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1.
Am J Physiol Renal Physiol ; 327(1): F37-F48, 2024 Jul 01.
Article in English | MEDLINE | ID: mdl-38779752

ABSTRACT

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.


Subject(s)
Amiloride , Diabetes Mellitus, Type 2 , Epithelial Sodium Channel Blockers , Hypertension , Interleukin-17 , Interleukin-6 , Tumor Necrosis Factor-alpha , Amiloride/pharmacology , Amiloride/therapeutic use , Humans , Interleukin-17/blood , Animals , Diabetes Mellitus, Type 2/drug therapy , Diabetes Mellitus, Type 2/blood , Diabetes Mellitus, Type 2/immunology , Interleukin-6/blood , Male , Middle Aged , Hypertension/drug therapy , Hypertension/blood , Female , Epithelial Sodium Channel Blockers/pharmacology , Tumor Necrosis Factor-alpha/blood , Aged , Mice , Epithelial Sodium Channels/metabolism , Epithelial Sodium Channels/drug effects , Mice, Inbred C57BL , Antihypertensive Agents/pharmacology , Macrophages/metabolism , Macrophages/drug effects , Blood Pressure/drug effects , Diabetes Mellitus, Type 1/drug therapy , Diabetes Mellitus, Type 1/blood
2.
Am J Physiol Renal Physiol ; 327(1): F158-F170, 2024 Jul 01.
Article in English | MEDLINE | ID: mdl-38779755

ABSTRACT

Diabetes is closely associated with K+ disturbances during disease progression and treatment. However, it remains unclear whether K+ imbalance occurs in diabetes with normal kidney function. In this study, we examined the effects of dietary K+ intake on systemic K+ balance and renal K+ handling in streptozotocin (STZ)-induced diabetic mice. The control and STZ mice were fed low or high K+ diet for 7 days to investigate the role of dietary K+ intake in renal K+ excretion and K+ homeostasis and to explore the underlying mechanism by evaluating K+ secretion-related transport proteins in distal nephrons. K+-deficient diet caused excessive urinary K+ loss, decreased daily K+ balance, and led to severe hypokalemia in STZ mice compared with control mice. In contrast, STZ mice showed an increased daily K+ balance and elevated plasma K+ level under K+-loading conditions. Dysregulation of the NaCl cotransporter (NCC), epithelial Na+ channel (ENaC), and renal outer medullary K+ channel (ROMK) was observed in diabetic mice fed either low or high K+ diet. Moreover, amiloride treatment reduced urinary K+ excretion and corrected hypokalemia in K+-restricted STZ mice. On the other hand, inhibition of SGLT2 by dapagliflozin promoted urinary K+ excretion and normalized plasma K+ levels in K+-supplemented STZ mice, at least partly by increasing ENaC activity. We conclude that STZ mice exhibited abnormal K+ balance and impaired renal K+ handling under either low or high K+ diet, which could be primarily attributed to the dysfunction of ENaC-dependent renal K+ excretion pathway, despite the possible role of NCC.NEW & NOTEWORTHY Neither low dietary K+ intake nor high dietary K+ intake effectively modulates renal K+ excretion and K+ homeostasis in STZ mice, which is closely related to the abnormality of ENaC expression and activity. SGLT2 inhibitor increases urinary K+ excretion and reduces plasma K+ level in STZ mice under high dietary K+ intake, an effect that may be partly due to the upregulation of ENaC activity.


Subject(s)
Diabetes Mellitus, Experimental , Epithelial Sodium Channels , Potassium, Dietary , Potassium , Animals , Diabetes Mellitus, Experimental/metabolism , Potassium/metabolism , Potassium/urine , Male , Potassium, Dietary/metabolism , Epithelial Sodium Channels/metabolism , Mice, Inbred C57BL , Sodium-Glucose Transporter 2 Inhibitors/pharmacology , Potassium Channels, Inwardly Rectifying/metabolism , Potassium Channels, Inwardly Rectifying/genetics , Mice , Diabetic Nephropathies/metabolism , Diabetic Nephropathies/etiology , Diabetic Nephropathies/physiopathology , Kidney/metabolism , Kidney/drug effects , Kidney/physiopathology , Hypokalemia/metabolism , Amiloride/pharmacology , Renal Elimination/drug effects , Homeostasis , Solute Carrier Family 12, Member 3/metabolism , Solute Carrier Family 12, Member 3/genetics , Glucosides/pharmacology , Streptozocin , Benzhydryl Compounds , Sodium-Glucose Transporter 2
3.
Lab Chip ; 24(12): 3093-3100, 2024 Jun 11.
Article in English | MEDLINE | ID: mdl-38779981

ABSTRACT

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.


