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International Journal of Molecular... Jul 2022Inflammation and hypoxia impair alveolar barrier tightness, inhibit Na- and fluid reabsorption, and cause edema. We tested whether stimulated alveolar macrophages affect...
Inflammation and hypoxia impair alveolar barrier tightness, inhibit Na- and fluid reabsorption, and cause edema. We tested whether stimulated alveolar macrophages affect alveolar Na-transport and whether hypoxia aggravates the effects of inflammation, and tested for involved signaling pathways. Primary rat alveolar type II cells (rA2) were co-cultured with rat alveolar macrophages (NR8383) or treated with NR8383-conditioned media after stimulation with lipopolysaccharide (LPS; 1 µg/mL) and exposed to normoxia and hypoxia (1.5% O). LPS caused a fast, transient increase in TNFα and IL-6 mRNA in macrophages and a sustained increase in inducible nitric oxide synthase (NOS2) mRNA in macrophages and in rA2 cells resulting in elevated nitrite levels and secretion of TNF-α and IL-6 into culture media. In normoxia, 24 h of LPS treated NR8383 decreased the transepithelial electrical resistance (TEER) of co-cultures, of amiloride-sensitive short circuit current (ISC); whereas Na/K-ATPase activity was not affected. Inhibition was also seen with conditioned media from LPS-stimulated NR8383 on rA2, but was less pronounced after dialysis to remove small molecules and nitrite. The effect of LPS-stimulated macrophages on TEER and Na-transport was fully prevented by the iNOS-inhibitor L-NMMA applied to co-cultures and to rA2 mono-cultures. Hypoxia in combination with LPS-stimulated NR8383 totally abolished TEER and ISCΔamil. These results indicate that the LPS-stimulation of alveolar macrophages impairs alveolar epithelial Na-transport by NO-dependent mechanisms, where part of the NO is produced by rA2 induced by signals from LPS stimulated alveolar macrophages.
Topics: Animals; Culture Media, Conditioned; Hypoxia; Inflammation; Interleukin-6; Lipopolysaccharides; Macrophages, Alveolar; Nitrites; RNA, Messenger; Rats; Sodium; Tumor Necrosis Factor-alpha
PubMed: 35955448
DOI: 10.3390/ijms23158315 -
Viruses Feb 2022(1) Background: Porcine deltacoronavirus (PDCoV) is a newly emerged enteric virus affecting pig breeding industries worldwide, and its pathogenic mechanism remains...
(1) Background: Porcine deltacoronavirus (PDCoV) is a newly emerged enteric virus affecting pig breeding industries worldwide, and its pathogenic mechanism remains unclear. (2) Methods: In this study, we preliminarily identified the endocytic pathway of PDCoV in PK-15 cells, using six chemical inhibitors (targeting clathrin-mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis pathway and endosomal acidification), overexpression of dominant-negative (DN) mutants to treat PK-15 cells and proteins knockdown. (3) Results: The results revealed that PDCoV entry was not affected after treatment with chlorpromazine (CPZ), 5-(N-ethyl-N-isopropyl) amiloride (EIPA)or ammonium chloride (NHCl), indicating that the entry of PDCoV into PK-15 cells were clathrin-, micropinocytosis-, PH-independent endocytosis. Conversely, PDCoV infection was sensitive to nystatin, dynasore and methyl-β-cyclodextrin (MβCD) with reduced PDCoV internalization, indicating that entry of PDCoV into PK-15 cells was caveolae-mediated endocytosis that required dynamin and cholesterol; indirect immunofluorescence and shRNA interference further validated these results. (4) Conclusions: In conclusion, PDCoV entry into PK-15 cells depends on caveolae-mediated endocytosis, which requires cholesterol and dynamin. Our finding is the first initial identification of the endocytic pathway of PDCoV in PK-15 cells, providing a theoretical basis for an in-depth understanding of the pathogenic mechanism of PDCoV and the design of new antiviral targets.
