Biomedical subjects

Baoxue Yang

Publications and source records attributed to Baoxue Yang.

At least 19 recordsLinked to original sources

PubMed2006

Comparative efficacy of HgCl2 with candidate aquaporin-1 inhibitors DMSO, gold, TEA+ and acetazolamide.

Aquaporin-1 (AQP1) inhibitors are predicted to have multiple clinical applications. Hg(++) is a non-specific and toxic AQP1 blocker. We compared compounds with reported AQP1 inhibition activity, including DMSO, Au(+++), Ag(+), tetraethylammonium and acetazolamide. Water permeability was measured by stopped-flow light scattering in erythrocytes and volume marker dilution in epithelial cells. Au(+++) inhibited AQP1 with IC50 approximately 14 microM, similar to 10 microM for Hg(++). DMSO slowed osmotic equilibration; however, the apparent inhibition was due to 'osmotic clamp' rather than AQP1 inhibition. Neither tetraethylammonium nor acetazolamide (to 10 mM) inhibited AQP1. Our data indicate the need to identify new AQP1 inhibitors.

Acetazolamide↗

PubMed2006

Hypertonic saline therapy in cystic fibrosis: Evidence against the proposed mechanism involving aquaporins.

Recent data indicate the clinical benefit of nebulized hypertonic saline in cystic fibrosis lung disease, with a proposed mechanism involving sustained increase in airway surface liquid volume. To account for the paradoxical observation that amiloride suppresses the beneficial effect of hypertonic saline, it has been previously concluded (Donaldson, S. H., Bennett, W. D., Zeman, K. L., Knowles, M. R., Tarran, R., and Boucher, R. C. (2006) N. Engl. J. Med. 354, 241-250) that amiloride-inhibitable aquaporin (AQP) water channels in airway epithelia modulate airway surface liquid volume. Here, we have characterized water permeability and amiloride effects in well differentiated, primary cultures of human airway epithelial cells, stably transfected Fisher rat thyroid epithelial cells expressing individual airway/lung AQPs, and perfused mouse lung. We found high transepithelial water permeability (P(f), 54 +/- 5 microm/s) in airway epithelial cells that was weakly temperature-dependent and inhibited by >90% by reduced pH in the basal membrane-facing solution. Reverse transcription-PCR and immunofluorescence suggested the involvement of AQPs 3, 4, and 5 in high airway water permeability. Experiments using several sensitive measurement methods indicated that amiloride does not inhibit water permeability in non-cystic fibrosis (non-CF) or CF airway epithelia, AQP-transfected Fisher rat thyroid cells, or intact lung. Our data provide evidence against the mechanism proposed by Donaldson et al. to account for the effects of amiloride and hypertonic saline in CF lung disease, indicating the need to identify alternate mechanisms.

Amiloride↗

PubMed2006

Evidence against functionally significant aquaporin expression in mitochondria.

Recent reports suggest the expression of aquaporin (AQP)-type water channels in mitochondria from liver (AQP8) (Calamita, G., Ferri, D., Gena, P., Liquori, G. E., Cavalier, A., Thomas, D., and Svelto, M. (2005) J. Biol. Chem. 280, 17149-17153) and brain (AQP9) (Amiry-Moghaddam, M., Lindland, H., Zelenin, S., Roberg, B. A., Gundersen, B. B., Petersen, P., Rinvik, E., Torgner, I. A., and Ottersen, O. P. (2005) FASEB J. 19, 1459-1467), where they were speculated to be involved in metabolism, apoptosis, and Parkinson disease. Here, we systematically examined the functional consequence of AQP expression in mitochondria by measurement of water and glycerol permeabilities in mitochondrial membrane preparations from rat brain, liver, and kidney and from wild-type versus knock-out mice deficient in AQPs -1, -4, or -8. Osmotic water permeability, measured by stopped-flow light scattering, was similar in all mitochondrial preparations, with a permeability coefficient P(f) approximately 0.009 cm/s. Glycerol permeability was also similar ( approximately 5 x 10(-6) cm/s) in the various preparations. HgCl(2) slowed osmotic equilibration comparably in mitochondria from wild-type and AQP-deficient mice, although the slowing was explained by altered mitochondrial size rather than reduced P(f). Immunoblot analysis of mouse liver mitochondria failed to detect AQP8 expression, with liver homogenates from wild-type/AQP8 null mice as positive/negative controls. Our results provide evidence against functionally significant AQP expression in mitochondria, which is consistent with the high mitochondrial surface-to-volume ratio producing millisecond osmotic equilibration, even when intrinsic membrane water permeability is not high.

