Effects of furosemide, acetazolamide and amiloride on renal cortical and medullary tissue oxygenation in non-anaesthetised healthy sheep.

It has been proposed that diuretics can improve renal tissue oxygenation through inhibition of tubular sodium reabsorption and reduced metabolic demand. However, the impact of clinically used diuretic drugs on the renal cortical and medullary microcirculation is unclear. Therefore, we examined the effects of three commonly used diuretics, at clinically relevant doses, on renal cortical and medullary perfusion and oxygenation in non-anaesthetised healthy sheep. Merino ewes received acetazolamide (250 mg; n = 9), furosemide (20 mg; n = 10) or amiloride (10 mg; n = 7) intravenously. Systemic and renal haemodynamics, renal cortical and medullary tissue perfusion and P O 2 ${P_{{{\mathrm{O}}_{\mathrm{2}}}}}$ , and renal function were then monitored for up to 8 h post-treatment. The peak diuretic response occurred 2 h (99.4 ± 14.8 mL/h) after acetazolamide, at which stage cortical and medullary tissue perfusion and P O 2 ${P_{{{\mathrm{O}}_{\mathrm{2}}}}}$ were not significantly different from their baseline levels. The peak diuretic response to furosemide occurred at 1 h (196.5 ± 12.3 mL/h) post-treatment but there were no significant changes in cortical and medullary tissue oxygenation during this period. However, cortical tissue P O 2 ${P_{{{\mathrm{O}}_{\mathrm{2}}}}}$ fell from 40.1 ± 3.8 mmHg at baseline to 17.2 ± 4.4 mmHg at 3 h and to 20.5 ± 5.3 mmHg at 6 h after furosemide administration. Amiloride did not produce a diuretic response and was not associated with significant changes in cortical or medullary tissue oxygenation. In conclusion, clinically relevant doses of diuretic agents did not improve regional renal tissue oxygenation in healthy animals during the 8 h experimentation period. On the contrary, rebound renal cortical hypoxia may develop after dissipation of furosemide-induced diuresis.

Role of paraoxonase 3 in regulating ENaC-mediated Na+ transport in the distal nephron.

Paraoxonase 3 (PON3) is expressed in the aldosterone-sensitive distal nephron, where filtered Na+ is reabsorbed mainly via the epithelial Na+ channel (ENaC) and Na+ -coupled co-transporters. We previously showed that PON3 negatively regulates ENaC through a chaperone mechanism. The present study aimed to determine the physiological role of PON3 in renal Na+ and K+ homeostasis. Pon3 knockout (KO) mice had higher amiloride-induced natriuresis and lower plasma [K+ ] at baseline. Single channel recordings in split-open tubules showed that the number of active channels per patch was significantly higher in KO mice, resulting in a higher channel activity in the absence of PON3. Although whole kidney abundance of ENaC subunits was not altered in Pon3 KOs, ENaC gamma subunit was more apically distributed within the connecting tubules and cortical collecting ducts of Pon3 KO kidneys. Additionally, small interfering RNA-mediated knockdown of PON3 in cultured mouse cortical collecting duct cells led to an increased surface abundance of ENaC gamma subunit. As a result of lower plasma [K+ ], sodium chloride co-transporter phosphorylation was enhanced in the KO kidneys, a phenotype that was corrected by a high K+ diet. Finally, PON3 expression was upregulated in mouse kidneys under dietary K+ restriction, potentially providing a mechanism to dampen ENaC activity and associated K+ secretion. Taken together, our results show that PON3 has a role in renal Na+ and K+ homeostasis through regulating ENaC functional expression in the distal nephron. KEY POINTS: Paraoxonase 3 (PON3) is expressed in the distal nephron of mouse kidneys and functions as a molecular chaperone to reduce epithelial Na+ channel (ENaC) expression and activity in heterologous expression systems. We examined the physiological role of PON3 in renal Na+ and K+ handling using a Pon3 knockout (KO) mouse model. At baseline, Pon3 KO mice had lower blood [K+ ], more functional ENaC in connecting tubules/cortical collecting ducts, higher amiloride-induced natriuresis, and enhanced sodium chloride co-transporter (NCC) phosphorylation. Upon challenge with a high K+ diet, Pon3 KO mice had normalized blood [K+ ] and -NCC phosphorylation but lower circulating aldosterone levels compared to their littermate controls. Kidney PON3 abundance was altered in mice under dietary K+ loading or K+ restriction, providing a potential mechanism for regulating ENaC functional expression and renal Na+ and K+ homeostasis in the distal nephron.