Topics: Animals; Caveolae; Cell Line; Cholesterol; Clathrin; Deltacoronavirus; Dynamins; Endocytosis; Swine; Virus Internalization
PubMed: 35336903
DOI: 10.3390/v14030496 -
Frontiers in Pediatrics 2022Liddle syndrome (LS) is a monogenic hypertension consistent with autosomal dominant inheritance, often with early onset high blood pressure in childhood or adolescence....
OBJECTIVE
Liddle syndrome (LS) is a monogenic hypertension consistent with autosomal dominant inheritance, often with early onset high blood pressure in childhood or adolescence. This study aimed to identify the pathogenicity of a nonsense mutation in in a Chinese family with LS and the long-term outcomes of tailored treatment with amiloride.
METHODS
To explore the pathogenicity of candidate variant reported in 2015 by our team, we constructed mutant and wild-type models and measured amiloride-sensitive current in () cells using patch clamp technique. Participants were followed up for 7 years after tailored treatment with amiloride.
RESULTS
A nonsense variant was detected in six members, two of whom were pediatric patients. This mutation resulted in a termination codon at codon 572, truncating the Pro-Pro-Pro-X-Tyr motif. The mutant epithelial sodium channels displayed higher amiloride-sensitive currents than the wild-type channels ( < 0.05). Tailored treatment with amiloride achieved ideal blood pressure control in all patients with normal cardiorenal function, and no adverse events occurred during follow-up.
CONCLUSION
We found the pathogenicity of a nonsense mutation (p.Glu571*) with enhanced amiloride-sensitive currents in a LS family with young patients. Tailored treatment with amiloride may be an effective strategy for the long-term control of blood pressure and protection from target organ damage or cardiovascular events, including children and youth patients with LS.
PubMed: 35685915
DOI: 10.3389/fped.2022.887214 -
Frontiers in Physiology 2021Nephron loss initiates compensatory hemodynamic and cellular effects on the remaining nephrons. Increases in single nephron glomerular filtration rate and tubular flow...
Nephron loss initiates compensatory hemodynamic and cellular effects on the remaining nephrons. Increases in single nephron glomerular filtration rate and tubular flow rate exert higher fluid shear stress (FSS) on tubules. In principal cell (PC) culture models FSS induces ERK, and ERK is implicated in the regulation of transepithelial sodium (Na) transport, as well as, proliferation. Thus, we hypothesize that high tubular flow and FSS mediate ERK activation in the cortical collecting duct (CCD) of solitary kidney which regulates amiloride sensitive Na transport and affects CCD cell number. Immunoblotting of whole kidney protein lysate was performed to determine phospho-ERK (pERK) expression. Next, sham and unilateral nephrectomized mice were stained with anti-pERK antibodies, and dolichos biflorus agglutinin (DBA) to identify PCs with pERK. Murine PCs (mpkCCD) were grown on semi-permeable supports under static, FSS, and FSS with U0126 (a MEK1/2 inhibitor) conditions to measure the effects of FSS and ERK inhibition on amiloride sensitive Na short circuit current (). pERK abundance was greater in kidney lysate of unilateral vs. sham nephrectomies. The total number of cells in CCD and pERK positive PCs increased in nephrectomized mice (9.3 ± 0.4 vs. 6.1 ± 0.2 and 5.1 ± 0.5 vs. 3.6 ± 0.3 cell per CCD nephrectomy vs. sham, respectively, > 6 per group, < 0.05). However, Ki67, a marker of proliferation, did not differ by immunoblot or immunohistochemistry in nephrectomy samples at 1 month compared to sham. Next, amiloride sensitive in static mpkCCD cells was 25.3 ± 1.7 μA/cm ( = 21), but after exposure to 24 h of FSS the increased to 41.4 ± 2.8 μA/cm ( = 22; < 0.01) and returned to 19.1 ± 2.1 μA/cm ( = 18, < 0.01) upon treatment with U0126. Though FSS did not alter α- or γ-ENaC expression in mpkCCD cells, γ-ENaC was reduced in U0126 treated cells. In conclusion, pERK increases in whole kidney and, specifically, CCD cells after nephrectomy, but pERK was not associated with active proliferation at 1-month post-nephrectomy. studies suggest high tubular flow induces ERK dependent ENaC Na absorption and may play a critical role in Na balance post-nephrectomy.
