Inhibition of the Aquaporin-1 Cation Conductance by Selected Furan Compounds Reduces Red Blood Cell Sickling

In sickle cell disease (SCD), the pathological shift of red blood cells (RBCs) into distorted morphologies under hypoxic conditions follows activation of a cationic leak current (Psickle) and cell dehydration. Prior work showed sickling was reduced by 5-hydroxylmethyl-2-furfural (5-HMF), which stabilized mutant hemoglobin and also blocked the Psickle current in RBCs, though the molecular basis of this 5-HMF-sensitive cation current remained a mystery. Work here is the first to test the hypothesis that Aquaporin-1 (AQP1) cation channels contribute to the monovalent component of Psickle. Human AQP1 channels expressed in Xenopus oocytes were evaluated for sensitivity to 5-HMF and four derivatives known to have differential efficacies in preventing RBC sickling. Ion conductances were measured by two-electrode voltage clamp, and osmotic water permeability by optical swelling assays. Compounds tested were: 5-HMF; 5-PMFC (5-(phenoxymethyl)furan-2-carbaldehyde); 5-CMFC (5-(4-chlorophenoxymethyl)furan-2-carbaldehyde); 5-NMFC (5-(2-nitrophenoxymethyl)-furan-2-carbaldehyde); and VZHE006 (tert-butyl (5-formylfuran-2-yl)methyl carbonate). The most effective anti-sickling agent, 5-PMFC, was the most potent inhibitor of the AQP1 ion conductance (98% block at 100 µM). The order of sensitivity of the AQP1 conductance to inhibition was 5-PMFC > VZHE006 > 5-CMFC ≥ 5-NMFC, which corresponded with effectiveness in protecting RBCs from sickling. None of the compounds altered AQP1 water channel activity. Combined application of a selective AQP1 ion channel blocker AqB011 (80 µM) with a selective hemoglobin modifying agent 5-NMFC (2.5 mM) increased anti-sickling effectiveness in red blood cells from human SCD patients. Another non-selective cation channel known to be expressed in RBCs, Piezo1, was unaffected by 2 mM 5-HMF. Results suggest that inhibition of AQP1 ion channels and capacity to modify hemoglobin are combined features of the most effective anti-sickling agents. Future therapeutics aimed at both targets could hold promise for improved treatments for SCD.

Interactive Dynamics of Cell Volume and Cell Death in Human Erythrocytes Exposed to α-Hemolysin from Escherichia coli

α-hemolysin (HlyA) of E. coli binds irreversibly to human erythrocytes and induces cell swelling, ultimately leading to hemolysis. We characterized the mechanism involved in water transport induced by HlyA and analyzed how swelling and hemolysis might be coupled. Osmotic water permeability (Pf) was assessed by stopped-flow light scattering. Preincubation with HlyA strongly reduced Pf in control- and aquaporin 1-null red blood cells, although the relative Pf decrease was similar in both cell types. The dynamics of cell volume and hemolysis on RBCs was assessed by electrical impedance, light dispersion and hemoglobin release. Results show that HlyA induced erythrocyte swelling, which is enhanced by purinergic signaling, and is coupled to osmotic hemolysis. We propose a mathematical model of HlyA activity where the kinetics of cell volume and hemolysis in human erythrocytes depend on the flux of osmolytes across the membrane, and on the maximum volume that these cells can tolerate. Our results provide new insights for understanding signaling and cytotoxicity mediated by HlyA in erythrocytes.

Cerebrospinal fluid formation is controlled by membrane transporters to modulate intracranial pressure

Disturbances in the brain fluid balance can lead to life-threatening elevation in the intracranial pressure (ICP), which represents a vast clinical challenge. Nevertheless, the molecular mechanisms governing cerebrospinal fluid (CSF) secretion are largely unresolved, thus preventing targeted and efficient pharmaceutical therapy of cerebral pathologies involving elevated ICP. Here, we employed experimental rats to demonstrate low osmotic water permeability of the choroid plexus, lack of an osmotic gradient across this tissue, and robust CSF secretion against osmotic gradients. Together, these results illustrate that CSF secretion occurs independently of conventional osmosis, which challenges the existing assumption that CSF production is driven entirely by bulk osmotic forces across the CSF-secreting choroid plexus. Instead, we reveal that the choroidal Na+/K+/Cl− cotransporter NKCC1, Na+/HCO3− cotransporter NBCe2, and Na+/K+-ATPase are actively involved in CSF production and propose a molecular mode of water transport supporting CSF production in this secretory tissue. Further, we demonstrate that inhibition of NKCC1 directly reduces the ICP, illustrating that altered CSF secretion may be employed as a strategy to modulate ICP. These insights identify new promising therapeutic targets against brain pathologies associated with elevated ICP.