Opioid Analgesic as a Positive Allosteric Modulator of Acid-Sensing Ion Channels.

Tafalgin (Taf) is a tetrapeptide opioid used in clinical practice in Russia as an analgesic drug for subcutaneous administration as a solution (4 mg/mL; concentration of 9 mM). We found that the acid-sensing ion channels (ASICs) are another molecular target for this molecule. ASICs are proton-gated sodium channels that mediate nociception in the peripheral nervous system and contribute to fear and learning in the central nervous system. Using electrophysiological methods, we demonstrated that Taf could increase the integral current through heterologically expressed ASIC with half-maximal effective concentration values of 0.09 mM and 0.3 mM for rat and human ASIC3, respectively, and 1 mM for ASIC1a. The molecular mechanism of Taf action was shown to be binding to the channel in the resting state and slowing down the rate of desensitization. Taf did not compete for binding sites with both protons and ASIC3 antagonists, such as APETx2 and amiloride (Ami). Moreover, Taf and Ami together caused an unusual synergistic effect, which was manifested itself as the development of a pronounced second desensitizing component. Thus, the ability of Taf to act as a positive allosteric modulator of these channels could potentially cause promiscuous effects in clinical practice. This fact must be considered in patients' treatment.

Amiloride Reduces Urokinase/Plasminogen-Driven Intratubular Complement Activation in Glomerular Proteinuria.

SIGNIFICANCE STATEMENT: Proteinuria predicts accelerated decline in kidney function in CKD. The pathologic mechanisms are not well known, but aberrantly filtered proteins with enzymatic activity might be involved. The urokinase-type plasminogen activator (uPA)-plasminogen cascade activates complement and generates C3a and C5a in vitro / ex vivo in urine from healthy persons when exogenous, inactive, plasminogen, and complement factors are added. Amiloride inhibits uPA and attenuates complement activation in vitro and in vivo . In conditional podocin knockout (KO) mice with severe proteinuria, blocking of uPA with monoclonal antibodies significantly reduces the urine excretion of C3a and C5a and lowers tissue NLRP3-inflammasome protein without major changes in early fibrosis markers. This mechanism provides a link to proinflammatory signaling in proteinuria with possible long-term consequences for kidney function. BACKGROUND: Persistent proteinuria is associated with tubular interstitial inflammation and predicts progressive kidney injury. In proteinuria, plasminogen is aberrantly filtered and activated by urokinase-type plasminogen activator (uPA), which promotes kidney fibrosis. We hypothesized that plasmin activates filtered complement factors C3 and C5 directly in tubular fluid, generating anaphylatoxins, and that this is attenuated by amiloride, an off-target uPA inhibitor. METHODS: Purified C3, C5, plasminogen, urokinase, and urine from healthy humans were used for in vitro / ex vivo studies. Complement activation was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and ELISA. Urine and plasma from patients with diabetic nephropathy treated with high-dose amiloride and from mice with proteinuria (podocin knockout [KO]) treated with amiloride or inhibitory anti-uPA antibodies were analyzed. RESULTS: The combination of uPA and plasminogen generated anaphylatoxins C3a and C5a from intact C3 and C5 and was inhibited by amiloride. Addition of exogenous plasminogen was sufficient for urine from healthy humans to activate complement. Conditional podocin KO in mice led to severe proteinuria and C3a and C5a urine excretion, which was attenuated reversibly by amiloride treatment for 4 days and reduced by >50% by inhibitory anti-uPA antibodies without altering proteinuria. NOD-, LRR- and pyrin domain-containing protein 3-inflammasome protein was reduced with no concomitant effect on fibrosis. In patients with diabetic nephropathy, amiloride reduced urinary excretion of C3dg and sC5b-9 significantly. CONCLUSIONS: In conditions with proteinuria, uPA-plasmin generates anaphylatoxins in tubular fluid and promotes downstream complement activation sensitive to amiloride. This mechanism links proteinuria to intratubular proinflammatory signaling. In perspective, amiloride could exert reno-protective effects beyond natriuresis and BP reduction. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: Increased Activity of a Renal Salt Transporter (ENaC) in Diabetic Kidney Disease, NCT01918488 and Increased Activity of ENaC in Proteinuric Kidney Transplant Recipients, NCT03036748 .

