Aquaporin-1 Null Mice Research Articles

Aquaporin‐3 deficiency slows renal cystogenesis via regulation of AMPK/ERK/mTOR signaling

OBJECTIVEHuman autosomal dominant polycystic kidney disease (ADPKD) is characterized by numerous bilateral renal cysts that lead to a decline in kidney function over time. Previous studies reported aquaporin‐3 (AQP3) expression in cysts derived from collecting ducts in ADPKD. The aim of this study is to study the effect of AQP3 on ADPKD development.METHODSAQP3 deficient PKD mice and inducible PKD mice were established in this study. Effect of AQP3 on PKD development was studied with pathological and molecular biological techniques.RESULTSIn both models, kidney size and cyst number were ~30% smaller in AQP3 null PKD mice than in AQP3‐expressing PKD mice, with the difference due mainly to smaller collecting ducts cysts. In matrix cultured MDCK cyst model, AQP3 transfection promoted cyst enlargement. To investigate the mechanism of AQP3‐dependent cyst development, we detected related signaling pathways. It was found that the AMPK/ERK/mTOR signaling, as well as intracellular ATP, were down‐regulated after AQP3 deletion. In addition, overexpression of AQP3 increased intracellular ATP synthesis, and HIF1α and glucose transporter 1 expression.CONCLUSIONWe conclude that AQP3 depletion retards cyst growth, primarily due to AQP3‐dependent energy metabolism. These findings identified the AQP3 as a potential therapeutic target for treating ADPKD.Support or Funding InformationThis work was supported by National Natural Science Foundation of China grants 31200869, 81261160507, 81330074 and 81170632, and the 111 Project.

Aquaporin-3 deletion in mice results in renal collecting duct abnormalities and worsens ischemia-reperfusion injury

Aquaporin-3 (AQP3), a transporter of water, glycerol and H2O2, is expressed in basolateral membranes of principal cells in kidney collecting duct. Here, we report that AQP3 deletion in mice affects renal function and modulates renal injury. We found collecting duct hyperplasia and cell swelling in kidneys of adult AQP3 null mice. After mild renal ischemia-reperfusion (IR), AQP3 null mice had significantly greater blood urea nitrogen (57mg/dl) and creatinine (136μM) than wild-type mice (35mg/dl and 48μM, respectively), and showed renal morphological changes, including tubular dilatation, erythrocyte diapedesis and collecting duct incompletion. MPO, MDA and SOD following IR in AQP3 null mice were significantly different from that in wild-type mice (1.7U/g vs 0.8U/g, 3.9μM/g vs 2.4μM/g, 6.4U/mg vs 11U/mg, respectively). Following IR, AQP3 deletion inhibited activation of mitogen-activated protein kinase (MAPK) signaling and produced an increase in the ratios of Bax/Bcl-2, cleaved caspase-3/caspase-3 and p-p53/p53. Studies in transfected MDCK cells showed that AQP3 expression attenuated reduced cell viability following hypoxia-reoxygenation, with reduced apoptosis and increased MAPK signaling. Our results support a novel role for AQP3 in modulating renal injury and suggest the mechanisms involved in protection against hypoxic injury.

Increased Lung Ischemia-Reperfusion Injury in Aquaporin 1-Null Mice Is Mediated via Decreased Hypoxia-Inducible Factor 2α Stability.

Aquaporin (AQP) 1, a water channel protein expressed widely in vascular endothelia, has been shown to regulate cell migration, angiogenesis, and organ regeneration. Even though its role in the pathogenesis of lung ischemia-reperfusion (IR) injury has been defined, the functional role of AQP1 during long-term IR resolution remains to be clarified. Here, we found that AQP1 expression was increased at late time points (7-14 d) after IR and colocalized with endothelial cell (EC) marker CD31. Compared with IR in wild-type mice, IR in Aqp1(-/-) mice had significantly enhanced leukocyte infiltration, collagen deposition, and microvascular permeability, as well as inhibited angiogenic factor expression. AQP1 knockdown repressed hypoxia-inducible factor (HIF)-2α protein stability. HIF-2α overexpression rescued the angiogenic factor expression in pulmonary microvascular ECs with AQP1 knockdown exposed to hypoxia-reoxygenation. Furthermore, AQP1 knockdown suppressed cellular viability and capillary tube formation, and enhanced permeability in pulmonary microvascular ECs, which were partly rescued by HIF-2α overexpression. Thus, this study demonstrates that AQP1 deficiency delays long-term IR resolution, partly through repressing angiogenesis mediated by destabilizing HIF-2α. These results suggest that AQP1 participates in long-term IR resolution, at least in part by promoting angiogenesis.

