REVIEW J Am Soc Nephrol 10: 2224–2236, 1999

Essential Role of Megalin in Renal Proximal Tubule for Vitamin Homeostasis

ERIK ILSØ CHRISTENSEN* and THOMAS E. WILLNOW† *Department of Cell Biology, Institute of Anatomy, University of Aarhus, Denmark; and †Max-Delbrueck- Center for Molecular Medicine, Berlin, Germany.

Proteins filtered in renal glomeruli are reabsorbed in the prox- kidney, the is also localized to epithelial cells of several imal tubule by endocytosis and subsequently degraded in ly- other organs (Table 1). In addition to its main function as an sosomes, as recently extensively reviewed (1). In the proximal endocytic receptor, it may also be involved in calcium sensing tubule, there is virtually no transcellular transport of intact (10), as originally suggested by its ability to bind calcium in protein (2), and the extreme efficiency of the process of re- the kidney (11). moval of protein from the tubular lumen is substantiated by the A variety of ligands have been found for megalin (Table 2). fact that human urine is virtually devoid of protein. For several of these ligands, the megalin-mediated uptake in Two major aspects concerning the reabsorption process are proximal tubules appears to be of significant physiologic im- ␤ still unclear, namely: (1) the molecular basis for the efficient portance. Thus, H/ 2--I is normally clearance of protein from the tubular fluid, and (2) the physi- filtered in the glomeruli and is excreted in the urine in several ologic rationale for it, since the amounts of amino acids lost in tubular proteinuric conditions (30), whereas the other lipopro- the urine due to glomerular filtration of peptides and tein ligands mentioned in the table are probably only to a would probably otherwise be of minor importance in the over- limited extent filtered due to their high molecular weight. In all protein metabolism. In this review, we shall try at least in this regard, it has been shown that (20) and part to answer these two questions, introducing a major recep- (14) are internalized by glomerular podo- tor for endocytic clearance of proteins in the tubular fluid and cytes, possibly by megalin-mediated endocytosis (31). demonstrating the general importance of this reabsorption for The polybasic drugs are important ligands (16) because vitamin homeostasis. many of them, such as gentamicin, are nephrotoxic and oto- With respect to the mechanisms responsible for the endo- toxic and this toxicity appears to be at least in part due to the cytic uptake, several more or less specific proposals were put megalin-mediated endocytotic uptake. This observation opens forward (reviewed in reference 3), because it was difficult to possibilities for producing drugs with similar antibiotic prop- imagine a single receptor being responsible for binding and erties but without binding affinity for megalin, which might uptake of the wide variety of proteins and peptides present in render them less toxic. The low molecular weight peptides, the glomerular ultrafiltrate. However, at present one protein including insulin (Table 2), recently shown to be ligands for immediately attracts attention as a major scavenger receptor, megalin appear to be physiologic ligands since most of them namely, megalin. Megalin is a large membrane protein, about are filtered normally in the renal glomeruli. The affinity for 600 kD in its glycosylated form, located in the endocytic megalin, however, apparently is low, as it is also for pathway in proximal tubule cells and probably one of the main since several of these ligands including albumin fail to bind to endocytic receptor proteins in these cells. Megalin was origi- purified megalin in BIAcore experiments (unpublished obser- nally described as one of the pathogenic antigens in Heymann vations). Receptor-associated protein (RAP) is another ligand nephritis (4), an experimental rat model of glomerulonephritis for megalin. RAP is a 40-kD protein localized to the rough (reviewed in reference 5), and was localized to the glomerular endoplasmic reticulum. It binds to megalin and the other mem- podocytes and to the endocytic pathway of proximal tubule bers of the LDL receptor family with high affinity and has been cells by immunohistochemistry and immunocytochemistry (6). used experimentally in inhibition experiments to study other Rat (7) and human (8) megalin have been cloned, and the ligands for these receptors; thus far it has been shown to inhibit protein belongs to the family of endocytic membrane receptors binding of all known ligands to megalin with the exception of designated the LDL receptor family (see below). Besides in the insulin (24). Studies by Willnow et al. using RAP knockout mice (32,33) and Bu and Rennke (34) have implicated RAP in the early processing of members of the LDL receptor family, Received January 29, 1999. Accepted March 1, 1999. including megalin, probably functioning as a kind of chaperone Correspondence to Dr. Erik Ilsø Christensen, Department of Cell Biology, in the biosynthetic pathway of these receptors. Since in the Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C, Denmark. Phone: ϩ45 89 42 30 57; Fax: ϩ45 86 19 86 64; E-mail: [email protected] kidney proximal tubule megalin appears to be the only major 1046-6673/1010-2224 protein binding RAP with high affinity (Figure 1), RAP has Journal of the American Society of Nephrology been particularly important in studying ligands for megalin in Copyright © 1999 by the American Society of Nephrology the proximal tubule. Calcium appears important for the func- J Am Soc Nephrol 10: 2224–2236, 1999 Megalin-Mediated Endocytosis 2225