Animals↗

PubMed2006

Evidence from knockout mice against physiologically significant aquaporin 8-facilitated ammonia transport.

Aquaporin (AQP)8-facilitated transport of NH(3) has been suggested recently by increased NH(3) permeability in Xenopus oocytes and yeast expressing human or rat AQP8. We tested the proposed roles of AQP8-facilitated NH(3) transport in mammalian physiology by comparative phenotype studies in wild-type vs. AQP8-null mice. AQP8-facilitated NH(3) transport was confirmed in mammalian cell cultures expressing rat or mouse AQP8, in which the fluorescence of a pH-sensing yellow fluorescent protein was measured in response to ammonia (NH(3)/NH(4)(+)) gradients. Relative AQP8 single-channel NH(3)-to-water permeability was approximately 0.03. AQP8-facilitated NH(3) and water permeability in a native tissue was confirmed in membrane vesicles isolated from testes of wild-type vs. AQP8-null mice, in which BCECF was used as an intravesicular pH indicator. A series of in vivo studies were done in mice, including 1) serum ammonia measurements before and after ammonia infusion, 2) renal ammonia clearance, 3) colonic ammonia absorption, and 4) liver ammonia accumulation and renal ammonia excretion after acute and chronic ammonia loading. Except for a small reduction in hepatic ammonia accumulation and increase in ammonia excretion in AQP8-null mice loaded with large amounts of ammonia, there were no significant differences in wild-type vs. AQP8-null mice. Our results support the conclusion that AQP8 can facilitate NH(3) transport but provide evidence against physiologically significant AQP8-facilitated NH(3) transport in mice.

Ammonia↗

PubMed2006

Cardiac aquaporin expression in humans, rats, and mice.

Water accumulation in the heart is important in ischemia-reperfusion injury and operations performed by using cardiopulmonary bypass, with cardiac dysfunction associated with myocardial edema being the principal determinant of clinical outcome. As an initial step in determining the role of aquaporin (AQP) water channels in myocardial edema, we have assessed the myocardial expression of AQPs in humans, rats, and mice. RT-PCR revealed expression of AQP-1, -4, -6, -7, -8, and -11 transcripts in the mouse heart. AQP-1, -6, -7, and -11 mRNAs were found in the rat heart as well as low levels of AQP-4 and -9. Human hearts contained AQP-1, -3, -4, -5, -7, -9, -10, and -11 mRNAs. AQP-1 protein expression was confirmed by Western blot analysis in all three species. AQP-4 protein was detected in the mouse heart but not in the rat or human heart. To determine the potential functional consequences of myocardial AQP expression, water permeability was measured in plasma membrane vesicles from myocardial cells of wild-type versus various AQP knockout mice. Water permeability was reduced by AQP-1 knockout but not by AQP-4 or AQP-8 knockout. With the use of a model of isolated rat heart perfusion, it was found that osmotic and ischemic stresses are not associated with changes in AQP-1 or AQP-4 expression. These studies support a possible functional role of AQP-1 in myocardium but indicate that early adaptations to osmotic and ischemic stress do not involve transcriptional or posttranslational AQP-1 regulation.

Animals↗

PubMed2006

Urea and urine concentrating ability in mice lacking AQP1 and AQP3.

