Endocytosis of water channels in rat kidney: cell specificity and correlation with in vivo antidiuresis

1990 ◽  
Vol 259 (6) ◽  
pp. C920-C932 ◽  
Author(s):  
W. I. Lencer ◽  
D. Brown ◽  
D. A. Ausiello ◽  
A. S. Verkman
Keyword(s):  
Water Permeability ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Water Channel ◽  
Water Channels ◽  
Intercalated Cells ◽  
Principal Cells ◽  
Osmotic Water ◽  
Principal And Intercalated Cells

Vasopressin action in the renal collecting duct is believed to be mediated by the cycling of water channels in principal and, possibly, intercalated cells. We used 6-carboxyfluorescein (6-CF) or fluorescein-labeled dextran (FITC-dextran) to determine the location and water permeability of endocytic vesicles from papilla and inner stripe of Brattleboro rats in different states of diuresis. Fifteen minutes after FITC-dextran infusion, fluorescent vesicles were concentrated at the apical pole of principal and intercalated cells. The osmotic water permeability (Pf) of these endosomes was measured by fluorescence quenching. In papillary endosomes, Pf was high (0.04 +/- 0.004 cm/s) when rats were in physiological states of antidiuresis or after treatment with vasopressin, 1-desamino-8-D-arginine vasopressin (DDAVP), or oxytocin; endosomes isolated from these regions of untreated animals had a low Pf. The number of papillary endosomes with high Pf increased with increasing doses of DDAVP. Endosomes from the inner stripe also had a high Pf only after vasopressin treatment. Confocal microscopy of sections of papilla showed that vasopressin significantly increased endocytosis in principal cells but had no effect on intercalated cells. Our data demonstrate that the bulk of fluorescently labeled vesicles from the papilla originate from the apical membrane of principal cells and contain water channels in their limiting membrane only when the rats are in physiological states of antidiuresis. In contrast, the majority of endocytosis in intercalated cells is not involved in water channel recycling.

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.

A rat kidney tubule suspension for the study of vasopressin-induced shuttling of AQP2 water channels

2002 ◽  
Vol 283 (5) ◽  
pp. F1160-F1166 ◽  
Author(s):  
Stephen Shaw ◽  
David Marples
Keyword(s):  
Plasma Membrane ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Primary Cultures ◽  
Water Channels ◽  
Apical Plasma Membrane ◽  
Cellular Mechanisms ◽  
Principal Cells ◽  
Microtubule Disruption ◽  
Osmotic Water

AVP increases the osmotic water permeability of renal collecting ducts by inducing the translocation of specific aquaporin-2 (AQP2) water channels from cytoplasmic vesicles to the apical plasma membrane of the principal cells. Here, we report a novel inner medullary tubule suspension for the study of this phenomenon that overcomes some of the drawbacks faced by present techniques; both primary cultures of inner medullary collecting duct cells and cell lines expressing AQP2 show aberrant trafficking and/or signaling pathways. The tubule suspensions were prepared by proteolytic digestion of inner medullas dissected from freshly isolated rat kidneys. After drug treatment, cellular distribution of AQP2 was determined by membrane fractionation and Western blotting or by immunocytochemistry. Treatment of suspensions with 1 nM AVP caused redistribution of AQP2 to the apical plasma membrane of the principal cells, a process inhibited by microtubule disruption or PKA inhibition. We conclude that this method provides a valuable new approach to the study of the cellular mechanisms involved in the response of the collecting duct to AVP.

scholarly journals Expression of multiple water channel activities in Xenopus oocytes injected with mRNA from rat kidney.

1993 ◽  
Vol 101 (6) ◽  
pp. 827-841 ◽  
Author(s):  
M Echevarria ◽  
G Frindt ◽  
G M Preston ◽  
S Milovanovic ◽  
P Agre ◽  
...  
Keyword(s):  
Water Permeability ◽  
Antisense Oligonucleotide ◽  
Xenopus Oocytes ◽  
Rat Kidney ◽  
Water Channel ◽  
Water Channels ◽  
Renal Tissue ◽  
Osmotic Water Permeability ◽  
Volume Increase ◽  
Osmotic Water

To test the hypothesis that renal tissue contains multiple distinct water channels, mRNA prepared from either cortex, medulla, or papilla of rat kidney was injected into Xenopus oocytes. The osmotic water permeability (Pf) of oocytes injected with either 50 nl of water or 50 nl of renal mRNA (1 microgram/microliter) was measured 4 d after the injection. Pf was calculated from the rate of volume increase on exposure to hyposmotic medium. Injection of each renal mRNA preparation increased the oocyte Pf. This expressed water permeability was inhibited by p-chloromercuriphenylsulfonate and had a low energy of activation, consistent with the expression of water channels. The coinjection of an antisense oligonucleotide for CHIP28 protein, at an assumed > 100-fold molar excess, with either cortex, medulla, or papilla mRNA reduced the expression of the water permeability by approximately 70, 100, and 30%, respectively. Exposure of the oocyte to cAMP for 1 h resulted in a further increase in Pf only in oocytes injected with medulla mRNA. This cAMP activation was not altered by the CHIP28 antisense oligonucleotide. These results suggest that multiple distinct water channels were expressed in oocytes injected with mRNA obtained from sections of rat kidney: (a) CHIP28 water channels in cortex and medulla, (b) cAMP-activated water channels in medulla, and (c) cAMP-insensitive water channels in papilla.

