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International Journal of Molecular... Apr 2023Epithelial sodium channels (ENaC) are part of a complex network of interacting biochemical pathways and as such are involved in several disease states. Dependent on site... (Review)
Review
Epithelial sodium channels (ENaC) are part of a complex network of interacting biochemical pathways and as such are involved in several disease states. Dependent on site and type of mutation, gain- or loss-of-function generated symptoms occur which span from asymptomatic to life-threatening disorders such as Liddle syndrome, cystic fibrosis or generalized pseudohypoaldosteronism type 1. Variants of ENaC which are implicated in disease assist further understanding of their molecular mechanisms in order to create models for specific pharmacological targeting. Identification and characterization of ENaC modifiers not only furthers our basic understanding of how these regulatory processes interact, but also enables discovery of new therapeutic targets for the disease conditions caused by ENaC dysfunction. Numerous test compounds have revealed encouraging results in vitro and in animal models but less in clinical settings. The EMA- and FDA-designated orphan drug solnatide is currently being tested in phase 2 clinical trials in the setting of acute respiratory distress syndrome, and the NOX1/ NOX4 inhibitor setanaxib is undergoing clinical phase 2 and 3 trials for therapy of primary biliary cholangitis, liver stiffness, and carcinoma. The established ENaC blocker amiloride is mainly used as an add-on drug in the therapy of resistant hypertension and is being studied in ongoing clinical phase 3 and 4 trials for special applications. This review focuses on discussing some recent developments in the search for novel therapeutic agents.
Topics: Animals; Epithelial Sodium Channels; Hypertension; Liddle Syndrome; Pseudohypoaldosteronism; Amiloride
PubMed: 37175488
DOI: 10.3390/ijms24097775 -
African Journal of Laboratory Medicine 2023Liddle syndrome is an autosomal dominantly inherited disorder usually arising from single mutations of the genes that encode for the alpha, beta and gamma epithelial...
INTRODUCTION
Liddle syndrome is an autosomal dominantly inherited disorder usually arising from single mutations of the genes that encode for the alpha, beta and gamma epithelial sodium channel (ENaC) subunits. This leads to refractory hypertension, hypokalaemia, metabolic alkalosis, hyporeninaemia and hypoaldosteronism, through over-activation of the ENaC.
CASE PRESENTATION
We describe a 5-day old neonate who presented with severe hypernatraemic dehydration requiring admission to Steve Biko Academic Hospital in South Africa in 2012. Further evaluation revealed features in keeping with Liddle syndrome. Two compound heterozygous mutations located at different subunits encoding the ENaC were detected following genetic sequencing done in 2020. The severe clinical phenotype observed here could be attributed to the synergistic effect of these known pathological mutations, but may also indicate that one of the other variants detected has hitherto undocumented pathological effects.
MANAGEMENT AND OUTCOME
This child's treatment course was complicated by poor adherence to therapy, requiring numerous admissions over the years. Adequate blood pressure control was achieved only after the addition of amiloride at the end of 2018, which raised the suspicion of an ENaC abnormality.
CONCLUSION
To our knowledge, this is the first Liddle syndrome case where a combined effect from mutations resulted in severe disease. This highlights the importance of early recognition and management of this highly treatable genetic disease to prevent the grave sequelae associated with long-standing hypertension. Whole exome sequencing may assist in the detection of known mutations, but may also unveil new potentially pathological variants.
WHAT THIS STUDY ADDS
This study highlights the importance of developing a high index of suspicion of tubulopathy such as Liddle syndrome for any child presenting with persistent hypertension associated with hypokalaemic metabolic alkalosis.
PubMed: 37151815
DOI: 10.4102/ajlm.v12i1.1998 -
Submersion and hypoxia inhibit alveolar epithelial Na transport through ERK/NF-κB signaling pathway.Respiratory Research Apr 2023Hypoxia is associated with many respiratory diseases, partly due to the accumulation of edema fluid and mucus on the surface of alveolar epithelial cell (AEC), which...
BACKGROUND
Hypoxia is associated with many respiratory diseases, partly due to the accumulation of edema fluid and mucus on the surface of alveolar epithelial cell (AEC), which forms oxygen delivery barriers and is responsible for the disruption of ion transport. Epithelial sodium channel (ENaC) on the apical side of AEC plays a crucial role to maintain the electrochemical gradient of Na and water reabsorption, thus becomes the key point for edema fluid removal under hypoxia. Here we sought to explore the effects of hypoxia on ENaC expression and the further mechanism related, which may provide a possible treatment strategy in edema related pulmonary diseases.
