Essential Role of Megalin in Renal Proximal Tubule for Vitamin Homeostasis (1999)

Proteins filtered in renal glomeruli are reabsorbed in the proximal tubule by endocytosis and subsequently degraded in lysosomes, as recently extensively reviewed (1). In the proximal tubule, there is virtually no transcellular transport of intact protein (2), and the extreme efficiency of the process of removal of protein from the tubular lumen is substantiated by the fact that human urine is virtually devoid of protein. Two major aspects concerning the reabsorption process are still unclear, namely: (1) the molecular basis for the efficient clearance of protein from the tubular fluid, and (2) the physiologic rationale for it, since the amounts of amino acids lost in the urine due to glomerular filtration of peptides and proteins would probably otherwise be of minor importance in the overall protein metabolism. In this review, we shall try at least in part to answer these two questions, introducing a major receptor for endocytic clearance of proteins in the tubular fluid and demonstrating the general importance of this reabsorption for vitamin homeostasis. With respect to the mechanisms responsible for the endocytic uptake, several more or less specific proposals were put forward (reviewed in reference 3), because it was difficult to imagine a single receptor being responsible for binding and uptake of the wide variety of proteins and peptides present in the glomerular ultrafiltrate. However, at present one protein immediately attracts attention as a major scavenger receptor, namely, megalin. Megalin is a large membrane protein, about 600 kD in its glycosylated form, located in the endocytic pathway in proximal tubule cells and probably one of the main endocytic receptor proteins in these cells. Megalin was originally described as one of the pathogenic antigens in Heymann nephritis (4), an experimental rat model of glomerulonephritis (reviewed in reference 5), and was localized to the glomerular podocytes and to the endocytic pathway of proximal tubule cells by immunohistochemistry and immunocytochemistry (6). Rat (7) and human (8) megalin have been cloned, and the protein belongs to the family of endocytic membrane receptors designated the LDL receptor family (see below). Besides in the kidney, the protein is also localized to epithelial cells of several other organs (Table 1). In addition to its main function as an endocytic receptor, it may also be involved in calcium sensing (10), as originally suggested by its ability to bind calcium in the kidney (11).Table 1: Expression of megalinaA variety of ligands have been found for megalin (Table 2). For several of these ligands, the megalin-mediated uptake in proximal tubules appears to be of significant physiologic importance. Thus, apolipoprotein H/β2-glycoprotein-I is normally filtered in the glomeruli and is excreted in the urine in several tubular proteinuric conditions (30), whereas the other lipoprotein ligands mentioned in the table are probably only to a limited extent filtered due to their high molecular weight. In this regard, it has been shown that apolipoprotein B (20) and apolipoprotein E (14) are internalized by glomerular podocytes, possibly by megalin-mediated endocytosis (31).Table 2: Substances reported to bind to megalinaThe polybasic drugs are important ligands (16) because many of them, such as gentamicin, are nephrotoxic and ototoxic and this toxicity appears to be at least in part due to the megalin-mediated endocytotic uptake. This observation opens possibilities for producing drugs with similar antibiotic properties but without binding affinity for megalin, which might render them less toxic. The low molecular weight peptides, including insulin (Table 2), recently shown to be ligands for megalin appear to be physiologic ligands since most of them are filtered normally in the renal glomeruli. The affinity for megalin, however, apparently is low, as it is also for albumin since several of these ligands including albumin fail to bind to purified megalin in BIAcore experiments (unpublished observations). Receptor-associated protein (RAP) is another ligand for megalin. RAP is a 40-kD protein localized to the rough endoplasmic reticulum. It binds to megalin and the other members of the LDL receptor family with high affinity and has been used experimentally in inhibition experiments to study other ligands for these receptors; thus far it has been shown to inhibit binding of all known ligands to megalin with the exception of 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, including megalin, probably functioning as a kind of chaperone in the biosynthetic pathway of these receptors. Since in the kidney proximal tubule megalin appears to be the only major protein binding RAP with high affinity (Figure 1), RAP has been particularly important in studying ligands for megalin in the proximal tubule. Calcium appears important for the functions of megalin not only for the reasons mentioned above, but calcium is also necessary for the binding of ligands to megalin.