Lens transcriptome profile during cataract development in Mip-null mice.

Major intrinsic protein or aquaporin-0 (MIP/AQP0) functions as a water channel and a cell-junction molecule in the vertebrate eye lens. Loss of MIP function in the lens leads to degraded optical quality and cataract formation by pathogenic mechanisms that are unclear. Here we have used microarray-hybridization analysis to detect lens transcriptome changes during cataract formation in mice that are functionally null for MIP (Mip-/-). In newborn Mip-/- lenses (P1) 11 genes were up-regulated and 18 were down-regulated (>2-fold, p=<0.05) and a similar number of genes was differentially regulated at P7. The most up-regulated genes (>6-fold) in the Mip-/- lens at P1 included those coding for a mitochondrial translocase (Timmdc1), a matrix metallopeptidase (Mmp2), a Rho GTPase-interacting protein (Ubxn11) and a transcription factor (Twist2). Apart from Mip, the most down-regulated genes (>4-fold) in the Mip-/- lens at P1 included those coding for a proteasome sub-unit (Psmd8), a ribonuclease (Pop4), and a heat-shock protein (Hspb1). Lens fiber cell degeneration in the Mip-/- lens was associated with increased numbers of TUNEL-positive cell nuclei and dramatically elevated levels of calpain-mediated proteolysis of αII-spectrin. However red-ox status, measured by glutathione and free-radical levels, was similar to that of wild-type. These data suggest that while relatively few genes (∼1.5% of the transcriptome) were differentially regulated >2-fold in the Mip-/- lens, calpain hyper-activation acts as a terminal pathogenic event during lens fiber cell death and cataract formation.

Enhanced Autophagy in Polycystic Kidneys of AQP11 Null Mice.

Aquaporin-11 (AQP11) is an intracellular water channel expressed at the endoplasmic reticulum (ER) of the proximal tubule. Its gene disruption in mice leads to intracellular vacuole formation at one week and the subsequent development of polycystic kidneys by three weeks. As the damaged proximal tubular cells with intracellular vacuoles form cysts later, we postulated that autophagy may play a role in the cyst formation and examined autophagy activity before and after cyst development in AQP11(-/-) kidneys. PCR analysis showed the increased expression of the transcript encoding LC3 (Map1lc3b) as well as other autophagy-related genes in AQP11(-/-) mice. Using green fluorescent protein (GFP)-LC3 transgenic mice and AQP11(-/-) mice, we found that the number of GFP-LC3-positive puncta was increased in the proximal tubule of AQP11(-/-) mice before the cyst formation. Interestingly, they were also observed in the cyst-lining epithelial cell. Further PCR analyses revealed the enhanced expression of apoptosis-related and ER stress-related caspase genes before and after the cyst formation, which may cause the enhanced autophagy. These results suggest the involvement of autophagy in the development and maintenance of kidney cysts in AQP11(-/-) mice.

Loss of aquaporin 9 expression adversely affects the survival of retinal ganglion cells.

Aquaporin 9 (AQP9), an aquaglyceroporin belonging to the AQP water channel family, is permeable not only to water but also to noncharged solutes such as lactate. In neurons, lactate presumably acts as an energy substrate and as a source of NADH (the reduced form of nicotinamide adenine dinucleotide), a scavenger of reactive oxygen species (ROS). We previously reported that retinal ganglion cells (RGCs) express AQP9 and that elevated intraocular pressure reduces AQP9 expression and increases death of neurons in the retinal ganglion cell layer of rodents. In the present study, we investigated the association of AQP9 expression with serum deprivation-induced death of RGC-5 cells and with death of neurons in the rat retinal ganglion cell layer after optic nerve transection (ONT). The effect of AQP9 RNA interference on serum deprivation-induced apoptosis, ROS accumulation, and the NAD(+)/NADH ratio in RGC-5 cells was examined. Both serum deprivation and ONT significantly reduced AQP9 protein expression in RGCs and increased the rate of RGC death. Retinal AQP9 gene expression also declined after ONT. Down-regulation of AQP9 significantly increased apoptosis, ROS accumulation, and the NAD(+)/NADH ratio in the RGC-5 cells. These findings suggest that AQP9 loss adversely affects survival of RGCs, at least partly because of decreased transport of lactate as a substrate for energy and/or ROS scavenger.