Aquaporin‐3 gene and protein expression in sun‐protected human skin decreases with skin ageing

Aquaporin 3 (AQP3) is a protein implicated in skin hydration. AQP3 null mice have relatively dry skin, reduced skin elasticity, and delayed recovery of barrier function after removal of the stratum corneum which is also present in skin of old people. A feature of skin aging is the change in both water content and barrier function of the skin. We investigated the expression of aquaporin 3 in non sun-exposed human skin, normal human keratinocytes and fibroblasts from different age groups to further understand the relationship between AQP3 and intrinsic skin aging. We investigated the expression of aquaporin3 (AQP3) in normal human skin, normal human epidermal keratinocytes (NHEK) and skin fibroblast of different ages by immunohistochemistry, immunocytochemistry, the reverse transcription-polymerase chain reaction (RT-PCR) and western blotting. The samples were derived from 60 patients of varying ages: <20 years of age, 30-45 years and >60 years of age. Twenty skin biopsies, 6 for keratinocyte/fibroblast cultures, were taken from each age group. AQP3 decreased with increasing age in both skin and NHEK samples. We demonstrated significant differences in AQP3 expression between the 3 age groups (P < 0.05). In fibroblasts, AQP3 expression levels were significantly lower in the >60 year olds compared to 30-45 year olds (P < 0.05) and <20 year olds (P < 0.05), there was no significant difference between the two younger groups (P > 0.05). AQP3 may be involved in the intrinsic aging process of non sun-exposed human skin.

The expression of differentiation markers in aquaporin-3 deficient epidermis

Aquaporin-3 (AQP3) is a water/glycerol transporting protein expressed strongly at the plasma membrane of keratinocytes. There is evidence for involvement of AQP3-facilitated water and glycerol transport in keratinocyte migration and proliferation, respectively. Here, we investigated the involvement of AQP3 in keratinocyte differentiation. Studies were done using AQP3 knockout mice, primary cultures of mouse keratinocytes (AQP3 knockout), neonatal human keratinocytes (AQP3 knockdown), and human skin. Cells were cultured with high Ca(2+) or 1alpha,25-dihydroxyvitamin D(3) (VD(3)) to induce differentiation. The expression of differentiation marker proteins and differentiating responses were comparable in control and AQP3-knockout or knockdown keratinocytes. Topical application of all-trans retinoic acid (RA), a known regulator of keratinocyte differentiation and proliferation, induced comparable expression of differentiation marker proteins in wildtype and AQP3 null epidermis, though with impaired RA-induced proliferation in AQP3 null mice. Immunostaining of human and mouse epidermis showed greater AQP3 expression in cells undergoing proliferation than differentiation. Our results showed little influence of AQP3 on keratinocyte differentiation, and provide further support for the proposed involvement of AQP3-facilitated cell proliferation.

Aquaporin-3 facilitates epidermal cell migration and proliferation during wound healing

Healing of skin wounds is a multi-step process involving the migration and proliferation of basal keratinocytes in epidermis, which strongly express the water/glycerol-transporting protein aquaporin-3 (AQP3). In this study, we show impaired skin wound healing in AQP3-deficient mice, which results from distinct defects in epidermal cell migration and proliferation. In vivo wound healing was approximately 80% complete in wild-type mice at 5 days vs approximately 50% complete in AQP3 null mice, with remarkably fewer proliferating, BrdU-positive keratinocytes. After AQP3 knock-down in keratinocyte cell cultures, which reduced cell membrane water and glycerol permeabilities, cell migration was slowed by more than twofold, with reduced lamellipodia formation at the leading edge of migrating cells. Proliferation of AQP3 knock-down keratinocytes was significantly impaired during wound repair. Mitogen-induced cell proliferation was also impaired in AQP3 deficient keratinocytes, with greatly reduced p38 MAPK activity. In mice, oral glycerol supplementation largely corrected defective wound healing and epidermal cell proliferation. Our results provide evidence for involvement of AQP3-facilitated water transport in epidermal cell migration and for AQP3-facilitated glycerol transport in epidermal cell proliferation.

