The exchange of functional domains among aquaporins with different transport characteristics

 Aquaporins are transmembrane proteins that contain six bilayer-spanning domains, connected by loops A to E. The hourglass model predicts that the conserved loops B and E are essential for the formation of the water pore. To test the importance of loops B and E in the determination of the transport characteristics, we exchanged loops B and/or E between AQP0, AQP2, and AQP3. Detailed functional, immunoblot and immunocytochemical analyses of expression in Xenopus oocytes revealed that six out of the nine chimeric aquaporin proteins were not functional, because of misrouting. AQP0 with loop E of AQP2 was not impaired in its routing and revealed a low water permeability equal to that of wild-type AQP0. AQP2 with loop B of AQP0 was also routed normally and gave a high water permeability, similar to that of wild-type AQP2. AQP0 with loops B and E of AQP2 (AQP0–2BE) did not result in an increase in water permeability and was partly misrouted. However, the plasma membrane expression was high enough to expect an increase in water permeability, as loops B and E of AQP2 confer AQP2’s water permeability to AQP0. Although it is unclear for the dual chimera (AQP0–2BE), the parental water permeabilities obtained in oocytes expressing AQP0 with loop E of AQP2 or AQP2 with loop B of AQP0 indicate that, besides loops B and E, other parts of the AQP protein are important in the determination of the characteristics of the channel. Received: 24 February 1998 / Received after revision: 20 April 1998 / Accepted: 28 April 1998     

NH3 and NH 4 + permeability in aquaporin-expressing Xenopus oocytes

We have shown recently, in a yeast expression system, that some aquaporins are permeable to ammonia. In the present study, we expressed the mammalian aquaporins AQP8, AQP9, AQP3, AQP1 and a plant aquaporin TIP2;1 in Xenopus oocytes to study the transport of ammonia (NH₃) and ammonium (NH₄⁺) under open-circuit and voltage-clamped conditions. TIP2;1 was tested as the wild-type and in a mutated version (tip2;1) in which the water permeability is intact. When AQP8-, AQP9-, AQP3- and TIP2;1-expressing oocytes were placed in a well-stirred bathing medium of low buffer capacity, NH₃ permeability was evident from the acidification of the bathing medium; the effects observed with AQP1 and tip2;1 did not exceed that of native oocytes. AQP8, AQP9, AQP3, and TIP2;1 were permeable to larger amides, while AQP1 was not. Under voltage-clamp conditions, given sufficient NH₃, AQP8, AQP9, AQP3, and TIP2;1 supported inwards currents carried by NH₄⁺. This conductivity increased as a sigmoid function of external [NH₃]: for AQP8 at a bath pH (pH_e) of 6.5, the conductance was abolished, at pH_e 7.4 it was half maximal and at pH_e 7.8 it saturated. NH₄⁺ influx was associated with oocyte swelling. In comparison, native oocytes as well as AQP1 and tip2;1-expressing oocytes showed small currents that were associated with small and even negative volume changes. We conclude that AQP8, AQP9, AQP3, and TIP2;1, apart from being water channels, also support significant fluxes of NH₃. These aquaporins could support NH₄⁺ transport and have physiological implications for liver and kidney function.     

Novel variants in human Aquaporin-4 reduce cellular water permeability

Cerebral edema contributes significantly to morbidity and mortality after brain injury and stroke. Aquaporin-4 (AQP4), a water channel expressed in astrocytes, plays a key role in brain water homeostasis. Genetic variants in other aquaporin family members have been associated with disease phenotypes. However, in human AQP4, only one non-synonymous single-nucleotide polymorphism (nsSNP) has been reported, with no characterization of protein function or disease phenotype. We analyzed DNA from an ethnically diverse cohort of 188 individuals to identify novel AQP4 variants. AQP4 variants were constructed by site-directed mutagenesis and expressed in cells. Water permeability assays in the cells were used to measure protein function. We identified 24 variants in AQP4 including four novel nsSNPs (I128T, D184E, I205L and M224T). We did not observe the previously documented M278T in our sample. The nsSNPs found were rare ( approximately 1-2% allele frequency) and heterozygous. Computational analysis predicted reduced function mutations. Protein expression and membrane localization were similar for reference AQP4 and the five AQP4 mutants. Cellular assays confirmed that four variant AQP4 channels reduced normalized water permeability to between 26 and 48% of the reference (P < 0.001), while the M278T mutation increased normalized water permeability (P < 0.001). We identified multiple novel AQP4 SNPs and showed that four nsSNPs reduced water permeability. The previously reported M278T mutation resulted in gain of function. Our experiments provide insight into the function of the AQP4 protein. These nsSNPs may have clinical implications for patients with cerebral edema and related disorders.     

