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shRNA‐knockdown of divalent metal transporter 1 (DMT1) attenuates cadmium‐metallothionein‐1 (CdMT‐1) cytotoxicity in rat renal proximal tubule (PT) cells
Author(s) -
Wolff Natascha A.,
Abouhamed Marouan,
Smith Craig P.,
Thévenod Frank
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a1325-b
Subject(s) - dmt1 , cytotoxicity , chemistry , transfection , gene knockdown , small hairpin rna , microbiology and biotechnology , cell culture , endocytosis , internalization , deferoxamine , metallothionein , transporter , biochemistry , cell , apoptosis , in vitro , biology , gene , genetics
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