Enhanced ammonia detoxification to urea in hepatocytes transduced with human aquaporin-8 gene.

Hepatic ammonia detoxification to urea is critical for the prevention of hyperammonemia and neurological damage. Hepatocyte mitochondrial aquaporin-8 (AQP8) channels have been involved in ammonia-derived ureagenesis. Herein, we studied whether the adenoviral gene transfer of human AQP8 (hAQP8) to hepatocyte mitochondria enhances ammonia conversion to urea. Using primary cultured rat hepatocytes, we first confirmed the mitochondrial expression of hAQP8 and then, using unlabeled or 15 N-labeled ammonia, we demonstrated that the urea synthesis was significantly enhanced in hAQP8-transduced hepatocytes. Studies using isolated hAQP8-expressing mitochondria also showed an increased ammonia metabolism. hAQP8 transduction was able to recover the impaired ammonia-derived ureagenesis in hepatotoxin-treated hepatocytes. Our data suggest that mitochondrially-expressed hAQP8 enhances and improves hepatocyte ammonia conversion to urea, a finding with potential therapeutic implications for liver disease with impaired ammonia detoxification.

Maternal Protein Restriction Modulates Angiogenesis and AQP9 Expression Leading to a Delay in Postnatal Epididymal Development in Rat.

The maternal nutritional status is essential to the health and well-being of the fetus. Maternal protein restriction during the perinatal stage causes sperm alterations in the offspring that are associated with epididymal dysfunctions. Vascular endothelial growth factor (VEGF) and its receptor, VEGFr-2, as well as aquaporins (AQPs) are important regulators of angiogenesis and the epididymal microenvironment and are associated with male fertility. We investigated the effects of maternal protein restriction on epididymal angiogenesis and AQP expression in the early stages of postnatal epididymal development. Pregnant rats were divided into two experimental groups that received either a normoprotein (17% protein) or low-protein diet (6% protein) during gestation and lactation. At postnatal day (PND)7 and PND14, male offspring were euthanized, the epididymides were subjected to morphometric and microvascular density analyses and to VEGF-A, VEGF-r2, AQP1 and AQP9 expression analyses. The maternal low-protein diet decreased AQP9 and VEGFr-2 expression, decreased epididymal microvascularity and altered the morphometric features of the epididymal epithelium; no changes in AQP1 expression were observed at the beginning of postnatal epididymal development. Maternal protein restriction alters microvascularization and affects molecules involved in the epidydimal microenvironment, resulting in morphometric alterations related to a delay in the beginning of epididymis postnatal development.

Aquaporin in different moult stages of a freshwater decapod crustacean: Expression and participation in muscle hydration control.

Crustaceans, during their moult cycle, at the stages of both pre-moult and post-moult, need water uptake. This movement of water creates a challenge for the regulation of cell volume. The cells of freshwater decapods require a high regulatory capacity to deal with hyposmotic stresses, given the need to face dilution of the haemolymph during their moult cycles. This study investigated the variation in the expression of water channels (aquaporins) along the moult cycle of a freshwater palaemonid shrimp, focusing on their role in cell volume regulation. Moults in Palaemonetes argentinus have been investigated along three stages of its moult cycle: intermoult, late pre-moult and recent post-moult. For the evaluation of tissue volume regulation, the weight of isolatedmuscle, subjected to isosmotic and hyposmotic salines, was followed for 60min. The expression of AQP during the different moult stages was evaluated by immunocytochemistry. Muscle from the three moult stages in isosmotic conditions showed the same pattern of tissue volume regulation. When muscle from animals in pre-moult and intermoult were submitted to hyposmotic stress they swell, followed by volume regulation, while in post-moult the regulation is compromised. The difference in volume regulatory control between pre-moult and post-moult may be related to a possible regulation of water channels, as AQP expression was equal at these stages. This study presents novel findings for crustaceans in general, in the demonstration that AQP expression changes during the moult cycle of a decapod crustacean, together with the regulation of cell volume with the participation of AQPs.

The expression patterns of aquaporin 9, vacuolar H+-ATPase, and cytokeratin 5 in the epididymis of the common vampire bat.