Transcriptomic Analysis Reveals Regulatory Networks for Osmotic Water Stress and Rewatering Response in the Leaves of Ginkgo biloba

To elucidate the transcriptomic regulation mechanisms that underlie the response of Ginkgo biloba to dehydration and rehydration, we used ginkgo saplings exposed to osmotically driven water stress and subsequent rewatering. When compared with a control group, 137, 1453, 1148, and 679 genes were differentially expressed in ginkgo leaves responding to 2, 6, 12, and 24 h of water deficit, and 796 and 1530 genes were differentially expressed responding to 24 and 48 h of rewatering. Upregulated genes participated in the biosynthesis of abscisic acid, eliminating reactive oxygen species (ROS), and biosynthesis of flavonoids and bilobalide, and downregulated genes were involved in water transport and cell wall enlargement in water stress-treated ginkgo leaves. Under rehydration conditions, the genes associated with water transport and cell wall enlargement were upregulated, and the genes that participated in eliminating ROS and the biosynthesis of flavonoids and bilobalide were downregulated in the leaves of G. biloba. Furthermore, the weighted gene coexpression networks were established and correlated with distinct water stress and rewatering time-point samples. Hub genes that act as key players in the networks were identified. Overall, these results indicate that the gene coexpression networks play essential roles in the transcriptional reconfiguration of ginkgo leaves in response to water stress and rewatering.

Aquaporin-1 Facilitates Transmesothelial Water Permeability: In Vitro and Ex Vivo Evidence and Possible Implications in Peritoneal Dialysis

We previously showed that mesothelial cells in human peritoneum express the water channel aquaporin 1 (AQP1) at the plasma membrane, suggesting that, although in a non-physiological context, it may facilitate osmotic water exchange during peritoneal dialysis (PD). According to the three-pore model that predicts the transport of water during PD, the endothelium of peritoneal capillaries is the major limiting barrier to water transport across peritoneum, assuming the functional role of the mesothelium, as a semipermeable barrier, to be negligible. We hypothesized that an intact mesothelial layer is poorly permeable to water unless AQP1 is expressed at the plasma membrane. To demonstrate that, we characterized an immortalized cell line of human mesothelium (HMC) and measured the osmotically-driven transmesothelial water flux in the absence or in the presence of AQP1. The presence of tight junctions between HMC was investigated by immunofluorescence. Bioelectrical parameters of HMC monolayers were studied by Ussing Chambers and transepithelial water transport was investigated by an electrophysiological approach based on measurements of TEA+ dilution in the apical bathing solution, through TEA+-sensitive microelectrodes. HMCs express Zo-1 and occludin at the tight junctions and a transepithelial vectorial Na+ transport. Real-time transmesothelial water flux, in response to an increase of osmolarity in the apical solution, indicated that, in the presence of AQP1, the rate of TEA+ dilution was up to four-fold higher than in its absence. Of note, we confirmed our data in isolated mouse mesentery patches, where we measured an AQP1-dependent transmesothelial osmotic water transport. These results suggest that the mesothelium may represent an additional selective barrier regulating water transport in PD through functional expression of the water channel AQP1.

Insight into Debye Hückel length (κ−1): smart gravimetric and swelling techniques reveals discrepancy of diffuse double layer theory at high ionic concentrations

Smart gravimetric and swelling techniques were utilized in this work to examine the validity of the Debye Hückel length (κ−1) equation when shale interacts with highly concentrated salt solutions. The swelling and shrinkage behavior of two different shales, when exposed to monovalent and divalent ionic solutions (NaCl, KCl and CaCl2) at concentrations ranging from 2 to 22%w/w was observed and measured. Shale swelling and shrinkage results show that Debye Hückel length (κ−1) equation seems to work adequately at low ionic concentrations where osmotic water flow out of shale plays a major role in decreasing the diffuse double layer thickness by withdrawing water out and thereby shrinking κ−1. At high ionic concentration levels, the flow of associated water into the diffuse double layer negates the withdrawal of osmotic water out of the diffuse double layer which could maintain κ−1 or possibly increase it. Data on measured ionic uptake into shale suggests that excessive ionic diffusion into shale, especially at high concentrations, leads to higher electrical repulsion between alike ions in the diffuse layer which could lead to the expansion of the diffuse double layer thickness. Furthermore, swelling and shrinkage data analysis for shale suggests the existence of a ‘critical concentration’ below which the Debye Hückel length equation works. Above the critical concentration, the validity of the Debye Hückel length equation might be in question. The critical concentration is different for all ions and depends on ionic valence, hydrated ion diameter, and clay type.

