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 approximately 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 microM. At 14.3 microM, metallothionein-1 uptake could be significantly attenuated by 30.9 +/- 6.6% (n = 4) by 1 muM 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 muM 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 microM RAP, 400 microg/ml anti-megalin antibody, or 5 microM 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.
Wolff
Natascha A
NA
Department of Physiology and Pathophysiology, University of Witten/Herdecke, Faculty of Medicine, Stockumer Strasse 12, D-58448 Witten, Germany.
Abouhamed
Marouan
M
Verroust
Pierre J
PJ
Thévenod
Frank
F
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology / Q000302:isolation & purification; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D026561:Low Density Lipoprotein Receptor-Related Protein-2 / Q000096:biosynthesis / Q000378:metabolism / Q000502:physiology*; D008301:Maleimides; D008668:Metallothionein / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D011956:Receptors, Cell Surface / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction
C504424:Alexa Fluor 488 C5-maleimide; D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D026561:Low Density Lipoprotein Receptor-Related Protein-2; D008301:Maleimides; D011956:Receptors, Cell Surface; C004203:cadmium-binding protein; C029486:intrinsic factor-cobalamin receptor; D008668:Metallothionein; D019204:GTP-Binding Proteins