Cathepsin K deficiency prevented stress-related thrombosis in a mouse FeCl3 model.

BACKGROUND: Exposure to chronic psychological stress (CPS) is a risk factor for thrombotic cardiocerebrovascular diseases (CCVDs). The expression and activity of the cysteine cathepsin K (CTSK) are upregulated in stressed cardiovascular tissues, and we investigated whether CTSK is involved in chronic stress-related thrombosis, focusing on stress serum-induced endothelial apoptosis. METHODS AND RESULTS: Eight-week-old wild-type male mice (CTSK+/+) randomly divided to non-stress and 3-week restraint stress groups received a left carotid artery iron chloride3 (FeCl3)-induced thrombosis injury for biological and morphological evaluations at specific timepoints. On day 21 post-stress/injury, the stress had enhanced the arterial thrombi weights and lengths, in addition to harmful alterations of plasma ADAMTS13, von Willebrand factor, and plasminogen activation inhibitor-1, plus injured-artery endothelial loss and CTSK protein/mRNA expression. The stressed CTSK+/+ mice had increased levels of injured arterial cleaved Notch1, Hes1, cleaved caspase8, matrix metalloproteinase-9/-2, angiotensin type 1 receptor, galactin3, p16IN4A, p22phox, gp91phox, intracellular adhesion molecule-1, TNF-α, MCP-1, and TLR-4 proteins and/or genes. Pharmacological and genetic inhibitions of CTSK ameliorated the stress-induced thrombus formation and the observed molecular and morphological changes. In cultured HUVECs, CTSK overexpression and silencing respectively increased and mitigated stressed-serum- and H2O2-induced apoptosis associated with apoptosis-related protein changes. Recombinant human CTSK degraded γ-secretase substrate in a dose-dependent manor and activated Notch1 and Hes1 expression upregulation. CONCLUSIONS: CTSK appeared to contribute to stress-related thrombosis in mice subjected to FeCl3 stress, possibly via the modulation of vascular inflammation, oxidative production and apoptosis, suggesting that CTSK could be an effective therapeutic target for CPS-related thrombotic events in patients with CCVDs.

[Transcriptional analysis of the molecular response of Arabidopsis to manganese stress and recovery].

Manganese (Mn) is an essential element for plants and plays a role in various metabolic processes. However, excess manganese can be toxic to plants. This study aimed to analyze the changes in various physiological activities and the transcriptome of Arabidopsis under different treatments: 1 mmol/L MnCl2 treatment for 1 day or 3 days, and 1 day of recovery on MS medium after 3 days of MnCl2 treatment. During the recovery phase, minor yellowing symptoms appeared on the leaves of Arabidopsis, and the content of chlorophyll and carotenoid decreased significantly, but the content of malondialdehyde and soluble sugar increased rapidly. Transcriptome sequencing data shows that the expression patterns of differentially expressed genes exhibit three major models: initial response model, later response model, recovery response model. Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis identified several affected metabolic pathways, including plant hormone signal transduction mitosolysis activates protein kinase (MAPK) phytohormone signaling, phenylpropanoid biosynthesis, ATP binding cassette transporters (ABC transporter), and glycosphingolipid biosynthesis. Differential expressed genes (DEGs) involved in phenylpropanoid biosynthesis, ABC transporter, and glycosphingolipid biosynthesis, were identified. Sixteen randomly selected DEGs were validated through qRT-PCR and showed consistent results with RNA-seq data. Our findings suggest that the phenylpropanoid metabolic pathway is activated to scavenge reactive oxygen species, the regulation of ABC transporter improves Mn transport, and the adjustment of cell membrane lipid composition occurs through glycerophospholipid metabolism to adapt to Mn stress in plants. This study provides new insights into the molecular response of plants to Mn stress and recovery, as well as theoretical cues for cultivating Mn-resistant plant varieties.

YabJ from Staphylococcus aureus entraps chlorides within its pocket.