Aquaporin-1–Independent Microvessel Proliferation in a Neonatal Mouse Model of Oxygen-Induced Retinopathy

Aquaporin-1 (AQP1) water channels are expressed widely in organ and tumor microvascular endothelia. Rapid microvessel proliferation occurs in growing tumors, diabetic and other retinopathies, and prenatal development. The purpose of this study was to investigate the role of AQP1 in retinal vessel proliferation. Comparative studies were performed on wild-type compared with AQP1 null mice using an established mouse model of oxygen-induced retinopathy. Neonatal mice were maintained in a 75% oxygen atmosphere for 5 days to suppress angiogenesis and then were returned to room air to induce vessel proliferation. AQP1 expression was also studied in extraocular microvessels and in primary endothelial cell cultures from pig retina. Surprisingly, AQP1 immunoreactivity was detected in only a small percentage of newly formed retinal microvessels, whereas AQP1 was strongly expressed in all choroidal and hyaloid vessels and in various extraocular microvessels in neonatal and prenatal mice. Oxygen-induced retinal microvessel proliferation was not significantly impaired in neonatal mice lacking AQP1, as quantified in flat-mounted retinas and thin sections. However, AQP1 was expressed in endothelial cells cultured from retinal microvessels. Microvessel proliferation in oxygen-induced retinopathy is AQP1-independent. Retinal endothelia have the capacity to express AQP1, though intact retinal vessels chronically suppress AQP1 expression.

NO Break-Ins at Water Gate

Nitric oxide (NO) is a gaseous free radical that functions as an endogenous mediator in diverse biological effects in numerous tissues. In the kidney and vasculature, these processes include the control of systemic and microvascular tone, the glomerular microcirculation, renal sodium excretion, and inflammatory responses in the glomerulus and tubulointerstitium, among many others.1 NO also impacts the renin–angiotensin and eicosanoid systems, endothelin, cytokines, and other key regulators of inflammation. Because of its potent chemical reactivity, high diffusibility, and the fact that, unlike most endogenous chemical signals, it cannot be stored or secreted, NO production by NO synthases is under complex, tight control to dictate specificity of its signaling and to limit toxicity to other cellular components. These controls serve to govern the timing, magnitude, and spatial distribution of NO release, and, in turn, specify the input signals that activate NO release and the effector functions of the molecule to target specific proteins. The biological role of NO as an inter- and intracellular messenger molecule is greatly dependent on its effective concentration and bioavailability at sites of action. As a hydrophobic gas, NO has traditionally been thought to traverse freely across cell membranes without the need for a specific transport protein to facilitate diffusion. It is also known that NO may accumulate in cellular lipids and preferentially interact with molecules in lipid environments. Since cellular entry of NO has generally been regarded to be near diffusion-limited, many of the reactions of NO are thought to depend on the rate of collision between NO and its target molecules or functional groups. Proteins that are structurally or …

The Role of the Tight Junction in Paracellular Fluid Transport across Corneal Endothelium. Electro-osmosis as a Driving Force