PubMed: 33633581
DOI: 10.3389/fphys.2021.583453 -
JCI Insight Feb 2022Exchange proteins directly activated by cAMP (Epacs) are abundantly expressed in the renal tubules. We used genetic and pharmacological tools in combination with...
Exchange proteins directly activated by cAMP (Epacs) are abundantly expressed in the renal tubules. We used genetic and pharmacological tools in combination with balance, electrophysiological, and biochemical approaches to examine the role of Epac1 and Epac2 in renal sodium handling. We demonstrate that Epac1-/- and Epac2-/- mice exhibit a delayed anti-natriuresis to dietary sodium restriction despite augmented aldosterone levels. This was associated with a significantly lower response to the epithelial Na+ channel (ENaC) blocker amiloride, reduced ENaC activity in split-opened collecting ducts, and defective posttranslational processing of α and γENaC subunits in the KO mice fed with a Na+-deficient diet. Concomitant deletion of both isoforms led to a marginally greater natriuresis but further increased aldosterone levels. Epac2 blocker ESI-05 and Epac1&2 blocker ESI-09 decreased ENaC activity in Epac WT mice kept on the Na+-deficient diet but not on the regular diet. ESI-09 injections led to natriuresis in Epac WT mice on the Na+-deficient diet, which was caused by ENaC inhibition. In summary, our results demonstrate similar but nonredundant actions of Epac1 and Epac2 in stimulation of ENaC activity during variations in dietary salt intake. We speculate that inhibition of Epac signaling could be instrumental in treatment of hypertensive states associated with ENaC overactivation.
Topics: Animals; Biomarkers; Calcium Channels; Cells, Cultured; Disease Models, Animal; Gene Expression Regulation; Guanine Nucleotide Exchange Factors; Kidney Diseases; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Natriuresis; RNA; Sodium; TRPV Cation Channels
PubMed: 34914636
DOI: 10.1172/jci.insight.145653 -
Research Square Mar 2023Acute lung injury is characterized by overwhelmingly elevated PAI-1 in both lung edema fluid and the circulating system. The role of increased PAI-1, encoded by...
Acute lung injury is characterized by overwhelmingly elevated PAI-1 in both lung edema fluid and the circulating system. The role of increased PAI-1, encoded by Serpine1 gene, in the regeneration of injured lung epithelium has not been understood completely. This study aimed to investigate the role of Serpine1 in the regulation of alveolar type 2 epithelial cell (AT2) fate in a humanized mouse line carrying diseased mutants (Serpine1Tg). Wild type (wt) and Serpine1Tg AT2 cells were either cultured as monolayers or 3D alveolospheres. Colony forming assay and total surface area of organoids were analyzed. AT1 and AT2 cells in organoids were counted by immunohistochemistry and fluorescence-activated cell sorting (FACS). To test the potential effects of elevated PAI-1 on the permeability in the epithelial monolayers, we digitized the biophysical properties of polarized AT2 monolayers grown at the air-liquid interface. A significant reduction in total AT2 cells harvested in Serpine1Tg mice was observed compared with wt controls. AT2 cells harvested from Serpine1Tg mice reduced significantly over the wt controls. Spheroids formed by Serpine1Tg AT2 cells were lesser than wt control. Similarly, the corresponding surface area, a readout of realveolarization of injured epithelium, was markedly reduced in Serpine1Tg organoids. FACS analysis revealed a significant suppression in the number of AT2 cells, in particular, the CD44+ subpopulation, in Serpine1Tg organoids. A lesser ratio of AT1:AT2 cells in Serpine1Tg organoids was observed compared with wt cultures. There was a significant increase in transepithelial resistance but not amiloride inhibition. Our study suggests elevated PAI-1 in injured lungs downregulates alveolar epithelial regeneration by reducing the AT2 self-renewal, particularly in the CD44+ cells.
