Megalin mediates renal uptake of heavy metal metallothionein complexes

Although several heavy metal toxins are delivered to the kidney on the carrier protein metallothionein (MT), uncertainty as to how MT enters proximal tubular cells limits treatment strategies. Prompted by reports that MT-I interferes with renal uptake of the megalin ligand beta(2)-microglobulin in conscious rats, we tested the hypothesis that megalin binds MT and mediates its uptake. Three lines of evidence suggest that binding of MT to megalin is critical in renal proximal tubular uptake of MT-bound heavy metals. First, MT binds megalin, but not cubilin, in direct surface plasmon resonance studies. Binding of MT occurs at a single site with a K(d) approximately 10(-4) and, as with other megalin ligands, depends on divalent cations. Second, antisera and various known megalin ligands inhibit the uptake of fluorescently labeled MT in model cell systems. Anti-megalin antisera, but not control sera, displace >90% bound MT from rat renal brush-border membranes. Megalin ligands including beta(2)-microglobulin and also recombinant MT fragments compete for uptake by megalin-expressing rat yolk sac BN-16 cells. Third, megalin and fluorescently labeled MT colocalize in BN-16 cells, as shown by fluorescent microscopic techniques. Follow-up surface plasmon resonance and flow cytometry studies using overlapping MT Peptides and recombinant MT fragments identify the hinge SCKKSCC region of MT as a critical site for megalin binding. These findings suggest that disruption of the SCKKSCC motif can inhibit proximal tubular MT uptake and thereby eliminate much of the renal accumulation and toxicity of heavy metals such as cadmium, gold, copper, and cisplatinum.