Aquaporin-1 (AQP1) and aquaporin-3 (AQP3) water channels expressed in the kidney play a critical role in the urine concentrating mechanism. Mice with AQP1 or AQP3 deletion have a urinary concentrating defect. To better characterize this defect, we studied the influence of an acute urea load (300 mumol ip) in conscious AQP1-null, AQP3-null, and wild-type mice. Urine was collected and assayed every 2 h, from 2 h before (baseline) to 8 h after the urea load. Mice of all genotypes excreted the urea load in approximately 4 h with the same time course. Interestingly, despite their low baseline, the AQP3-null mice raised their urine osmolality and urea concentration progressively after the urea load to values almost equal to those in wild-type mice at 8 h. In contrast, urine non-urea solute concentration did not change. Urine volume fell in the last 4 h to about one-fourth of basal values. AQP1-null mice increased their urine flow rate much more than AQP3-null mice and showed no change in urine osmolality and urea concentration. The urea load strongly upregulated urea transporter UT-A3 expression in all three genotypes. These observations show that the lack of AQP3 does not interfere with the ability of the kidney to concentrate urea but impairs its ability to concentrate other solutes. This solute-selective response could result from the capacity of AQP3 to transport not only water but also urea. The results suggest a novel role for AQP3 in non-urea solute concentration in the urine.

Animals↗

PubMed2006

Mouse model of inducible nephrogenic diabetes insipidus produced by floxed aquaporin-2 gene deletion.

Transgenic mouse models of defective urinary concentrating ability produced by deletion of various membrane transport or receptor proteins, including aquaporin-2 (AQP2), are associated with neonatal mortality from polyuria. Here, we report an inducible mouse model of AQP2 gene deletion with severe polyuria in adult mice. LoxP sequences were inserted into introns 1 and 2 in the mouse AQP2 gene by homologous recombination in embryonic stem cells. Mating of germ-line AQP2-loxP mice with tamoxifen-inducible Cre-expressing mice produced offspring with inducible homozygous Cre-AQP2-loxP, which had a normal phenotype. Tamoxifen injections over 10 days resulted in AQP2 gene excision, with undetectable full-length AQP2 transcript in kidney and a >95% reduction in immunoreactive AQP2 protein. Urine osmolality decreased from approximately 2,000 to <500 mosmol/kgH(2)O after 4-5 days, with urine output increasing from 2 to 25 ml/day. Urine osmolality did not increase after water deprivation. Interestingly, AQP3 protein expression in the collecting duct was increased by about fivefold after AQP2 gene excision. Mild renal damage was seen after 6 wk of polyuria, with collecting duct dilatation, yet normal creatinine clearance and serum chemistries. These results establish the first adult model of nephrogenic diabetes insipidus (NDI) caused by AQP2 deficiency, with daily urine output comparable to body weight, although remarkable preservation of renal function compared with non-inducible NDI models.

Animals↗

PubMed2006

Decreased saliva secretion and down-regulation of AQP5 in submandibular gland in irradiated rats.

The molecular mechanisms of radiation-induced xerostomia remain unclear. The purpose of this study was to investigate the alterations of aquaporins (AQPs) and Na(+)/K(+)-ATPase in irradiated rat submandibular glands and to test the hypothesis that down-regulation of AQP5 expression in irradiated salivary glands is one of the mechanisms of radiation-induced xerostomia. Saliva from control and irradiated rat submandibular glands was analyzed. The mRNA level of AQP5 in the submandibular glands was assessed by semi-quantitative RT-PCR and in situ hybridization. The protein expression of AQP5, AQP1 and Na(+)/K(+)-ATPase was determined by Western blotting and immunohistochemistry. The body weight, submandibular gland weight, and saliva secretion of irradiated rats significantly decreased by 12, 24 and 32% on day 3 and 24, 16 and 38% on day 30 postirradiation, respectively. There was a significant increase in the protein concentration and osmolality of saliva in irradiated rats on days 3 and 30 postirradiation. However, there was no significant difference between irradiated and control rats in total saliva protein secretion. RT-PCR analysis showed that mRNA expression of AQP5 was significantly down-regulated by 37 and 51% in irradiated rats on days 3 and 30 postirradiation, respectively. Immunoblotting showed that the AQP5 protein level was decreased by 40 and 60% in irradiated glands, in contrast to the slight reductions of AQP1 and Na(+)/K(+)-ATPase proteins. Immunohistochemical analysis demonstrated that loss of AQP5 protein occurred throughout the irradiated glands, while no significant reduction was detected in AQP1 and Na(+)/ K(+)-ATPase labeling density. These results suggest that the preferential down-regulation of AQP5 with minor effects on AQP1 and Na(+)/K(+)-ATPase may contribute to radiation-induced salivary dysfunction.

