ARTICLES Evidence for an Essential Role of Megalin in Transepithelial Transport of Retinol ERIK ILSØ CHRISTENSEN,* JAN ØIVIND MOSKAUG, ‡ HENRIK VORUM, † CHRISTIAN JACOBSEN, † THOMAS E. GUNDERSEN, ‡ ANDERS NYKJÆR, § RUNE BLOMHOFF, ‡ THOMAS E. WILLNOW § and SØREN K. MOESTRUP † *Department of Cell Biology, Institute of Anatomy and † Department of Medical Biochemistry, University of Aarhus, Denmark; ‡ Institute for Nutrition Research, University of Oslo, Norway; and § Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany. Abstract. Transepithelial transport of retinol is linked to reti- nol-binding protein (RBP), which is taken up and also synthe- sized in a number of epithelia. By immunocytochemistry of human, rat, and mouse renal proximal tubules, a strong staining in apical endocytic vacuoles, lysosomes, endoplasmic reticu- lum, Golgi, and basal vesicles was observed, in accordance with luminal endocytic uptake as well as a constitutive syn- thesis 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 tu- bules 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 glo- merular 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 sig- nificantly increased loss of RBP and retinol in the urine, demonstrating a crucial role of megalin in vitamin A homeosta- sis. Retinol-binding protein (RBP1) is a 21-kD plasma protein and the main carrier of vitamin A (retinol) in plasma. Retinol is coupled to RBP in the liver and the complex is circulating in plasma bound to transthyretin (TTR), previously named preal- bumin, which to a certain extent prevents the RBP-retinol complex from being filtered in the glomeruli. However, 4 to 5% of the circulating RBP-retinol complex is not bound to TTR (1), and the kidney appears to be a very important organ in the recycling of RBP-retinol, contributing about 50% of the total circulating pool of RBP-retinol as estimated in rat (2). In accordance with this, RBP has been intensively used clinically to determine proteinurias of tubular origin (3,4), further indi- cating that RBP is filtered to a major extent in the renal glomeruli and taken up in proximal tubules as confirmed by immunohistochemistry (5,6). The role of the kidney in retinol homeostasis is also substantiated by the finding that acute renal failure induces significant elevations of serum retinol concen- trations (7,8). Furthermore, RBP mRNA has been detected in rat kidney by in situ hybridization (9), and it is suggested that retinol subsequent to tubular uptake is being returned to the circulation again in complex with RBP. Because RBP is taken up in proximal tubules, and retinol stimulates the expression of megalin in a rat kidney proximal tubule cell line (10), we investigated if this highly expressed receptor in proximal tubules might mediate uptake of RBP. Megalin, a 600-kD protein localized in the endocytic pathway of renal proximal tubules (11), belongs to the LDL receptor family (12–14). The protein functions as an endocytic receptor for a wide variety of substances, including lipoproteins (15– 18), albumin (19), and basic drugs (20) (reviewed in reference (21)). In addition, megalin binds calcium and receptor-associ- ated protein (RAP), a chaperone-like protein (22). Interest- ingly, megalin also functions as a receptor for endocytic uptake of the two vitamin carrier proteins, vitamin B 12 -carrier transco- balamin (23) and vitamin D-binding protein (A. Nykjær, D. Dragun, D. Walther, et al., Cell 1999, in press). These findings demonstrate that megalin is fundamental for the retainment of substances vital for the organism. The general importance of megalin is supported by the findings that knockout of the megalin gene in mice results in a high mortality and develop- mental abnormalities (24). The present study was carried out to investigate the impor- tance of megalin for renal proximal tubular reabsorption and synthesis of RBP, which is suggested to control the transepi- thelial transport of retinol. Using different approaches includ- ing immunocytochemistry and urine analyses of megalin-defi- Received September 23, 1998. Accepted October 5, 1998. Correspondence to Dr. Erik Ilsø Christensen, Department of Cell Biology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C, Denmark. Phone: (45) 89 42 30 57; Fax: (45) 86 19 86 64; E-mail: EIC@ANA.AU.DK 1046-6673/1004-0685$03.00/0 Journal of the American Society of Nephrology Copyright © 1999 by the American Society of Nephrology J Am Soc Nephrol 10: 685– 695, 1999