Subject(s)
Trachea , Trachea/metabolism , Amiloride/pharmacology , Animals , Lab-On-A-Chip Devices , Humans , Microfluidic Analytical Techniques/instrumentation , Respiratory Mucosa/metabolism , Respiratory Mucosa/cytology
4.
Am J Physiol Renal Physiol ; 327(3): F435-F449, 2024 Sep 01.
Article in English | MEDLINE | ID: mdl-38779754

ABSTRACT

We have previously observed that prolonged administration of rapamycin, an inhibitor targeting the mammalian target of rapamycin complex (mTORC)1, partially reduced hypertension and alleviated kidney inflammation in Dahl salt-sensitive (SS) rats. In contrast, treatment with PP242, an inhibitor affecting both mTORC1/mTORC2, not only completely prevented hypertension but also provided substantial protection against kidney injury. Notably, PP242 exhibited potent natriuretic effects that were not evident with rapamycin. The primary objective of this study was to pinpoint the specific tubular sites responsible for the natriuretic effect of PP242 in SS rats subjected to either 0.4% NaCl (normal salt) or 4.0% NaCl (high salt) diet. Acute effects of PP242 on natriuretic, diuretic, and kaliuretic responses were determined in unanesthetized SS rats utilizing benzamil, furosemide, or hydrochlorothiazide [inhibitors of epithelial Na+ channel (ENaC), Na-K-2Cl cotransporter (NKCC2), or Na-Cl cotransporter (NCC), respectively] either administered alone or in combination. The findings indicate that the natriuretic effects of PP242 in SS rats stem predominantly from the inhibition of NCC and a reduction of ENaC open probability. Molecular analysis revealed that mTORC2 regulates NCC activity through protein phosphorylation and ENaC activity through proteolytic cleavage in vivo. Evidence also indicated that PP242 also prevents the loss of K+ associated with the inhibition of NCC. These findings suggest that PP242 may represent an improved therapeutic approach for antihypertensive intervention, potentially controlling blood pressure and mitigating kidney injury in salt-sensitive human subjects.NEW & NOTEWORTHY This study explored mechanisms underlying the natriuretic effects of mammalian target of rapamycin protein complex 2 inhibition using PP242 and revealed both epithelial Na+ channel and Na-Cl cotransporter in the distal tubular segments were potentially inhibited. These observations, with prior lab evidence, indicate that PP242 prevents hypertension via its potent inhibitory effects on these specific sodium transporters and by reducing renal immune responses. This dual action, coupled with potassium sparing effects, suggests an improved approach for managing hypertension and associated kidney damage.


Subject(s)
Epithelial Sodium Channels , Mechanistic Target of Rapamycin Complex 2 , Natriuresis , Rats, Inbred Dahl , Sodium Chloride, Dietary , Solute Carrier Family 12, Member 3 , Animals , Epithelial Sodium Channels/metabolism , Natriuresis/drug effects , Mechanistic Target of Rapamycin Complex 2/metabolism , Male , Solute Carrier Family 12, Member 3/metabolism , Hypertension/metabolism , Hypertension/drug therapy , Hypertension/physiopathology , Kidney/drug effects , Kidney/metabolism , Disease Models, Animal , Rats , Amiloride/pharmacology , Amiloride/analogs & derivatives , Blood Pressure/drug effects , Phosphorylation , Signal Transduction/drug effects , Indoles , Purines
5.
Arch Pharm (Weinheim) ; 357(8): e2400063, 2024 Aug.
Article in English | MEDLINE | ID: mdl-38704748

