Effect of antihypertensive agents on sleep apnea and ambulatory blood pressure in patients with hypertension: A randomized controlled trial.

BACKGROUND: Obstructive sleep apnea (OSA) and hypertension are common conditions that may be linked through sympathetic activation and water retention. We hypothesized that diuretics, which reduce the body water content, may be more effective than amlodipine, a blood pressure (BP)-lowering agent implicated with edema, in controlling OSA in patients with hypertension. We also aimed to compare the effects of these treatments on ambulatory blood pressure monitoring (ABPM). METHODS: In a randomized, double-blind clinical trial, we compared the effects of chlorthalidone/amiloride 25/5 mg with amlodipine 10 mg on OSA measured by portable sleep monitor and BP measured by ABPM. The study included participants older than 40 who had moderate OSA (10-40 apneas/hour of sleep) and BP within the systolic range of 140-159 mmHg or diastolic range of 90-99 mmHg. RESULTS: The individuals in the experimental groups were comparable in age, gender, and other relevant characteristics. Neither the combination of diuretics nor amlodipine alone reduced the AHI after 8 weeks of treatment (AHI 26.3 with diuretics and 25.0 with amlodipine. P = 0.713). Both treatments significantly lowered office, 24-h, and nighttime ABP, but the two groups had no significant difference. CONCLUSION: Chlorthalidone associated with amiloride and amlodipine are ineffective in decreasing the frequency of sleep apnea episodes in patients with moderate OSA and hypertension. Both treatments have comparable effects in lowering both office and ambulatory blood pressure. The notion that treatments could offer benefits for both OSA and hypertension remains to be demonstrated. TRIAL REGISTRATION CLINICALTRIALS. GOV IDENTIFIER: NCT01896661.

Dynamic measurement of airway surface liquid volume with an ex vivo trachea-chip.

The volume and composition of airway surface liquid (ASL) is regulated by liquid secretion and absorption across airway epithelia, controlling the pH, solute concentration, and biophysical properties of ASL in health and disease. Here, we developed a method integrating explanted tracheal tissue with a micro-machined device (referred to as "ex vivo trachea-chip") to study the dynamic properties of ASL volume regulation. The ex vivo trachea-chip allows real-time measurement of ASL transport (Jv) with intact airway anatomic structures, environmental control, high-resolution, and enhanced experimental throughput. Applying this technology to freshly excised tissue we observed ASL absorption under basal conditions. The apical application of amiloride, an inhibitor of airway epithelial sodium channels (ENaC), reduced airway liquid absorption. Furthermore, the basolateral addition of NPPB, a Cl- channel inhibitor, reduced the basal rate of ASL absorption, implicating a role for basolateral Cl- channels in ASL volume regulation. When tissues were treated with apical amiloride and basolateral methacholine, a cholinergic agonist that stimulates secretion from airway submucosal glands, the net airway surface liquid production shifted from absorption to secretion. This ex vivo trachea-chip provides a new tool to investigate ASL transport dynamics in pulmonary disease states and may aid the development of new therapies targeting ASL regulation.

Amiloride lowers plasma TNF and interleukin-6 but not interleukin-17A in patients with hypertension and type 2 diabetes.

Interleukin (IL)-17A contributes to hypertension in preclinical models. T helper 17 and dendritic cells are activated by NaCl, which could involve the epithelial Na+ channel (ENaC). We hypothesized that the ENaC blocker amiloride reduces plasma IL-17A and related cytokines in patients with hypertension. Concentrations of IL-17A, IFN-γ, TNF, IL-6, IL-1ß, and IL-10 were determined by immunoassays in plasma from two patient cohorts before and after amiloride treatment: 1) patients with type 2 diabetes mellitus (T2DM) and treatment-resistant hypertension (n = 69, amiloride 5-10 mg/day for 8 wk) and 2) patients with hypertension and type 1 diabetes mellitus (T1DM) (n = 29) on standardized salt intake (amiloride 20-40 mg/day, 2 days). Plasma and tissue from ANG II-hypertensive mice with T1DM treated with amiloride (2 mg/kg/day, 4 days) were analyzed. The effect of amiloride and benzamil on macrophage cytokines was determined in vitro. Plasma cytokines showed higher concentrations (IL-17A ∼40-fold) in patients with T2DM compared with T1DM. In patients with T2DM, amiloride had no effect on IL-17A but lowered TNF and IL-6. In patients with T1DM, amiloride had no effect on IL-17A but increased TNF. In both cohorts, blood pressure decline and plasma K+ increase did not relate to plasma cytokine changes. In mice, amiloride exerted no effect on IL-17A in the plasma, kidney, aorta, or left cardiac ventricle but increased TNF in cardiac and kidney tissues. In lipopolysaccharide-stimulated human THP-1 macrophages, amiloride and benzamil (from 1 nmol/L) decreased TNF, IL-6, IL-10, and IL-1ß. In conclusion, inhibition of ENaC by amiloride reduces proinflammatory cytokines TNF and IL-6 but not IL-17A in patients with T2DM, potentially by a direct action on macrophages.NEW & NOTEWORTHY ENaC activity may contribute to macrophage-derived cytokine release, since amiloride exerts anti-inflammatory effects by suppression of TNF and IL-6 cytokines in patients with resistant hypertension and type 2 diabetes and in THP-1-derived macrophages in vitro.