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, ×850.In this review, we shall describe in more detail three vitamin carrier proteins recently shown to be specific ligands for megalin. These three ligands emphasize the role of megalin in the proximal tubule as being responsible for the reabsorption of vital substances such as vitamins from the tubular fluid, which in part answers the second question concerning the importance of removing proteins from the tubular fluid. As described in the sections that follow, megalin knockout mice (35) have been invaluable tools in finding and describing a variety of ligands for megalin. Ultrastructure and Immunocytochemical Localization of Megalin in the Endocytic Pathway in Renal Proximal Tubule The avidity by which the proximal tubule reabsorbs macromolecules by endocytosis is illustrated by the extensively developed endocytic apparatus, especially in segments 1 and 2 (reviewed in references 1 and 36). In brief, it consists of coated pits located between the very densely packed microvilli. The apical cytoplasm is filled with small coated and noncoated vesicles, some of which are in fact cross-sectioned coated pits (37,38), large endosomes up to 1 μm in diameter, and dense apical tubules, apparently free in the cytoplasm or connected to small or large endosomes (38), responsible for the recycling of membrane and membrane receptors back to the apical plasma membrane (39). Deeper in the cytoplasm, a large number of lysosomes up to 1 μm in diameter and of varying electron density are present. The expression of megalin in the renal proximal tubule shown in Figure 2 has been studied extensively (6,11,40,41,42,43) and recently reviewed (3). The brush border expression has a certain segmental variation (43). In the initial part of the proximal tubule, there is no labeling of the brush border, which, however, is extensively labeled in segment 2. In segment 3, the brush border expression exhibits a distinctive spike-like appearance. The endocytic apparatus including coated pits, endosomes, and dense apical tubules is intensively labeled (Figure 2), and in addition many lysosomes are labeled in the matrix, a labeling that is due mainly to degradation products of megalin (43).Figure 2: . . Immunocytochemical localization of megalin in ultrathin cryosection from rat renal proximal tubule (segment 1) incubated with a sheep anti-megalin antibody 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, ×50,000.Megalin Is a Member of the LDL Receptor Gene Family When the first fragments of the rat megalin cDNA were cloned, it became apparent that the receptor shares structural similarities with the LDL receptor (44). This finding was confirmed when the complete cDNA sequences from rat and human megalin were elucidated (7,8). As seen in Figure 3, the deduced cDNA sequence encodes a protein of approximately 600 kD, which exhibits all of the hallmarks of an endocytic receptor of the LDL receptor gene family. Megalin is a type 1 cell surface receptor with a single transmembrane domain, a short cytoplasmic tail, and a large amino-terminal portion extending into the extracellular space. The amino-terminal region contains cysteine-rich ligands or complement-type repeats, stretches of approximately 40 amino acids each that are characterized by three internal disulfide bonds. These repeats constitute the binding sites for ligands, and it has been demonstrated that several ligands bind to the same or closely associated sites in the second cluster of ligand-binding repeats (45). Furthermore, megalin harbors cysteine-rich epidermal growth factor (EGF) precursor-type repeats, separated by cysteine-poor spacer regions. The spacer regions contain YWTD motifs responsible for pH-dependent release of ligands in endosomal compartments. YWTD repeats flanked by EGF precursor-type repeats are referred to as the EGF precursor homology domain. Finally, the cytoplasmic tail of megalin carries three copies of a NPXY motif, which directs receptors into coated pits. Megalin does not contain an O-linked sugar domain, which is found in some receptors of the gene family.