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

• Published in: American journal of physiology. Renal physiology 2007-09, Vol.293 (3), p.F705-F712
• Main Authors: Abouhamed, Marouan, Wolff, Natascha A, Lee, Wing-Kee, Smith, Craig P, Thévenod, Frank
• Format: Article
• Language: English
• Subjects: Animals; Apoptosis - drug effects; Apoptosis - physiology; Cation Transport Proteins - deficiency; Cation Transport Proteins - genetics; Cation Transport Proteins - metabolism; Cell Line; Chemical elements; Endocytosis; Heavy metals; Human exposure; Kidney Tubules, Proximal - cytology; Kidney Tubules, Proximal - drug effects; Metallothionein - toxicity; Rats; Ribonucleic acid; RNA; RNA Interference; RNA, Messenger - genetics; RNA, Messenger - metabolism; Toxicity
• ISSN: 1931-857X; 1522-1466
• DOI: 10.1152/ajprenal.00198.2007

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 microM 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.