Transepithelial water and urea permeabilities of isolated perfused Munich-Wistar rat inner medullary thin limbs of Henle's loop

To better understand the role that water and urea fluxes play in the urine concentrating mechanism, we determined transepithelial osmotic water permeability ( Pf) and urea permeability ( Purea) in isolated perfused Munich-Wistar rat long-loop descending thin limbs (DTLs) and ascending thin limbs (ATLs). Thin limbs were isolated either from 0.5 to 2.5 mm below the outer medulla (upper inner medulla) or from the terminal 2.5 mm of the inner medulla. Segment types were characterized on the basis of structural features and gene expression levels of the water channel aquaporin 1, which was high in the upper DTL (DTLupper), absent in the lower DTL (DTLlower), and absent in ATLs, and the Cl-1 channel ClCK1, which was absent in DTLs and high in ATLs. DTLupper Pf was high (3,204.5 ± 450.3 μm/s), whereas DTLlower showed very little or no osmotic Pf (207.8 ± 241.3 μm/s). Munich-Wistar rat ATLs have previously been shown to exhibit no Pf. DTLupper Purea was 40.0 ± 7.3 × 10−5 cm/s and much higher in DTLlower (203.8 ± 30.3 × 10−5 cm/s), upper ATL (203.8 ± 35.7 × 10−5 cm/s), and lower ATL (265.1 ± 49.8 × 10−5 cm/s). Phloretin (0.25 mM) did not reduce DTLupper Purea, suggesting that Purea is not due to urea transporter UT-A2, which is expressed in short-loop DTLs and short portions of some inner medullary DTLs close to the outer medulla. In summary, Purea is similar in all segments having no osmotic Pf but is significantly lower in DTLupper, a segment having high osmotic Pf. These data are inconsistent with the passive mechanism as originally proposed.

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Expression of Aquaporin-1 in the Peritoneal Tissues: Localization and Regulation by Hyperosmolality

Peritoneal Dialysis International ◽

10.1177/089686080202200303 ◽

2002 ◽

Vol 22 (3) ◽

pp. 307-315 ◽

Cited By ~ 12

Author(s):

Tomoko Ota ◽

Michio Kuwahara ◽

Shuling Fan ◽

Yoshio Terada ◽

Takashi Akiba ◽

...

Keyword(s):

Endothelial Cells ◽

Water Permeability ◽

Water Channel ◽

Mesothelial Cells ◽

Scattering Method ◽

Osmotic Water Permeability ◽

Peritoneal Mesothelial Cells ◽

Aquaporin 1 ◽

Osmotic Water ◽

Aqp1 Expression

Objective The purpose of this study was to determine the localization of the aquaporin-1 (AQP1) water channel in peritoneal tissues and the effect of hyperosmolality on the peritoneal expression and function of AQP1. Methods Immunohistochemical localization of AQP1 was identified in rat peritoneal tissues. Cultured rat peritoneal mesothelial cells (RPMCs) were exposed to hyperosmolality by adding 4% glucose to the culture medium. After 1 hour, 4 hours, 24 hours, and 48 hours, AQP1 was identified by semiquantitative immunoblot and immunocytochemistry. Osmotic water permeability was measured using a light-scattering method. Results Immunohistochemistry of rat peritoneal tissues showed the presence of AQP1 in mesothelial cells, venular endothelial cells, and capillary endothelial cells, but not in arteriole and interstitial cells. Semiquantitative immunoblot revealed that exposure to hyperosmolality significantly increased AQP1 expression after 24 hours in whole RPMC lysates (3.3-fold at 24 hours and 3.9-fold at 48 hours). Consistent with the immunoblot, osmotic water permeability of RPMC was augmented 1.7-fold and 2.7-fold after 1 hour and 24 hours, respectively, in a hyperosmotic environment. In RPMC membrane fractions, AQP1 expression was significantly increased after 1 hour of exposure to hyperosmolality (3.9-fold at 1 hour, 7.1-fold at 4 hours, and 8.7-fold at 24 hours). Immunocytochemistry of RPMCs showed that AQP1 was gradually redistributed from the perinuclear area to the peripheral cytoplasm, and then to the plasma membrane after a 1-hour hyperosmotic challenge, suggesting hyperosmolality-induced translocation of AQP1. Upregulation of AQP1 was also observed in the omentum of rats loaded intraperitoneally with hyperosmotic dialysate every day for 10 weeks. Conclusion AQP1 is widely distributed in the peritoneal cavity and may provide the major aqueous pathway across the peritoneal barrier. In addition, our findings suggested that hyperosmolality increases AQP1-dependent water permeability in peritoneal tissues by regulating the translocation and synthesis of AQP1 protein.

