Colon water transport in transgenic mice lacking aquaporin-4 water channels

Transgenic null mice were used to test the hypothesis that water channel aquaporin-4 (AQP4) is involved in colon water transport and fecal dehydration. AQP4 was immunolocalized to the basolateral membrane of colonic surface epithelium of wild-type (+/+) mice and was absent in AQP4 null (-/-) mice. The transepithelial osmotic water permeability coefficient (P(f)) of in vivo perfused colon of +/+ mice, measured using the volume marker (14)C-labeled polyethylene glycol, was 0.016 +/- 0.002 cm/s. P(f) of proximal colon was greater than that of distal colon (0.020 +/- 0.004 vs. 0. 009 +/- 0.003 cm/s, P < 0.01). P(f) was significantly lower in -/- mice when measured in full-length colon (0.009 +/- 0.002 cm/s, P < 0. 05) and proximal colon (0.013 +/- 0.002 cm/s, P < 0.05) but not in distal colon. There was no difference in water content of cecal stool from +/+ vs. -/- mice (0.80 +/- 0.01 vs. 0.81 +/- 0.01), but there was a slightly higher water content in defecated stool from -/- mice (0.68 +/- 0.01 vs. 0.65 +/- 0.01, P < 0.05). Despite the differences in water permeability with AQP4 deletion, theophylline-induced secretion was not impaired (50 +/- 9 vs. 51 +/- 8 microl. min(-1). g(-1)). These results provide evidence that transcellular water transport through AQP4 water channels in colonic epithelium facilitates transepithelial osmotic water permeability but has little or no effect on colonic fluid secretion or fecal dehydration.     

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 approximately 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.     

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 (microS), p < 0.001), as well as in a CD1 genetic background (209 +/- 21 versus 469 +/- 11 microS). 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 microS) 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 +/- 13 versus 441 +/- 34 microS). 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.     

Impaired pain sensation in mice lacking Aquaporin-1 water channels

Aquaporin-1 (AQP1), a membrane water channel, is expressed in choroid plexus where it contributes to cerebrospinal fluid production. Here, we show that AQP1 is also expressed in the dorsal horn of the spinal cord and the trigeminal nucleus caudalis, regions that process pain information. Within the dorsal root and trigeminal sensory ganglia, AQP1 is concentrated in small diameter cell bodies, most of which give rise to unmyelinated C-fibers. To study the role of AQP1 in pain signaling, we compared acute pain responses in wild-type mice and in mice lacking AQP1. AQP1^−/− mice had reduced responsiveness to thermal and capsaicin chemical stimuli, but not to mechanical stimuli or formalin. These results provide evidence for AQP1 expression in nociceptive neurons and suggest that AQP1 may play a role in pain signal transduction.     

Defective dietary fat processing in transgenic mice lacking aquaporin-1 water channels

Immunocytochemistry showed expression of aquaporin-1 (AQP1) water channels at sites involved in dietary fat processing, including intrahepatic cholangiocytes, gallbladder, pancreatic microvascular endothelium, and intestinal lacteals. To determine whether AQP1 has a role in dietary fat digestion and/or absorption, mice were placed on a diet that contained 50% fat. Whereas wild-type mice (3-3.5 wk of age, 10-12 g) gained 49 +/- 5% (SE, n = 50) body weight in 8 days, and heterozygous mice gained 46 +/- 4%, AQP1 null mice gained only 4 +/- 3%; weights became similar after return to a 6% fat diet after 6 days. The null mice on a high-fat diet acquired an oily appearance, developed steatorrhea with increased stool triglyceride content, and manifested serum hypotriglyceridemia. Supplementation of the high-fat diet with pancreatic enzymes partially corrected the decreased weight gain in null mice. Absorption of [(14)C]oleic acid from small intestine was not affected by AQP1 deletion, as determined by blood radioactivity after duodenal infusion. Lipase activity in feces and small intestine was remarkably greater in AQP1 null than wild-type mice on low- and high-fat diets. Fluid collections done in older mice (that are less sensitive to a high-fat diet) by ductal cannulation showed threefold increased pancreatic fluid flow in response to secretin/cholecystokinin, but volumes, pH, and amylase activities were affected little by AQP1 deletion, nor were bile flow rates and bile salt concentrations. Together, these results establish a dietary fat misprocessing defect in AQP1 null mice.     

Heterotetrameric composition of aquaporin-4 water channels.

