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

Chronic exposure to [Cd.sup.2+] causes renal proximal tubular (PT) damage. [Cd.sup.2+] 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.sup.2+] is thought to be released in the cytosol to activate cell death pathways. The proton-coupled divalent metal transporter DMT1 also transports [Cd.sup.2+] 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 C1.2, we tested the hypothesis that endosomal/lysosomal DMTI is involved in CdMT-1 nephrotoxicity. One out of 5 shRNAs tested (sh3) significantly reduced expression of DMTI protein detected by immunoblotting and DMT1 mRNA as determined by RT-PCR by 45.1 [+ or -] 9.6 and 36.9 [+ or -] 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 [micro]M CdMT-1 by 35.6 [+ or -] 4.2% (17 = 13). In contrast, neither fluorescently labeled metallothionein-1 (MT-1) uptake nor free [Cd.sup.2+] toxicity was altered by the effective DMT1 shRNA (sh3), indicating that cellular uptake of metal-MT-1 complexes and [Cd.sup.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. DMT1; endocytosis; heavy metals; metallothionein