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

International Journal of Molecular Sciences ◽

10.3390/ijms222212535 ◽

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.

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Inhibition of the Aquaporin-1 Cation Conductance by Selected Furan Compounds Reduces Red Blood Cell Sickling

Frontiers in Pharmacology ◽

10.3389/fphar.2021.794791 ◽

2022 ◽

Vol 12 ◽

Author(s):

Pak Hin Chow ◽

Charles D. Cox ◽

Jinxin V. Pei ◽

Nancy Anabaraonye ◽

Saeed Nourmohammadi ◽

...

Keyword(s):

Red Blood Cells ◽

Blood Cells ◽

Water Channel ◽

Ion Conductance ◽

Aquaporin 1 ◽

Hypoxic Conditions ◽

Combined Application ◽

Cation Conductance ◽

Osmotic Water ◽

Combined Features

In sickle cell disease (SCD), the pathological shift of red blood cells (RBCs) into distorted morphologies under hypoxic conditions follows activation of a cationic leak current (Psickle) and cell dehydration. Prior work showed sickling was reduced by 5-hydroxylmethyl-2-furfural (5-HMF), which stabilized mutant hemoglobin and also blocked the Psickle current in RBCs, though the molecular basis of this 5-HMF-sensitive cation current remained a mystery. Work here is the first to test the hypothesis that Aquaporin-1 (AQP1) cation channels contribute to the monovalent component of Psickle. Human AQP1 channels expressed in Xenopus oocytes were evaluated for sensitivity to 5-HMF and four derivatives known to have differential efficacies in preventing RBC sickling. Ion conductances were measured by two-electrode voltage clamp, and osmotic water permeability by optical swelling assays. Compounds tested were: 5-HMF; 5-PMFC (5-(phenoxymethyl)furan-2-carbaldehyde); 5-CMFC (5-(4-chlorophenoxymethyl)furan-2-carbaldehyde); 5-NMFC (5-(2-nitrophenoxymethyl)-furan-2-carbaldehyde); and VZHE006 (tert-butyl (5-formylfuran-2-yl)methyl carbonate). The most effective anti-sickling agent, 5-PMFC, was the most potent inhibitor of the AQP1 ion conductance (98% block at 100 µM). The order of sensitivity of the AQP1 conductance to inhibition was 5-PMFC > VZHE006 > 5-CMFC ≥ 5-NMFC, which corresponded with effectiveness in protecting RBCs from sickling. None of the compounds altered AQP1 water channel activity. Combined application of a selective AQP1 ion channel blocker AqB011 (80 µM) with a selective hemoglobin modifying agent 5-NMFC (2.5 mM) increased anti-sickling effectiveness in red blood cells from human SCD patients. Another non-selective cation channel known to be expressed in RBCs, Piezo1, was unaffected by 2 mM 5-HMF. Results suggest that inhibition of AQP1 ion channels and capacity to modify hemoglobin are combined features of the most effective anti-sickling agents. Future therapeutics aimed at both targets could hold promise for improved treatments for SCD.

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UT-A2: a 55-kDa urea transporter in thin descending limb whose abundance is regulated by vasopressin

AJP Renal Physiology ◽

10.1152/ajprenal.2000.278.1.f52 ◽

2000 ◽

Vol 278 (1) ◽

pp. F52-F62 ◽

Cited By ~ 112

Author(s):

J. B. Wade ◽

A. J. Lee ◽

J. Liu ◽

C. A. Ecelbarger ◽

C. Mitchell ◽

...