Aquaporin (AQP) water channel proteins are tetrameric assemblies of individually active approximately 30 kDa subunits. AQP4 is the predominant water channel protein in brain, but immunoblotting of native tissues has previously yielded multiple poorly resolved bands. AQP4 is known to encode two distinct mRNAs with different translation initiating methionines, M1 or M23. Using SDS-PAGE urea gels and immunoblotting with anti-peptide antibodies, four polypeptides were identified in brain and multiple other rat tissues with the following levels of expression: 32 kDa > 34 kDa > 36 kDa > 38 kDa. The 34 and 38 kDa polypeptides react with an antibody specific for the N-terminus of the M1 isoform, and 32 and 36 kDa correspond to the shorter M23 isoform. Immunogold electron microscopic studies with rat cerebellum cryosections demonstrated that the 34 kDa polypeptide colocalizes in perivascular astrocyte endfeet where the 32 kDa polypeptide is abundantly expressed. Velocity sedimentation, cross-linking, and immunoprecipitation analyses of detergent-solubilized rat brain revealed that the 32 and 34 kDa polypeptides reside within heterotetramers. Immunoprecipitation of AQP4 expressed in Xenopus laevis oocytes demonstrated that heterotetramer formation reflects the relative expression levels of the 32 and 34 kDa polypeptides; however, tetramers containing different compositions of the two polypeptides exhibit similar water permeabilities. These studies demonstrate that AQP4 heterotetramers are formed from two overlapping polypeptides and indicate that the 22-amino acid sequence at the N-terminus of the 34 kDa polypeptide does not influence water permeability but may contribute to membrane trafficking or assembly of arrays.     

Impaired osmotic sensation in mice lacking TRPV4

The Ca²⁺-permeable cation channel TRPV4, which is part of the Trp family located in the circumventricular organs, is activated by cell swelling. To investigate the role of TRPV4 in osmotic sensation, we disrupted the TRPV4 gene in mice and examined the effect on osmotic metabolism. Disruption of the mouse TRPV4 gene did not influence either water intake behavior or serum osmolality. Short-term salt ingestion, however, seemed to impair the transient free water clearance. The level of serum arginine vasopressin (AVP) of TRPV4–/– mice was not significantly changed under normal conditions but was significantly increased under stimulated conditions. Incubation of brain slices with graded hyperosmolality suggested an exaggerated response of AVP secretion in TRPV4–/– mice. Thus TRPV4 in the brain may transmit a negative signal to AVP secretion similar to an inhibitory pass through the baroregulatory system. Thus, in the regulation of serum osmolality, TRPV4 is a swell-activated channel that appears to play a role in reversion toward hyposmolality. Trp; calcium channel; vasopressin; mechanosensitive channel     

Impaired pressure sensation in mice lacking TRPV4

The sensation of pressure, mechanosensation, in vertebrates remains poorly understood on the molecular level. The ion channel TRPV4 is in the TRP family and is a candidate for a mechanosensitive calcium-permeable channel. It is located in dorsal root ganglia. In the present study, we show that disrupting the Trpv4 gene in mice markedly reduced the sensitivity of the tail to pressure and acidic nociception. The threshold to noxious stimuli and the conduction velocity of myelinated nerve responding to stimuli were also impaired. Activation of unmyelinated nerve was undetected. However, the mouse still retained olfaction, taste sensation, and heat avoidance. The TRPV4 channel expressed in vitro in Chinese hamster ovary cells was opened by low pH, citrate, and inflation but not by heat or capsaicin. These data identify the TRPV4 channel as essential for the normal detection of pressure and as a receptor of the high-threshold mechanosensory complex.     

Erythrocyte water permeability and renal function in double knockout mice lacking aquaporin-1 and aquaporin-3

Aquaporin (AQP) water channel AQP3 has been proposed to be the major glycerol and non-AQP1 water transporter in erythrocytes. AQP1 and AQP3 are also expressed in the kidney where their deletion in mice produces distinct forms of nephrogenic diabetes insipidus. Here AQP1/AQP3 double knockout mice were generated and analyzed to investigate the functional role of AQP3 in erythrocytes and kidneys. 53 double knockout mice were born out of 756 pups from breeding double heterozygous mice. The double knockout mice had reduced survival and impaired growth compared with the single knockout mice. Erythrocyte water permeability was 7-fold reduced by AQP1 deletion but not further reduced in AQP1/AQP3 null mice. AQP3 deletion did not affect erythrocyte glycerol permeability or its inhibition by phloretin. Daily urine output in AQP1/AQP3 double knockout mice (15 ml) was 9-fold greater than in wild-type mice, and urine osmolality (194 mosm) was 8.4-fold reduced. The mice remained polyuric after DDAVP administration or water deprivation. The renal medulla in most AQP1/AQP3 null mice by age 4 weeks was atrophic and fluid-filled due to the severe polyuria and hydronephrosis. Our data provide direct evidence that AQP3 is not functionally important in erythrocyte water or glycerol permeability. The renal function studies indicate independent roles of AQP1 and AQP3 in countercurrent exchange and collecting duct osmotic equilibration, respectively.     

