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.

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.

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.

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.

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.

ATP depletion alters myosin I beta cellular location in LLC-PK1 cells

1997 ◽  
Vol 272 (5) ◽  
pp. C1680-C1690 ◽  
Author(s):  
M. C. Wagner ◽  
B. A. Molitoris
Keyword(s):  
Structure And Function ◽  
Atp Depletion ◽  
Cell Periphery ◽  
Proximal Tubule Cell ◽  
Tubule Cell ◽  
Dynamic Membrane ◽  
Binding Domains ◽  
Myosin I ◽  
And Function ◽  
Triton X

The brush border (BB) of the proximal tubule cell (PTC) requires dynamic membrane events for function. The actin cytoskeleton is necessary for structure and function in this region. ATP depletion disrupts both structure and function. In this report, myosin 1 beta location in LLC-PK1 cells was followed during ATP depletion and repletion using immunofluorescence and Western blot techniques. Myosin I beta colocalized with F-actin in the microvilli and cell periphery, but no colocalization was observed with stress fibers. ATP depletion increased the apical F-actin, and myosin I beta was colocalized there. In addition, after ATP depletion, myosin I beta was extracted less by Triton X-100. These changes were reversed after ATP repletion. Finally, immunofluorescence of kidney sections shows myosin I beta in the BB. These results place this motor in a dynamic region of the PTC where its actin and membrane binding domains can contribute to PTC function.

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

scholarly journals Metabolic aspects of protection by glycine against hypoxic injury to isolated proximal tubules.

1991 ◽  
Vol 1 (7) ◽  
pp. 949-958
Author(s):  
J M Weinberg ◽  
D N Buchanan ◽  
J A Davis ◽  
M Abarzua
Keyword(s):  
Gas Chromatography ◽  
Cell Injury ◽  
Potent Inhibitor ◽  
Proximal Tubule Cell ◽  
Protective Effects ◽  
Glycolytic Metabolism ◽  
Tubule Cell ◽  
Hypoxic Injury ◽  
Hypoxic Conditions ◽  
Gas Chromatography Mass Spectroscopy

To clarify the roles of butyrate and acylglycine formation in hypoxic proximal tubule cell injury and protection by glycine and to test the contribution of iodoacetate-suppressible metabolism to protection, (1) it was determined whether protection by glycine is fully expressed when glucose, lactate, alanine, and butyrate are replaced by alpha-ketoglutarate as the sole substrate for the tubules, (2) butyrate metabolism and acylglycine formation were directly measured in control and hypoxic preparations, and (3) it was assessed whether injury produced by iodoacetate, a potent inhibitor of glycolytic metabolism, is subject to protection by glycine. Susceptibility to hypoxic injury in medium with alpha-ketoglutarate as the sole substrate was similar to that seen in medium containing glucose, lactate, alanine, and butyrate. Tubules in alpha-ketoglutarate medium showed high degrees of protection by glycine against injury produced by 30-min of hypoxia, by iodoacetate alone, and by iodoacetate combined with hypoxia. Protection did not require preservation of cell ATP or glutathione. In glucose-lactate-alanine-butyrate medium, butyrate, measured by gas chromatography, was rapidly metabolized by oxygenated tubules and fully accounted for basal rates of oxygen consumption. Butyrate utilization stopped during hypoxia. Neither aspect of butyrate metabolism was altered by glycine. Formation of acylglycines was assessed by gas chromatography/mass spectroscopy. In preparations treated with glycine, butyrylglycine was detected under both oxygenated and hypoxic conditions; the quantities, however, were small and no other acylglycines were found. These observations indicate that protective effects of glycine are independent of short-chain acylglycine formation and glycolytic metabolism.

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