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, Caenorhabditis elegans; LRP, LDL receptor-related protein; VLDL, very low density lipoprotein.Members of the LDL receptor superfamily can be divided into two subgroups according to the structural organization of their extracellular domains. “Low molecular weight” receptors (95 to 150 kD in size) such as the LDL receptor (46), the very low density lipoprotein (VLDL) receptor (47), and the apolipoprotein (apo) E receptor-2 (48) are composed of one aminoterminal stretch of seven to eight ligand binding-type repeats followed by one EGF precursor homology domain. In contrast, the “high molecular weight” receptors such as megalin and the LDL receptor-related protein (49) consist of several such regions, each harboring one cluster of ligand binding-type repeats followed by one to four EGF precursor homology domains. Thus, their extracellular portions resemble multiple copies of the LDL receptor domain. The overall amino acid sequence identity between megalin and other family members varies between 30 and 50%. The human megalin gene is located on chromosome 2q24-q31 (50). All available information on the structure and subcellular localization of megalin indicates that the protein is a membrane-anchored receptor. However, the existence of soluble receptor fragments in the kidney and in the medium of megalin-expressing cell lines has been reported (51,52). Whether these fragments have a distinct physiologic role remains to be elucidated. By amino acid sequence, megalin is the largest receptor of the LDL receptor gene family known to date. It may also be the phylogenetically oldest member in this gene family, because it represents the mammalian homologue of an endocytic receptor found in the nematode Caenorhabditis elegans (53). Analysis of Megalin-Deficient Mice The distinct localization of megalin on the surface of absorptive epithelia both in the embryo and the adult organism (Table 1) suggested that the receptor is involved in uptake of ligands from the extracellular space. A number of macromolecules have been identified that bind to the receptor (Table 2). Which of these macromolecules constitutes endogenous ligands needs to be confirmed in vivo. To uncover the functions of megalin and to identify its endogenous ligands, we used targeted gene disruption to generate megalin knockout mice (35). Megalin-deficient animals are born alive but most of them die perinatally. They are characterized by an abnormal formation of the forebrain (prosencephalon) and forebrain-derived structures. Malformations include incomplete development of the eyes, lack of olfactory bulbs and corpus callosum, a fused ventricular system, and incomplete separation of the forebrain hemispheres. These defects are hallmarks of a syndrome known as holoprosencephaly, the fusion of the prosencephalic hemispheres. Holoprosencephaly is observed in patients and in animal models (54). In humans it affects 1 in 16,000 live born children. Multiple cytogenetic aberrations on different chromosomes have been associated with this malformation. Whether any of these genetic defects map to the megalin gene locus is unclear. In addition to genetic lesions, various infections (e.g., cytomegalovirus) and toxic agents (e.g., alcohol) applied during pregnancy also give rise to holoprosencephalic phenotypes. Common to most causes of holoprosencephaly is that they affect the viability of the neuroepithelium, a single layer of highly mitotic ectodermal cells that constitute the neural plate. During development, the neural plate forms the neural tube, which differentiates into the various parts of the central nervous system (CNS). Because the forebrain is the most rapidly expanding part of the forming CNS, insults to the viability of the neuroepithelium are likely to affect the forebrain and derived tissues. Defects inducing holoprosencephaly are known to act at a time before neural tube closure. During this time, megalin present on the apical membranes of neuroepithelial cells is exposed to the fluids surrounding the embryo. This observation suggests that megalin is involved in the clearance of ligands from extraembryonic fluids into the neuroepithelium and that deficiency in ligand uptake impairs development of this tissue. Although the gene-targeting experiment had uncovered an important role for megalin in brain development, the ligands taken up by this receptor in the embryo and in the adult organism remained unclear. A breakthrough was achieved when we observed that not all megalin-deficient newborns die after birth. The severity of brain malformations varies among individual animals, and 1 in 50 of the megalin -/- mice survives to adulthood. These mice enabled us to identify some of the endogenous receptor ligands in the kidney. The ultrastructure of mouse proximal tubules (Figure 4A) is essentially as described above; however, in proximal tubules from megalin-deficient animals, the ultrastructure is significantly changed but apparently only with respect to development of the endocytic apparatus. Thus, although