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Introduccion: In nephrotic syndrome, increased podocyturia accompanies pathologic proteinuria. The therapeutic regimen with enalapril, losartan and amiloride could reduce both variables., Objetives: Evaluate the anti-proteinuric effect of 2 non-immunological therapeutic regimens, the quantitative relationship between podocyturia and proteinuria., Material and Methods: We included children aged 4 to 12 years with corticoresistant nephrotic syndrome, using 2 different schemes: group A, enalapril+losartan, and group B, enalapril+losartan+amiloride., Results: In group A, 17 patients completed the study, the initial mean proteinuria was 39mg/m 2 /h and mean proteinuria at the end was 24mg/m 2 /h, while in group B 14 patients were treated and the initial average proteinuria was 36mg/m 2 /h and the end average proteinuria was 13mg/m 2 /h. The paired T test showed significant differences in the decrease in proteinuria, for patients in group B without variation in podocyturia. The 2 factors associated with an increase in proteinuria were podocyturia and the time elapsed from the diagnosis of cortico-resistant nephrotic syndrome to the start of treatment anti-proteinuric., Conclusions: The use of amiloride decreased proteinuria, without significantly modifying podocyturia; we did not observe a positive relationship between both variables., (Copyright © 2021 Sociedad Española de Nefrología. Published by Elsevier España, S.L.U. All rights reserved.)

Acidosis in the brain plays an important role in neuronal injury and is a common feature of several neurological diseases. It has been reported that the sodium-hydrogen exchanger-1 (NHE-1) is a key mediator of acidosis-induced neuronal injury. It modulates the concentration of intra- and extra-cellular sodium and hydrogen ions. During the ischemic state, excessive sodium ions enter neurons and inappropriately activate the sodium-calcium exchanger (NCX). Zinc can also enter neurons through voltage-gated calcium channels and NCX. Here, we tested the hypothesis that zinc enters the intracellular space through NCX and the subsequent zinc accumulation induces neuronal cell death after global cerebral ischemia (GCI). Thus, we conducted the present study to confirm whether inhibition of NHE-1 by amiloride attenuates zinc accumulation and subsequent hippocampus neuronal death following GCI. Mice were subjected to GCI by bilateral common carotid artery (BCCA) occlusion for 30 min, followed by restoration of blood flow and resuscitation. Amiloride (10 mg/kg, intraperitoneally ( i.p. )) was immediately injected, which reduced zinc accumulation and neuronal death after GCI. Therefore, the present study demonstrates that amiloride attenuates GCI-induced neuronal injury, likely via the prevention of intracellular zinc accumulation. Consequently, we suggest that amiloride may have a high therapeutic potential for the prevention of GCI-induced neuronal death.

Background: Distal diuretics are considered less effective than loop diuretics in CKD. However, data to support this perception are limited., Methods: To investigate whether distal diuretics are noninferior to dietary sodium restriction in reducing BP in patients with CKD stage G3 or G4 and hypertension, we conducted a 6-week, randomized, open-label crossover trial comparing amiloride/hydrochlorothiazide (5 mg/50 mg daily) with dietary sodium restriction (60 mmol per day). Antihypertension medication was discontinued for a 2-week period before randomization. We analyzed effects on BP, kidney function, and fluid balance and related this to renal clearance of diuretics., Results: A total of 26 patients (with a mean eGFR of 39 ml/min per 1.73 m 2 ) completed both treatments. Dietary sodium restriction reduced sodium excretion from 160 to 64 mmol per day. Diuretics produced a greater reduction in 24-hour systolic BP (SBP; from 138 to 124 mm Hg) compared with sodium restriction (from 134 to 129 mm Hg), as well as a significantly greater effect on extracellular water, eGFR, plasma renin, and aldosterone. Both interventions resulted in a similar decrease in body weight and NT-proBNP. Neither approaches decreased albuminuria significantly, whereas diuretics did significantly reduce urinary angiotensinogen and β 2-microglobulin excretion. Although lower eGFR and higher plasma indoxyl sulfate correlated with lower diuretic clearance, the diuretic effects on body weight and BP at lower eGFR were maintained. During diuretic treatment, higher PGE2 excretion correlated with lower free water clearance, and four patients developed mild hyponatremia., Conclusions: Distal diuretics are noninferior to dietary sodium restriction in reducing BP and extracellular volume in CKD. Diuretic sensitivity in CKD is maintained despite lower diuretic clearance., Clinical Trial Registry Name and Registration Number: DD-study: Diet or Diuretics for Salt-sensitivity in Chronic Kidney Disease (DD), NCT02875886., (Copyright © 2020 by the American Society of Nephrology.)

Background: Thiazide diuretics have demonstrated favorable blood pressure lowering efficacy, but the equivalent doses of their more common agents, chlorthalidone and hydrochlorothiazide, are still unclear. Further, concerns exist regarding adverse metabolic effects, which may be attenuated with the concomitant administration of a potassium-sparing diuretic, such as amiloride. This trial aims to investigate the efficacy of chlorthalidone and hydrochlorothiazide, in combination with amiloride at different doses, for initial management of patients with primary hypertension., Methods/design: This is a factorial (2 × 2) randomized double-blinded clinical trial comparing the association of a thiazide diuretic (chlorthalidone 25 mg/day or hydrochlorothiazide 50 mg/day) with a potassium-sparing diuretic (amiloride 10 mg/day or amiloride 20 mg/day) in patients with primary hypertension. The primary outcome will be the mean change from baseline in 24-h systolic and diastolic blood pressure measured by ambulatory blood pressure monitoring. The secondary outcomes will be the mean change from baseline in daytime and nighttime systolic and diastolic blood pressure measured by ambulatory blood pressure monitoring, mean change from baseline in systolic and diastolic blood pressure measured by office blood pressure, incidence of adverse events, variation of laboratory parameters, and proportion of patients who achieved blood pressure control. The follow-up will last 12 weeks. For a P alpha of 0.05, power of 80%, standard deviation of 9 mmHg, and absolute difference of 6 mmHg on systolic blood pressure on 24-h ambulatory blood pressure monitoring, it will be necessary to study a total of 76 patients. The sample size will be increased by 10% to compensate for losses, resulting in 84 patients being randomized., Discussion: Diuretics are pivotal drugs for the treatment of hypertension. Chlorthalidone and hydrochlorothiazide, in combination with amiloride in multiple doses, will be tested in terms of blood pressure lowering efficacy and safety. Since the intensity of blood pressure reduction is the major determinant of reduction in cardiovascular risk in hypertensive patients, this study will help to determine which combination of diuretics represents the most appropriate treatment for this population., Trial Registration: ClinicalTrials.gov, NCT03928145. Registered on 25 April 2019. Last update on 29 April 2019.

Whether aldosterone itself contributes directly to macro- or microcirculatory disease in man or to adverse cardiovascular outcomes is not fully known. We report our long-term single-practice experience in 5 patients with chronic hyperaldosteronism (HA, including 3 with glucocorticoid remediable aldosteronism, GRA) treated with low-dose amiloride (a specific epithelial sodium channel [ENaC] blocker) 5-10 (mean 7) mg daily for 14-28 (mean 20) years. Except for 1 GRA diagnosed in infancy, all had severe resistant hypertension. In each case, BP was normal or near-normal within 1-4 weeks after starting amiloride and office BP's were well controlled for 20 years thereafter. Vascular studies and 24-hour ambulatory BP monitoring with pulse wave analysis (cardiac output, vascular resistance, augmentation index, and reflection magnitude) were assessed after a mean of 18 years as were regional pulse wave velocities, pulse stiffening ratio, ankle-brachial index, serum creatinine, estimated glomerular filtration rate, and spot urinary albumin:creatinine ratio. All indicators were completely normal in all patients after 18 years of amiloride, and none had a cardiovascular event during the 20-year mean follow-up. We conclude that long-term ENaC blockade can normalize BP and protect macro- and microvascular function in patients with HA. This suggests that (a) any vasculopathic effects of aldosterone are mediated via ENaC, not MR activation itself, and are fully preventable or reversible with ENaC blockade or (b) aldosterone may not play a major BP-independent role in human macro- and microcirculatory diseases. These and other widely divergent results in the literature underscore the need for additional studies regarding aldosterone, ENaC, and vascular disease., (©2019 Wiley Periodicals, Inc.)

