Protection of NRK-52E cells, a rat renal proximal tubular cell line, from chemical-induced apoptosis by overexpression of a mitochondrial glutathione transporter

被引:67
作者
Lash, LH
Putt, DA
Matherly, LH
机构
[1] Wayne State Univ, Sch Med, Barbara Ann Karmanos Canc Inst, Dept Pharmacol, Detroit, MI 48201 USA
[2] Wayne State Univ, Sch Med, Barbara Ann Karmanos Canc Inst, Expt & Clin Therapeut Program, Detroit, MI 48201 USA
关键词
D O I
10.1124/jpet.102.040220
中图分类号
R9 [药学];
学科分类号
1007 ;
摘要
The dicarboxylate carrier (DCC) is one of two carriers responsible for glutathione (GSH) transport into rat kidney mitochondria. The central hypothesis of the present study was that overexpression of this carrier in renal proximal tubular cells increases content of mitochondrial GSH, which in turn can protect these cells from chemical-induced injury. We first cloned the carrier protein and verified its properties. This was accomplished by reverse transcribing total rat kidney RNA and polymerase chain reaction amplification with primers based on the complete cDNA sequence for the mitochondrial DCC protein. DCC was expressed as a His(6) tagged protein, purified from Escherichia coli inclusion bodies, and reconstituted into proteoliposomes for transport assays. Time- and concentration-dependent uptake of both L-[H-3-glycyl] GSH and [2-C-14] malonate was observed with kinetics, substrate specificity, and inhibitor sensitivities similar to those observed in rat kidney proximal tubules. We next transiently transfected NRK-52E cells with the cDNA for rat kidney DCC to overexpress the protein. The presence of the recombinant DCC-His(6) protein was confirmed by immunoblots. Transport of both GSH and malonate into the mitochondrial fraction of transfected cells was enhanced 2.45- to 11.3-fold, compared with that in wild-type cells. Transfected cells exhibited markedly less apoptosis from tert-butyl hydroperoxide or S-(1,2-dichlorovinyl)-L-cysteine than did wild-type cells, validating the central hypothesis and providing us with a valuable and novel tool with which to further study GSH and thiol redox status in renal mitochondria, and the function of GSH transport in regulation of processes such as apoptosis and oxidative phosphorylation.
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页码:476 / 486
页数:11
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