Cadmium Complexed with β2-Microglubulin, Albumin and Lipocalin-2 rather than Metallothionein Cause Megalin:Cubilin Dependent Toxicity of the Renal Proximal Tubule

Cadmium (Cd(2+)) in the environment is a significant health hazard. Chronic low Cd(2+) exposure mainly results from food and tobacco smoking and causes kidney damage, predominantly in the proximal tubule. Blood Cd(2+) binds to thiol-containing high (e.g., albumin, transferrin) and low molecular weight proteins (e.g., the high-affinity metal-binding protein metallothionein, β2-microglobulin, α1-microglobulin and lipocalin-2). These plasma proteins reach the glomerular filtrate and are endocytosed at the proximal tubule via the multiligand receptor complex megalin:cubilin. The current dogma of chronic Cd(2+) nephrotoxicity claims that Cd(2+)-metallothionein endocytosed via megalin:cubilin causes renal damage. However, a thorough study of the literature strongly argues for revision of this model for various reasons, mainly: (i) It relied on studies with unusually high Cd(2+)-metallothionein concentrations; (ii) the K(D) of megalin for metallothionein is ~10(5)-times higher than (Cd(2+))-metallothionein plasma concentrations. Here we investigated the uptake and toxicity of ultrafiltrated Cd(2+)-binding protein ligands that are endocytosed via megalin:cubilin in the proximal tubule. Metallothionein, β2-microglobulin, α1-microglobulin, lipocalin-2, albumin and transferrin were investigated, both as apo- and Cd(2+)-protein complexes, in a rat proximal tubule cell line (WKPT-0293 Cl.2) expressing megalin:cubilin at low passage, but is lost at high passage. Uptake was determined by fluorescence microscopy and toxicity by MTT cell viability assay. Apo-proteins in low and high passage cells as well as Cd(2+)-protein complexes in megalin:cubilin deficient high passage cells did not affect cell viability. The data prove Cd(2+)-metallothionein is not toxic, even at >100-fold physiological metallothionein concentrations in the primary filtrate. Rather, Cd(2+)-β2-microglobulin, Cd(2+)-albumin and Cd(2+)-lipocalin-2 at concentrations present in the primary filtrate are taken up by low passage proximal tubule cells and cause toxicity. They are therefore likely candidates of Cd(2+)-protein complexes damaging the proximal tubule via megalin:cubilin at concentrations found in the ultrafiltrate.