Development of UTX-143, a selective sodium-hydrogen exchange subtype 5 inhibitor, using amiloride as a lead compound.

NHE5, an isoform of the Na+/H+ exchanger (NHE) protein, is an ion-transporting membrane protein that regulates intracellular pH and is highly expressed in colorectal adenocarcinoma. Therefore, we hypothesized that NHE5 inhibitors can be used as anticancer drugs. However, because NHE1 is ubiquitously expressed in all cells, it is extremely important to demonstrate its selective inhibitory activity against NHE5. We used amiloride, an NHE non-selective inhibitor, as a lead compound and created UTX-143, which has NHE5-selective inhibitory activity, using a structure-activity relationship approach. UTX-143 showed selective cytotoxic effects on cancer cells and reduced the migratory and invasive abilities of cancer cells. These results suggest a new concept wherein drugs exhibit cancer-specific cytotoxic effects through selective inhibition of NHE5 and the possibility of UTX-143 as a lead NHE5-selective inhibitor.

High-fat Western diet alters crystalline silica-induced airway epithelium ion transport but not airway smooth muscle reactivity.

OBJECTIVES: Silicosis is an irreversible occupational lung disease resulting from crystalline silica inhalation. Previously, we discovered that Western diet (HFWD)-consumption increases susceptibility to silica-induced pulmonary inflammation and fibrosis. This study investigated the potential of HFWD to alter silica-induced effects on airway epithelial ion transport and smooth muscle reactivity. METHODS: Six-week-old male F344 rats were fed a HFWD or standard rat chow (STD) and exposed to silica (Min-U-Sil 5®, 15 mg/m3, 6 h/day, 5 days/week, for 39 d) or filtered air. Experimental endpoints were measured at 0, 4, and 8 weeks post-exposure. Transepithelial potential difference (Vt), short-circuit current (ISC) and transepithelial resistance (Rt) were measured in tracheal segments and ion transport inhibitors [amiloride, Na+ channel blocker; NPPB; Cl- channel blocker; ouabain, Na+, K+-pump blocker] identified changes in ion transport pathways. Changes in airway smooth muscle reactivity to methacholine (MCh) were investigated in the isolated perfused trachea preparation. RESULTS: Silica reduced basal ISC at 4 weeks and HFWD reduced the ISC response to amiloride at 0 week compared to air control. HFWD + silica exposure induced changes in ion transport 0 and 4 weeks after treatment compared to silica or HFWD treatments alone. No effects on airway smooth muscle reactivity to MCh were observed.

Single-cell transcriptomics and chromatin accessibility profiling elucidate the kidney-protective mechanism of mineralocorticoid receptor antagonists.

Mineralocorticoid excess commonly leads to hypertension (HTN) and kidney disease. In our study, we used single-cell expression and chromatin accessibility tools to characterize the mineralocorticoid target genes and cell types. We demonstrated that mineralocorticoid effects were established through open chromatin and target gene expression, primarily in principal and connecting tubule cells and, to a lesser extent, in segments of the distal convoluted tubule cells. We examined the kidney-protective effects of steroidal and nonsteroidal mineralocorticoid antagonists (MRAs), as well as of amiloride, an epithelial sodium channel inhibitor, in a rat model of deoxycorticosterone acetate, unilateral nephrectomy, and high-salt consumption-induced HTN and cardiorenal damage. All antihypertensive therapies protected against cardiorenal damage. However, finerenone was particularly effective in reducing albuminuria and improving gene expression changes in podocytes and proximal tubule cells, even with an equivalent reduction in blood pressure. We noted a strong correlation between the accumulation of injured/profibrotic tubule cells expressing secreted posphoprotein 1 (Spp1), Il34, and platelet-derived growth factor subunit b (Pdgfb) and the degree of fibrosis in rat kidneys. This gene signature also showed a potential for classifying human kidney samples. Our multiomics approach provides fresh insights into the possible mechanisms underlying HTN-associated kidney disease, the target cell types, the protective effects of steroidal and nonsteroidal MRAs, and amiloride.