METHODS
Excess volume of culture medium was added on the surface of AEC to simulate the hypoxic environment of alveoli in the state of pulmonary edema, supported by the evidence of increased hypoxia-inducible factor-1 expression. The protein/mRNA expressions of ENaC were detected, and extracellular signal-regulated kinase (ERK)/nuclear factor κB (NF-κB) inhibitor was applied to explore the detailed mechanism about the effects of hypoxia on epithelial ion transport in AEC. Meanwhile, mice were placed in chambers with normoxic or hypoxic (8%) condition for 24 h, respectively. The effects of hypoxia and NF-κB were assessed through alveolar fluid clearance and ENaC function by Ussing chamber assay.
RESULTS
Hypoxia (submersion culture mode) induced the reduction of protein/mRNA expression of ENaC, whereas increased the activation of ERK/NF-κB signaling pathway in parallel experiments using human A549 and mouse alveolar type 2 cells, respectively. Moreover, the inhibition of ERK (PD98059, 10 µM) alleviated the phosphorylation of IκB and p65, implying NF-κB as a downstream pathway involved with ERK regulation. Intriguingly, the expression of α-ENaC could be reversed by either ERK or NF-κB inhibitor (QNZ, 100 nM) under hypoxia. The alleviation of pulmonary edema was evidenced by the administration of NF-κB inhibitor, and enhancement of ENaC function was supported by recording amiloride-sensitive short-circuit currents.
CONCLUSIONS
The expression of ENaC was downregulated under hypoxia induced by submersion culture, which may be mediated by ERK/NF-κB signaling pathway.
Topics: Mice; Humans; Animals; NF-kappa B; Pulmonary Edema; Extracellular Signal-Regulated MAP Kinases; Immersion; Pulmonary Alveoli; Hypoxia; Signal Transduction; Epithelial Sodium Channels; Sodium; RNA, Messenger; Epithelial Cells
PubMed: 37095538
DOI: 10.1186/s12931-023-02428-z -
Sheng Li Xue Bao : [Acta Physiologica... Apr 2023Virtually all of the dietary potassium intake is absorbed in the intestine, over 90% of which is excreted by the kidneys regarded as the most important organ of... (Review)
Review
Virtually all of the dietary potassium intake is absorbed in the intestine, over 90% of which is excreted by the kidneys regarded as the most important organ of potassium excretion in the body. The renal excretion of potassium results primarily from the secretion of potassium by the principal cells in the aldosterone-sensitive distal nephron (ASDN), which is coupled to the reabsorption of Na by the epithelial Na channel (ENaC) located at the apical membrane of principal cells. When Na is transferred from the lumen into the cell by ENaC, the negativity in the lumen is relatively increased. K efflux, H efflux, and Cl influx are the 3 pathways that respond to Na influx, that is, all these 3 pathways are coupled to Na influx. In general, Na influx is equal to the sum of K efflux, H efflux, and Cl influx. Therefore, any alteration in Na influx, H efflux, or Cl influx can affect K efflux, thereby affecting the renal K excretion. Firstly, Na influx is affected by the expression level of ENaC, which is mainly regulated by the aldosterone-mineralocorticoid receptor (MR) pathway. ENaC gain-of-function mutations (Liddle syndrome, also known as pseudohyperaldosteronism), MR gain-of-function mutations (Geller syndrome), increased aldosterone levels (primary/secondary hyperaldosteronism), and increased cortisol (Cushing syndrome) or deoxycorticosterone (hypercortisolism) which also activate MR, can lead to up-regulation of ENaC expression, and increased Na reabsorption, K excretion, as well as H excretion, clinically manifested as hypertension, hypokalemia and alkalosis. Conversely, ENaC inactivating mutations (pseudohypoaldosteronism type 1b), MR inactivating mutations (pseudohypoaldosteronism type 1a), or decreased aldosterone levels (hypoaldosteronism) can cause decreased reabsorption of Na and decreased excretion of both K and H, clinically manifested