Table 1. Expression of megalina membrane and membrane receptors back to the apical plasma membrane (39). Deeper in the cytoplasm, a large number of Group Megalin lysosomes up to 1 ␮m in diameter and of varying electron Adult density are present. kidney The expression of megalin in the renal proximal tubule proximal tubule ϩϩϩϩ shown in Figure 2 has been studied extensively (6,11,40–43) glomerulus and recently reviewed (3). The brush border expression has a podocytes (rat) ϩ certain segmental variation (43). In the initial part of the lung proximal tubule, there is no labeling of the brush border, pneumocytes type II ϩϩ which, however, is extensively labeled in segment 2. In seg- epididymis ϩϩϩ ment 3, the brush border expression exhibits a distinctive endometrium ϩϩϩ spike-like appearance. The endocytic apparatus including oviduct ϩϩ coated pits, endosomes, and dense apical tubules is intensively thyroid ϩϩ labeled (Figure 2), and in addition many lysosomes are labeled parathyroid ϩϩϩ in the matrix, a labeling that is due mainly to degradation ependyma ϩϩ products of megalin (43). ileal enterocytes ϩ eye ciliary epithelium ϩϩ Megalin Is a Member of the LDL Receptor inner ear Gene Family epithelial cells ϩϩ When the first fragments of the rat megalin cDNA were Embryonic cloned, it became apparent that the receptor shares structural trophectoderm ϩϩ similarities with the LDL receptor (44). This finding was placenta confirmed when the complete cDNA sequences from rat and syncytiotrophoblast ϩ human megalin were elucidated (7,8). As seen in Figure 3, the yolk sac ϩϩϩ deduced cDNA sequence encodes a protein of approximately neuroectoderm ϩϩϩ 600 kD, which exhibits all of the hallmarks of an endocytic receptor of the LDL receptor gene family. Megalin is a type 1 a Data for megalin was modified from Zheng et al. (9). with a single transmembrane domain, a short cytoplasmic tail, and a large amino-terminal portion extending into the extracellular space. The amino-terminal tions of megalin not only for the reasons mentioned above, but region contains cysteine-rich ligands or complement-type re- calcium is also necessary for the binding of ligands to megalin. peats, stretches of approximately 40 amino acids each that are In this review, we shall describe in more detail three vitamin characterized by three internal bonds. These repeats carrier proteins recently shown to be specific ligands for mega- constitute the binding sites for ligands, and it has been dem- lin. These three ligands emphasize the role of megalin in the onstrated that several ligands bind to the same or closely proximal tubule as being responsible for the reabsorption of associated sites in the second cluster of ligand-binding repeats vital substances such as vitamins from the tubular fluid, which (45). Furthermore, megalin harbors cysteine-rich epidermal in part answers the second question concerning the importance growth factor (EGF) precursor-type repeats, separated by cys- of removing proteins from the tubular fluid. As described in the teine-poor spacer regions. The spacer regions contain YWTD sections that follow, megalin knockout mice (35) have been motifs responsible for pH-dependent release of ligands in en- invaluable tools in finding and describing a variety of ligands dosomal compartments. YWTD repeats flanked by EGF pre- for megalin. cursor-type repeats are referred to as the EGF precursor ho- mology domain. Finally, the cytoplasmic tail of megalin carries Ultrastructure and Immunocytochemical three copies of a NPXY motif, which directs receptors into Localization of Megalin in the Endocytic coated pits. Megalin does not contain an O-linked sugar do- Pathway in Renal Proximal Tubule main, which is found in some receptors of the gene family. The avidity by which the proximal tubule reabsorbs macro- Members of the LDL receptor superfamily can be divided molecules by endocytosis is illustrated by the extensively de- into two subgroups according to the structural organization of veloped endocytic apparatus, especially in segments 1 and 2 their extracellular domains. “Low molecular weight” receptors (reviewed in references 1 and 36). In brief, it consists of coated (95 to 150 kD in size) such as the LDL receptor (46), the very pits located between the very densely packed microvilli. The low density (VLDL) receptor (47), and the apoli- apical cytoplasm is filled with small coated and noncoated poprotein (apo) E receptor-2 (48) are composed of one amino- vesicles, some of which are in fact cross-sectioned coated pits terminal stretch of seven to eight ligand binding-type repeats (37,38), large endosomes up to 1 ␮m in diameter, and dense followed by one EGF precursor homology domain. In contrast, apical tubules, apparently free in the cytoplasm or connected to the “high molecular weight” receptors such as megalin and the small or large endosomes (38), responsible for the recycling of LDL receptor-related protein (49) consist of several such re- 2226 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 2224–2236, 1999

