Heterogeneity among β-adrenoreceptor blockers in the modulation of energy-dependent uptake of the organic cation amantadine by rat renal tubules

1999 ◽  
Vol 77 (6) ◽  
pp. 407-413 ◽  
Author(s):  
Dwayne G Stupack ◽  
Miguel R Escobar ◽  
Maxine Carlisle ◽  
Thomas Davie ◽  
Daniel S Sitar
Keyword(s):  
Organic Cation ◽  
Renal Tubule ◽  
Competitive Inhibition ◽  
Kinetic Studies ◽  
Distal Tubule ◽  
Renal Tubules ◽  
Dependent Inhibition ◽  
Presence And Absence ◽  
Energy Dependent

Eight representative β-adrenoreceptor blocking drugs, acebutolol, atenolol, labetalol, metoprolol, nadolol, pindolol, propranolol, and timolol, were studied in vitro with respect to their potential to block energy-dependent uptake of [3H]amantadine into proximal and distal rat renal tubule fragments in the presence and absence of bicarbonate. Five of the eight β-adrenoreceptor blockers showed a dose-dependent inhibition of renal tubule accumulation of amantadine: labetalol, metoprolol, pindolol, propranolol, and timolol. Labetalol was the only β-adrenoreceptor blocker with greater inhibitory potency in phosphate-based buffer than in bicarbonate-based buffer. Propranolol was the most potent inhibitor of renal tubule amantadine accumulation with IC50values of 15 ± 10 and 31 ± 11 µM for proximal and distal tubule fragments, respectively, in a bicarbonate-based buffer environment. Inhibition in a phosphate-based buffer was less potent only in proximal tubules, with an IC50of 76 ± 30 µM. Kinetic studies of propranolol inhibition of amantadine uptake were consistent with both uncompetitive and competitive inhibition mechanisms in bicarbonate-based buffer in both proximal and distal renal tubule segments. There was no chiral preference between the R and S forms of propranolol for the inhibitory effects observed. These data suggest that there is potential for selection among the β-adrenoreceptor blocking drugs to minimize or restrict the inhibition of amantadine energy-dependent uptake at the organic cation ion uptake sites characterized by amantadine in the presence and absence of bicarbonate.Key words: organic cation transport, kidney, amantadine, β-adrenoreceptor blockers, bicarbonate.

scholarly journals Effects of 2-mercaptoacetate in isolated liver mitochondria in vitro. Competitive inhibition of 3-hydroxybutyrate dehydrogenase and depression of the β-oxidation pathway

1982 ◽  
Vol 206 (1) ◽  
pp. 53-59 ◽  
Author(s):  
F Bauché ◽  
D Sabourault ◽  
Y Giudicelli ◽  
J Nordmann ◽  
R Nordmann
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Oxidation ◽  
Competitive Inhibition ◽  
Liver Mitochondria ◽  
Inhibitory Effect ◽  
Acid Oxidation ◽  
Membrane Bound ◽  
Energy Dependent ◽  
Isolated Liver

The effects of 2-mercaptoacetate on the respiration rates induced by different substrates were studied in vitro in isolated liver mitochondria. With palmitoyl-L-carnitine or 2-oxoglutarate as the substrate, the ADP-stimulated respiration (State 3) was dose-dependently inhibited by 2-mercaptoacetate. with glutamate or succinate as the substrate. State-3 respiration was only slightly inhibited by 2-mercaptoacetate. In contrast, the oxidation rate of 3-hydroxybutyrate was competitively inhibited by 2-mercaptoacetate in both isolated mitochondria and submitochondrial particles. In uncoupled mitochondria and in mitochondria in which ATP- and GTP-dependent acyl-CoA biosynthesis was inhibited, the inhibitory effect of 2-mercaptoacetate on palmitoyl-L-carnitine oxidation was abolished; under the same conditions, however, inhibition of 3-hydroxybutyrate oxidation by 2-mercaptoacetate still persisted. These results led to the following conclusions: 2-mercaptoacetate itself enters the mitochondrial matrix, inhibits fatty acid oxidation through a mechanism requiring an energy-dependent activation of 2-mercaptoacetate and itself inhibits 3-hydroxybutyrate oxidation through a competitive inhibition of the membrane-bound 3-hydroxybutyrate dehydrogenase. This study also strongly suggests that the compound responsible for the inhibition of fatty acid oxidation is 2-mercaptoacetyl-CoA.