as hypotension, hyperkalemia, and acidosis. The ENaC inhibitors amiloride and Triamterene can cause manifestations resembling pseudohypoaldosteronism type 1b; MR antagonist spironolactone causes manifestations similar to pseudohypoaldosteronism type 1a. Secondly, Na influx is regulated by the distal delivery of water and sodium. Therefore, when loss-of-function mutations in Na-K-2Cl cotransporter (NKCC) expressed in the thick ascending limb of the loop and in Na-Cl cotransporter (NCC) expressed in the distal convoluted tubule (Bartter syndrome and Gitelman syndrome, respectively) occur, the distal delivery of water and sodium increases, followed by an increase in the reabsorption of Na by ENaC at the collecting duct, as well as increased excretion of K and H, clinically manifested as hypokalemia and alkalosis. Loop diuretics acting as NKCC inhibitors and thiazide diuretics acting as NCC inhibitors can cause manifestations resembling Bartter syndrome and Gitelman syndrome, respectively. Conversely, when the distal delivery of water and sodium is reduced (e.g., Gordon syndrome, also known as pseudohypoaldosteronism type 2), it is manifested as hypertension, hyperkalemia, and acidosis. Finally, when the distal delivery of non-chloride anions increases (e.g., proximal renal tubular acidosis and congenital chloride-losing diarrhea), the influx of Cl in the collecting duct decreases; or when the excretion of hydrogen ions by collecting duct intercalated cells is impaired (e.g., distal renal tubular acidosis), the efflux of H decreases. Both above conditions can lead to increased K secretion and hypokalemia. In this review, we focus on the regulatory mechanisms of renal potassium excretion and the corresponding diseases arising from dysregulation.
Topics: Humans; Bartter Syndrome; Pseudohypoaldosteronism; Potassium; Aldosterone; Hypokalemia; Gitelman Syndrome; Hyperkalemia; Clinical Relevance; Epithelial Sodium Channels; Kidney Tubules, Distal; Sodium; Hypertension; Alkalosis; Water; Kidney
PubMed: 37089096
DOI: No ID Found -
ENeuro Apr 2023Taste cells are maintained by continuous turnover throughout a lifetime, yet the mechanisms of taste cell differentiation, and how taste sensations remain constant...
Taste cells are maintained by continuous turnover throughout a lifetime, yet the mechanisms of taste cell differentiation, and how taste sensations remain constant despite this continuous turnover, remain poorly understood. Here, we report that a transcription factor Etv1 (also known as Er81) is involved in the differentiation of taste cells responsible for the preference for sweet, umami, and salty tastes. Molecular analyses revealed that is expressed by a subset of taste cells that depend on Skn-1a (also known as Pou2f3) for their generation and express T1R genes (responsible for sweet and umami tastes) or (responsible for amiloride-sensitive salty taste). mice express Etv1 isoform(s) but not Etv1 in putative proprioceptive neurons as comparable to wild-type mice, yet lack expression of Etv1 or an isoform in taste cells. These mice have the same population of Skn-1a-dependent cells in taste buds as wild-type mice but have altered gene expression in taste cells, with regional differences. They have markedly decreased electrophysiological responses of chorda tympani nerves to sweet and umami tastes and to amiloride-sensitive salty taste evoked by sodium cation, but they have unchanged responses to bitter or sour tastes. Our data thus show that Etv1 is involved in the differentiation of the taste cells responsible for sweet, umami, and salty taste preferences.
Topics: Animals; Mice; Amiloride; Cell Differentiation; Sodium; Taste; Taste Buds; Transcription Factors
PubMed: 37045597
DOI: 10.1523/ENEURO.0236-22.2023 -
Frontiers in Pharmacology 2023The human organic cation transporter 2 (OCT2) is involved in the transport of endogenous quaternary amines and positively charged drugs across the basolateral membrane...
