Megalin is essential for renal proximal tubule reabsorption and accumulation of transcobalamin-B12

Henrik Birn1, Thomas E. Willnow2, Rikke Nielsen1, Anthony G. W. Norden3, Christian Bönsch2, Søren K. Moestrup4, Ebba Nexø5, and Erik I. Christensen1

1 Department of Cell Biology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C; 2 Max Delbrueck Center for Molecular Medicine, 13125 Berlin, Germany; 3 Department of Clinical Biochemistry, Addenbrooke's Hospital, Cambridge CB2 2QR, United Kingdom; 4 Department of Medical Biochemistry, University of Aarhus, DK-8000 Aarhus C; and 5 Department of Clinical Biochemistry, Aarhus University Hospital, DK-8000 Aarhus C, Denmark


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Megalin has previously been shown to bind and mediate endocytosis of transcobalamin (TC)-B12. However, the physiological significance of this has not been established, and other TC-B12 binding proteins have been suggested to mediate renal uptake of this vitamin complex. The present study demonstrates by the use of megalin-deficient mice that megalin is, in fact, essential for the normal renal reabsorption of TC-vitamin B12 and for renal accumulation of this highly conserved vitamin. Megalin-deficient mice excrete increased amounts of TC and B12 in the urine, revealing a defective renal tubular uptake of TC-B12. The urinary B12 excretion is increased ~4-fold, resulting in an ~28-fold higher renal B12 clearance. This is associated with an ~4-fold decrease in B12 content in megalin-deficient kidney cortex. Thus megalin is important to prevent urinary loss of vitamin B12. In addition, light- and electron-microscopic immunocytochemistry demonstrate lysosomal accumulation of B12 in rat and mouse proximal tubules. In rats this accumulation is correlated with vitamin intake. Thus renal lysosomal B12 accumulation is dependent on vitamin status, indicating a possible reserve function of this organelle in the rat kidney.

kidney; receptor-mediated endocytosis; cobalamin; lysosomes; immunocytochemistry


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

VITAMIN B12 serves as an essential cofactor for important mammalian enzymes, including the cytoplasmic enzyme methionine synthetase and the mitochondrial enzyme methylmalonyl-CoA mutase. Vitamin deficiency is associated with severe hematological and neurological defects. Vitamin B12 supplied with food is bound to intrinsic factor and absorbed in the intestine by a process involving the receptor cubilin (1, 26, 40). Absorbed B12 bound to transcobalamin C (TC) is transported to the tissues. The TC-B12 complex (mol mass ~50 kDa) is filtered in the glomeruli. This is followed by tubular reabsorption to prevent urinary losses of this highly conserved vitamin (25). The rat kidney accumulates large amounts of vitamin B12 (2, 15, 17, 23, 32, 39), concentrated in the proximal tubules (38). Membrane fractionation studies in rat kidneys have suggested accumulation of vitamin B12 in lysosomal-enriched fractions, although no morphological support has been provided (27, 28).

Two receptors for the renal tubular uptake of TC-B12 have been reported: the multiligand, endocytic receptor megalin (25) and a 62-kDa protein of yet unknown structure localized by immunoblotting to kidney cortical membranes (4, 5). Megalin (600 kDa) belongs to the low-density lipoprotein receptor family (36). It is expressed in kidney proximal tubules (7, 9, 19, 20, 24, 33) and several other absorptive epithelia (reviewed in Refs. 6 and 13). Megalin is able to bind and mediate the endocytosis of TC-B12 (25). Whereas megalin is expressed in the apical endocytic apparatus facing the ultrafiltrate, the 62-kDa protein apparently is expressed mainly in the basolateral membranes (4). The significance of these receptors for the renal uptake of TC-B12 has not been clarified so far.

