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

Marouan Abouhamed, Natascha A. Wolff, Wing Kee Lee, Craig P. Smith, Frank Thévenod

    Research output: Contribution to journalArticlepeer-review

    Abstract

    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, Cd 2+ 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 Cd 2+-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. Copyright © 2007 the American Physiological Society.
    Original languageEnglish
    Pages (from-to)F705-F712
    JournalAmerican Journal of Physiology: Renal Physiology
    Volume293
    Issue number3
    DOIs
    Publication statusPublished - Sept 2007

    Keywords

    • DMT1
    • Endocytosis
    • Heavy metals
    • Metallothionein

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