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Journal of Ocular Pharmacology and... Jun 2024Antibody-drug conjugates (ADCs) are a relatively recent advance in the delivery of chemotherapeutics that improve targeting of cytotoxic agents. However, despite their...
Antibody-drug conjugates (ADCs) are a relatively recent advance in the delivery of chemotherapeutics that improve targeting of cytotoxic agents. However, despite their antitumor activity, severe ocular adverse effects, including vision loss, have been reported for several ADCs. The nonspecific uptake of ADCs into human corneal epithelial cells (HCECs) and their precursors via macropinocytosis has been proposed to be the primary mechanism of ocular toxicity. In this study, we evaluated the ability of a novel polymer, poly(l-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG), to decrease the ADC rituximab-mc monomethylauristatin F (MMAF) (RIX) uptake into human corneal epithelial (HCE-T) cells. HCE-T cells were exposed to increasing concentrations of RIX to determine inhibition of cell proliferation. HCE-T cells were treated with PLL-g-PEG, the macropinocytosis inhibitor 5-(N-ethyl-N-isopropyl) amiloride (EIPA), or vehicle. After 30 min of incubation, RIX was added. ADC was detected by fluorescent anti-human immunoglobulin G and fluorescently conjugated dextran as viewed by microscopy. RIX caused dose-dependent inhibition of HCE-T cell proliferation. EIPA significantly reduced RIX uptake and decreased macropinocytosis as assessed by direct quantification of RIX using a fluorescently conjugated anti-human antibody as well as quantification of macropinocytosis using fluorescently conjugated dextran. PLL-g-PEG resulted in a dose-dependent inhibition of RIX uptake with half-maximal inhibitory concentrations of 0.022%-0.023% PLL-g-PEG. The data show PLL-g-PEG to be a potent inhibitor of RIX uptake by corneal epithelial cells and support its use as a novel therapeutic approach for the prevention of ocular adverse events associated with ADC therapy.
PubMed: 38935528
DOI: 10.1089/jop.2024.0019 -
IScience Jun 2024The kidney is a vital organ responsible for water and sodium metabolism, while the primary function of amiloride is to promote the excretion of water and sodium. We...
The kidney is a vital organ responsible for water and sodium metabolism, while the primary function of amiloride is to promote the excretion of water and sodium. We investigated amiloride enhanced the sunitinib sensitivity in RCC. We found both sunitinib and amiloride displayed cytotoxicity and exerted the synergy effect in RCC cells and arrays. Protein expression profiles were screened via MS/TMT, revealing that FN3K was upregulated in the sunitinib group, and rescued in amiloride and the combination administration. Exogenous FN3K could promote proliferation, invasion and metastasis and decrease the sensitivity of Caki-1 cells to sunitinib, also, exogenous FN3K up-regulated VEGFR2 expression and activated AKT/mTOR signal pathway. More FN3K and VEGFR2 accumulated in R-Caki-1 cells and rescued by amiloride treatment. Co-IP and IF confirmed the interaction between FN3K and VEGFR2. In conclusion, FN3K depletion mediated VEGFR2 disruption promotes amiloride synergized the anti-RCC activity of sunitinib.
PubMed: 38868177
DOI: 10.1016/j.isci.2024.109997 -
American Journal of Physiology. Renal... Jun 2024Ubiquitination influences the expression of the epithelial Na channel (ENaC). We assessed the mechanisms of selective ubiquitination of the mature, cleaved form of...
Ubiquitination influences the expression of the epithelial Na channel (ENaC). We assessed the mechanisms of selective ubiquitination of the mature, cleaved form of γENaC in both native rodent kidneys and in Fisher Rat Thyroid (FRT) cells expressing the channel heterologously. In both models, singly cleaved and fully cleaved γENaC were both strongly ubiquitinated, implying that the second cleavage releasing an inhibitory peptide was not essential for the process. To see if location of the protein in or near the apical membrane rather than cleavage influences ubiquitination we studied mutants of γENaC in which cleavage sites are abolished. These subunits were ubiquitinated only when co-expressed with α and βENaC, facilitating trafficking through the Golgi apparatus. To test whether reaching the apical surface is necessary we performed surface biotinylation and measured ENaC ubiquitination in the apical membrane of rat kidney. Ubiquitination of cleaved γENaC was similar in whole-kidney and surface fractions, implying that both apical and subapical channels could be modified. In FRT cells, inhibiting clathrin-mediated endocytosis with Dyngo-4a increased both total the ubiquitinated γENaC at the cell surface. Finally, we tested the idea that increased intracellular Na could stimulate ubiquitination. Administration of amiloride to block Na entry through the channels did not affect ubiquitination of γENaC in either FRT cells or rat kidney. However, presumed large increases in cellular Na produced by monensin in FRT cells or acute Na repletion in rats increased ubiquitination and decreased overall ENaC expression.