To examine the importance of megalin to renal B12 uptake and vitamin status in vivo, we analyzed the uptake of TC-B12 and vitamin B12 accumulation in megalin-deficient mice. The data show megalin deficiency to be associated with increased urinary excretion of TC and B12 as well as decreased renal B12 accumulation. We also investigated renal accumulation of vitamin B12 in proximal tubule cells from rats subjected to different levels of vitamin intake. In conclusion, megalin-mediated TC-B12 uptake is essential to prevent urinary loss of the vitamin and to maintain normal renal B12 status. Furthermore, renal accumulation of B12 in rat proximal tubule lysosomes is dependent on the vitamin status, indicating a possible reserve function of this organelle in the kidney.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Tissues and urine samples. Megalin-deficient mice were produced by gene targeting as described in Willnow et al. (44). Sex-matched wild-type and heterozygous littermates were used as controls. Mice were kept on a normal diet (0.08 mg vitamin B12/kg food) with free access to drinking water. Urine was collected during a 16-h period from mice aged 10-16 wk. Collections were performed simultaneously from megalin-deficient and sex-matched wild-type littermates. Urine was kept on ice during the sampling period to minimize protein degradation. For determination of vitamin B12 concentration, the kidney cortices were removed from anesthetized mice and immediately frozen. For immunocytochemistry, mouse kidneys were fixed by perfusion of 4% paraformaldehyde in 0.1 M cacodylate buffer, pH = 7.4, through the left ventricle of the heart.

Newly weened male Wistar rats were kept on a normal (0.041 mg/kg food; Altromin, Lage, Germany) or low-vitamin-B12 (0.011 mg/kg food; Altromin) diet for 60 days. Vitamin B12-overloaded rats were fed a normal diet for 60 days and injected subcutaneously with cyanocobalamine (10 µg/day; SAD) for 17 days before death. All rats were at the same age when killed. The left renal vessels were ligated, and the kidney was removed and frozen for analysis of vitamin B12. The liver, intestines, and right kidney were fixed by retrograde perfusion with 4% paraformaldehyde in 0.1 M cacodylate buffer, pH = 7.4, through the abdominal aorta. Random urine specimens were collected from three male patients suffering from Dent's disease, as well as from three control subjects, and frozen until processed for immunoblotting. All patients had clinical and laboratory features of the disease and a serum creatinine level <200 µmol/l.

Antibodies. A monoclonal anti-B12 antibody (ascites; Sigma) and a polyclonal sheep anti-B12 antibody (Biogenesis) were used to identify B12 by immunocytochemistry. The affinity of the monoclonal anti-B12 antibody for different cobalamin forms [cyanocobalamin (Sigma), hydroxycobalamin (Gea), methylcobalamin (Sigma), 5'-deoxyadenosylcobalamin (Sigma), and dicyanocobinamide (Sigma)] was analyzed by their ability to displace 57Co-cyanocobalamin (0.39 MBq/ml; Amersham) from the monoclonal anti-B12. One hundred microliters of the anti-B12 antibody diluted 1:1,000 with 0.1 M PBS and 0.1% human albumin (ORHA 20/21; Aventis Behring) were incubated with 100 µl of each of the cobalamins (412-1,000 nM) and 50 µl of 57Co-cyanocobalamin. After 20 h of incubation at 4°C, free and bound cobalamin were separated using hemoglobin (Difco)-coated charcoal (Sigma). Bound 57Co-cyanocobalamin was counted in a Wallac 1480 gamma counter. The analyses showed that hydroxycobalamin, methylcobalamin, and cyanocobalamin were able to displace labeled cyanocobalamin from the monoclonal antibody, whereas 5'-deoxyadenosylcobalamin and dicyanocobinamide were unable to displace cyanocobalamin from the antibody (Fig. 1).


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Fig. 1.   Competition-inhibition assay showing the ability of cyanocobalamin (black-triangle), hydroxycobalamin (), methylcobalamin (), 5'-deoxyadenosylcobalamin (), and dicyanocobinamide () to displace 57Co-cyanocobalamin from the monoclonal anti-vitamin B12 antibody. A comparable affinity of the monoclonal anti-vitamin B12 antibody for cyanocobalamin, hydroxycobalamin, and methylcobalamin is observed, whereas 5'-deoxyadenosylcobalamin and dicyanocobinamide are unable to displace labeled cyanocobalamin.