ABSTRACT

Lithium induces nephrogenic diabetes insipidus (NDI) and microcystic chronic kidney disease (CKD). As previous clinical studies suggest that NDI is dose-dependent and CKD is time-dependent, we investigated the effect of low exposition to lithium in a long-term experimental rat model. Rats were fed with a normal diet (control group), with the addition of lithium (Li+ group), or with lithium and amiloride (Li+/Ami group) for 6 months, allowing obtaining low plasma lithium concentrations (0.25 ± 0.06 and 0.43 ± 0.16 mmol/L, respectively). Exposition to low concentrations of plasma lithium levels prevented NDI but not microcystic dilations of kidney tubules, which were identified as collecting ducts (CDs) on immunofluorescent staining. Both hypertrophy, characterized by an increase in the ratio of nuclei per tubular area, and microcystic dilations were observed. The ratio between principal cells and intercalated cells was higher in microcystic than in hypertrophied tubules. There was no correlation between AQP2 messenger RNA levels and cellular remodeling of the CD. Additional amiloride treatment in the Li+/Ami group did not allow consistent morphometric and cellular composition changes compared to the Li+ group. Low exposition to lithium prevented overt NDI but not microcystic dilations of the CD, with differential cellular composition in hypertrophied and microcystic CDs, suggesting different underlying cellular mechanisms.


Subject(s)
Amiloride , Aquaporin 2 , Diabetes Insipidus, Nephrogenic , Disease Models, Animal , Kidney Tubules, Collecting , Animals , Diabetes Insipidus, Nephrogenic/chemically induced , Diabetes Insipidus, Nephrogenic/prevention & control , Kidney Tubules, Collecting/drug effects , Kidney Tubules, Collecting/pathology , Kidney Tubules, Collecting/metabolism , Male , Rats , Aquaporin 2/metabolism , Amiloride/pharmacology , Rats, Wistar , Time Factors , Renal Insufficiency, Chronic/prevention & control , Renal Insufficiency, Chronic/chemically induced , Lithium/pharmacology , Dose-Response Relationship, Drug
6.
Am J Physiol Renal Physiol ; 326(6): F1066-F1077, 2024 Jun 01.
Article in English | MEDLINE | ID: mdl-38634134

ABSTRACT

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.


Subject(s)
Epithelial Sodium Channels , Proteolysis , Sodium , Animals , Epithelial Sodium Channels/metabolism , Epithelial Sodium Channels/genetics , Male , Female , Sodium/metabolism , Kidney Tubules, Collecting/metabolism , Homeostasis , Furin/metabolism , Furin/genetics , Mice , Colon/metabolism , Potassium/metabolism , Diet, Sodium-Restricted , Mice, 129 Strain , Mutation , Amiloride/pharmacology
7.
Exp Physiol ; 109(5): 766-778, 2024 May.
Article in English | MEDLINE | ID: mdl-38551893

ABSTRACT

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.


Subject(s)
Acetazolamide , Amiloride , Diuretics , Furosemide , Kidney Cortex , Kidney Medulla , Animals , Furosemide/pharmacology , Acetazolamide/pharmacology , Amiloride/pharmacology , Diuretics/pharmacology , Sheep , Female , Kidney Cortex/drug effects , Kidney Cortex/metabolism , Kidney Medulla/drug effects , Kidney Medulla/metabolism , Oxygen/metabolism , Hemodynamics/drug effects , Oxygen Consumption/drug effects
8.
J Physiol ; 602(4): 737-757, 2024 Feb.
Article in English | MEDLINE | ID: mdl-38345534

ABSTRACT

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.


Subject(s)
Amiloride , Symporters , Mice , Animals , Amiloride/pharmacology , Aryldialkylphosphatase/metabolism , Epithelial Sodium Channels/metabolism , Aldosterone/metabolism , Sodium Chloride/metabolism , Sodium/metabolism , Nephrons/metabolism
9.
Int J Mol Sci ; 25(3)2024 Jan 24.
Article in English | MEDLINE | ID: mdl-38338690

ABSTRACT

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.


