Ca2+ uptake, fatty acid, and LDH release during proximal tubule hypoxia: effects of mepacrine and dibucaine

1994 ◽  
Vol 266 (2) ◽  
pp. F196-F201 ◽  
Author(s):  
D. Bunnachak ◽  
A. R. Almeida ◽  
J. F. Wetzels ◽  
P. Gengaro ◽  
R. A. Nemenoff ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Lactate Dehydrogenase ◽  
Proximal Tubule ◽  
Uptake Rate ◽  
Cell Injury ◽  
Potential Effect ◽  
Proximal Tubules ◽  
Membrane Injury ◽  
Ldh Release ◽  
Higher Doses

In freshly isolated hypoxic rat proximal tubules, Ca2+ uptake rate increases promptly, within 1 min, and remains significantly elevated throughout a 20-min period of hypoxia. Lactate dehydrogenase (LDH) release, a sign of membrane injury, increases only after 5 min of hypoxia and thereafter rises progressively. The potential effect of increased Ca2+ uptake rate to activate phospholipases, which would then initiate membrane injury, was evaluated by treating hypoxic tubules with three dissimilar phospholipase inhibitors, i.e., mepacrine, dibucaine, or p-bromophenacyl bromide (PBPB). LDH release averaged 11.9 and 13.8% after 10 and 20 min of normoxia, respectively. With 10 or 20 min of hypoxia LDH release increased to 46.0 and 65.2%, respectively (P < 0.01), and Ca2+ uptake rate increased from 2.56 in normoxia to 4.71 nmol.mg-1 x min-1 at 10 min of hypoxia (P < 0.01) and from 2.82 in normoxia to 3.76 nmol/mg at 20 min of hypoxia (P < 0.05). In a separate series of tubules, after 10 min of hypoxia LDH release was reduced by pretreatment with 50 microM mepacrine (66.1 to 47.3%, P < 0.01) or 50 microM dibucaine (53.1 to 38.5%, P < 0.02). The increase in Ca2+ uptake rate also was significantly reduced. After 20 min of hypoxia neither mepacrine nor dibucaine reduced Ca2+ uptake rate; LDH release was modestly reduced by dibucaine but not mepacrine. Higher doses of mepacrine (500 microM) and dibucaine (250 microM) also reduced cell injury at 10 min of hypoxia as assessed by LDH release.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential effects of respiratory inhibitors on glycolysis in proximal tubules

1990 ◽  
Vol 258 (6) ◽  
pp. F1608-F1615 ◽  
Author(s):  
K. G. Dickman ◽  
L. J. Mandel
Keyword(s):  
Oxidative Metabolism ◽  
Cell Injury ◽  
Lactate Production ◽  
Proximal Tubules ◽  
Antimycin A ◽  
Transport Function ◽  
Atp Production ◽  
Rotenone Treatment ◽  
Glycolytic Atp ◽  
Ldh Release

The effects of inhibition of mitochondrial energy production at various points along the respiratory chain on glycolytic lactate production and transport function were examined in a suspension of purified rabbit renal proximal tubules. Paradoxically, partial blockage at site 3 by hypoxia (1% O2) induced lactate production, whereas total site 3 blockage by anoxia (0% O2) failed to stimulate glycolysis. Compared with anoxia, hypoxic tubules exhibited greater preservation of ATP and K+ contents during O2 deprivation and more fully recovered oxidative metabolism and transport function during reoxygenation. The mitochondrial site 1 inhibitor rotenone and the uncoupler carbonyl cyanide-p-trifluorome-thoxyphenylhydrazone (FCCP) were equipotent stimuli for lactate production, whereas the site 2 inhibitor antimycin A failed to stimulate glycolysis despite a 90% inhibition of O2 consumption. Compared with antimycin A, treatment with rotenone or FCCP resulted in less cell injury [measured by lactate dehydrogenase (LDH) release] and greater preservation of cell K+ and ATP contents. 2-Deoxyglucose blocked lactate production by 50% in the presence of rotenone and increased LDH release, suggesting that glycolytic ATP is partially protective. Addition of ouabain during rotenone treatment reduced lactate production by 50%, indicating that glycolytic ATP can be used to fuel the Na pump when mitochondrial ATP production is inhibited. We conclude that 1) proximal tubules can generate lactate during inhibition of oxidative metabolism by hypoxia, rotenone, or FCCP; 2) mitochondrial inhibition is not obligatorily linked to activation of glycolysis, since neither anoxia nor antimycin A stimulate lactate production; 3) when ATP can be produced through anaerobic glycolysis it serves to protect cell viability and transport function during respiratory inhibition.