Table 2. Substances reported to bind to megalina

Substance Study Reference and inhibitors PAI-1 (Stefansson et al., 1995) (12) PAI-1-urokinase (Moestrup et al., 1993) (13) PAI-1-tPA (Willnow et al., 1992) (14) (Moestrup et al., 1993) (13) prourokinase (Stefansson et al., 1995) (12) (Kounnas et al., 1993) (15) aprotinin (Moestrup et al., 1995) (16) Vitamin binding proteins transcobalamin-vitamin B12 (Moestrup et al., 1996) (17) vitamin D binding protein (Nykjær et al., 1999) (18) retinol binding protein (Christensen et al., 1999) (19) apolipoprotein B (Stefansson et al., 1995) (20) apolipoprotein E (Willnow et al., 1992) (14) apolipoprotein J/ (Kounnas et al., 1995) (21) ␤ / 2-glycoprotein-I (Moestrup et al., 1998) (22) Polybasic drugs aminoglycosides (Moestrup et al., 1995) (16) polymyxin B (Moestrup et al., 1995) (16) aprotinin (Moestrup et al., 1995) (16) Low molecular weight peptides and hormones PTH (Hilpert et al., 1999) (23) insulin (Orlando et al., 1998) (24) ␤ 2- (Orlando et al., 1998) (24) epidermal growth factor (Orlando et al., 1998) (24) prolactin (Orlando et al., 1998) (24) lysozyme (Orlando et al., 1998) (24) cytochrome c (Orlando et al., 1998) (24) Other (Zheng et al., 1998) (25) plasminogen (Kanalas and Makker, 1991) (26) albumin (Cui et al., 1996) (27) (Willnow et al., 1992) (14) RAP (Christensen et al., 1992) (11) (Orlando et al., 1992) (28) (Kounnas et al., 1992) (29) (Willnow et al., 1992) (14) Ca2ϩ (Christensen et al., 1992) (11)

a PAI, plasminogen activator inhibitor; tPA, tissue-type plasminogen activator; PTH, parathyroid hormone; RAP, receptor-associated protein.