Background: Osteosarcoma (OSA) is a common bone tumor of mesenchymal origin in dogs. Chemotherapy delays metastasis, yet most dogs die of this disease within 1 year of diagnosis. The high metabolic demand of cancer cells promotes proton pump upregulation, leading to acidification of the tumor microenvironment and chemoresistance. The potassium-sparing diuretic amiloride is among a class of proton pump inhibitors prescribed for refractory heart failure treatment in dogs., Objective: We hypothesized that amiloride treatment improves chemotherapy response by reducing acidification in canine OSA cells. Our objective was to assess the in vitro effects of amiloride on cell viability, apoptosis, and metabolism., Methods: In vitro study. Assessments of cell viability and apoptosis were performed after single agent or combination treatment, along with calculations of pharmacological synergism using the combination index. Protein signaling during apoptosis was evaluated by Western blotting. Metabolic profiling was performed using a Seahorse bioanalyzer., Results: Amiloride strongly synergized with doxorubicin in combination treatment and exhibited additive or antagonistic effects with carboplatin in canine OSA cells. Combination treatment with doxorubicin significantly upregulated p53-mitochondrial signaling to activate apoptosis and downregulate Akt phosphorylation. Amiloride-treated cells further exhibited metabolic switching with reductions in glycolytic capacity and maximal respiration., Conclusion and Clinical Importance: Amiloride synergized with doxorubicin to potentiate apoptosis in canine OSA cells. These results justify further investigation into repurposing of amiloride as an oncology drug for the treatment of OSA in dogs., (© 2018 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Background and Purpose: Acid-sensing ion channels (ASICs) are neuronal proton sensors emerging as potential therapeutic targets in pain of the orofacial region. Amiloride, a non-specific ASIC blocker, has been shown to exert beneficial effects in animal models of migraine and in patients. We explored the involvement of the ASIC1-subtype in cutaneous allodynia, a hallmark of migraine affecting cephalic and extra-cephalic regions in about 70% of migrainers., Experimental Approach: We investigated the effects of systemic injections of amiloride and mambalgin-1, a specific inhibitor of ASIC1a- and ASIC1b-containing channels, on cephalic and extra-cephalic mechanical sensitivity in a rodent model of acute and chronic migraine induced by i.p. injections of isosorbide dinitrate., Key Results: I.v. injections of these inhibitors reversed cephalic and extra-cephalic acute cutaneous mechanical allodynia in rats, a single injection inducing a delay in the subsequent establishment of chronic allodynia. Both mambalgin-1 and amiloride also reversed established chronic allodynia. The anti-allodynic effects of mambalgin-1 were not altered in ASIC1a-knockout mice, showing the ASIC1a subtype is not involved in these effects which were comparable to those of the anti-migraine drug sumatriptan and of the preventive drug topiramate on acute and chronic allodynia respectively. A single daily injection of mambalgin-1 also had a significant preventive effect on allodynia chronification., Conclusions and Implications: These pharmacological data support the involvement of peripheral ASIC1-containing channels in migraine cutaneous allodynia as well as in its chronification. They highlight the therapeutic potential of ASIC1 inhibitors as both an acute and prophylactic treatment for migraine., (© 2018 The British Pharmacological Society.)

Wheatley, Courtney M., Sarah E. Baker, Bryan J. Taylor, Manda L. Keller-Ross, Steven C. Chase, Alex R. Carlson, Robert J. Wentz, Eric M. Snyder, and Bruce D. Johnson. Influence of inhaled amiloride on lung fluid clearance in response to normobaric hypoxia in healthy individuals. High Alt Med Biol 18:343-354, 2017., Aim: To investigate the role of epithelial sodium channels (ENaC) on lung fluid clearance in response to normobaric hypoxia, 20 healthy subjects were exposed to 15 hours of hypoxia (fraction of inspired oxygen [FiO 2 ] = 12.5%) on two randomized occasions: (1) inhaled amiloride (A) (1.5 mg/5 mL saline); and (2) inhaled saline placebo (P). Changes in lung fluid were assessed through chest computed tomography (CT) for lung tissue volume (TV), and the diffusion capacity of the lungs for carbon monoxide (DLCO) and nitric oxide (DLNO) for pulmonary capillary blood volume (V C ). Extravascular lung water (EVLW) was derived as TV-V C and changes in the CT attenuation distribution histograms were reviewed., Results: Normobaric hypoxia caused (1) a reduction in EVLW (change from baseline for A vs. P, -8.5% ± 3.8% vs. -7.9% ± 5.2%, p < 0.05), (2) an increase in V C (53.6% ± 28.9% vs. 53.9% ± 52.3%, p < 0.05), (3) a small increase in DLCO (9.6% ± 29.3% vs. 9.9% ± 23.9%, p > 0.05), and (4) CT attenuation distribution became more negative, leftward skewed, and kurtotic (p < 0.05)., Conclusion: Acute normobaric hypoxia caused a reduction in lung fluid that was unaffected by ENaC inhibition through inhaled amiloride. Although possible amiloride-sensitive ENaC may not be necessary to maintain lung fluid balance in response to hypoxia, it is more probable that normobaric hypoxia promotes lung fluid clearance rather than accumulation for the majority of healthy individuals. The observed reduction in interstitial lung fluid means alveolar fluid clearance may not have been challenged.

Purpose: The search for new drugs that control the continuous relapses of multiple myeloma is still required. Here, we report for the first time the potent antimyeloma activity of amiloride, an old potassium-sparing diuretic approved for the treatment of hypertension and edema due to heart failure. Experimental Design: Myeloma cell lines and primary samples were used to evaluate cytotoxicity of amiloride. In vivo studies were carried out in a xenograft mouse model. The mechanisms of action were investigated using RNA-Seq experiments, qRT-PCR, immunoblotting, and immunofluorescence assays. Results: Amiloride-induced apoptosis was observed in a broad panel of multiple myeloma cell lines and in a xenograft mouse model. Moreover, amiloride also had a synergistic effect when combined with dexamethasone, melphalan, lenalidomide, and pomalidomide. RNA-Seq experiments showed that amiloride not only significantly altered the level of transcript isoforms and alternative splicing events, but also deregulated the spliceosomal machinery. In addition, disruption of the splicing machinery in immunofluorescence studies was associated with the inhibition of myeloma cell viability after amiloride exposure. Although amiloride was able to induce apoptosis in myeloma cells lacking p53 expression, activation of p53 signaling was observed in wild-type and mutated TP53 cells after amiloride exposure. On the other hand, we did not find a significant systemic toxicity in mice treated with amiloride. Conclusions: Overall, our results demonstrate the antimyeloma activity of amiloride and provide a mechanistic rationale for its use as an alternative treatment option for relapsed multiple myeloma patients, especially those with 17p deletion or TP53 mutations that are resistant to current therapies. Clin Cancer Res; 23(21); 6602-15. ©2017 AACR ., (©2017 American Association for Cancer Research.)

"Broiler-type" chickens are fast-grow-ing, heavy-bodied birds with high demands on bone quality. Phenamil increased mineralization in cultured murine mesenchymal stem cells. Phenamil effects were tested in 2 groups of weight and gender matched day-old broiler chickens (n = 13). Oral administration of 30 mg phenamil/kg body weight d 1 to 13 reduced growth of chicks d 5 to 14 (P = 0.002); with phenamil-treated (PT) chick body weight being 84% of vehicle-treated (VT) chicks' body weight on d 14. Tissues collected on d 15 showed that femur lengths and widths did not differ, but tibias from PT chicks were 6% shorter (P = 0.002) and 13% narrower (P = 0.012) with 18% thinner tibial cross-sections (P < 0.008) than in VT chicks. Angles of the caudal aspect of the anterior surface of keeled-sternums were 166° in PT chicks, flatter than the 148° found in VT chicks (P = 0.000). Total mineral content of both tibia and femur were lower in PT chicks (P = 0.005 for both). Bone Ca, P, and Mg (ppm) in ash were similar, but Ca:P was lower (1.70 vs 1.75) in PT versus VT chicks (P < 0.05). Osteocalcin was ∼20% lower (P = 0.020), PINP was ∼45% higher (P = 0.000) in PT chicks. Carboxy-terminal telopeptide type I collagen (ICTP) and cross-linked N-telopeptide of type I collagen (NTX1) were similar in the 2 groups. Phenamil had unexpected and detrimental effects on bone formation in growing broiler chicks, reducing linear skeletal growth and markedly changing bone architecture., (© 2017 Poultry Science Association Inc.)

Background: Prehypertension is associated with higher cardiovascular risk, target organ damage, and incidence of hypertension. The Prevention of Hypertension in Patients with PreHypertension (PREVER-Prevention) trial aimed to evaluate the efficacy and safety of a low-dose diuretic for the prevention of hypertension and end-organ damage., Methods and Results: This randomized, parallel, double-blind, placebo-controlled trial was conducted in 21 Brazilian academic medical centers. Participants with prehypertension who were aged 30 to 70 years and who did not reach optimal blood pressure after 3 months of lifestyle intervention were randomized to a chlorthalidone/amiloride combination pill or placebo and were evaluated every 3 months during 18 months of treatment. The primary outcome was incidence of hypertension. Development or worsening of microalbuminuria, new-onset diabetes mellitus, and reduction of left ventricular mass were secondary outcomes. Participant characteristics were evenly distributed by trial arms. The incidence of hypertension was significantly lower in 372 study participants allocated to diuretics compared with 358 allocated to placebo (hazard ratio 0.56, 95% CI 0.38-0.82), resulting in a cumulative incidence of 11.7% in the diuretic arm versus 19.5% in the placebo arm (P=0.004). Adverse events; levels of blood glucose, glycosylated hemoglobin, creatinine, and microalbuminuria; and incidence of diabetes mellitus were no different between the 2 arms. Left ventricular mass assessed through Sokolow-Lyon voltage and voltage-duration product decreased to a greater extent in participants allocated to diuretic therapy compared with placebo (P=0.02)., Conclusions: A combination of low-dose chlorthalidone and amiloride effectively reduces the risk of incident hypertension and beneficially affects left ventricular mass in patients with prehypertension., Clinical Trial Registration: URL: http://www.ClinicalTrials.gov, www.ensaiosclinicos.gov. Unique identifiers: NCT00970931, RBR-74rr6s., (© 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