Cardiovascular-metabolic impact of adiponectin and aquaporin.

Visceral fat accumulation is located upstream of metabolic syndrome. Recent progress in adipocyte biology has clarified the molecular mechanism for pathophysiology of metabolic syndrome and its related disorders. In this review we summarize adiponectin and aquaporin 7 (AQP7) in the role of metabolic syndrome and cardiovascular diseases.

Aquaporin-9 and urea transporter-A gene deletions affect urea transmembrane passage in murine hepatocytes.

In mammals, the majority of nitrogen from protein degradation is disposed of as urea. Several studies have partly characterized expression of urea transporters (UTs) in hepatocytes, where urea is produced. Nevertheless, the contribution of these proteins to hepatocyte urea permeability (P(urea)) and their role in liver physiology remains unknown. The purpose of this study was to biophysically examine hepatocyte urea transport. We hypothesized that the water, glycerol, and urea channel aquaporin-9 (AQP9) is involved in hepatocyte urea release. Stopped-flow light-scattering measurements determined that the urea channel inhibitors phloretin and dimethylurea reduced urea permeability of hepatocyte basolateral membranes by 70 and 40%, respectively. In basolateral membranes isolated from AQP9(-/-) and UT-A1/3(-/-) single-knockout and AQP9(-/-):UT-A1/3(-/-) double-knockout mice, P(urea) was decreased by 30, 40, and 76%, respectively, compared with AQP9(+/-):UT-A1/3(+/-) mice. However, expression analysis by RT-PCR did not identify known UT-A transcripts in liver. High-protein diet followed by 24-h fasting affected the concentrations of urea and ammonium ions in AQP9(-/-) mouse liver and plasma without generating an apparent tissue-to-plasma urea gradient. We conclude that AQP9 and unidentified UT-A urea channels constitute primary but redundant urea facilitators in murine hepatocytes.

Reduced arsenic clearance and increased toxicity in aquaglyceroporin-9-null mice.

Expressed in liver, aquaglyceroporin-9 (AQP9) is permeated by glycerol, arsenite, and other small, neutral solutes. To evaluate a possible protective role, AQP9-null mice were evaluated for in vivo arsenic toxicity. After injection with NaAsO(2), AQP9-null mice suffer reduced survival rates (LD(50), 12 mg/kg) compared with WT mice (LD(50), 15 mg/kg). The highest tissue level of arsenic is in heart, with AQP9-null mice accumulating 10-20 times more arsenic than WT mice. Within hours after NaAsO(2) injection, AQP9-null mice sustain profound bradycardia, despite normal serum electrolytes. Increased arsenic levels are also present in liver, lung, spleen, and testis of AQP9-null mice. Arsenic levels in the feces and urine of AQP9-null mice are only approximately 10% of the WT levels, and reduced clearance of multiple arsenic species by the AQP9-null mice suggests that AQP9 is involved in the export of multiple forms of arsenic. Immunohistochemical staining of liver sections revealed that AQP9 is most abundant in basolateral membrane of hepatocytes adjacent to the sinusoids. AQP9 is not detected in heart or kidney by PCR or immunohistochemistry. We propose that AQP9 provides a route for excretion of arsenic by the liver, thereby providing partial protection of the whole animal from arsenic toxicity.

Effects of acetaldehyde on hepatocyte glycerol uptake and cell size: implication of aquaporin 9.