The human organic cation transporter 2 (OCT2) is involved in the transport of endogenous quaternary amines and positively charged drugs across the basolateral membrane of proximal tubular cells. In the absence of a structure, the progress in unraveling the molecular basis of OCT2 substrate specificity is hampered by the unique complexity of OCT2 binding pocket, which seemingly contains multiple allosteric binding sites for different substrates. Here, we used the thermal shift assay (TSA) to better understand the thermodynamics governing OCT2 binding to different ligands. Molecular modelling and docking of different ligands revealed two distinct binding sites at OCT2 outer part of the cleft. The predicted interactions were assessed by -inhibition assay using [H]1-methyl-4-phenylpyridinium ([H]MPP) as a model substrate, or by measuring the uptake of radiolabeled ligands in intact cells. Crude membranes from HEK293 cells harboring human OCT2 (OCT2-HEK293) were solubilized in n-Dodecyl-β-D-Maltopyranoside (DDM), incubated with the ligand, heated over a temperature gradient, and then pelleted to remove heat-induced aggregates. The OCT2 in the supernatant was detected by western blot. Among the compounds tested, -inhibition and TSA assays showed partly overlapping results. Gentamicin and methotrexate (MTX) did not inhibit [H]MPP uptake but significantly increased the thermal stabilization of OCT2. Conversely, amiloride completely inhibited [H]MPP uptake but did not affect OCT2 thermal stabilization. [H]MTX intracellular level was significantly higher in OCT2-HEK293 cells than in wild type cells. The magnitude of the thermal shift (ΔT) did not provide information on the binding. Ligands with similar affinity showed markedly different ΔT, indicating different enthalpic and entropic contributions for similar binding affinities. The ΔT positively correlated with ligand molecular weight/chemical complexity, which typically has high entropic costs, suggesting that large ΔT reflect a larger displacement of bound water molecules. In conclusion, TSA might represent a viable approach to expand our knowledge on OCT2 binding descriptors.
PubMed: 37007010
DOI: 10.3389/fphar.2023.1154213 -
Frontiers in Molecular Neuroscience 2023Itch is a common symptom of many skin and systemic diseases. Identifying novel endogenous itch mediators and the downstream signaling pathways involved will contribute...
INTRODUCTION
Itch is a common symptom of many skin and systemic diseases. Identifying novel endogenous itch mediators and the downstream signaling pathways involved will contribute to the development of new strategies for the treatment of chronic itch. In the present study, we adopted behavioral testing, patch clamp recording and metabonomics analysis to investigate the role of agmatine in itch and the underlying mechanism.
METHODS
Behavioral analysis was used to evaluate the establishing of acute and chronic itch mice model, and to test the effects of different drugs or agents on mice itch behavior. Western blotting analysis was used to test the effect of agmatine on phosphorylation of ERK (p-ERK) expression in the spinal cord. Patch clamp recording was used to determine the effect agmatine on the excitability of DRG neurons and the role of ASIC3. Finally, the metabonomics analysis was performed to detect the concentration of agmatine in the affected skin under atopic dermatitis or psoriasis conditions.
RESULTS
We fused a mouse model and found that an intradermal injection of agmatine (an endogenous polyamine) into the nape of the neck or cheek induced histamine-independent scratching behavior in a dose-dependent manner. In addition, the ablation of nociceptive C-fibers by resiniferatoxin (RTX) abolished agmatine-induced scratching behavior. However, agmatine-induced itch was not affected by the pharmacological inhibition of either transient receptor potential vanilloid 1 (TRPV1) or transient receptor potential ankyrin 1 (TRPA1); similar results were obtained from TRPV1 or TRPA1 mice. Furthermore, agmatine-induced itch was significantly suppressed by the administration of acid-sensing ion channel 3 (ASIC3) inhibitors, APETx2 or amiloride. Agmatine also induced the upregulation of p-ERK in the spinal cord; this effect was inhibited by amiloride. Current clamp recording showed that the acute perfusion of agmatine reduced the rheobase and increased the number of evoked action potentials in acute dissociated dorsal root ganglion (DRG) neurons while amiloride reversed agmatine-induced neuronal hyperexcitability. Finally, we identified significantly higher levels of agmatine in the affected skin of a mouse model of atopic dermatitis (AD) when compared to controls, and the scratching behavior of AD mice was significantly attenuated by blocking ASIC3.
DISCUSSION
Collectively, these results provide evidence that agmatine is a novel mediator of itch and induces itch via the activation of ASIC3. Targeting neuronal ASIC3 signaling may represent a novel strategy for the treatment of itch.