Chlorination is a potent disinfectant against various microorganisms, including bacteria and viruses, by inducing protein modifications and functional changes. Chlorine, in the form of sodium hypochlorite, stands out as the predominant sanitizer choice due to its cost-effectiveness and powerful antimicrobial properties. Upon exposure to chlorination, proteins undergo modifications, with amino acids experiencing alterations through the attachment of chloride or oxygen atoms. These modifications lead to shifts in protein function and the modulation of downstream signaling pathways, ultimately resulting in a bactericidal effect. However, certain survival proteins, such as chaperones or transcription factors, aid organisms in overcoming harsh chlorination conditions. The expression of YabJ, a highly conserved protein from Staphylococcus aureus, is regulated by a stress-activated sigma factor called sigma B (σB). This research revealed that S. aureus YabJ maintains its structural integrity even under intense chlorination conditions and harbors sodium hypochlorite molecules within its surface pocket. Notably, the pocket of S. aureus YabJ is primarily composed of amino acids less susceptible to chlorination-induced damage, rendering it resistant to such effects. This study elucidates how S. aureus YabJ evades the detrimental effects of chlorination and highlights its role in sequestering sodium hypochlorite within its structure. Consequently, this process enhances resilience and facilitates adaptation to challenging environmental conditions.

WNK kinase is a vasoactive chloride sensor in endothelial cells.

Endothelial cells (ECs) line the wall of blood vessels and regulate arterial contractility to tune regional organ blood flow and systemic pressure. Chloride (Cl-) is the most abundant anion in ECs and the Cl- sensitive With-No-Lysine (WNK) kinase is expressed in this cell type. Whether intracellular Cl- signaling and WNK kinase regulate EC function to alter arterial contractility is unclear. Here, we tested the hypothesis that intracellular Cl- signaling in ECs regulates arterial contractility and examined the signaling mechanisms involved, including the participation of WNK kinase. Our data obtained using two-photon microscopy and cell-specific inducible knockout mice indicated that acetylcholine, a prototypical vasodilator, stimulated a rapid reduction in intracellular Cl- concentration ([Cl-]i) due to the activation of TMEM16A, a Cl- channel, in ECs of resistance-size arteries. TMEM16A channel-mediated Cl- signaling activated WNK kinase, which phosphorylated its substrate proteins SPAK and OSR1 in ECs. OSR1 potentiated transient receptor potential vanilloid 4 (TRPV4) currents in a kinase-dependent manner and required a conserved binding motif located in the channel C terminus. Intracellular Ca2+ signaling was measured in four dimensions in ECs using a high-speed lightsheet microscope. WNK kinase-dependent activation of TRPV4 channels increased local intracellular Ca2+ signaling in ECs and produced vasodilation. In summary, we show that TMEM16A channel activation reduces [Cl-]i, which activates WNK kinase in ECs. WNK kinase phosphorylates OSR1 which then stimulates TRPV4 channels to produce vasodilation. Thus, TMEM16A channels regulate intracellular Cl- signaling and WNK kinase activity in ECs to control arterial contractility.

Chloride/Multiple Anion Exchanger SLC26A Family: Systemic Roles of SLC26A4 in Various Organs.

Solute carrier family 26 member 4 (SLC26A4) is a member of the SLC26A transporter family and is expressed in various tissues, including the airway epithelium, kidney, thyroid, and tumors. It transports various ions, including bicarbonate, chloride, iodine, and oxalate. As a multiple-ion transporter, SLC26A4 is involved in the maintenance of hearing function, renal function, blood pressure, and hormone and pH regulation. In this review, we have summarized the various functions of SLC26A4 in multiple tissues and organs. Moreover, the relationships between SLC26A4 and other channels, such as cystic fibrosis transmembrane conductance regulator, epithelial sodium channel, and sodium chloride cotransporter, are highlighted. Although the modulation of SLC26A4 is critical for recovery from malfunctions of various organs, development of specific inducers or agonists of SLC26A4 remains challenging. This review contributes to providing a better understanding of the role of SLC26A4 and development of therapeutic approaches for the SLC26A4-associated hearing loss and SLC26A4-related dysfunction of various organs.