Amiloride alleviates morphine tolerance by suppressing ASIC3-dependent neuroinflammation in the spinal cord.

BACKGROUND: The use of morphine in clinical medicine is severely constrained by tolerance. Therefore, it is essential to examine pharmacological therapies that suppress the development of morphine tolerance. Amiloride suppressed the expression of inflammatory cytokines by inhibiting microglial activation. Microglia play a crucial role in the establishment of morphine tolerance. Thus, we anticipated that amiloride might suppress the development of morphine tolerance. During this investigation, we assessed the impact of amiloride on mouse morphine tolerance. METHODS: Mice received morphine (10 mg/kg, s.c.) twice daily with intrathecally injected amiloride (0.3 µg/5 µl, 1 µg/5 µl, and 3 µg/5 µl) for nine continuous days. To assess morphine tolerance, mice underwent the tail-flick and hot plate tests. BV-2 cells were used to investigate the mechanism of amiloride. By using Western blotting, real-time PCR, and immunofluorescence labeling methods, the levels of acid-sensing ion channels (ASICs), nuclear factor kappa B (NF-kB) p65, p38 mitogen-activated protein kinase (MAPK) proteins, and neuroinflammation-related cytokines were determined. RESULTS: The levels of ASIC3 in the spinal cord were considerably increased after long-term morphine administration. Amiloride was found to delay the development of tolerance to chronic morphine assessed via tail-flick and hot plate tests. Amiloride reduced microglial activation and downregulated the cytokines IL-1ß and TNF-a by inhibiting ASIC3 in response to morphine. Furthermore, amiloride reduced p38 MAPK phosphorylation and inhibited NF-κB expression. CONCLUSIONS: Amiloride effectively reduces chronic morphine tolerance by suppressing microglial activation caused by morphine by inhibiting ASIC3.

Discovery and development of BI 1265162, an ENaC inhibitor for the treatment of cystic fibrosis.

Lung selective inhibition of the endothelial sodium channel (ENaC) is a potential mutation agnostic treatment of Cystic Fibrosis (CF). We describe the discovery and development of BI 1265162, the first ENaC inhibitor devoid of the amiloride structural motif that entered clinical trials. The design of BI 1265162 focused on its suitability for inhalation via the Respimat® Soft Mist™ Inhaler and a long duration of action. A convergent and scalable route for the synthesis of BI 1265162 as dihydrogen phosphate salt is presented, that was applied to support clinical trials. A phase 2 study with BI 1265162 did not provide a clear sign of clinical benefit. Whether ENaC inhibition will be able to hold its promise for CF patients remains an open question.

Study of Amiloride Binding to Human Serum Albumin: Insights from Thermodynamic, Spectroscopic, and Molecular Docking Investigations.

This study was undertaken to investigate the interaction between the sodium channel blocker amiloride (AML) and human serum albumin (HSA). A combination of multi-spectroscopic techniques and computational methods were employed to identify the AML binding site on HSA and the forces responsible for the formation of the HSA-AML complex. Our findings revealed that AML specifically binds to Sudlow's site II, located in subdomain IIIA of HSA, and that the complex formed is stabilized using van der Waals hydrogen-bonding and hydrophobic interactions. FRET analysis showed that the distance between AML and Trp214 was optimal for efficient quenching. UV-Vis spectroscopy and circular dichroism indicated minor changes in the structure of HSA after AML binding, and molecular dynamics simulations (MDS) conducted over 100 ns provided additional evidence of stable HSA-AML-complex formation. This study enhances understanding of the interaction between AML and HSA and the mechanism responsible.

Capsazepine activates amiloride-insensitive ENaC-like channels in human leukemia cells.