MO068A KIDNEY-ON-THE-CHIP APPROACH USING PRIMARY HUMAN TUBULAR CELLS IN A 3D CO-CULTURE SYSTEM

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Gregor J Wilken ◽  
Julian Aurelio Marschner ◽  
Paola Romagnani ◽  
Hans-Joachim Anders
Keyword(s):  
Cell Culture ◽  
Immune Cells ◽  
Blood Cells ◽  
Culture System ◽  
Renal Tubule ◽  
Tubular Structure ◽  
Renal Tubules ◽  
Tubular Cells

Abstract Background and Aims Conventional 2D mono-culture in vitro models using immortalized cell lines are still widely used in experimental nephrology, although their value is limited by poor translatability and predictive value for the in vivo or even human situation. The implementation of more sophisticated in vitro assays as routine cell culture systems is often limited by complex protocols and long lasting procedures. We aimed to establish and validate a relatively easy-to-use but yet (patho-) physiologically relevant cell culture assay that mimics key aspects of the in vivo situation of renal tubules, including a leak-thight epithelium with a luminal and baso-lateral side, interstitial matrix, a peri-tubular capillary and circulating blood cells inside its lumen. Method We utilized the 3-lane OrganoPlate® system (Mimetas, Leiden, Netherlands) as a scaffold. After infusing a collagen I matrix in the middle channel (C2), primary human renal progenitor cells are seeded into the upper channel (C1), adhering to the C2-matrix. The plate is put on a perfusion rocker (Mimetas), that facilitates continuous gravity-triggered bi-directional perfusion of C1. Within 48h the cells form a leak-tight tubular structure with a continuous lumen. Next, human endothelial cells are seeded into the bottom channel (C3), which adhered to the opposite site of the C2-matrix and – upon continuous perfusion – formed a vessel-like structure with a continuous lumen, as well. Finally, primary human white blood cells were isolated and seeded into C3 (see figure). Results Establishing the whole tubule-on-the-chip as described above takes on average three days. We investigated its operational life span by monitoring the barrier integrity of the tubular structure in C1 using a fluorescence-labeled dextran (150 kDa). Over a course of 5 days the tubular integrity did not decline, suggesting that the co-culture system remains stable and functional for at least 5 days. In accordance with other studies, the primary human tubular cells constituting the 3D tubule-on-the-chip expressed higher levels of functionally relevant proteins, e..g the tight-junction protein ZO-1 and the sodium-potassium-pump Na-K-ATPase, compared to standard 2D settings without perfusion. This emphasizes, that even primary cells show a physiologically reduced phenotype in standard 2D settings, which possibly impedes the identification and representative quantification of physiologically and hence also patho-physiologically relevant mechanisms in vitro. To study the interaction of cells in the tubule-on-the-chip, we investigated the recruitment of immune cells from C3 (vessel) across C2 (interstitium) to C1 (renal tubule), which - in vivo - represents a detrimental mechanism of action in intrarenal forms of AKI. Under baseline conditions the immune cells inserted into C3 did not leave their compartment. Upon damaging the tubular cells in C1 with extracellular histones, neutrophils and monocytes left C3 (extravasation), migrated through C2 and could be found in close contact with epithelial cells of C1. This serves as a proof of principle, that the tubule-on-the-chip is applicable to study complex cell-cell and cell-substrate interactions, such as chemokine-mediate immune cell homing. Measuring lactate dehydrogenase release for a number of known nephrotoxic agents revealed, that tubular cells forming a 3D-structure while kept under perfusion show significantly different responses to the same dose compared to standard 2D conditions, suggesting that dose-response studies using target cells out of their tissues context can be misleading when extrapolating results from in vitro to in vivo. Conclusion The results of this study suggest, that sophisticated 3D co-culture models of a renal tubule including an interstitial compartment, a peri-tubluar capillary and circulating immune cells are feasible and potentially suited to allow for in depth mechanistic studies in vitro.

Cimetidine and procainamide secretion by proximal tubules in vitro

1982 ◽  
Vol 242 (6) ◽  
pp. F672-F680 ◽  
Author(s):  
T. D. McKinney ◽  
K. V. Speeg
Keyword(s):  
Competitive Inhibition ◽  
Proximal Tubules ◽  
Renal Tubules ◽  
Co2 Absorption ◽  
Organic Base ◽  
Organic Bases ◽  
Straight Tubules ◽  
Tubule Lumen ◽  
High Concentrations