The mechanism of epithelial fluid transport is controversial and remains unsolved. Experimental difficulties pose obstacles for work on a complex phenomenon in delicate tissues. However, the corneal endothelium is a relatively simple system to which powerful experimental tools can be applied. In recent years our laboratory has developed experimental evidence and theoretical insights that illuminate the mechanism of fluid transport across this leaky epithelium. Our evidence points to fluid being transported via the paracellular route by a mechanism requiring junctional integrity, which we attribute to electro-osmotic coupling at the junctions. Fluid movements can be produced by electrical currents. The direction of the movement can be reversed by current reversal or by changing junctional electrical charges by polylysine. Aquaporin 1 (AQP1) is the only AQP present in these cells, and its deletion in AQP1 null mice significantly affects cell osmotic permeability but not fluid transport, which militates against the presence of sizable water movements across the cell. By contrast, AQP1 null mice cells have reduced regulatory volume decrease (only 60% of control), which suggests a possible involvement of AQP1 in either the function or the expression of volume-sensitive membrane channels/transporters. A mathematical model of corneal endothelium predicts experimental results only when based on paracellular electro-osmosis, and not when transcellular local osmosis is assumed instead. Our experimental findings in corneal endothelium have allowed us to develop a novel paradigm for this preparation that includes: (1) paracellular fluid flow; (2) a crucial role for the junctions; (3) hypotonicity of the primary secretion; (4) an AQP role in regulation and not as a significant water pathway. These elements are remarkably similar to those proposed by the Hill laboratory for leaky epithelia.

Proximal tubule water transport-lessons from aquaporin knockout mice

THE PROXIMAL TUBULE REABSORBS essentially all the filtered organic solutes, most of the filtered phosphate, 80% of the filtered bicarbonate, and 60% of the filtered sodium chloride. Approximately 70% of the filtered water is also reabsorbed by this segment. Despite these very high rates of proximal tubule solute transport, the osmolality of the luminal fluid decreases by only 5 mosmol/kgH2O from Bowman’s space to the end of the proximal tubule accessible by micropuncture (3). Furthermore, the luminal osmolality was found to be 7.5 mosmol/ kgH2O lower than that in the peritubular plasma in MunichWistar rats (3). This luminal hypotonicity provides the driving force for water reabsorption in the proximal tubule. The fact that there is only a very small decrease in luminal osmolality is consistent with a very high diffusional water permeability in this nephron segment (10). Despite much debate, physiological studies showed that the vast majority of water is transported across the proximal tubular cell and not across the paracellular pathway (1, 8, 10). The mechanism for this high rate of transcellular water flow was elusive until the cloning of aquaporin-1 (9), which was found to be expressed in high abundance on the apical and basolateral membranes of the proximal tubule and also in the thin descending limb (7). Since these seminal observations, a family of aquaporins has been cloned. At least seven aquaporin isoforms are expressed in the kidney, and each different nephron segment has a unique aquaporin isoform expression (6). In addition to aquaporin-1, aquaporin-7 is also expressed on the proximal tubule but only on the apical membrane of the distal portion of the proximal straight tubule, the S3 segment. The role of aquaporin-1 in mediating proximal tubule water transport has been delineated by elegant studies by Schnermann, Verkman, and co-workers (11). Aquaporin-1 null mice have an 50% lower rate of proximal tubule volume absorption measured in vivo and in vitro than do wild-type mice (11). These data indicate that water permeability can be a limiting factor in proximal tubule solute transport. These mice also have a lower brush-border membrane vesicle osmotic water permeability compared with wild-type mice (4). In addition, aquaporin-1 null mice had an 80% reduction in transepithelial water permeability consistent with most of water movement occurring transcellularly via aquaporin-1 (11). Aquaporin-1 null mice generate a higher proximal tubule transepithelial osmotic gradient despite the lower rates of volume reabsorption, indicating that the nearly isotonic reabsorption of proximal tubular fluid is largely due to water movement through aquaporin-1 (13). Aquaporin-1 null mice have polydipsia and polyuria and an impaired concentrating ability despite the fact that the collecting duct expresses the vasopressin-responsive aquaporin-2 on the apical membrane and aquaporins-3 and -4 are present on the basolateral membrane (5, 11). The impaired concentrating mechanism in aquaporin-1 null mice is probably not secondary to an increased solute load from impaired proximal tubule transport flooding the distal nephron as tubular glomerular feedback decreases single-nephron GFR in these mice (11) but is most likely the result of an inability to maximally form a hypertonic medulla due to the lack of aquaporin-1 in the thin descending limb. The study by Sohara et al. (12) examined the importance of aquaporin-7 in water and glycerol transport using aquaporin-7 knockout mice. Aquaporin-7, unlike aquaporin-1, also facilitates glycerol as well as water transport. Brush-border membrane vesicles from the outer medulla, which includes the S3 proximal tubule, of aquaporin-7 knockout mice had only a 10% reduction in osmotic water permeability. Thus even in the late proximal straight tubule, aquaporin-1 appeared to be the channel responsible for most water transport. Furthermore, unlike aquaporin-1 knockout mice (4), aquaporin-7 null mice did not have a reduction in urinary concentrating ability. Rather than assume that aquaporin-7 had no significant role in proximal tubule water reabsorption, the authors generated an aquaporin1/aquaporin-7 double-knockout mouse. They found that brush-border membrane vesicles from the outer medulla of double-knockout mice had a lower osmotic permeability than that of aquaporin-1 knockout mice. Furthermore, the doubleknockout mouse had a greater impairment in urinary concentrating ability compared with the aquaporin-1 knockout mouse, indicating that the normal urinary concentrating ability in the aquaporin-7 knockout was due to compensation by aquaporin-1 and that aquaporin-7 does indeed play an important role in proximal straight tubule water reabsorption.

