title: Knockdown of endosomal/lysosomal divalent metal transporter 1 by RNA interference prevents cadmium-metallothionein-1 cytotoxicity in renal proximal tubule cells.
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, 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.
authors.affiliation: Department of Physiology and Pathophysiology, University of Witten/Herdecke, D-58448 Witten, Germany.
authors.forename: Marouan
authors.initials: M
authors.lastname: Abouhamed
authors.forename: Natascha A
authors.initials: NA
authors.lastname: Wolff
authors.forename: Wing-Kee
authors.initials: WK
authors.lastname: Lee
authors.forename: Craig P
authors.initials: CP
authors.lastname: Smith
authors.forename: Frank
authors.initials: F
authors.lastname: Thévenod
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D027682:Cation Transport Proteins / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004705:Endocytosis; D007687:Kidney Tubules, Proximal / Q000166:cytology* / Q000187:drug effects*; D008668:Metallothionein / Q000633:toxicity*; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D000099236:Solute Carrier Family 11, Member 2
chemical_list: D027682:Cation Transport Proteins; D008668:Metallothionein; D012333:RNA, Messenger; D000099236:Solute Carrier Family 11, Member 2; C116509:cadmium-metallothionein complex