Lead induces increased water permeability in astrocytes expressing aquaporin 4

The water channel aquaporin 4 (AQP4) is abundantly expressed in astrocytes. There is now compelling evidence that AQP4 may contribute to an unfavorable course in brain edema. Acute lead intoxication is a condition that causes brain damage preceded by brain edema. Here we report that lead increases AQP4 water permeability (P(f)) in astrocytes. A rat astrocyte cell line that does not express aquaporin 4 was transiently transfected with aquaporin 4 tagged with green fluorescent protein (GFP). Using confocal laser scanning microscopy we measured water permeability in these cells and in AQP4-negative cells located on the same plate. AQP4-expressing astrocytes had a three-fold higher water permeability than astrocytes not expressing AQP4. Lead exposure induced a significant, 40%, increase in water permeability in astrocytes expressing AQP4, but had no effect on P(f) in astrocytes not expressing AQP4. The increase in water permeability persisted after lead washout, while treatment with a lead chelator, meso-2,3-dimercaptosuccinic acid, abolished the lead-induced increase in P(f). The effect of lead was attenuated in the presence of a calcium (Ca(2+))/calmodulin-dependent protein kinase II (CaMKII) inhibitor, but not in the presence of a protein kinase C inhibitor. In cells expressing AQP4 where the consensus site for CaMKII phosphorylation was mutated, lead failed to increase water permeability. Lead exposure also increased P(f) in rat astroglial cells in primary culture, which express endogenous AQP4. Lead had no effect on P(f) in astrocytes transfected with aquaporin 3. In situ hybridization studies on rat brain after oral lead intake for three days showed no change in distribution of AQP4 mRNA. It is suggested that lead-triggered stimulation of water transport in AQP4-expressing astrocytes may contribute to the pathology of acute lead intoxication.     

Water permeability through aquaporin-4 is regulated by protein kinase C and becomes rate-limiting for glioma invasion

Glial-derived tumors, gliomas, are highly invasive cancers that invade normal brain through the extracellular space. To navigate the tortuous extracellular spaces, cells undergo dynamic changes in cell volume, which entails water flux across the membrane through aquaporins (AQPs). Two members of this family, AQP1 and AQP4 are highly expressed in primary brain tumor biopsies and both have a consensus phosphorylation site for protein kinase C (PKC), which is a known regulator of glioma cell invasion. AQP4 colocalizes with PKC to the leading edge of invading processes and clustered with chloride channel (ClC2) and K⁺–Cl⁻ cotransporter 1 (KCC1), believed to provide the pathways for Cl⁻ and K⁺ secretion to accomplish volume changes. Using D54MG glioma cells stably transfected with either AQP1 or AQP4, we show that PKC activity regulates water permeability through phosphorylation of AQP4. Activation of PKC with either phorbol 12-myristate 13-acetate or thrombin enhanced AQP4 phosphorylation, reduced water permeability and significantly decreased cell invasion. Conversely, inhibition of PKC activity with chelerythrine reduced AQP4 phosphorylation, enhanced water permeability and significantly enhanced tumor invasion. PKC regulation of AQP4 was lost after mutational inactivation of the consensus PKC phosphorylation site S180A. Interestingly, AQP1 expressing glioma cells, by contrast, were completely unaffected by changes in PKC activity. To demonstrate a role for AQPs in glioma invasion in vivo, cells selectively expressing AQP1, AQP4 or the mutated S180A-AQP4 were implanted intracranially into SCID mice. AQP4 expressing glioma cells showed significantly reduced invasion compared to AQP1 and S180 expressing tumors as determined by quantitative stereology, consistent with a differential role for AQP1 and AQP4 in this process.     

