Contribution of actin cytoskeletal alterations to ATP depletion and calcium-induced proximal tubule cell injury

1996 ◽  
Vol 270 (1) ◽  
pp. F39-F52 ◽  
Author(s):  
S. Nurko ◽  
K. Sogabe ◽  
J. A. Davis ◽  
N. F. Roeser ◽  
M. Defrain ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Proximal Tubule ◽  
Laser Scanning ◽  
Structural Changes ◽  
Cell Injury ◽  
Membrane Damage ◽  
Atp Depletion ◽  
Similar Proportion ◽  
Proximal Tubule Cell ◽  
Tubule Cell

The actin cytoskeleton of rabbit proximal tubules was assessed by deoxyribonuclease (DNase) binding, sedimentability of detergent-insoluble actin, laser-scanning confocal microscopy, and ultrastructure during exposure to hypoxia, antimycin, or antimycin plus ionomycin. One-third of total actin was DNase reactive in control cells prior to deliberate depolymerization, and a similar proportion was unsedimentable from detergent lysates during 2.5 h at 100,000 g. Tubules injured by hypoxia or antimycin alone, without glycine, showed Ca(2+)-dependent pathology of the cytoskeleton, consisting of increases in DNase-reactive actin, redistribution of pelletable actin, and loss of microvilli concurrent with lethal membrane damage. In contrast, tubules similarly depleted of ATP and incubated with glycine showed no significant changes of DNase-reactive actin or actin sedimentability for up to 60 min, but, nevertheless, developed substantial loss of basal membrane-associated actin within 15 min and disruption of actin cores and clubbing of microvilli at durations > 30 min. These structural changes that occurred in the presence of glycine were not prevented by limiting Ca2+ availability or pH 6.9. Very rapid and extensive cytoskeletal disruption followed antimycin-plus-ionomycin treatment. In this setting, glycine and pH 6.9 decreased lethal membrane damage but did not ameliorate pathology in the cytoskeleton or microvilli; limiting Ca2+ availability partially protected the cytoskeleton but did not prevent lethal membrane damage. The data suggest that both ATP depletion-dependent but Ca(2+)-independent, as well as Ca(2+)-mediated, processes can disrupt the actin cytoskeleton during acute proximal tubule cell injury; that both types of change occur, despite protection afforded by glycine and reduced pH against lethal membrane damage; and that Ca(2+)-independent processes primarily account for prelethal actin cytoskeletal alterations during simple ATP depletion of proximal tubule cells.

Calcium dependence of integrity of the actin cytoskeleton of proximal tubule cell microvilli

1996 ◽  
Vol 271 (2) ◽  
pp. F292-F303 ◽  
Author(s):  
K. Sogabe ◽  
N. F. Roeser ◽  
J. A. Davis ◽  
S. Nurko ◽  
M. A. Venkatachalam ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Proximal Tubule ◽  
High Speed ◽  
Membrane Damage ◽  
Defined Medium ◽  
Atp Depletion ◽  
Metabolic Inhibitor ◽  
Proximal Tubule Cell ◽  
Tubule Cell ◽  
Order Of Magnitude

To better define the role of Ca2+ in pathophysiological alterations of the proximal tubule microvillus actin cytoskeleton, we studied freshly isolated tubules in which intracellular free Ca2+ was equilibrated with highly buffered, precisely defined medium Ca2+ levels using a combination of the metabolic inhibitor, antimycin, and the ionophore, ionomycin, in the presence of glycine, to prevent lethal membrane damage and resulting nonspecific changes. Increases of Ca2+ to > or = 10 microM were sufficient to initiate concurrent actin depolymerization, fragmentation of F-actin into forms requiring high-speed centrifugation for recovery, redistribution of villin to sedimentable fractions, and structural microvillar damage consisting of severe swelling and fragmentation of actin cores. These observations implicate Ca(2+)-dependent, villin-mediated actin cytoskeletal disruption in tubule cell microvillar damage under conditions conceivably present during pathophysiological states. However, despite prior evidence for cytosolic free Ca2+ increases of the same order of magnitude and similar structural microvillar alterations, Ca(2+)- and villin-mediated events did not appear to account for the initial microvillar damage that occurs during ATP depletion induced by antimycin alone or hypoxia.

scholarly journals The actin cytoskeleton and integrin expression in the recovery of cell adhesion after oxidant stress to a proximal tubule cell line (JTC-12).