Impaired host defense in mice lacking ONZIN

ONZIN is a small, cysteine-rich peptide of unique structure that is conserved in all vertebrates examined to date. We show that ONZIN is expressed at high levels in epithelial cells of the intestinal tract, the lung, and in cells of the immune system including macrophages and granulocytes. Because this pattern of expression is suggestive of a role in innate immune function, we have generated mice lacking this protein and examined their ability to respond to challenge with infectious agents. Onzin(-/-) mice show a heightened innate immune response after induction of acute peritonitis with Klebsiella pneumoniae. This increased response is consistent with an increased bacterial burden in the Onzin(-/-) mice. Ex vivo studies show that, whereas phagocytosis is not altered in Onzin(-/-) neutrophils, phagocytes lacking this protein kill bacteria less effectively. This result identifies ONZIN as a novel class of intracellular protein required for optimal function of the neutrophils after uptake of bacteria.     

Skeletal muscle function and water permeability in aquaporin-4 deficient mice

It has beenproposed that aquaporin-4 (AQP4), a water channel expressed at theplasmalemma of skeletal muscle cells, is important in normal musclephysiology and in the pathophysiology of Duchenne's musculardystrophy. To test this hypothesis, muscle water permeability andfunction were compared in wild-type and AQP4 knockout mice. Immunofluorescence and freeze-fracture electron microscopy showed AQP4protein expression in plasmalemma of fast-twitch skeletal muscle fibersof wild-type mice. Osmotic water permeability was measured inmicrodissected muscle fibers from the extensor digitorum longus (EDL) and fractionated membrane vesicles from EDLhomogenates. With the use of spatial-filtering microscopy to measureosmotically induced volume changes in EDL fibers, half times(t_1/2) for osmotic equilibration (7.5-8.5 s)were not affected by AQP4 deletion. Stopped-flow light-scatteringmeasurements of osmotically induced volume changes in plasmalemmavesicles also showed no significant differences in water permeability.Similar water permeability, yet ~90% decreased AQP4 proteinexpression was found in EDL from mdx mice that lack dystrophin.Skeletal muscle function was measured by force generation in isolatedEDL, treadmill performance time, and in vivo muscle swelling inresponse to water intoxication. No differences were found in EDL forcegeneration after electrical stimulation [42 ± 2 (wild-type) vs. 41 ± 2 (knockout) g/s], treadmill performance time (22 vs. 26 min; 29 m/min, 13° incline), or muscle swelling (2.8 vs. 2.9% increasedwater content at 90 min after intraperitoneal water infusion). Togetherthese results provide evidence against a significant role of AQP4 inskeletal muscle physiology in mice.

     

Progressive hearing loss in mice lacking the cyclin-dependent kinase inhibitor Ink4d

Maintenance of the post-mitotic state in the post-natal mammalian brain is an active process that requires the cyclin-dependent kinase inhibitors (CKIs) p19Ink4d (Ink4d) and p27Kip1 (Kip1). In animals with targeted deletions of both Ink4d and Kip1, terminally differentiated, post-mitotic neurons are observed to re-enter the cell cycle, divide and undergo apoptosis. However, when either Ink4d or Kip1 alone are deleted, the post-mitotic state is maintained, suggesting a redundant role for these genes in mature neurons. In the organ of Corti--the auditory sensory epithelium of mammals--sensory hair cells and supporting cells become post-mitotic during embryogenesis and remain quiescent for the life of the animal. When lost as a result of environmental insult or genetic abnormality, hair cells do not regenerate, and this loss is a common cause of deafness in humans. Here, we report that targeted deletion of Ink4d alone is sufficient to disrupt the maintenance of the post-mitotic state of sensory hair cells in post-natal mice. In Ink4d-/- animals, hair cells are observed to aberrantly re-enter the cell cycle and subsequently undergo apoptosis, resulting in progressive hearing loss. Our results identify a novel mechanism underlying a non-syndromic form of progressive hearing loss in mice.     

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.     