Upregulation of AQP3 and AQP5 induced by dexamethasone and ambroxol in A549 cells

Aquaporins (AQPs) are membrane channel proteins that play roles in the regulation of water permeability in many tissues. AQP1 and AQP5 expressed in lung provide the principal route for osmotically driven water transport. In the airways, AQP3 and AQP4 facilitate water transport. Dexamethasone and ambroxol are often used to treat patients with pulmonary diseases accompanied by airway hypersecretion. The role of AQPs in these effective treatments has not been addressed. In this study, we analyzed the expression of AQPs in a human airway epithelial cell line (A549 cells) and showed that AQP3 and 5, but not AQP1 and 4, were expressed in A549 cells. Both dexamethasone and ambroxol stimulated the expression of AQP3 and 5 at the mRNA and protein levels. The data suggest potential roles of AQP3 and 5 in the regulation of airway hypersecretion, perhaps ultimately providing a target for treating such diseases.     

Role of water channels in fluid transport studied by phenotype analysis of aquaporin knockout mice

Aquaporin-type water channels are expressed widely in mammalian tissues, particularly in the kidney, lung, eye and gastrointestinal tract. To define the role of aquaporins in organ physiology, we have generated and analysed transgenic mice lacking aquaporins (AQP) 1, 3, 4 and 5. Multiple phenotype abnormalities were found in the null mice. For example, in kidney, deletion of AQP1 or AQP3 produced marked polyuria whereas AQP4 deletion produced only a mild concentrating defect. Deletion of AQP5, the apical membrane water channel in the salivary gland, caused defective saliva production. Deletion of AQP1 or AQP5, water channels in lung endothelia and epithelia, resulted in a 90% decrease in airspace-capillary water permeability. In the brain, deletion of AQP4 conferred marked protection from brain swelling induced by acute water intoxication and ischaemic stroke. The general paradigm that has emerged from these phenotype studies is that aquaporins facilitate rapid near-isosmolar transepithelial fluid absorption/secretion, as well as rapid vectorial water movement driven by osmotic gradients. However, we have found many examples in which the tissue-specific expression of an aquaporin is not associated with any apparent phenotypic abnormality. The physiological data on aquaporin null mice suggest the utility of aquaporin blockers and aquaporin gene replacement in selected human diseases.     

Identification and characterization of aquaporin-2 water channel mutations causing nephrogenic diabetes insipidus with partial vasopressin response

Congenital nephrogenic diabetes insipidus (NDI) is a rare disease caused most often by mutations in the vasopressin V2 receptor (AVPR2). We studied a family which included a female patient with NDI with symptoms dating from infancy. The patient responded to large doses of desmopressin (dDAVP) which decreased urine volume from 10 to 4 I/day. Neither the parents nor the three sisters were polyuric. The patient was found to be a compound heterozygote for two novel recessive point mutations in the aquaporin-2 (AQP2) gene: L22V in exon 1 and C181W in exon 3. Residue Cys181 in AQP2 is the site for inhibition of water permeation by mercurial compounds and is located near to the NPA motif conserved in all aquaporins. Osmotic water permeability (Pf) in Xenopus oocytes injected with cRNA encoding C181W-AQP2 was not increased over water control, while expression of L22V cRNA increased the Pf to approximately 60% of that for wild-type AQP2. Co-injection of the mutant cRNAs with the wild-type cRNA did not affect the function of the wild-type AQP2. Immunolocalization of AQP2-transfected CHO cells showed that the C181W mutant had an endoplasmic reticulum-like intracellular distribution, whereas L22V and wild-type AQP2 showed endosome and plasma membrane staining. Water permeability assays showed a high Pf in cells expressing wild-type and L22V AQP2. This study indicates that AQP2 mutations can confer partially responsive NDI.      