Background: In CF patients, the defective ion transport causes a simultaneous reduction of fluid, Cl(-) and HCO3(-) secretion. We aimed to demonstrate that the resulting altered properties of mucus can be recovered using lumacaftor, a CFTR corrector., Methods: The micro-rheology of non-CF and CF mucus was analysed using Multiple Particle Tracking., Results: The diffusion coefficient of nano-beads imbedded in mucus from CF human bronchial epithelium was lower than in non-CF mucus, and the elastic and viscous moduli were higher. We found that 25% correction of F508del-CFTR mutation with lumacaftor was enough to improve significantly CF mucus properties. Surprisingly, also incubation with amiloride, a compound that reduces fluid absorption but might not change the secretion of HCO3(-) towards the airway surface fluid, improved CF mucus properties., Conclusion: CF mucus properties can be recovered by either improving the hydration of the airways or recovering Cl(-) and HCO3(-) secretion across the mutated protein treated with a corrector compound., (Copyright © 2015 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

We aimed to evaluate mineralocorticoid receptor (MR) expression in rat bladder and the physiological role of the MR-epithelial sodium channel (ENaC) pathway in controlling bladder function in 10-12-week-old, male Sprague-Dawley rats. First, we examined the mRNA expression of MR and localization of MR and ENaC-α proteins in the urinary bladder. MR mRNA expression was observed in untreated-rat urinary bladders, and MR and ENaC-α proteins were localized in the epithelium. Next, rats were treated with vehicle (controls) or fludrocortisone (an MR agonist) for 3 days, and ENaC-α protein expression levels and bladder function were evaluated on day 4. ENaC-α protein expression was significantly higher in fludrocortisone-treated rats than in controls. In addition, cystometry was performed during intravesical infusion of saline and amiloride (an ENaC inhibitor). While intercontraction intervals (ICIs) during saline infusion were significantly shorter in the fludrocortisone group than in the controls, infusion of amiloride normalized the ICIs in the fludrocortisone group. However, no intra- or inter-group differences in maximum intravesical pressure were observed. Taken together, MR protein is localized in the rat urinary bladder epithelium, and may regulate ENaC expression and bladder afferent input. The MR-ENaC pathway may be a therapeutic target for ameliorating storage symptoms., (Copyright © 2016 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Introduction: Neurodegeneration is a widely accepted contributor to the development of long-term disability in multiple sclerosis (MS). While current therapies in MS predominantly target inflammation and reduce relapse rate they have been less effective at preventing long-term disability. The identification and evaluation of effective neuroprotective therapies within a trial paradigm are key unmet needs. Emerging evidence supports amiloride, a licenced diuretic, as a neuroprotective agent in MS through acid sensing ion channel blockade. Optic neuritis (ON) is a common manifestation of MS with correlates of inflammation and neurodegeneration measurable within the visual pathways. Amiloride Clinical Trial In Optic Neuritis (ACTION) will utilise a multimodal approach to assess the neuroprotective efficacy of amiloride in acute ON., Methods and Analysis: 46 patients will be recruited within 28 days from onset of ON visual symptoms and randomised on a 1:1 basis to placebo or amiloride 10 mg daily. Double-blinded treatment groups will be balanced for age, sex and visual loss severity by a random-deterministic minimisation algorithm. The primary objective is to demonstrate that amiloride is neuroprotective in ON as assessed by scanning laser polarimetry of the peripapillary retinal nerve fibre layer (RNFL) thickness at 6 months in the affected eye compared to the unaffected eye at baseline. RNFL in combination with further retinal measures will also be assessed by optical coherence tomography. Secondary outcome measures on brain MRI will include cortical volume, diffusion-weighted imaging, resting state functional MRI, MR spectroscopy and magnetisation transfer ratio. In addition, high and low contrast visual acuity, visual fields, colour vision and electrophysiology will be assessed alongside quality of life measures., Ethics and Dissemination: Ethical approval was given by the south central Oxford B research ethics committee (REC reference: 13/SC/0022). The findings from ACTION will be disseminated through peer-reviewed publications and at scientific conferences., Trial Registration Number: EudraCT2012-004980-39, ClinicalTrials.gov Identifier: NCT01802489., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

This study examined the mechanism of action of amiloride, a urokinase-type plasminogen activator receptor inhibitor, in lowering proteinuria. Podocytes were resuscitated to allow for their proliferation and were observed for morphological changes. In the in vitro experiment, control, lipopolysaccharide, and lipopolysaccharide + amiloride groups were established. The expression of urokinase-type plasminogen activator receptor (uPAR) in podocytes was detected with a flow cytometer and cell motility was detected with the transwell migration assay. In the in vivo test, the urine protein volume of the model was detected at 24 h using Coomassie brilliant blue staining and the morphological changes of the podocytes were detected with immunofluorescence. The protein expression rate of uPAR in the lipopolysaccharide group was significantly higher than those in the control and lipopolysaccharide + amiloride groups (P < 0.05). The viability of cells in the lipopolysaccharide group was significantly higher than those in the control and lipopolysaccharide + amiloride groups (P < 0.05). Compared with the urine protein level in the control group at 24 h, the level in the lipopolysaccharide group increased significantly (P < 0.05), whereas compared with the urine protein level in the lipopolysaccharide group, the level in the lipopolysaccharide + amiloride group decreased (P < 0.05). uPAR expression was significantly downregulated, and the fusion of the podocyte-specific skelemin synaptopodin on the glomerulus podocytes was significantly decreased in the lipopolysaccharide + amiloride group. These results suggest that amiloride is able to reduce cell motility and thus lower proteinuria by inhibiting the expression of uPAR in podocytes.

Introduction: Thiazide diuretics are associated with increased risk of diabetes mellitus. This risk may arise from K(+)-depletion. We hypothesised that a K(+)-sparing diuretic will improve glucose tolerance, and that combination of low-dose thiazide with K(+)-sparing diuretic will improve both blood pressure reduction and glucose tolerance, compared to a high-dose thiazide., Methods and Analysis: This is a parallel-group, randomised, double-blind, multicentre trial, comparing hydrochlorothiazide 25-50 mg, amiloride 10-20 mg and combination of both diuretics at half these doses. A single-blind placebo run-in of 1 month is followed by 24 weeks of blinded active treatment. There is forced dose-doubling after 3 months. The Primary end point is the blood glucose 2 h after oral ingestion of a 75 g glucose drink (OGTT), following overnight fasting. The primary outcome is the difference between 2 h glucose at weeks 0, 12 and 24. Secondary outcomes include the changes in home systolic blood pressure (BP) and glycated haemoglobin and prediction of response by baseline plasma renin. Eligibility criteria are: age 18-79, systolic BP on permitted background treatment ≥ 140 mm Hg and home BP ≥ 130 mm Hg and one component of the metabolic syndrome additional to hypertension. Principal exclusions are diabetes, estimated-glomerular filtration rate <45 mL/min, abnormal plasma K(+), clinic SBP >200 mm Hg or DBP >120 mm Hg (box 2). The sample size calculation indicates that 486 patients will give 80% power at α=0.01 to detect a difference in means of 1 mmol/L (SD=2.2) between 2 h glucose on hydrochlorothiazide and comparators., Ethics and Dissemination: PATHWAY-3 was approved by Cambridge South Ethics Committee, number 09/H035/19. The trial results will be published in a peer-reviewed scientific journal., Trial Registration Numbers: Eudract number 2009-010068-41 and clinical trials registration number: NCT02351973., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Background: People with cystic fibrosis (CF) have increased transport of the salt, sodium across their airway lining. Over-absorption of sodium results in the dehydration of the liquid that lines the airway surface and (along with defective chloride secretion) is a primary defect in people with CF., Objectives: To determine whether the topical administration of drugs that block sodium transport improves the respiratory condition of people with CF., Search Methods: We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group Trials Register comprising references identified from comprehensive electronic database searches, handsearching relevant journals and abstract books of conference proceedings. We contacted principal investigators known to work in the field, previous authors and pharmaceutical companies who manufacture ion transport agents for unpublished or follow-up data.Most recent search of the Group's register: 19 December 2013., Selection Criteria: Published or unpublished randomised controlled trials (RCTs) or quasi-randomised controlled trials of sodium channel blockers compared to placebo or another sodium channel blocker or the same sodium channel blocker at a different dosing regimen., Data Collection and Analysis: Two authors independently extracted data. Meta-analysis was limited due to differing study designs., Main Results: Five RCTs, with a total of 226 participants, examining the topical administration of the short-acting sodium channel blocker, amiloride, compared to placebo were identified as eligible for inclusion in the review. In three studies over six months, there was a significant difference found in the difference in relative change in FVC in favour of placebo (weighted mean difference 1.51% (95% confidence interval -2.77 to -0.25), although heterogeneity was evident. A two-week study demonstrated that hypertonic saline with amiloride pre-treatment did not result in a significant improvement in respiratory function or mucus clearance, in contrast to pre-treatment with placebo. There were no significant differences identified in other clinically relevant outcomes., Authors' Conclusions: We found no evidence that the topical administration of a short-acting sodium channel blocker improves respiratory condition in people with cystic fibrosis and some limited evidence of deterioration in lung function.