1998 ◽  
Vol 9 (10) ◽  
pp. 1787-1797
Author(s):  
S Nigam ◽  
C E Weston ◽  
C H Liu ◽  
E E Simon
Keyword(s):  
Cell Adhesion ◽  
Actin Cytoskeleton ◽  
Proximal Tubule ◽  
Cell Line ◽  
Oxidant Stress ◽  
Proximal Tubule Cell ◽  
Integrin Expression ◽  
Tubule Cell ◽  
Essentially Normal ◽  
Atp Levels

This study examines the role of the actin cytoskeleton and integrin expression in the recovery of cell adhesion in the proximal tubule cell line JTC-12 after peroxide injury. The cells were exposed to 10, 20, or 50 mM hydrogen peroxide for 10 min and then allowed to recover. Viability measurements by trypan blue exclusion confirmed that the injury was largely nonlethal with 85% viability at 1 h even at 50 mM peroxide. ATP levels fell immediately after the peroxide incubation in all groups to approximately 10% of normal, but already showed some recovery by 1 h and full recovery in the 10 and 20 mM groups by 24 h. Cell adhesion to extracellular matrix immediately after injury was depressed at 20 and 50 mM peroxide, but by 12 h was abnormal only at 50 mM peroxide and at 24 h was essentially normal at all peroxide concentrations. Immediately after exposure to 10 mM peroxide, there were subtle abnormalities in the actin cytoskeleton (thickening of fibrils) as assessed by phalloidin staining, with more pronounced effects at 20 and 50 mM. At 1 h, many cells showed collapse of the actin cytoskeleton to the periphery. There was some recovery at 4 h; by 12 h, the actin cytoskeleton showed further recovery, although was still abnormal (coarsened microfilaments), especially at 20 and 50 mM peroxide. By 24 h, the actin cytoskeleton showed only subtle coarsening. Integrin surface expression was assessed by flow cytometry. The alpha6 subunit on cells exposed to 20 mM peroxide was unchanged at 1 h and 4 h, but by 12 h had increased to 118.5+/-4.5% and by 24 h to 146+/-13.4% of control levels. The expression of the beta1 and alphaVbeta3 integrins remained unchanged. Thus, despite coarsening of the actin cytoskeleton and depressed ATP levels, cell adhesion recovered from oxidant stress. Abnormal cell adhesion after injury was not a consequence of a decrease in integrin expression, and recovery of cell adhesion was not a consequence of the modest and selective increase in integrin expression.

ER stress contributes to renal proximal tubule injury by increasing SREBP-2-mediated lipid accumulation and apoptotic cell death

2012 ◽  
Vol 303 (2) ◽  
pp. F266-F278 ◽  
Author(s):  
Šárka Lhoták ◽  
Sudesh Sood ◽  
Elise Brimble ◽  
Rachel E. Carlisle ◽  
Stephen M. Colgan ◽  
...  
Keyword(s):  
Er Stress ◽  
Proximal Tubule ◽  
Lipid Accumulation ◽  
Cell Injury ◽  
Apoptotic Cell ◽  
Renal Proximal Tubule ◽  
Proximal Tubule Cell ◽  
Tubule Cell ◽  
Proximal Tubule Cells ◽  
Tubule Cells

Renal proximal tubule injury is induced by agents/conditions known to cause endoplasmic reticulum (ER) stress, including cyclosporine A (CsA), an immunosuppressant drug with nephrotoxic effects. However, the underlying mechanism by which ER stress contributes to proximal tubule cell injury is not well understood. In this study, we report lipid accumulation, sterol regulatory element-binding protein-2 (SREBP-2) expression, and ER stress in proximal tubules of kidneys from mice treated with the classic ER stressor tunicamycin (Tm) or in human renal biopsy specimens showing CsA-induced nephrotoxicity. Colocalization of ER stress markers [78-kDa glucose regulated protein (GRP78), CHOP] with SREBP-2 expression and lipid accumulation was prominent within the proximal tubule cells exposed to Tm or CsA. Prolonged ER stress resulted in increased apoptotic cell death of lipid-enriched proximal tubule cells with colocalization of GRP78, SREBP-2, and Ca2+-independent phospholipase A2 (iPLA2β), an SREBP-2 inducible gene with proapoptotic characteristics. In cultured HK-2 human proximal tubule cells, CsA- and Tm-induced ER stress caused lipid accumulation and SREBP-2 activation. Furthermore, overexpression of SREBP-2 or activation of endogenous SREBP-2 in HK-2 cells stimulated apoptosis. Inhibition of SREBP-2 activation with the site-1-serine protease inhibitor AEBSF prevented ER stress-induced lipid accumulation and apoptosis. Overexpression of the ER-resident chaperone GRP78 attenuated ER stress and inhibited CsA-induced SREBP-2 expression and lipid accumulation. In summary, our findings suggest that ER stress-induced SREBP-2 activation contributes to renal proximal tubule cell injury by dysregulating lipid homeostasis.