Aquaporin water channels in gastrointestinal physiology

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

Functional analysis of novel aquaporins from Fasciola gigantica

Fascioliasis, caused by liver flukes of the genus Fasciola, is an important disease of ruminants. In order to identify a potential new drug target we have studied aquaporin (AQP) in Fasciola gigantica. AQPs facilitate the transport of water, glycerol and other small solutes across biological membranes. The structure, function, and pathology of AQPs have been extensively studied in mammals but data for AQPs from trematodes is still limited. In the present study, we have functionally characterized two closely related AQP isoforms, FgAQP-1 and FgAQP-2, from the trematode F. gigantica. Immunohistochemical analysis located the FgAQPs in the tegumental cells, their processes and the tegument itself. In addition, they were present in the epithelial linings of testes and ovary. Expression in Xenopus oocytes of these FgAQPs increased osmotic water permeability 3-4-fold but failed to increase glycerol and urea permeability. AQPs have two highly conserved NPA motifs that are important for the function of the channel pore. In FgAQP-1 and FgAQP-2 the first NPA motif is changed to TAA. Substitution of Thr with Asn in the TAA motif of FgAQP-1 increased its water permeability twofold but did not affect urea and glycerol impermeability while the substitution at the pore mouth of Cys204 by Tyr caused loss of water permeability. In addition, the FgAQPs did not increase methylamine and ammonia permeability after expression in yeast. In comparison to rat AQP-1 the described FgAQPs showed low water permeability and further in vivo analyses are necessary to determine their contribution to osmoregulation in Fasciola.     

Expression and localization of epithelial aquaporins in the adult human lung

Aquaporins (AQPs) facilitate water transport across epithelia and play an important role in normal physiology and disease in the human airways. We used in situ hybridization and immunofluorescence to determine the expression and cellular localization of AQPs 5, 4, and 3 in human airway sections. In nose and bronchial epithelia, AQP5 is expressed at the apical membrane of columnar cells of the superficial epithelium and submucosal gland acinar cells. AQP4 was detected in basolateral membranes in ciliated ducts and by in situ in gland acinar cells. AQP3 is present on basal cells of both superficial epithelium and gland acinus. In these regions AQPs 5, 4, and 3 are appropriately situated to permit transepithelial water permeability. In the small airways (proximal and terminal bronchioles) AQP3 distribution shifts from basal cell to surface expression (i.e., localized to the apical membrane of proximal and terminal bronchioles) and is the only AQP identified in this region of the human lung. The alveolar epithelium has all three AQPs represented, with AQP5 and AQP4 localized to type I pneumocytes and AQP3 to type II cells. This study describes an intricate network of AQP expression that mediates water transport across the human airway epithelium.     

Aquaporins in the kidney: from molecules to medicine.

The discovery of aquaporin-1 (AQP1) answered the long-standing biophysical question of how water specifically crosses biological membranes. In the kidney, at least seven aquaporins are expressed at distinct sites. AQP1 is extremely abundant in the proximal tubule and descending thin limb and is essential for urinary concentration. AQP2 is exclusively expressed in the principal cells of the connecting tubule and collecting duct and is the predominant vasopressin-regulated water channel. AQP3 and AQP4 are both present in the basolateral plasma membrane of collecting duct principal cells and represent exit pathways for water reabsorbed apically via AQP2. Studies in patients and transgenic mice have demonstrated that both AQP2 and AQP3 are essential for urinary concentration. Three additional aquaporins are present in the kidney. AQP6 is present in intracellular vesicles in collecting duct intercalated cells, and AQP8 is present intracellularly at low abundance in proximal tubules and collecting duct principal cells, but the physiological function of these two channels remains undefined. AQP7 is abundant in the brush border of proximal tubule cells and is likely to be involved in proximal tubule water reabsorption. Body water balance is tightly regulated by vasopressin, and multiple studies now have underscored the essential roles of AQP2 in this. Vasopressin regulates acutely the water permeability of the kidney collecting duct by trafficking of AQP2 from intracellular vesicles to the apical plasma membrane. The long-term adaptational changes in body water balance are controlled in part by regulated changes in AQP2 and AQP3 expression levels. Lack of functional AQP2 is seen in primary forms of diabetes insipidus, and reduced expression and targeting are seen in several diseases associated with urinary concentrating defects such as acquired nephrogenic diabetes insipidus, postobstructive polyuria, as well as acute and chronic renal failure. In contrast, in conditions with water retention such as severe congestive heart failure, pregnancy, and syndrome of inappropriate antidiuretic hormone secretion, both AQP2 expression levels and apical plasma membrane targetting are increased, suggesting a role for AQP2 in the development of water retention. Continued analysis of the aquaporins is providing detailed molecular insight into the fundamental physiology and pathophysiology of water balance and water balance disorders.     