Experiments were carried out to validate the use of F. glumosa extract as a diuretic in the treatment of hypertension as claimed by traditional healers. The experiments were performed under the same conditions with two synthetic pharmacological diuretics considered as check (Furosemide and Amiloride hydrochlorothiazide). The aqueous extract leaves of F. glumosa accelerated the elimination of overloaded fluid. At the maximum of diuretic response, urinary osmolarity decreased significantly when compared with controls. The single dose treatment of the aqueous extract leaves of F. glumosa has significantly increased urine volume 24 h after administration of the extract. The stability of aldosterone level, the absence of correlation with the plasma levels of sodium, and the increased clearance of free water in the animals receiving the extract show that increased diuresis and natriuresis moderate elevation are tubular in origin. The increase in Na(+), K(+), and Cl(-) induced by the extract caused alkalinization of the urine and showed a strong inhibitory effect of carbonic anhydrase and saluretic. These effects were mainly observed at the dose of 375 mg/kg. These observations confirm the traditional use in the treatment of hypertension and support the importance of the conservation of local knowledge as well as the conservation of Cameroonian biodiversity.

Hypertonic saline (HS) inhalation therapy benefits cystic fibrosis (CF) patients [Donaldson SH, Bennet WD, Zeman KL, Knowles MR, Tarran R, Boucher RC. N Engl J Med 354: 241-250, 2006; Elkins MR, Robinson M, Rose BR, Harbour C, Moriarty CP, Marks GB, Belousova EG, Xuan W, Bye PT; the National Hypertonic Saline in Cystic Fibrosis (NHSCF) Study Group. N Engl J Med 354: 229-240, 2006]. Surprisingly, these benefits are long-lasting and are diminished by the epithelial Na(+) channel blocker amiloride (Donaldson SH, Bennet WD, Zeman KL, Knowles MR, Tarran R, Boucher RC. N Engl J Med 354: 241-250, 2006). Our aim was to explain these effects. Human bronchial epithelial (hBE) cells from CF lungs were grown in inserts and were used in three experimental approaches: 1) Ussing chambers to measure amiloride-sensitive short-circuit currents (INa); 2) continuous perfusion Ussing chambers; and 3) near "thin-film" conditions in which the airway surface of the inserts was exposed to a small volume (30 μl) of isosmotic or HS solution as the inserts were kept in their incubation tray and were subsequently used to measure INa under isosmotic conditions (near thin-film experiments; Tarran R, Boucher RC. Methods Mol Med 70: 479-492, 2002). HS solutions (660 mosmol/kgH2O) were prepared by adding additional NaCl to the isosmotic buffer. The transepithelial short-circuit current (ISC), conductance (GT), and capacitance (CT) were measured by transepithelial impedance analysis (Danahay H, Atherton HC, Jackson AD, Kreindler JL, Poll CT, Bridges RJ. Am J Physiol Lung Cell Mol Physiol 290: L558-L569, 2006; Singh AK, Singh S, Devor DC, Frizzell RA, van Driessche W, Bridges RJ. Methods Mol Med 70: 129-142, 2002). Exposure to apical HS inhibited INa, GT, and CT. The INa inhibition required 60 min of reexposure to the isosmotic solution to recover 75%. The time of exposure to HS required to inhibit INa was <2.5 min. Under near thin-film conditions, apical exposure to HS inhibited INa, but as osmotically driven water moved to the apical surface, the aqueous apical volume increased, leading to an amiloride-insensitive decrease in its osmolality and to recovery of INa that lagged behind the osmotic recovery. Amiloride significantly accelerated the recovery of INa following exposure to HS. Our conclusions are that exposure to HS inhibits hBE INa and that amiloride diminishes this effect.

Background: Recent phase-contrast X-ray imaging studies suggest that inspiration primarily drives lung aeration and airway liquid clearance at birth, which questions the role of adrenaline-induced activation of epithelial sodium channels (ENaCs). We hypothesized that pressures generated by inspiration have a greater role in airway liquid clearance than do ENaCs after birth., Methods: Rabbit pups (30 d of gestation) were delivered and sedated, and 0.1 ml of saline (S) or amiloride (Am; an ENaC inhibitor) was instilled into the lungs before mechanical ventilation. Two other groups (30 d of gestation) were treated similarly but were also given adrenaline (S/Ad and Am/Ad) before mechanical ventilation., Results: Amiloride and adrenaline did not affect functional residual capacity (FRC) recruitment (P > 0.05). Amiloride increased the rate of FRC loss between inflations (Am: -5.2 ± 0.6 ml/kg/s), whereas adrenaline reduced the rate of FRC loss (S/Ad: -1.9 ± 0.3 ml/kg/s) as compared with saline-treated controls (S: -3.5 ± -0.6 ml/kg/s; P < 0.05)., Conclusion: These data indicate that inspiration is a major determinant of airway liquid clearance and FRC development during positive pressure ventilation. Although ENaC inhibition and adrenaline administration had no detectable effect on FRC development, ENaC may help to prevent liquid from re-entering the airways during expiration.

Background: Burkholderia dolosa can result in chronic airway infection and rapid decline in lung function in patients with cystic fibrosis (CF). Amiloride has antibacterial properties that may be synergistic with aminoglycosides against other species belonging to the Burkholderia cepacia complex (Bcc). We attempted to eradicate B. dolosa using a combination of nebulized tobramycin and nebulized amiloride in infected CF patients., Methods: A 6-month, open-label trial of continuous inhaled amiloride, delivered via nebulization four times daily, and continuous inhaled tobramycin (TIS or TOBI®) nebulized twice daily, was offered to all CF patients at our institution who are chronically infected with B. dolosa., Results: Twenty two of 27 patients with B. dolosa were eligible and twelve elected to participate. Eradication of B. dolosa was not noted in any study subject. While patients tolerated treatment with no adverse effects, there was also no apparent impact on other secondary outcome measures., Conclusions: Concurrent, continuous inhalation of amiloride and tobramycin for 6 months was not effective for the eradication of chronic B. dolosa airway infection in CF patients., (Copyright © 2012 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Background: Potassium-sparing diuretics, which block the epithelial sodium channel (ENaC), are widely prescribed for hypertension as a second-line drug in patients taking other diuretics (e.g. thiazide diuretics) and much less commonly prescribed as monotherapy. Therefore, it is essential to determine the effects of ENaC blockers on blood pressure (BP), heart rate and withdrawals due to adverse effects (WDAEs) when given as a first-line or second-line therapy., Objectives: To quantify the dose-related reduction in systolic blood pressure (SBP) and diastolic blood pressure (DBP) of ENaC blocker therapy as a first-line or second-line drug in patients with primary hypertension., Search Methods: We searched CENTRAL (The Cochrane Library 2012), MEDLINE (1950 to August 2012), EMBASE (1980 to August 2012) and reference lists of articles., Selection Criteria: Double-blind, randomized, controlled trials in patients with primary hypertension that evaluate, for a duration of 3 to 12 weeks, the BP lowering efficacy of: 1) fixed-dose monotherapy with an ENaC blocker compared with placebo; or 2) an ENaC blocker in combination with another class of anti-hypertensive drugs compared with the respective monotherapy (without an ENaC blocker)., Data Collection and Analysis: Two authors independently assessed the risk of bias and extracted data. Study authors were contacted for additional information. WDAE information was also collected from the trials., Main Results: No trials evaluating the BP lowering efficacy of ENaC blockers as monotherapy in patients with primary hypertension were identified. Only 6 trials evaluated the BP lowering efficacy of low doses of amiloride and triamterene as a second drug in 496 participants with a baseline BP of 151/102 mm Hg. The additional BP reduction caused by the ENaC blocker as a second drug was estimated by comparing the difference in BP reduction between the combination and monotherapy groups. The addition of low doses of amiloride and triamterene in these trials did not reduce BP. An estimate of the dose-related BP lowering efficacy for ENaC blockers was not possible because of a lack of trial data at higher doses., Authors' Conclusions: ENaC blockers do not have a statistically or clinically significant BP lowering effect at low doses but trials at higher doses are not available. The review did not provide a good estimate of the incidence of harms associated with ENaC blockers.

