Evidence for an essential role of megalin in transepithelial transport of retinol.

Transepithelial transport of retinol is linked to retinol-binding protein (RBP), which is taken up and also synthesized in a number of epithelia. By immunocytochemistry of human, rat, and mouse renal proximal tubules, a strong staining in apical endocytic vacuoles, lysosomes, endoplasmic reticulum, Golgi, and basal vesicles was observed, in accordance with luminal endocytic uptake as well as a constitutive synthesis and basal secretion of RBP. Analysis of mice with target disruption of the gene for the major endocytic receptor of proximal tubules, megalin, revealed no RBP in proximal tubules of these mice. Western blotting and HPLC of the urine of the megalin-deficient mice instead revealed a highly increased urinary excretion of RBP and retinol, demonstrating that glomerular filtered RBP-retinol of megalin-deficient mice escapes uptake by proximal tubules. A direct megalin-mediated uptake of purified RBP-retinol was indicated by surface plasmon resonance analysis and uptake in immortalized rat yolk sac cells. Uptake was partially inhibited by a polyclonal megalin antibody and the receptor-associated protein. The present data show that the absence of RBP-binding megalin causes a significantly increased loss of RBP and retinol in the urine, demonstrating a crucial role of megalin in vitamin A homeostasis.