Subject(s)
Acid Sensing Ion Channels , Analgesics, Opioid , Rats , Humans , Animals , Acid Sensing Ion Channels/metabolism , Analgesics, Opioid/pharmacology , Amiloride/pharmacology , Protons , Binding Sites
10.
J Am Soc Nephrol ; 35(4): 410-425, 2024 Apr 01.
Article in English | MEDLINE | ID: mdl-38254266

ABSTRACT

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 .


Subject(s)
Diabetic Nephropathies , Urokinase-Type Plasminogen Activator , Humans , Mice , Animals , Urokinase-Type Plasminogen Activator/metabolism , Plasminogen/metabolism , Amiloride/pharmacology , Fibrinolysin/metabolism , Inflammasomes , Mice, Inbred NOD , Proteinuria/metabolism , Complement Activation , Anaphylatoxins , Fibrosis
11.
BMC Res Notes ; 17(1): 13, 2024 Jan 03.
Article in English | MEDLINE | ID: mdl-38172968

ABSTRACT

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.


Subject(s)
Amiloride , Silicon Dioxide , Male , Rats , Animals , Amiloride/metabolism , Amiloride/pharmacology , Silicon Dioxide/pharmacology , Diet, Western , Rats, Inbred F344 , Epithelium/metabolism , Ion Transport , Methacholine Chloride/pharmacology , Methacholine Chloride/metabolism , Muscle, Smooth/metabolism
12.
Bioorg Med Chem ; 99: 117603, 2024 Feb 01.
Article in English | MEDLINE | ID: mdl-38246115

ABSTRACT

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.


Subject(s)
Amiloride , Sodium , Amiloride/pharmacology , Sodium/metabolism , Sodium-Hydrogen Exchangers/metabolism , Membrane Proteins/metabolism , Hydrogen , Hydrogen-Ion Concentration
13.
Eur J Med Chem ; 265: 116038, 2024 Feb 05.
Article in English | MEDLINE | ID: mdl-38157597

ABSTRACT

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.


Subject(s)
Cystic Fibrosis , Humans , Cystic Fibrosis/drug therapy , Cystic Fibrosis/genetics , Sodium Channel Blockers/therapeutic use , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Epithelial Sodium Channels/genetics , Epithelial Sodium Channels/therapeutic use , Amiloride/pharmacology , Amiloride/therapeutic use , Sodium/metabolism , Sodium/therapeutic use
14.
Arq. bras. cardiol ; 112(1): 87-90, Jan. 2019. tab
Article in English | LILACS | ID: biblio-1038534

ABSTRACT

Abstract Blood pressure (BP)-lowering therapy improves left ventricular (LV) parameters of hypertensive target-organ damage in stage II hypertension, but whether there is a drug-class difference in echocardiographic parameters in stage I hypertension patients is less often studied. In the PREVER treatment study, where individuals with stage I hypertension were randomized for treatment with diuretics (chlorthalidone/amiloride) or losartan, 110 participants accepted to participate in a sub-study, where two-dimensional echocardiograms were performed at baseline and after 18 months of antihypertensive treatment. As in the general study, systolic BP reduction was similar with diuretics or with losartan. Echocardiographic parameters showed small but significant changes in both treatment groups, with a favorable LV remodeling with antihypertensive treatment for 18 months when target blood pressure was achieved either with chlorthalidone/amiloride or with losartan as the initial treatment strategy. In conclusion, even in stage I hypertension, blood pressure reduction is associated with improvement in echocardiographic parameters, either with diuretics or losartan as first-drug regimens.


Resumo A terapia de redução da pressão arterial (PA) melhora os parâmetros do ventrículo esquerdo (VE) na lesão a órgãos-alvo causada pela condição hipertensiva na hipertensão de estágio II; no entanto, se existem ou não diferenças relacionadas à classe de medicamentos nos parâmetros ecocardiográficos de pacientes com hipertensão estágio I é menos frequentemente estudado. No estudo PREVER-treatment, em que indivíduos com hipertensão estágio I foram randomizados para tratamento com diuréticos (clortalidona/amilorida) ou losartana, 110 participantes aceitaram participar de um subestudo, no qual foram realizados ecocardiogramas bidimensionais basais e após 18 meses de tratamento anti-hipertensivo. Como no estudo geral, a redução da PA sistólica foi semelhante com diuréticos ou com losartana. Os parâmetros ecocardiográficos mostraram pequenas mas significativas alterações em ambos os grupos de tratamento, com um remodelamento favorável do VE com tratamento anti-hipertensivo por 18 meses, quando a pressão arterial alvo foi atingida com clortalidona/amilorida ou com losartana como estratégia inicial de tratamento. Em conclusão, mesmo na hipertensão estágio I, a redução da pressão arterial está associada à melhora nos parâmetros ecocardiográficos tanto com o uso de diuréticos ou losartana como primeiro esquema de tratamento farmacológico.


