Tonghui Ma

Aquaporin-4 deletion in mice reduces brain edema after acute water intoxication and ischemic stroke.

Cerebral edema contributes significantly to morbidity and death associated with many common neurological disorders. However, current treatment options are limited to hyperosmolar agents and surgical decompression, therapies introduced more than 70 years ago. Here we show that mice deficient in aquaporin-4 (AQP4), a glial membrane water channel, have much better survival than wild-type mice in a model of brain edema caused by acute water intoxication. Brain tissue water content and swelling of pericapillary astrocytic foot processes in AQP4-deficient mice were significantly reduced. In another model of brain edema, focal ischemic stroke produced by middle cerebral artery occlusion, AQP4-deficient mice had improved neurological outcome. Cerebral edema, as measured by percentage of hemispheric enlargement at 24 h, was decreased by 35% in AQP4-deficient mice. These results implicate a key role for AQP4 in modulating brain water transport, and suggest that AQP4 inhibition may provide a new therapeutic option for reducing brain edema in a wide variety of cerebral disorders.

Thiazolidinone CFTR inhibitor identified by high-throughput screening blocks cholera toxin–induced intestinal fluid secretion

Secretory diarrhea is the leading cause of infant death in developing countries and a major cause of morbidity in adults. The cystic fibrosis transmembrane conductance regulator (CFTR) protein is required for fluid secretion in the intestine and airways and, when defective, causes the lethal genetic disease cystic fibrosis. We screened 50,000 chemically diverse compounds for inhibition of cAMP/flavone-stimulated Cl(-) transport in epithelial cells expressing CFTR. Six CFTR inhibitors of the 2-thioxo-4-thiazolidinone chemical class were identified. The most potent compound discovered by screening of structural analogs, CFTR(inh)-172, reversibly inhibited CFTR short-circuit current in less than 2 minutes in a voltage-independent manner with K(I) approximately 300 nM. CFTR(inh)-172 was nontoxic at high concentrations in cell culture and mouse models. At concentrations fully inhibiting CFTR, CFTR(inh)-172 did not prevent elevation of cellular cAMP or inhibit non-CFTR Cl(-) channels, multidrug resistance protein-1 (MDR-1), ATP-sensitive K(+) channels, or a series of other transporters. A single intraperitoneal injection of CFTR(inh)-172 (250 micro g/kg) in mice reduced by more than 90% cholera toxin-induced fluid secretion in the small intestine over 6 hours. Thiazolidinone CFTR inhibitors may be useful in developing large-animal models of cystic fibrosis and in reducing intestinal fluid loss in cholera and other secretory diarrheas.

Defective Secretion of Saliva in Transgenic Mice Lacking Aquaporin-5 Water Channels

Aquaporin-5 (AQP5) is a water-selective transporting protein expressed in epithelial cells of serous acini in salivary gland. We generated AQP5 null mice by targeted gene disruption. The genotype distribution from intercross of founder AQP5 heterozygous mice was 70:69:29 wild-type:heterozygote:knockout, indicating impaired prenatal survival of the null mice. The knockout mice had grossly normal appearance, but grew ∼20% slower than litter-matched wild-type mice when placed on solid food after weaning. Pilocarpine-stimulated saliva production was reduced by more than 60% in AQP5 knockout mice. Compared with the saliva from wild-type mice, the saliva from knockout mice was hypertonic (420 mosm) and dramatically more viscous. Amylase and protein secretion, functions of salivary mucous cells, were not affected by AQP5 deletion. Water channels AQP1 and AQP4 have also been localized to salivary gland; however, pilocarpine stimulation studies showed no defect in the volume or composition of saliva in AQP1 and AQP4 knockout mice. These results implicate a key role for AQP5 in saliva fluid secretion and provide direct evidence that high epithelial cell membrane water permeability is required for active, near-isosmolar fluid transport.

Generation and phenotype of a transgenic knockout mouse lacking the mercurial-insensitive water channel aquaporin-4.

Aquaporin-4 (AQP4) is a mercurial-insensitive, water-selective channel that is expressed in astroglia and basolateral plasma membranes of epithelia in the kidney collecting duct, airways, stomach, and colon. A targeting vector for homologous recombination was constructed using a 7-kb SacI AQP4 genomic fragment in which part of the exon 1 coding sequence was deleted. Analysis of 164 live births from AQP4[+/-] matings showed 41 [+/+], 83 [+/-], and 40 [-/-] genotypes. The [-/-] mice expressed small amounts of a truncated AQP4 transcript and lacked detectable AQP4 protein by immunoblot analysis and immunocytochemistry. Water permeability in an AQP4-enriched brain vesicle fraction in [+/+] mice was high and mercurial insensitive, and was decreased by 14-fold in [-/-] mice. AQP4 deletion did not affect growth or tissue morphology at the light microscopic level. Northern blot analysis showed that tissue-specific expression of AQPs 1, 2, 3, and 5 was not affected by AQP4 deletion. Maximum urine osmolality after a 36-h water deprivation was (in mosM, n = 15) [+/+] 3,342+/-209, [+/-] 3, 225+/-167, and [-/-] 2,616+/-229 (P < 0.025), whereas urine osmolalities before water deprivation did not differ among the genotypes. Rotorod analysis of 35- 38-d-old mice revealed no differences in neuromuscular function (performance time in s, n = 8): [+/+] 297+/-25, [+/-] 322+/-28, [-/-] 288+/-37. These results indicate that AQP4 deletion in CD1 mice has little or no effect on development, survival, growth, and neuromuscular function, but produces a small defect in urinary concentrating ability consistent with its expression in the medullary collecting duct.

