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.

scholarly journals Functional colocalization of water channels and proton pumps in endosomes from kidney proximal tubule.

1989 ◽  
Vol 93 (5) ◽  
pp. 885-902 ◽  
Author(s):  
R G Ye ◽  
L B Shi ◽  
W I Lencer ◽  
A S Verkman
Keyword(s):  
Proximal Tubule ◽  
Time Course ◽  
Apical Membrane ◽  
Water Channels ◽  
Fluorescence Signal ◽  
Proton Pumps ◽  
Osmotic Gradient ◽  
Proton Atpase ◽  
Osmotic Water

The apical membrane of mammalian proximal tubule undergoes rapid membrane cycling by exocytosis and endocytosis. Osmotic water and ATP-driven proton transport were measured in endocytic vesicles from rabbit and rat proximal tubule apical membrane labeled in vivo with the fluid phase marker fluorescein-dextran. Osmotic water permeability (Pf) was determined from the time course of fluorescein-dextran fluorescence after exposure of endosomes to an inward osmotic gradient in a stopped-flow apparatus. Pf was 0.009 (rabbit) and 0.029 cm/s (rat) (23 degrees C) and independent of osmotic gradient size. Pf in rabbit endosomes was inhibited reversibly by HgCl2 (KI = 0.2 mM) and had an activation energy of 6.4 +/- 0.5 kcal/mol (15-35 degrees C). Endosomal proton ATPase activity was measured from the time course of internal pH, measured by fluorescein-dextran fluorescence, after the addition of external ATP. Endosomes contained an ATP-driven proton pump that was sensitive to N-ethylmaleimide and insensitive to vanadate and oligomycin. In response to saturating [ATP] the pump acidified the endosomal compartment at a rate of 0.17 (rat) and 0.029 pH unit/s (rabbit); at an external pH of 7.4, the steady-state pH was 6.4 (rat) and 6.5 (rabbit). To examine whether water channels and the proton ATPase were present in the same endosome, the time course of fluorescein-dextran fluorescence was measured in response to an osmotic gradient in the presence and absence of ATP. ATP did not alter endosome Pf, but decreased the amplitude of the fluorescence signal by 43 +/- 3% (rabbit) and 47 +/- 4% (rat).(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Quantitation and topography of membrane proteins in highly water-permeable vesicles from ADH-stimulated toad bladder

1991 ◽  
Vol 261 (1) ◽  
pp. C143-C153 ◽  
Author(s):  
H. W. Harris ◽  
M. L. Zeidel ◽  
C. Hosselet
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Lipid Bilayer ◽  
Apical Membrane ◽  
Water Channel ◽  
Protein S ◽  
Water Channels ◽  
Toad Bladder ◽  
Western Blots ◽  
Vesicle Protein

Antidiuretic hormone (ADH) stimulation of toad bladder granular cells rapidly increases the osmotic water permeability (Pf) of their apical membranes by insertion of highly selective water channels. Before ADH stimulation, these water channels are stored in large cytoplasmic vesicles called aggrephores. ADH causes aggrephores to fuse with the apical membrane. Termination of ADH stimulation results in prompt endocytosis of water channel-containing membranes via retrieval of these specialized regions of apical membrane. Protein components of the ADH water channel contained within these retrieved vesicles would be expected to be integral membrane protein(s) that span the vesicle's lipid bilayer to create narrow aqueous channels. Our previous work has identified proteins of 55 (actually a 55/53-kDa doublet), 17, 15, and 7 kDa as candidate ADH water channel components. We now have investigated these candidate ADH water channel proteins in purified retrieved vesicles. These vesicles do not contain a functional proton pump as assayed by Western blots of purified vesicle protein probed with anti-H(+)-ATPase antisera. Approximately 60% of vesicle protein is accounted for by three protein bands of 55, 53, and 46 kDa. Smaller contributions to vesicle protein are made by the 17- and 15-kDa proteins. Triton X-114-partitioning analysis shows that the 55, 53, 46, and 17 kDa are integral membrane proteins. Vectorial labeling analysis with two membrane-impermeant reagents shows that the 55-, 53-, and 46-kDa protein species span the lipid bilayer of these vesicles. Thus the 55-, 53-, and 46-kDa proteins possess characteristics expected for ADH water channel components. These data show that the 55- and 53- and perhaps the 46-, 17-, and 15-kDa proteins are likely components of aqueous transmembrane pores that constitute ADH water channels contained within these vesicles.