Subject(s)
Humans , Male , Female , Adult , Middle Aged , Aged , Losartan/therapeutic use , Diuretics/therapeutic use , Amiloride/therapeutic use , Hypertension/drug therapy , Antihypertensive Agents/therapeutic use , Time Factors , Blood Pressure/drug effects , Echocardiography , Double-Blind Method , Follow-Up Studies , Treatment Outcome , Losartan/pharmacology , Ventricular Remodeling/drug effects , Diuretics/pharmacology , Amiloride/pharmacology , Hypertension/diagnostic imaging , Antihypertensive Agents/pharmacology
15.
Article in English | WPRIM (Western Pacific) | ID: wpr-215794

ABSTRACT

PURPOSE: The effects of amiloride on cellular toxicity caused by tissue plasminogen activator (tPA) in mouse primary retinal cells were investigated. METHODS: Primary retinal cell cultures were maintained using glial conditioned medium. Commercial tPA and L-arginine were added, and the level of cyclic guanosine monophosphate (cyclic-GMP) in the culture supernatant was assessed using an ELISA assay. We measured the cell viability of cultured retinal cells pretreated with three different concentrations of amiloride (1, 10, and 100 microm) in addition to commercial tPA or L-arginine treatment. RESULTS: After exposing the cultured mouse retinal cells to tPA plus L-arginine or L-arginine alone, cyclic-GMP concentrations were 61.9 +/- 5.1 pmole/mL and 63.1 +/- 6.1 pmole/mL, respectively. However, the control group had a significantly lower concentration of cyclic-GMP (37.2 +/- 3.4 pmole/mL, p < 0.01). The cyclic GMP-dissolved solution did not cause retinal cell death. In the control group and the group treated with 1 microm amiloride and tPA containing L-arginine, the cell viability was 43.7% and 44.5%, respectively. However, cell viability increased to 70.6% with 10 microm amiloride and 78.4% with 100 microm amiloride (p = 0.015). CONCLUSIONS: L-arginine increases intracellular cyclic-GMP and may give rise to retinal cells through this mechanism. In addition, amiloride in concentrations greater than 10 microm protects against L-arginine-induced retinal cell death.


Subject(s)
Animals , Mice , Amiloride/pharmacology , Analysis of Variance , Arginine/toxicity , Cell Death/drug effects , Cells, Cultured , Cyclic GMP/pharmacology , Enzyme-Linked Immunosorbent Assay , Retina/cytology , Tissue Plasminogen Activator/toxicity
16.
Acta gastroenterol. latinoam ; 31(3): 123-130, 2001. ilus, tab, gra
Article in English | BINACIS | ID: bin-8910

ABSTRACT

In normal rat distal colon isolated mucosa, basal short-circuit current (Isc) is mostly due to chloride secretion. Isc is depressed by a brief (5 min) acute hypoxia and overshoots above baseline during reoxygenation. Sodium deprivation raises serum aldosterone levels and leads to expression of functional epithelial sodium channels which are amiloride-sensitive. Thus, in sodium-deprived rats (SDRs) Isc is dependent on electrogenic sodium absorption. Since the ion primarily responsible for the Isc is different in each functional condition, it is not known whether hypoxia and reoxygenation affect SDRs epithelial response in the same way as in normal rats. Therefore the electrical behavior of isolated mucosa preparations from normal and SDRs was studied in an Ussing chamber, and the effect of the epithelial sodium channel blocker, amiloride sensitive, basal Isc than controls. Their response to hypoxia (expressed as a fraction of basal Isc) was similar to controls but upon reoxygenation their recovery was incomplete. SDRs response to hypoxia was not affected by amiloride at any concentration tested. However, post-hypoxic recovery was modified by amiloride in a concentration-dependent way: it was incomplete at 10(-8) M, complete at 10(-6) M, and at 10(-4) M it overshooted above baseline values. Therefore, in sodium-deprived rats, sodium channel blockade reverts the pattern of blunted recovery to the overshooting pattern seen normal rats. These results may be explained by two non-mutually exclusive hypotheses: Epithelial sodium channel blockade in sodium-deprived rats might (1) unmask a basal chloride conductance, and (2) interfere with a negative interaction between sodium chloride conductances. (AU)