Aquaporin-1 Facilitates Epithelial Cell Migration in Kidney Proximal Tubule

Aquaporin-1 (AQP1) is the principal water-transporting protein in cell plasma membranes in kidney proximal tubule, where it facilitates transepithelial water transport. Here, a novel role for AQP1 in kidney involving the migration of proximal tubule cells is reported. Migration was compared in primary cultures of proximal tubule cells from wild-type and AQP1 null mice. Cell cultures from AQP1 null mice were indistinguishable from those of wild-type mice in their appearance, growth/proliferation, and adhesiveness, although, as expected, they had reduced plasma membrane water permeability. Migration of AQP1-deficient cells was reduced by >50% compared with wild-type cells, as measured in a Boyden chamber in the presence of a chemotactic stimulus. Comparable slowing of migration of AQP1-deficient cells was also found in an in vitro scratch assay of wound healing, with reduced appearance of lamella-like membrane protrusions at the cell leading edge. Adenoviral-mediated expression of AQP1 in the AQP1-deficient cells, which increased their water permeability to that of wild-type cells, corrected their migration defect. The potential relevance of these in vitro findings to the intact kidney was tested in an in vivo model of acute tubular injury caused by 30 min of renal artery occlusion. At 3 to 5 d after ischemia-reperfusion, kidneys in AQP1 null mice showed remarkably greater tubular injury and cellular actin disorganization than kidneys in wild-type mice. These results provide evidence for the involvement of AQP1 in migration of proximal tubule cells and possibly in the response of the proximal tubule to injury.

Decreased expression of AQP2 and AQP4 water channels and Na, K‐ATPase in kidney collecting duct in AQP3 null mice

Phenotype analysis has demonstrated that AQP3 (aquaporin 3) null mice are polyuric and manifest a urinary concentration defect. In the present study, we report that deletion of AQP3 is also associated with an increased urinary sodium excretion. To investigate further the mechanism of the decreased urinary concentration and significant natriuresis, we examined the segmental and subcellular localization of collecting duct AQPs [AQP2, p-AQP2 (phosphorylated AQP2), AQP3 and AQP4], ENaC (epithelial sodium channel) subunits and Na,K-ATPase by immunoperoxidase and immunofluorescence microscopy in AQP3 null (-/-), heterozygous (+/-) mice, wild-type and unrelated strain of normal mice. The present study confirms that AQP3 null mice exhibit severe polyuria and polydipsia and demonstrated that they exhibit increased urinary sodium excretion. In AQP3 null mice, there is a marked down-regulation of AQP2 and p-AQP2 both in CNT (connecting tubule) and CCD (cortical collecting duct). Moreover, AQP4 is virtually absent from CNT and CCD in AQP3 null mice. Basolateral AQP2 was virtually absent from AQP3 null mice and normal mice in contrast with rat. Thus the above results demonstrate that no basolateral AQPs are expressed in CNT and CCD of AQP3 null mice. However, in the medullary-collecting ducts, there is no difference in the expression levels and subcellular localization of AQP2, p-AQP2 and AQP4 between AQP3 +/- and AQP3 null mice. Moreover, a striking decrease in the immunolabelling of the alpha1 subunit of Na,K-ATPase was observed in CCD in AQP3 null mice, whereas a medullary-collecting duct exhibited normal labelling. Immunolabelling of all the ENaC subunits in the collecting duct was comparable between the two groups. The results improve the possibility that the severe urinary concentrating defect in AQP3 null mice may in part be caused by the decreased expression of AQP2, p-AQP2 and AQP4 in CNT and CCD, whereas the increased urinary sodium excretion may in part be accounted for by Na,K-ATPase in CCD in AQP3 null mice.