scholarly journals Aquaporin-1 Facilitates Transmesothelial Water Permeability: In Vitro and Ex Vivo Evidence and Possible Implications in Peritoneal Dialysis

2021 ◽  
Vol 22 (22) ◽  
pp. 12535
Author(s):  
Francesca Piccapane ◽  
Andrea Gerbino ◽  
Monica Carmosino ◽  
Serena Milano ◽  
Arduino Arduini ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Peritoneal Dialysis ◽  
Tight Junctions ◽  
Water Transport ◽  
Water Flux ◽  
Water Channel ◽  
Bathing Solution ◽  
Ussing Chambers ◽  
Aquaporin 1 ◽  
Osmotic Water

We previously showed that mesothelial cells in human peritoneum express the water channel aquaporin 1 (AQP1) at the plasma membrane, suggesting that, although in a non-physiological context, it may facilitate osmotic water exchange during peritoneal dialysis (PD). According to the three-pore model that predicts the transport of water during PD, the endothelium of peritoneal capillaries is the major limiting barrier to water transport across peritoneum, assuming the functional role of the mesothelium, as a semipermeable barrier, to be negligible. We hypothesized that an intact mesothelial layer is poorly permeable to water unless AQP1 is expressed at the plasma membrane. To demonstrate that, we characterized an immortalized cell line of human mesothelium (HMC) and measured the osmotically-driven transmesothelial water flux in the absence or in the presence of AQP1. The presence of tight junctions between HMC was investigated by immunofluorescence. Bioelectrical parameters of HMC monolayers were studied by Ussing Chambers and transepithelial water transport was investigated by an electrophysiological approach based on measurements of TEA+ dilution in the apical bathing solution, through TEA+-sensitive microelectrodes. HMCs express Zo-1 and occludin at the tight junctions and a transepithelial vectorial Na+ transport. Real-time transmesothelial water flux, in response to an increase of osmolarity in the apical solution, indicated that, in the presence of AQP1, the rate of TEA+ dilution was up to four-fold higher than in its absence. Of note, we confirmed our data in isolated mouse mesentery patches, where we measured an AQP1-dependent transmesothelial osmotic water transport. These results suggest that the mesothelium may represent an additional selective barrier regulating water transport in PD through functional expression of the water channel AQP1.

Evidence that aquaporin-1 mediates NaCl-induced water flux across descending vasa recta

1997 ◽  
Vol 272 (5) ◽  
pp. F587-F596 ◽  
Author(s):  
T. L. Pallone ◽  
B. K. Kishore ◽  
S. Nielsen ◽  
P. Agre ◽  
M. A. Knepper
Keyword(s):  
Water Permeability ◽  
Water Movement ◽  
Water Flux ◽  
Water Channel ◽  
Enzyme Linked Immunosorbent Assay ◽  
Aquaporin 1 ◽  
Vasa Recta ◽  
Water Efflux

Outer medullary descending vasa recta (OMDVR) were perfused in vitro, and volume efflux was measured by driving water movement with transmural gradients of NaCl or albumin. Consistent with mediation by water channels, p-chloromercuribenzenesulfonic acid (pCMBS) markedly inhibited volume flux induced by NaCl. Dithiothreitol reversed the inhibition, pCMBS did not significantly alter water flux induced by albumin. Osmotic water permeability (Pf) of the pCMBS-sensitive pathway of glutaraldehyde-fixed and nonfixed OMDVR was 1,102 +/- 449 and 1,257 +/- 718 microns/s (means +/- SD), respectively. pCMBS reduced Pf to near zero, whereas diffusional water permeability in the same vessels was only slightly inhibited. Immunoreactive aquaporin-1 (AQP1) measured by enzyme-linked immunosorbent assay in collagenase-treated and untreated OMDVR was 5.2 +/- 1.0 and 4.2 +/- 0.4 fmol/mm, respectively, values that account well for the experimental Pf. We conclude that OMDVR water flux driven by NaCl gradients is most likely mediated by the AQP1 water channel and that NaCl and urea gradients drive water efflux in vivo by this route.

scholarly journals Water, proton, and urea transport in toad bladder endosomes that contain the vasopressin-sensitive water channel.

