Aquaporin Inhibitors Research Articles

Can Stabilization and Inhibition of Aquaporins Contribute to Future Development of Biomimetic Membranes?

In recent years, the use of biomimetic membranes that incorporate membrane proteins, i.e., biomimetic-hybrid membranes, has increased almost exponentially. Key membrane proteins in these systems have been aquaporins, which selectively permeabilize cellular membranes to water. Aquaporins may be incorporated into synthetic lipid bilayers or to more stable structures made of block copolymers or solid-state nanopores. However, translocation of aquaporins to these alien environments has adverse consequences in terms of performance and stability. Aquaporins incorporated in biomimetic membranes for use in water purification and desalination should also withstand the harsh environment that may prevail in these conditions, such as high pressure, and presence of salt or other chemicals. In this respect, modified aquaporins that can be adapted to these new environments should be developed. Another challenge is that biomimetic membranes that incorporate high densities of aquaporin should be defect-free, and this can only be efficiently ascertained with the availability of completely inactive mutants that behave otherwise like the wild type aquaporin, or with effective non-toxic water channel inhibitors that are so far inexistent. In this review, we describe approaches that can potentially be used to overcome these challenges.

A generic high-throughput assay to detect aquaporin functional mutants: Potential application to discovery of aquaporin inhibitors

BackgroundThe discovery of stable, yet functional, protein mutants is a limiting factor in the development of biotechnological applications, structural studies or in drug discovery. Rapid detection of functional mutants is especially challenging for water channel aquaporins, as they do not have a directly measurable enzymatic or binding activity. Current methods available are time consuming and only applicable to specific aquaporins. MethodsHerein we describe an assay based on the protective effect of aquaporins on yeast S. cerevisiae in response to rapid freezing. ResultsYeast overexpressing a functional water-permeable aquaporin of choice are rescued after the challenge, while inactive or blocked aquaporins confer no protection and lead to cell death. The potential of this assay is shown by screening a small number of E. coli aquaporin Z (AQPZ) mutants. Additionally, a library of ~10,000 drug-like compounds was tested against human AQP1 (hAQP1). ConclusionsSince rescue is only dependent on transmembrane water flux, the assay is applicable to water-permeable aquaporins of any origin. General significanceMapping of permissive mutations on the aquaporin structure can help delineate the minimal requirements for effective water transport. Alternatively, the assay can be potentially used to discover compounds that inhibit aquaporin water transport. When additionally screened for thermostability, functional aquaporin mutants can be useful in the development of biomimetic membranes for water purification, or to improve the likelihood of producing well-diffracting crystals, enabling rational design of much needed aquaporin inhibitors.

Silicon improves salt tolerance by increasing root water uptake in Cucumis sativus L.

Silicon enhances root water uptake in salt-stressed cucumber plants through up-regulating aquaporin gene expression. Osmotic adjustment is a genotype-dependent mechanism for silicon-enhanced water uptake in plants. Silicon can alleviate salt stress in plants. However, the mechanism is still not fully understood, and the possible role of silicon in alleviating salt-induced osmotic stress and the underlying mechanism still remain to be investigated. In this study, the effects of silicon (0.3 mM) on Na accumulation, water uptake, and transport were investigated in two cucumber (Cucumis sativus L.) cultivars ('JinYou 1' and 'JinChun 5') under salt stress (75 mM NaCl). Salt stress inhibited the plant growth and photosynthesis and decreased leaf transpiration and water content, while added silicon ameliorated these negative effects. Silicon addition only slightly decreased the shoot Na levels per dry weight in 'JinYou 1' but not in 'JinChun 5' after 10 days of stress. Silicon addition reduced stress-induced decreases in root hydraulic conductivity and/or leaf-specific conductivity. Expressions of main plasma membrane aquaporin genes in roots were increased by added silicon, and the involvement of aquaporins in water uptake was supported by application of aquaporin inhibitor and restorative. Besides, silicon application decreased the root xylem osmotic potential and increased root soluble sugar levels in 'JinYou 1.' Our results suggest that silicon can improve salt tolerance of cucumber plants through enhancing root water uptake, and silicon-mediated up-regulation of aquaporin gene expression may in part contribute to the increase in water uptake. In addition, osmotic adjustment may be a genotype-dependent mechanism for silicon-enhanced water uptake in plants.