Accelerated Stability Assessment of Extemporaneously Compounded Amiloride Nasal Spray.

Amiloride is a U.S. Food and Drug Administration-approved diuretic agent used to treat hypertension and congestive heart failure. Recent human and animal studies have suggested that amiloride may also have a role in treating anxiety through its acid-sensing ion channel antagonism. Intranasal administration of amiloride nasal spray via an extemporaneously compounded preparation has the potential for rapid delivery to the site of action to achieve therapeutic outcomes in individual patients with anxiety disorders. However, these patient-specific preparations do not have the pre-formulation characterization, including chemical stability, that conventional manufactured dosage forms have. The objective of this study was to assess the estimated chemical stability of compounded amiloride nasal spray over 6 months and 12 months utilizing accelerated degradation with high heat and the Arrhenius equation. A stability-indicating highperformance liquid chromatography analytical method was employed at appropriate intervals over a 12-month period to reveal that amiloride remained chemically stable over the period tested and by extrapolation. Physical stability and compatibility with the preservative benzyl alcohol were also confirmed via visual inspection, pH monitoring, and measurement of turbidity.

Influence of proteolytic cleavage of ENaC's γ subunit upon Na+ and K+ handling.

The epithelial Na+ channel (ENaC) γ subunit is essential for homeostasis of Na+, K+, and body fluid. Dual γ subunit cleavage before and after a short inhibitory tract allows dissociation of this tract, increasing channel open probability (PO), in vitro. Cleavage proximal to the tract occurs at a furin recognition sequence (143RKRR146, in the mouse γ subunit). Loss of furin-mediated cleavage prevents in vitro activation of the channel by proteolysis at distal sites. We hypothesized that 143RKRR146 mutation to 143QQQQ146 (γQ4) in 129/Sv mice would reduce ENaC PO, impair flow-stimulated flux of Na+ (JNa) and K+ (JK) in perfused collecting ducts, reduce colonic amiloride-sensitive short-circuit current (ISC), and impair Na+, K+, and body fluid homeostasis. Immunoblot of γQ4/Q4 mouse kidney lysates confirmed loss of a band consistent in size with the furin-cleaved proteolytic fragment. However, γQ4/Q4 male mice on a low Na+ diet did not exhibit altered ENaC PO or flow-induced JNa, though flow-induced JK modestly decreased. Colonic amiloride-sensitive ISC in γQ4/Q4 mice was not altered. γQ4/Q4 males, but not females, exhibited mildly impaired fluid volume conservation when challenged with a low Na+ diet. Blood Na+ and K+ were unchanged on a regular, low Na+, or high K+ diet. These findings suggest that biochemical evidence of γ subunit cleavage should not be used in isolation to evaluate ENaC activity. Furthermore, factors independent of γ subunit cleavage modulate channel PO and the influence of ENaC on Na+, K+, and fluid volume homeostasis in 129/Sv mice, in vivo.NEW & NOTEWORTHY The epithelial Na+ channel (ENaC) is activated in vitro by post-translational proteolysis. In vivo, low Na+ or high K+ diets enhance ENaC proteolysis, and proteolysis is hypothesized to contribute to channel activation in these settings. Using a mouse expressing ENaC with disruption of a key proteolytic cleavage site, this study demonstrates that impaired proteolytic activation of ENaC's γ subunit has little impact upon channel open probability or the ability of mice to adapt to low Na+ or high K+ diets.

4-(Azolyl)-Benzamidines as a Novel Chemotype for ASIC1a Inhibitors.