1990 ◽  
Vol 95 (5) ◽  
pp. 941-960 ◽  
Author(s):  
L B Shi ◽  
D Brown ◽  
A S Verkman
Keyword(s):  
Urinary Bladder ◽  
Permeability Coefficient ◽  
Water Channel ◽  
Water Channels ◽  
Water Proton ◽  
Toad Urinary Bladder ◽  
Osmotic Gradient ◽  
Urea Transport ◽  
Proton Permeability ◽  
Quenching Assay

Vasopressin (VP) increases the water permeability of the toad urinary bladder epithelium by inducing the cycling of vesicles containing water channels to and from the apical membrane of granular cells. In this study, we have measured several functional characteristics of the endosomal vesicles that participate in this biological response to hormonal stimulation. The water, proton, and urea permeabilities of endosomes labeled in the intact bladder with fluorescent fluid-phase markers were measured. The diameter of isolated endosomes labeled with horse-radish peroxidase was 90-120 nm. Osmotic water permeability (Pf) was measured by a stopped-flow fluorescence quenching assay (Shi, L.-B., and A. S. Verkman. 1989. J. Gen. Physiol. 94:1101-1115). The number of endosomes formed when bladders were labeled in the absence of a transepithelial osmotic gradient increased with serosal [VP] (0-50 mU/ml), and endosome Pf was very high and constant (0.08-0.10 cm/s, 18 degrees C). When bladders were labeled in the presence of serosal-to-mucosal osmotic gradient, the number of functional water channels per endosome decreased (at [VP] = 0.5 mU/ml, Pf = 0.09 cm/s, 0 osmotic gradient; Pf = 0.02 cm/s, 180 mosmol gradient). Passive proton permeability was measured from the rate of pH decrease in voltage-clamped endosomes in response to a 1 pH unit gradient (pHin = 7.5, pHout = 6.5). The proton permeability coefficient (PH) was 0.051 cm/s at 18 degrees C in endosomes containing the VP-sensitive water channel; PH was not different from that measured in vesicles not containing water channels. Measurement of urea transport by the fluorescence quenching assay gave a urea reflection coefficient of 0.97 and a permeability coefficient of less than 10(-6) cm/s. These results demonstrate: (a) VP-induced endosomes from toad urinary bladder have extremely high Pf. (b) In states of submaximal bladder Pf, the density of functional water channels in endosomes in constant in the absence of an osmotic gradient, but decreases in the presence of a serosal-to-mucosal gradient, suggesting that the gradient has a direct effect on the efficiency of packaging of water channels into endosomes. (c) The VP-sensitive water channel does not have a high proton permeability. (d) Endosomes that cycle the water channel do not contain urea transporters. These results establish a labeling procedure in which greater than 85% of labeled vesicles from toad urinary bladder are endosomes that contain the VP-sensitive water channel in a functional form.

Proton/hydroxyl permeability of proximal tubule brush border vesicles

1985 ◽  
Vol 248 (1) ◽  
pp. F78-F86 ◽  
Author(s):  
H. E. Ives
Keyword(s):  
Activation Energy ◽  
Proximal Tubule ◽  
Brush Border ◽  
Buffer Capacity ◽  
Gradient Centrifugation ◽  
Sulfhydryl Group ◽  
Membrane Vesicles ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Ph Gradients

The net H+/OH- permeability of rabbit renal proximal tubule brush border membrane vesicles was determined by measuring the rate of collapse of preformed pH gradients using acridine orange. The membranes were voltage clamped using valinomycin and [K+]in = [K+]out. Internal buffer capacity was determined by titration of lysed vesicles and by titration of measured Na+/H+ exchange rates with exogenously added buffers. Both methods revealed an intravesicular buffer capacity of 125-135 mM/pH unit at pH 6.0 and 20 degrees C. Using this buffer capacity, the net H+/OH- permeability was found to be 5 X 10(-3) cm/s in brush border vesicles prepared by Mg2+ aggregation. The rate of collapse of pH gradients in brush border vesicles prepared by sucrose density gradient centrifugation was virtually identical to the rate in vesicles prepared with Mg2+, indicating that the high H+/OH- permeability was not an artifact of Mg2+ preparation. Activation energy of the H+/OH- permeability pathway was 4.9 kcal/mol, whereas activation energy of the Na+/H+ antiporter was 11.4 kcal/mol. Since the rate of H+/OH- diffusion was not affected by amiloride, it is concluded that H+/OH- permeate through brush border membranes by a pathway separate from the Na+/H+ antiporter. This pathway is not inhibited by dicyclohexylcarbodiimide at concentrations up to 2 mM but is inhibited by 0.2-5 mM p-chloromercuribenzenesulfonate, suggesting the presence of a sulfhydryl group in the pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Micropuncture study of proximal tubule pH in avian kidney

