Aquaporin-3 deficiency slows cyst enlargement in experimental mouse models of autosomal dominant polycystic kidney disease.

Human autosomal dominant polycystic kidney disease (ADPKD) is characterized by bilateral renal cysts that lead to a decline in kidney function. Previous studies reported aquaporin (AQP)-3 expression in cysts derived from collecting ducts in ADPKD. To study the role of AQP3 in cyst development, we generated 2 polycystic kidney disease (PKD) mouse models: kidney-specific Pkd1 knockout mice and inducible Pkd1 knockout mice, each without and with AQP3 deletion. In both models, kidney sizes and cyst indexes were significantly reduced in AQP3-null PKD mice compared with AQP3-expressing PKD mice, with the difference seen mainly in collecting duct cysts. AQP3-deficient kidneys showed significantly reduced ATP content, increased phosphorylated (p)-AMPK, and decreased p-ERK and p-mammalian target of rapamycin (mTOR). In a matrix-grown Madin-Darby canine kidney cyst model, AQP3 expression promoted cyst enlargement and was associated with increased expression of hypoxia-inducible factor 1-α and glucose transporter 1 and increased glucose uptake. Our data suggest that the slowed renal cyst enlargement in AQP3 deficiency involves impaired energy metabolism in the kidney through AMPK and mTOR signaling and impaired cellular glucose uptake. These findings implicate AQP3 as a novel determinant of renal cyst enlargement and hence a potential drug target in ADPKD.-Wang, W., Geng, X., Lei, L., Jia, Y., Li, Y., Zhou, H., Verkman, A. S., Yang, B. Aquaporin-3 deficiency slows cyst enlargement in experimental mouse models of autosomal dominant polycystic kidney disease.

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.

Aquaporin-3 mediates hydrogen peroxide-dependent responses to environmental stress in colonic epithelia.

The colonic epithelium provides an essential barrier against the environment that is critical for protecting the body and controlling inflammation. In response to injury or gut microbes, colonic epithelial cells produce extracellular hydrogen peroxide (H2O2), which acts as a potent signaling molecule affecting barrier function and host defense. In humans, impaired regulation of H2O2 in the intestine has been associated with early-onset inflammatory bowel disease and colon cancer. Here, we show that signal transduction by H2O2 depends on entry into the cell by transit through aquaporin-3 (AQP3), a plasma membrane H2O2-conducting channel. In response to injury, AQP3-depleted colonic epithelial cells showed defective lamellipodia, focal adhesions, and repair after wounding, along with impaired H2O2 responses after exposure to the intestinal pathogen Citrobacter rodentium Correspondingly, AQP3-/- mice showed impaired healing of superficial wounds in the colon and impaired mucosal innate immune responses against C. rodentium infection, manifested by reduced crypt hyperplasia, reduced epithelial expression of IL-6 and TNF-α, and impaired bacterial clearance. These results elucidate the signaling mechanism of extracellular H2O2 in the colonic epithelium and implicate AQP3 in innate immunity at mucosal surfaces.

Impaired self-healing capacity in airway epithelia lacking aquaporin-3.

In this study, we utilized AQP3-knockout mice as the in vivo model and AQP3-knockdown human bronchial epithelial cells (HBECs) as the in vitro model. Airway injury was experimentally induced by intra-tracheal injection of naphthalene. HE staining, transmission and scanning electron microscope were performed to evaluate self-healing capacity in vivo. Transwell and wound-healing assays were performed to evaluate epithelial cell migration in vitro. We found that both the airway epithelial cells of AQP3-knockout mice and AQP3-knockdown HBECs exhibited an obviously impaired self-healing capacity with defective epithelial cell migration through AQP3-facilitated glycerol transport. In addition, glycerol supplementation could largely correct defective injury healing and epithelial cell migration. For the first time, we found evidence for distinct defects in AQP3-deficient airway epithelial cell migration. Mechanistic analysis showed AQP3-facillitated glycerol transport plays a role in airway epithelial self-healing after injury.

The effect of AQP3 deficiency on fuel selection during a single bout of exhausting exercise.