the brush border and the ultrastructure of the cells appears otherwise essentially unchanged, the number of coated pits, endosomes, dense apical tubules, and lysosomes in megalin-deficient mice is significantly decreased (Figure 4B), indicating the general importance of megalin for the apical endocytic process in these cells. The megalin expression in mouse proximal tubules is shown in Figure 5A, demonstrating at the light microscope level the apical localization of the protein, and at the subcellular level the localization on the brush border, in coated pits, endosomes, dense apical tubules, and lysosomes. In megalindeficient mice, no labeling was seen, demonstrating also no cross-reactivity with other proteins of the polyclonal antibody used (Figure 5B).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: ×31,000 in A; ×23,000 in B.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: ×35,000 in A and B; ×750 in lower left inset in A and B; ×53,000 in upper right inset in A.Because a number of studies have demonstrated the role of megalin in the uptake of macromolecules from the glomerular ultrafiltrate, we speculated that megalin-deficient mice should exhibit tubular reabsorption deficiency and excrete receptor ligands in the urine. This hypothesis was confirmed when the protein profile of urine samples was analyzed (Figure 6). Megalin -/- mice excrete in the urine a distinct pattern of relatively low molecular weight proteins, indicating an inability of the proximal tubules to reabsorb filtered macromolecules. Similar phenotypes are observed in patients with Fanconi renotubular syndrome, a tubular reabsorption deficiency caused by various genetic as well as environmental factors (e.g., heavy metal poisoning). We applied amino acid sequence analysis to identify some of the proteins excreted in the urine of knockout animals. Two of the proteins identified were particularly interesting because they represent plasma carriers for lipophilic vitamins, the vitamin D binding protein (DBP), and the retinol binding protein (RBP) (Figure 6).Figure 6: . . Urinary protein profile of wild-type and megalin -/- mice. Fifteen microliters of urine from mice of the indicated genotypes were subjected to 4 to 15% nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and staining with Coomassie blue. Protein bands corresponding to serum albumin (asterisk), vitamin D binding protein (DBP), and retinol binding protein (RBP) are highlighted. F, female; M, male.Proximal Tubular Handling of Three Vitamins and Their Carrier Proteins In the sections that follow, we shall emphasize the role of megalin in kidney proximal tubule, describing the reabsorption of three vitamins (vitamin D, vitamin A [retinol], and vitamin B12) and their binding proteins (DBP, RBP, and transcobalamin [TC II]). It should be noted that these three proteins most likely are representatives of a group of more or less related carrier proteins, which in the be identified for reabsorption in the renal proximal tubule by or similar endocytic uptake. D Protein The is the main for vitamin in the It exhibits affinity for vitamin to to this binding affinity and the high plasma of to virtually all vitamin in are present in with A central in vitamin D is the of vitamin to vitamin an important of the calcium metabolism. This in the epithelial cells of the proximal The cells up the precursor vitamin and it into the vitamin by of the vitamin 1 in the mitochondria. has on the specific mechanisms that vitamin to this renal cell In the of uptake of the and the role of in this process are still unclear. of in the urine of megalin-deficient mice suggested an important role for megalin in the tubular uptake of vitamin This hypothesis was confirmed in studies We were to that of vitamin and are filtered the and reabsorbed by megalin from the lumen of the proximal tubule. Tubular of the is to of the vitamin and to the precursor for of vitamin As a of the receptor gene megalin -/- mice exhibit vitamin deficiency and from formation Protein is a retinol carrier protein in the The protein is filtered in the glomeruli and is as a for tubular We have recently shown that the protein binds to purified megalin by BIAcore experiments and that the protein and retinol is found in the urine of megalin-deficient mice but is in mice (Figure 6). Furthermore, endogenous was found by immunocytochemistry in the proximal tubules of mice but was in megalin knockout mice (Figure There was an segmental of uptake, the uptake was very in the initial part of the proximal tubule and decreased in segments with virtually no uptake in segment 3, indicating that the tubule fluid is for the protein rapidly after glomerular The cytoplasmic labeling observed in the initial parts of the proximal