Previous studies have shown that organic bases, including some drugs, are secreted by renal proximal tubules. The present studies examined the transport of the organic bases cimetidine and procainamide by rabbit proximal straight tubules perfused in vitro. Both drugs were secreted into the tubule lumen. [3H]cimetidine secretion was reduced by quinidine, procainamide, and N-acetylprocainamide. Previous studies showed that cimetidine secretion was reduced by other organic bases. Hypothermia and ouabain inhibited [3H]procainamide secretion as was shown previously for cimetidine secretion. [3H]procainamide secretion was also reduced by quinidine, cimetidine, procainamide, and N-acetylprocainamide but not by probenecid. High concentrations of cimetidine (10(-3) M) had no effect on the rates of fluid or total CO2 absorption. When analyzed in terms of Michaelis-Menten kinetics, the effect of cimetidine on procainamide secretion and procainamide on cimetidine secretion was consistent with competitive inhibition. The results suggest that both cimetidine and procainamide are secreted into the lumen of proximal straight tubules predominately by an organic base transport mechanism. These studies raise the possibility that some of these drugs might compete for a common secretory mechanism in renal tubules and reduce the elimination of each other.

scholarly journals Chemical profiling, in vitro antioxidant and antidiabetic assessment of flavonoids from the ethanol extract of Hermannia geniculata root

2021 ◽  
Vol 19 (1) ◽  
pp. 1-13
Author(s):  
L.A. Adeniran ◽  
C.P. Palanisamy ◽  
A.O.T. Ashafa
Keyword(s):  
Competitive Inhibition ◽  
Antioxidant Properties ◽  
Reactive Oxygen ◽  
Ethanol Extract ◽  
Kinetic Studies ◽  
Retention Factor ◽  
Chemical Profiling ◽  
Ic50 Values ◽  
Inhibitory Potential

Determination of the in vitro antioxidant and the inhibitory potential of flavonoids from Hermannia geniculata (FHG) roots on diabetes-linked enzymes was carried out. The chemical profiling of FHG roots extract was investigated using High Pressure Thin Layer Chromatography (HPTLC) fingerprint analysis. The reactive oxygen scavenging potential of the extract was analyzed. Starch solution (1%) was reacted with different concentrations of FHG extract to determine the α-amylase inhibitory potential of the extract while α- glucosidase inhibition assay was carried out through incubation of different concentrations of the extract followed by addition of p-ntrophenyl-α-Dglucopyranoside solution. HPTLC results indicated the presence of flavonoids/ phenolcarboxylic acid, and Kaemferol (Rf 0.80) were detected in the extract with retention factor Rf. ranging from 0.08 to 0.95. FHG extract showed commendable antioxidant properties with IC50 values (3.07± 0.12, 2.13± 0.67) µg/mL for 1, 1-diphenyl-2- picrylhydrazyl (DPPH) and 2, 2-azino-bis (3- ethylbenzothiazoline-6-sulphonic) acid (ABTS) radicals which were lower and significantly different (p<0.05) compared to standard silymarin with IC50: (3.55± 0.10, 2.77± 0.75) µg/mL for DPPH and ABTS respectively. The results indicated mild inhibition of α-amylase with IC50: (5.55± 0.37) µg/mL which was higher and significantly different (p<0.05) from acarbose with IC50: (3.81± 0.29) µg/mL. Moreover, the extract showed 73% inhibition of α-glucosidase. Kinetic studies of FHG extract revealed competitive and mixed non-competitive inhibition of α- amylase and α-glucosidase respectively. This study indicated FHG capabilities of scavenging reactive oxygen species and reducing hydrolysis of starch responsible for post-prandial hyperglyceamia seen in type 2 diabetes mellitus. Keywords: Antidiabetic, Antioxidant, Flavonoids, Hermannia geniculate, HPTLC

scholarly journals Kinetics of acetylcholinesterase inhibition by an aqueous extract of Cuminum cyminum seeds.

2014 ◽  
Vol 2 (1) ◽  
pp. 64-68 ◽  
Author(s):  
Suresh Kumar ◽  
Suman Chowdhury
Keyword(s):  
Aqueous Extract ◽  
Side Effect ◽  
Competitive Inhibition ◽  
Kinetic Studies ◽  
Symptomatic Treatment ◽  
Acetylcholinesterase Inhibition ◽  
Ache Inhibitors ◽  
Maximum Inhibition ◽  
Ellman’S Method