Aquaporins as diagnostic and therapeutic targets in cancer: how far we are?

Aquaporins (AQPs) are a family of water channel proteins distributed in various human tissues, responsible for the transport of small solutes such as glycerol, even gas and ions. The expression of AQPs has been found in more than 20 human cancer types and is significantly correlated with the severity of histological tumors and prognosis of patients with cancer. More recent evidence showed that AQPs could also play a role in tumor-associated edema, tumor cell proliferation and migration, and tumor angiogenesis in solid and hematological tumors. Inhibitors of AQPs in tumor cells and microvessels have been suggested as new therapeutic strategies. The present review overviews AQPs structures, expression variation among normal tissues and tumors, AQPs functions and roles in the development of cancer with special focuses on lung, colorectal, liver, brain and breast cancers, and potential AQPs-target inhibitors. We call the special attention to consider AQPs important as diagnostic and therapeutic biomarkers. It may be a novel anticancer therapy by the AQPs inhibition.

Prostaglandin E2 inhibits the proinflammatory phenotype induced by TGF-β on monocyte-derived dendritic cells

Prostaglandin E2 inhibits the proinflammatory phenotype induced by TGF-β on monocyte-derived dendritic cells

Cardioprotective effect of valsartan in mice with short-term high-salt diet by regulating cardiac aquaporin 1 and angiogenic factor expression.

Hypertension is the most common risk factor for various cardiovascular and cerebrovascular diseases that affects approximately 61 million, or 25% of the population in United States. The dietary salt intake is one of the most important but modifiable factors for hypertension. In the current study, we aim to elucidate the role of aquaporin 1 in high-salt-induced hypertension and cardiac injuries and whether angiotensin II receptor blocker valsartan could ameliorate the effect of high salt on blood pressure. Mice were fed with normal diet, high-salt diet in the presence or absence of valsartan for 4 weeks. The body weight gain, feeding behavior, blood pressure, and cardiac pathology changes were monitored after 4 weeks. The expression of aquaporin 1, vascular endothelial growth factor, transforming growth factor β1, and basic fibroblast growth factor were analyzed using quantitative real-time polymerase chain reaction, Western blot, and immunohistochemical staining. Valsartan partially reversed the effects of high-salt diet on hypertension, cardiac injuries such as fibrosis and inflammatory cell infiltration, and inhibition of aquaporin 1 and angiogenic factors; valsartan alone did not exert such effects. The current data demonstrated that the reduction of cardiac aquaporin 1 and angiogenic factor expression level might be associated with high-salt-induced hypertension and cardiac injuries in mice, which could be ameliorated by angiotensin II receptor blocker treatment.

Regulation of salmonid fish sperm motility by osmotic shock-induced water influx across the plasma membrane

The motility of salmonid fish sperm is initiated by a decrease in the extracellular K+ concentration. However, our previous studies revealed that salmonid fish sperm motility could be initiated in the presence of an inhibitory concentration of K+ by drastic osmotic shock induced by suspension in a hypertonic glycerol solution and subsequent dilution in a hypotonic solution (glycerol-treatment). In the present study, we examined if an osmotic shock-induced water influx is involved in the regulation of salmonid fish sperm motility. HgCl2, a common inhibitor of aquaporins (AQPs), decreased the duration of salmonid fish sperm motility. Dilution of sperm cells in a hypotonic solution increased the cellular volume, whereas HgCl2 inhibited such an increase in cellular volume. Furthermore, the expression of AQP 1a and 10 in rainbow trout testes was confirmed. In contrast, HgCl2 did not affect glycerol-treated sperm motility, indicating that AQPs are not involved in glycerol-treated sperm motility. We also explored the possibility of aquaporin-independent water influx in glycerol-treated sperm by assessing the sperm membrane permeability using propidium iodide. The plasma membrane of glycerol-treated sperm was considerably permeabilized. The cellular volume was decreased in a hypertonic glycerol solution and increased upon subsequent hypoosmotic shock, indicating an AQP-independent water flux across the plasma membrane upon glycerol-treatment. Taken together, these results showed that water influx across the plasma membrane via AQP is crucial for the maintenance of salmonid fish sperm motility under normal conditions, whereas water influx by osmotic shock-induced membrane permeation is critical for the initiation of glycerol-treated sperm motility.