scholarly journals The Vasopressin Receptor 2 Mutant R137L Linked to the Nephrogenic Syndrome of Inappropriate Antidiuresis (NSIAD) Signals through an Alternative Pathway that Increases AQP2 Membrane Targeting Independently of S256 Phosphorylation

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1354
Author(s):  
Marianna Ranieri ◽  
Maria Venneri ◽  
Tommaso Pellegrino ◽  
Mariangela Centrone ◽  
Annarita Di Mise ◽  
...  
Keyword(s):  
Water Permeability ◽  
Collecting Duct ◽  
Alternative Pathway ◽  
Water Channel ◽  
Rho Kinase ◽  
Membrane Targeting ◽  
Rock Inhibitor ◽  
Activating Mutations ◽  
Osmotic Water

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

Three distinct cell populations in rat kidney collecting duct

1987 ◽  
Vol 253 (2) ◽  
pp. C323-C328 ◽  
Author(s):  
H. Holthofer ◽  
B. A. Schulte ◽  
G. Pasternack ◽  
G. J. Siegel ◽  
S. S. Spicer
Keyword(s):  
Collecting Duct ◽  
Rat Kidney ◽  
Morphological Characteristics ◽  
Anion Channel ◽  
Band 3 ◽  
Cell Populations ◽  
Intercalated Cells ◽  
Distinct Cell ◽  
Collecting Ducts ◽  
Principal And Intercalated Cells

The morphologically heterogeneous cell populations in the collecting ducts of the rat kidney were studied using immunocytochemical detection of Na+-K+-ATPase and the anion channel (band 3) glycoprotein. Both enzymes were localized to the basal aspect of separate and morphologically distinct subpopulations of cells in various segments of the collecting duct. Na+-K+-ATPase appeared to be present exclusively in principal cells as identified by their morphology, whereas band 3 antibodies reacted only with intercalated cells. However, 5-20% of cells with the morphological characteristics of intercalated cells failed to react with either antisera in various segments of collecting ducts. As band 3 glycoprotein serves in exchanging intracellular bicarbonate for chloride, it is highly likely that the cells positive for this antigen secrete protons. The method introduced here appears thus useful for distinguishing between principal and intercalated cells by differences in their enzyme content and further for revealing two subpopulations of intercalated cells. This method promises to provide a useful approach for studying the principal and intercalated cell populations in various metabolic states.

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.

Permeability properties of rat renal lysosomes

1994 ◽  
Vol 266 (1) ◽  
pp. C121-C133 ◽  
Author(s):  
A. I. Piqueras ◽  
M. Somers ◽  
T. G. Hammond ◽  
K. Strange ◽  
H. W. Harris ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Lipid Bilayer ◽  
Gradient Centrifugation ◽  
Water Flux ◽  
Water Channel ◽  
Water Channels ◽  
Water Proton ◽  
Ph Gradients ◽  
Osmotic Water

Although lysosomes maintain large pH gradients and may be subjected to significant osmotic gradients in vivo, little is known about their passive permeability properties. In recent studies, vacuolar H(+)-adenosine-triphosphatases (ATPases), such as those found in lysosomes, have been suggested to act as water channels. In addition, the erythrocyte and proximal tubule water channel CHIP28 is present on the plasma membrane of proximal tubule cells and may undergo endocytosis so that it is incorporated in lysosomes. We therefore examined water, proton, and small nonelectrolyte permeabilities in freshly purified lysosomes from rat renal proximal tubule. Lysosomes were purified by differential and Percoll gradient centrifugation. The preparation contained only lysosomes when examined by electron microscopy. Moreover, analysis by flow cytometry showed virtually all particles to be positive for acid phosphatase and cathepsin B activities. Permeabilities were measured on a stopped-flow fluorimeter by monitoring the self-quenching or pH-sensitive quenching of entrapped fluorescein derivatives. Osmotic water permeability (Pf) averaged 0.011 +/- 0.003 cm/s (n = 6), a value similar to that of biological membranes containing water channels. However, Pf was insensitive to the organic mercurial reagent p-chloromercuribenzene-sulfonate and to HgCl2 and exhibited an activation energy of 10.8 +/- 0.8 kcal/mol. These results indicate that water flux in lysosomes occurred via the lipid bilayer, and not via water channels. Addition of ATP led to lysosomal acidification (proton flux = 4.6 +/- 0.8 x 10(-11) mmol H+.s-1.cm-2), which was completely inhibited by 0.1 microM bafilomycin. Pf was insensitive to this agent as was the passive proton permeability (0.36 +/- 0.18 cm/s, n = 4). Permeabilities to small nonelectrolytes varied in proportion to the oil-water partition coefficient, confirming the applicability of Overton's rule to lysosomes. We conclude that proximal tubular lysosomes exhibit high Pf, which occurs via the lipid bilayer and not via vacuolar H(+)-ATPase.

