Baoxue Yang

WATER AND GLYCEROL PERMEABILITIES OF AQUAPORINS 1-5 AND MIP DETERMINED QUANTITATIVELY BY EXPRESSION OF EPITOPE-TAGGED CONSTRUCTS IN XENOPUS OOCYTES

The goal of this study was to compare single channel water and glycerol permeabilities of mammalian aquaporins (AQP) 1–5 and the major intrinsic protein of lens fiber (MIP). Each of the six cloned cDNAs from rat was left untagged or was epitope-tagged with c-Myc or FLAG at either the N or C terminus so that results would not depend on epitope identity or location. The constructs were expressed in Xenopus oocytes for measurement of osmotic water permeability (P f ), [3H]glycerol uptake, and protein expression. Each of the 30 epitope-tagged constructs was expressed strongly at the oocyte plasma membrane. The 10-min uptake of [3H]glycerol was increased significantly (range of 4.5–8-fold over control) in oocytes expressing untagged AQP3 (GLIP) and each of the four tagged AQP3 constructs; [3H]glycerol uptake was not increased in oocytes expressing AQP1, AQP2, AQP4, AQP5, or MIP. In oocytes microinjected with 5 ng of cRNA, average P f values (in cm/s × 10−3) were 0.67 ± 0.06 (control), 19 ± 2 (AQP1), 10 ± 1 (AQP2), 8 ± 2 (AQP3), 29 ± 1 (AQP4), 10 ± 1 (AQP5), and 1.3 ± 0.2 (MIP), and they were relatively insensitive to the presence, identity, or location of the epitope tag. P f values were not affected by protein kinase A or C activation. After normalization for plasma membrane expression by immunoprecipitation of microdissected plasma membranes, single channel water permeabilities (p f , referenced to the AQP1 p f of 6 × 10−14cm3/s) were (in cm3/s × 10−14) 3.3 ± 0.2 (AQP2), 2.1 ± 0.3 (AQP3), 24 ± 0.6 (AQP4), 5.0 ± 0.4 (AQP5), and 0.25 ± 0.05 (MIP); p f values were insensitive to epitope identity and location. These results indicate very different intrinsic water permeabilities for the mammalian aquaporin homologs, with thep f value for AQP4 remarkably higher than those for the others. The p f values establish limits on aquaporin tissue densities required for physiological function and suggest significant structural and functional differences among the aquaporins.

Urea-selective Concentrating Defect in Transgenic Mice Lacking Urea Transporter UT-B

Urea transporter UT-B has been proposed to be the major urea transporter in erythrocytes and kidney-descending vasa recta. The mouse UT-B cDNA was isolated and encodes a 384-amino acid urea-transporting glycoprotein expressed in kidney, spleen, brain, ureter, and urinary bladder. The mouse UT-B gene was analyzed, and UT-B knockout mice were generated by targeted gene deletion of exons 3–6. The survival and growth of UT-B knockout mice were not different from wild-type littermates. Urea permeability was 45-fold lower in erythrocytes from knockout mice than from those in wild-type mice. Daily urine output was 1.5-fold greater in UT-B- deficient mice (p < 0.01), and urine osmolality (U osm) was lower (1532 ± 71versus 2056 ± 83 mosm/kgH2O, mean ± S.E., p < 0.001). After 24 h of water deprivation, U osm (in mosm/kgH2O) was 2403 ± 38 in UT-B null mice and 3438 ± 98 in wild-type mice (p < 0.001). Plasma urea concentration (P urea) was 30% higher, and urine urea concentration (U urea) was 35% lower in knockout mice than in wild-type mice, resulting in a much lowerU urea/P urea ratio (61 ± 5 versus 124 ± 9, p < 0.001). Thus, the capacity to concentrate urea in the urine is more severely impaired than the capacity to concentrate other solutes. Together with data showing a disproportionate reduction in the concentration of urea compared with salt in homogenized renal inner medullas of UT-B null mice, these data define a novel “urea-selective” urinary concentrating defect in UT-B null mice. The UT-B null mice generated for these studies should also be useful in establishing the role of facilitated urea transport in extrarenal organs expressing UT-B.

