Modeling ischemia in vitro: selective depletion of adenine and guanine nucleotide pools

Dagher, Pierre C.

doi:10.1152/ajpcell.2000.279.4.c1270

Cited by 75 publications

(59 citation statements)

References 31 publications

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“…One cellular event that follows low oxygen concentrations is ATP depletion, which is reflected in disruptions of cell polarity and intercellular junctions through changes in actin cytoskeleton dynamics (Dagher, 2000). Since this organization of actin structures is mediated by Rho proteins such as Cdc42, Rac1 and RhoA (Raman and Atkinson, 1999;Bokoch, 2000), we decided to investigate the role of these key regulators of cytoskeletal rearrangement during hypoxic conditions in a renal carcinoma model using Caki-1 cells.…”

Section: Discussionmentioning

confidence: 99%

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HIF-1α mRNA and protein upregulation involves Rho GTPase expression during hypoxia in renal cell carcinoma

Turcotte

Desrosiers

Béliveau

2003

Journal of Cell Science

139

126

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The small G proteins of the Rho family are involved in reorganization of the actin cytoskeleton, cell migration and in the regulation of gene transcription. Hypoxia-induced ATP depletion results in the disruption of actin organization which could affect Rho functions. In solid tumors, regions with low oxygen tension stimulate angiogenesis in order to increase oxygen and nutrient supply. This process is mediated by stabilization of the transcriptional factor hypoxia inducible factor 1 (HIF-1), which increases vascular endothelial growth factor (VEGF) production. In this study, we investigated the activities of Rho proteins, which are key regulators of cytoskeleton organization during hypoxia in renal cell carcinoma. Caki-1 cells were exposed to hypoxia (1% O2) and exhibited increased Cdc42, Rac1 and RhoA protein expression. Immunoprecipitation of metabolically labelled RhoA showed that overexpression was at least due to neo-synthesis. The Rho GTPases overexpressed during hypoxia were mainly located at membranes and pull-down assays demonstrated that they were active since they bound GTP. RT-PCR analysis indicated that the increase in RhoA protein expression was also reflected at the mRNA level. Overexpression and activation of Rho proteins were downstream of, and dependent on, the production of reactive oxygen species (ROS) since, in the presence of an inhibitor, both the rise of ROS and upregulation of Rho proteins were abolished. Importantly,preincubation of cells with the toxin C3, which inhibits RhoA, reduced HIF-1α protein accumulation by 84% during hypoxia. Together, these results support a model where ROS upregulate Rho protein expression and where active RhoA is required for HIF-1α accumulation during hypoxia.

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Section: Discussionmentioning

confidence: 99%

“…1B). ATP depletion has been well documented as causing a disorganization of the actin cytoskeleton (Dagher, 2000). Since regulation of the actin cytoskeleton is mediated by Rho proteins, we next investigated the effects of hypoxia on expression and activation of these proteins.…”

Section: Atp Depletion and Cell Survival During Hypoxiamentioning

confidence: 99%

HIF-1α mRNA and protein upregulation involves Rho GTPase expression during hypoxia in renal cell carcinoma

Turcotte

Desrosiers

Béliveau

2003

Journal of Cell Science

139

126

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“…The role of other cellular nucleotide pools in ischemic conditions was not investigated. Recent reports indicate that depletion of GTP occurs concurrently with depletion of ATP in in vitro and in vivo models of ischemic renal injury (44). Selective depletion of GTP induced apoptosis in renal tubular cells.…”

Section: Depletion Of Gtpmentioning

confidence: 98%

“…Selective depletion of GTP induced apoptosis in renal tubular cells. Supplementation of guanosine to renal tubular cells subjected to chemical anoxia selectively enhanced GTP levels close to normal values and significantly reduced apoptotic cell death (44). Administration of guanosine before inducement of ischemic renal injury improved renal functions and significantly reduced the number of cells undergoing apoptotic cell death (98).…”

Section: Depletion Of Gtpmentioning

confidence: 99%

Cell death induced by acute renal injury: a perspective on the contributions of apoptosis and necrosis