Escape from vasopressin-induced antidiuresis: role of vasopressin resistance of the collecting duct

1998 ◽  
Vol 274 (6) ◽  
pp. F1161-F1166 ◽  
Author(s):  
Carolyn A. Ecelbarger ◽  
Chung-Lin Chou ◽  
Alanna J. Lee ◽  
Susan R. DiGiovanni ◽  
Joseph G. Verbalis ◽  
...  
Keyword(s):  
Water Permeability ◽  
Collecting Duct ◽  
Water Channel ◽  
Phosphodiesterase Inhibitor ◽  
Intracellular Camp ◽  
Protein Subunit ◽  
Aquaporin 2 ◽  
Camp Generation ◽  
Osmotic Water ◽  
Camp Levels

Previously, we demonstrated that escape from vasopressin-induced antidiuresis (“vasopressin escape”) in rats is associated with a large, selective decrease in whole kidney expression of aquaporin-2, the vasopressin-regulated water channel. Here, we show that isolated perfused inner medullary collecting ducts (IMCDs) from vasopressin-escape rats {desamino-[d-arginine]vasopressin (DDAVP)/water-loaded} have dramatically reduced vasopressin-dependent osmotic water permeabilities [46% of control rats (DDAVP alone)], which coincides with a fall in inner medullary aquaporin-2 protein abundance as measured by immunoblotting in the opposite kidney. Furthermore, we demonstrate in IMCD suspensions that cAMP accumulation in response to DDAVP is substantially reduced in the vasopressin-escape rats both in the presence and absence of the phosphodiesterase inhibitor IBMX. By immunoblotting, we show that the abundance of two proteins important in cAMP generation: the stimulatory heterotrimeric G protein subunit Gsα and adenylyl cyclase type VI, do not change. We conclude that vasopressin escape is associated with relative vasopressin resistance of the collecting duct cells manifested by decreased intracellular cAMP levels. The decreased cAMP levels can contribute to the demonstrated decrease in collecting duct water permeability in two ways: 1) by causing a decrease in aquaporin-2 expression and 2) by limiting the acute action of vasopressin to increase collecting duct water permeability.

scholarly journals Tolvaptan as a tool in renal physiology

2014 ◽  
Vol 306 (3) ◽  
pp. F359-F366 ◽  
Author(s):  
Carlos A. Miranda ◽  
Jae Wook Lee ◽  
Chung-Lin Chou ◽  
Mark A. Knepper
Keyword(s):  
Collecting Duct ◽  
Rat Kidney ◽  
Water Channel ◽  
The United States ◽  
Competitive Inhibitor ◽  
Renal Physiology ◽  
Brattleboro Rat ◽  
V2 Receptor

For decades, the Brattleboro rat has been a useful model in kidney physiology. These animals manifest central diabetes insipidus (lack of circulating vasopressin) due to a mutation in the vasopressin-neurophysin gene. V2 receptor-mediated vasopressin actions in the kidney can be assessed in these animals by infusing the V2-selective vasopressin analog 1-desamino-8-d-arginine vasopressin (dDAVP). However, the major commercial supplier in the United States has ceased production, creating the need for another reliable experimental model of V2 receptor-mediated vasopressin action in rodents. We designed an in vivo protocol to investigate vasopressin responses in the rat kidney using osmotic minipumps loaded with tolvaptan, a nonpeptide competitive inhibitor of the vasopressin V2 receptor. Tolvaptan-infused rats had a mean urinary osmolality of <300 vs. >2,000 mosmol/kgH2O in vehicle-infused rats. The tolvaptan infusion produced large decreases in the renal abundance of aquaporin-2 (AQP2), aquaporin-3 (AQP3), the β-subunit of the epithelial sodium channel (β-ENaC), and γ-ENaC that were comparable to the differences seen in vehicle-infused vs. vasopressin-infused Brattleboro rats. Thus we conclude that tolvaptan infusion in rats provides an additional model (besides dDAVP-infusion in the Brattleboro rat) for the assessment of V2 receptor-mediated vasopressin actions in the kidney. We also provide ancillary in vitro data in rat inner-medullary-collecting-duct suspensions showing that tolvaptan can block vasopressin's effects on phosphorylation of the water channel AQP2 in vitro. Specifically, tolvaptan almost completely inhibited the ability of vasopressin to increase AQP2 phosphorylation at Ser256, Ser264, and Ser269, while strongly inhibiting a vasopressin-induced decrease in AQP2 phosphorylation at Ser261.

Sign in / Sign up

Export Citation Format

Share Document