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This Article Full Text Full Text (PDF) All Versions of this Article: 293/3/F705    most recent 00198.2007v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via ISI Web of Science (2) Citing Articles via Google Scholar Google Scholar Articles by Abouhamed, M. Articles by Thévenod, F. Search for Related Content PubMed PubMed Citation Articles by Abouhamed, M. Articles by Thévenod, F.

Knockdown of endosomal/lysosomal divalent metal transporter 1 by RNA interference prevents cadmium-metallothionein-1 cytotoxicity in renal proximal tubule cells

¹Department of Physiology and Pathophysiology, University of Witten/Herdecke, Witten, Germany; and ²Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom

Submitted 25 April 2007 ; accepted in final form 21 June 2007

Chronic exposure to Cd²⁺ causes renal proximal tubular (PT) damage. Cd²⁺ reaches the PT mainly as cadmium-metallothionein 1 (CdMT-1) complexes that are filtered at the glomerulus and then internalized in part via endocytosis mediated by megalin and cubulin. Subsequently, Cd²⁺ is thought to be released in the cytosol to activate cell death pathways. The proton-coupled divalent metal transporter DMT1 also transports Cd²⁺ and is expressed exclusively in endosomes/lysosomes in rat PT cells. Using vector-based RNA interference with short-hairpin small-interfering RNAs (shRNAs) to downregulate DMT1 in the rat renal PT cell line WKPT-0293 Cl.2, we tested the hypothesis that endosomal/lysosomal DMT1 is involved in CdMT-1 nephrotoxicity. One out of 5 shRNAs tested (sh3) significantly reduced expression of DMT1 protein detected by immunoblotting and DMT1 mRNA as determined by RT-PCR by 45.1 ± 9.6 and 36.9 ± 14.4% (n = 5–6), respectively. Similarly, sh3 reduced perinuclear DMT1 immunostaining in transfected cells. Consistent with the assumed role of DMT1 in CdMT-1 cytotoxicity, sh3, but not the empty vector or sh5, significantly attenuated cell death induced by a 24-h exposure to 14.3 µM CdMT-1 by 35.6 ± 4.2% (n = 13). In contrast, neither fluorescently labeled metallothionein-1 (MT-1) uptake nor free Cd²⁺ toxicity was altered by the effective DMT1 shRNA (sh3), indicating that cellular uptake of metal-MT-1 complexes and Cd²⁺-induced cell death signaling are not affected by DMT1 knockdown. Thus we conclude that endosomal/lysosomal DMT1 plays a role in renal PT CdMT-1 toxicity.

DMT1; endocytosis; heavy metals; metallothionein