Lung fluid transport in aquaporin-5 knockout mice

The mammalian lung expresses water channel aquaporin-1 (AQP1) in microvascular endothelia, AQP4 in airway epithelia, and AQP5 at the apical plasma membrane in type I cells of alveolar epithelia. We previously studied the role of AQP1 and AQP4 in lung fluid transport using knockout mice. Here, we examined the role of AQP5 using AQP5 knockout mice, which were recently shown to manifest defective saliva secretion. AQP5 deletion did not affect lung morphology at the light microscopic level, nor did it affect the distribution or expression of aquaporins 1, 3, or 4. Airspace-capillary osmotic water permeability (P(f)) was measured in isolated perfused lungs by pleural surface fluorescence and gravimetric methods. P(f) was reduced 10-fold by AQP5 deletion and was further reduced by 2- to 3-fold in AQP1/AQP5 double-knockout mice. Hydrostatic lung edema in response to acute increases in pulmonary artery pressure was not affected by AQP5 deletion. Active alveolar fluid absorption was measured in an in situ lung model from the increase in concentration of a volume marker in an isosmolar alveolar instillate. Interestingly, fluid absorption did not differ in litter-matched AQP5 knockout mice, nor was there an effect of AQP5 deletion when fluid absorption was maximally stimulated by pretreatment of mice with keratinocyte growth factor. These results indicate that AQP5 is responsible for the majority of water transport across the apical membrane of type I alveolar epithelial cells. The unimpaired alveolar fluid clearance in AQP5-null mice indicates that high alveolar water permeability is not required for active, near-isosmolar fluid transport.

Aquaporin water channels in gastrointestinal physiology

Fluid transport is a major function of the gastrointestinal (GI) tract with more than 9 litres of fluid being absorbed or secreted across epithelia in human salivary gland, stomach, the hepatobiliary tract, pancreas, small intestine and colon. This review evaluates the evidence that aquaporin‐type water channels are involved in GI fluid transport. The aquaporins are a family of small (≈30 kDa) integral membrane proteins that function as water channels. At least seven aquaporins are expressed in various tissues in the GI tract: AQP1 in intrahepatic cholangiocytes, AQP4 in gastric parietal cells, AQP3 and AQP4 in colonic surface epithelium, AQP5 in salivary gland, AQP7 in small intestine, AQP8 in liver, pancreas and colon, and AQP9 in liver. There are functional data suggesting that some GI cell types expressing aquaporins have high or regulated water permeability; however, there has been no direct evidence for a role of aquaporins in GI physiology. Recently, transgenic mice have been generated with selective deletions of various aquaporins. Preliminary evaluation of GI function suggests a role for AQP1 in dietary fat processing and AQP4 in colonic fluid absorption. Further study of aquaporin function in the GI tract should provide new insights into normal GI physiology and disease mechanisms, and may yield novel therapies to regulate fluid movement in GI diseases.

Lung fluid transport in aquaporin-1 and aquaporin-4 knockout mice

The mammalian lung expresses water channel aquaporin-1 (AQP1) in microvascular endothelia and aquaporin-4 (AQP4) in airway epithelia. To test whether these water channels facilitate fluid movement between airspace, interstitial, and capillary compartments, we measured passive and active fluid transport in AQP1 and AQP4 knockout mice. Airspace-capillary osmotic water permeability (Pf) was measured in isolated perfused lungs by a pleural surface fluorescence method. Pf was remarkably reduced in AQP1 (-/-) mice (measured in cm/s x 0.001, SE, n = 5-10: 17 +/- 2 [+/+]; 6.6 +/- 0.6 AQP1 [+/-]; 1.7 +/- 0.3 AQP1 [-/-]; 12 +/- 1 AQP4 [-/-]). Microvascular endothelial water permeability, measured by a related pleural surface fluorescence method in which the airspace was filled with inert perfluorocarbon, was reduced more than 10-fold in AQP1 (-/-) vs. (+/+) mice. Hydrostatically induced lung interstitial and alveolar edema was measured by a gravimetric method and by direct measurement of extravascular lung water. Both approaches indicated a more than twofold reduction in lung water accumulation in AQP1 (-/-) vs. (+/+) mice in response to a 5- to 10-cm H2O increase in pulmonary artery pressure for five minutes. Active, near-isosmolar alveolar fluid absorption (Jv) was measured in in situ perfused lungs using 125I-albumin as an airspace fluid volume marker. Jv (measured in percent fluid uptake at 30 min, n = 5) in (+/+) mice was 6.0 +/- 0.6 (37 degrees C), increased to 16 +/- 1 by beta-agonists, and inhibited to less than 2.0 by amiloride, ouabain, or cooling to 23 degrees C. Jv (with isoproterenol) was not affected by aquaporin deletion (18.9 +/- 2.2 [+/+]; 16.4 +/- 1.5 AQP1 [-/-]; 16.3 +/- 1.7 AQP4 [-/-]). These results indicate that osmotically driven water transport across microvessels in adult lung occurs by a transcellular route through AQP1 water channels and that the microvascular endothelium is a significant barrier for airspace-capillary osmotic water transport. AQP1 facilitates hydrostatically driven lung edema but is not required for active near-isosmolar absorption of alveolar fluid.