Desmodus rotundus is a vampire bat species that inhabits Latin America. Some basic aspects of this species' biology are still unknown, as the histophysiological characteristics of the male reproductive tract. Our study has focused on its epididymis, which is an important organ for performing a variety of functions, especially the sperm maturation and storage. The aim of this study was to identify principal, narrow, clear, and basal cells using cell-specific markers such as aquaporin 9 (AQP9), vacuolar H+-ATPase (V-ATPase), and cytokeratin 5 (KRT5). Principal cells were labeled by AQP9 from initial segment to cauda region in their stereocilia. They were shown with a columnar shape, whereas V-ATPase-rich cells were identified with a goblet-shaped body along the entire epididymis, including the initial segment, which were named as clear cells. Pencil-shaped V-ATPase-rich cells (narrow cells) were not detected in the initial segment of the bat epididymis, unlike in the rodent. Basal cells were labeled by KRT5 and were located at the basal portion of the epithelium forming a dense network. However, no basal cells with a luminal-reaching body extension were observed in the bat epididymis. In summary, epithelial cells were identified by their specific markers in the vampire bat epididymis. Principal and basal cells were labeled by AQP9 and KRT5, respectively. Narrow cells were not observed in the vampire bat epididymis, whereas clear cells were identified by V-ATPase labeling along the entire duct in a goblet-shaped body. In addition, no luminal-reaching basal cells were observed in the vampire bat epididymis.

Effects of exogenous 5-aminolevulinic acid on PIP1 and NIP aquaporin gene expression in seedlings of cucumber cultivars subjected to salinity stress.

Aquaporins play a direct role in plant water relation under salt stress, but the effects of 5-aminolevulinic acid (ALA) on aquaporin gene expression in salt-treated plants remain unknown. This study investigated the potential effects of exogenous ALA (50 mg/dm3) on aquaporin expression levels under salt stress (75 mM NaCl) in the salt-sensitive (Jinchun No.4) and the relatively salt-tolerant cucumber (Jinyou No.1) seedlings. The expressions of cucumber PIP aquaporin gene (CsPIP1:1) and cucumber NIP aquaporin gene (CsNIP) were analyzed in 20-day-old seedling leaves at 2, 4, 8, 16, and 24 h after ALA treatment. After treatment with saline alone and ALA alone, CsPIP1:1 and CsNIP gene expression levels in the 2 cucumber cultivars increased to maximum at 2 h. The aquaporin gene expression in salt-treated cucumber seedling leaves was considerably higher than that in leaves subjected to exogenous ALA. Further, the aquaporin expression levels in Jinchun No.4 were higher than those in Jinyou No.1, reaching 5.20- and 2-fold induction levels, respectively. After treatment with both ALA and NaCl, the CsNIP gene expression was downregulated in both the cucumber cultivars, while that of CsPIP1:1 decreased at 2 h and then increased to 3.8-fold in Jinchun No.4. In Jinyou No.1, CsPIP1:1 gene expression gradually increased to 2.3-fold at 4 h, followed by a decline in expression. The results indicated that ALA might delay and counteract the upregulated expression of CsPIP1:1 and CsNIP genes in cucumber seedlings under NaCl stress. Thus, salt tolerance of cucumber seedlings might be enhanced by ALA application.

Oxygen tension modulates AQP9 expression in human placenta.