BACKGROUND: The effects of ethanol and acetaldehyde on uptake of glycerol and on cell size of hepatocytes and a role Aquaporin 9 (AQP9), a glycerol transport channel, were evaluated. METHODS: The studies were done in primary rat and mouse hepatocytes. The uptake of [(14) C] glycerol was determined with hepatocytes in suspension. For determination of cell size, rat hepatocytes on coated dishes were incubated with a lipophilic fluorochrome that is incorporated into the cell membrane and examined by confocal microscopy. A three-dimensional z scan of the cell was performed, and the middle slice of the z scan was used for area measurements. RESULTS: Acute exposure to acetaldehyde, but not to ethanol, causes a rapid increase in the uptake of glycerol and an increase in hepatocyte size, which was inhibited by HgCl(2) , an inhibitor of aquaporins. This was not observed in hepatocytes from AQP9 knockout mice, nor observed by direct application of acetaldehyde to AQP9 expressed in Xenopus Laevis oocytes. Prolonged 24-hour exposure to either acetaldehyde or ethanol did not result in an increase in glycerol uptake by rat hepatocytes. Acetaldehyde decreased AQP9 mRNA and AQP9 protein, while ethanol decreased AQP9 mRNA but not AQP9 protein. Ethanol, but not acetaldehyde, increased the activities of glycerol kinase and phosphoenolpyruvate carboxykinase. CONCLUSIONS: The acute effects of acetaldehyde, while mediated by AQP9, are probably influenced by binding of acetaldehyde to hepatocyte membranes and changes in cell permeability. The effects of ethanol in enhancing glucose kinase, and phosphoenolpyruvate carboxykinase leading to increased formation of glycerol-3-phosphate most likely contribute to alcoholic fatty liver.

Impairment of angiogenesis and cell migration by targeted aquaporin-1 gene disruption.

Aquaporin-1 (AQP1) is a water channel protein expressed widely in vascular endothelia, where it increases cell membrane water permeability. The role of AQP1 in endothelial cell function is unknown. Here we show remarkably impaired tumour growth in AQP1-null mice after subcutaneous or intracranial tumour cell implantation, with reduced tumour vascularity and extensive necrosis. A new mechanism for the impaired angiogenesis was established from cell culture studies. Although adhesion and proliferation were similar in primary cultures of aortic endothelia from wild-type and from AQP1-null mice, cell migration was greatly impaired in AQP1-deficient cells, with abnormal vessel formation in vitro. Stable transfection of non-endothelial cells with AQP1 or with a structurally different water-selective transporter (AQP4) accelerated cell migration and wound healing in vitro. Motile AQP1-expressing cells had prominent membrane ruffles at the leading edge with polarization of AQP1 protein to lamellipodia, where rapid water fluxes occur. Our findings support a fundamental role of water channels in cell migration, which is central to diverse biological phenomena including angiogenesis, wound healing, tumour spread and organ regeneration.

Pregnant phenotype in aquaporin 8-deficient mice.

AIM: Aquaporin 8 (AQP8) is expressed within the female reproductive system but its physiological function reminds to be elucidated. This study investigates the role of AQP8 during pregnancy using AQP8-knockout (AQP8-KO) mice. METHODS: Homozygous AQP8-KO mice were mated, and the conception rate was recorded. AQP8-KO pregnant mice or their offspring were divided into 5 subgroups according to fetal gestational day (7, 13, 16, 18 GD) and newborn. Wild type C57 pregnant mice served as the control group. The number of pregnant mice, total embryos and atrophic embryos, as well as fetal weight, placental weight and placental area were recorded for each subgroup. The amount of amniotic fluid in each sac at 13, 16, and 18 GD was calculated. Statistical significance was determined by analysis of variance of factorial design and chi-square tests. RESULTS: Conception rates did not differ significantly between AQP8-KO and wild type mice. AQP8-KO pregnant mice had a significantly higher number of embryos compared to wild type controls. Fetal/neonatal weight was also significantly greater in the AQP8-KO group compared to age-matched wild type controls. The amount of amniotic fluid was greater in AQP8-KO pregnant mice than wild type controls, although the FM/AFA (fetal weight/amniotic fluid amount) did not differ. While AQP8-KO placental weight was significantly larger than wild type controls, there was no evidence of placental pathology in either group. CONCLUSION: The results suggest that AQP8 deficiency plays an important role in pregnancy outcome.