Hypertension guidelines advise limiting the dose of thiazide diuretics and avoiding combination with β-blockade, because of increased risk of diabetes mellitus. We tested whether changes in the 2-hour oral glucose tolerance test could be detected after 4 weeks of treatment with a thiazide and could be avoided by switching to amiloride. Two double-blind, placebo-controlled, crossover studies were performed. In study 1 (41 patients), we found that changes in glucose during a 2-hour oral glucose tolerance test could be detected after 4 weeks of treatment with bendroflumethiazide. In study 2, 37 patients with essential hypertension received, in random order, 4 weeks of once-daily treatment with hydrochlorothiazide (HCTZ) 25 to 50 mg, nebivolol 5 to 10 mg, combination (HCTZ 25-50 mg+nebivolol 5-10 mg), amiloride (10-20 mg), and placebo. Each drug was force titrated at 2 weeks and separated by a 4-week placebo washout. At each visit, we recorded blood pressure and performed a 75-g oral glucose tolerance test. Primary outcome was the difference in glucose (over the 2 hours of the oral glucose tolerance test) between 0 and 4 weeks, when HCTZ and amiloride were compared by repeated-measures analysis. For similar blood pressure reductions, there were opposite changes in glucose between the 2 diuretics (P<0.0001). Nebivolol did not impair glucose tolerance, either alone or in combination. There was a negative correlation between Δpotassium and Δ2-hour glucose (r=-0.28; P<0.0001). In 2 crossover studies, 4 weeks of treatment with a thiazide diuretic impaired glucose tolerance. No impairment was seen with K(+)-sparing diuretic or β(1)-selective blockade. Substitution or addition of amiloride may be the solution to preventing thiazide-induced diabetes mellitus.

Background: People with cystic fibrosis (CF) have increased transport of the salt, sodium across their airway lining. Over-absorption of sodium results in the dehydration of the liquid that lines the airway surface and is a primary defect in people with CF., Objectives: To determine whether the topical administration of drugs that block sodium transport improves the respiratory condition of people with CF., Search Methods: We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group Trials Register comprising references identified from comprehensive electronic database searches, handsearching relevant journals and abstract books of conference proceedings. We contacted principal investigators known to work in the field, previous authors and pharmaceutical companies who manufacture ion transport agents for unpublished or follow-up data.Most recent search of the Group's register: 22nd August 2011., Selection Criteria: Published or unpublished randomised controlled trials (RCTs) or quasi-randomised controlled trials of sodium channel blockers compared to placebo or another sodium channel blocker or the same sodium channel blocker at a different dosing regimen., Data Collection and Analysis: Two authors independently extracted data. Meta-analysis was limited due to differing study designs., Main Results: Five RCTs, with a total of 226 participants, examining the topical administration of the short-acting sodium channel blocker, amiloride, compared to placebo were identified as eligible for inclusion in the review. In three studies over six months, there was a significant difference found in the difference in relative change in FVC in favour of placebo (weighted mean difference 1.51% (95% confidence interval -2.77 to -0.25), although heterogeneity was evident. A two-week study demonstrated that hypertonic saline with amiloride pre-treatment did not result in a significant improvement in respiratory function or mucus clearance, in contrast to pre-treatment with placebo. There were no significant differences identified in other clinically relevant outcomes., Authors' Conclusions: We found no evidence that the topical administration of a short-acting sodium channel blocker improves respiratory condition in people with cystic fibrosis and some limited evidence of deterioration in lung function.

Unlabelled: In this study, we aimed to evaluate the antinociceptive interaction between intrathecally administered amiloride and morphine or clonidine. Using rats chronically implanted with lumbar intrathecal catheters, we examined the ability of intrathecal amiloride, morphine, clonidine, and mixtures of amiloride-morphine and amiloride-clonidine to alter tail-flick latency. To characterize any interactions, isobolographic analysis was performed. The effects of pretreatment with intrathecally administered naloxone or yohimbine were tested. Intrathecal administration of amiloride (25-150 μg), morphine (.25-10 μg), or clonidine (.5-10 μg) alone produced significant dose-dependent antinociception in the tail-flick test. The median effective dose (ED(50)) values for intrathecally administered amiloride, morphine, and clonidine were 120.5 μg, 5.0 μg, and 4.4 μg, respectively. Isobolographic analysis exhibited a synergistic interaction after coadministration of amiloride-morphine and amiloride-clonidine. Intrathecal pretreatment with naloxone (10 μg) completely blocked the antinociceptive effects of morphine and the amiloride-morphine mixture. Intrathecal pretreatment with yohimbine (20 μg) completely blocked the antinociceptive effect of clonidine and antagonized the effect of the amiloride-clonidine mixture. There was no motor dysfunction or significant change in blood pressure or heart rate after the intrathecal administration of amiloride, amiloride-morphine, and amiloride-clonidine. The synergistic effect observed after the coadministration of amiloride and morphine or clonidine suggests a functional interaction among calcium channels, μ-receptors and α(2)-receptors at the spinal cord level of the nociceptive processing system., Perspective: Although intrathecal morphine and clonidine produces pronounced analgesia, antinociceptive doses of intrathecal morphine and clonidine produce several side effects, including hypotension, bradycardia, sedation, and tolerance. This article presents antinociceptive synergistic interaction between amiloride and morphine, amiloride, and clonidine on thermal nociceptive tests in the rat., (Copyright © 2012 American Pain Society. Published by Elsevier Inc. All rights reserved.)

Na absorption across the cornified, multilayered, and squamous rumen epithelium is mediated by electrogenic amiloride-insensitive transport and by electroneutral Na transport. High concentrations of amiloride (>100 μM) inhibit Na transport, indicating Na(+)/H(+) exchange (NHE) activity. The underlying NHE isoform for transepithelial Na absorption was characterized by mucosal application of the specific inhibitor HOE642 for NHE1 and S3226 for NHE3 in Ussing chamber studies with isolated epithelia from bovine and sheep forestomach. S3226 (1 μM; NHE3 inhibitor) abolished electroneutral Na transport under control conditions and also the short-chain fatty acid-induced increase of Na transport via NHE. However, HOE642 (30 μM; NHE1 inhibitor) did not change Na transport rates. NHE3 was immunohistochemically localized in membranes of the upper layers toward the lumen. Expression of NHE1 and NHE3 has been previously demonstrated by RT-PCR, and earlier experiments with isolated rumen epithelial cells have shown the activity of both NHE1 and NHE3. Obviously, both isoforms are involved in the regulation of intracellular pH, pH(i). However, transepithelial Na transport is only mediated by apical uptake via NHE3 in connection with extrusion of Na by the basolaterally located Na-K-ATPase. The missing involvement of NHE1 in transepithelial Na transport suggests that the proposed "job sharing" in epithelia between these two isoforms probably also applies to forestomach epithelia: NHE3 for transepithelial transport and NHE1 for, among others, pH(i) and volume regulation.

Purpose: To investigate the presence of epithelial sodium channels (ENaC) in rabbit and human conjunctival epithelium and to test the effects of topical amiloride, a potassium-sparing diuretic that blocks the ENaC, on tear quantity in rabbits., Methods: Both healthy normal human and rabbit conjunctival tissues underwent immunohistochemistry staining for ENaC-α and γ subunits as well as for reverse transcription-polymerase chain reaction (RT-PCR) for detection of ENaC-α and ENaC-γ subunit mRNA expression. Rabbits were instilled topical amiloride eye drops and tear function tests were performed before and after instillations., Results: Immunohistochemical staining for ENaC-α subunit in all rabbit eyes showed positive staining in apical and basal conjunctival epithelial cells. Human conjunctival epithelia revealed positive staining with ENaC-α antibody especially in the apical and basal layers. Immunohistochemistry staining with ENaC- γ antibody also revealed positive staining of the conjunctival epithelial cells especially in the basal layers. The ENaC-α mRNA was detected in samples from healthy white rabbit conjunctival epithelia, and ENaC-α and ENaC- γ mRNAs were detected in samples from healthy human conjunctival epithelia. The mean tear quantity showed a significant increase at 15 and 30 min compared to the pre-instillation value in eyes assigned to amiloride eye drops. The mean tear quantity at 15 and 30 min was significantly higher in the amiloride group compared to the control eyes., Conclusions: Topical amiloride application appears to increase the quantity of preocular tears owing to inhibition of conjunctival epithelial sodium channels.

Background and Purpose: The epithelial sodium channel (ENaC) regulates airway mucosal hydration and mucus clearance. The lack of such regulation in cystic fibrosis patients leads to desiccation of the airway lumen, resulting in mucostasis that establishes the environment for infections. Osmotic agents and negative ENaC regulators can be used to restore mucosal hydration. We aimed to assess whether: (i) osmotically driven fluid flux into the rat lung could be quantified in vivo by magnetic resonance imaging (MRI); and (ii) the MRI signals could be modulated through the regulation of ENaC function., Experimental Approach: Lung images from spontaneously breathing rats were acquired following intra-tracheal (i.t.) administration of physiological or hypertonic saline (HS). Compounds known to modulate the ENaC function were given i.t. prior to saline. Volumes of fluid signals were quantified on the images., Key Results: A tonicity-dependent increase in lung fluid was demonstrated following HS administration. Pretreatment with the ENaC blockers, amiloride or 552-02, resulted in an enhancement of HS-induced lung fluid signals, which were detectable for up to 4 h, consistent with a role for ENaC in fluid clearance. Aprotinin, a serine protease inhibitor that attenuates ENaC function, likewise enhanced the HS-induced increase in lung fluid signal, while alpha(1)-anti-trypsin was without significant effect., Conclusions and Implications: Proton MRI provides a non-invasive technique for studying modulators of lung fluid hydration in rat lung in vivo. The pharmacological sensitivity of MRI-detected fluid signals is consistent with ENaC-mediated fluid reabsorption after HS. This target-related readout may be used to characterize new ENaC modulators.