Keyword(s):

Polyclonal Antibodies ◽

Renal Medulla ◽

Brattleboro Rats ◽

Urea Transporter ◽

Outer Medulla ◽

Aquaporin 1 ◽

Henle's Loop ◽

Collecting Ducts ◽

Chronic Infusion ◽

Immunofluorescence Labeling

The renal urea transporter gene (UT-A) produces different transcripts in the inner medullary collecting ducts (UT-A1) and thin descending limbs of Henle's loop (UT-A2), coding for distinct proteins. Peptide-directed rabbit polyclonal antibodies were used to identify the UT-A2 protein in renal medulla of mouse and rat. In the inner stripe of outer medulla, an antibody directed to the COOH terminus of UT-A recognized a membrane protein of 55 kDa. The abundance of this 55-kDa protein was strongly increased in response to chronic infusion of the vasopressin analog 1-deamino-[8-d-arginine]vasopressin (DDAVP) in Brattleboro rats, consistent with previous evidence that UT-A2 mRNA abundance is markedly increased. Immunofluorescence labeling with the COOH-terminal antibody in Brattleboro rats revealed labeling in the lower portion of descending limbs from short-looped nephrons (in the aquaporin-1-negative portion of this segment). This UT-A labeling was increased in response to DDAVP. Increased labeling was also seen in descending limbs of long-looped nephrons in the base of the inner medulla. These results indicate that UT-A2 is expressed as a 55-kDa protein in portions of the thin descending limbs of Henle's loop and that the abundance of this protein is strongly upregulated by vasopressin.

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Urea transporter UT-A1 and aquaporin-2 proteins decrease in response to angiotensin II or norepinephrine-induced acute hypertension

AJP Renal Physiology ◽

10.1152/ajprenal.00173.2006 ◽

2006 ◽

Vol 291 (5) ◽

pp. F952-F959 ◽

Cited By ~ 27

Author(s):

Janet D. Klein ◽

Brian P. Murrell ◽

Suzanne Tucker ◽

Young-Hee Kim ◽

Jeff M. Sands

Keyword(s):

Water Channel ◽

Urine Volume ◽

Urine Osmolality ◽

Ang Ii ◽

Urea Transporter ◽

Outer Medulla ◽

Acute Hypertension ◽

Water Excretion ◽

Transporter Protein ◽

Aquaporin 2

The kidney responds to high levels of ANG II, as may occur during malignant hypertension, by increasing sodium and water excretion. To study whether kidney medullary transporters contribute to this response, rats were made hypertensive using ANG II. Within 3 days of being given ANG II, systolic blood pressure (BP) was increased (200 mmHg), vs control (130 mmHg), and remained high through day 14. Kidney inner medullary (IM) tip and base and outer medulla were analyzed for transporter protein abundance. There were significant decreases in UT-A1 urea transporter, aquaporin-2 (AQP2) water channel, and NKCC2/BSC1 Na+-K+-2Cl− cotransporter. To determine whether the decreases were a response to hypertension, ANG II, or an ANG II-induced increase in aldosterone, rats were given 1) norepinephrine (to increase BP) and 2) ANG II plus spironolactone (to block the mineralocorticoid receptor). Norepinephrine (7 days) increased BP, urine volume, sodium excretion, and decreased urine osmolality and UT-A1, AQP2, and NKCC2/BSC1 abundances, similar to ANG II. ANG II alone or with spironolactone yielded similar increases in BP, urine volume, and urine osmolality, and decreases in UT-A1 and AQP2 proteins in the IM tip. Plasma vasopressin was unaffected by treatment. Water diuresis did not change UT-A1 but decreased AQP2 and NKCC2/BSC1 abundances. We conclude that decreases in UT-A1, AQP2, and NKCC2/BSC1 proteins may contribute to the diuresis and natriuresis that occur following ANG II or norepinephrine-induced acute hypertension and do not appear to involve ANG II stimulation of aldosterone or thirst.

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Aquaporin-1 water channel expression in human kidney.

Journal of the American Society of Nephrology ◽

10.1681/asn.v811 ◽

1997 ◽

Vol 8 (1) ◽

pp. 1-14

Author(s):

A B Maunsbach ◽

D Marples ◽

E Chin ◽

G Ning ◽

C Bondy ◽

...