K+-Driven Cl-/HCO3- Exchange Mediated by Slc4a8 and Slc4a10.

Slc4a genes encode various types of transporters, including Na+-HCO3- cotransporters, Cl-/HCO3- exchangers, or Na+-driven Cl-/HCO3- exchangers. Previous research has revealed that Slc4a9 (Ae4) functions as a Cl-/HCO3- exchanger, which can be driven by either Na+ or K+, prompting investigation into whether other Slc4a members facilitate cation-dependent anion transport. In the present study, we show that either Na+ or K+ drive Cl-/HCO3- exchanger activity in cells overexpressing Slc4a8 or Slc4a10. Further characterization of cation-driven Cl-/HCO3- exchange demonstrated that Slc4a8 and Slc4a10 also mediate Cl- and HCO3--dependent K+ transport. Full-atom molecular dynamics simulation on the recently solved structure of Slc4a8 supports the coordination of K+ at the Na+ binding site in S1. Sequence analysis shows that the critical residues coordinating monovalent cations are conserved among mouse Slc4a8 and Slc4a10 proteins. Together, our results suggest that Slc4a8 and Slc4a10 might transport K+ in the same direction as HCO3- ions in a similar fashion to that described for Na+ transport in the rat Slc4a8 structure.

Kalium channelrhodopsins effectively inhibit neurons.

The analysis of neural circuits has been revolutionized by optogenetic methods. Light-gated chloride-conducting anion channelrhodopsins (ACRs)-recently emerged as powerful neuron inhibitors. For cells or sub-neuronal compartments with high intracellular chloride concentrations, however, a chloride conductance can have instead an activating effect. The recently discovered light-gated, potassium-conducting, kalium channelrhodopsins (KCRs) might serve as an alternative in these situations, with potentially broad application. As yet, KCRs have not been shown to confer potent inhibitory effects in small genetically tractable animals. Here, we evaluated the utility of KCRs to suppress behavior and inhibit neural activity in Drosophila, Caenorhabditis elegans, and zebrafish. In direct comparisons with ACR1, a KCR1 variant with enhanced plasma-membrane trafficking displayed comparable potency, but with improved properties that include reduced toxicity and superior efficacy in putative high-chloride cells. This comparative analysis of behavioral inhibition between chloride- and potassium-selective silencing tools establishes KCRs as next-generation optogenetic inhibitors for in vivo circuit analysis in behaving animals.

Chloride ions in health and disease.

Chloride is a key anion involved in cellular physiology by regulating its homeostasis and rheostatic processes. Changes in cellular Cl- concentration result in differential regulation of cellular functions such as transcription and translation, post-translation modifications, cell cycle and proliferation, cell volume, and pH levels. In intracellular compartments, Cl- modulates the function of lysosomes, mitochondria, endosomes, phagosomes, the nucleus, and the endoplasmic reticulum. In extracellular fluid (ECF), Cl- is present in blood/plasma and interstitial fluid compartments. A reduction in Cl- levels in ECF can result in cell volume contraction. Cl- is the key physiological anion and is a principal compensatory ion for the movement of the major cations such as Na+, K+, and Ca2+. Over the past 25 years, we have increased our understanding of cellular signaling mediated by Cl-, which has helped in understanding the molecular and metabolic changes observed in pathologies with altered Cl- levels. Here, we review the concentration of Cl- in various organs and cellular compartments, ion channels responsible for its transportation, and recent information on its physiological roles.

Coupling and regulation mechanisms of the flavin-dependent halogenase PyrH observed by infrared difference spectroscopy.