Reduced water permeability and altered ultrastructure in thin descending limb of Henle in aquaporin-1 null mice

It has been controversial whether high water permeability in the thin descending limb of Henle (TDLH) is required for formation of a concentrated urine by the kidney. Freeze-fracture electron microscopy (FFEM) of rat TDLH has shown an exceptionally high density of intramembrane particles (IMPs), which were proposed to consist of tetramers of aquaporin-1 (AQP1) water channels. In this study, transepithelial osmotic water permeability (Pf) was measured in isolated perfused segments (0.5-1 mm) of TDLH in wild-type (+/+), AQP1 heterozygous (+/-), and AQP1 null (-/-) mice. Pf was measured at 37 degrees C using a 100 mM bath-to-lumen osmotic gradient of raffinose, and fluorescein isothiocyanate (FITC)-dextran as the luminal volume marker. Pf was (in cm/s): 0.26 +/- 0.02 ([+/+]; SE, n = 9 tubules), 0.21 +/- 0.01 ([+/-]; n = 12), and 0.031 +/- 0.007 ([-/-]; n = 6) (P < 0.02, [+/+] vs. [+/-]; P < 0.0001, [+/+] vs. [-/-]). FFEM of kidney medulla showed remarkably fewer IMPs in TDLH from (-/-) vs. (+/+) and (+/-) mice. IMP densities were (in microm-2, SD, 5-12 micrographs): 5,880 +/- 238 (+/+); 5,780 +/- 450 (+/-); and 877 +/- 420 (-/-). IMP size distribution analysis revealed mean IMP diameters of 8.4 nm ([+/+] and [+/-]) and 5.2 nm ([-/-]). These results demonstrate that AQP1 is the principal water channel in TDLH and support the view that osmotic equilibration along TDLH by water transport plays a key role in the renal countercurrent concentrating mechanism. The similar Pf and AQP1 expression in TDLH of (+/+) and (+/-) mice was an unexpected finding that probably accounts for the unimpaired urinary concentrating ability in (+/-) mice.

Carbon Dioxide Permeability of Aquaporin-1 Measured in Erythrocytes and Lung of Aquaporin-1 Null Mice and in Reconstituted Proteoliposomes

Measurements of CO2permeability in oocytes and liposomes containing water channel aquaporin-1 (AQP1) have suggested that AQP1 is able to transport both water and CO2. We studied the physiological consequences of CO2 transport by AQP1 by comparing CO2permeabilities in erythrocytes and intact lung of wild-type and AQP1 null mice. Erythrocytes from wild-type mice strongly expressed AQP1 protein and had 7-fold greater osmotic water permeability than did erythrocytes from null mice. CO2 permeability was measured from the rate of intracellular acidification in response to addition of CO2/HCO3 − in a stopped-flow fluorometer using 2′,7′-bis-(2-carboxyethyl)-5-(and -6)-carboxyfluorescein (BCECF) as a cytoplasmic pH indicator. In erythrocytes from wild-type mice, acidification was rapid (t 1 2 , 7.3 ± 0.4 ms, S.E., n = 11 mice) and blocked by acetazolamide and increasing external pH (to decrease CO2/HCO3 − ratio). Apparent CO2permeability (PCO2 ) was not different in erythrocytes from wild-type (0.012 ± 0.0008 cm/s)versus null (0.011 ± 0.001 cm/s) mice. Lung CO2 transport was measured in anesthetized, ventilated mice subjected to a decrease in inspired CO2 content from 5% to 0%, producing an average decrease in arterial bloodpCO2 from 77 ± 4 to 39 ± 3 mm Hg (14 mice) with a t 1 2 of 1.4 min. ThepCO2 values and kinetics of decreasing pCO2 were not different in wild-type versusnull mice. Because AQP1 deletion did not affect CO2transport in erythrocytes and lung, we re-examined CO2permeability in AQP1-reconstituted liposomes containing carbonic anhydrase (CA) and a fluorescent pH indicator. Whereas osmotic water permeability in AQP1-reconstituted liposomes was >100-fold greater than that in control liposomes, apparent PCO2 (∼10−3 cm/s) did not differ. Measurements using different CA concentrations and HgCl2 indicated that liposome PCO2 is unstirred layer-limited and that HgCl2 slows acidification because of inhibition of CA rather than AQP1. These results provide direct evidence against physiologically significant AQP1-mediated CO2 transport and establish an upper limit to the CO2 permeability through single AQP1 water channels.