Effects of dexamthasone with different doses on aquaporin-4 in brain of intracerebral hemorrhage rats

Objective:To determine the relationship between the expression of aquaporin-4(AQP4) after intracerebral hemorrhage and dexamethasone treated. Methods:Collagenase Ⅶ was injected in caudate nucleus in a stereotaxis frame to establish the intracerebral hemorrhage(ICH) animal models. The intracerebral hemorrhage(ICH) rats were randomly divided into four groups: the sham group (group A), the ICH group(group B), low dosertreated group(group C), moderate dose group(group D) and high dose group(group E). The groups were respectively received an intraperitoneal dexamethasone injection with 1 mg/kg, 15 mg/kg, 30 mg/kg, twice a day for three days. The brain water content(BWC), the permeability of blood-brain barrier(BBB) and the expression of AQP4 were observed. Results:Both the BBB disruption and AQP4 expression decreased in treated groups, and the AQP4 expression had a dose-dependent manner in the dexamethasone treatment. And it seemed that low dose dexamethasone was in favor of brain swelling elimination, but the higher dosage had not similar effect. Conclusion:Dexamethesone may play a critical role on expression of AQP4 in the physiopathology of hemorrhagic edema.     

Bronchiolar expression of aquaporin-3 (AQP3) in rat lung and its dynamics in pulmonary oedema

Aquaporins (AQPs) are water channel proteins that permit osmotically driven water movement. To determine their dynamics in pulmonary oedema, we examined the expression of mRNA and protein for AQP1, AQP3, AQP4, and AQP5 in the lungs of normal and thiourea-treated rats. In the thiourea group, lung water content increased significantly (vs. controls) with the peak at around 4 h. Semi-quantitative RT-PCR showed that AQP3 mRNA in the thiourea group rose significantly, peaking at around 4–8 h. The expression of AQP1, AQP4, AQP5, ENaC and CFTR mRNA each decreased significantly some time after the peak in lung water content. Immunoblot analysis showed that glycosylated AQP3 protein was increased 4–10 h after treatment. Expression of the other AQP proteins was not significantly altered, except for that of AQP4. Immunohistochemical examination revealed that AQP1 was expressed in endothelia, AQP3 in the basal cells of the large airways and in cuboidal cells in the bronchioles, AQP4 in the basolateral membrane of airway cells and AQP5 in type-I pneumocytes. Our results suggest that AQP3 is expressed not only in large airways, but also in bronchioles, and is related to water movement in pulmonary oedema.     

Aquaporin water channels and endothelial cell function

The aquaporins (AQP) are a family of homologous water channels expressed in many epithelial and endothelial cell types involved in fluid transport. AQP1 protein is strongly expressed in most microvascular endothelia outside of the brain, as well as in endothelial cells in cornea, intestinal lacteals, and other tissues. AQP4 is expressed in astroglial foot processes adjacent to endothelial cells in the central nervous system. Transgenic mice lacking aquaporins have been useful in defining their role in mammalian physiology. Mice lacking AQP1 manifest defective urinary concentrating ability, in part because of decreased water permeability in renal vasa recta microvessels. These mice also show a defect in dietary fat processing that may involve chylomicron absorption by intestinal lacteals, as well as defective active fluid transport across the corneal endothelium. AQP1 might also play a role in tumour angiogenesis and in renal microvessel structural adaptation. However, AQP1 in most endothelial tissues does not appear to have a physiological function despite its role in osmotically driven water transport. For example, mice lacking AQP1 have low alveolar-capillary water permeability but unimpaired lung fluid absorption, as well as unimpaired saliva and tear secretion, aqueous fluid outflow, and pleural and peritoneal fluid transport. In the central nervous system mice lacking AQP4 are partially protected from brain oedema in water intoxication and ischaemic models of brain injury. Therefore, although the role of aquaporins in epithelial fluid transport is in most cases well-understood, there remain many questions about the role of aquaporins in endothelial cell function. It is unclear why many leaky microvessels strongly express AQP1 without apparent functional significance. Improved understanding of aquaporin-endothelial biology may lead to novel therapies for human disease, such as pharmacological modulation of corneal fluid transport, renal fluid clearance and intestinal absorption.     

