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(The FASEB Journal. 2007;21:934.1)
© 2007 FASEB
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934.1

shRNA-knockdown of divalent metal transporter 1 (DMT1) attenuates cadmium-metallothionein-1 (CdMT-1) cytotoxicity in rat renal proximal tubule (PT) cells

Natascha A. Wolff1, Marouan Abouhamed1, Craig P. Smith2 and Frank Thévenod1

1 Physiology and Pathophysiology, University of Witten/Herdecke, Stockumer Str. 12, Witten, 58453, Germany,
2 Faculty of Life Sciences, The University of Manchester, Oxford Road, Manchester, M13 9PT, United Kingdom

ABSTRACT

The nephrotoxic metal cadmium (Cd) is delivered to the kidneys mainly as CdMT-1 complexes, which are internalized by kidney PT cells in part via megalin/cubilin-mediated endocytosis. In these cells, the proton-coupled transporter DMT1, which also transports Cd, is localized in late endo-/lysosomal compartments. In the present study, we thus investigated the involvement of DMT1 in CdMT-1 cytotoxicity. A cell line derived from the S1 segment of rat PT was transiently transfected with vectors coding for DMT1-targeted short hairpin RNAs. 18 h post transfection, the cells were either exposed to CdMT-1 for a further 24 h, or processed for immunocytochemistry or immunoblotting. shRNA3 significantly reduced DMT1 protein expression - as determined by immunoblotting using a polyclonal anti-DMT1 antibody - by ~45% relative to cells transfected with empty vector. In cytotoxicity tests carried out in parallel using the MTT assay, shRNA3 significantly attenuated cytotoxicity induced by a 24 h CdMT-incubation by ~30%, while toxicity of free Cd and AlexaFluor546-conjugated MT-1 internalization remained unaltered, indicating that pro-apoptotic signaling and uptake mechanisms for CdMT-1 were not affected by DMT1 knockdown. These data support a role for DMT1 in CdMT-1-induced PT cytotoxicity, by possibly mediating efflux of Cd released from MT-1 from endo-/lysosomal compartments into the cytosol.

Funded by DFG TH 345/8-1





This Article
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