No abstract

Although myeloma light chains are known to undergo receptor-mediated endocytosis in the kidney, the molecular identity of the receptor has not been characterized. We examined the interaction between cubilin (gp280) and four species of light chains isolated from the urine of patients with multiple myeloma. Four lines of evidence identify cubilin, a giant glycoprotein receptor, which is restricted in distribution to endocytic scavenger pathways and which has potent effects on endosomal trafficking, as a potentially physiologically relevant binding site for light chains: 1) light chains coeluted during immunoaffinity purification of cubilin; 2) polyclonal antisera to cubilin but not control sera, displaced human light chain binding from rat renal brush-border membranes; 3) cubilin bound to multiple species of light chains during surface plasmon resonance; 4) anti-cubilin antiserum interfered with light chain endocytosis by visceral yolk sac epithelial cells. However, both binding of light chains to brush-border membranes and endocytosis of light chains by yolk sac epithelial cells were only partially inhibited by anticubilin antibodies, suggesting presence of additional or alternate binding sites for light chains. Excess light chain had a potent inhibitory effect on endosomal fusion in vitro. Binding showed dose and time-dependent saturability with low-affinity, high-capacity equilibrium binding parameters. These data demonstrate that cubilin plays a role in the endocytosis and trafficking of light chains in renal proximal tubule cells.

The rotating wall vessel has gained popularity as a clinical cell culture tool to produce hormonal implants. It is desirable to understand the mechanisms by which the rotating wall vessel induces genetic changes, if we are to prolong the useful life of implants. During rotating wall vessel culture gravity is balanced by equal and opposite hydrodynamic forces including shear stress. The current study provides the first evidence that shear stress response elements, which modulate gene expression in endothelial cells, are also active in epithelial cells. Rotating wall culture of renal cells changes expression of select gene products including the giant glycoprotein scavenger receptors cubulin and megalin, the structural microvillar protein villin, and classic shear stress response genes ICAM, VCAM and MnSOD. Using a putative endothelial cell shear stress response element binding site as a decoy, we demonstrate the role of this sequence in the regulation of selected genes in epithelial cells. However, many of the changes observed in the rotating wall vessel are independent of this response element. It remains to define other genetic response elements modulated during rotating wall vessel culture, including the role of hemodynamics characterized by 3-dimensionality, low shear and turbulence, and cospatial relation of dissimilar cell types.