Background: Potassium-sparing diuretics, which block the epithelial sodium channel (ENaC), are widely prescribed for hypertension as a second-line drug in patients taking other diuretics (e.g. thiazide diuretics) and much less commonly prescribed as monotherapy. Therefore, it is essential to determine the effects of ENaC blockers on blood pressure (BP), heart rate and withdrawals due to adverse effects (WDAEs) when given as a first-line or second-line therapy., Objectives: To quantify the dose-related reduction in systolic blood pressure (SBP) and diastolic blood pressure (DBP) of ENaC blocker therapy as a first-line or second-line drug in patients with primary hypertension., Search Strategy: We searched CENTRAL (The Cochrane Library 2009, Issue 3), MEDLINE (1950 to August 2009), EMBASE (1980 to August 2009) and reference lists of articles., Selection Criteria: Double-blind, randomized, controlled trials in patients with primary hypertension that evaluate, for a duration of 3 to 12 weeks, the BP lowering efficacy of: 1) fixed-dose monotherapy with an ENaC blocker compared with placebo; or 2) an ENaC blocker in combination with another class of anti-hypertensive drugs compared with the respective monotherapy (without an ENaC blocker)., Data Collection and Analysis: Two authors independently assessed the risk of bias and extracted data. Study authors were contacted for additional information. WDAE information was also collected from the trials., Main Results: No trials evaluating the BP lowering efficacy of ENaC blockers as monotherapy in patients with primary hypertension were identified. Only 6 trials evaluated the BP lowering efficacy of low doses of amiloride and triamterene as a second drug in 496 participants with a baseline BP of 151/102 mm Hg. The additional BP reduction caused by the ENaC blocker as a second drug was estimated by comparing the difference in BP reduction between the combination and monotherapy groups. The addition of low doses of amiloride and triamterene in these trials did not reduce BP. An estimate of the dose-related BP lowering efficacy for ENaC blockers was not possible because of a lack of trial data at higher doses., Authors' Conclusions: ENaC blockers do not have a statistically or clinically significant BP lowering effect at low doses but trials at higher doses are not available. The review did not provide a good estimate of the incidence of harms associated with ENaC blockers.

Dietary sodium restriction coupled with axotomy of the rat chorda tympani nerve (CTX) results in selectively attenuated taste responses to sodium salts in the contralateral, intact chorda tympani nerve. Converging evidence indicates that sodium deficiency also diminishes the activated macrophage response to injury on both the sectioned and contralateral, intact sides of the tongue. Because a sodium-restricted diet causes a robust increase in circulating aldosterone, we tested the hypothesis that changes in neurophysiological and immune responses contralateral to the CTX could be mimicked by aldosterone administration instead of the low-sodium diet. Taste responses in rats with CTX and supplemental aldosterone for 4-6 days were similar to rats with CTX and dietary sodium restriction. Responses to sodium salts were as much as 50% lower compared with sham-operated and vehicle-supplemented rats. The group-related functional differences were eliminated with lingual application of amiloride, suggesting that a major transduction pathway affected was through epithelial sodium channels. Consistent with the functional results, few macrophages were observed on either side of the tongue in rats with CTX and aldosterone. In contrast, macrophages were elevated on both sides of the tongue in rats with CTX and the vehicle. These results show that sodium deficiency or administration of aldosterone suppresses the immune response to neural injury, resulting in attenuation of peripheral gustatory function. They also show a potential key link among downstream consequences of sodium imbalance, taste function, and immune activity.

Renal Na(+) and K(+) excretion was measured in rats with varying dietary K(+) intake. The requirement for channel-mediated distal nephron Na(+) reabsorption was assessed by infusing the animals with the K(+)-sparing diuretic amiloride via osmotic minipumps. At infusion rates of 2 nmol/min, the concentration of amiloride in the urine was 38 microM, corresponding to concentrations of 9-23 microM in the distal tubular fluid, sufficient to block >98% of Na(+) transport through apical Na(+) channels (ENaC). With a control K(+) intake (0.6% KCl), amiloride reduced K(+) excretion rates (U(K)V) from 0.85 +/- 0.15 to 0.05 +/- 0.01 micromol/min during the first 2 h of infusion, suggesting that distal nephron K(+) secretion was completely dependent on the activity of Na(+) channels. When K(+) intake was increased by feeding overnight with a diet containing 10% KCl, amiloride reduced U(K)V from 7.5 +/- 0.7 to 1.3 +/- 0.1 micromol/min despite an increased plasma K(+) of 9 mM, again suggesting a major but not exclusive role for the Na(+) channel-dependent pathway of K(+) secretion. The maximal measured rates of amiloride-sensitive K(+) excretion correspond well with estimates based on apical K(+) channel activity in distal nephron segments. However, when the animals were adapted to the high-K(+) diet for 7-9 days, the diuretic decreased U(K)V less, from 6.1 +/- 0.6 to 3.0 +/- 0.8 micromol/min, indicating an increasing fraction of K(+) excretion that was independent of Na(+) channels. This indicates the upregulation of a Na(+) channel-independent mechanism for secreting K(+).

We investigated the effects of the selective inhibitor of Na(+)/Ca(2+) exchanger (NCX), 2',4'- and 3',4'-dichlorobenzamil (DCB), on large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels in cultured human umbilical vein endothelial cells (HUVECs) and fresh isolated mouse aortic smooth muscle cells (MASMCs) using the patch clamp techniques. Both kinds of DCB reversibly activated BK(Ca) currents in whole-cell clamped HUVECs or MASMCs. The EC(50) of 2',4'-DCB for BK(Ca) current activation in HUVECs was 2.64 +/- 0.10 muM. In inside-out and outside-out patches, 2',4'-DCB remarkably increased BK(Ca) channels activity. 2',4'-DCB increased open frequency, but had no significant effect on mean open time. In inside-out patches, 2',4'-DCB shifted the relationship curve between [Ca(2+)](i) and open probability (NP(o)) to the left; the [Ca(2+)](i) required to evoke half-maximal activation changed from 1087.45 +/- 142.91 nM to 500.24 +/- 66.83 nM by 10 muM 2',4'-DCB. In addition, 2',4'-DCB shifted the relationship curve between membrane potential and NP(o) to the left; the membrane potential to evoke half-maximal activation changed from 81.1 +/- 2.4 to 64.7 +/- 3.1 mV by 10 muM 2',4'-DCB. 3',4'-DCB also increased BK(Ca) channels activity. There was no significant difference in the effect of DCB on BK(Ca) channels between both excised patches. These results suggested that 2',4'- and 3',4'-DCB activate BK(Ca) channels activity in HUVECs and MASMCs by increasing the sensitivity of BK(Ca) channels to cytosolic free Ca(2+) and membrane potential. Our report would provide a consideration if they are used as NCX blocker in vascular endothelial cells or smooth muscle cells.

Background and Purpose: Protein transduction domains (PTDs), such as Tat, antennapedia homeoprotein (Antp), Rev and VP22, have been extensively utilized for intracellular delivery of biologically active macromolecules in vitro and in vivo. There is little known, however, about the relative transduction efficacy, cytotoxicity and internalization mechanism of individual PTDs., Experimental Approach: We examined the cargo delivery efficacies of four major PTDs (Tat, Antp, Rev and VP22) and evaluated their toxicities and cell internalizing pathways in various cell lines., Key Results: The relative order of the transduction efficacy of these PTDs conjugated to fluorescein was Rev>Antp>Tat>VP22, independent of cell type (HeLa, HaCaT, A431, Jurkat, MOLT-4 and HL60 cells). Antp produced significant toxicity in HeLa and Jurkat cells, and Rev produced significant toxicity in Jurkat cells. Flow cytometric analysis demonstrated that the uptake of PTD-fluorescein conjugate was dose-dependently inhibited by methyl-beta-cyclodextrin, cytochalasin D and amiloride, indicating that all four PTDs were internalized by the macropinocytotic pathway. Accordingly, in cells co-treated with 'Tat-fused' endosome-disruptive HA2 peptides (HA2-Tat) and independent PTD-fluorescent protein conjugates, fluorescence spread throughout the cytosol, indicating that all four PTDs were internalized into the same vesicles as Tat., Conclusions and Implications: These findings suggest that macropinocytosis-dependent internalization is a crucial step in PTD-mediated molecular transduction. From the viewpoint of developing effective and safe protein transduction technology, although Tat was the most versatile carrier among the peptides studied, PTDs should be selected based on their individual characteristics.