UNLABELLED: Placental hypoxia has been implicated in pregnancy pathologies such as preeclampsia. We have previously reported that AQP9 is highly expressed in syncytiotrophoblast from normal placentas and shows an overexpression in preeclamptic placentas, with a lack of functionality for water transport. Up to now, the response of AQP9 to changes in the oxygen tension in trophoblast cells is still unknown. OBJECTIVE: Our aim was to establish whether alterations in oxygen levels may modulate AQP9 expression in human placenta. METHODS: A theoretical analysis of the human AQP9 gene to find conserved DNA regions that could serve as putative HIF-1 binding sites. Then, explants from normal placentas were cultured at different concentrations of oxygen or with 250 µM CoCl2. AQP9 molecular expression and water uptake was determined. RESULTS: Fourteen consensus HIF-1 binding sites were found in the human AQP9 gene, but none of them in the promoter region. However, placental AQP9 decreased abruptly when HIF-1α is expressed by deprivation of oxygen or CoCl2 stabilization. In contrast, after reoxygenation, HIF-1α was undetectable while AQP9 increased significantly and changed its cellular distribution, showing the same pattern as that previously described in preeclamptic placentas. Accordingly with the decrease in AQP9 expression, water uptake decreased in explants exposed to hypoxia or treated with CoCl2. Conversely as we expected, after reoxygenation, water uptake decreased dramatically compared to the control and was not sensitive to HgCl2. CONCLUSION: Our findings suggest that oxygen tension may modulate AQP9 expression in human placenta. However, the role of AQP9 still remains uncertain.

Evidence for insulin-mediated control of AQP9 expression in human placenta.

UNLABELLED: The AQP9 gene contains a negative insulin response element, suggesting that it may be modulated by insulin. Previously, we reported AQP9 overexpression in preeclamptic placentas but a lack of functionality of AQP9 in water and mannitol transport. We also observed high serum levels of insulin and TNF-α in preeclamptic women. OBJECTIVE: To evaluate whether AQP9 expression is regulated by insulin in the human placenta, and whether the dysregulation of AQP9 observed in preeclamptic placentas may be related to the inability to respond to insulin stimuli. METHODS: Explants from normal and preeclamptic placentas were cultured at different concentrations of insulin. Treatment with TNF-α was used to induce phosphorylation of insulin receptor substrate (IRS), which may desensitize insulin action. AQP9 molecular expression and water uptake was determined. RESULTS: Insulin decreased the molecular expression of AQP9 exclusively in explants from normal placentas in a concentration-dependent manner. Treatment with TNF-α previous to insulin addition prevented these changes. Moreover, insulin treatment did not modify water uptake neither its sensitivity to HgCl(2.) CONCLUSION: AQP9 water permeability seems to be independent of its molecular expression, strongly suggesting that AQP9 might not have a key role in water transport in human placenta. We also propose another mechanism of down-regulation of AQP9 molecular expression mediated by insulin in a concentration-dependent manner in human placenta and provide new evidence that in preeclamptic placentas the mechanisms of insulin signaling may be altered, producing an overexpression of AQP9 that does not correlate with an increase in its functionality.

High levels of human chorionic gonadotropin (hCG) correlate with increased aquaporin-9 (AQP9) expression in explants from human preeclamptic placenta.

Trophoblastic abnormalities have a central role in the pathophysiology of preeclampsia, and some placental hormones, such as human chorionic gonadotropin (hCG), could affect the placental function. Here, we hypothesized that the elevated serum levels of hCG may be involved in the increased aquaporin-9 (AQP9) protein expression in preeclamptic placentas via adenosine 3('),5(')-cyclic phosphate (cAMP) pathways. Normal placental explants were cultured with different concentrations of recombinant hCG or 8-Br-cAMP, a potent analogue of cAMP. We evaluated AQP9 protein expression and localization. After both treatments, we localized AQP9 in the apical membrane of syncytiotrophoblast and in the cytoplasm. We also observed a concentration-dependent effect on AQP9 protein expression. In addition, water uptake increased 1.6-fold in explants treated with hCG. Our results suggest that hCG may increase AQP9 protein expression and functionality via cAMP pathways. Although, in preeclamptic placentas high levels of hCG may upregulate AQP9 protein expression, AQP9 functionality was reduced possibly by other factors.

AtTIP1;3 and AtTIP5;1, the only highly expressed Arabidopsis pollen-specific aquaporins, transport water and urea.