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 α2-macroglobulin (α2M), which targets to late endosomes. In pulse label-chase experiments, [Cl–] was 19 mm just after internalization in α2M-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 α2M-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 α2M-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.

Role of water channels in fluid transport studied by phenotype analysis of aquaporin knockout mice

Aquaporin‐type water channels are expressed widely in mammalian tissues, particularly in the kidney, lung, eye and gastrointestinal tract. To define the role of aquaporins in organ physiology, we have generated and analysed transgenic mice lacking aquaporins (AQP) 1, 3, 4 and 5. Multiple phenotype abnormalities were found in the null mice. For example, in kidney, deletion of AQP1 or AQP3 produced marked polyuria whereas AQP4 deletion produced only a mild concentrating defect. Deletion of AQP5, the apical membrane water channel in the salivary gland, caused defective saliva production. Deletion of AQP1 or AQP5, water channels in lung endothelia and epithelia, resulted in a 90% decrease in airspace‐capillary water permeability. In the brain, deletion of AQP4 conferred marked protection from brain swelling induced by acute water intoxication and ischaemic stroke. The general paradigm that has emerged from these phenotype studies is that aquaporins facilitate rapid near‐isosmolar transepithelial fluid absorption/secretion, as well as rapid vectorial water movement driven by osmotic gradients. However, we have found many examples in which the tissue‐specific expression of an aquaporin is not associated with any apparent phenotypic abnormality. The physiological data on aquaporin null mice suggest the utility of aquaporin blockers and aquaporin gene replacement in selected human diseases.

Fourfold reduction of water permeability in inner medullary collecting duct of aquaporin-4 knockout mice

Aquaporin (AQP)-3 and AQP4 water channels are expressed at the basolateral membrane of mammalian collecting duct epithelium. To determine the contribution of AQP4 to water permeability in the initial inner medullary collecting duct (IMCD), osmotic water permeability (Pf) was compared in isolated perfused IMCD segments from wild-type and AQP4 knockout mice. The AQP4 knockout mice were previously found to have normal gross appearance, survival, growth, and kidney morphology and a mild urinary concentrating defect (T. Ma, B. Yang, A. Gillespie, E. J. Carlson, C. J. Epstein, and A. S. Verkman, J. Clin. Invest. 100: 957-962, 1997). Transepithelial Pf was measured in microdissected IMCDs after 18-48 h of water deprivation and in the presence of 0.1 nM arginine vasopressin (to make basolateral Pf rate limiting). Pf values (37 degrees C; means +/- SE in cm/s x 10(-3)) were 56.0 +/- 8.5 for wild-type mice (n = 5) and 13.1 +/- 3.7 for knockout mice (n = 6) (P < 0.001). Northern blot analysis of kidney showed that transcript expression of AQP1, AQP2, AQP3, and AQP6 were not affected by AQP4 deletion. Immunoblot analysis indicated no differences in protein expression of AQP1, AQP2, or AQP3, and immunoperoxidase showed no differences in staining patterns. Coexpression of AQP3 and AQP4 in Xenopus laevis oocytes showed additive water permeabilities, suggesting that AQP4 deletion does not affect AQP3 function. These results indicate that AQP4 is responsible for the majority of basolateral membrane water movement in IMCD but that its deletion is associated with a very mild defect in urinary concentrating ability.Aquaporin (AQP)-3 and AQP4 water channels are expressed at the basolateral membrane of mammalian collecting duct epithelium. To determine the contribution of AQP4 to water permeability in the initial inner medullary collecting duct (IMCD), osmotic water permeability ( P f) was compared in isolated perfused IMCD segments from wild-type and AQP4 knockout mice. The AQP4 knockout mice were previously found to have normal gross appearance, survival, growth, and kidney morphology and a mild urinary concentrating defect (T. Ma, B. Yang, A. Gillespie, E. J. Carlson, C. J. Epstein, and A. S. Verkman. J. Clin. Invest. 100: 957-962, 1997). Transepithelial P f was measured in microdissected IMCDs after 18-48 h of water deprivation and in the presence of 0.1 nM arginine vasopressin (to make basolateral P f rate limiting). P fvalues (37°C; means ± SE in cm/s × 10-3) were 56.0 ± 8.5 for wild-type mice ( n = 5) and 13.1 ± 3.7 for knockout mice ( n = 6) ( P < 0.001). Northern blot analysis of kidney showed that transcript expression of AQP1, AQP2, AQP3, and AQP6 were not affected by AQP4 deletion. Immunoblot analysis indicated no differences in protein expression of AQP1, AQP2, or AQP3, and immunoperoxidase showed no differences in staining patterns. Coexpression of AQP3 and AQP4 in Xenopus laevis oocytes showed additive water permeabilities, suggesting that AQP4 deletion does not affect AQP3 function. These results indicate that AQP4 is responsible for the majority of basolateral membrane water movement in IMCD but that its deletion is associated with a very mild defect in urinary concentrating ability.