Expression of aquaporin-4 water channels in the digestive tract of the guinea pig

Expression of the aquaporin-4 (AQP4) water channel was systematically studied in the digestive tract of the guinea pig using Western blot and immunofluorescence techniques. The results showed that AQP4 was expressed widely in different segments of the guinea pig digestive tract. AQP4-immunoreactivity was confined to parietal cells in the stomach, and absorptive and glandular epithelial cells of small and large intestine. AQP4 protein was also expressed by enteric glial cells of submucosal and myenteric ganglia and primary nerve trunks. AQP4 was expressed by both type I and type II enteric gliocytes, but not by type III or type IV enteric gliocytes, indicating that enteric gliocytes have a heterogeneous distribution in the gut wall. In addition, different patterns of AQP4 expression in the enteric nervous system of human, guinea pig, rat and mouse colon mucosa were identified: in rat and mouse AQP4 was localised to a small subpopulation of neurons; in the guinea pig AQP4 was localised to enteric glial cells; and in the human colon mucosa, AQP4 was also detected mainly in the glial cells. It has been speculated that AQP4 may be involved in water transport in the gastrointestinal tract. Its role in enteric neurons and glia is unknown, but, by analogy with the brain, AQP4 may be involved in the formation and resolution of edema.     

Impaired cytokine signaling in mice lacking the IL-1 receptor-associated kinase.

Stimulation of the type 1 IL-1R (IL-1R1) and the IL-18R by their cognate ligands induces recruitment of the IL-1R-associated kinase (IRAK). Activation of IRAK leads in turn to nuclear translocation of NF-kappaB, which directs expression of innate and adaptive immune response genes. To study IRAK function in cytokine signaling, we generated cells and mice lacking the IRAK protein. IRAK-deficient fibroblasts show diminished activation of NF-kappaB when stimulated with IL-1. Immune effector cells without IRAK exhibit a defective IFN-gamma response to costimulation with IL-18. Furthermore, mice lacking the Irak gene demonstrate an attenuated response to injected IL-1. Deletion of Irak, however, does not affect the ability of mice to develop delayed-type hypersensitivity or clear infection with the intracellular parasite, Listeria monocytogenes. These results demonstrate that although IRAK participates in IL-1 and IL-18 signal transduction, residual cytokine responsiveness operates through an IRAK-independent pathway.     

Impaired fertility in female mice lacking urinary trypsin inhibitor

Urinary trypsin inhibitor (UTI) is a serine proteinase inhibitor that is found in blood and urine. To investigate the physiological functions of UTI in vivo, we generated UTI-deficient mice by gene targeting. The mice showed no obvious abnormalities and appeared healthy. However, the females displayed a severe reduction in fertility. Wild-type embryos developed normally when transplanted into UTI-deficient female mice, suggesting that UTI-deficient females have a normal ability to maintain pregnancy. The number of naturally ovulated oocytes from UTI-deficient mice was greatly reduced compared with that from wild-type mice. Histologically, oocytes with disorganized corona radiata were frequently seen in the ovaries of UTI-deficient mice after hormonal stimulation. When ovaries from UTI-deficient mice were transplanted into wild-type mice, pups derived from the transplanted ovaries were obtained, suggesting that the ovary of UTI-deficient mice functions normally if UTI is supplied from the systemic circulation. These results demonstrate that UTI plays an important role in the formation of the stable cumulus-oocyte complex that is essential for oocyte maturation and ovulation.     

Sour taste responses in mice lacking PKD channels

Background

The polycystic kidney disease-like ion channel PKD2L1 and its associated partner PKD1L3 are potential candidates for sour taste receptors. PKD2L1 is expressed in type III taste cells that respond to sour stimuli and genetic elimination of cells expressing PKD2L1 substantially reduces chorda tympani nerve responses to sour taste stimuli. However, the contribution of PKD2L1 and PKD1L3 to sour taste responses remains unclear.

Methodology/Principal Findings

We made mice lacking PKD2L1 and/or PKD1L3 gene and investigated whole nerve responses to taste stimuli in the chorda tympani or the glossopharyngeal nerve and taste responses in type III taste cells. In mice lacking PKD2L1 gene, chorda tympani nerve responses to sour, but not sweet, salty, bitter, and umami tastants were reduced by 25–45% compared with those in wild type mice. In contrast, chorda tympani nerve responses in PKD1L3 knock-out mice and glossopharyngeal nerve responses in single- and double-knock-out mice were similar to those in wild type mice. Sour taste responses of type III fungiform taste cells (GAD67-expressing taste cells) were also reduced by 25–45% by elimination of PKD2L1.

Conclusions/Significance

These findings suggest that PKD2L1 partly contributes to sour taste responses in mice and that receptors other than PKDs would be involved in sour detection.