1987 ◽  
Vol 253 (4) ◽  
pp. R587-R591 ◽  
Author(s):  
G. Laverty ◽  
M. Alberici
Keyword(s):  
Proximal Tubule ◽  
Proximal Tubules ◽  
Ph Gradient ◽  
Chloride Diffusion ◽  
Ph Gradients ◽  
Single Lumen ◽  
Micropuncture Study ◽  
Gradient Based ◽  
Luminal Ph

Transepithelial potentials (PD) and pH gradients were measured by in vivo micropuncture in superficial proximal tubules of anesthetized European starlings. The average PD for 46 tubules was 2.24 +/- 1.17 mV (mean +/- SD), lumen negative. Only a single lumen-positive potential was recorded, even though late proximal segments are accessible to micropuncture. Proximal luminal pH, measured with single-barrel pH-sensitive microelectrodes, averaged 7.62 +/- 0.26. The pH of peritubular blood, also measured with microelectrodes, was 7.58 +/- 0.15. Correction of the luminal pH for the average PD resulted in a value of 7.58. A few measurements made with double-barrel microelectrodes indicated a pH in proximal tubules of 7.65 +/- 0.08 with a PD of -3.32 +/- 1.47 mV. Thus there appears to be no transepithelial pH gradient across these tubules. On the other hand, pH measurements of cortical collecting ducts averaged 6.40 +/- 0.37, indicating significant acidification in more distal segments. Starlings made acutely acidotic by HCl infusion had significantly reduced arterial and renal cortical pH values, but there was still no significant pH gradient across the proximal tubule. In conclusion, these superficial "reptilian-type" proximal tubules do not appear to have the capacity to maintain a pH gradient. Based on this and on the lack of lumen-positive chloride diffusion potentials, there appears to be no early proximal preferential bicarbonate reabsorption by these nephrons.

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.

Quantification of osmotic water transport in vivo using fluorescent albumin

2014 ◽  
Vol 307 (8) ◽  
pp. F981-F989 ◽  
Author(s):  
Johann Morelle ◽  
Amadou Sow ◽  
Didier Vertommen ◽  
François Jamar ◽  
Bengt Rippe ◽  
...  
Keyword(s):  
Peritoneal Dialysis ◽  
Water Transport ◽  
Molecular Mechanisms ◽  
Water Channel ◽  
Transgenic Animals ◽  
Peritoneal Membrane ◽  
Intraperitoneal Pressure ◽  
Alexa Fluor ◽  
Osmotic Water

Osmotic water transport across the peritoneal membrane is applied during peritoneal dialysis to remove the excess water accumulated in patients with end-stage renal disease. The discovery of aquaporin water channels and the generation of transgenic animals have stressed the need for novel and accurate methods to unravel molecular mechanisms of water permeability in vivo. Here, we describe the use of fluorescently labeled albumin as a reliable indicator of osmotic water transport across the peritoneal membrane in a well-established mouse model of peritoneal dialysis. After detailed evaluation of intraperitoneal tracer mass kinetics, the technique was validated against direct volumetry, considered as the gold standard. The pH-insensitive dye Alexa Fluor 555-albumin was applied to quantify osmotic water transport across the mouse peritoneal membrane resulting from modulating dialysate osmolality and genetic silencing of the water channel aquaporin-1 (AQP1). Quantification of osmotic water transport using Alexa Fluor 555-albumin closely correlated with direct volumetry and with estimations based on radioiodinated (125I) serum albumin (RISA). The low intraperitoneal pressure probably accounts for the negligible disappearance of the tracer from the peritoneal cavity in this model. Taken together, these data demonstrate the appropriateness of pH-insensitive Alexa Fluor 555-albumin as a practical and reliable intraperitoneal volume tracer to quantify osmotic water transport in vivo.

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