Animals↗

PubMed2005

Sulfamoyl-4-oxoquinoline-3-carboxamides: novel potentiators of defective DeltaF508-cystic fibrosis transmembrane conductance regulator chloride channel gating.

The synthesis of a small collection of sulfamoyl-4-oxoquinoline-3-carboxamides is described for use as correctors of defective gating of the DeltaF508-cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. Several compounds with submicromolar potency were obtained. N-Ethyl 6-(ethylphenylsulfamoyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (7b) was found to be the most effective sulfonamide corrector of defective DeltaF508-CFTR gating.

Chloride Channels↗

PubMed2005

Phenotype analysis of aquaporin-8 null mice.

Aquaporin-8 (AQP8) is a water-transporting protein expressed in organs of the mammalian gastrointestinal tract (salivary gland, liver, pancreas, small intestine, and colon) and in the testes, heart, kidney, and airways. We studied the phenotype of AQP8-null mice, and mice lacking AQP8, together with AQP1 or AQP5. AQP8-knockout mice lacked detectable AQP8 transcript and protein, and had reduced water permeability in plasma membranes from testes. Breeding of AQP8 heterozygous mice yielded AQP8-null mice, whose number, survival, and growth were not different from those of wild-type mice. Organ weight and serum/urine chemistries were similar in wild-type and AQP8-null mice, except for increased testicular weight in the null mice (4.8 +/- 0.7 vs. 7.3 +/- 0.3 mg/g body wt). Urinary concentrating ability in AQP8-null mice was unimpaired as assessed by urine osmolality (3,590 +/- 360 mosmol/kgH(2)O) and weight loss (22 +/- 2%) after 36-h water deprivation; urinary concentrating ability was similarly impaired in AQP1-null mice vs. AQP8/AQP1 double-knockout mice. Agonist-driven fluid secretion in salivary gland was not different in AQP8 vs. wild-type mice ( approximately 1 microl.min(-1).g body wt(-1)) or in AQP5-null mice vs. AQP8/AQP5 double-knockout mice. Closed intestinal loop measurements in vivo indicated unimpaired osmotically driven water transport, active fluid absorption, and cholera toxin-driven fluid secretion in AQP8-null mice. After 21 days on a 50% fat diet, wild-type and AQP8-null mice had similar weight gain ( approximately 15 g), with no evidence of steatorrhea or abnormalities in blood chemistries, except for mild hypertriglyceridemia in the null mice. The mild phenotype of AQP8-null mice was surprising in view of the multiple phenotype abnormalities found in mouse models of AQP1-5 deficiency.

Animals↗

PubMed2005

Urea and urine concentrating ability: new insights from studies in mice.

Urea is the most abundant solute in the urine in humans (on a Western-type diet) and laboratory rodents. It is far more concentrated in the urine than in plasma and extracellular fluids. This concentration depends on the accumulation of urea in the renal medulla, permitted by an intrarenal recycling of urea among collecting ducts, vasa recta and thin descending limbs, all equipped with specialized, facilitated urea transporters (UTs) (UT-A1 and 3, UT-B, and UT-A2, respectively). UT-B null mice have been recently generated by targeted gene deletion. This review describes 1) the renal handling of urea by the mammalian kidney; 2) the consequences of UT-B deletion on urinary concentrating ability; and 3) species differences among mice, rats, and humans related to their very different body size and metabolic rate, leading to considerably larger needs to excrete and to concentrate urea in smaller species (urea excretion per unit body weight in mice is 5 times that in rats and 23 times that in humans). UT-B null mice have a normal glomerular filtration rate but moderately reduced urea clearance. They exhibit a 30% reduction in urine concentrating ability with a more severe defect in the capacity to concentrate urea (50%) than other solutes, despite a twofold enhanced expression of UT-A2. The urea content of the medulla is reduced by half, whereas that of chloride is almost normal. When given an acute urea load, UT-B null mice are unable to raise their urinary osmolality, urine urea concentration (Uurea), and the concentration of non-urea solutes, as do wild-type mice. When fed diets with progressively increasing protein content (10, 20, and 40%), they cannot prevent a much larger increase in plasma urea than wild-type mice because they cannot raise Uurea. In both wild-type and UT-B null mice, urea clearance was higher than creatinine clearance, suggesting the possibility that urea could be secreted in the mouse kidney, thus allowing more efficient excretion of the disproportionately high urea load. On the whole, studies in UT-B null mice suggest that recycling of urea by countercurrent exchange in medullary vessels plays a more crucial role in the overall capacity to concentrate urine than its recycling in the loops of Henle.