Small-Molecule CFTR Inhibitors Slow Cyst Growth in Polycystic Kidney Disease

Cyst expansion in polycystic kidney disease (PKD) involves progressive fluid accumulation, which is believed to require chloride transport by the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Herein is reported that small-molecule CFTR inhibitors of the thiazolidinone and glycine hydrazide classes slow cyst expansion in in vitro and in vivo models of PKD. More than 30 CFTR inhibitor analogs were screened in an MDCK cell model, and near-complete suppression of cyst growth was found by tetrazolo-CFTR(inh)-172, a tetrazolo-derived thiazolidinone, and Ph-GlyH-101, a phenyl-derived glycine hydrazide, without an effect on cell proliferation. These compounds also inhibited cyst number and growth by >80% in an embryonic kidney cyst model involving 4-d organ culture of embryonic day 13.5 mouse kidneys in 8-Br-cAMP-containing medium. Subcutaneous delivery of tetrazolo-CFTR(inh)-172 and Ph-GlyH-101 to neonatal, kidney-specific PKD1 knockout mice produced stable, therapeutic inhibitor concentrations of >3 microM in urine and kidney tissue. Treatment of mice for up to 7 d remarkably slowed kidney enlargement and cyst expansion and preserved renal function. These results implicate CFTR in renal cyst growth and suggest that CFTR inhibitors may hold therapeutic potential to reduce cyst growth in PKD.

Glial Cell Aquaporin-4 Overexpression in Transgenic Mice Accelerates Cytotoxic Brain Swelling

Aquaporin-4 (AQP4) is a water transport protein expressed in glial cell plasma membranes, including glial cell foot processes lining the blood-brain barrier. AQP4 deletion in mice reduces cytotoxic brain edema produced by different pathologies. To determine whether AQP4 is rate-limiting for brain water accumulation and whether altered AQP4 expression, as occurs in various pathologies, could have functional importance, we generated mice that overexpressed AQP4 in brain glial cells by a transgenic approach using the glial fibrillary acid protein promoter. Overexpression of AQP4 protein in brain by ∼2.3-fold did not affect mouse survival, appearance, or behavior, nor did it affect brain anatomy or intracranial pressure (ICP). However, following acute water intoxication produced by intraperitoneal water injection, AQP4-overexpressing mice had an accelerated progression of cytotoxic brain swelling, with ICP elevation of 20 ± 2 mmHg at 10 min, often producing brain herniation and death. In contrast, ICP elevation was 14 ± 2 mmHg at 10 min in control mice and 9.8 ± 2 mmHg in AQP4 knock-out mice. The deduced increase in brain water content correlated linearly with brain AQP4 protein expression. We conclude that AQP4 expression is rate-limiting for brain water accumulation, and thus, that altered AQP4 expression can be functionally significant.