Amiloride-sensitive Na+ absorption is a well-described feature of numerous transporting epithelia in vertebrates. Yet, very little is known about this important physiological process regarding invertebrates. In the present paper, we compare vertebrate Na+ absorption mediated by the amiloride-sensitive epithelial Na+ channel (ENaC) and its invertebrate counterpart. We used the dorsal skin of the annelid Hirudo medicinalis as a model for the Na+ absorption of invertebrate epithelia. In applying electrophysiological, molecular, and biochemical techniques we found striking functional and structural differences between vertebrate and invertebrate amiloride-sensitive Na+ absorption. Using modified Ussing chambers, we analyzed the influence of different known blockers and effectors of vertebrate ENaC on leech epithelial Na+ absorption. We demonstrate that the serine protease trypsin had no effect on the Na+ transport across leech integument, while it strongly activates vertebrate ENaC. While protons, and the divalent cations Ni2+ and Zn2+ stimulate vertebrate ENaC, amiloride-sensitive Na+ currents in leech integument were substantially reduced. For molecular studies, we constructed a cDNA library of Hirudo medicinalis and screened it with specific ENaC antibodies. We performed numerous PCR approaches using a vast number of different degenerated and specific ENaC primers to identify ENaC-like structures. Yet, both strategies did not reveal any ENaC-like sequence in leech integument. From these data we conclude that amiloride-sensitive Na+ absorption in leech skin is not mediated by an ENaC-like Na+ channel but by a still unknown invertebrate member of the ENaC/DEG family that we termed lENaTP (leech epithelial Na+ transporting protein).

Amiloride is a nonspecific blocker of acid-sensing ion channels (ASICs) that have been recently implicated in the mediation of mechanical and chemical/inflammatory nociception. Preliminary data using a transgenic model are suggestive of sex differences in the role of ASICs. We report here that systemic administration of amiloride (10-70 mg/kg ip) produces a robust, dose-dependent blockade of late/tonic phase nociceptive behavior on the mouse formalin test (5%; 20 microl) in female but not male mice, completely abolishing the known sex difference in formalin test response. Adult gonadectomy produced a "switching" of sex differences in amiloride efficacy, with castrated males displaying an amiloride blockade and ovariectomized females rendered less sensitive to amiloride. Gonadectomized mice could be switched back to their intact status using chronic estrogen benzoate or testosterone propionate replacement via osmotic minipump (6 microg/day or 250 microg/day, respectively). It is unclear whether this striking sex difference is due to sex-specific involvement of ASICs in pain processing, but the present data represent one of the first demonstrations of pain-related sex differences with no obvious opioid involvement.

We tested the hypothesis that oxytocin-induced labor augmented IL-1beta-induced/-stimulated lung fluid absorption in preterm guinea pig fetuses. IL-1beta was administered subcutaneously daily to timed-pregnant guinea pigs for 3 days with and without simultaneous cortisol synthesis inhibition by metyrapone. At day 3, oxytocin was administered, and fetuses were delivered by abdominal hysterotomy at 61 and by oxytocin-induced birth at 68 days gestation. Delivered fetuses were instilled with isosmolar 5% albumin into the lungs, and lung fluid movement was measured over 1 h by mass balance. Lung fluid absorption was induced in 61-day and stimulated in 68-day gestation lungs by IL-1beta. Labor induction by oxytocin augmented IL-1beta-induced/-stimulated lung fluid absorption. Metyrapone pretreatment did not affect oxytocin-induced/-stimulated lung fluid absorption, while completely blocking IL-1beta-induced/-stimulated fluid absorption. Fetal lung fluid absorption, when present, was always propranolol and amiloride sensitive, suggesting that beta-adrenoceptor stimulation and amiloride-sensitive sodium channels were critical for fluid absorption. Epithelial sodium channel and Na-K-ATPase subunit expressions were both increased by IL-1beta, but not further by oxytocin. Our results indicate that IL-1beta release into the maternal blood circulation positively affects lung maturation due to the IL-1beta-induced release of cortisol and thus prepares the lungs for the epinephrine surge associated with labor.

Burkholderia cepacia infection in cystic fibrosis (CF) patients is associated with significant morbidity and mortality, yet no definitive treatment is currently available. This report describes a new approach to treat B. cepacia infection in CF patients, using a combination of amiloride and tobramycin aerosols. Four adults with the typical clinical syndrome of CF were recruited after repeated positive sputum cultures for B. cepacia. Aerosols of amiloride and tobramycin were given three times daily for 1-6 months, and repeated sputum cultures were collected to assess efficacy. Three of the four patients treated with the combined therapy eradicated B. cepacia from their sputum cultures for at least 2 yrs, and there were no adverse events. This novel combination may provide a new therapeutic option for Burkholderia cepacia infections. Furthermore, the strategy of combining antibiotics with ion transport agents may have ramifications for the treatment of other multi-resistant organisms.

Background: Recently, we demonstrated that the amiloride derivative ethylisopropylamiloride (EIPA) limits vascular smooth muscle cell growth and migration. The purpose of the present experiments was to determine whether EIPA can also reduce the inflammatory component of atherogenesis and stent neointima formation., Methods and Results: To determine the effect of EIPA on the early inflammatory stages of atherogenesis, apolipoprotein E null mice (apoE-/-) fed an atherogenic diet received a subcutaneous pump infusion of either EIPA (3 mg x kg(-1)d(-1)) or the control vehicle for 4 weeks. The en face aortic area of atherosclerotic lesions and the subendothelial accumulation of macrophages were reduced by 46% and 38%, respectively, in EIPA-treated mice. Moreover, the number of vascular cell adhesion molecule-1 (VCAM-1) immunopositive lumenal endothelial cells was 59% less in the EIPA treatment group. In vitro, there was a concentration-dependent inhibition of lipopolysaccharide (LPS)-induced VCAM-1 expression with a corresponding 37% reduction in U-937 cell adhesion to endothelial cells. EIPA also reduced LPS-stimulated nuclear factor-kappaB (NF-kappaB) activation as reflected by a 66% reduction in NF-kappaB nuclear translocation. Finally, to test the effect of EIPA on the early inflammatory reaction to stent implantation, stents coated with jelly alone or jelly plus EIPA were implanted into rabbit iliac arteries. Four weeks later, the stent neointimal area, abundance of peristrut macrophages, and density of intimal smooth muscle cells were reduced by 38%, 47%, and 37%, respectively, for EIPA stents., Conclusions: EIPA downregulates endothelial cell activation of NF-kappaB and VCAM-1 expression and attenuates the early inflammatory stages of atherogenesis and stent intimal formation.

Despite the discovery of ion channels that are activated by protons, we still know relatively little about the signaling of acid pain. We used a novel technique, iontophoresis of protons, to investigate acid-induced pain in human volunteers. We found that transdermal iontophoresis of protons consistently caused moderate pain that was dose-dependent. A marked desensitization occurred with persistent stimulation, with a time constant of approximately 3 min. Recovery from desensitization occurred slowly, over many hours. Acid-induced pain was significantly augmented in skin sensitized by acute topical application of capsaicin. However, skin desensitized by repeated capsaicin application showed no significant reduction in acid-induced pain, suggesting that both capsaicin-sensitive and insensitive sensory neurons contribute to acid pain. Furthermore, topical application of non-steroidal anti-inflammatory drugs (NSAIDs) significantly attenuated acid-evoked pain but did not affect the heat pain threshold, suggesting a specific interaction between NSAIDs and peripheral acid sensors. Subcutaneous injection of amiloride (1 mm) also significantly inhibited the pain induced by iontophoresis of acid, suggesting an involvement of acid-sensing ion channel (ASIC) receptors. Conversely, iontophoresis of acid over a wide range of skin temperatures from 4 to 40 degrees C produced only minor changes in the induced pain. Together these data suggest a prominent role for ASIC channels and only a minor role for transient receptor potential vanilloid receptor-1 as mediators of cutaneous acid-induced pain.

Sodium taste transduction is thought to occur via an amiloride-sensitive, sodium-selective pathway and an amiloride-insensitive, cation nonselective, anion-dependent pathway(s). It has been shown by others that amiloride, an epithelial sodium channel (ENaC) blocker, significantly reduces the chorda tympani nerve response to lingually applied NaCl in C57BL/6 (B6) mice but not in DBA/2 (D2) mice, suggesting that the latter strain might not possess functional ENaCs in taste receptor cells. We psychophysically measured and compared taste detection thresholds of NaCl and sodium gluconate (NaGlu) prepared with and without 100 microM amiloride in these two strains (eight/strain). Mice were trained and tested in a two-response operant signal detection procedure conducted in a gustometer. Surprisingly, no strain effect was found for the detection thresholds of both salts (approximately 0.05-0.06 M). Moreover, these thresholds were increased by almost an order of magnitude by amiloride adulteration of the solutions. This marked effect of amiloride on sodium detection thresholds suggests that ENaCs are necessary for normal sensitivity to sodium salts in both strains. In addition, because NaGlu is thought to stimulate primarily the amiloride-sensitive pathway, especially at low concentrations, the similarity of NaCl and NaGlu thresholds (r > 0.81 both strains) suggests that ENaCs are also sufficient to support the detection of sodium in weak solutions by B6 and D2 mice.