Polyclonal anti-rabbit TC antibody was produced by immunization of a guinea pig by five consecutive injections totaling 0.5 mg of rabbit TC purified from rabbit serum (29). Polyclonal anti-human TC was raised against recombinant human TC. For double-labeling experiments to identify lysosomal structures, we used polyclonal rabbit anti-rat LAMP-1 (kindly provided by Dr. Ira Mellman) or anti-rat cathepsin B (Upstate Biotechnology). The following secondary antibodies were used: horseradish peroxidase (HRP)-conjugated rabbit anti-guinea pig, goat anti-rabbit, goat anti-mouse, or rabbit anti-sheep IgG (DAKO), FITC-conjugated goat anti-mouse IgG (DAKO), rhodamine-conjugated swine anti-rabbit IgG (DAKO), gold-conjugated goat anti-mouse, and goat anti-rabbit IgG (British BioCell).

Determination of vitamin B12 concentration in serum, urine, and renal tissues. Between 17 and 41 mg kidney cortex was homogenized in 0.4 ml of 10 mM PIPES buffer containing (in mM) 1 EGTA, 3 MgCl2, 0.4 NaCl, and 1 PMSF, pH 7.4, followed by centrifugation at 20,000 g for 40 min at 4°C. The total amount of B12 in the supernatant, serum, and urine was analyzed by an automated protein binding assay (ACS 180 Plus; CHIRON/Diagnostics). Unsaturated serum B12 binding capacity was determined as described in Gottlieb et al. (14). Creatinine and urea in mouse serum were measured using automatic equipment (Cobas-Integra; Hoffmann-La Roche).

SDS-PAGE and immunoblotting of urine samples. Urine samples were concentrated using Centricon YM-10 centrifugal filter devices (Millipore) and mixed with Laemmli sample buffer containing 2.5% SDS and subjected to SDS-PAGE using polyacrylamide minigels (Bio-Rad Mini Protean II). The gels were transferred to nitrocellulose paper. Blots were blocked with 5% milk in PBS-T (80 mM Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl, 0.1% Tween 20, pH 7.5) for 1 h and incubated overnight at 4°C with anti-TC antibody in PBS-T with 1% BSA. After being washed in PBS-T, the blots were incubated for 1 h with HRP-conjugated anti-guinea pig or anti-rabbit IgG. After the final wash, antibody binding was visualized by using an enhanced chemiluminescence system (Amersham). Controls involving incubation without primary antibody and incubation with preimmune serum revealed no significant labeling.