PubMed: 38867672
DOI: 10.1152/ajprenal.00037.2024 -
Acta Physiologica (Oxford, England) Jun 2024In rodent models of nephrotic syndrome (NS), edema formation was prevented by blockade of the epithelial sodium channel ENaC with amiloride. However, apart from case...
AIM
In rodent models of nephrotic syndrome (NS), edema formation was prevented by blockade of the epithelial sodium channel ENaC with amiloride. However, apart from case reports, there is no evidence favoring ENaC blockade in patients with NS.
METHODS
The monocentric randomized controlled AMILOR study investigated the antiedematous effect of amiloride (starting dose 5 mg/day, max. 15 mg/day) in comparison to standard therapy with the loop diuretic furosemide (40 mg/day, max. 120 mg/day) over 16 days. Overhydration (OH) was measured by bioimpedance spectroscopy (BCM, Fresenius). Depending on the OH response, diuretic dose was adjusted on days 2, 5, 8 and 12, and if necessary, hydrochlorothiazide (HCT) was added from d8 (12.5 mg/day, max. 25 mg/day). The primary endpoint was the decrease in OH on d8. The study was terminated prematurely due to insufficient recruitment and a low statistical power due to a low actual effect size.
RESULTS
Median baseline OH was +26.4 (interquartile range 15.5-35.1)% extracellular water (ECW) in the amiloride arm and + 27.9 (24.1-29.4)% ECW in the furosemide arm and decreased by 1.95 (0.80-6.40) and 5.15 (0.90-8.30)% ECW after 8 days, respectively, and by 10.10 (1.30-14.40) and 7.40 (2.80-10.10)% ECW after 16 days, respectively. OH decrease on d8 and d16 was not significantly different between both arms.
CONCLUSION
The AMILOR study is the first randomized controlled pilot study suggesting a similar antiedematous effect as furosemide. Further studies are required to better define the role of amiloride in NS (EudraCT 2019-002607-18).
PubMed: 38822593
DOI: 10.1111/apha.14183 -
Sleep Medicine Jul 2024Obstructive sleep apnea (OSA) and hypertension are common conditions that may be linked through sympathetic activation and water retention. We hypothesized that... (Randomized Controlled Trial)
Randomized Controlled Trial
BACKGROUND
Obstructive sleep apnea (OSA) and hypertension are common conditions that may be linked through sympathetic activation and water retention. We hypothesized that diuretics, which reduce the body water content, may be more effective than amlodipine, a blood pressure (BP)-lowering agent implicated with edema, in controlling OSA in patients with hypertension. We also aimed to compare the effects of these treatments on ambulatory blood pressure monitoring (ABPM).
METHODS
In a randomized, double-blind clinical trial, we compared the effects of chlorthalidone/amiloride 25/5 mg with amlodipine 10 mg on OSA measured by portable sleep monitor and BP measured by ABPM. The study included participants older than 40 who had moderate OSA (10-40 apneas/hour of sleep) and BP within the systolic range of 140-159 mmHg or diastolic range of 90-99 mmHg.
RESULTS
The individuals in the experimental groups were comparable in age, gender, and other relevant characteristics. Neither the combination of diuretics nor amlodipine alone reduced the AHI after 8 weeks of treatment (AHI 26.3 with diuretics and 25.0 with amlodipine. P = 0.713). Both treatments significantly lowered office, 24-h, and nighttime ABP, but the two groups had no significant difference.
CONCLUSION
Chlorthalidone associated with amiloride and amlodipine are ineffective in decreasing the frequency of sleep apnea episodes in patients with moderate OSA and hypertension. Both treatments have comparable effects in lowering both office and ambulatory blood pressure. The notion that treatments could offer benefits for both OSA and hypertension remains to be demonstrated. TRIAL REGISTRATION CLINICALTRIALS.
GOV IDENTIFIER
NCT01896661.
Topics: Humans; Male; Female; Double-Blind Method; Hypertension; Middle Aged; Blood Pressure Monitoring, Ambulatory; Antihypertensive Agents; Chlorthalidone; Amlodipine; Sleep Apnea, Obstructive; Amiloride; Diuretics; Blood Pressure; Polysomnography; Aged
PubMed: 38781664
DOI: 10.1016/j.sleep.2024.05.035 -
Lab on a Chip Jun 2024The volume and composition of airway surface liquid (ASL) is regulated by liquid secretion and absorption across airway epithelia, controlling the pH, solute...
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 " trachea-chip") to study the dynamic properties of ASL volume regulation. The trachea-chip allows real-time measurement of ASL transport () 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 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.