Involvement of TRPV1 and AQP2 in hypertonic stress by xylitol in odontoblast cells

Aim: To examine the responses of mouse odontoblast-lineage cell line (OLC) cultures to xylitol-induced hypertonic stress.Methodology: OLCs were treated with xylitol, sucrose, sorbitol, mannitol, arabinose and lyxose. Cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium assay. The expression of transient receptor potential vanilloids (TRPV) 1, 3 and 4 was detected using a reverse transcriptase-polymerase chain reaction (RT-PCR) assay. The expression of aquaporin (AQP) 2 was detected using immunofluorescence and Western blotting analysis. The expression of interleukin-6 (IL-6) under xylitol-induced hypertonic stress was assessed using an enzyme-linked immunosorbent assay (ELISA). Small interfering ribonucleic acid (siRNA) for AQP-2 was used to inhibition assay.Results: Xylitol-induced hypertonic stress did not decrease OLC viability, unlike the other sugars tested. OLCs expressed TRPV1, 3 and 4 as well as AQP2. Xylitol inhibited lipopolysaccharide (LPS)-induced IL-6 expression after 3 h of hypertonic stress. TRPV1 mRNA expression was upregulated by xylitol. Costimulation with HgCl2 (AQP inhibitor) and Ruthenium red (TRPV1 inhibitor) decreased cell viability with xylitol stimulation. OLCs treated with siRNA against TRPV1 exhibited decreased cell viability with xylitol stimulation.Conclusion: OLCs have high-cell viability under xylitol-induced hypertonic stress, which may be associated with TRPV1 and AQP2 expressions.

The role of water channel proteins in facilitating recovery of leaf hydraulic conductance from water stress in Populus trichocarpa.

Gas exchange is constrained by the whole-plant hydraulic conductance (K plant). Leaves account for an important fraction of K plant and may therefore represent a major determinant of plant productivity. Leaf hydraulic conductance (K leaf) decreases with increasing water stress, which is due to xylem embolism in leaf veins and/or the properties of the extra-xylary pathway. Water flow through living tissues is facilitated and regulated by water channel proteins called aquaporins (AQPs). Here we assessed changes in the hydraulic conductance of Populus trichocarpa leaves during a dehydration-rewatering episode. While leaves were highly sensitive to drought, K leaf recovered only 2 hours after plants were rewatered. Recovery of K leaf was absent when excised leaves were bench-dried and subsequently xylem-perfused with a solution containing AQP inhibitors. We examined the expression patterns of 12 highly expressed AQP genes during a dehydration-rehydration episode to identify isoforms that may be involved in leaf hydraulic adjustments. Among the AQPs tested, several genes encoding tonoplast intrinsic proteins (TIPs) showed large increases in expression in rehydrated leaves, suggesting that TIPs contribute to reversing drought-induced reductions in K leaf. TIPs were localized in xylem parenchyma, consistent with a role in facilitating water exchange between xylem vessels and adjacent living cells. Dye uptake experiments suggested that reversible embolism formation in minor leaf veins contributed to the observed changes in K leaf.

Inhibitor screen for limited-transpiration trait among maize hybrids

A plant trait to minimize the impact of drought on crop yield is limited-transpiration rate (TR) under high ambient vapor pressure deficit (VPD) so that soil water is conserved to sustain grain fill. Variation among maize (Zea mays L.) hybrids has been identified for the existence of the limited-TR trait at high atmospheric VPD, and the VPD at which TR becomes limited. Further, it has been shown that the TR limitation at high VPD is related to plant hydraulic conductance, which may be due to differences in aquaporin expression. This paper reports studies to relate the TR response of 21 maize hybrids to treatment of leaves and intact plants with cycloheximide (CHX) and four aquaporin inhibitors: silver (AgNO3), gold (HAuCl4), zinc (ZnCl2), and mercury (HgCl2). There was no discrimination among hybrids based on treatment with Hg or CHX. Segregation between hybrids for response to increasing VPD corresponded with differences in leaf response to Ag and Au treatment and intact plant response to Zn. The highest correlation (r=0.90) between VPD breakpoint and TR response to inhibitor was with Ag treatment of leaves. These results indicate that Ag may be an effective initial screen for expression of the limited-TR trait under high VPD.