Animals↗

PubMed2005

A novel mutation at the JK locus causing Jk null phenotype in a Chinese family.

Urea transporters are a group of proteins that facilitate urea movement across biological membranes. Kidd blood group (Jk antigen) and urea transporter of human erythrocytes are carried by the same protein UT-B. To investigate the molecular basis of the Jk null phenotype in the Chinese population, blood samples from Chinese individuals were screened using the 2 mol/L urea solution hemolysis test. Urea and water permeability of erythrocytes membrane was measured by stopped-flow light scattering. Genomic DNA was extracted from lymphocytes. UT-B gene of JKnnu's family was analyzed using genomic PCR by primers designed to cover sequences of all exons and exon-intron boundaries in human UT-B gene. One Jk null subject was found from twenty thousand screened Chinese individuals, and it was confirmed that this individual did not express the erythrocyte urea transporter. Genomic sequence analysis of the Jk null individual showed that there were two point mutations, G-->C, which is novel, and G-->A, at the 3'-acceptor splice site (AG) of intron 5 of UT-B gene. Exon 6 is spliced out in the UT-B transcript due to either of these mutations. Water permeability in Jk null erythrocytes (Pf, -0.00037 cm/s) was significantly lower than that in normal erythrocytes (Pf, -0.00062 cm/s) after HgCl2 incubation, providing evidence for UT-B facilitated water transport in human erythrocytes.

Asian People↗

PubMed2004

ClC-3 chloride channels facilitate endosomal acidification and chloride accumulation.

We investigated the involvement of ClC-3 chloride channels in endosomal acidification by measurement of endosomal pH and chloride concentration [Cl-] in control versus ClC-3-deficient hepatocytes and in control versus ClC-3-transfected Chinese hamster ovary cells. Endosomes were labeled with pH or [Cl-]-sensing fluorescent transferrin (Tf), which targets to early/recycling endosomes, or alpha2-macroglobulin (alpha2M), which targets to late endosomes. In pulse label-chase experiments, [Cl-] was 19 mM just after internalization in alpha2M-labeled endosomes in primary cultures of hepatocytes from wild-type mice, increasing to 58 mM over 45 min, whereas pH decreased from 7.1 to 5.4. Endosomal acidification and [Cl-] accumulation were significantly impaired in hepatocytes from ClC-3 knock-out mice, with [Cl-] increasing from 16 to 43 mM and pH decreasing from 7.1 to 6.0. Acidification and Cl- accumulation were blocked by bafilomycin. In Tf-labeled endosomes, [Cl-] was 46 mM in wild-type versus 35 mM in ClC-3-deficient hepatocytes at 15 min after internalization, with corresponding pH of 6.1 versus 6.5. Approximately 4-fold increased Cl- conductance was found in alpha2M-labeled endosomes isolated from hepatocytes of wild-type versus ClC-3 null mice. In contrast, Golgi acidification was not impaired in ClC-3-deficient hepatocytes. In transfected Chinese hamster ovary cells expressing ClC-3A, endosomal acidification and [Cl-] accumulation were enhanced. [Cl-] in alpha2M-labeled endosomes was 42 mM (control) versus 53 mM (ClC-3A) at 45 min, with corresponding pH 5.8 versus 5.2; [Cl-] in Tf-labeled endosomes at 15 min was 37 mM (control) versus 49 mM (ClC-3A) with pH 6.3 versus 5.9. Our results provide direct evidence for involvement of ClC-3 in endosomal acidification by Cl- shunting of the interior-positive membrane potential created by the vacuolar H+ pump.

Acids↗

PubMed2004

Upregulation of urea transporter UT-A2 and water channels AQP2 and AQP3 in mice lacking urea transporter UT-B.

