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CELLULAR AND MOLECULAR

Extracellular Signal-Regulated Kinase Activation Mediates Mitochondrial Dysfunction and Necrosis Induced by Hydrogen Peroxide in Renal Proximal Tubular Cells

Department of Medicine, Brown University School of Medicine, Providence, Rhode Island (S.Z.); Departments of Pharmaceutical Sciences (G.R.K., R.G.S.) and Surgery (Y.Y.), Medical University of South Carolina, Charleston, South Carolina; and Department of Immunology, the Scripps Research Institute, La Jolla, California (J.H.)

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.

Address correspondence to: Dr. Shougang Zhuang, Department of Medicine Rhode Island Hospital, Middle House 301, 593 Eddy Street, Providence, RI 02903. E-mail: szhuang{at}lifespan.org