Glycine protection against hypoxic but not phospholipase A2-induced injury in rat proximal tubules

1993 ◽  
Vol 264 (1) ◽  
pp. F94-F99 ◽  
Author(s):  
J. F. Wetzels ◽  
X. Wang ◽  
P. E. Gengaro ◽  
R. A. Nemenoff ◽  
T. J. Burke ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Arachidonic Acid ◽  
Lactate Dehydrogenase ◽  
Phospholipase A2 ◽  
Cell Injury ◽  
Proximal Tubules ◽  
Exogenous Arachidonic Acid ◽  
Induced Changes ◽  
Phospholipid Degradation ◽  
Isolated Rat

We studied the effects of glycine (2 mM) on hypoxia-induced changes in phospholipids and fatty acids in isolated rat proximal tubules. In this preparation, 25 min of hypoxia caused cell injury, as reflected by the release of lactate dehydrogenase (LDH) (13.1 +/- 0.8 vs. 43.5 +/- 3.2%; P < 0.01). Hypoxia caused increases in fatty acids and in lysophospholipids. Glycine prevented the hypoxia-induced cell injury (LDH 13.1 +/- 0.8 vs. 11 +/- 0.7%; not significant) but did not attenuate the increases in fatty acids or lysophospholipids. In additional experiments, the effects of glycine on phospholipid changes and cell injury induced by exogenous phospholipase A2 (PLA2) were studied. PLA2 caused dramatic increases in fatty acids and lysophospholipids and mild cell injury; these effects were not influenced by glycine. In contrast, glycine attenuated increases in LDH release induced by exposing the tubules to exogenous arachidonic acid. In conclusion, glycine does not prevent the phospholipid degradation induced by either exogenous PLA2 or hypoxia in isolated proximal tubules and yet affords protection against hypoxia and exogenous arachidonic acid.

scholarly journals Kidney Proximal Tubule Lipoapoptosis Is Regulated by Fatty Acid Transporter-2 (FATP2)

2017 ◽  
Vol 29 (1) ◽  
pp. 81-91 ◽  
Author(s):  
Shenaz Khan ◽  
Pablo D. Cabral ◽  
William P. Schilling ◽  
Zachary W. Schmidt ◽  
Asif N. Uddin ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Epithelial Cells ◽  
Proximal Tubule ◽  
Proximal Tubules ◽  
Proximal Tubule Cell ◽  
Apical Surface ◽  
Wild Type ◽  
Ckd Progression ◽  
Fatty Acid Transporter ◽  
Acid Transporter

Albuminuria and tubular atrophy are among the highest risks for CKD progression to ESRD. A parsimonious mechanism involves leakage of albumin-bound nonesterified fatty acids (NEFAs) across the damaged glomerular filtration barrier and subsequent reabsorption by the downstream proximal tubule, causing lipoapoptosis. We sought to identify the apical proximal tubule transporter that mediates NEFA uptake and cytotoxicity. We observed transporter-mediated uptake of fluorescently labeled NEFA in cultured proximal tubule cells and microperfused rat proximal tubules, with greater uptake from the apical surface than from the basolateral surface. Protein and mRNA expression analyses revealed that kidney proximal tubules express transmembrane fatty acid transporter-2 (FATP2), encoded by Slc27a2, but not the other candidate transporters CD36 and free fatty acid receptor 1. Kidney FATP2 localized exclusively to proximal tubule epithelial cells along the apical but not the basolateral membrane. Treatment of mice with lipidated albumin to induce proteinuria caused a decrease in the proportion of tubular epithelial cells and an increase in the proportion of interstitial space in kidneys from wild-type but not Slc27a2−/−mice. Ex vivo microperfusion and in vitro experiments with NEFA-bound albumin at concentrations that mimic apical proximal tubule exposure during glomerular injury revealed significantly reduced NEFA uptake and palmitate-induced apoptosis in microperfused Slc27a2−/− proximal tubules and Slc27a2−/− or FATP2 shRNA-treated proximal tubule cell lines compared with wild-type or scrambled oligonucleotide–treated cells, respectively. We conclude that FATP2 is a major apical proximal tubule NEFA transporter that regulates lipoapoptosis and may be an amenable target for the prevention of CKD progression.