The UT-B urea transporter is the major urea transporter in red blood cells and kidney descending vasa recta. Humans and mice that lack UT-B have a mild urine-concentrating defect. Whether deletion of UT-B altered the expression of other transporter proteins involved in urinary concentration was tested. Fluorescence-based real-time reverse transcription-PCR and Northern blot analysis showed upregulation of the UT-A2 urea transporter and the aquaporin 2 (AQP2) and AQP3 water channel transcripts but no change in other urea transporters or AQP. Western blot analysis showed that UT-A2 protein abundance in the outer medulla of UT-B null mice increased to 122 +/- 6% of wild-type control. AQP2 protein abundance increased to 177 +/- 32% and 127 +/- 7% in the outer and inner medulla, respectively, of UT-B null versus wild-type mice. The abundance of UT-A1, AQP1, renal outer medullary potassium channel, and NKCC2/BSC1 proteins were not significantly different between UT-B null and wild-type mice. The increases in AQP2 and AQP3 would reduce water loss and improve concentrating ability. The lack of UT-B does not result in a change in expression of urea transporters involved in urea reabsorption from the inner medullary collecting duct (UT-A1 and UT-A3). However, UT-B null mice have a selective increase in UT-A2 protein abundance. This may be an adaptive response to the loss of UT-B, because UT-B and UT-A2 are involved in different intrarenal urea recycling pathways.

Animals↗

PubMed2004

[Reconstruction and expression of green fluorescent protein and aquaporin 7 fusion recombinant vector].

OBJECTIVE: To study the membrane mobility of aquaporin 7 (AQP7) by cloning stably transfected CHO cells with expression of pEGFP-C1-AQP7, in which AQP7 cDNA was fused downstream and in frame to pEGFP-C1 gene. METHODS: The full sequence of AQP7 was amplified by RT-PCR and then recombined in the downstream of the green fluorescent protein gene in the pEGFP-C1 vector. The recombinant vector pEGFP-C1-AQP7 was stably transfected into CHO cells. With fluorescent microscopy, immunocytochemical stain and Western blot, pEGFP-C1-AQP7 showed a predominant intracellular vesicular localization. RESULTS: (1) The sequence of AQP7 cDNA of the Wistar rat was logged into the GenBank (access number: AY157737). (2) Identification demonstrated that pEGFP-C1-AQP7 fusion protein stably expressed in CHO cells. (3) With fluorescence microscopy, pEGFP-C1-AQP7 showed a predominant intracellular vesicular localization. CONCLUSION: The CHO cell line with stable pEGFP-C1-AQP7 expression was set up successfully for advanced research.

Animals↗

PubMed2003

Lack of UT-B in vasa recta and red blood cells prevents urea-induced improvement of urinary concentrating ability.

Recycling of urea within the renal medulla is known to play an important role in the capacity of the kidney to concentrate urine. This recycling occurs simultaneously through a tubular and a vascular route (i.e., through the loops of Henle and vasa recta, respectively). In the present study, transgenic mice with a selective deficiency in UT-B (the urea transporter protein expressed in descending vasa recta and red blood cells), were used to evaluate the specific contribution of vascular urea recycling to overall urine-concentrating ability (UCA). The renal handling of urea was studied in normal conditions and after acute or chronic alterations in urea excretion (acute urea loading or variations in protein intake, respectively). In normal conditions, UT-B null mice exhibited a 44% elevation in plasma urea (Purea), a normal creatinine clearance, but a 25% decrease in urea clearance, with no change in that of sodium and potassium. Acute urea loading induced a progressive increase in urinary urea concentration (Uurea) in wild-type mice and a subsequent improvement in their UCA in contrast to UT-B null mice, in which urinary osmolality and Uurea did not rise, due to the failure to accumulate urea in the medulla. With increasing protein intake (from 10 to 40% protein in diet, leading to a 5-fold increase in urea excretion), Purea was further increased in null mice while little change was observed in wild-type mice, and null mice were not able to increase Uurea as did wild-type mice. In conclusion, this study in UT-B-deficient mice reveals that countercurrent exchange of urea in renal medullary vessels and red blood cells accounts for a major part of the kidney's concentrating ability and for the adaptation of renal urea handling during a high-protein intake.