Aquaporin-3 (AQP3) is an integral membrane protein that facilitates the transport of water and glycerol across cell membranes. However, the precise localization and function of AQP3 in skeletal muscles is currently unknown. In this study, we investigated the capacity of AQP3 knockout mice to perform a single bout of exhausting exercise and analyzed the parameters related to skeletal muscle energy metabolism during exhausting exercise. Mice were exposed to a single bout of treadmill running at a speed of 12 m/min with 10° inclination until exhaustion, and sacrificed immediately, 24 h and 48 h after exercise. Both immunohistochemistry and double immunofluorescence staining revealed that AQP3 is expressed at the cell surface with no evidence of colocalization with either AQP1 or AQP4 in hamstring skeletal muscles. When exposed to a single bout of exhaustive exercise, AQP3 knockout mice fatigued more easily with the average time to exhaustion shorter than the wild-type mice. After exhausting exercise, plasma glucose, muscle glycogen, muscle triglyceride, and muscle free fatty acid levels decreased compared with the values before exercise in both AQP3 knockout and wild-type mice. However, muscle glycerol concentration after exercise decreased in the wild-type mice, but rather increased in AQP3 knockout mice. These findings suggest that decreased glycerol efflux from the skeletal muscles in AQP3 knockout mice may result in low exercise capacity, presumably due to the limitations in the constant energy supply through hepatic gluconeogenesis from glycerol during the prolonged endurance exercise.

Aquaporin-3 potentiates allergic airway inflammation in ovalbumin-induced murine asthma.

Oxidative stress plays a pivotal role in the pathogenesis of asthma. Aquaporin-3 (AQP3) is a small transmembrane water/glycerol channel that may facilitate the membrane uptake of hydrogen peroxide (H2O2). Here we report that AQP3 potentiates ovalbumin (OVA)-induced murine asthma by mediating both chemokine production from alveolar macrophages and T cell trafficking. AQP3 deficient (AQP3(-/-)) mice exhibited significantly reduced airway inflammation compared to wild-type mice. Adoptive transfer experiments showed reduced airway eosinophilic inflammation in mice receiving OVA-sensitized splenocytes from AQP3(-/-) mice compared with wild-type mice after OVA challenge, consistently with fewer CD4(+) T cells from AQP3(-/-) mice migrating to the lung than from wild-type mice. Additionally, in vivo and vitro experiments indicated that AQP3 induced the production of some chemokines such as CCL24 and CCL22 through regulating the amount of cellular H2O2 in M2 polarized alveolar macrophages. These results imply a critical role of AQP3 in asthma, and AQP3 may be a novel therapeutic target.

The AQP-3 water channel is a pivotal modulator of glycerol-induced chloride channel activation in nasopharyngeal carcinoma cells.

Aquaporin (AQP) and chloride channels are ubiquitous in virtually all living cells, playing pivotal roles in cell proliferation, migration and apoptosis. We previously reported that AQP-3 aquaglyceroporin and ClC-3 chloride channels could form complexes to regulate cell volume in nasopharyngeal carcinoma cells. In this study, the roles of AQP-3 in their hetero-complexes were further investigated. Glycerol entered the cells via AQP-3 and induced two different Cl(-) currents through cell swelling-dependent or -independent pathways. The swelling-dependent Cl(-) current was significantly inhibited by pretreatment with CuCl2 and AQP-3-siRNA. After siRNA-induced AQP-3 knock-down, the 140 mM glycerol isoosmotic solution swelled cells by 22% (45% in AQP-3-intact cells) and induced a smaller Cl(-) current; this current was smaller than that activated by 8% cell volume swelling, which induced by the 140 mM glycerol hyperosmotic solution in AQP-3-intact cells. This suggests that the interaction between AQP-3 and ClC-3 plays an important role in cell volume regulation and that AQP-3 may be a modulator that opens volume-regulated chloride channels. The swelling-independent Cl(-) current, which was activated by extracellular glycerol, was reduced by CuCl2 and AQP-3-siRNA pretreatment. Dialyzing glycerol into cells via the pipette directly induced the swelling-independent Cl(-) current; however this current was blocked by AQP-3 down-regulation, suggesting AQP-3 is essential for the opening of chloride channels. In conclusion, AQP-3 is the pathway for water, glycerol and other small solutes to enter cells, and it may be an essential modulator for the gating of chloride channels.