gions, each harboring one cluster of ligand binding-type re- anchored receptor. However, the existence of soluble receptor peats followed by one to four EGF precursor homology do- fragments in the kidney and in the medium of megalin-expressing mains. Thus, their extracellular portions resemble multiple cell lines has been reported (51,52). Whether these fragments have copies of the LDL receptor domain. The overall amino acid a distinct physiologic role remains to be elucidated. By amino acid sequence identity between megalin and other family members sequence, megalin is the largest receptor of the LDL receptor gene varies between 30 and 50%. The human megalin gene is family known to date. It may also be the phylogenetically oldest located on 2q24-q31 (50). member in this gene family, because it represents the mammalian All available information on the structure and subcellular lo- homologue of an endocytic receptor found in the nematode Cae- calization of megalin indicates that the protein is a membrane- norhabditis elegans (53). J Am Soc Nephrol 10: 2224–2236, 1999 Megalin-Mediated Endocytosis 2227

Figure 1. Light microscope autoradiography of cryosections from renal cortex of wild-type (A) and megalin-deficient (B) mice incubated with 125I-labeled receptor-associated protein. Specific labeling is seen exclusively apically in the proximal tubule cells of wild-type mice (arrowheads), corresponding to the localization of megalin (compare with Figure 5A, inset). Magnification, ϫ850.

Figure 2. Immunocytochemical localization of megalin in ultrathin cryosection from rat renal proximal tubule (segment 1) incubated with a sheep anti-megalin visualized by 10-nm gold particles. The labeling is confined to microvilli (MV), coated pits and coated endosomes (arrows), larger endosomes (E), and dense apical tubules (arrowheads). Labeling is also seen in the matrix of a lysosomal-like body (L). Magnification, ϫ50,000. 2228 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 2224–2236, 1999

Figure 3. The LDL receptor superfamily. The structural organization of some members of the LDL receptor gene family is depicted. Ligand binding-type repeats constitute the binding sites for ligands. Epidermal growth factor (EGF) precursor homology domains, consisting of EGF precursor-type repeats and YWTD spacer regions, are involved in the pH-dependent release of ligands in endosomes. NPXY designates the tetra-amino acid motif asparagine-proline-X-tyrosine, which directs the receptors into coated pits. apo, apolipoprotein; C. elegans, Caeno- rhabditis elegans; LRP, LDL receptor-related protein; VLDL, very low density lipoprotein.

Analysis of Megalin-Deficient Mice pregnancy also give rise to holoprosencephalic phenotypes. The distinct localization of megalin on the surface of ab- Common to most causes of holoprosencephaly is that they sorptive epithelia both in the embryo and the adult organism affect the viability of the neuroepithelium, a single layer of (Table 1) suggested that the receptor is involved in uptake of highly mitotic ectodermal cells that constitute the neural plate. ligands from the extracellular space. A number of macromol- During development, the neural plate forms the neural tube, ecules have been identified that bind to the receptor (Table 2). which differentiates into the various parts of the central ner- Which of these macromolecules constitutes endogenous li- vous system (CNS). Because the forebrain is the most rapidly gands needs to be confirmed in vivo. expanding part of the forming CNS, insults to the viability of To uncover the functions of megalin and to identify its the neuroepithelium are likely to affect the forebrain and de- endogenous ligands, we used targeted gene disruption to gen- rived tissues. Defects inducing holoprosencephaly are known erate megalin knockout mice (35). Megalin-deficient animals to act at a time before neural tube closure. During this time, are born alive but most of them die perinatally. They are megalin present on the apical membranes of neuroepithelial characterized by an abnormal formation of the forebrain cells is exposed to the fluids surrounding the embryo. This (prosencephalon) and forebrain-derived structures. Malforma- observation suggests that megalin is involved in the clearance tions include incomplete development of the eyes, lack of of ligands from extraembryonic fluids into the neuroepithelium olfactory bulbs and corpus callosum, a fused ventricular sys- and that deficiency in ligand uptake impairs development of tem, and incomplete separation of the forebrain hemispheres. this tissue. These defects are hallmarks of a syndrome known as holo- Although the gene-targeting experiment had uncovered an prosencephaly, the fusion of the prosencephalic hemispheres. important role for megalin in brain development, the ligands Holoprosencephaly is observed in patients and in animal mod- taken up by this receptor in the embryo and in the adult els (54). In humans it affects 1 in 16,000 live born children. organism remained unclear. A breakthrough was achieved Multiple cytogenetic aberrations on different when we observed that not all megalin-deficient newborns die have been associated with this malformation. Whether any of after birth. The severity of brain malformations varies among these genetic defects map to the megalin gene locus is unclear. individual animals, and 1 in 50 of the megalin Ϫ/Ϫ mice In addition to genetic lesions, various infections (e.g., cyto- survives to adulthood. These mice enabled us to identify some megalovirus) and toxic agents (e.g., alcohol) applied during of the endogenous receptor ligands in the kidney. J Am Soc Nephrol 10: 2224–2236, 1999 Megalin-Mediated Endocytosis 2229