Reduced osmotic water permeability of the peritoneal barrier in aquaporin-1 knockout mice

Aquaporin-1 (AQP1) water channels are expressed widely in epithelia and capillary endothelia involved in fluid transport. To test whether AQP1 facilitates water movement from capillaries into the peritoneal cavity, osmotically induced water transport rates were compared in AQP1 knockout [(-/-)], heterozygous [(+/-)], and wild-type [(+/+)] mice. In (+/+) mice, RT-PCR showed detectable transcripts for AQP1, AQP3, AQP4, AQP7, and AQP8. Immunofluorescence showed AQP1 protein in capillary endothelia and mesangium near the peritoneal surface and AQP4 in adherent muscle plasmalemma. For measurement of water transport, 2 ml of saline containing 300 mM sucrose (600 mosM) were infused rapidly into the peritoneal cavity via a catheter. Serial fluid samples (50 μl) were withdrawn over 60 min, with albumin as a volume marker. The albumin dilution data showed significantly decreased initial volume influx in AQP1 (-/-) mice: 101 ± 8, 107 ± 5, and 42 ± 4 (SE) μl/min in (+/+), (+/-), and (-/-) mice, respectively [ n = 6-10, P < 0.001, (-/-) vs. others]. Volume influx for AQP4 knockout mice was 100 ± 8 μl/min. In the absence of an osmotic gradient,3H2O uptake [half time = 2.3 and 2.2 min in (+/+) and (-/-) mice, respectively], [14C]urea uptake [half time = 7.9 and 7.7 min in (+/+) and (-/-) mice, respectively], and spontaneous isosmolar fluid absorption from the peritoneal cavity [0.47 ± 0.05 and 0.46 ± 0.04 ml/h in (+/+) and (-/-) mice, respectively] were not affected by AQP1 deletion. Therefore, AQP1 provides a major route for osmotically driven water transport across the peritoneal barrier in peritoneal dialysis.Aquaporin-1 (AQP1) water channels are expressed widely in epithelia and capillary endothelia involved in fluid transport. To test whether AQP1 facilitates water movement from capillaries into the peritoneal cavity, osmotically induced water transport rates were compared in AQP1 knockout [(-/-)], heterozygous [(+/-)], and wild-type [(+/+)] mice. In (+/+) mice, RT-PCR showed detectable transcripts for AQP1, AQP3, AQP4, AQP7, and AQP8. Immunofluorescence showed AQP1 protein in capillary endothelia and mesangium near the peritoneal surface and AQP4 in adherent muscle plasmalemma. For measurement of water transport, 2 ml of saline containing 300 mM sucrose (600 mosM) were infused rapidly into the peritoneal cavity via a catheter. Serial fluid samples (50 microliter) were withdrawn over 60 min, with albumin as a volume marker. The albumin dilution data showed significantly decreased initial volume influx in AQP1 (-/-) mice: 101 +/- 8, 107 +/- 5, and 42 +/- 4 (SE) microliter/min in (+/+), (+/-), and (-/-) mice, respectively [n = 6-10, P < 0.001, (-/-) vs. others]. Volume influx for AQP4 knockout mice was 100 +/- 8 microliters/min. In the absence of an osmotic gradient, 3H2O uptake [half time = 2.3 and 2.2 min in (+/+) and (-/-) mice, respectively], [14C]urea uptake [half time = 7.9 and 7.7 min in (+/+) and (-/-) mice, respectively], and spontaneous isosmolar fluid absorption from the peritoneal cavity [0.47 +/- 0.05 and 0.46 +/- 0.04 ml/h in (+/+) and (-/-) mice, respectively] were not affected by AQP1 deletion. Therefore, AQP1 provides a major route for osmotically driven water transport across the peritoneal barrier in peritoneal dialysis.