of the vacuolar H(+)-adenosinetriphosphatase (H(+)-ATPase) was used to identify intercalated cells, while the AQP2 water channel was used as a specific marker for principal cells of the collecting duct. The CAII deficiency of the CAR2-null mice was first confirmed by the absence of immunofluorescent staining of kidney sections exposed to an anti-CAII antibody. Cells positive for AE1 and H(+)-ATPase were common in all collecting duct regions in normal mice but were virtually absent from the inner stripe of the outer medulla and the inner medulla of CAR2-null mice. The number of positive cells was also reduced threefold in the cortical collecting duct of CAR2-null animals compared with normal mice. In parallel, the percentage of AQP2-positive cells was correspondingly increased in the collecting tubules of CAII-deficient mice, whereas the total number of cells per tubule remained unchanged. These results suggest that intercalated cells are severely depleted and are replaced by principal cells in CAII-deficient mice. Quantitative analysis and double staining showed that, in the cortex, both type A and type B intercalated cells are equally affected. Elucidation of the mechanism(s) responsible for this phenotype will be of importance in understanding the origin and development of intercalated cells in the kidney.

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