Figure 4. Ultrastructure of mouse proximal tubules from wild-type (A) and megalin-deficient mice (B). In the wild-type mouse, the endocytic apparatus is extensively developed, consisting of coated pits (arrows), endosomes (E), dense apical tubules (arrowheads), and lysosomes (L). M, mitochondria. In the knockout mouse, the endocytic apparatus is much less developed (B). The apical cytoplasm appears empty with only a few dense apical tubules (arrowheads), very few endosome-like structures, and instead the cytoplasm contains ribosomes and dilated rough endoplasmic reticulum cisternae (arrows). G, Golgi apparatus. Magnification: ϫ31,000 in A; ϫ23,000 in B.

The ultrastructure of mouse proximal tubules (Figure 4A) is cross-reactivity with other proteins of the polyclonal antibody essentially as described above; however, in proximal tubules used (Figure 5B). from megalin-deficient animals, the ultrastructure is signifi- Because a number of studies have demonstrated the role of cantly changed but apparently only with respect to develop- megalin in the uptake of macromolecules from the glomerular ment of the endocytic apparatus. Thus, although the brush ultrafiltrate, we speculated that megalin-deficient mice should border and the ultrastructure of the cells appears otherwise exhibit tubular reabsorption deficiency and excrete receptor essentially unchanged, the number of coated pits, endosomes, ligands in the urine. This hypothesis was confirmed when the dense apical tubules, and lysosomes in megalin-deficient mice protein profile of urine samples was analyzed (Figure 6). is significantly decreased (Figure 4B), indicating the general Megalin Ϫ/Ϫ mice excrete in the urine a distinct pattern of importance of megalin for the apical endocytic process in these relatively low molecular weight proteins, indicating an inabil- cells. The megalin expression in mouse proximal tubules is ity of the proximal tubules to reabsorb filtered macromole- shown in Figure 5A, demonstrating at the light microscope cules. Similar phenotypes are observed in patients with Fan- level the apical localization of the protein, and at the subcel- coni renotubular syndrome, a tubular reabsorption deficiency lular level the localization on the brush border, in coated pits, caused by various genetic as well as environmental factors endosomes, dense apical tubules, and lysosomes. In megalin- (e.g., heavy metal poisoning). We applied amino acid sequence deficient mice, no labeling was seen, demonstrating also no analysis to identify some of the proteins excreted in the urine 2230 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 2224–2236, 1999 J Am Soc Nephrol 10: 2224–2236, 1999 Megalin-Mediated Endocytosis 2231

to 0.5 mg/ml), virtually all 25-(OH) vitamin D3 molecules in circulation are present in complex with DBP. A central step in vitamin D homeostasis is the conversion of

25-(OH) vitamin D3 to 1,25-(OH)2 vitamin D3, an important regulator of the systemic calcium metabolism. This conversion takes place in the epithelial cells of the proximal tubules. The

cells take up the precursor 25-(OH) vitamin D3 and convert it into the active vitamin by action of the 25-(OH) vitamin D3 1␣-hydroxylase in the mitochondria. Considerable interest has focused on elucidating the specific mechanisms that deliver