Aquaporin-mediated increase in root hydraulic conductance is involved in silicon-induced improved root water uptake under osmotic stress in Sorghum bicolor L.

The fact that silicon application alleviates water deficit stress has been widely reported, but the underlying mechanism remains unclear. Here the effects of silicon on water uptake and transport of sorghum seedlings (Sorghum bicolor L.) growing under polyethylene glycol-simulated osmotic stress in hydroponic culture and water deficit stress in sand culture were investigated. Osmotic stress dramatically decreased dry weight, photosynthetic rate, transpiration rate, stomatal conductance, and leaf water content, but silicon application reduced these stress-induced decreases. Although silicon application had no effect on stem water transport capacity, whole-plant hydraulic conductance (Kplant) and root hydraulic conductance (Lp) were higher in silicon-treated seedlings than in those without silicon treatment under osmotic stress. Furthermore, the extent of changes in transpiration rate was similar to the changes in Kplant and Lp. The contribution of aquaporin to Lp was characterized using the aquaporin inhibitor mercury. Under osmotic stress, the exogenous application of HgCl2 decreased the transpiration rates of seedlings with and without silicon to the same level; after recovery induced by dithiothreitol (DTT), however, the transpiration rate was higher in silicon-treated seedlings than in untreated seedlings. In addition, transcription levels of several root aquaporin genes were increased by silicon application under osmotic stress. These results indicate that the silicon-induced up-regulation of aquaporin, which was thought to increase Lp, was involved in improving root water uptake under osmotic stress. This study also suggests that silicon plays a modulating role in improving plant resistance to osmotic stress in addition to its role as a mere physical barrier.

Critical Role of Aquaporins in Interleukin 1β (IL-1β)-induced Inflammation

Rapid changes in cell volume characterize macrophage activation, but the role of water channels in inflammation remains unclear. We show here that, in vitro, aquaporin (AQP) blockade or deficiency results in reduced IL-1β release by macrophages activated with a variety of NLRP3 activators. Inhibition of AQP specifically during the regulatory volume decrease process is sufficient to limit IL-1β release by macrophages through the NLRP3 inflammasome axis. The immune-related activity of AQP was confirmed in vivo in a model of acute lung inflammation induced by crystals. AQP1 deficiency is associated with a marked reduction of both lung IL-1β release and neutrophilic inflammation. We conclude that AQP-mediated water transport in macrophages constitutes a general danger signal required for NLRP3-related inflammation. Our findings reveal a new function of AQP in the inflammatory process and suggest a novel therapeutic target for anti-inflammatory therapy.

Gold compounds as aquaporin inhibitors: new opportunities for therapy and imaging

A review on the development of gold-based compounds as aquaglyceroporin inhibitors with potential as therapeutic agents or as chemical probes.

Defensive slime formation in Pacific hagfish requires Ca2+- and aquaporin-mediated swelling of released mucin vesicles.

Hagfishes defend themselves from fish predators via the rapid deployment of a fibrous slime that adheres to and clogs gills. The slime transforms from a thick glandular exudate to a fully hydrated product in a fraction of a second through a process that involves the swelling and rupture of numerous mucin vesicles. Here we demonstrate that the vesicle membrane plays an important role in regulating the swelling of mucin granules, and provide evidence that the membrane contains proteins that facilitate the movement of ions and water molecules. By exposing isolated mucin vesicles to varying combinations of inorganic ions, organic compounds and membrane channel inhibitors, we found that the majority of hagfish mucin vesicles require Ca(2+) to rupture. We also show that Ca(2+)-dependent rupture can be pharmacologically inhibited, which suggests a role for Ca(2+)-activated membrane transporters. We demonstrate that the aquaporin inhibitor mercuric chloride reduces the rate of vesicle swelling by an order of magnitude, which suggests that aquaporins facilitate the influx of water during vesicle deployment. Molecular evidence of two aquaporin homologues expressed in the slime glands further supports this idea. We propose a model of hagfish slime mucin vesicle rupture that involves Ca(2+)-activated transporters and aquaporins, and suggest that the presence of these proteins is an adaptation for increasing the speed of vesicle rupture and, consequently, the speed of the sliming response of hagfishes.

