Knockdown of endosomal/lysosomal divalent metal transporter 1 by RNA interference prevents cadmium-metallothionein-1 cytotoxicity in renal proximal tubule cells
| Autor: | Frank Thévenod, Natascha A. Wolff, Marouan Abouhamed, Wing-Kee Lee, Craig P. Smith |
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| Rok vydání: | 2007 |
| Předmět: |
Physiology
Apoptosis Glomerulus (kidney) Endocytosis Cell Line Kidney Tubules Proximal RNA interference medicine Metallothionein Animals RNA Messenger Cytotoxicity Cation Transport Proteins Kidney Gene knockdown biology digestive oral and skin physiology DMT1 Cell biology Rats medicine.anatomical_structure Biochemistry biology.protein RNA Interference |
| Zdroj: | American journal of physiology. Renal physiology. 293(3) |
| ISSN: | 1931-857X |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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