Reduced cerebrospinal fluid production and intracranial pressure in mice lacking choroid plexus water channel Aquaporin‐1

Aquaporin-1 (AQP1) is a water channel expressed strongly at the ventricular-facing surface of choroid plexus epithelium. We developed novel methods to compare water permeability in isolated choroid plexus of wild-type vs. AQP1 null mice, as well as intracranial pressure (ICP) and cerebrospinal fluid (CSF) production and absorption. Osmotically induced water transport was rapid in choroid plexus from wild-type mice and reduced by fivefold by AQP1 deletion. AQP1 deletion did not affect choroid plexus size or structure. By stereotaxic puncture of the lateral ventricle with a microneedle, ICP was 9.5 +/- 1.4 cm H2O in wild-type mice and 4.2 +/- 0.4 cm H2O in AQP1 null mice. CSF production, an isosmolar fluid secretion process, was measured by a dye dilution method involving fluid collections using a second microneedle introduced into the cisterna magna. CSF production in wild-type mice was (in microl min(-1)) 0.37 +/- 0.04 (control), 0.16 +/- 0.03 (acetazolamide-treated), and 1.14 +/- 0.15 (forskolin-treated), and reduced by approximately 25% in AQP1 null mice. Pressure-dependent CSF outflow, measured from steady-state ICP at different ventricular infusion rates, was not affected by AQP1 deletion. In a model of focal brain injury, AQP1 null mice had remarkably reduced ICP and improved survival compared with wild-type mice. The reduced ICP and CSF production in AQP1 null mice provides direct functional evidence for the involvement of AQP1 in CSF dynamics, suggesting AQP1 inhibition as a novel option for therapy of elevated ICP.

Fluid transport across cultured layers of corneal endothelium from aquaporin-1 null mice

We explored the role of AQP1, the only known aquaporin in corneal endothelium, on active fluid transport and passive osmotic water movements across corneal endothelial layers cultured from AQP1 null mice and wildtype mice. AQP1 null mice had grossly transparent corneas, just as wildtype mice. Endothelial cell layers grown on permeable supports transported fluid at rates of (in μl h −1 cm −2, n=9, mean± s.e.): 4·3±0·6, wildtype mice (MCE); 3·5±0·6, AQP1 null mice (KMCE; difference not significant). The osmotic water flow (also in μl h −1 cm −2) induced by a 100 mOsm sucrose gradient across MCE cell layers (8·7±0·6, n=8) was significantly greater than that across KMCE (5·7±0·7, n=6, p=0·007). When plated on glass coverslips, plasma membrane osmotic water permeability determined by light scattering was significantly higher for cells from wildtype vs. AQP1 null mice (in μm sec −1: 74±4, n=19 vs. 44±4 μm sec −1, n=11, p<0·001). Unexpectedly, after 10% hypo-osmotic challenge, the extent of the regulatory volume recovery was significantly reduced for AQP1 null mice cells (in%: MCE controls, 99±1, n=19, vs. KMCE: 64±5, n=11, p<0·001). Thus, as in other ‘low rate’ fluid transporting epithelia, deletion of AQP1 in mice corneal endothelium reduces osmotic water permeability but not active transendothelial fluid transport. However, that deletion impaired the extent of regulatory volume decrease after a hypo-osmotic challenge, suggesting a novel role for AQP1 in corneal endothelium.