Modulation by Gly, Ca, and acidosis of injury-associated unesterified fatty acid accumulation in proximal tubule cells

1995 ◽  
Vol 268 (1) ◽  
pp. F110-F121 ◽  
Author(s):  
J. M. Weinberg ◽  
M. A. Venkatachalam ◽  
H. Goldberg ◽  
N. F. Roeser ◽  
J. A. Davis
Keyword(s):  
Fatty Acid ◽  
Proximal Tubule ◽  
Cell Injury ◽  
Membrane Damage ◽  
Suppressive Effect ◽  
Independent Component ◽  
Unesterified Fatty Acid ◽  
Proximal Tubule Cell ◽  
Fatty Acid Accumulation ◽  
Acid Accumulation

We have examined the dependence of unesterified fatty acid accumulation by intact, freshly isolated proximal tubules on Ca2+, pH, and the cytoprotective amino acid, glycine, during injury induced by hypoxia, antimycin, or antimycin plus ionomycin. In the absence of glycine, similarly high levels of fatty acid accumulation were seen during all three injury conditions irrespective of whether tubules were incubated in normal 1.25 mM Ca2+ medium or in medium where Ca2+ was buffered to 0.1 microM, a maneuver which prevented injury-associated increase of cytosolic-free Ca2+ as measured with fura 2. In the presence of glycine, which strongly suppressed development of lethal membrane damage for at least 60 min and did not have any apparent direct effects on fatty acid accumulation, both Ca(2+)-independent and Ca(2+)-dependent components of fatty acid accumulation were discernible. The Ca(2+)-independent component accounted for approximately 2/3 of fatty acid accumulation and did not vary as Ca2+ ranged from 10 nM to 1 microM. Unequivocal Ca(2+)-dependent accumulation occurred when Ca2+ exceeded 10 microM. Lowering pH to 6.9 had a moderate, generalized suppressive effect on fatty acid accumulation, including the major Ca(2+)-independent component, irrespective of the presence of glycine. These data emphasize the role of Ca(2+)-independent fatty acid accumulation during proximal tubule cell injury, clarify the modulatory actions of the potent, intrinsic cytoprotective factors, glycine and reduced pH, and provide insight into the relationship between fatty acid accumulation and lethal membrane damage.

Absence of endonuclease activation during acute cell death in renal proximal tubules

1993 ◽  
Vol 265 (2) ◽  
pp. C485-C490 ◽  
Author(s):  
R. G. Schnellmann ◽  
A. R. Swagler ◽  
M. M. Compton
Keyword(s):  
Cell Death ◽  
Proximal Tubule ◽  
Calcium Ionophore ◽  
Renal Proximal Tubule ◽  
Proximal Tubules ◽  
Free Calcium ◽  
Proximal Tubule Cell ◽  
Antimycin A ◽  
Aurintricarboxylic Acid ◽  
Ldh Release

The role of endonuclease and poly(ADP-ribose) polymerase activation in various types of cell injuries and death to rabbit renal proximal tubule suspensions was examined. Proximal tubules were exposed to the mitochondrial inhibitor antimycin A (0.1 microM), the protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP, 1 microM), the calcium ionophore ionomycin (5 microM), or the oxidant t-butyl hydroperoxide (TBHP, 0.5 mM) in the absence or presence of the endonuclease inhibitor aurintricarboxylic acid or the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide. Lactate dehydrogenase (LDH) release was used as a marker of cell death and analysis of genomic DNA for internucleosomal cleavage was used as a marker of endonuclease activation. Aurintricarboxylic acid and 3-aminobenzamide had no effect on the proximal tubule LDH release produced by 1 h exposure to antimycin A, FCCP, or ionomycin, or 2 h exposure to TBHP. Furthermore, there was no evidence of DNA fragmentation with any compound prior to or after cell death began. As a positive control, proximal tubules exposed to digitonin in the absence of metabolic substrates resulted in the chelator-inhibitable fragmentation of DNA, indicating that the endonuclease is present in proximal tubules. These results show that endonuclease activation did not occur prior to or after cell death began. Furthermore, these results suggest that endonuclease and poly(ADP-ribose) polymerase activation do not play a role in this model of acute renal proximal tubule cell injury and death induced by agents that cause oxidative stress, mitochondrial dysfunction, or increases in cytosolic free calcium.