Flavin-dependent halogenases are central enzymes in the production of halogenated secondary metabolites in various organisms and they constitute highly promising biocatalysts for regioselective halogenation. The mechanism of these monooxygenases includes formation of hypohalous acid from a reaction of fully reduced flavin with oxygen and halide. The hypohalous acid then diffuses via a tunnel to the substrate-binding site for halogenation of tryptophan and other substrates. Oxidized flavin needs to be reduced for regeneration of the enzyme, which can be performed in vitro by a photoreduction with blue light. Here, we employed this photoreduction to study characteristic structural changes associated with the transition from oxidized to fully reduced flavin in PyrH from Streptomyces rugosporus as a model for tryptophan-5-halogenases. The effect of the presence of bromide and chloride or the absence of any halides on the UV-vis spectrum of the enzyme demonstrated a halide-dependent structure of the flavin-binding pocket. Light-induced FTIR difference spectroscopy was applied and the signals assigned by selective isotope labeling of the protein moiety. The identified structural changes in α-helix and ß-sheet elements were strongly dependent on the presence of bromide, chloride, the substrate tryptophan, and the product 5-chloro-tryptophan, respectively. We identified a clear allosteric coupling in solution at ambient conditions between cofactor-binding site and substrate-binding site that is active in both directions, despite their separation by a tunnel. We suggest that this coupling constitutes a fine-tuned mechanism for the promotion of the enzymatic reaction of flavin-dependent halogenases in dependence of halide and substrate availability.

Multi-session transcutaneous spinal cord stimulation prevents chloride homeostasis imbalance and the development of hyperreflexia after spinal cord injury in rat.

Spasticity is a complex and multidimensional disorder that impacts nearly 75% of individuals with spinal cord injury (SCI) and currently lacks adequate treatment options. This sensorimotor condition is burdensome as hyperexcitability of reflex pathways result in exacerbated reflex responses, co-contractions of antagonistic muscles, and involuntary movements. Transcutaneous spinal cord stimulation (tSCS) has become a popular tool in the human SCI research field. The likeliness for this intervention to be successful as a noninvasive anti-spastic therapy after SCI is suggested by a mild and transitory improvement in spastic symptoms following a single stimulation session, but it remains to be determined if repeated tSCS over the course of weeks can produce more profound effects. Despite its popularity, the neuroplasticity induced by tSCS also remains widely unexplored, particularly due to the lack of suitable animal models to investigate this intervention. Thus, the basis of this work was to use tSCS over multiple sessions (multi-session tSCS) in a rat model to target spasticity after SCI and identify the long-term physiological improvements and anatomical neuroplasticity occurring in the spinal cord. Here, we show that multi-session tSCS in rats with an incomplete (severe T9 contusion) SCI (1) decreases hyperreflexia, (2) increases the low frequency-dependent modulation of the H-reflex, (3) prevents potassium-chloride cotransporter isoform 2 (KCC2) membrane downregulation in lumbar motoneurons, and (4) generally augments motor output, i.e., EMG amplitude in response to single pulses of tSCS, particularly in extensor muscles. Together, this work displays that multi-session tSCS can target and diminish spasticity after SCI as an alternative to pharmacological interventions and begins to highlight the underlying neuroplasticity contributing to its success in improving functional recovery.

Chloride intracellular channel (CLIC) proteins function as fusogens.

Chloride Intracellular Channel (CLIC) family members uniquely transition between soluble and membrane-associated conformations. Despite decades of extensive functional and structural studies, CLICs' function as ion channels remains debated, rendering our understanding of their physiological role incomplete. Here, we expose the function of CLIC5 as a fusogen. We demonstrate that purified CLIC5 directly interacts with the membrane and induces fusion, as reflected by increased liposomal diameter and lipid and content mixing between liposomes. Moreover, we show that this activity is facilitated by acidic pH, a known trigger for CLICs' transition to a membrane-associated conformation, and that increased exposure of the hydrophobic inter-domain interface is crucial for this process. Finally, mutation of a conserved hydrophobic interfacial residue diminishes the fusogenic activity of CLIC5 in vitro and impairs excretory canal extension in C. elegans in vivo. Together, our results unravel the long-sought physiological role of these enigmatic proteins.