The cholinergic hypothesis of Alzheimer’s disease (AD) has provided the rationale for the current pharmacotherapy of this disease. Acetylcholinesterase (AChE) inhibitors are currently the only approved therapy for the symptomatic treatment of AD. The current drugs available in the market has shown various side effect which prompted scientist to search for new and potent AChE inhibitors which exerts minimal side effect in AD patient. In present study, an aqueous extract of Cumin cyminum was tested for in vitro acetylcholinesterase inhibitory activity based on Ellman’s method. C. cyminum showed maximum inhibition of 76.90±0.003% in an aqueous extract at 50μg/ml final concentration. Further studies were conducted to elucidate the mode of AChE inhibition by kinetic studies. Competitive inhibition was observed at lower concentrations (12.5μg/ml & 25μg/ml) and mixed inhibition was observed at higher concentrations (50μg/ml & 100μg/ml). DOI: http://dx.doi.org/10.3126/ijasbt.v2i1.9348   Int J Appl Sci Biotechnol, Vol 2(1): 64-68

scholarly journals Basolateral LPS inhibits NHE3 and HCO3− absorption through TLR4/MyD88-dependent ERK activation in medullary thick ascending limb

2011 ◽  
Vol 301 (6) ◽  
pp. C1296-C1306 ◽  
Author(s):  
Bruns A. Watts ◽  
Thampi George ◽  
Edward R. Sherwood ◽  
David W. Good
Keyword(s):  
Renal Tubule ◽  
Interleukin 1 ◽  
Mitogen Activated Protein Kinase ◽  
Renal Tubules ◽  
Thick Ascending Limb ◽  
Maximal Velocity ◽  
Erk Pathway ◽  
Medullary Thick Ascending Limb ◽  
Erk Phosphorylation

Sepsis is associated with defects in renal tubule function, but the underlying mechanisms are incompletely understood. Recently, we demonstrated that Gram-negative bacterial lipopolysaccharide (LPS) inhibits HCO3− absorption in the medullary thick ascending limb (MTAL) through activation of Toll-like receptor 4 (TLR4). Here, we examined the mechanisms responsible for inhibition of HCO3− absorption by basolateral LPS. Adding LPS to the bath decreased HCO3− absorption by 30% in rat and mouse MTALs perfused in vitro. The inhibition of HCO3− absorption was eliminated by the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK)/ERK inhibitors U0126 and PD98059. LPS induced a rapid (<15 min) and sustained (up to 60 min) increase in ERK phosphorylation in microdissected MTALs that was blocked by PD98059. The effects of basolateral LPS to activate ERK and inhibit HCO3− absorption were eliminated in MTALs from TLR4−/− and myeloid differentiation factor 88 (MyD88)−/− mice but were preserved in MTALs from TIR (Toll/interleukin-1 receptor) domain-containing adapter-inducing interferon-β (Trif)−/− mice. Basolateral LPS decreased apical Na+/H+ exchanger 3 NHE3 activity through a decrease in maximal velocity ( Vmax). The inhibition of NHE3 by LPS was eliminated by MEK/ERK inhibitors. LPS inhibited HCO3− absorption despite the presence of physiological stimuli that activate ERK in the MTAL. We conclude that basolateral LPS inhibits HCO3− absorption in the MTAL through activation of a TLR4/MyD88/MEK/ERK pathway coupled to inhibition of NHE3. These studies identify NHE3 as a target of TLR4 signaling in the MTAL and show that bacterial molecules can impair the absorptive functions of renal tubules through inhibition of this exchanger. The ERK pathway links TLR4 to downstream modulation of ion transport proteins and represents a potential target for treatment of sepsis-induced renal tubule dysfunction.

scholarly journals Successful Introduction of Human Renovascular Units into the Mammalian Kidney

2020 ◽  
Vol 31 (12) ◽  
pp. 2757-2772 ◽  
Author(s):  
Oren Pleniceanu ◽  
Orit Harari-Steinberg ◽  
Dorit Omer ◽  
Yehudit Gnatek ◽  
Bat-El Lachmi ◽  
...  
Keyword(s):  
Renal Tubule ◽  
Potential Interaction ◽  
Therapeutic Effects ◽  
Renal Tubules ◽  
Vascular Networks ◽  
Paracrine Effects ◽  
Endothelial Colony Forming Cells ◽  
Renal Vascular