Mechanism of protection by verapamil and nifedipine from anoxic injury in isolated cardiac myocytes

1984 ◽  
Vol 246 (3) ◽  
pp. C323-C329 ◽  
Author(s):  
J. Y. Cheung ◽  
A. Leaf ◽  
J. V. Bonventre
Keyword(s):  
Lactate Dehydrogenase ◽  
Cardiac Myocytes ◽  
Cell Injury ◽  
Ionic Composition ◽  
Myocardial Cell ◽  
Significant Loss ◽  
Membrane Injury ◽  
Lactate Dehydrogenase Release ◽  
Atp Levels ◽  
Anoxic Injury

This study was designed to determine whether slow Ca2+ channel blocking agents exert a direct protective effect on the anoxic myocardial cell and, if so, what the mechanism of protection is. Isolated Ca2+-tolerant rat cardiac myocytes were incubated under aerobic or anaerobic conditions, with or without verapamil or nifedipine, in the resting and contractile state. Protection against cell injury was assessed by preservation of rod-shaped morphology, cellular ATP levels, intracellular ionic composition, and lactate dehydrogenase release. Resting myocytes incubated anaerobically lost their rod-shaped appearance, accumulated Na+ and lost K+, and suffered a significant loss of cellular ATP. The release of lactate dehydrogenase into the medium was increased twofold, indicating significant membrane injury. Verapamil (1 microM) or nifedipine (1 microM) did not afford any protection against anoxic injury as measured by these parameters. Furthermore, on reoxygenation, anoxic verapamil- and nifedipine-treated myocytes had significantly higher cellular Ca2+ levels than control aerobic cells. When anoxic myocytes were paced at a rate of 300/min for 10 min, there were marked decreases in the number of rod-shaped cells and cellular ATP levels, whereas identically paced aerobic cells sustained no significant injury. Verapamil (1 microM) or nifedipine (1 microM) protected cells paced at 300/min from anoxic injury, but the cells were unable to sustain contraction rates at the frequency of the imposed pacing.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals A novel method of inducing and assuring total anoxia during in vitro studies of O2 deprivation injury.

1990 ◽  
Vol 1 (5) ◽  
pp. 837-840
Author(s):  
J K Joseph ◽  
D Bunnachak ◽  
T J Burke ◽  
R W Schrier
Keyword(s):  
Lactate Dehydrogenase ◽  
Anaerobic Bacteria ◽  
Proximal Tubules ◽  
Membrane Injury ◽  
Lactate Dehydrogenase Release ◽  
Novel Method ◽  
Unique Ability ◽  
Krebs Henseleit Buffer ◽  
Isolated Rat

Oxyrase is an enzyme mixture coveted by microbiologists for its unique ability to remove O2 from media in which anaerobic bacteria are grown. The study reported here examined the potential usefulness of Oxyrase as an adjunct to gassing freshly isolated rat proximal tubules (RPT) with 95% N2-5% CO2 in an attempt to achieve totally O2-free conditions (anoxia) before initiating studies on the mechanism of O2 deprivation injury in vitro. RPT, in 6 ml of Krebs-Henseleit buffer (KHB), were initially gassed with 95% N2-5% CO2 at 1.5 liters/min for 5 min and incubated for 15 to 30 min at 37 degrees C in a shaking water bath, pO2 decreased from approximately 400 to 80 mm Hg. If RPT were present in the KHB, pO2 was even lower, i.e., approximately 50 mm Hg. Addition of increasing concentrations of Oxyrase (300 to 1,500 mU) to KHB alone, that is, without RPT, reduced pO2 from 80 mm Hg to less than 5 mm Hg; increasing the gas rate from 1.5 to 3.0 liter/min of 95% N2-5% CO2, the concentration of Oxyrase to 1,800 mU, and adding RPT reduced pO2 to zero. In this latter condition, pO2 remained unmeasurable during the 20 min of study and neither pH nor pCO2 changed compared with control values. Oxyrase (1,800 mU) had no effect on lactate dehydrogenase release, a sign of membrane injury, in normoxic RPT in KHB. We conclude that anoxia can easily be achieved by the addition of Oxyrase to KHB in which RPT are suspended, if the appropriate concentration of Oxyrase is added and if the RPT are gassed with 95% N2-5% CO2. This concentration of Oxyrase exerts no detrimental effects on RPT gassed with 95% O2-5% CO2.