Pollination includes processes where water and/or solute movements must be finely regulated, suggesting participation of aquaporins. Using information available from different transcriptional profilings of Arabidopsis thaliana mature pollen, we showed that the only aquaporins that are selectively and highly expressed in mature pollen are two TIPs: AtTIP1;3 and AtTIP5;1. Pollen exhibited a lower number and more exclusive type of aquaporin expressed genes when compared to other single cell transcriptional profilings. When characterized using Xenopus oocyte swelling assays, AtTIP1;3 and AtTIP5;1 showed intermediate water permeabilities. Although they displayed neither glycerol nor boric acid permeability they both transported urea. In conclusion, these results suggest a function for AtTIP1;3 and AtTIP5;1 as specific water and urea channels in Arabidopsis pollen.

Effects of 3-beta-diol, an androgen metabolite with intrinsic estrogen-like effects, in modulating the aquaporin-9 expression in the rat efferent ductules.

BACKGROUND: Fluid homeostasis is critical for normal function of the male reproductive tract and aquaporins (AQP) play an important role in maintenance of this water and ion balance. Several AQPs have been identified in the male, but their regulation is not fully comprehended. Hormonal regulation of AQPs appears to be dependent on the steroid in the reproductive tract region. AQP9 displays unique hormonal regulation in the efferent ductules and epididymis, as it is regulated by both estrogen and dihydrotestosterone (DHT) in the efferent ductules, but only by DHT in the initial segment epididymis. Recent data have shown that a metabolite of DHT, 5-alpha-androstane-3-beta-17-beta-diol (3-beta-diol), once considered inactive, is also present in high concentrations in the male and indeed has biological activity. 3-beta-diol does not bind to the androgen receptor, but rather to estrogen receptors ER-alpha and ER-beta, with higher affinity for ER-beta. The existence of this estrogenic DHT metabolite has raised the possibility that estradiol may not be the only estrogen to play a major role in the male reproductive system. Considering that both ER-alpha and ER-beta are highly expressed in efferent ductules, we hypothesized that the DHT regulation of AQP9 could be due to the 3-beta-diol metabolite. METHODS: To test this hypothesis, adult male rats were submitted to surgical castration followed by estradiol, DHT or 3-beta-diol replacement. Changes in AQP9 expression in the efferent ductules were investigated by using immunohistochemistry and Western blotting assay. RESULTS: Data show that, after castration, AQP9 expression was significantly reduced in the efferent ductules. 3-beta-diol injections restored AQP9 expression, similar to DHT and estradiol. The results were confirmed by Western blotting assay. CONCLUSION: This is the first evidence that 3-beta-diol has biological activity in the male reproductive tract and that this androgen metabolite has estrogen-like activity in the efferent ductules, whose major function is the reabsorption of luminal fluid.

Functional and molecular expression of AQP9 channel and UT-A transporter in normal and preeclamptic human placentas.

Transcellular flux of urea across human placenta may be facilitated by aquaglyceroporins and/or by urea transporters (UT). Previously we have reported the presence of AQP3 and AQP9 in the syncytiotrophoblast of human term placenta (hST). In the present study, we detected a significant uptake of water, urea and mannitol sensitive to mercury and phloretin in explants from normal term placenta, which indicates a functional AQP9. In addition, we observed an increase of AQP9 expression in preeclamptic placenta with a lack of functionality of AQP9 for water and mannitol. Our data showed a molecular and functional expression of UT-A in hST from normal and preeclamptic placentas. In the last case, urea uptake sensitive to phloretin and mercury increased and the UT-A protein seems to be augmented. These results suggest that the increase of urea uptake in preeclamptic pregnancies could be attributed to an increase of expression of UT-A more than AQP9 proteins. In conclusion, our results provide new evidences that suggest the involvement of AQP9 and UT-A in the urea excretion mechanism across hST from mother to fetus in physiological conditions. Much further work is needed to define whether the overexpression of AQP9 plays a direct role either in the pathogenesis or in the adaptative response of preeclampsia.

Regulation of the immunoexpression of aquaporin 9 by ovarian hormones in the rat oviductal epithelium.