25-(OH) vitamin D3 to this renal cell type. In particular, the mode of cellular uptake of the sterol and the role of DBP in this process are still unclear. Excretion of DBP in the urine of megalin-deficient mice suggested an important role for mega-

lin in the tubular uptake of 25-(OH) vitamin D3. This hypoth- esis was confirmed in recent studies (18). We were able to

Figure 6. Urinary protein profile of wild-type and megalin Ϫ/Ϫ mice. demonstrate that complexes of 25-(OH) vitamin D3 and DBP Fifteen microliters of urine from mice of the indicated genotypes were are continuously filtered through the glomerulus and reab- subjected to 4 to 15% nonreducing sodium dodecyl sulfate-polyacryl- sorbed by megalin from the lumen of the proximal tubule. amide gel electrophoresis and staining with Coomassie blue. Protein Tubular retrieval of the complexes is essential to prevent bands corresponding to (asterisk), vitamin D binding constant urinary loss of the vitamin and to deliver the precursor protein (DBP), and retinol binding protein (RBP) are highlighted. F, for generation of 1,25-(OH) vitamin D . As a consequence of female; M, male. 2 3 the receptor gene defect, megalin Ϫ/Ϫ mice exhibit severe

vitamin D3 deficiency and suffer from bone formation defects. of knockout animals. Two of the proteins identified were Retinol Binding Protein particularly interesting because they represent plasma carriers RBP is a 21-kD retinol carrier protein in the blood circula- for lipophilic vitamins, the vitamin D binding protein (DBP), tion. The protein is filtered in the glomeruli and is widely and the retinol binding protein (RBP) (Figure 6). recognized as a marker for tubular proteinuria. We have re- cently shown that the protein binds to purified megalin by Proximal Tubular Handling of Three Vitamins BIAcore experiments and that the protein and retinol is found and Their Carrier Proteins in the urine of megalin-deficient mice but is absent in control In the sections that follow, we shall emphasize the role of mice (19) (Figure 6). Furthermore, endogenous RBP was found megalin in kidney proximal tubule, describing the reabsorption by immunocytochemistry in the proximal tubules of control of three vitamins (vitamin D, vitamin A [retinol], and vitamin mice but was absent in megalin knockout mice (Figure 7).

B12) and their binding proteins (DBP, RBP, and transcobal- There was an obvious segmental gradient of uptake, i.e., the amin [TC II]). It should be noted that these three proteins most uptake was very intense in the initial part of the proximal likely are representatives of a group of more or less related tubule and decreased in later segments with virtually no uptake carrier proteins, which undoubtedly in the future will be iden- in segment 3, indicating that the tubule fluid is cleared effi- tified for reabsorption processes in the renal proximal tubule by ciently for the protein rapidly after glomerular filtration. The identical or similar receptor-mediated endocytic uptake. cytoplasmic labeling observed in the initial parts of the prox- imal tubule (Figure 7) will be discussed later. In vitro uptake Vitamin D Binding Protein studies of RBP using a rat yolk sac carcinoma cell line ex-

The 58-kD DBP is the main transporter for vitamin D3 pressing megalin (55) demonstrated that RAP and anti-megalin metabolites in the circulation. It exhibits highest affinity for antibody in part inhibited uptake and degradation. In conclu- ϭ Ϫ10 Ϫ12 25-(OH) vitamin D3 (Kd 10 to 10 M). Due to this tight sion, these experiments demonstrate the importance of megalin binding affinity and the high plasma concentration of DBP (0.3 in the tubular reabsorption of RBP and thereby the conserva-

Figure 5. Immunocytochemical labeling for megalin as in Figure 2 of wild-type mouse (A) and megalin-deficient mouse (B). Labeling is seen on microvilli (MV), in coated pits, and in structures of the apical endocytic apparatus. Lysosomal labeling is also seen (L). (A, Lower Left Inset) Light microscope (horseradish peroxidase) visualization of megalin. Reaction is seen on the brush border, in apical endosomes (arrows), and in lysosome-like structures (arrowheads). (A, Upper Right Inset) High magnification of megalin expression. Labeling is found in coated pits (large arrowheads), in endosomes (arrows), and in dense apical tubules (small arrowheads). There is no labeling for megalin in the megalin-deficient mouse either at the electron microscope level (B) or at the light microscope level (inset in B). Magnification: ϫ35,000 in A and B; ϫ750 in lower left inset in A and B; ϫ53,000 in upper right inset in A. 2232 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 2224–2236, 1999