Glycerol replacement corrects defective skin hydration, elasticity, and barrier function in aquaporin-3-deficient mice.

Mice deficient in the epidermal water/glycerol transporter aquaporin-3 (AQP3) have reduced stratum corneum (SC) hydration and skin elasticity, and impaired barrier recovery after SC removal. SC glycerol content is reduced 3-fold in AQP3 null mice, whereas SC structure, protein/lipid composition, and ion/osmolyte content are not changed. We show here that glycerol replacement corrects each of the defects in AQP3 null mice. SC water content, measured by skin conductance and 3H2O accumulation, was 3-fold lower in AQP3 null vs. wild-type mice, but became similar after topical or systemic administration of glycerol in quantities that normalized SC glycerol content. SC water content was not corrected by glycerol-like osmolytes such as xylitol, erythritol, and propanediol. Orally administered glycerol fully corrected the reduced skin elasticity in AQP3 null mice as measured by the kinetics of skin displacement after suction, and the delayed barrier recovery as measured by transepidermal water loss after tape-stripping. Analysis of [14C]glycerol kinetics indicated reduced blood-to-SC transport of glycerol in AQP3 null mice, resulting in slowed lipid biosynthesis. These data provide functional evidence for a physiological role of glycerol transport by an aquaglyceroporin, and indicate that glycerol is a major determinant of SC water retention, and mechanical and biosynthetic functions. Our findings establish a scientific basis for the >200-yr-old empirical practice of including glycerol in cosmetic and medicinal skin formulations.

Increased expression of H+-ATPase in inner medullary collecting duct of aquaporin-1-deficient mice.

Phenotype analysis has demonstrated that aquaporin-1 (AQP1) null mice are polyuric and manifest a urinary concentrating defect because of an inability to create a hypertonic medullary interstitium. We report here that deletion of AQP1 is also associated with a decrease in urinary pH from 6.15 +/- (SE) 0.1 to 5.63 +/- 0.07. To explore the mechanism of the decrease in urinary pH, we examined the expression of H+-ATPase in kidneys of AQP1 null mice. There was strong labeling for H+-ATPase in intercalated cells and proximal tubule cells in both AQP1 null and wild-type mice. Strong H+-ATPase immunostaining was also present in the apical plasma membrane of inner medullary collecting duct (IMCD) cells in AQP1 null mice, whereas no H+-ATPase labeling was observed in IMCD cells in wild-type mice. In addition, there was an increase in the prevalence of type A intercalated cells in the IMCD of AQP1 null mice, suggesting that the deletion of intercalated cells from the IMCD, which normally occurs during postnatal kidney development, was impaired. Western blot analysis of H+-ATPase expression in the different regions of the kidney demonstrated a significant increase in H+-ATPase protein in the inner medulla of AQP1 null mice compared with wild-type mice. There were no changes in H+-ATPase expression in the cortex or outer medulla. These results represent the first demonstration of apical H+-ATPase immunoreactivity in IMCD cells in vivo and suggest that the decrease in urinary pH observed in AQP1 null mice is due to upregulation of H+-ATPase in the IMCD. The induction of H+-ATPase expression in IMCD cells of AQP1 null mice may be related to the chronically low interstitial osmolality in these animals. The challenge will be to identify the molecular signal(s) responsible for the de novo H+-ATPase expression.

Aquaporin-1 deletion reduces osmotic water permeability and cerebrospinal fluid production.