Subject(s)
Animals , Male , Rats , RESEARCH SUPPORT, NON-U.S. GOVT , Aldosterone/blood , Cell Hypoxia , Colon/physiopathology , Sodium/deficiency , Amiloride/pharmacology , Colon/drug effects , Diuretics/pharmacology , Dose-Response Relationship, Drug , Electrophysiology , Intestinal Mucosa/physiopathology , Intestinal Mucosa/drug effects , Rats, Wistar
17.
Biocell ; 24(3): 233-237, Dec. 2000.
Article in English | BINACIS | ID: bin-6424

ABSTRACT

In red cells from umbilical cord blood it has been referred the existence of lithium fluxes (contralateral sodium dependent) asymmetry. On account of the relevancy of this transport system in some pathologies it is pertinent the study of its kinetics to relate normal with pathological states in which it is affected. Lithium fluxes--contralateral sodium dependent were determined in N-ethylmaleimide treated neonatal red blood cells. Experimental data were fitted by simple Michaelis-Menten kinetics, finding Km and Vmax variables. It was shown the persistency of asymmetry. The independence of sulfhydryl groups (or the occultation of the groups involved to this inhibitor) could explain asymmetry persistence.(AU)


Subject(s)
Humans , Female , Pregnancy , RESEARCH SUPPORT, NON-U.S. GOVT , Cell Membrane/metabolism , Erythrocytes/metabolism , Ion Transport/physiology , Lithium/metabolism , Sodium-Potassium-Exchanging ATPase/metabolism , Amiloride/pharmacology , Cell Membrane/drug effects , Culture Media , Erythrocytes/drug effects , Ethylmaleimide/pharmacology , Fetal Blood/cytology , Fetal Blood/drug effects , Fetal Blood/metabolism , Hematopoiesis/physiology , Ion Transport/drug effects , Kinetics , Sodium-Potassium-Exchanging ATPase/drug effects , Sodium/metabolism
18.
Acta cient. venez ; 50(1): 48-58, 1999. graf, tab
Article in Spanish | LILACS | ID: lil-240254

ABSTRACT

Se estudiaron los efectos cronotrópico e inotrópico de la Amilorida (AMI) y la dicloro-benzamil-Amilorida (DCB-AMI) sobre las aurículas aislada del acure, así como la interacción de estas drogas con la beta-metil-digoxina (BM_DIGO), la epinefrina y la disminución del potasio extracelular (de 4 a 1 mM). La AMI (1 mM) causa un efecto inotrópico positivo y cronotrópico negativo, independientes del sistema autonómico. La DCB-AMI causa um efecto bimodal sobre la fuerza de contracción: la aumenta a bajas dosis pero la disminuye a concentraciones mayores de 10(-6) M. También disminuye levemente la frecuencia sinusal. El efecto de la AMI sobre el automatismo sinusal no es alterado por la BM-DIGO. En cambio, la AMI ((10(-3 M) disminuye el efecto inotrópico positivo de la BM-DIGO e incrementa la dosis tóxica en preparaciones aisladas. La curva dosis-respuesta a la epinefrina no varía en presencia de AMI. Resultados similares se obtuvieron con DCB-AMI (2 x 10(-7 M). El incremento de contractilidad que se observa al disminuir la concentración extracelular de potasio a 1 mM no se altera en presencia de AMI. La actividad de la Na+/K+ ATPasa dependiente de Mg++ de la fracción microsomal obtenida del corazón del acure disminuye en 10 por ciento aproximadamente en presencia de AMI (1nM). Por otra parte, el efecto inhibitorio sobre la enzima obtenido con ouabaína no varía con esta droga. En conclusión, nuestros resultados sugieren múltiples efectos de la AMI y DCB-AMI sobre el corazón del acure. La inhibición del intercambiador Na+/Ca++ explica solo parte de ellos; el bloqueo de los canales lentos parece fundamental para explicar nuestras observaciones.