The proliferation impairment induced by AQP3 deficiency is the result of glycerol uptake and metabolism inhibition in gastric cancer cells.

Gastric cancer is a big threat to human health. Effective therapeutic cancer target remains to be discovered. Aquaporin 3 (AQP3) belongs to a family of transmembrane channels that are important in transporting water, glycerol, and other small molecules across the cell membrane. Glycerol that is transported by AQP3 is necessary for cell energy generation and lipid synthesis which fulfill the cell biological processes. Previous studies have shown that AQP3 is implicated in disease progression in several cancer types. However, whether AQP3-regulated glycerol uptake and metabolism were involved in cancer progression remains to be further studied. Our study demonstrated that the expression of AQP3 was positively correlated with glycerol level in human gastric cancer tissues. AQP3 inhibition induced proliferation impairment in gastric cancer cells both in vitro and in vivo. AQP3 inhibition that induced glycerol uptake reduction and glycerol administration would rehabilitate the cell proliferation. The energy and lipid production decreased when AQP3 was knocked down since the cellular glycerol level and several lipogenesis enzymes were downregulated. PI3K/Akt signaling pathway, which was involved in the impaired lipid and ATP production, was also inhibited after AQP3 knockdown. Our study indicated that the energy and lipid production inhibition, which were responsible for gastric cancer cell proliferation impairment, were induced by glycerol uptake reduction after AQP3 knockdown.

Defective macrophage function in aquaporin-3 deficiency.

Macrophages play an essential role in innate immunity. We found that mouse resident peritoneal macrophages (mRPMs) express the aquaglyceroporin aquaporin-3 (AQP3) in a plasma membrane pattern. AQP3-deficient (AQP3(-/-)) mice showed significantly greater mortality than wild-type (AQP3(+/+)) mice in a model of bacterial peritonitis. To establish the cellular mechanism of the peritonitis phenotype, measurements were made of mRPM phagocytosis, migration, and water/glycerol permeability. We found significantly impaired engulfment of Escherichia coli and chicken erythrocytes in AQP3(-/-) vs. AQP3(+/+) mRPMs, as well as impaired migration of AQP3(-/-) mRPMs in response to a chemotactic stimulus. In AQP3(+/+) mRPMs, AQP3 was polarized to pseudopodia at the leading edge during migration and around the phagocytic cup during engulfment. Water and glycerol permeabilities in mRPMs from AQP3(-/-) mice were reduced compared to mRPMs from AQP3(+/+) mice. Cellular glycerol and ATP content were remarkably lower in AQP3(-/-) vs. AQP3(+/+) mRPMs, and glycerol supplementation partially rescued the reduced ATP content and impaired function of AQP3(-/-) mRPMs. These data implicate AQP3 as a novel determinant in macrophage immune function by a cellular mechanism involving facilitated water and glycerol transport, and consequent phagocytic and migration activity. This is the first study demonstrating involvement of an aquaporin in innate immunity. Our results suggest AQP3 as a novel therapeutic target in modulating the immune response in various infectious and inflammatory conditions.

In situ fluorescence measurement of tear film [Na+], [K+], [Cl-], and pH in mice shows marked hypertonicity in aquaporin-5 deficiency.