Sodium influx carried out by ion channels is one of the main regulators of water-salt and volume balance in cells of blood origin. Previously, we described amiloride-insensitive ENaC-like channels in human myeloid leukemia K562 cells; the intracellular regulatory mechanisms of the channels are associated with actin cytoskeleton dynamics. Recently, an extracellular mechanism of ENaC-like channels activation in K562 cells by the action of serine protease trypsin has been revealed. The other extracellular pathways that modulate ENaC (epithelial Na+ channel) activity and sodium permeability in transformed blood cells are not yet fully investigated. Here, we study the action of capsazepine (CPZ), as δ-ENaC activator, on single channel activity in K562 cells in whole-cell patch clamp experiments. Addition of CPZ (2 µM) to the extracellular solution caused an activation of sodium channels with typical features; unitary conductance was 15.1 ± 0.8 pS. Amiloride derivative benzamil (50 µM) did not inhibit their activity. Unitary currents and conductance of CPZ-activated channels were higher in Na+-containing extracellular solution than in Li+, that is one of the main fingerprints of δ-ENaC. The results of RT-PCR analysis and immunofluorescence staining also confirmed the expression of δ-hENaC (as well as α-, ß-, γ-ENaC) at the mRNA and protein level. These findings allow us to speculate that CPZ activates amiloride-insensitive ENaC-like channels that contain δ-ENaC in К562 cells. Our data reveal a novel extracellular mechanism for ENaC-like activation in human leukemia cells.

Enhanced harm detection following maternal separation: Transgenerational transmission and reversibility by inhaled amiloride.

Early-life adversities are associated with altered defensive responses. Here, we demonstrate that the repeated cross-fostering (RCF) paradigm of early maternal separation is associated with enhancements of distinct homeostatic reactions: hyperventilation in response to hypercapnia and nociceptive sensitivity, among the first generation of RCF-exposed animals, as well as among two successive generations of their normally reared offspring, through matrilineal transmission. Parallel enhancements of acid-sensing ion channel 1 (ASIC1), ASIC2, and ASIC3 messenger RNA transcripts were detected transgenerationally in central neurons, in the medulla oblongata, and in periaqueductal gray matter of RCF-lineage animals. A single, nebulized dose of the ASIC-antagonist amiloride renormalized respiratory and nociceptive responsiveness across the entire RCF lineage. These findings reveal how, following an early-life adversity, a biological memory reducible to a molecular sensor unfolds, shaping adaptation mechanisms over three generations. Our findings are entwined with multiple correlates of human anxiety and pain conditions and suggest nebulized amiloride as a therapeutic avenue.

Mice lacking γENaC palmitoylation sites maintain benzamil-sensitive Na+ transport despite reduced channel activity.

Epithelial Na+ channels (ENaCs) control extracellular fluid volume by facilitating Na+ absorption across transporting epithelia. In vitro studies showed that Cys-palmitoylation of the γENaC subunit is a major regulator of channel activity. We tested whether γ subunit palmitoylation sites are necessary for channel function in vivo by generating mice lacking the palmitoylated cysteines (γC33A,C41A) using CRISPR/Cas9 technology. ENaCs in dissected kidney tubules from γC33A,C41A mice had reduced open probability compared with wild-type (WT) littermates maintained on either standard or Na+-deficient diets. Male mutant mice also had higher aldosterone levels than WT littermates following Na+ restriction. However, γC33A,C41A mice did not have reduced amiloride-sensitive Na+ currents in the distal colon or benzamil-induced natriuresis compared to WT mice. We identified a second, larger conductance cation channel in the distal nephron with biophysical properties distinct from ENaC. The activity of this channel was higher in Na+-restricted γC33A,C41A versus WT mice and was blocked by benzamil, providing a possible compensatory mechanism for reduced prototypic ENaC function. We conclude that γ subunit palmitoylation sites are required for prototypic ENaC activity in vivo but are not necessary for amiloride/benzamil-sensitive Na+ transport in the distal nephron or colon.

Hexamethylene amiloride binds the SARS-CoV-2 envelope protein at the protein-lipid interface.