The volume of oviductal fluid fluctuates during the estrous cycle, suggesting that water availability is under hormonal control. It has been postulated that sex-steroid hormones may regulate aquaporin (AQP) channels involved in water movement across cell membranes. Using a functional assay (oocytes of Xenopus laevis), we demonstrated that the rat oviductal epithelium contains mRNAs coding for water channels, and we identified by RT-PCR the mRNAs for AQP5, -8, and -9, but not for AQP2 and -3. The immunoreactivity for AQP5, -8, and -9 was localized only in epithelial cells of the oviduct. The distribution of AQP5 and -8 was mainly cytoplasmic, whereas we confirmed, by confocal microscopy, that AQP9 localized to the apical plasma membrane. Staining of AQP5, -8, and -9 was lost after ovariectomy, and only AQP9 immunoreactivity was restored after estradiol and/or progesterone treatments. The recovery of AQP9 reactivity after ovariectomy correlated with increased mRNA and protein levels after treatment with estradiol alone or progesterone administration after estradiol priming. Interestingly, progesterone administration after progesterone priming also induced AQP9 expression but without a change in mRNA levels. Levels of AQP9 varied along the estrous cycle with their highest levels during proestrus and estrus. These results indicate that steroid hormones regulate AQP9 expression at the mRNA and protein level and that other ovarian signals are involved in the expression of AQP5 and -8. Thus hormonal regulation of the type and quantity of water channels in this epithelium might control water transport in the oviductal lumen.

Aquaporin-2, a regulated water channel, is expressed in apical membranes of rat distal colon epithelium.

Aquaporin-2 (AQP-2) is the vasopressin-regulated water channel expressed in the apical membrane of principal cells in the collecting duct and is involved in the urinary concentrating mechanism. In the rat distal colon, vasopressin stimulates water absorption through an unknown mechanism. With the hypothesis that AQP-2 could contribute to this vasopressin effect, we studied its presence in rat colonic epithelium. We used RT-PCR, in situ hybridization, immunoblotting, and immunocytochemistry to probe for AQP-2 expression. An AQP-2 amplicon was obtained through RT-PCR of colon epithelium RNA, and in situ hybridization revealed AQP-2 mRNA in colonic crypts and, to a lesser extent, in surface absorptive epithelial cells. AQP-2 protein was localized to the apical membrane of surface absorptive epithelial cells, where it colocalized with H(+)-K(+)-ATPase but not with Na(+)-K(+)-ATPase. AQP-2 was absent from the small intestine, stomach, and liver. Water deprivation increased the hybridization signal and the protein level (assessed by Western blot analysis) for AQP-2 in distal colon. This was accompanied by increased p-chloromercuriphenylsulfonic acid-sensitive water absorption. These results indicate that AQP-2 is present in the rat distal colon, where it might be involved in a water-sparing mechanism. In addition, these results support the idea that AQP-2, and probably other aquaporins, are involved in water absorption in the colon.

Water permeability in rat oocytes at different maturity stages: aquaporin-9 expression.

Important functional and structural modifications occur in mammalian oocytes during their arrival to maturity. In this process, oocytes switch from a high activity level, implying an important metabolic rate and a coordinated movement of water and solutes, to a lower functional state. The aim of this work was to study the mechanisms involved in water movements during oocyte arrival to maturity. Volume changes, induced by an osmotic gradient, were followed by video microscopy in rat oocytes. The water osmotic permeability (P(osm)) of immature oocytes (proestrus) was sensitive to HgCl(2) and phloretin. In contrast, mature oocytes (estrus) had a reduced P(osm) that was not sensitive to these compounds. When proestrus oocytes were incubated in vitro at 37 degrees C they spontaneously arrived at maturity and its P(osm) decreased between four and six hours of incubation. RT-PCR experiments were performed using specific primers for all rat aquaporins that had been cloned. We found that aquaporin-9 transcript (AQP9) is present in proestrus oocytes but not in estrus oocytes. AQP9 has been recently described as a "broad selective channel" responsible for solute and water transfers in highly active cells. Our experiments showed that proestrus oocytes, but not estrus, are permeable to mannitol. It is concluded that during the process of maturation, P(osm) decreases and AQP9 transcripts disappear. We report here the first study correlating water permeability and aquaporin mRNA expression in mammalian oocytes.