Reduction of Potassium in Kidney Proximal Tubules to Aid in Electron Probe X-Ray Microanalysis of Calcium

1985 ◽  
Vol 43 ◽  
pp. 474-475
Author(s):  
A. LeFurgey ◽  
P. Ingram ◽  
L.J. Mandel
Keyword(s):  
Smooth Muscle ◽  
Proximal Tubule ◽  
Electron Probe ◽  
Clinical Applications ◽  
Proximal Tubules ◽  
Rabbit Kidney ◽  
Target Cells ◽  
X Ray ◽  
Freeze Dried

For quantitative determination of subcellular Ca distribution by electron probe x-ray microanalysis, decreasing (and/or eliminating) the K content of the cell maximizes the ability to accurately separate the overlapping K Kß and Ca Kα peaks in the x-ray spectra. For example, rubidium has been effectively substituted for potassium in smooth muscle cells, thus giving an improvement in calcium measurements. Ouabain, a cardiac glycoside widely used in experimental and clinical applications, inhibits Na-K ATPase at the cell membrane and thus alters the cytoplasmic ion (Na,K) content of target cells. In epithelial cells primarily involved in active transport, such as the proximal tubule of the rabbit kidney, ouabain rapidly (t1/2= 2 mins) causes a decrease2 in intracellular K, but does not change intracellular total or free Ca for up to 30 mins. In the present study we have taken advantage of this effect of ouabain to determine the mitochondrial and cytoplasmic Ca content in freeze-dried cryosections of kidney proximal tubule by electron probe x-ray microanalysis.

Proximal tubule dysfunction in cystine-loaded tubules: effect of phosphate and metabolic substrates

1996 ◽  
Vol 271 (3) ◽  
pp. F717-F722
Author(s):  
G. Bajaj ◽  
M. Baum
Keyword(s):  
Oxygen Consumption ◽  
Proximal Tubule ◽  
Tricarboxylic Acid Cycle ◽  
Dimethyl Ester ◽  
Proximal Tubules ◽  
Intracellular Atp ◽  
Metabolic Substrates ◽  
Rate Of Oxygen Consumption ◽  
Intracellular Phosphate

Intracellular cystine loading by use of cystine dimethyl ester (CDME) results in a generalized inhibition in proximal tubule transport due, in part, to a decrease in intracellular ATP. The present study examined the importance of phosphate and metabolic substrates in the proximal tubule dysfunction produced by cystine loading. Proximal tubule intracellular phosphorus was 1.8 +/- 0.1 in control tubules and 1.1 +/- 0.1 nmol/mg protein in proximal tubules incubated in vitro with CDME P < 0.001). Infusion of sodium phosphate in rabbits and subsequent incubation of proximal tubules with a high-phosphate medium attenuated the decrease in proximal tubule respiration and prevented the decrease in intracellular ATP with cystine loading. Tricarboxylic acid cycle intermediates have been shown to preserve oxidative metabolism in phosphate-depleted proximal tubules. In proximal tubules incubated with either 1 mM valerate or butyrate, there was a 42 and 34% reduction (both P < 0.05) in the rate of oxygen consumption with cystine loading. However, tubules incubated with 1 mM succinate or citrate had only a 13 and 14% P = NS) reduction in the rate of oxygen consumption, respectively. These data are consistent with a limitation of intracellular phosphate in the pathogenesis of the proximal tubule dysfunction with cystine loading.

scholarly journals Evaluation of Cytotoxic Effects of Dichloromethane Extract of Guduchi (Tinospora cordifolia Miers ex Hook F & THOMS) on Cultured HeLa Cells

2006 ◽  
Vol 3 (2) ◽  
pp. 267-272 ◽  
Author(s):  
Ganesh Chandra Jagetia ◽  
Shaival Kamalaksha Rao
Keyword(s):  
Lipid Peroxidation ◽  
Lactate Dehydrogenase ◽  
Hela Cells ◽  
Cytotoxic Effect ◽  
Tinospora Cordifolia ◽  
Cytotoxic Effects ◽  
Optimal Duration ◽  
Ldh Release ◽  
Gst Activity

Extracts ofTinospora cordifolia(TCE) have been shown to possess anti-tumor properties, but the mechanism of the anti-tumor function of TCE is poorly understood. This investigation elucidates the possible mechanism underlying the cytotoxic effects of dichlormethane extracts of TCE, after selecting optimal duration and concentration for treatment. HeLa cells were exposed to various concentrations of TCE, which has resulted in a concentration-dependent decline in the clonogenicity, glutathione-S-transferase (GST) activity and a concentration-dependent increase in lipid peroxidation (TBARS) with a peak at 4 h and lactate dehydrogenase (LDH) release with a peak at 2 h. Our results suggest that the cytotoxic effect of TCE may be due to lipid peroxidation and release of LDH and decline in GST.

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