Ouabain-induced lethal proximal tubule cell injury is prevented by glycine

1990 ◽  
Vol 258 (2) ◽  
pp. F346-F355 ◽  
Author(s):  
J. M. Weinberg ◽  
J. A. Davis ◽  
M. Abarzua ◽  
R. K. Smith ◽  
R. Kunkel
Keyword(s):  
Cell Injury ◽  
Atp Depletion ◽  
Cell Swelling ◽  
Proximal Tubule Cell ◽  
Cell Water ◽  
Tubule Cell ◽  
Lactate Dehydrogenase Release ◽  
High K ◽  
Early Effects ◽  
K Concentration

Exposure to 1 mM ouabain for greater than 30 min caused lethal cell injury to isolated rabbit proximal tubules as measured by increased lactate dehydrogenase release. Addition of 2 mM glycine or glutathione to the incubation medium prevented this injury and a sharp fall of cell ATP that accompanied it. Glycine and glutathione did not alter rapid, early effects of ouabain to deplete cell K+ and inhibit respiration. Preservation of cellular glutathione was not required for protection. Glycine did not ameliorate ouabain-induced increases of cell water and did not prevent lethal cell injury associated with cell swelling produced by incubation in a high K+ concentration medium. In contrast, 100 mM mannitol, which at least partially ameliorated swelling in both ouabain and high-K+ medium, prevented lethal injury in high-K+ medium and decreased it in the presence of ouabain. The combination of glycine and mannitol completely prevented ouabain-induced lethal injury and cell water increases. These observations indicate that glycine, unlike mannitol, does not protect against primary volume-induced insults. Ouabain-induced lethal cell injury results from a process that includes both a volume component ameliorated by mannitol and a volume-independent component that is prevented by glycine and is closely associated with accelerated ATP depletion.

In Vitro to In Vivo Concordance of Toxicity Using the Human Proximal Tubule Cell Line HK-2

2020 ◽  
Vol 39 (5) ◽  
pp. 452-464
Author(s):  
Miriam E. Mossoba ◽  
Robert L. Sprando
Keyword(s):  
Proximal Tubule ◽  
Cell Line ◽  
Cell Injury ◽  
Culture Media ◽  
Sugar Transport ◽  
Molecular Characteristics ◽  
Proximal Tubule Cell ◽  
Tubule Cell

The renal proximal tubule cell line, human kidney 2 (HK-2), recapitulates many of the functional cellular and molecular characteristics of differentiated primary proximal tubule cells. These features include anchorage dependence, gluconeogenesis capability, and sodium-dependent sugar transport. In order to ascertain how well HK-2 cells can reliably reveal the toxicological profile of compounds having a potential to cause proximal tubule injury in vivo, we sought to evaluate the effects of known proximal tubule toxicants using the HK-2 cell line. We selected 20 pure nephrotoxic compounds that included chemotherapeutic drugs, antibiotics, and heavy metal-containing compounds and evaluated their ability to induce HK-2 cell injury relative to 10 innocuous pure compounds or cell culture media alone. We performed a comprehensive set of in vitro cellular toxicological assays to evaluate cell viability, oxidative stress, mitochondrial integrity, and a specific biomarker of renal injury, Kidney Injury Molecule 1. For each of our selected compounds, we were able to establish a reproducible profile of toxicological outcomes. We compared our results to those described in peer-reviewed publications to understand how well the HK-2 cellular model agrees with overall in vivo rat or human toxicological outcomes. This study begins to address the question of how well in vitro data generated with HK-2 cells can mirror in vivo animal and human outcomes.

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.

scholarly journals Microfilament disruption occurs very early in ischemic proximal tubule cell injury

1992 ◽  
Vol 42 (4) ◽  
pp. 896-902 ◽  
Author(s):  
Paul S. Kellerman ◽  
Ronald T. Bogusky
Keyword(s):  
Proximal Tubule ◽  
Cell Injury ◽  
Proximal Tubule Cell ◽  
Tubule Cell
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