Megalin-dependent internalization of cadmium-metallothionein and cytotoxicity in cultured renal proximal tubule cells

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 similar to 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 mu M. At 14.3 mu M, metallothionein-1 uptake could be significantly attenuated by 30.9 +/- 6.6% (n = 4) by 1 mu M 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 mu M 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 mu M RAP, 400 mu g/ml anti-megalin antibody, or 5 mu M 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.