The Rectal Gland of the Shark: The Road to Understanding the Mechanism and Regulation of Transepithelial Chloride Transport.

Pictured, described, and speculated on, for close to 400 years, the function of the rectal gland of elasmobranchs remained unknown. In the late 1950s, Burger discovered that the rectal gland of Squalus acanthias secreted an almost pure solution of sodium chloride, isosmotic with blood, which could be stimulated by volume expansion of the fish. Twenty five years later, Stoff discovered that the secretion of the gland was mediated by adenyl cyclase. Studies since then have shown that vasoactive intestinal peptide (VIP) is the neurotransmitter responsible for activating adenyl cyclase; however, the amount of circulating VIP does not change in response to volume expansion. The humoral factor involved in activating the secretion of the gland is C-type natriuretic peptide, secreted from the heart in response to volume expansion. C-type natriuretic peptide circulates to the gland where it stimulates the release of VIP from nerves within the gland, but it also has a direct effect, independent of VIP. Sodium, potassium, and chloride are required for the gland to secrete, and the secretion of the gland is inhibited by ouabain or furosemide. The current model for the secretion of chloride was developed from this information. Basolateral NaKATPase maintains a low intracellular concentration of sodium, which establishes the large electrochemical gradient for sodium directed into the cell. Sodium moves from the blood into the cell (together with potassium and chloride) down this electrochemical gradient, through a coupled sodium, potassium, and two chloride cotransporter (NKCC1). On activation, chloride moves from the cell into the gland lumen, down its electrical gradient through apical cystic fibrosis transmembrane regulator. The fall in intracellular chloride leads to the phosphorylation and activation of NKCC1 that allows more chloride into the cell. Transepithelial sodium secretion into the lumen is driven by an electrical gradient through a paracellular pathway. The aim of this review was to examine the history of the origin of this model for the transport of chloride and suggest that it is applicable to many epithelia that transport chloride, both in resorptive and secretory directions.

Involvement of sodium-glucose cotransporter-1 activities in maintaining oscillatory Cl- currents from mouse submandibular acinar cells.

In salivary acinar cells, cholinergic stimulation induces elevations of cytosolic [Ca2+]i to activate the apical exit of Cl- through TMEM16A Cl- channels, which acts as a driving force for fluid secretion. To sustain the Cl- secretion, [Cl-]i must be maintained to levels that are greater than the electrochemical equilibrium mainly by Na+-K+-2Cl- cotransporter-mediated Cl- entry in basolateral membrane. Glucose transporters carry glucose into the cytoplasm, enabling the cells to produce ATP to maintain Cl- and fluid secretion. Sodium-glucose cotransporter-1 is a glucose transporter highly expressed in acinar cells. The salivary flow is suppressed by the sodium-glucose cotransporter-1 inhibitor phlorizin. However, it remains elusive how sodium-glucose cotransporter-1 contributes to maintaining salivary fluid secretion. To examine if sodium-glucose cotransporter-1 activity is required for sustaining Cl- secretion to drive fluid secretion, we analyzed the Cl- currents activated by the cholinergic agonist, carbachol, in submandibular acinar cells while comparing the effect of phlorizin on the currents between the whole-cell patch and the gramicidin-perforated patch configurations. Phlorizin suppressed carbachol-induced oscillatory Cl- currents by reducing the Cl- efflux dependent on the Na+-K+-2Cl- cotransporter-mediated Cl- entry in addition to affecting TMEM16A activity. Our results suggest that the sodium-glucose cotransporter-1 activity is necessary for maintaining the oscillatory Cl- secretion supported by the Na+-K+-2Cl- cotransporter activity in real time to drive fluid secretion. The concerted effort of sodium-glucose cotransporter-1, Na+-K+-2Cl- cotransporter, and apically located Cl- channels might underlie the efficient driving of Cl- secretion in different secretory epithelia from a variety of animal species.

Astrocytic chloride regulates brain function in health and disease.