Keyword(s):

Proximal Tubule ◽

Water Channel ◽

Neck Region ◽

Human Kidney ◽

Proximal Tubules ◽

Mrna Levels ◽

Outer Medulla ◽

Aquaporin 1 ◽

Peritubular Capillaries

The pattern of aquaporin-1 water channel protein (AQP1) expression in the human kidney was analyzed by immunocytochemistry using semi-thin and optimized high-resolution immunoelectron microscopy based on freeze-substituted and Lowicryl HM20 embedded tissue. In addition, in situ hybridization was used to determine AQP1 mRNA distribution. Immunoblots revealed a 28-kd band and a 35- to 45-kd band corresponding to unglycosylated and glycosylated AQP1. Glomerular capillary endothelium exhibited extensive AQP1 labeling, whereas glomerular podocytes and Bowman's capsule epithelium were unlabeled. AQP1 was localized in the proximal tubule, including the neck region directly connected to the glomerulus. However, there was a marked difference in the level of expression between cross-sections of the convoluted part and the proximal straight tubules, the latter displaying the most intense labeling. AQP1 labeling continued uninterrupted from the proximal straight tubule into descending thin limbs in outer medulla. Abrupt transitions from heavily labeled to unlabeled segments of thin limbs were observed, primarily in the inner medulla. This may represent the transition from the water-permeable thin descending limb to the water-impermeable thin ascending limb. In addition, heavy labeling of fenestrated endothelium was also observed in peritubular capillaries in cortex, outer medulla, and inner medulla. Immunolabeling controls were negative. In situ hybridization documented a marked difference in AQP1 mRNA levels within the proximal tubule, with the greatest AQP1 mRNA expression in straight proximal tubules. Glomeruli also showed marked signals, and descending thin limbs exhibited extensive expression in exact concordance with the immunocytochemical results. It was concluded that: (1) AQP1 is present in all proximal tubule segments, including segment 1 and the neck region, but there is a pronounced difference in expression levels with respect to both protein and mRNA levels; (2) AQP1 labeling is observed in the endothelium of fenestrated peritubular capillaries, as well as fenestrated glomerular capillaries; (3) AQP1 labeling continues directly from proximal tubules to descending thin limbs; and (4) abrupt transitions from labeled to unlabeled thin limb epithelium are noted.

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Addendum to ?Regulation of the water channel aquaporin-1: isolation and reconstitution of the regulatory complex? 6Cell Boil. Int. 28(1) (2004) 7?719

Cell Biology International ◽

10.1016/s1065-6995(04)00053-8 ◽

2004 ◽

Author(s):

R ABUHAMDAH

Keyword(s):

Water Channel ◽

Aquaporin 1 ◽

Regulatory Complex

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Molecular sieving of small solutes by outer medullary descending vasa recta

AJP Renal Physiology ◽

10.1152/ajprenal.1997.272.5.f579 ◽

1997 ◽

Vol 272 (5) ◽

pp. F579-F586 ◽

Cited By ~ 7

Author(s):

T. L. Pallone ◽

M. R. Turner

Keyword(s):

Water Channel ◽

Fluorescein Isothiocyanate ◽

Pressure Gradients ◽

Volume Flux ◽

Aquaporin 1 ◽

A Value ◽

Vasa Recta ◽

Mathematical Simulations ◽

Molecular Sieving ◽

Hydrophilic Solutes

Molecular sieving of small solutes by outer medullary descending vasa recta (OMDVR). Descending vasa recta (DVR) plasma equilibrates with the medullary interstitium by volume efflux (Jv), as well as by influx of solutes. Jv is driven by transmural osmotic pressure gradients due to small hydrophilic solutes (delta pi s), NaCl and urea. DVR endothelium probably contains a "water-only" pathway most likely mediated by the aquaporin-1 (AQP1) water channel. We measured the ability of microperfused OMDVR to concentrate lumenal 22Na and [3H]raffinose when Jv was driven by transmural NaCl gradients. Collectate-to-perfusate ratios of 2 x 10(6) M(r) fluorescein isothiocyanate-labeled dextran volume marker (RDx), 22Na (RNa), and [3H]raffinose (Rraf) were measured in the absence and presence of Jv. During volume efflux (Jv > 0), RDx was 1.37 +/- 0.31. RNa increased from 0.64 +/- 0.03 when Jv = 0 to 0.82 +/- 0.05 when Jv > 0 and Rraf increased from 0.83 +/- 0.03 to 1.13 +/- 0.05: Mathematical simulations predict RNa and Rraf most accurately when the OMDVR reflection coefficient to the tracers is assigned a value near unity. This indicates that the OMDVR wall contains a pathway for osmotic volume flux that excludes small hydrophilic solutes, a behavior consistent with that of aquaporins.

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