Ablation of Aquaporin-9 Ameliorates the Systemic Inflammatory Response of LPS-Induced Endotoxic Shock in Mouse.

Septic shock is the most severe complication of sepsis, being characterized by a systemic inflammatory response following bacterial infection, leading to multiple organ failure and dramatically high mortality. Aquaporin-9 (AQP9), a membrane channel protein mainly expressed in hepatocytes and leukocytes, has been recently associated with inflammatory and infectious responses, thus triggering strong interest as a potential target for reducing septic shock-dependent mortality. Here, we evaluated whether AQP9 contributes to murine systemic inflammation during endotoxic shock. Wild type (Aqp9+/+; WT) and Aqp9 gene knockout (Aqp9-/-; KO) male mice were submitted to endotoxic shock by i.p. injection of lipopolysaccharide (LPS; 40 mg/kg) and the related survival times were followed during 72 h. The electronic paramagnetic resonance and confocal microscopy were employed to analyze the nitric oxide (NO) and superoxide anion (O2-) production, and the expression of inducible NO-synthase (iNOS) and cyclooxigenase-2 (COX-2), respectively, in the liver, kidney, aorta, heart and lung of the mouse specimens. LPS-treated KO mice survived significantly longer than corresponding WT mice, and 25% of the KO mice fully recovered from the endotoxin treatment. The LPS-injected KO mice showed lower inflammatory NO and O2- productions and reduced iNOS and COX-2 levels through impaired NF-κB p65 activation in the liver, kidney, aorta, and heart as compared to the LPS-treated WT mice. Consistent with these results, the treatment of FaO cells, a rodent hepatoma cell line, with the AQP9 blocker HTS13268 prevented the LPS-induced increase of inflammatory NO and O2-. A role for AQP9 is suggested in the early acute phase of LPS-induced endotoxic shock involving NF-κB signaling. The modulation of AQP9 expression/function may reveal to be useful in developing novel endotoxemia therapeutics.

The role of mammalian superaquaporins inside the cell: An update.

The progress on mammalian superaquaporin (sAQP), AQP11 and AQP12, in the past seven years is brought up to date from the previous review. This subfamily is separated because of the very low homology with other AQP subfamilies and it is present only in multicellular organisms excluding fungi and plants. Its unique intracellular localization, specifically in the ER has made its functional studies challenging, but it may function as glyceroporin, aquaporin and peroxiporin, H2O2 transporter. Knowledge on AQP11 has been expanded by tissue specific conditional knockout mice and by the identification of a SNP associated with kidney diseases. Moreover, the functional identification of AQP11 as a peroxiporin has expanded the role of AQP11 to the regulation of intracellular H2O2 homeostasis to prevent ER stress, which awaits further in vivo studies. As kidney-specific AQP11 knockout of developed kidney has produced little phenotype, AQP11 is critical for kidney development but its physiological significance remains to be clarified. On the other hand, little has been known on pancreas-specific AQP12. To move this field forward, the results of sAQP in lower animals will be necessary to obtain the insights into the role of mammalian sAQP, which hopefully will lead to the discovery of therapeutic targets.

Increased female fertility in aquaporin 8-deficient mice.

Aquaporin-8 (AQP8) is a water channel expressed extensively in male and female reproductive systems. But its physiological functions are largely unknown. In the present study, we first found significantly increased number of offspring delivered by AQP8(-/-) mothers compared with wild-type mothers in cross-mating experiments. Comparison of ovulation in the two genotypes demonstrated that AQP8(-/-) ovaries released more oocytes (9.5 ± 1.9 vs. 7.1 ± 2.1 in normal ovulation and 37.8 ± 6.7 vs. 27.9 ± 5.7 in superovulation). Histological analysis showed increased number of corpus luteums in mature AQP8(-/-) ovaries, suggesting increased maturation and ovulation of follicles. By RT-PCR, western blot and immunohistochemistry analyses, we determined the expression of AQP8 in mouse ovarian granulosa cells. Granulosa cells isolated from AQP8(-/-) mice showed 45% of decreased membrane water permeability than wild-type mice. As the atresia of ovarian follicles is primarily due to apoptosis of granulosa cells, we analyzed the apoptosis of isolated granulosa cells from wild-type and AQP8(-/-) mice. The results indicated significantly lower apoptosis rate in AQP8(-/-) granulosa cells (21.3 ± 3.6% vs. 32.6 ± 4.3% in AQP8(+/+) granulosa cells). Taken together, we conclude that AQP8 deficiency increases the number of mature follicles by reducing the apoptosis of granulosa cells, thus increasing the fertility of female mice. This discovery may offer new insight of improving female fertility by reducing granulosa cell apoptosis through AQP8 inhibition.