Aquaporin-1 (AQP1) is a water channel that is strongly expressed at the ventricular-facing surface of choroid plexus epithelium. Using wildtype and AQP1 null mice, we developed novel methods to compare the water permeability in isolated choroid plexus, and cerebrospinal fluid (CSF) production in living mice. Osmotically-induced water transport was rapid in freshly isolated choroid plexus from wildtype mice as measured by a spatial-filtering optical method, and reduced by 5-fold by AQP1 deletion. CSF production, an isosmolar fluid secretion process, was measured by a dye dilution method involving fluid collections using a second microneedle introduced into the cisterna magna. CSF production in wildtype mice was (in microl/min) 0.37 +/- 0.04 microl/min (control), 0.16 +/- 0.03 microl/min (acetazolamide-treated) and 1.14 +/- 0.15 microl/min (forskolin-treated), and reduced by up to 25% in AQP1 null mice. The impaired CSF production in AQP1 null mice provides direct functional evidence for the involvement of AQP1 in CSF formation.

Selectively Reduced Glycerol in Skin of Aquaporin-3-deficient Mice May Account for Impaired Skin Hydration, Elasticity, and Barrier Recovery

Deletion of the epidermal water/glycerol transporter aquaporin-3 (AQP3) in mice reduced superficial skin conductance by approximately 2-fold (Ma, T., Hara, M., Sougrat, R., Verbavatz, J. M., and Verkman, A. S. (2002) J. Biol. Chem. 277, 17147-17153), suggesting defective stratum corneum (SC) hydration. Here, we demonstrate significant impairment of skin hydration, elasticity, barrier recovery, and wound healing in AQP3 null mice in a hairless (SKH1) genetic background and investigate the cause of the functional defects by analysis of SC morphology and composition. Utilizing a novel (3)H(2)O distribution method, SC water content was reduced by approximately 50% in AQP3 null mice. Skin elasticity measured by cutometry was significantly reduced in AQP3 null mice with approximately 50% reductions in elasticity parameters Uf, Ue, and Ur. Although basal skin barrier function was not impaired, AQP3 deletion produced an approximately 2-fold delay in recovery of barrier function as measured by transepidermal water loss after tape stripping. Another biosynthetic skin function, wound healing, was also approximately 2-fold delayed by AQP3 deletion. By electron microscopy AQP3 deletion did not affect the structure of the unperturbed SC. The SC content of ions (Na(+), K(+), Ca(2+), Mg(2+)) and small solutes (urea, lactic acid, glucose) was not affected by AQP3 deletion nor was the absolute amount or profile of lipids and free amino acids. However, AQP3 deletion produced significant reductions in glycerol content in SC and epidermis (in nmol/microg protein: 5.5 +/- 0.4 versus 2.3 +/- 0.7 in SC; 0.037 +/- 0.007 versus 0.022 +/- 0.005 in epidermis) but not in dermis or blood. These results establish hydration, mechanical, and biosynthetic defects in skin of AQP3-deficient mice. The selective reduction in epidermal and SC glycerol content in AQP3 null mice may account for these defects, providing the first functional evidence for physiologically important glycerol transport by an aquaporin.

Unimpaired osmotic water permeability and fluid secretion in bile duct epithelia of AQP1 null mice.

The mechanisms by which fluid moves across the luminal membrane of cholangiocyte epithelia are uncertain. Previous studies suggested that aquaporin-1 (AQP1) is an important determinant of water movement in rat cholangiocytes and that cyclic AMP mediates the movement of these water channels from cytoplasm to apical membrane, thereby increasing the osmotic water permeability. To test this possibility we measured agonist-stimulated fluid secretion and osmotically driven water transport in isolated bile duct units (IBDUs) from AQP1 wild-type (+/+) and null (-/-) mice. AQP1 expression was confirmed in a mouse cholangiocyte cell line and +/+ liver. Forskolin-induced fluid secretion, measured from the kinetics of IBDU luminal expansion, was 0.05 fl/min and was not impaired in -/- mice. Osmotic water permeability (P(f)), measured from the initial rate of IBDU swelling in response to a 70-mosM osmotic gradient, was 11.1 x 10(-4) cm/s in +/+ mice and 11.5 x 10(-4) cm/s in -/- mice. P(f) values increased by approximately 50% in both +/+ and -/- mice following preincubation with forskolin. These findings provide direct evidence that AQP1 is not rate limiting for water movement in mouse cholangiocytes and does not appear to be regulated by cyclic AMP in this species.

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.