Conservative water use under high evaporative demand associated with smaller root metaxylem and limited trans-membrane water transport in wheat.

Efficient breeding of drought-tolerant wheat (Triticum spp.) genotypes requires identifying mechanisms underlying exceptional performances. Evidence indicates that the drought-tolerant breeding line RAC875 is water-use conservative, limiting its transpiration rate (TR) sensitivity to increasing vapour pressure deficit (VPD), thereby saving soil water moisture for later use. However, the physiological basis of the response remains unknown. The involvement of leaf and root developmental, anatomical and hydraulic features in regulating high-VPD, whole-plant TR was investigated on RAC875 and a drought-sensitive cultivar (Kukri) in 12 independent hydroponic and pot experiments. Leaf areas and stomatal densities were found to be identical between lines and de-rooted plants didn't exhibit differential TR responses to VPD or TR sensitivity to four aquaporin (AQP) inhibitors that included mercury chloride (HgCl2). However, intact plants exhibited a differential sensitivity to HgCl2 that was partially reversed by β-mercaptoethanol. Further, root hydraulic conductivity of RAC875 was found to be lower than Kukri's and root cross-sections of RAC875 had significantly smaller stele and central metaxylem diameters. These findings indicate that the water-conservation of RAC875 results from a root-based hydraulic restriction that requires potentially heritable functional and anatomical features. The study revealed links between anatomical and AQP-based processes in regulating TR under increasing evaporative demand.

Divergence in Drought‐resistance Traits among Parents of Recombinant Peanut Inbred Lines

ABSTRACTPeanut (Arachis hypogaea L.) is often grown in climates of intermittent drought on sandy soils. Plants expressing water‐conservative traits would minimize exposure to end‐of‐season, severe drought. Two traits resulting in conservative transpiration rates (TRs) are limitations on TR with soil drying and with increasing vapor pressure deficit (VPD). This study focused on parents of existing recombinant inbred line (RIL) populations as sources of divergent expression of these two traits. If divergence is found, their derived RIL population could be used in identifying genetic markers. Since both water‐conservation traits are laborious to document, a key extension of this study was to explore the possibility of using aquaporin inhibitors as practical tools in marker identification. Tifrunner had a lower soil water threshold for a decline in TR than NC 3033 and N08082olJCT. Tifrunner also had a higher VPD breakpoint than three genotypes, including NC 3033 and N08082olJCT. The difference between Tifrunner and these other two genotypes extended to their response to aquaporin inhibitors. The decrease in TR of Tifrunner when exposed to aquaporin inhibitors was much larger than NC 3033 when treated with silver and N08082olJCT when treated with zinc. This study indicates that an effort to develop drought markers in peanut RIL population should focus on Tifrunner × NC 3033 using the silver inhibitor and/or Tifrunner × N08082olJCT using the zinc inhibitor.

The Role of Aquaporin and Tight Junction Proteins in the Regulation of Water Movement in Larval Zebrafish (Danio rerio)

Teleost fish living in freshwater are challenged by passive water influx; however the molecular mechanisms regulating water influx in fish are not well understood. The potential involvement of aquaporins (AQP) and epithelial tight junction proteins in the regulation of transcellular and paracellular water movement was investigated in larval zebrafish (Danio rerio). We observed that the half-time for saturation of water influx (K u) was 4.3±0.9 min, and reached equilibrium at approximately 30 min. These findings suggest a high turnover rate of water between the fish and the environment. Water influx was reduced by the putative AQP inhibitor phloretin (100 or 500 μM). Immunohistochemistry and confocal microscopy revealed that AQP1a1 protein was expressed in cells on the yolk sac epithelium. A substantial number of these AQP1a1-positive cells were identified as ionocytes, either H+-ATPase-rich cells or Na+/K+-ATPase-rich cells. AQP1a1 appeared to be expressed predominantly on the basolateral membranes of ionocytes, suggesting its potential involvement in regulating ionocyte volume and/or water flux into the circulation. Additionally, translational gene knockdown of AQP1a1 protein reduced water influx by approximately 30%, further indicating a role for AQP1a1 in facilitating transcellular water uptake. On the other hand, incubation with the Ca2+-chelator EDTA or knockdown of the epithelial tight junction protein claudin-b significantly increased water influx. These findings indicate that the epithelial tight junctions normally act to restrict paracellular water influx. Together, the results of the present study provide direct in vivo evidence that water movement can occur through transcellular routes (via AQP); the paracellular routes may become significant when the paracellular permeability is increased.

