Abstract
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 His6-tagged protein, purified from Escherichia coli inclusion bodies, and reconstituted into proteoliposomes for transport assays. Time- and concentration-dependent uptake of bothl-[3H-glycyl]GSH and [2-14C]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-His6 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 orS-(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.
Footnotes
-
This work was funded by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK40725 (to L.H.L. and L.H.M.). Core facilities funded by the National Institute of Environmental Health Sciences Center for Molecular Toxicology with Human Applications (Grant P30-ES06639) at Wayne State University were used for some of these studies.
-
DOI: 10.1124/jpet.102.040220
- Abbreviations:
- GSH
- glutathione
- DCVC
- S-(1,2-dichlorovinyl)-l-cysteine
- DCC
- dicarboxylate carrier
- OGC
- oxoglutarate carrier
- NRK
- normal rat kidney
- tBH
- tert-butyl hydroperoxide
- PCR
- polymerase chain reaction
- PBS
- phosphate-buffered saline
- FACS
- fluorescence-activated cell sorting
- IPTG
- isopropyl β-d-thiogalactoside
- Received June 11, 2002.
- Accepted July 9, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
JPET articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|