Magnesium sulphate treatment decreases blood-brain barrier permeability during acute hypertension in pregnant rats

Eclampsia is associated with increased blood–brain barrier (BBB) permeability and formation of cerebral oedema. Magnesium sulphate is used to treat eclampsia despite an unclear mechanism of action. This study was to determine the effect of magnesium sulphate on in vivo BBB permeability and formation of cerebral oedema during acute hypertension and on brain aquaporin-4 (AQP4) protein expression. An in vivo model of hypertensive encephalopathy was used in late-pregnant (LP) rats following magnesium sulphate treatment, 270 mg kg⁻¹ i.p. injection every 4 h for 24 h. Permeability of the BBB was determined by in situ brain perfusion of Evan's Blue (EB) and sodium fluorescein (NaFl), and dye clearance determined by fluorescence spectrophotometry. Cerebral oedema was determined following acute hypertension by measuring brain water content. The effect of magnesium treatment on AQP4 expression was determined by Western blot analysis. Acute hypertension with autoregulatory breakthrough increased BBB permeability to EB in both brain regions studied ( P < 0.05). Magnesium attenuated BBB permeability to EB during acute hypertension by 41% in the posterior cerebrum ( P < 0.05) but had no effect in the anterior cerebrum ( P > 0.05). Treatment with magnesium did not change NaFl permeability, cerebral oedema formation or AQP4 expression. In summary, BBB permeability to Evan's Blue was increased by acute hypertension in LP rats, and this was attenuated by treatment with magnesium sulphate. The greatest effect on BBB permeability to EB was in the posterior cerebrum, an area particularly susceptible to oedema formation during eclampsia.     

The role of aquaporin-1 (AQP1) expression in a murine model of lipopolysaccharide-induced acute lung injury

A murine model of lipopolysaccharide (LPS)-induced acute lung injury (ALI) was used to evaluate whether aquaporin-1 (AQP1) is involved in lung inflammation and lung edema formation. Swiss strain mice (n = 122) had LPS (5 mg/kg) instilled intratracheally (IT), and were then treated with either 0.9 % saline or dexamethasone (5 mg/kg/day). Mice were euthanized at 2 days and 7 days after treatment. Inflammatory cytokines (TNF-alpha, IL-6), protein concentration in bronchoalveolar lavage (BAL) fluid, lung wet-to-dry weight ratio, histology, immunohistochemistry, and AQP1 Western blot were performed. Lung wet-to-dry weight ratio and lung vascular permeability were also measured in the AQP1 knockout mice (n = 9) that received IT LPS (5 mg/kg) at 2 days. Intratracheal instillation of LPS produced a severe lung injury at 2 days, characterized by elevation of TNF-alpha, IL-6 in the BAL fluid, and by histological changes consistent with increased lung vascular permeability and neutrophil infiltration. AQP1-immunoreactivity in the pulmonary capillary endothelium was reduced at 2 days and 7 days. Administration of dexamethasone improved LPS-induced ALI and retained expression of AQP1. However, depletion of AQP1 did not affect lung edema formation, lung vascular permeability, or lung histology. The results suggest that although AQP1 expression is decreased after lung injury, depletion of AQP1 does not alter lung inflammation and lung edema induced by LPS.     

Increased differentiation capacity of bone marrow-derived mesenchymal stem cells in aquaporin-5 deficiency

Mesenchymal stem cells (MSCs) are adult stem cells with a self-renewal and multipotent capability and express extensively in multitudinous tissues. We found that water channel aquaporin-5 (AQP5) is expressed in bone marrow-derived MSCs (BMMSCs) in the plasma membrane pattern. BMMSCs from AQP5(-/-) mice showed significantly lower plasma membrane water permeability than those from AQP5(+/+) mice. In characterizing the cultured BMMSCs from AQP5(-/-) and AQP5(+/+) mice, we found no obvious differences in morphology and proliferation between the 2 genotypes. However, the multiple differentiation capacity was significantly higher in AQP5(-/-) than AQP5(+/+) BMMSCs as revealed by representative staining by Oil Red O (adipogenesis); Alizarin Red S and alkaline phosphatase (ALP; osteogenesis); and type II collagen and Safranin O (chondrogenesis) after directional induction. Relative mRNA expression levels of 3 lineage differentiation markers, including PPARγ2, C/EBPα, adipsin, collagen 1a, osteopontin, ALP, collagen 11a, collagen 2a, and aggrecan, were significantly higher in AQP5(-/-) -differentiating BMMSCs, supporting an increased differentiation capacity of AQP5(-/-) BMMSCs. Furthermore, a bone-healing process was accelerated in AQP5(-/-) mice in a drill-hole injury model. Mechanistic studies indicated a significantly lower apoptosis rate in AQP5(-/-) than AQP5(+/+) BMMSCs. Apoptosis inhibitor Z-VAD-FMK increased the differentiation capacity to a greater extent in AQP5(+/+) than AQP5(-/-) BMMSCs. We conclude that AQP5-mediated high plasma membrane water permeability enhances the apoptosis rate of differentiating BMMSCs, thus decreasing their differentiation capacity. These data implicate AQP5 as a novel determinant of differentiation of BMMSCs and therefore a new molecular target for regulating differentiation of BMMSCs during tissue repair and regeneration.     