Topics: Trachea; Amiloride; Animals; Lab-On-A-Chip Devices; Humans; Microfluidic Analytical Techniques; Respiratory Mucosa
PubMed: 38779981
DOI: 10.1039/d4lc00134f -
American Journal of Physiology. Renal... May 2024Diabetes is closely associated with K disturbances during disease progression and treatment. However, it remains unclear whether K imbalance occurs in diabetes with...
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 to 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), epithelia 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 level 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.
PubMed: 38779755
DOI: 10.1152/ajprenal.00240.2023 -
American Journal of Physiology. Renal... Jul 2024Interleukin (IL)-17A contributes to hypertension in preclinical models. T helper 17 and dendritic cells are activated by NaCl, which could involve the epithelial Na...
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-1β, and IL-10 were determined by immunoassays in plasma from two patient cohorts before and after amiloride treatment: ) patients with type 2 diabetes mellitus (T2DM) and treatment-resistant hypertension ( = 69, amiloride 5-10 mg/day for 8 wk) and ) patients with hypertension and type 1 diabetes mellitus (T1DM) ( = 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-1β. 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. 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.
Topics: Amiloride; Humans; Interleukin-17; Animals; Diabetes Mellitus, Type 2; Interleukin-6; Male; Middle Aged; Hypertension; Female; Epithelial Sodium Channel Blockers; Tumor Necrosis Factor-alpha; Aged; Mice; Epithelial Sodium Channels; Mice, Inbred C57BL; Antihypertensive Agents; Macrophages; Blood Pressure; Diabetes Mellitus, Type 1
PubMed: 38779752
DOI: 10.1152/ajprenal.00268.2023 -
Hypertension Research : Official... May 2024Hypertension remains the leading cause of morbidity and mortality worldwide. Despite its prevalence, the development of novel antihypertensive therapies has only... (Review)
Review
Hypertension remains the leading cause of morbidity and mortality worldwide. Despite its prevalence, the development of novel antihypertensive therapies has only recently accelerated, with novel agents not yet commercialized, leaving a substantial proportion of individuals resistant to existing treatments. The intricate pathophysiology of hypertension is now understood to involve chronic low-grade inflammation, which places the immune system in the spotlight as a potential target for new therapeutics. This review explores the factors that initiate and sustain an immune response in hypertension, offering insights into potential targets for new treatments. Several factors contribute to immune activation in hypertension, including diet and damage-associated molecular pattern (DAMP) generation. Diets rich in fat or sodium can promote inflammation by inducing intestinal barrier dysfunction and triggering salt-sensitive receptors in T cells and dendritic cells. DAMPs, such as extracellular adenosine triphosphate and heat-shock protein 70, are released during episodes of increased blood pressure, contributing to immune cell activation and inflammation. Unconventional innate-like γδ T cells contribute to initiating and maintaining an immune response through their potential involvement in antigen presentation and regulating cytokine-mediated responses. Immunologic memory, sustained through the formation of effector memory T cells after exposure to hypertensive insults, likely contributes to maintaining an immune response in hypertension. When exposed to hypertensive insults, these memory cells are rapidly activated and contribute to elevated blood pressure and end-organ damage. Evidence from human hypertension, although limited, supports the relevance of distinct immune pathways in hypertension, and highlights the potential of targeted immune interventions in human hypertension. Diet and acute bouts of high blood pressure result in the release of dietary triggers, neoantigens, and damage-associated molecular patterns (DAMPs), which promote immune system activation. Elements such as lipopolysaccharides (LPS), sodium, heat-shock protein (HSP)70, extracellular adenosine triphosphate (eATP), and growth arrest-specific 6 (GAS6) promote activation of innate immune cells such as dendritic cells (DCs) and monocytes (Mo) through their respective receptors (toll-like receptor [TLR]4, amiloride-sensitive epithelial sodium channel [ENaC], TLR2/4, P2X7 receptor [P2RX7], and Axl) leading to costimulatory molecule expression and interleukin (IL)-1β and IL-23 production. The neoantigens HSP70 and isolevuglandins (IsoLGs) are presented to T cells by DCs and possibly γδ T cells, triggering T cell activation, IL-17 and interferon (IFN)-γ production, and the formation of T effector memory (T) cells in the kidney, perivascular adipose tissue, bone marrow, and spleen. Exposure of T cells to their cognate antigen or previous activating stimuli causes these cells rapid expansion and activation. Cumulatively, this inflammatory state contributes to hypertension and end-organ damage. The figure was created using images from smart.servier.com and is licensed under a Creative Commons Attribution 4.0 license (CC BY 4.0).
PubMed: 38778172
DOI: 10.1038/s41440-024-01731-6