Abstract
Although tubular necrosis in acute renal failure is associated with excessive production of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), the mechanism of ROS-induced cell necrosis remains poorly understood. In this study, we examined the role of the extracellular signaling-regulated kinase (ERK) pathway in H2O2-induced necrosis of renal proximal tubular cells (RPTC) in primary culture. Exposure of 60 to 70% confluent RPTC to 1 mM H2O2 for 3 h resulted in 44% necrotic cell death, as measured by trypan blue uptake, and inactivation of mitogen-activated protein kinase kinase (MEK), the upstream activator of ERK, by either 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126) or 2-(2′-amino-3′-methoxyphenyl)-oxanaphthalen-4-one (PD98059) or overexpression of dominant-negative mutant of MEK1, inhibited cell death. In contrast, overexpression of active MEK1 enhanced H2O2-induced cell death. H2O2 treatment led to the loss of mitochondrial membrane potential (MMP) in RPTC, which was decreased by U0126 and PD98059. Furthermore, inhibition of the MEK/ERK pathway decreased oxidant-mediated ERK1/2 activation and mitochondrial swelling in isolated renal cortex mitochondria. However, treatment with cyclosporin A (CsA), a mitochondrial permeability transition blocker, did not suppress RPTC necrotic cell death, loss of MMP, and mitochondrial swelling. We suggest that ERK is a critical mediator of mitochondrial dysfunction and necrotic cell death of renal epithelial cells following oxidant injury. Oxidant-induced necrotic cell death was mediated by a CsA-insensitive loss of MMP that is regulated by the ERK pathway.
Footnotes
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This work was supported by National Institutes of Health (NIH)/National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-071997 (to S.Z.) and DK-62028 (to R.G.S.). G.R.K. was supported by a training grant from NIH/National Institute on Environmental Health Sciences (ES-012878).
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.108.136358.
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ABBREVIATIONS: ARF, acute renal failure; MPT, mitochondrial permeability transition; MMP, mitochondrial membrane potential; PT, permeability transition; CsA, cyclosporin A; ROS, reactive oxygen species; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase kinase; RPTC, renal proximal tubular cells; U0126, 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]-butadiene; SB203580, 4-(4-fluorophenyl)-2-(4 methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; SP600125, anthra[1,9-cd]pyrazol-6(2H)-one; PD98059, 2-(2′-amino-3′-methoxyphenyl)-oxanaphthalen-4-one; Ad-caMEK1, active MEK1; Ad-dnMEK, dominant-negative mutant of MEK1; MOI, multiplicity of infection; pfu, plaque-forming units; LDH, lactate dehydrogenase; LDH, lactate dehydrogenase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; PBS, phosphate-buffered saline; PI, propidium iodide; MMP, matrix metalloproteinase; RCM, renal cortical mitochondria; BHA, antioxidant butylated hydroxyanisole; cPLA2, cytosolic phospholipase A2; iPLA2γ, independent phospholipase A2γ.
- Received January 10, 2008.
- Accepted March 12, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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