Chloride ions (Cl-) play a pivotal role in synaptic inhibition in the central nervous system, primarily mediated through ionotropic mechanisms. A recent breakthrough emphathizes the significant influence of astrocytic intracellular chloride concentration ([Cl-]i) regulation, a field still in its early stages of exploration. Typically, the [Cl-]i in most animal cells is maintained at lower levels than the extracellular chloride [Cl-]o, a critical balance to prevent cell swelling due to osmotic pressure. Various Cl- transporters are expressed differently across cell types, fine-tuning the [Cl-]i, while Cl- gradients are utilised by several families of Cl- channels. Although the passive distribution of ions within cells is governed by basic biophysical principles, astrocytes actively expend energy to sustain [Cl-]i at much higher levels than those achieved passively, and much higher than neuronal [Cl-]i. Beyond the role in volume regulation, astrocytic [Cl-]i is dynamically linked to brain states and influences neuronal signalling in actively behaving animals. As a vital component of brain function, astrocytic [Cl-]i also plays a role in the development of disorders where inhibitory transmission is disrupted. This review synthesises the latest insights into astrocytic [Cl-]i, elucidating its role in modulating brain function and its implications in various pathophysiological conditions.

Structural identification of a selectivity filter in CFTR.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that regulates transepithelial salt and fluid homeostasis. CFTR dysfunction leads to reduced chloride secretion into the mucosal lining of epithelial tissues, thereby causing the inherited disease cystic fibrosis. Although several structures of CFTR are available, our understanding of the ion-conduction pathway is incomplete. In particular, the route that connects the cytosolic vestibule with the extracellular space has not been clearly defined, and the structure of the open pore remains elusive. Furthermore, although many residues have been implicated in altering the selectivity of CFTR, the structure of the "selectivity filter" has yet to be determined. In this study, we identify a chloride-binding site at the extracellular ends of transmembrane helices 1, 6, and 8, where a dehydrated chloride is coordinated by residues G103, R334, F337, T338, and Y914. Alterations to this site, consistent with its function as a selectivity filter, affect ion selectivity, conductance, and open channel block. This selectivity filter is accessible from the cytosol through a large inner vestibule and opens to the extracellular solvent through a narrow portal. The identification of a chloride-binding site at the intra- and extracellular bridging point leads us to propose a complete conductance path that permits dehydrated chloride ions to traverse the lipid bilayer.

Effects of naringenin on oxidative damage and apoptosis in liver and kidney in rats subjected to chronic mercury chloride.

Mercury chloride is a type of heavy metal that causes the formation of free radicals, causing hepatotoxicity, nephrotoxicity and apoptosis. In this study, the effects of naringenin on oxidative stress and apoptosis in the liver and kidney of rats exposed to mercury chloride were investigated. In the study, 41 2-month-old male Wistar-Albino rats were divided into five groups. Accordingly, group 1 was set as control group, group 2 as naringenin-100, group 3 as mercury chloride, group 4 as mercury chloride + naringenin-50, and group 5 as mercury chloride + naringenin-100. For the interventions, 1 mL/kg saline was administered to the control, 0.4 mg/kg/day mercury (II) chloride to the mercury chloride groups by i.p., and 50 and 100 mg/kg/day naringenin prepared in corn oil to the naringenin groups by gavage. All the interventions lasted for 20 days. Mercury chloride administration was initiated 1 h following the administration of naringenin. When mercury chloride and the control group were compared, a significant increase in plasma urea, liver and kidney malondialdehyde (MDA) levels, in kidney superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione-S-transferase (GST) activities (p < .001), and a significant decrease in liver and kidney glutathione (GSH) levels (p < .001), in liver catalase (CAT) activity (p < .01) were observed. In addition, histopathological changes and a significant increase in caspase-3 levels were detected (p < .05). When mercury chloride and treatment groups were compared, the administration of naringenin caused a decrease aspartate transaminase (AST), alanine transaminase (ALT), lactate dehydrogenase (LDH) (p < .01), urea, creatinine levels (p < .001) in plasma, MDA levels in liver and kidney, SOD, GSH-Px, GST activities in kidney (p < .001), and increased GSH levels in liver and kidney. The addition of naringenin-100 increased GSH levels above the control (p < .001). The administration of naringenin was also decreased histopathological changes and caspase-3 levels (p < .05). Accordingly, it was determined that naringenin is protective and therapeutic against mercury chloride-induced oxidative damage and apoptosis in the liver and kidney, and 100 mg/kg naringenin is more effective in preventing histopathological changes and apoptosis.