Acid-sensing ion channels (ASICs) play a key role in the perception and response to extracellular acidification changes. These proton-gated cation channels are critical for neuronal functions, like learning and memory, fear, mechanosensation and internal adjustments like synaptic plasticity. Moreover, they play a key role in neuronal degeneration, ischemic neuronal injury, seizure termination, pain-sensing, etc. Functional ASICs are homo or heterotrimers formed with (ASIC1-ASIC3) homologous subunits. ASIC1a, a major ASIC isoform in the central nervous system (CNS), possesses an acidic pocket in the extracellular region, which is a key regulator of channel gating. Growing data suggest that ASIC1a channels are a potential therapeutic target for treating a variety of neurological disorders, including stroke, epilepsy and pain. Many studies were aimed at identifying allosteric modulators of ASIC channels. However, the regulation of ASICs remains poorly understood. Using all available crystal structures, which correspond to different functional states of ASIC1, and a molecular dynamics simulation (MD) protocol, we analyzed the process of channel inactivation. Then we applied a molecular docking procedure to predict the protein conformation suitable for the amiloride binding. To confirm the effect of its sole active blocker against the ASIC1 state transition route we studied the complex with another MD simulation run. Further experiments evaluated various compounds in the Enamine library that emerge with a detectable ASIC inhibitory activity. We performed a detailed analysis of the structural basis of ASIC1a inhibition by amiloride, using a combination of in silico approaches to visualize its interaction with the ion pore in the open state. An artificial activation (otherwise, expansion of the central pore) causes a complex modification of the channel structure, namely its transmembrane domain. The output protein conformations were used as a set of docking models, suitable for a high-throughput virtual screening of the Enamine chemical library. The outcome of the virtual screening was confirmed by electrophysiological assays with the best results shown for three hit compounds.

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.

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.

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.

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 .

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.

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.

Mouse TMC4 is involved in the detection of chloride taste of salts.

Licking behavior with various salts in transmembrane channel-like 4 (Tmc4) knockout (KO) mice was observed. In Tmc4 KO mice, a significant decrease in sensitivity to chloride salts, such as NaCl, KCl, and NH4Cl, was observed, while no significant decrease in sensitivity to Na-gluconate was observed. This finding suggests that TMC4 may be involved in the detection of chloride taste.

Hereditary causes of hypertension due to increased sodium transport.

PURPOSE OF REVIEW: Hypertension, commonly known as high blood pressure, is a widespread health condition affecting a large number of individuals across the globe. Although lifestyle choices and environmental factors are known to have a significant impact on its development, there is growing recognition of the influence of genetic factors in the pathogenesis of hypertension. This review specifically focuses on the hereditary causes of hypertension that are associated with increased sodium transport through the thiazide-sensitive NaCl cotransporter (NCC) or amiloride-sensitive epithelial sodium channel (ENaC), crucial mechanisms involved in regulating blood pressure in the kidneys. By examining genetic mutations and signaling molecules linked to the dysregulation of sodium transport, this review aims to deepen our understanding of the hereditary causes of hypertension and shed light on potential therapeutic targets. RECENT FINDINGS: Liddle syndrome (LS) is a genetic disorder that typically manifests early in life and is characterized by hypertension, hypokalemic metabolic alkalosis, hyporeninemia, and suppressed aldosterone secretion. This condition is primarily caused by gain-of-function mutations in ENaC. In contrast, Pseudohypoaldosteronism type II (PHAII) is marked by hyperkalemia and hypertension, alongside other clinical features such as hyperchloremia, metabolic acidosis, and suppressed plasma renin levels. PHAII results from overactivations of NCC, brought about by gain-of-function mutations in its upstream signaling molecules, including WNK1 (with no lysine (K) 1), WNK4, Kelch-like 3 (KLHL3), and cullin3 (CUL3). SUMMARY: NCC and ENaC are integral components, and their malfunctions lead to disorders like LS and PHAII, hereditary causes of hypertension. Current treatments for LS involve ENaC blockers (e.g., triamterene and amiloride) in conjunction with low-sodium diets, effectively normalizing blood pressure and potassium levels. In PHAII, thiazide diuretics, which inhibit NCC, are the mainstay treatment, albeit with some limitations and potential side effects. Ongoing research in developing alternative treatments, including small molecules targeting key regulators, holds promise for more effective and tailored hypertension solutions.

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.

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.

The role of acid-sensing ion channels in monosodium urate-induced gouty pain in mice.

Acid-sensing ion channels (ASICs) in dorsal root ganglion (DRG) neurons play an important role in inflammatory pain. The objective of this study is to observe the regulatory role of ASICs in monosodium urate (MSU) crystal-induced gout pain and explore the basis for ASICs in DRG neurons as a target for gout pain treatment. The gout arthritis model was induced by injecting MSU crystals into the ankle joint of mice. The circumference of the ankle joint was used to evaluate the degree of swelling; the von Frey filaments were used to determine the withdrawal threshold of the paw. ASIC currents and action potentials (APs) were recorded by patch clamp technique in DRG neurons. The results displayed that injecting MSU crystals caused ankle edema and mechanical hyperalgesia of the paw, which was relieved after amiloride treatment. The ASIC currents in DRG neurons were increased to a peak on the second day after injecting MSU crystals, which were decreased after amiloride treatment. MSU treatment increased the current density of ASICs in different diameter DRG cells. MSU treatment does not change the characteristics of AP. The results suggest that ASICs in DRG neurons participate in MSU crystal-induced gout pain.