Aquaporin Inhibition by Gold(III) Compounds: New Insights

Aquaporins (AQPs) are membrane water/glycerol channels with essential roles in biological systems, as well as being promising targets for therapy and imaging. Using a stopped-flow method, a series of gold(III), platinum(II) and copper(II) complexes bearing nitrogen donor ligands, such as 1,10-phenatroline, 2,2'-bipyridine, 4,4'-dimethyl-2,2'-bipyridine, 4,4'-diamino-2,2'-bipyridine and 2,2';6',2"-terpyridine, were evaluated in human red blood cells expressing AQP1 and AQP3, responsible for water and glycerol movement, respectively. The results showed that the gold(III) complexes selectively modulate AQP3 over AQP1. Molecular modeling and density functional theory (DFT) calculations were subsequently performed to rationalize the observations and to investigate the possible molecular mechanism through which these gold compounds act on their putative target (AQP3). In the absence of any crystallographic data, a previously reported homology model was used for this purpose. Combined, the findings of this study show that potent and selective modulation of these solute channels is possible, however further investigation is required into the selectivity of this class of agents against all AQP isoforms and their potential therapeutic uses.

The identification of novel, high affinity AQP9 inhibitors in an intracellular binding site

The involvement of aquaporin (AQP) water and small solute channels in the etiology of several diseases, including cancer, neuromyelitis optica and body fluid imbalance disorders, has been suggested previously. Furthermore, results obtained in a mouse model suggested that AQP9 function contributes to hyperglycemia in type-2 diabetes. In addition, the physiological role of several AQP family members remains poorly understood. Small molecule inhibitors of AQPs are therefore desirable to further study AQP physiological and pathophysiological functions. The binding of recently established AQP9 inhibitors to a homology model of AQP9 was investigated by molecular dynamics simulations and molecular docking. Putative inhibitor binding sites identified with this procedure were modified by site-directed mutagenesis. Active compounds were measured in a mammalian cell water permeability assay of mutated AQP9 isoforms and tested for changes in inhibitory effects. Three independent cell lines were established for each mutated AQP9 isoform and functionality of mutant isoforms was established. We have identified putative binding sites of recently established AQP9 inhibitors. This information facilitated successful identification of novel AQP9 inhibitors with low micromolar IC50 values in a cell based assay by in silico screening of a compound library targeting specifically this binding site. We have established a successful strategy for AQP small molecule inhibitor identification. AQP inhibitors may be relevant as experimental tools, to enhance our understanding of AQP function, and in the treatment of various diseases.

Mechanistic studies of perfluorooctane sulfonate, perfluorooctanoic acid uptake by maize (Zea mays L. cv. TY2)

Perfluorinated compounds (PFCs) are of particular environmental concern. The migration of PFCs from soil to plants is a likely pathway for PFCs to enter the human food chain. This study aimed to investigate the uptake mechanisms of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) by maize (Zea mays L. cv. TY2). Hydroponic greenhouse experiments were performed. The kinetics of PFOS and PFOA uptake fitted Mechaelis-Menten equation well, suggesting their carrier-mediated influx processes. Uptake of PFOS was insensitive to metabolic inhibitors (NaN3 and Na3VO4). In contrast, treated with NaN3 and Na3VO4 reduced the uptake of PFOA by 83 and 43 % respectively. PFOS uptake was decreased by 31 % and 25 % when plants were treated with aquaporin inhibitors, AgNO3 and glycerol, respectively, while aquaporin inhibitors had no effect on PFOA uptake. Anion channel blockers, 4, 4′-diisothiocyanostibene-2,2′-disolfonate (DID) and 5-nitro 2-(3-phenylpropylamine) benzoic acid (NPPB) inhibited the uptake of PFOS by 33 % and 30 %, respectively. Anion channel blocker anthracene-9-carboxylic acid (9-AC) decreased the uptake of PFOA by 28 %. No competitive uptake was found between PFOS and PFOA. Uptake of PFOS and PFOA by maize may have different mechanisms.