Protection of NRK-52E cells, a rat renal proximal tubular cell line, from chemical-induced apoptosis by overexpression of a mitochondrial glutathione transporter.
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-[(3)H-glycyl]GSH and [2-(14)C]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.
Lash
Lawrence H
LH
Department of Pharmacology, Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, 540 East Canfield Avenue, Detroit, MI 48201, USA. l.h.lash@wayne.edu
Putt
David A
DA
Matherly
Larry H
LH
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002460:Cell Line; D003545:Cysteine / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000633:toxicity; D029382:Dicarboxylic Acid Transporters / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D005978:Glutathione / Q000378:metabolism*; D007687:Kidney Tubules, Proximal / Q000187:drug effects / Q000473:pathology*; D007700:Kinetics; D008081:Liposomes; D008297:Male; D008314:Malonates / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D051381:Rats; D011916:Rats, Inbred F344; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D020122:tert-Butylhydroperoxide / Q000037:antagonists & inhibitors / Q000633:toxicity
D029382:Dicarboxylic Acid Transporters; D008081:Liposomes; D008314:Malonates; C039961:S-(1,2-dichlorovinyl)cysteine; D020122:tert-Butylhydroperoxide; D005978:Glutathione; D003545:Cysteine