Impaired Stratum Corneum Hydration in Mice Lacking Epidermal Water Channel Aquaporin-3

The water and solute transporting properties of the epidermis have been proposed to be important determinants of skin moisture content and barrier properties. The water/small solute-transporting protein aquaporin-3 (AQP3) was found by immunofluorescence and immunogold electron microscopy to be expressed at the plasma membrane of epidermal keratinocytes in mouse skin. We studied the role of AQP3 in stratum corneum (SC) hydration by comparative measurements in wild-type and AQP3 null mice generated in a hairless SKH1 genetic background. The hairless AQP3 null mice had normal perinatal survival, growth, and serum chemistries but were polyuric because of defective urinary concentrating ability. AQP3 deletion resulted in a >4-fold reduced osmotic water permeability and >2-fold reduced glycerol permeability in epidermis. Epidermal, dermal, and SC thickness and morphology were not grossly affected by AQP3 deletion. Surface conductance measurements showed remarkably reduced SC water content in AQP3 null mice in the hairless genetic background (165 ± 10 versus 269 ± 12 microsiemens (μS), p < 0.001), as well as in a CD1 genetic background (209 ± 21 versus 469 ± 11 μS). Reduced SC hydration was seen from 3 days after birth. SC hydration in hairless wild-type and AQP3 null mice was reduced to comparable levels (90–100 μS) after a 24-h exposure to a dry atmosphere, but the difference was increased when surface evaporation was prevented by occlusion or exposure to a humidified atmosphere (179 ± 13versus 441 ± 34 μS). Conductance measurements after serial tape stripping suggested reduced water content throughout the SC in AQP3 null mice. Water sorption-desorption experiments indicated reduced water holding capacity in the SC of AQP3 null mice. The impaired skin hydration in AQP3 null mice provides the first functional evidence for the involvement of AQP3 in skin physiology. Modulation of AQP3 expression or function may thus alter epidermal moisture content and water loss in skin diseases.

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.

High-affinity activators of cystic fibrosis transmembrane conductance regulator (CFTR) chloride conductance identified by high-throughput screening.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) protein that reduce cAMP-stimulated Cl− conductance in airway and other epithelia. The purpose of this investigation was to identify new classes of potent CFTR activators. A collection of 60,000 diverse drug-like compounds was screened at 10 μmtogether with a low concentration of forskolin (0.5 μm) in Fisher rat thyroid epithelial cells co-expressing human CFTR and a green fluorescent protein-based Cl− sensor. Primary screening yielded 57 strong activators (greater activity than reference compound apigenin), most of which were unrelated in chemical structure to known CFTR activators, and 284 weaker activators. Secondary analysis of the strong activators included analysis of CFTR specificity, forskolin requirement, transepithelial short-circuit current, activation kinetics, dose response, toxicity, and activation mechanism. Three compounds, the most potent being a dihydroisoquinoline, activated CFTR by elevating cellular cAMP, probably by phosphodiesterase inhibition. Fourteen compounds activated CFTR without cAMP elevation or phosphatase inhibition, suggesting direct CFTR interaction. The most potent compounds had tetrahydrocarbazol, hydroxycoumarin, and thiazolidine core structures. These compounds induced CFTR Cl− currents rapidly (<5 min) with Kd down to 200 nm and were CFTR-selective, reversible, and nontoxic. Several compounds, the most potent being a trifluoromethylphenylbenzamine, activated the CF-causing mutant G551D, but with much weaker affinity (Kd > 10 μm). When added for 10 min, none of the compounds activated ΔPhe508-CFTR in transfected cells grown at 37 °C (with ΔPhe508-CFTR trapped in the endoplasmic reticulum). However, after correction of trafficking by 48 h of growth at 27 °C, tetrahydrocarbazol andN-phenyltriazine derivatives strongly stimulated Cl− conductance with Kd < 1 μm. The new activators identified here may be useful in defining molecular mechanisms of CFTR activation and as lead compounds in CF drug development.