PubMed: 36937045
DOI: 10.3389/fnmol.2023.1086285 -
Clinical and Translational Science Jun 2023Anxiety and panic disorders are the most common mental illnesses in the United States and lack effective treatment options. Acid-sending ion channels (ASICs) in the... (Clinical Trial)
Clinical Trial
Anxiety and panic disorders are the most common mental illnesses in the United States and lack effective treatment options. Acid-sending ion channels (ASICs) in the brain were shown to be associated with fear conditioning and anxiety responses and therefore are potential targets for treating panic disorder. Amiloride is an inhibitor of the ASICs in the brain and was shown to reduce panic symptoms in preclinical animal models. An intranasal formulation of amiloride will be highly beneficial to treat acute panic attacks due to advantages such as the rapid onset of action and patient compliance. The aim of this single-center, open-label trial was to evaluate the basic pharmacokinetics (PKs) and safety of amiloride after intranasal administration in healthy human volunteers at three doses (0.2, 0.4, and 0.6 mg). Amiloride was detected in plasma within 10 min of intranasal administration and showed a biphasic PK profile with an initial peak within 10 min of administration followed by a second peak between 4 and 8 h of administration. The biphasic PKs indicate an initial rapid absorption via the nasal pathway and later slower absorption by non-nasal pathways. Intranasal amiloride exhibited a dose-proportional increase in the area under the curve and did not exhibit any systemic toxicity. These data indicate that intranasal amiloride is rapidly absorbed and safe at the doses evaluated and can be further considered for clinical development as a portable, rapid, noninvasive, and nonaddictive anxiolytic agent to treat acute panic attacks.
Topics: Animals; Humans; Administration, Intranasal; Amiloride; Anti-Anxiety Agents; Anxiety; Healthy Volunteers
PubMed: 36932683
DOI: 10.1111/cts.13514 -
Frontiers in Cellular and Infection... 2023Fungal infections have become an increasing threat as a result of growing numbers of susceptible hosts and diminishing effectiveness of antifungal drugs due to...
Fungal infections have become an increasing threat as a result of growing numbers of susceptible hosts and diminishing effectiveness of antifungal drugs due to multi-drug resistance. This reality underscores the need to develop novel drugs with unique mechanisms of action. We recently identified 5-(,-hexamethylene)amiloride (HMA), an inhibitor of human Na/H exchanger isoform 1, as a promising scaffold for antifungal drug development. In this work, we carried out susceptibility testing of 45 6-substituted HMA and amiloride analogs against a panel of pathogenic fungi. A series of 6-(2-benzofuran)amiloride and HMA analogs that showed up to a 16-fold increase in activity against were identified. Hits from these series showed broad-spectrum activity against both basidiomycete and ascomycete fungal pathogens, including multidrug-resistant clinical isolates.
Topics: Humans; Amiloride; Antifungal Agents; Fungi; Mycoses; Microbial Sensitivity Tests; Cryptococcus neoformans
PubMed: 36923593
DOI: 10.3389/fcimb.2023.1101568 -
Research Square Mar 2023Acute lung injury is characterized by overwhelmingly elevated PAI-1 in both lung edema fluid and the circulating system. The role of increased PAI-1, encoded by...
Acute lung injury is characterized by overwhelmingly elevated PAI-1 in both lung edema fluid and the circulating system. The role of increased PAI-1, encoded by Serpine1 gene, in the regeneration of injured lung epithelium has not been understood completely. This study aimed to investigate the role of Serpine1 in the regulation of alveolar type 2 epithelial cell (AT2) fate in a humanized mouse line carrying diseased mutants (Serpine1Tg). Wild type (wt) and Serpine1Tg AT2 cells were either cultured as monolayers or 3D alveolospheres. Colony forming assay and total surface area of organoids were analyzed. AT1 and AT2 cells in organoids were counted by immunohistochemistry and fluorescence-activated cell sorting (FACS). To test the potential effects of elevated PAI-1 on the permeability in the epithelial monolayers, we digitized the biophysical properties of polarized AT2 monolayers grown at the air-liquid interface. A significant reduction in total AT2 cells harvested in Serpine1Tg mice was observed compared with wt controls. AT2 cells harvested from Serpine1Tg mice reduced significantly over the wt controls. Spheroids formed by Serpine1Tg AT2 cells were lesser than wt control. Similarly, the corresponding surface area, a readout of realveolarization of injured epithelium, was markedly reduced in Serpine1Tg organoids. FACS analysis revealed a significant suppression in the number of AT2 cells, in particular, the CD44+ subpopulation, in Serpine1Tg organoids. A lesser ratio of AT1:AT2 cells in Serpine1Tg organoids was observed compared with wt cultures. There was a significant increase in transepithelial resistance but not amiloride inhibition. Our study suggests elevated PAI-1 in injured lungs downregulates alveolar epithelial regeneration by reducing the AT2 self-renewal, particularly in the CD44+ cells.
PubMed: 36909505
DOI: 10.21203/rs.3.rs-2289657/v1