Insight on the inhibitive property of potassium ion on the stability of shale: a diffuse double-layer thickness (κ−1) perspective

It is believed that potassium ions reduce the hydration energy and swelling of clays and thus promoting stability to shales. This belief was made based on volumetric and linear expansion data obtained from shale and KCl solutions interactions. However, swelling data alone is not adequate to mitigate wellbore instability in shale. Such data must be incorporated with mechanical and physicochemical data for complete and accurate wellbore instability analysis.This paper presents clear experimental evidence showing that concentrated potassium chloride solutions tend to suppress shale swelling as higher concentration of potassium ions collapses the diffuse double layer of clay particles causing shale shrinkage which confirms the notion that the Debye length (κ−1) decreases as the ionic concentration increases.Results show that there exists a KCl concentration threshold above which shale’s compressive strength deteriorates significantly. This concentration threshold was found to hover around 5% by weight. The amount of water and ions uptake into shale was quantified using gravimetric measurements. Significant potassium ions invasion into shale was experimentally measured as KCl solution concentration increased which proved the leaky nature of shale’s membrane. The reduction of shale’s compressive strength seems to be well correlated with the amount of ions uptake into shale. Moreover, data suggests that shale’s compressive strength was not significantly impacted by swelling. It was possible to gravimetrically separate osmotic water from associated water as shale interacted with KCl solutions. Results suggest that osmotic water is responsible for shale swelling since it is unattached to ions which makes it free to move around inside shale. On the other hand, data suggest that associated water does not contribute to shale swelling as it is bound to potassium ions which makes it unfree to move around. It is fair to state, based on our experimental data, that osmotic water is responsible for shale swelling while associated water contributes to shale’s compressive strength alteration.

Preharvest Nutrient Deprivation Reconfigures Nitrate, Mineral and Phytochemical Content of Microgreens

While imparting gastronomic novelty and sensory delight, microgreens also constitute rudimentary leafy greens packed with nutrients and phytochemicals. As such, they comprise an upcoming class of functional foods. However, apart from bioactive secondary metabolites, microgreens also accumulate antinutritive agents such as nitrate, especially under conducive protected cultivation conditions. The current work examined nutrient deprivation before harvest (DBH), applied by replacing nutrient solution with osmotic water for six and twelve days, as a strategy for reducing microgreen nitrate levels in different species (lettuce, mustard, and rocket). The three species were sown on a peat-based substrate, cultivated in a controlled climate chamber, and harvested 18 days after sowing, when the first two true leaves emerged. DBH impact on major constituents of the secondary metabolome, mineral content, colorimetric, and yield traits was appraised. Nitrate and mineral content were determined through ion chromatography, phenolic composition through UHPLC-Q-Orbitrap HRMS, and carotenoid composition through HPLC-DAD. Nutrient deprivation was effective in reducing nitrate content; however, effective treatment duration differed between species and decline was more precipitous in nitrate hyperaccumulating species such as rocket. Quercetin and kaempferol glycosides were the flavonol glycosides most abundant in brassicaceous microgreens, whereas lettuce microgreens were steeped in caffeoyl quinic acid. DBH interacted with species as it increased the total phenolic content of lettuce, decreased that of rocket, but did not affect mustard. Further research to link changes in phenolic composition to the sensory and in vivo bioactive profile of microgreens is warranted. Notably, brief (≤6 days) DBH can be applied across species with moderate or no impact on the phenolic, carotenoid, and mineral composition of microgreens. Brief DBH applications also have limited impact on microgreens’ yield and colorimetric traits hence on the commercial value of the product. They can therefore be applied for reducing microgreen nitrate levels without significantly impacting key secondary metabolic constituents and their potential bioactive role.

Strawberry fruit skins are far more permeable to osmotic water uptake than to transpirational water loss

Water movements through the fruit skin play critical roles in many disorders of strawberry (Fragaria × ananassa Duch.) such as water soaking, cracking and shriveling. The objective was to identify the mechanisms of fruit water loss (dry skin, transpiration) and water uptake (wet skin, osmosis). Fruits were held above dried silica gel or incubated in deionized water. Water movements were quantified gravimetrically. Transpiration and osmotic uptake increased linearly with time. Abrading the thin cuticle (0.62 g m-2) increased rates of transpiration 2.6–fold, the rates of osmotic uptake 7.9-fold. The osmotic potential of the expressed juice was nearly the same for green and for white fruit but decreased in red fruit stages. Fruit turgor was low throughout development, except for green fruit. There was no relationship between the rates of water movement and fruit osmotic potential. The skin permeance for transpiration and for osmotic uptake were both high (relative to other fruit species) but were two orders of magnitude greater for osmotic uptake than for transpiration. Incubating fruit in isotonic solutions of osmolytes of different sizes resulted in increases in fruit mass that depended on the osmolyte. The rate of osmotic uptake decreased asymptotically as molecular size of the osmolyte increased. When transpiration and osmotic uptake experiments were conducted sequentially on the same fruit, the rates of transpiration were higher for fruit previously incubated in water. Fluorescence microscopy revealed considerable microcracking in a fruit previously incubated in water. Our findings indicate that the high permeance for osmotic uptake is accounted for by an extremely thin cuticle and by viscous water flow through microcracks and along polar pathways.