Activation of farnesoid X receptor retards expansion of renal collecting duct cell-derived cysts via inhibition of CFTR-mediated Cl- secretion.

The transcription factor farnesoid X receptor (FXR) regulates energy metabolism. Specifically, FXR functions to regulate cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl- secretion in intestinal epithelial cells. Therefore, this study aimed to investigate the role of FXR in CFTR-mediated Cl- secretion in renal tubular cells and to further elucidate its effects on renal cyst formation and growth. CFTR-mediated Cl- transport was evaluated via short-circuit current (ISC) measurements in Madin-Darby canine kidney (MDCK) cell monolayers and primary rat inner medullary collecting duct cells. The role of FXR in renal cyst formation and growth was determined by the MDCK cell-derived cyst model. Incubation with synthesized (GW4064) and endogenous (CDCA) FXR ligands reduced CFTR-mediated Cl- secretion in a concentration- and time-dependent manner. The inhibitory effect of FXR ligands was not due to the result of reduced cell viability and was attenuated by cotreatment with an FXR antagonist. FXR activation significantly decreased CFTR protein but not its mRNA. In addition, FXR activation inhibited CFTR-mediated Cl- secretion in primary renal collecting duct cells. FXR activation decreased ouabain-sensitive ISC without altering Na+-K+-ATPase mRNA and protein levels. Furthermore, FXR activation significantly reduced the number of cysts and renal cyst expansion. These inhibitory effects were correlated with a decrease in the expression of protein synthesis regulators mammalian target of rapamycin/S6 kinase. This study shows that FXR activation inhibits Cl- secretion in renal cells via inhibition of CFTR expression and retards renal cyst formation and growth. The discoveries point to a physiological role of FXR in the regulation of CFTR and a potential therapeutic application in polycystic kidney disease treatment.NEW & NOTEWORTHY The present study reveals that farnesoid X receptor (FXR) activation reduces microcyst formation and enlargement. This inhibitory effect of FXR activation is involved with decreased cell proliferation and cystic fibrosis transmembrane conductance regulator-mediated Cl- secretion in renal collecting duct cells. FXR might represent a novel target for the treatment of autosomal dominant polycystic kidney disease.

Stress stimulation promotes the injury repair process of airway epithelial cells through the [Cl-]i-FAK signaling axis.

The airway epithelium serves as a critical interface with the external environment, making it vulnerable to various external stimuli. Airway epithelial stress acts as a catalyst for the onset of numerous pulmonary and systemic diseases. Our previous studies have highlighted the impact of acute stress stimuli, especially bacterial lipopolysaccharide (LPS) and hydrogen peroxide (H2O2), on the continuous elevation of intracellular chloride concentration ([Cl-]i). However, the precise mechanism behind this [Cl-]i elevation and the consequential effects of such stress on the injury repair function of airway epithelial cells remain unclear. Our findings indicate that H2O2 induces an elevation in [Cl-]i by modulating the expression of CF transmembrane conductance regulator (CFTR) and Ca-activated transmembrane protein 16 A (TMEM16A) in airway epithelial cells (BEAS-2B), whereas LPS achieves this solely through CFTR. Subsequently, the elevated [Cl-]i level facilitated the injury repair process of airway epithelial cells by activating focal adhesion kinase (FAK). In summary, the [Cl-]i-FAK axis appears to play a promoting effect on the injury repair process triggered by stress stimulation. Furthermore, our findings suggest that abnormalities in the [Cl-]i-FAK signaling axis may play a crucial role in the pathogenesis of chronic airway diseases. Therefore, controlling the structure and function of airway epithelial barriers through the modulation of [Cl-]i holds promising prospects for future applications in managing and treating such conditions.