The SARS-CoV-2 envelope (E) protein forms a five-helix bundle in lipid bilayers whose cation-conducting activity is associated with the inflammatory response and respiratory distress symptoms of COVID-19. E channel activity is inhibited by the drug 5-(N,N-hexamethylene) amiloride (HMA). However, the binding site of HMA in E has not been determined. Here we use solid-state NMR to measure distances between HMA and the E transmembrane domain (ETM) in lipid bilayers. 13 C, 15 N-labeled HMA is combined with fluorinated or 13 C-labeled ETM. Conversely, fluorinated HMA is combined with 13 C, 15 N-labeled ETM. These orthogonal isotopic labeling patterns allow us to conduct dipolar recoupling NMR experiments to determine the HMA binding stoichiometry to ETM as well as HMA-protein distances. We find that HMA binds ETM with a stoichiometry of one drug per pentamer. Unexpectedly, the bound HMA is not centrally located within the channel pore, but lies on the lipid-facing surface in the middle of the TM domain. This result suggests that HMA may inhibit the E channel activity by interfering with the gating function of an aromatic network. These distance data are obtained under much lower drug concentrations than in previous chemical shift perturbation data, which showed the largest perturbation for N-terminal residues. This difference suggests that HMA has higher affinity for the protein-lipid interface than the channel pore. These results give insight into the inhibition mechanism of HMA for SARS-CoV-2 E.

Amiloride evokes significant natriuresis and weight loss in kidney transplant recipients with and without albuminuria.

Albuminuria in kidney transplant recipients (KTRs) is associated with hypertension and aberrant glomerular filtration of serine proteases that may proteolytically activate the epithelial Na+ channel (ENaC). The present nonrandomized, pharmacodynamic intervention study aimed to investigate if inhibition of ENaC increases Na+ excretion and reduces extracellular volume in KTRs dependent on the presence of albuminuria. KTRs with and without albuminuria (albumin-to-creatinine ratio > 300 mg/g, n = 7, and <30 mg/g, n = 7, respectively) were included and ingested a diet with fixed Na+ content (150 mmol/day) for 5 days. On the last day, amiloride at 10 mg was administered twice. Body weight, 24-h urine electrolyte excretion, body water content, and ambulatory blood pressure as well as plasma renin, angiotensin II, and aldosterone concentrations were determined before and after amiloride. Amiloride led to a significant decrease in body weight, increase in 24-h urinary Na+ excretion, and decrease in 24-h urinary K+ excretion in both groups. Urine output increased in the nonalbuminuric group only. There was no change in plasma renin, aldosterone, and angiotensin II concentrations after amiloride, whereas a significant decrease in nocturnal systolic blood pressure and increase in 24-h urine aldosterone excretion was observed in albuminuric KTRs only. There was a significant correlation between 24-h urinary albumin excretion and amiloride-induced 24-h urinary Na+ excretion. In conclusion, ENaC activity contributes to Na+ and water retention in KTRs with and without albuminuria. ENaC is a relevant pharmacological target in KTRs; however, larger and long-term studies are needed to evaluate whether the magnitude of this effect depends on the presence of albuminuria.NEW & NOTEWORTHY Amiloride has a significant natriuretic effect in kidney transplant recipients (KTRs) that relates to urinary albumin excretion. The epithelial Na+ channel may be a relevant direct pharmacological target to counter Na+ retention and hypertension in KTRs. Epithelial Na+ channel blockers should be further investigated as a mean to mitigate Na+ and water retention and to potentially obtain optimal blood pressure control in KTRs.

Effects of chlorthalidone plus amiloride compared with amlodipine on short-term blood pressure variability in individuals with hypertension and obstructive sleep apnea: a randomized controlled trial.