Nicotinamide attenuates aquaporin 3 overexpression induced by retinoic acid through inhibition of EGFR/ERK in cultured human skin keratinocytes

The most common adverse effects that are related to all-trans retinoic acid (atRA) treatment are irritation and dryness of the skin. atRA therapy is reported to impair barrier function as achieved by trans-epidermal water loss (TEWL). Treatment with nicotinamide prior to initiation of atRA therapy provides additional barrier protection and thus reduces susceptibility of retinoic acid. Our previous studies showed that atRA upregulates aquaporin 3 (AQP3) in cultured human skin keratinocytes and fibroblasts. Others have demonstrated that in atopic dermatitis, overexpression of AQP3 is linked to elevated TEWL and that nicotinamide treatment reduces skin TEWL. In this study, we observed that while atRA upregulates AQP3 expression in cultured human skin keratinocytes (HaCaT cells), nicotinamide attenuates the effect of atRA in a concentration-dependent manner. atRA treatment induces EGFR and ERK activation. PD153035, an EGFR inhibitor, and U0126, an ERK inhibitor, inhibit atRA-induced upregulation of AQP3. Nicotinamide also inhibits atRA-induced activation of EGFR/ERK signal transduction and decreases water permeability by downregulating AQP3 expression. Collectively, our results indicate that the effect of atRA on AQP3 expression is at least partly mediated by EGFR/ERK signaling in cultured human skin keratinocytes. Nicotinamide attenuates atRA-induced AQP3 expression through inhibition of EGFR/ERK signal transduction and eventually decreases water permeability and water loss. Our study provides insights into the molecular mechanism through which nicotinamide reverses the side effects of dryness in human skin after treatment with atRA.     

Functionality of aquaporin-2 missense mutants in recessive nephrogenic diabetes insipidus

Aquaporin-2 (AQP2) missense mutants in recessive nephrogenic diabetes insipidus (NDI) are all retained in the endoplasmic reticulum (ER), but some could function as water channels. No conclusions could be drawn about the water permeability (Pf) of others, because there was no method for quantifying AQP2 expression in the plasma membrane. We recently developed such a method, which has allowed us to study the functionality of these AQP2 mutants. Immunoblot analysis of membranes of injected oocytes revealed that all mutants (AQP2-G64R, AQP2-N68S, AQP2 T126M, AQP2-A147T, AQP2-R187C, AQP2-S216P) are expressed as unglycosylated and high-mannose glycosylated AQP2. The level of the high-mannose form of AQP2-A147T in the plasma membranes was low, indicating that this mutation has a less severe effect on proper folding. Analysis of Pf values and plasma membrane expression levels reveals that AQP2-N68S, AQP2-R187C and AQP2-S216P are non-functional, AQP2-A147T is as functional as wt-AQP2, while AQP2-T126M and AQP2-G64R retain 20% of the permeability of wt-AQP2. Since G64 is highly conserved between AQPs and expected to form essential interactions with other amino acids within AQP1, the residual functionality of AQP2-G64R is surprising. Our data furthermore indicate that an eventual therapy with chemical chaperones that restores the routing of AQP2 mutants to the apical membrane of collecting ducts cells might relieve NDI in patients encoding AQP2-A147T, and to a lesser extent AQP2-T126M and AQP2-G64R, but not in patients encoding AQP2-N68S, AQP2-R187C or AQP2-S216P.