Abstract
Chronic cadmium (Cd2+) exposure results in renal proximal tubular cell damage. Delivery of Cd2+ to the kidney occurs mainly as complexes with metallothionein-1 (molecular mass ∼ 7 kDa), freely filtered at the glomerulus. For Cd2+ to gain access to the proximal tubule cells, these complexes are thought to be internalized via receptors for small protein ligands, such as megalin and cubilin, followed by release of Cd2+ from metallothionein-1 in endosomal/lysosomal compartments. To investigate the role of megalin in renal cadmium-metallothionein-1 reabsorption, megalin expression and dependence of cadmium-metallothionein-1 internalization and cytotoxicity on megalin were studied in a renal proximal tubular cell model (WKPT-0293 Cl.2 cells). Expression of megalin was detected by reverse transcriptase-polymerase chain reaction and visualized by immunofluorescence both at the cell surface (live staining) and intracellularly (permeabilized cells). Internalization of Alexa Fluor 488-coupled metallothionein-1 was concentration-dependent, saturating at approximately 15 μM. At 14.3 μM, metallothionein-1 uptake could be significantly attenuated by 30.9 ± 6.6% (n = 4) by 1 μM of the receptor-associated protein (RAP) used as a competitive inhibitor of cadmium-metallothionein-1 binding to megalin and cubilin. Consistently, cytotoxicity of a 24-h treatment with 7.14 μM cadmium-metallothionein-1 was significantly reduced by 41.0 ± 7.6%, 61.6 ± 3.4%, and 26.2 ± 1.8% (n = 4-5 each) by the presence of 1 μM RAP, 400 μg/ml anti-megalin antibody, or 5 μM of the cubilin-specific ligand, apo-transferrin, respectively. Cubilin expression in proximal tubule cells was also confirmed at the mRNA and protein level. The data indicate that renal proximal tubular cadmium-metallothionein-1 uptake and cell death are mediated at least in part by megalin.
Footnotes
-
This work was supported by the Deutsche Forschungsgemeinschaft (TH 345/8-1 and 8-2) and by start-up funds from the University of Witten/Herdecke to F.T. and by Association pour la Recherche sur le Cancer Grant 3443 to P.J.V.
-
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
-
doi:10.1124/jpet.106.102574.
-
ABBREVIATIONS: LY294002, 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride; DMT1, divalent metal transporter 1; RAP, receptor-associated protein; RT, reverse transcriptase; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; H-33342, 2′-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5′-bi-1H-benzimidazole, 3HCl; MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ANOVA, analysis of variance.
-
↵1 Both authors contributed equally to this work.
- Received February 13, 2006.
- Accepted May 9, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
JPET articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|