OBJECTIVE: To compare the effects of chlortalidone plus amiloride and amlodipine on blood pressure (BP) variability in patients with hypertension and obstructive sleep apnea syndrome (OSA). METHODS: A randomized, controlled, double-blind trial enrolled men and women aged 40 years or older with a diagnosis of OSA (apnea-hypopnea index 10-40 apneas/h of sleep) confirmed by overnight laboratory polysomnography and systolic BP 140-159 mmHg or diastolic BP 90-99 mmHg. Participants were randomized to receive chlortalidone 25 mg plus amiloride 5 mg daily or amlodipine 10 mg daily for 8 weeks. BP variability was calculated from 24-hour ambulatory BP monitoring at baseline and follow-up using the following indices: SD, coefficient of variation, average real variability (ARV), time-rate index, and variability independent of the mean (VIM). RESULTS: The study included 65 patients, with 33 assigned to the chlortalidone plus amiloride group and 32 to the amlodipine group. Participants in both groups had similar baseline characteristics. Short-term BP variability decreased within groups for SD and ARV indexes for 24-hour systolic BP and daytime systolic BP, but statistically significant time*group interactions were found for sleep systolic SD and VIM, with greater reduction in patients treated with amlodipine. CONCLUSION: In brief, our study has shown that the use of chlorthalidone in combination with amiloride and amlodipine produces comparable effects on short-term BP variability in patients with hypertension and OSA. Therefore, our findings suggest that BP variability may not be a significant factor when choosing between these medications for the treatment of hypertension and OSA.

Automated Patch Clamp Screening of Amiloride and 5-N,N-Hexamethyleneamiloride Analogs Identifies 6-Iodoamiloride as a Potent Acid-Sensing Ion Channel Inhibitor.

Acid-sensing ion channels (ASICs) are transmembrane sensors of extracellular acidosis and potential drug targets in several disease indications, including neuropathic pain and cancer metastasis. The K+-sparing diuretic amiloride is a moderate nonspecific inhibitor of ASICs and has been widely used as a probe for elucidating ASIC function. In this work, we screened a library of 6-substituted and 5,6-disubstituted amiloride analogs using a custom-developed automated patch clamp protocol and identified 6-iodoamiloride as a potent ASIC1 inhibitor. Follow-up IC50 determinations in tsA-201 cells confirmed higher ASIC1 inhibitory potency for 6-iodoamiloride 94 (hASIC1 94 IC50 = 88 nM, cf. amiloride 11 IC50 = 1.7 µM). A similar improvement in activity was observed in ASIC3-mediated currents from rat dorsal root ganglion neurons (rDRG single-concentration 94 IC50 = 230 nM, cf. 11 IC50 = 2.7 µM). 6-Iodoamiloride represents the amiloride analog of choice for studying the effects of ASIC inhibition on cell physiology.

The Epithelial Sodium Channel-An Underestimated Drug Target.

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.

Antifungal activity of 6-substituted amiloride and hexamethylene amiloride (HMA) analogs.

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-(N,N-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 Cryptococcus neoformans were identified. Hits from these series showed broad-spectrum activity against both basidiomycete and ascomycete fungal pathogens, including multidrug-resistant clinical isolates.

Resveratrol downregulates ENaCs through the activation of AMPK in human colon cancer cells.

Epithelial sodium channels (ENaCs) are critically engaged in a number of hallmarks of cancer progression, including proliferation, migration, invasion and apoptosis. Thus, the inhibition of ENaCs possesses therapeutic potential in cancer. Resveratrol, a natural polyphenol with anti-carcinogenic activity, is a potent activator of 5' AMP-activated protein kinase (AMPK) which reduces the abundance of ENaCs in the cell membrane by causing the internalization of the ß subunit. However, the effect of resveratrol on ENaCs in cancer cells is unknown. Therefore, in this study, we aimed to investigate the effects of resveratrol on ENaCs in human colon cancer cells HCT116 and HT29. The influences of resveratrol either alone or together with AMPK inhibitor compound C (CC), and ENaC inhibitor amiloride on cell viability were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The expression levels of phospho-AMPK Thr172 and ßENaC in cells were determined by immunofluorescence staining, and the expression of apoptotic markers Caspase-3 and Caspase-9 were analyzed by qRT-PCR. Resveratrol was found to activate AMPK in a dose-dependent manner. Both AMPK activation by resveratrol and ENaC inhibition by amiloride decreased cell viability and increased apoptosis significantly. AMPK activation also reduced ßENaC expression in cells. Our results suggest that ENaC inhibition through AMPK activation might be a potential mechanism underlying the anti-cancer effects of resveratrol.