Padanilam

2003

American Journal of Physiology-Renal Physiology

334

268

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In humans and experimental models of renal ischemia, tubular cells in various nephron segments undergo necrotic and/or apoptotic cell death. Various factors, including nucleotide depletion, electrolyte imbalance, reactive oxygen species, endonucleases, disruption of mitochondrial integrity, and activation of various components of the apoptotic machinery, have been implicated in renal cell vulnerability. Several approaches to limit the injury and augment the regeneration process, including nucleotide repletion, administration of growth factors, reactive oxygen species scavengers, and inhibition of inducers and executioners of cell death, proved to be effective in animal models. Nevertheless, an effective approach to limit or prevent ischemic renal injury in humans remains elusive, primarily because of an incomplete understanding of the mechanisms of cellular injury. Elucidation of cell death pathways in animal models in the setting of renal injury and extrapolation of the findings to humans will aid in the design of potential therapeutic strategies. This review evaluates our understanding of the molecular signaling events in apoptotic and necrotic cell death and the contribution of various molecular components of these pathways to renal injury.

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“…Despite numerous experimental and clinical studies on renal ischemia, the prognosis for patients with ischemic acute renal failure remains poor (Devarajan, 2006). Ischemia/reperfusion injury is characterized by ATP depletion, followed by subsequent activation of a number of critical alterations in metabolism (Dagher, 2000;Devarajan et al, 2003;Nangaku & Eckardt, 2007).…”

Section: Introductionmentioning

confidence: 99%

Alterations in Membrane Transport Function and Cell Viability Induced by ATP Depletion in Primary Cultured Rabbit Renal Proximal Tubular Cells

Lee

Kwon

Kim

2009

Korean J Physiol Pharmacol

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This study was undertaken to elucidate the underlying mechanisms of ATP depletion-induced membrane transport dysfunction and cell death in renal proximal tubular cells. ATP depletion was induced by incubating cells with 2.5 mM potassium cyanide (KCN)/0.1 mM iodoacetic acid (IAA), and membrane transport function and cell viability were evaluated by measuring Na ＋ -dependent phosphate uptake and trypan blue exclusion, respectively. ATP depletion resulted in a decrease in Na ＋ -dependent phosphate uptake and cell viability in a time-dependent manner. ATP depletion inhibited Na ＋ -dependent phosphate uptake in cells, when treated with 2 mM ouabain, a Na ＋ pump-specific inhibitor, suggesting that ATP depletion impairs membrane transport functional integrity. Alterations in Na ＋ -dependent phosphate uptake and cell viability induced by ATP depletion were prevented by the hydrogen peroxide scavenger such as catalase and the hydroxyl radical scavengers (dimethylthiourea and thiourea), and amino acids (glycine and alanine). ATP depletion caused arachidonic acid release and increased mRNA levels of cytosolic phospholipase A2 (cPLA2). The ATP depletion-dependent arachidonic acid release was inhibited by cPLA2 specific inhibitor AACOCF3. ATP depletion-induced alterations in Na ＋ -dependent phosphate uptake and cell viability were prevented by AACOCF3. Inhibition of Na ＋ -dependent phosphate uptake by ATP depletion was prevented by antipain and leupetin, serine/cysteine protease inhibitors, whereas ATP depletion-induced cell death was not altered by these agents. These results indicate that ATP depletion-induced alterations in membrane transport function and cell viability are due to reactive oxygen species generation and cPLA2 activation in renal proximal tubular cells. In addition, the present data suggest that serine/cysteine proteases play an important role in membrane transport dysfunction, but not cell death, induced by ATP depletion.

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Modeling ischemia in vitro: selective depletion of adenine and guanine nucleotide pools

Cited by 75 publications

References 31 publications

HIF-1α mRNA and protein upregulation involves Rho GTPase expression during hypoxia in renal cell carcinoma

HIF-1α mRNA and protein upregulation involves Rho GTPase expression during hypoxia in renal cell carcinoma

Cell death induced by acute renal injury: a perspective on the contributions of apoptosis and necrosis

Alterations in Membrane Transport Function and Cell Viability Induced by ATP Depletion in Primary Cultured Rabbit Renal Proximal Tubular Cells

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