Background: The National Service Framework (NSF) sets standards for the management of heart failure in the UK. Loop diuretics are commonly first prescribed in primary care. Some patients taking these drugs have heart failure and may benefit from other treatments including ACE inhibitors. Accurate diagnosis in primary care is essential for the aims of the NSF to be realised., Aims: To investigate loop diuretic prescribing in general practice, to analyse recorded clinical features, patient investigations and ACE inhibitor use in this population., Method: One thousand three hundred and one patients taking loop diuretics were identified from prescription records of seven general practices. Demographic details, clinical features, investigations and drug treatments were extracted from patient records., Results: The prevalence of loop diuretic prescribing increased with age. Twenty percent of patients were attributed a diagnosis of heart failure but relevant clinical features were recorded in less than 50% of patient records. Open access echocardiography was used in 8.9% of patients. ACE inhibitors were prescribed in 39.8% of patients considered to have heart failure. 18.2% of these were taking the recommended target dose., Conclusion: Loop diuretics are prescribed commonly, particularly in the elderly. There is no clear pattern of documented clinical features that leads to prescription of these drugs. Open access echocardiography is rarely used to aid diagnosis. ACE inhibitors are under-prescribed and under-dosed in patients diagnosed with heart failure in this study population.

Purpose: To test whether blocking the Na+/H+ antiport reduces intraocular pressure (IOP) in the mouse., Methods: The electrophysiologic approach (the servo-null micropipette system, SNMS) that had been adapted for continuously monitoring IOP in the mouse was used in a study of the effects of a series of transport inhibitors., Results: Topical application of three direct blockers of Na+/H+ exchangers produced comparable reductions in mouse IOP: dimethylamiloride (DMA, -5.0 +/- 0.7 mm Hg), ethylisopropylamiloride (EIPA, -4.1 +/- 1.0), and BIIB723 (-4.9 +/- 1.7 mm Hg). These effects were mediated locally, not systemically, because adding DMA to one eye had no effect on IOP in the contralateral eye. In contrast to the actions of selective inhibitors of Na+/H+ exchange, neither the low-potency inhibitor amiloride nor the inhibitor of Na+-K+-2Cl- cotransport bumetanide by itself was effective. Dorzolamide, which slows delivery of H+ and HCO3- to Na+/H+ and Cl-/HCO3- antiports, also reduced IOP by 2.9 +/- 0.6 mm Hg. After first blocking Na+/H+ exchange with DMA, EIPA, BIIB723, or dorzolamide, application of bumetanide produced an additional reduction in IOP of 3.8 to 4.0 mm Hg., Conclusions: The first step in formation of aqueous humor is uptake of NaCl by the ciliary epithelial cells from the stroma, possibly by both paired Na+/H+ and Cl-/HCO3- antiports and a bumetanide-sensitive Na+-K+-2Cl- symport. The present data are consistent with electron probe x-ray microanalyses of rabbit ciliary epithelium indicating that the antiports are the dominant mechanism. That bumetanide can produce a previously unobserved lowering of IOP when the Na+/H+ antiport is also inhibited substantiates a dominant antiport mechanism.

The epithelial sodium channel (ENaC) is a principal site for sodium reabsorption and as such may participate importantly in blood pressure (BP) regulation. Amiloride, a direct inhibitor of ENaC, characteristically has mild antihypertensive properties, consistent with ENaC having more minor influences on BP regulation. Counter-regulatory influences may, however, prevent amiloride from effectively lowering BP. Aldosterone secretion is known to increase in response to the reduced sodium reabsorption that follows amiloride inhibition of ENaC, and because aldosterone upregulates ENaC function, we considered the possibility that secondary hyperaldosteronism mitigates the ability of amiloride to reduce BP. In the present study, the BP responses to amiloride (5 mg per day), spironolactone (25 mg per day), the combination of the 2 drugs, and placebo were studied in healthy normotensive subjects. Over 4 weeks of treatment, the combination of amiloride and spironolactone lowered systolic BP by 4.6+/-1.6 (mean+/-SEM) mm Hg (P=0.022) and diastolic BP by 2.2+/-1.2 mm Hg (P=0.30), whereas either drug alone had no significant effect on BP. The findings suggest that the 2 drugs with different modes of action-amiloride, a direct inhibitor of ENaC, and spironolactone, a mineralocorticoid receptor antagonist-may compliment each other's ability to inhibit ENaC and thereby reduce sodium reabsorption to a point at which BP decreases. On the other hand, we cannot rule out that the BP response resulted from the greater dose of total drug. The lowering of BP with small doses of inhibitors of ENaC serves as additional evidence for the importance of ENaC to the tonic maintenance of BP.

Because tumor necrosis factor (TNF)-alpha can upregulate alveolar fluid clearance (AFC) in pneumonia or septic peritonitis, the mechanisms responsible for the TNF-alpha-mediated increase in epithelial fluid transport were studied. In rats, 5 microg of TNF-alpha in the alveolar instillate increased AFC by 67%. This increase was inhibited by amiloride but not by propranolol. We also tested a triple-mutant TNF-alpha that is deficient in the lectinlike tip portion of the molecule responsible for its membrane conductance effect; the mutant also has decreased binding affinity to both TNF-alpha receptors. The triple-mutant TNF-alpha did not increase AFC. Perfusion of human A549 cells, patched in the whole cell mode, with TNF-alpha (120 ng/ml) resulted in a sustained increase in Na(+) currents from 82 +/- 9 to 549 +/- 146 pA (P < 0.005; n = 6). The TNF-alpha-elicited Na(+) current was inhibited by amiloride, and there was no change when A549 cells were perfused with the triple-mutant TNF-alpha or after preincubation with blocking antibodies to the two TNF-alpha receptors before perfusion with TNF-alpha. In conclusion, although TNF- alpha can initiate acute inflammation and edema formation in the lung, TNF-alpha can also increase AFC by an amiloride-sensitive, cAMP-independent mechanism that enhances the resolution of alveolar edema in pathological conditions by either binding to its receptors or activating Na(+) channels by means of its lectinlike domain.

We employed ultrasonic nebulization for homogeneous alveolar tracer deposition into ventilated perfused rabbit lungs. (22)Na and (125)I-albumin transit kinetics were monitored on-line with gamma detectors placed around the lung and the perfusate reservoir. [(3)H]mannitol was measured by repetitive counting of perfusion fluid samples. Volume of the alveolar epithelial lining fluid was estimated with bronchoalveolar lavage with sodium-free isosmolar mannitol solutions. Sodium clearance rate was -2.2 +/- 0.3%/min. This rate was significantly reduced by preadministration of ouabain/amiloride and enhanced by pretreatment with aerosolized terbutaline. The (125)I-albumin clearance rate was -0.40 +/- 0.05%/min. The appearance of [(3)H]mannitol in the perfusate was not influenced by ouabain/amiloride or terbutaline but was markedly enhanced by pretreatment with aerosolized protamine. An epithelial lining fluid volume of 1.22 +/- 0.21 ml was calculated in control lungs. Fluid absorption rate was 1.23 microl x g lung weight(-1) x min(-1), which was blunted after pretreatment with ouabain/amiloride. We conclude that alveolar tracer loading by aerosolization is a feasible technique to assess alveolar epithelial barrier properties in aerated lungs. Data on active and passive sodium flux, paracellular solute transit, and net fluid absorption correspond well to those in previous studies in fluid-filled lungs; however, albumin clearance rates were markedly higher in the currently investigated aerated lungs.

In order to evaluate the degree and type of gas exchange impairment in cystic fibrosis, ventilation/perfusion relationships in ten patients (mean age 26 yrs, mean Shwachman score 86) were examined. Pulmonary gas exchange was studied using the multiple inert gas elimination technique. High-resolution computed tomography (HRCT) and spirometry, including diffusing capacity, were performed after each gas exchange study for comparison. Examinations were done before and after home i.v. antibiotic treatment (HIVAT, 14 days) and after inhaled amiloride and placebo (14 days), in crossover fashion, clinical status after HIVAT serving as the baseline for the crossover study. Before HIVAT, the mean residual volume was 182% of the predicted value, the mean vital capacity 72% pred and the mean forced expiratory volume in one second 53% pred (p<0.001). The dispersion of pulmonary blood flow at different ventilation/perfusion ratios (V'/Q') ((logarithmic SD of the perfusion distribution (log SDQ)), used as an index for gas exchange impairment, was increased to a mean of 0.72. No linear correlation was seen between ventilation/perfusion inequality, spirometry and HRCT (p>0.05). After HIVAT, log SDQ was significantly improved to 0.66 (p<0.05). After placebo, but not after amiloride, log SDQ, arterial oxygen tension, alveolar-arterial oxygen tension difference and maximal expiratory flows when 50% and 25% of the forced vital capacity tension remain to be exhaled were significantly worse (p<0.05, respectively). Areas with a low V'/Q' were significantly lower after amiloride compared to after the placebo period (p<0.05). Moderate ventilation/perfusion inequality was present in the majority of the studied cystic fibrosis patients. The degree of ventilation/perfusion inequality cannot be estimated from spirometry or high-resolution computed tomography. The low proportion of low ventilation/perfusion ratios indicates that the regular treatment directed towards mucus plugging of small airways is beneficial. An improvement in the ventilation/perfusion relationship was seen after home i.v. antibiotic treatment and inhaled amiloride may possibly have a further positive effect on gas exchange.