Predicting Mutational Effects on Ca2+-Activated Chloride Conduction of TMEM16A Based on a Simulation Study.

The dysfunction and defects of ion channels are associated with many human diseases, especially for loss-of-function mutations in ion channels such as cystic fibrosis transmembrane conductance regulator mutations in cystic fibrosis. Understanding ion channels is of great current importance for both medical and fundamental purposes. Such an understanding should include the ability to predict mutational effects and describe functional and mechanistic effects. In this work, we introduce an approach to predict mutational effects based on kinetic information (including reaction barriers and transition state locations) obtained by studying the working mechanism of target proteins. Specifically, we take the Ca2+-activated chloride channel TMEM16A as an example and utilize the computational biology model to predict the mutational effects of key residues. Encouragingly, we verified our predictions through electrophysiological experiments, demonstrating a 94% prediction accuracy regarding mutational directions. The mutational strength assessed by Pearson's correlation coefficient is -0.80 between our calculations and the experimental results. These findings suggest that the proposed methodology is reliable and can provide valuable guidance for revealing functional mechanisms and identifying key residues of the TMEM16A channel. The proposed approach can be extended to a broad scope of biophysical systems.

Neurohormonal Activation and Renal Chloride Avidity in Acute Heart Failure: Clinical Evidence Supporting the "Chloride Theory".

INTRODUCTION: Heart failure (HF) progression according to changes in the serum chloride concentration ([sCl-]) was recently proposed as the "chloride (Cl) theory" for HF pathophysiology. The present study examined the association of neurohormones and renal Cl avidity to determine their contribution to acute HF and their involvement to the "Cl theory." METHODS: Data from 29 patients with acute HF (48% men; 80.3 ± 12 years) were analyzed. Blood and urine samples were obtained before decongestive therapy. Clinical tests included peripheral blood, serum and spot urinary electrolytes, b-type natriuretic peptide (BNP), and plasma neurohormones. RESULTS: In the 29 patients, urinary Cl concentrations ([uCl-]) inversely correlated with log (plasma renin activity [PRA]) (r = -0.64, p = 0.0002) and log (plasma aldosterone concentration) (r = -0.50, p = 0.006). The [sCl-]‒[uCl-] difference positively correlated with log PRA (r = 0.63, p = 0.0002) and log (plasma aldosterone concentration) (r = 0.49, p = 0.008). Patients were divided into 2 groups according to the [sCl-]‒[uCl-] difference, an excretion (low renal Cl avidity) group and an absorption (high renal Cl avidity) group. Compared with the excretion group (-77 to ‒5 mEq/L; n = 14), the absorption group (1-84 mEq/L; n = 15) exhibited greater renal impairment (serum creatinine; 1.45 ± 0.63 vs. 1.00 ± 0.38 mg/d, p = 0.029) and cardiac burden (log BNP; 2.99 ± 0.3 vs. 2.66 ± 0.32 pg/mL, p = 0.008), higher log PRA (0.20 ± 0.58 vs. -0.25 ± 0.35 ng/mL/h, p = 0.018), and lower fractional urinary Cl excretion (1.34 ± 1.3 vs. 5.33 ± 4.1%, p < 0.001). CONCLUSION: Renal Cl avidity differs in acute HF, i.e., excretion (low renal Cl avidity) versus absorption (high renal Cl avidity) types, involving renin-aldosterone-angiotensin activity as the underlying mechanism, which provides the neurohormonal background for the "Cl theory." A version of this study was presented in part at the annual international scientific assembly (ACC.23) of the American College of Cardiology, March 4-6, 2023.