BackgroundCell-based therapies aimed at replenishing renal parenchyma have been proposed as an approach for treating CKD. However, pathogenic mechanisms involved in CKD such as renal hypoxia result in loss of kidney function and limit engraftment and therapeutic effects of renal epithelial progenitors. Jointly administering vessel-forming cells (human mesenchymal stromal cells [MSCs] and endothelial colony-forming cells [ECFCs]) may potentially result in in vivo formation of vascular networks.MethodsWe administered renal tubule–forming cells derived from human adult and fetal kidneys (previously shown to exert a functional effect in CKD mice) into mice, alongside MSCs and ECFCs. We then assessed whether this would result in generation of “renovascular units” comprising both vessels and tubules with potential interaction.ResultsDirectly injecting vessel-forming cells and renal tubule–forming cells into the subcutaneous and subrenal capsular space resulted in self-organization of donor-derived vascular networks that connected to host vasculature, alongside renal tubules comprising tubular epithelia of different nephron segments. Vessels derived from MSCs and ECFCs augmented in vivo tubulogenesis by the renal tubule–forming cells. In vitro coculture experiments showed that MSCs and ECFCs induced self-renewal and genes associated with mesenchymal–epithelial transition in renal tubule–forming cells, indicating paracrine effects. Notably, after renal injury, renal tubule–forming cells and vessel-forming cells infused into the renal artery did not penetrate the renal vascular network to generate vessels; only administering them into the kidney parenchyma resulted in similar generation of human renovascular units in vivo.ConclusionsCombined cell therapy of vessel-forming cells and renal tubule–forming cells aimed at alleviating renal hypoxia and enhancing tubulogenesis holds promise as the basis for new renal regenerative therapies.

Intracellular Accumulation as an Active Process in a Mammalian Renal Transport System in Vitro

1956 ◽  
Vol 186 (1) ◽  
pp. 167-171 ◽  
Author(s):  
Roy P. Forster ◽  
J. H. Copenhaver
Keyword(s):  
Renal Cortex ◽  
Competitive Inhibition ◽  
Proximal Tubules ◽  
Renal Tubules ◽  
Phenol Red ◽  
Active Process ◽  
Intracellular Accumulation ◽  
Thin Slices ◽  
Intact Kidney

Studies on thin slices of rabbit renal cortex indicate that substances which accumulate in vitro are concentrated intracellularly without further discernible concentration in the lumen. Movement into cells of the proximal tubules is an active process dependent upon aerobic phosphorylation and subject to competitive inhibition involving p-aminohippurate, phenol red, chlorphenol red and benemid. All experimental indications are that this is the first step in the over-all transfer of these substances by renal tubules in the intact kidney.

scholarly journals Lumen LPS inhibits HCO3− absorption in the medullary thick ascending limb through TLR4-PI3K-Akt-mTOR-dependent inhibition of basolateral Na+/H+ exchange

2013 ◽  
Vol 305 (4) ◽  
pp. F451-F462 ◽  
Author(s):  
Bruns A. Watts ◽  
Thampi George ◽  
David W. Good
Keyword(s):  
Renal Tubule ◽  
Mammalian Target Of Rapamycin ◽  
Mtor Pathway ◽  
Thick Ascending Limb ◽  
Medullary Thick Ascending Limb ◽  
Dependent Inhibition ◽  
Phosphoinositide 3 Kinase ◽  
Apical Membranes ◽  
Molecular Components

Sepsis and endotoxemia induce defects in renal tubule function, but the mechanisms are poorly understood. Recently, we demonstrated that lipopolysaccharide (LPS) inhibits HCO3− absorption in the medullary thick ascending limb (MTAL) through activation of different Toll-like receptor 4 (TLR4) signaling pathways in the basolateral and apical membranes. Basolateral LPS inhibits HCO3− absorption through ERK-dependent inhibition of the apical Na+/H+ exchanger NHE3. Here, we examined the mechanisms of inhibition by lumen LPS. Adding LPS to the lumen decreased HCO3− absorption by 29% in rat and mouse MTALs perfused in vitro. Inhibitors of phosphoinositide 3-kinase (PI3K) or its effectors Akt and mammalian target of rapamycin (mTOR) eliminated inhibition of HCO3− absorption by lumen LPS but had no effect on inhibition by bath LPS. Exposure to LPS for 15 min induced increases in phosphorylation of Akt and mTOR in microdissected MTALs that were blocked by wortmannin, consistent with activation of Akt and mTOR downstream of PI3K. The effects of lumen LPS to activate Akt and inhibit HCO3− absorption were eliminated in MTALs from TLR4−/− and MyD88−/− mice but preserved in tubules lacking Trif or CD14. Inhibition of HCO3− absorption by lumen LPS was eliminated under conditions that inhibit basolateral Na+/H+ exchange and prevent inhibition of HCO3− absorption mediated through NHE1. Lumen LPS decreased basolateral Na+/H+ exchange activity through PI3K. We conclude that lumen LPS inhibits HCO3− absorption in the MTAL through TLR4/MyD88-dependent activation of a PI3K-Akt-mTOR pathway coupled to inhibition of NHE1. Molecular components of the TLR4-PI3K-mTOR pathway represent potential therapeutic targets for sepsis-induced renal tubule dysfunction.

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