Subject(s)
Animals , Female , Guinea Pigs , Cardiotonic Agents/pharmacology , Diuretics/pharmacology , Amiloride/analogs & derivatives , Amiloride/pharmacology , Heart Atria/drug effects , Heart Rate/drug effects , Medigoxin/pharmacology , Drug Interactions , Myocardial Contraction/drug effects
19.
Article in English | WPRIM (Western Pacific) | ID: wpr-192960

ABSTRACT

We have shown that hyaluronic acid stimulates the proliferation of quiescent NIH 3T3 cells. We have shown that treatment of 1 mg/ml hyaluronic acid results in increase of tyrosine phosphorylation of two proteins, MW 124 kDa and 60 kDa as detected by anti-tyrosine antibodies by Western blot analysis. Maximum phosphorylation occurred within 2 h after addition of 1 mg/ml hyaluronic acid. Stimulation of proliferation was also accompanied by increase in c-Myc protein, which was inhibited by amlloride, an inhibitor of Na+/H+ antiporter and EGTA and increase in the steady state level of pRb, the RB gene product. These results suggest that the intracellular signal transduction pathways that mediate the stimulatory effects of hyaluronic acid on cellular proliferation are similar to those of growth factors.


Subject(s)
Mice , 3T3 Cells , Amiloride/pharmacology , Animals , Cell Division , Dose-Response Relationship, Drug , Egtazic Acid/pharmacology , Hyaluronic Acid/pharmacology , Mitogens/pharmacology , Phosphoproteins/metabolism , Phosphorylation , Proto-Oncogene Proteins c-myc/metabolism , Retinoblastoma Protein/metabolism , Signal Transduction , Sodium-Hydrogen Exchangers/antagonists & inhibitors , Tyrosine
20.
Yonsei Medical Journal ; : 278-286, 1995.
Article in English | WPRIM (Western Pacific) | ID: wpr-52252

ABSTRACT

Cardiac dysfunctions such as myocardial functional failure and ventricular arrhythmia have been largely attributed to intracellular Ca2+ overload. One of the mechanisms of intracellular Ca2+ overload involves a rapid influx of Ca2+ via Na(+)-Ca2+ exchange during the reperfusion which utilizes the accumulation of Na+ in myocytes during ischemic cardiac arrest. Possible sources of the intracellular Na+ accumulation include Na+ channel, Na(+)-H+ exchange, Na(+)-Ca2+ exchange, and Na+ background current. In this study, we studied the role of the Na+ background current in intracellular Na+ accumulation during the cardiac arrest by measuring the Na+ background current in guinea pig ventricular myocytes with whole cell clamp method and evaluating the effects of cardioprotective drugs on the Na+ background current. The results were as follows: (1) The Na+ background inward current at -40 mV membrane potential was larger at Ca2+ free solution than 1.8 mM Ca2+ solution. (2) The Na+ background current was not affected by verapamil. (3) 2 microM O-(N, N-hexamethylene)-amiloride (HMA) decreased the Na+ background current at negative membrane potential. (4) The new cardioprotective drug, R 56865, decreased the Na+ background current. These results suggest that the Na+ background current plays a role in increasing the intracellular Na+ activity during high K+ cardioplegia and the blocking effect of myoprotective drugs, such as R 56865, on the Na+ background current may contribute to myocardial protection after cardioplegia.


Subject(s)
Amiloride/pharmacology , Animals , Guinea Pigs , Heart/drug effects , Heart Arrest, Induced , Myocardium/metabolism , Piperidines/pharmacology , Potassium/pharmacology , Sodium/metabolism , Thiazoles/pharmacology , Verapamil/pharmacology
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