ATP-mediated increase in H+ efflux from retinal Müller cells of the axolotl.

Previous work has shown that activation of tiger salamander retinal radial glial cells by extracellular ATP induces a pronounced extracellular acidification, which has been proposed to be a potent modulator of neurotransmitter release. This study demonstrates that low micromolar concentrations of extracellular ATP similarly induce significant H+ effluxes from Müller cells isolated from the axolotl retina. Müller cells were enzymatically isolated from axolotl retina and H+ fluxes were measured from individual cells using self-referencing H+-selective microelectrodes. The increased H+ efflux from axolotl Müller cells induced by extracellular ATP required activation of metabotropic purinergic receptors and was dependent upon calcium released from internal stores. We further found that the ATP-evoked increase in H+ efflux from Müller cells of both tiger salamander and axolotl were sensitive to pharmacological agents known to interrupt calmodulin and protein kinase C (PKC) activity: chlorpromazine (CLP), trifluoperazine (TFP), and W-7 (all calmodulin inhibitors) and chelerythrine, a PKC inhibitor, all attenuated ATP-elicited increases in H+ efflux. ATP-initiated H+ fluxes of axolotl Müller cells were also significantly reduced by amiloride, suggesting a significant contribution by sodium-hydrogen exchangers (NHEs). In addition, α-cyano-4-hydroxycinnamate (4-cin), a monocarboxylate transport (MCT) inhibitor, also reduced the ATP-induced increase in H+ efflux in both axolotl and tiger salamander Müller cells, and when combined with amiloride, abolished ATP-evoked increase in H+ efflux. These data suggest that axolotl Müller cells are likely to be an excellent model system to understand the cell-signaling pathways regulating H+ release from glia and the role this may play in modulating neuronal signaling.NEW & NOTEWORTHY Glial cells are a key structural part of the tripartite synapse and have been suggested to regulate synaptic transmission, but the regulatory mechanisms remain unclear. We show that extracellular ATP, a potent glial cell activator, induces H+ efflux from axolotl retinal Müller (glial) cells through a calcium-dependent pathway that is likely to involve calmodulin, PKC, Na+/H+ exchange, and monocarboxylate transport, and suggest that such H+ release may play a key role in modulating neuronal transmission.

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.

Increased ENaC-mediated liquid absorption across vitamin-D deficient human airway epithelia.

Vitamin D deficiency is a risk factor for exacerbation of obstructive airway disease, a hallmark of which is mucus dehydration and plugging. Calcitriol (the active form of vitamin D) deficiency in cultured human airway epithelia resulted in increased SCNN1G and ATP1B1 mRNAs encoding subunits of ENaC and the Na-K pump compared with supplemented epithelia. These drive the absorption of airway surface liquid. Consistently, calcitriol-deficient epithelia absorbed liquid faster than supplemented epithelia. Calcitriol deficiency also increased amiloride-sensitive Isc and Gt without altering Na-K pump activity, indicating the changes in amiloride-sensitivity arose from ENaC. ENaC activity can be regulated by trafficking, proteases, and channel abundance. We found the effect was likely not induced by changes to endocytosis of ENaC given that calcitriol did not affect the half-lives of amiloride-sensitive Isc and Gt. Furthermore, trypsin nominally increased Isc produced by epithelia ± calcitriol, suggesting calcitriol did not affect proteolytic activation of ENaC. Consistent with mRNA and functional data, calcitriol deficiency resulted in increased γENaC protein. These data indicate that the vitamin D receptor response controls ENaC function and subsequent liquid absorption, providing insight into the relationship between vitamin D deficiency and respiratory disease.NEW & NOTEWORTHY It is unknown why calcitriol (active vitamin D) deficiency worsens pulmonary disease outcomes. Results from mRNA, immunoblot, Ussing chamber, and absorption experiments indicate that calcitriol deficiency increases ENaC activity in human airway epithelia, decreasing apical hydration. Given that epithelial hydration is required for mucociliary transport and airway innate immune function, the increased ENaC activity observed in calcitriol-deficient epithelia may contribute to respiratory pathology observed in vitamin D deficiency.