Figure 7. Labeling for RBP in wild-type mouse (A), megalin-deficient mouse (B), and rat (C). A granular labeling may be noted in the early part of the proximal tubule of wild-type mouse including the very initial part seen surrounding the glomerulus (G). No labeling is found in the knockouts (B). In the rat (C), there is in the early parts of the proximal tubule (S1), in addition to the granular labeling, a cytoplasmic labeling that is also obvious in the very early part (arrow) connected to the Bowman’s capsule of the glomerulus (G). In the later parts of the proximal tubule, only the granular labeling is seen (S2). Magnification, ϫ500.

tion of retinol that will otherwise be lost in the urine as sensor chips (17). Megalin-mediated cellular uptake, intracel- illustrated in the megalin knockout mice. Since megalin is lular transport, and degradation as well as RAP inhibition were normally expressed in the yolk sac of rodents (56) and in the demonstrated using intravenous injections and micropuncture placenta (Table 1), the possible reduced transfer of retinol into studies (proximal tubules) of rats as well as studies on rat yolk the megalin-deficient mouse embryos may also contribute to sac carcinoma cells. Preliminary unpublished results have also the developmental deficiencies observed (35). shown that megalin-deficient mice excrete increased amounts

of TC II and vitamin B12 in the urine compared with controls. Transcobalamin Again, these experiments demonstrate a crucial role of megalin

TC II is one of the major cobalamin (vitamin B12) carrier in maintaining vitamin B12 homeostasis. plasma proteins, with a molecular weight of 43 kD. The protein is filtered to a large extent in glomeruli and reabsorbed in the Intracellular Destiny of the Vitamins proximal tubule (57). It has been calculated (58) that the renal Figure 8 depicts the megalin-mediated uptake of the three glomerular filtration and subsequent tubular uptake of vitamin vitamin carrier proteins into renal proximal tubule cells. In ␮ B12 corresponds to about 1.5 g, which equals the amount of early and late endosomes, the proteins dissociate from megalin, vitamin B12 absorbed in the distal ileum bound to intrinsic and while the receptor returns to the apical plasma membrane factor via cubilin (the receptor). TC II binding via dense apical tubules, the proteins are transferred to lyso- to megalin was demonstrated by ligand blotting of renal cortex somes for subsequent degradation. Although this part of the and purified megalin, binding to megalin on cryosections and reabsorption pathway for the vitamins to a large extent appears surface plasmon resonance measurements on megalin BIAcore to be clarified, major questions remain concerning the transport J Am Soc Nephrol 10: 2224–2236, 1999 Megalin-Mediated Endocytosis 2233

of vitamins from the lysosomes and back to the circulation. It is highly unlikely that the general mechanisms for the three vitamins are identical. Vitamin D and vitamin A (retinol) are

both lipophilic, whereas vitamin B12 is hydrophilic. Small amounts of the apically reabsorbed vitamins probably also to a minor extent are used in the proximal tubule cells, and they may also to some extent undergo biochemical changes in the cells. Thus, vitamin D is being reabsorbed mainly as 25-OH-