Pancreas-specific aquaporin 12 null mice showed increased susceptibility to caerulein-induced acute pancreatitis.

Aquaporin 12 (AQP12) is the most recently identified member of the mammalian AQP family and is specifically expressed in pancreatic acinar cells. In vitro expression studies have revealed that AQP12 is localized at intracellular sites. To determine the physiological roles of AQP12 in the pancreas, we generated knockout mice for this gene (AQP12-KO). No obvious differences were observed under normal conditions between wild-type (WT) and AQP12-KO mice in terms of growth, blood chemistry, pancreatic fluid content, or histology. However, when we induced pancreatitis through the administration of a cholecystokinin-8 (CCK-8) analog, the AQP12-KO mice showed more severe pathological damage to this organ than WT mice. Furthermore, when we analyzed exocytosis in the pancreatic acini using a two-photon excitation imaging method, the results revealed larger exocytotic vesicles (vacuoles) in the acini of AQP12-KO mice at a high CCK-8 dose (100 nM). From these results, we conclude that AQP12 may function in the mechanisms that control the proper secretion of pancreatic fluid following rapid and intense stimulation.

Knock-out models reveal new aquaporin functions.

Knockout mice have been informative in the discovery of unexpected biological functions of aquaporins. Knockout mice have confirmed the predicted roles of aquaporins in transepithelial fluid transport, as in the urinary concentrating mechanism and glandular fluid secretion. A less obvious, though predictable role of aquaporins is in tissue swelling under stress, as in the brain in stroke, tumor and infection. Phenotype analysis of aquaporin knockout mice has revealed several unexpected cellular roles of aquaporins whose mechanisms are being elucidated. Aquaporins facilitate cell migration, as seen in aquaporin-dependent tumor angiogenesis and tumor metastasis, by a mechanism that may involve facilitated water transport in lamellipodia of migrating cells. The ' aquaglyceroporins', aquaporins that transport both glycerol and water, regulate glycerol content in epidermis, fat and other tissues, and lead to a multiplicity of interesting consequences of gene disruption including dry skin, resistance to skin carcinogenesis, impaired cell proliferation and altered fat metabolism. An even more surprising role of a mammalian aquaporin is in neural signal transduction in the central nervous system. The many roles of aquaporins might be exploited for clinical benefit by modulation of aquaporin expression/function - as diuretics, and in the treatment of brain swelling, glaucoma, epilepsy, obesity and cancer.

Mammalian aquaporins: diverse physiological roles and potential clinical significance.

Aquaporins have multiple distinct roles in mammalian physiology. Phenotype analysis of aquaporin-knockout mice has confirmed the predicted role of aquaporins in osmotically driven transepithelial fluid transport, as occurs in the urinary concentrating mechanism and glandular fluid secretion. Aquaporins also facilitate water movement into and out of the brain in various pathologies such as stroke, tumour, infection and hydrocephalus. A major, unexpected cellular role of aquaporins was revealed by analysis of knockout mice: aquaporins facilitate cell migration, as occurs in angiogenesis, tumour metastasis, wound healing, and glial scar formation. Another unexpected role of aquaporins is in neural function - in sensory signalling and seizure activity. The water-transporting function of aquaporins is likely responsible for these roles. A subset of aquaporins that transport both water and glycerol, the 'aquaglyceroporins', regulate glycerol content in epidermal, fat and other tissues. Mice lacking various aquaglyceroporins have several interesting phenotypes, including dry skin, resistance to skin carcinogenesis, impaired cell proliferation, and altered fat metabolism. The various roles of aquaporins might be exploited clinically by development of drugs to alter aquaporin expression or function, which could serve as diuretics, and in the treatment of brain swelling, glaucoma, epilepsy, obesity and cancer.