PURPOSE: Tear film composition depends on water and ion transport across ocular surface epithelia and on fluid secretion by lacrimal glands. The purpose of this study was to establish in situ fluorescence methods to measure tear film ionic concentrations and pH in mice and to determine whether tear film composition is sensitive to deficiency of the major ocular surface aquaporin water channels. METHODS: Tear film ionic concentrations and pH were measured in anesthetized mice by ratio imaging fluorescence microscopy after topical application of ion/pH-sensing, dual-wavelength fluorescent indicators. [Na(+)], [K(+)], and [Cl(-)] were measured with membrane-impermeant indicators developed by our laboratory, and pH was measured with bis(carboxyethyl)-carboxyfluorescein fluorescence-conjugated dextran. Measurements were performed on wild-type mice and on knockout mice lacking aquaporins AQP1, AQP3, and AQP5. RESULTS: In wild-type mice, tear film [Na(+)] was 139 +/- 8 mM, [K(+)] was 48 +/- 1 mM, [Cl(-)] was 127 +/- 4 mM, and pH was 7.59 +/- 0.2 (SE; n = 5-8). pH did not differ significantly in the AQP knockout mice. [Na(+)] was increased by approximately twofold in AQP5 null mice (230 +/- 20 mM) and was greatly reduced after exposure of the ocular surface to a humidified atmosphere. [K(+)] was mildly reduced in AQP1 null mice. CONCLUSIONS: These results establish an in situ optical methodology to measure tear film [Na(+)], [K(+)], [Cl(-)], and pH in living mice, without the need for fluid sampling. Tear film hypertonicity in AQP5 deficiency is likely caused by reduced transcorneal water secretion in response to evaporative water loss.

Prevention of skin tumorigenesis and impairment of epidermal cell proliferation by targeted aquaporin-3 gene disruption.

Aquaporin-3 (AQP3) is a water/glycerol-transporting protein expressed strongly at the plasma membranes of basal epidermal cells in skin. We found that human skin squamous cell carcinoma strongly overexpresses AQP3. A novel role for AQP3 in skin tumorigenesis was discovered using mice with targeted AQP3 gene disruption. We found that AQP3-null mice were remarkably resistant to the development of skin tumors following exposure to a tumor initiator and phorbol ester promoter. Though tumor initiator challenge produced comparable apoptotic responses in wild-type and AQP3-null mice, promoter-induced cell proliferation was greatly impaired in the AQP3-null epidermis. Reductions of epidermal cell glycerol, its metabolite glycerol-3-phosphate, and ATP were found in AQP3 deficiency without impairment of mitochondrial function. Glycerol supplementation corrected the reduced proliferation and ATP content in AQP3 deficiency, with cellular glycerol, ATP, and proliferative ability being closely correlated. Our data suggest involvement of AQP3-facilitated glycerol transport in epidermal cell proliferation and tumorigenesis by a novel mechanism implicating cellular glycerol as a key determinant of cellular ATP energy. AQP3 may thus be an important determinant in skin tumorigenesis and hence a novel target for tumor prevention and therapy.

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.

Impaired enterocyte proliferation in aquaporin-3 deficiency in mouse models of colitis.

BACKGROUND/AIMS: Recent evidence has implicated the involvement of aquaporins (AQPs) in cellular functions that are unrelated to transepithelial water transport. Although AQPs are expressed in the gastrointestinal tract, their importance has so far been unclear. AQP3 is a water/glycerol transporter expressed at the basolateral membrane of colonic epithelial cells. The aim of this study was to investigate the involvement of AQP3 in enterocyte proliferation using mouse models of inflammatory bowel disease. METHODS: Expression and function of AQP3 in mouse colonic epithelium were established. Colitis was induced in wild-type and AQP3 null mice by oral dextran sulphate administration or intracolonic acetic acid administration. Outcome measures included clinical disease severity, survival, pathology and cellular responses. Some mice were administered glycerol to test whether disease progression could be altered. RESULTS: AQP3 null mice given dextran sulphate developed severe colitis after 3 days, with colonic haemorrhage, marked epithelial cell loss and death. Wild-type mice, which had comparable initial colonic damage as assessed by cell apoptosis, developed remarkably less severe colitis, surviving to >8 days. Cell proliferation was greatly reduced in AQP3 null mice. Oral glycerol administration significantly improved survival and reduced the severity of colitis in AQP3 null mice. Survival was also reduced in AQP3 null mice in the acetic acid model. CONCLUSIONS: The results implicate a novel role for AQP3 in enterocyte proliferation that is probably related to its glycerol-transporting function. AQP3 is thus a potential target for therapy of intestinal diseases associated with enterocyte destruction.