D3, and we demonstrated that the hydroxylation into 1,25- diOH-D3 is dependent on megalin-mediated uptake (18). To our knowledge, no evidence has been presented indicat- ing proximal tubular synthesis of DBP. However, it appears highly unlikely that vitamin D in a nonregulated way should diffuse across the basolateral plasma membrane to meet apo- lipoprotein DBP in the interstitium. For retinol handling in the kidney, there are indications that RBP is being synthesized in proximal tubules. Thus, we showed by light microscope immunohistochemistry a strong apparent cytoplasmic labeling for RBP in the early part of the proximal tubules (Figure 7) and in addition a basal granular labeling (19). By electron microscope immunocytochemistry, we found labeling of nonlysosomal granules, granular endo- plasmic reticulum, and vesicles in the Golgi region (19). North- ern blot analysis has demonstrated RBP mRNA in the kidney at a level of 5 to 10% of that in the liver (59), and by in situ hybridization RBP has been localized to proximal tubules (60), although the labeling appeared confined to segment 3. The kidney appears to be important for the recycling of RBP– retinol complexes, and it has been estimated for rat that about 50% of the circulating pool originates from the kidney (61). Taken together, these results would indicate lysosomal degra- dation of reabsorbed RBP, coupling of retinol to newly syn- thesized RBP in the rough endoplasmic reticulum, and then basolateral secretion using the normal secretory pathway through the Golgi apparatus.

With respect to vitamin B12, it appears well established that TC II-CN-B12 is taken up by mammalian cells by endocytosis, and while TC II is broken down in the lysosomes, vitamin B12 is released into the cytoplasm (62). A magnesium- and pH- dependent transport system has been further characterized us- ing membrane vesicles from purified lysosomal fractions from

rat liver (63), and a defect in vitamin B12 release from lyso- somes has been ascribed to an inborn error (64). TC II mRNA has been found in high amounts in the kidney by Northern blot, in human 14-fold higher in the kidney than in the liver (65). It therefore appears reasonable to suggest a secretory pathway in

the proximal tubule for vitamin B12 similar to the one de- scribed above for retinol. Alternatively, as suggested by Figure 8. The schematic drawing illustrates the megalin-mediated Rothenberg and Quadros, in the intestine vitamin B12 might be proximal tubular reabsorption of the three vitamin carrier protein coupled in the interstitial fluid to TC II produced by endothelial complexes: DBP (vitamin D binding protein/vitamin D complexes), 3 cells (66). TC (transcobalamin/vitamin B12 complexes), and RBP (retinol bind- ing protein/vitamin A complexes). The lysosomal degradation of the three carrier proteins is depicted in the lower part as well as the Conclusion The megalin-mediated reabsorption of the three vitamin hydroxylation of 25-OH-D3 to 1,25-diOH-D3. The mechanisms of basolateral secretion of the three vitamins remain to be clarified. carrier proteins described in this article substantiates the role of megalin not only as a mediator of protein reabsorption in the renal proximal tubule, but also in capturing vital substances 2234 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 2224–2236, 1999 from the tubular fluid, which would otherwise be lost in the 11. Christensen EI, Gliemann J, Moestrup SK: Renal tubule gp330 is urine. The uptake of lipophilic substances such as retinol and a calcium binding receptor for endocytic uptake of protein. J Histochem Cytochem 40: 1481–1490, 1992 vitamin D3 bound to their carrier proteins also suggests that similar mechanisms may apply for other important regulators 12. Stefansson S, Kounnas MZ, Henkin J, Mallampalli RK, Chappell such as glucocorticoids and other steroid hormones. Further- DA, Strickland DK, Argraves WS: gp330 on type II pneumo- cytes mediates endocytosis leading to degradation of pro-uroki- more, evidence is presented for retinol and vitamin B that 12 nase, plasminogen activator inhibitor-1 and urokinase–plasmino- subsequent to lysosomal degradation of the carrier proteins, the gen activator inhibitor-1 complex. J Cell Sci 108: 2361–2368, vitamins may be coupled to newly synthesized carriers and 1995 secreted at the basolateral plasma membrane. The intracellular 13. Moestrup SK, Nielsen S, Andreasen P, Jørgensen KE, Nykjær A, events leading to this secretion, however, await further clari- Røigaard H, Gliemann J, Christensen EI: Epithelial glycoprotein- fication. 330 mediates endocytosis of plasminogen activator–plasminogen activator inhibitor type-1 complexes. J Biol Chem 268: 16564– Acknowledgments 16570, 1993 14. 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