Disruption of aquaporin-11 produces polycystic kidneys following vacuolization of the proximal tubule.

Aquaporin-11 (AQP11) has been identified with unusual pore-forming NPA (asparagine-proline-alanine) boxes, but its function is unknown. We investigated its potential contribution to the kidney. Immunohistochemistry revealed that AQP11 was localized intracellularly in the proximal tubule. When AQP11 was transfected in CHO-K1 cells, it was localized in intracellular organelles. AQP11-null mice were generated; these mice exhibited vacuolization and cyst formation of the proximal tubule. AQP11-null mice were born normally but died before weaning due to advanced renal failure with polycystic kidneys, in which cysts occupied the whole cortex. Remarkably, cyst epithelia contained vacuoles. These vacuoles were present in the proximal tubules of newborn mice. In 3-week-old mice, these tubules contained multiple cysts. Primary cultured cells of the proximal tubule revealed an endosomal acidification defect in AQP11-null mice. These data demonstrate that AQP11 is essential for the proximal tubular function. AQP11-null mice are a novel model for polycystic kidney diseases and will provide a new mechanism for cystogenesis.

Aquaporin-7 and glycerol permeability as novel obesity drug-target pathways.

Advances in determining the mechanisms that underlie the control of energy balance in mammals have recently been provided by the discovery and characterization of aquaporin-7 (AQP7), a water-glycerol transporter that is present in the plasma membrane of fat-storing cells (adipocytes). Recent studies have shown that the absence of AQP7 expression in fat cells increases glycerol kinase activity, boosting triacylglycerol synthesis and ultimately leading to obesity. Thus, AQP7 operates as a glycerol channel in vivo, whereby adipocyte glycerol permeability has a key role in the regulation of fat accumulation.

Glycerol transport: an additional target for obesity therapy?

There is increasing evidence that the glycerol channel, aquaporin 7(AQP7), has an important role in adipose tissue formation and function--deletion of the gene in a mouse strain leads to obesity and diabetes type 2 if the mice are aged or fed earlier with a high-fat or sucrose diet. Can increased levels of AQP7 in adipose tissue protect against obesity? New studies on AQP7 highlight the important role of glycerol transport in the development of obesity and metabolic disease.

A novel missense mutation of Mip causes semi-dominant cataracts in the Nat mouse.

Major intrinsic protein of lens fiber (MIP) is one of the proteins essential for maintaining lens transparency while also contributing to dominant cataracts in humans. The Nodai cataract (Nat) mice harbor a spontaneous mutation in Mip and develop early-onset nuclear cataracts. The Nat mutation is a c.631G>A mutation (MipNat), resulting in a glycine-to-arginine substitution (p.Gly211Arg) in the sixth transmembrane domain. The MipNat/Nat homozygotes exhibit congenital cataracts caused by the degeneration of lens fiber cells. MIP normally localizes to the lens fiber cell membranes. However, the MipNat/Nat mice were found to lack an organelle-free zone, and the MIP was mislocalized to the nuclear membrane and perinuclear region. Furthermore, the MipNat/+ mice exhibited milder cataracts than MipNat/Nat mice due to the slight degeneration of the lens fiber cells. Although there were no differences in the localization of MIP to the membranes of lens fiber cells in MipNat/+ mice compared to that in wild-type mice, the protein levels of MIP were significantly reduced in the eyes. These findings suggest that cataractogenesis in MipNat mutants are caused by defects in MIP expression. Overall, the MipNat mice offer a novel model to better understand the phenotypes and mechanisms for the development of cataracts in patients that carry missense mutations in MIP.