tubule (Figure be In uptake studies of using a rat cell megalin demonstrated that RAP and anti-megalin antibody in part uptake and In these experiments the importance of megalin in the tubular reabsorption of and the of retinol that otherwise be lost in the urine as illustrated in the megalin knockout mice. Since megalin is normally in the of and in the (Table 1), the of retinol into the megalin-deficient mouse may also to the observed . . Labeling for in wild-type mouse megalin-deficient mouse and rat A labeling may be noted in the early part of the proximal tubule of wild-type mouse including the very initial part seen surrounding the labeling is found in the (B). In the rat there is in the early parts of the proximal tubule in addition to the a cytoplasmic labeling that is also in the very early part connected to the of the In the parts of the proximal tubule, only the labeling is seen Magnification, is one of the major (vitamin B12) carrier plasma proteins, with a molecular weight of The protein is filtered to a large extent in glomeruli and reabsorbed in the proximal tubule It has been that the renal glomerular filtration and tubular uptake of vitamin to about which the of vitamin in the to factor factor binding to megalin was demonstrated by ligand of renal cortex and purified megalin, binding to megalin on cryosections and surface on megalin BIAcore uptake, and degradation as well as RAP inhibition were demonstrated using and studies of as well as studies on rat cells. have also shown that megalin-deficient mice excrete amounts of and vitamin in the urine with these experiments a role of megalin in vitamin homeostasis. of the Vitamins Figure the megalin-mediated uptake of the three vitamin carrier proteins into renal proximal tubule cells. In early and endosomes, the proteins from megalin, and the receptor to the apical plasma membrane dense apical tubules, the proteins are to lysosomes for Although this part of the reabsorption pathway for the vitamins to a large extent appears to be major concerning the transport of vitamins from the lysosomes and back to the It is highly that the general mechanisms for the three vitamins are D and vitamin A are both whereas vitamin is amounts of the apically reabsorbed vitamins probably also to a minor extent are used in the proximal tubule and they may also to some extent in the cells. Thus, vitamin D is being reabsorbed mainly as and we demonstrated that the into is on megalin-mediated uptake . . The the megalin-mediated proximal tubular reabsorption of the three vitamin carrier protein (vitamin D binding and binding A The degradation of the three carrier proteins is in the lower part as well as the of to The mechanisms of of the three vitamins to be no has been indicating proximal tubular of However, it appears highly that vitamin D in a should the plasma membrane to apolipoprotein in the For retinol in the kidney, there are that is being in proximal Thus, we by light microscope immunohistochemistry a apparent cytoplasmic labeling for in the early part of the proximal tubules (Figure and in addition a labeling By electron microscope we found labeling of endoplasmic and in the Golgi region analysis has demonstrated in the kidney at a level of to of that in the and by in has been localized to proximal tubules although the labeling confined to segment The kidney appears to be important for the recycling of and it has been for rat that about of the from the kidney these would degradation of reabsorbed RBP, of retinol to in the rough endoplasmic and using the pathway the Golgi apparatus. With respect to vitamin it appears well that is taken up by mammalian cells by and is in the lysosomes, vitamin is into the cytoplasm A and pH-dependent transport system has been characterized using membrane from purified from rat and a in vitamin release from lysosomes has been to an has been found in high amounts in the kidney by in human in the kidney in the It appears to a pathway in the proximal tubule for vitamin similar to the one described for as suggested by and in the vitamin might be in the fluid to by cells The megalin-mediated reabsorption of the three vitamin carrier proteins described in this the role of megalin not only as a of protein reabsorption in the renal proximal tubule, but also in vital substances from the tubular fluid, which would otherwise be lost in the urine. The uptake of lipophilic substances such as retinol and vitamin to their carrier proteins also suggests that similar mechanisms may for other important such as and other Furthermore, is for retinol and vitamin that to degradation of the carrier proteins, the vitamins may be to carriers and at the plasma The to this however, This was by from the the the the of the and a to We and for