PubMed: 36909505
DOI: 10.21203/rs.3.rs-2289657/v1 -
American Journal of Physiology. Renal... Aug 2020We investigated the regulation of Na and K excretion and the epithelial Na channel (ENaC) in mice lacking the gene for aldosterone synthase (AS) using clearance methods...
We investigated the regulation of Na and K excretion and the epithelial Na channel (ENaC) in mice lacking the gene for aldosterone synthase (AS) using clearance methods to assess excretion and electrophysiology and Western blot analysis to test for ENaC activity and processing. After 1 day of dietary Na restriction, AS mice lost more Na in the urine than AS mice did. After 1 wk on this diet, both genotypes strongly reduced urinary Na excretion, but creatinine clearance decreased only in AS mice. Only AS animals exhibited increased ENaC function, assessed as amiloride-sensitive whole cell currents in collecting ducts or cleavage of αENaC and γENaC in Western blots. To assess the role of aldosterone in the excretion of a K load, animals were fasted overnight and refed with high-K or low-K diets for 5 h. Both AS and AS mice excreted a large amount of K during this period. In both phenotypes the excretion was benzamil sensitive, indicating increased K secretion coupled to ENaC-dependent Na reabsorption. However, the increase in plasma K under these conditions was much larger in AS animals than in AS animals. In both groups, cleavage of αENaC and γENaC increased. However, Na current measured ex vivo in connecting tubules was enhanced only in AS mice. We conclude that in the absence of aldosterone, mice can conserve Na without ENaC activation but at the expense of diminished glomerular filtration rate. Excretion of a K load can be accomplished through aldosterone-independent upregulation of ENaC, but aldosterone is required to excrete the excess K without hyperkalemia.
Topics: Animals; Cytochrome P-450 CYP11B2; Epithelial Sodium Channels; Kidney Tubules, Collecting; Mice; Natriuresis; Potassium; Sodium; Sodium, Dietary
PubMed: 32628540
DOI: 10.1152/ajprenal.00204.2020 -
International Journal of Molecular... Dec 2020The inversion of the pH gradient in malignant tumors, known as the pH paradigm, is increasingly becoming accepted by the scientific community as a hallmark of cancer.... (Review)
Review
The inversion of the pH gradient in malignant tumors, known as the pH paradigm, is increasingly becoming accepted by the scientific community as a hallmark of cancer. Accumulated evidence shows that this is not simply a metabolic consequence of a dysregulated behavior, but rather an essential process in the physiopathology of accelerated proliferation and invasion. From the over-simplification of increased lactate production as the cause of the paradigm, as initially proposed, basic science researchers have arrived at highly complex and far-reaching knowledge, that substantially modified that initial belief. These new developments show that the paradigm entails a different regulation of membrane transporters, electrolyte exchangers, cellular and membrane enzymes, water trafficking, specialized membrane structures, transcription factors, and metabolic changes that go far beyond fermentative glycolysis. This complex world of dysregulations is still shuttered behind the walls of experimental laboratories and has not yet reached bedside medicine. However, there are many known pharmaceuticals and nutraceuticals that are capable of targeting the pH paradigm. Most of these products are well known, have low toxicity, and are also inexpensive. They need to be repurposed, and this would entail shorter clinical studies and enormous cost savings if we compare them with the time and expense required for the development of a new molecule. Will targeting the pH paradigm solve the "cancer problem"? Absolutely not. However, reversing the pH inversion would strongly enhance standard treatments, rendering them more efficient, and in some cases permitting lower doses of toxic drugs. This article's goal is to describe how to reverse the pH gradient inversion with existing drugs and nutraceuticals that can easily be used in bedside medicine, without adding toxicity to established treatments. It also aims at increasing awareness among practicing physicians that targeting the pH paradigm would be able to improve the results of standard therapies. Some clinical cases will be presented as well, showing how the pH gradient inversion can be treated at the bedside in a simple manner with repurposed drugs.
Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Biomarkers; Clinical Decision-Making; Disease Management; Extracellular Space; Humans; Hydrogen-Ion Concentration; Intracellular Space; Molecular Targeted Therapy; Neoplasms; Prognosis; Sodium-Hydrogen Exchanger 1; Sodium-Hydrogen Exchanger 3; Voltage-Gated Sodium Channel Blockers; Voltage-Gated Sodium Channels
PubMed: 33287221
DOI: 10.3390/ijms21239221 -
Research (Washington, D.C.) 2023Cell replacement therapy using neural progenitor cells (NPCs) has been shown to be an effective treatment for ischemic stroke. However, the therapeutic effect is...
Cell replacement therapy using neural progenitor cells (NPCs) has been shown to be an effective treatment for ischemic stroke. However, the therapeutic effect is unsatisfactory due to the imbalanced homeostasis of the local microenvironment after ischemia. Microenvironmental acidosis is a common imbalanced homeostasis in the penumbra and could activate acid-sensing ion channels 1a (ASIC1a), a subunit of proton-gated cation channels following ischemic stroke. However, the role of ASIC1a in NPCs post-ischemia remains elusive. Here, our results indicated that ASIC1a was expressed in NPCs with channel functionality, which could be activated by extracellular acidification. Further evidence revealed that ASIC1a activation inhibited NPC migration and neurogenesis through RhoA signaling-mediated reorganization of filopodia formation, which could be primarily reversed by pharmacological or genetic disruption of ASIC1a. In vivo data showed that the knockout of the ASIC1a gene facilitated NPC migration and neurogenesis in the penumbra to improve behavioral recovery after stroke. Subsequently, ASIC1a gain of function partially abrogated this effect. Moreover, the administration of ASIC1a antagonists (amiloride or Psalmotoxin 1) promoted functional recovery by enhancing NPC migration and neurogenesis. Together, these results demonstrate targeting ASIC1a is a novel strategy potentiating NPC migration toward penumbra to repair lesions following ischemic stroke and even for other neurological diseases with the presence of niche acidosis.
PubMed: 37275123
DOI: 10.34133/research.0105 -
International Journal of Molecular... Mar 2021The K-sparing diuretic amiloride shows off-target anti-cancer effects in multiple rodent models. These effects arise from the inhibition of two distinct cancer targets:...
The K-sparing diuretic amiloride shows off-target anti-cancer effects in multiple rodent models. These effects arise from the inhibition of two distinct cancer targets: the trypsin-like serine protease urokinase-type plasminogen activator (uPA), a cell-surface mediator of matrix degradation and tumor cell invasiveness, and the sodium-hydrogen exchanger isoform-1 (NHE1), a central regulator of transmembrane H that supports carcinogenic progression. In this study, we co-screened our library of 5- and 6-substituted amilorides against these two targets, aiming to identify single-target selective and dual-targeting inhibitors for use as complementary pharmacological probes. Closely related analogs substituted at the 6-position with pyrimidines were identified as dual-targeting (pyrimidine uPA IC = 175 nM, NHE1 IC = 266 nM, uPA selectivity ratio = 1.5) and uPA-selective (methoxypyrimidine uPA IC = 86 nM, NHE1 IC = 12,290 nM, uPA selectivity ratio = 143) inhibitors, while high NHE1 potency and selectivity was seen with 5-morpholino ( NHE1 IC = 129 nM, uPA IC = 10,949 nM; NHE1 selectivity ratio = 85) and 5-(1,4-oxazepine) ( NHE1 IC = 85 nM, uPA IC = 5715 nM; NHE1 selectivity ratio = 67) analogs. Together, these amilorides comprise a new toolkit of chemotype-matched, non-cytotoxic probes for dissecting the pharmacological effects of selective uPA and NHE1 inhibition versus dual-uPA/NHE1 inhibition.
Topics: Amiloride; Breast Neoplasms; Cell Line, Tumor; Diuretics; Female; Humans; Models, Molecular; Neoplasm Invasiveness; Sodium-Hydrogen Exchanger 1; Structure-Activity Relationship; Urokinase-Type Plasminogen Activator
PubMed: 33804289
DOI: 10.3390/ijms22062999