Animals↗

PubMed2002

Analysis of double knockout mice lacking aquaporin-1 and urea transporter UT-B. Evidence for UT-B-facilitated water transport in erythrocytes.

We reported increased water permeability and a low urea reflection coefficient in Xenopus oocytes expressing urea transporter UT-B (former name UT3), suggesting that water and urea share a common aqueous pathway (Yang, B., and Verkman, A. S. (1998) J. Biol. Chem. 273, 9369-9372). Although increased water permeability was confirmed in the Xenopus oocyte expression system, it has been argued (Sidoux-Walter, F., Lucien, N., Olives, B., Gobin, R., Rousselet, G., Kamsteeg, E. J., Ripoche, P., Deen, P. M., Cartron, J. P., and Bailly, P. (1999) J. Biol. Chem. 274, 30228-30235) that UT-B does not transport water when expressed at normal levels in mammalian cells such as erythrocytes. To quantify UT-B-mediated water transport, we generated double knockout mice lacking UT-B and the major erythrocyte water channel, aquaporin-1 (AQP1). The mice had reduced survival, retarded growth, and defective urinary concentrating ability. However, erythrocyte size and morphology were not affected. Stopped-flow light scattering measurements indicated erythrocyte osmotic water permeabilities (in cm/s x 0.01, 10 degrees C): 2.1 +/- 0.2 (wild-type mice), 2.1 +/- 0.05 (UT-B null), 0.19 +/- 0.02 (AQP1 null), and 0.045 +/- 0.009 (AQP1/UT-B null). The low water permeability found in AQP1/UT-B null erythrocytes was also seen after HgCl(2) treatment of UT-B null erythrocytes or phloretin treatment of AQP1 null erythrocytes. The apparent activation energy for UT-B-mediated water transport was low, <2 kcal/mol. Estimating 14,000 UT-B molecules per mouse erythrocyte, the UT-B-dependent P(f) of 0.15 x 10(-4) cm/s indicated a substantial single channel water permeability of UT-B of 7.5 x 10(-14) cm(3)/s, similar to that of AQP1. These results provide direct functional evidence for UT-B-facilitated water transport in erythrocytes and suggest that urea traverses an aqueous pore in the UT-B protein.

Animals↗

PubMed2002

Evidence against aquaporin-1-dependent CO2 permeability in lung and kidney.

AQP1-dependent CO2 transport has been suggested from the increased CO2 permeability in Xenopus oocytes expressing AQP1. Potential implications of this finding include AQP1-facilitated CO2 exchange in mammalian lung and HCO3-/CO2 transport in kidney proximal tubule. We reported previously that: (a) CO2 permeability in erythrocytes was not affected by AQP1 deletion, (b) CO2 permeability in liposomes was not affected by AQP1 reconstitution despite a 100-fold increased water permeability, and (c) CO2 blow-off by the lung in living mice was not impaired by AQP1 deletion. We extend these observations by direct measurement of CO2 permeabilities in lung and kidney. CO2 transport across the air-space-capillary barrier in isolated perfused lungs was measured from changes in air-space fluid pH in response to addition/removal of HCO3-/CO2 from the pulmonary artery perfusate. The pH was measured by pleural surface fluorescence of a pH indicator (BCECF-dextran) in the air-space fluid. Air-space fluid pH equilibrated rapidly (t(1/2) approximately 6 s) in response to addition/removal of HCO3-/CO2. However, the kinetics of pH change was not different in lungs of mice lacking AQP1, AQP5 or AQP1/AQP5 together, despite an up to 30-fold reduction in water permeability. CO2 transport across BCECF-loaded apical membrane vesicles from kidney proximal tubule was measured from the kinetics of intravesicular acidification in response to rapid mixing with a HCO3-/CO2 solution. Vesicles rapidly acidified (t(1/2) approximately 10 ms) in response to HCO3-/CO2 addition. However the acidification rate was not different in kidney vesicles from AQP1-null mice despite a 20-fold reduction in water permeability. The results provide direct evidence against physiologically significant transport of CO2 by AQP1 in mammalian lung and kidney.

Algorithms↗