Immunocytochemistry on renal tissue. Blocks of all perfusion-fixed tissues were postfixed in the same fixative for 2 h. For ultra- and semithin cryosections, tissue was infiltrated for 30 min in 2.3 M sucrose containing 2% paraformaldehyde and rapidly frozen in liquid N2. For cryostat sections, tissue was infiltrated in 30% sucrose in 150 mM Sørensen's buffer (123 mM Na2HPO4, 27 mM KH2PO4) and rapidly frozen using CO2. For light-microscopic immunocytochemistry, cryostat (10-15 µm) or semithin cryosections (~1 µm) were cut on a Walter Dittes Kryostat or Reichert Ultracut S and placed on coated glass slides. Cryostat sections were heated, dehydrated/rehydrated in graded alcohols, and permeabilized with 0.05% saponin before incubation with the primary antibody. Sections were incubated with a primary anti-B12 antibody in 0.01 M PBS, 0.15 M NaCl, 0.1% skim milk, and 0.02 M NaN3, followed by incubation with florescent or HRP-labeled anti-mouse, anti-rabbit, or anti-sheep IgG. Peroxidase labeling was visualized by incubation with diaminobenzidine and 0.03% H2O2 for 10 min. For double labeling, both mouse anti-B12 and rabbit anti-cathepsin B antibodies were applied, followed by conjugated anti-mouse and anti-rabbit IgG. All incubations were performed at room temperature, and sections were counterstained with Meiers hematoxylin before examination in a Leica DMR microscope equipped with a Sony SCCD color video camera or a Zeiss LSM510 laser confocal microscope. Electron-microscopic immunocytochemistry was performed on 90-nm cryosections incubated overnight at 5°C with anti-B12 (IgG concentration: 450 µg/ml), followed by incubation with gold-conjugated anti-mouse IgG secondary antibody in 0.05 M Tris buffer, 0.15 M NaCl, 0.1% BSA, 0.06% polyethylene glycol, 1% fish gelatin, and 0.02% NaN3 for 2 h at room temperature. For double-labeling experiments, sections were incubated with anti-B12 and anti-LAMP-1 (1:8,000) followed by incubation with gold-conjugated anti-mouse (10 nm) and anti-rabbit (5 nm) secondary antibodies. Controls involving incubation without primary antibody and incubation with preimmune serum, nonspecific rabbit serum, or mouse IgG1 revealed no significant labeling.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Megalin is important for normal uptake of filtered TC-B12 and accumulation of the vitamin. In megalin-deficient mice, increased urinary excretion of both TC (Fig. 2) and vitamin B12 (Table 1) was observed. Urinary B12 concentration was increased approximately fourfold despite significant lower serum B12 levels. As a result, urinary B12 clearance was increased ~28-fold in megalin-deficient mice. Furthermore, no vitamin B12 could be identified by immunocytochemistry in the proximal tubules from megalin-knockout mice, indicating defective cellular uptake of the vitamin (Fig. 3). Little or no TC was identified in urine from control littermates (Fig. 2), whereas cellular B12 uptake was evident in proximal tubules (Fig. 3). The defect in vitamin accumulation in megalin-deficient mice was further substantiated by the determination of vitamin B12 concentration in kidney cortical tissue from two megalin-knockout mice, showing a fourfold reduction compared with normal controls (Table 1).


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Fig. 2.   Identification of transcobalamin (TC) in urine from megalin-deficient and control mice. Immunoblotting was done using an anti-rabbit TC antibody. Two bands corresponding to the size of purified rabbit TC (left-arrow ) are visible in all megalin-deficient mice. The dual bands probably represent isoforms, as has previously been described (12). The amount of urine loaded in each lane is adjusted proportionally to the urinary output volume. Mouse pairs 1 and 2, 3 and 4, 5 and 6, and 7 and 8 represent littermates. Pairs 1 and 2, 3 and 4, and 5 and 6 are male and pair 7 and 8 are female.


                              
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Table 1.   Urea, creatinine, and vitamin B12 in serum, urine, and renal tissue from megalin-deficient and normal control mice



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Fig. 3.   Immunocytochemistry using a polyclonal sheep anti-B12 antibody (1:10,000) on sections of kidney cortex of normal control and megalin-deficient mice. a: In normal mice the vitamin can be identified by a vesicular labeling in proximal tubules. b: In contrast, no tubules containing the vitamin can be identified in megalin-deficient mice, indicating defective uptake and accumulation. Only some tubular cross sections are labeled in the normal mice (a). Labeled cross sections probably represent the first part of the proximal tubule, as the reabsorbed vitamin B12 appears to be concentrated there (see Fig. 6). Findings were consistent, showing no labeling in tubules from 5 megalin-deficient mice and labeling in tubules from all 5 wild-type or heterozygous normal controls. Magnification: ×500.

Although there was no significant difference in urinary output or in urinary creatinine or urea excretion between megalin-deficient and wild-type or heterozygous mice (Table 1), a reduction in urea clearance was calculated in megalin-deficient mice, indicating reduced filtration in these animals.

TC-B12 is normally filtered and reabsorbed in the human kidney. The possibility of tubular reabsorption of filtered TC in humans was investigated by analyzing urine from the patients with Dent's disease. This Fanconi-like syndrome, caused by mutations in the renal chloride channel ClC-5 (41), is characterized by tubular proteinuria, hypercalciuria, nephrolithiasis, and, possibly, a decrease in proximal tubule megalin expression (34). In analyzed urine samples from all three patients, TC could be clearly identified by immunoblotting, whereas only traces of TC were observed in urine from control subjects (Fig. 4). This shows that excretion of TC in human urine is strongly increased when tubular reabsorption is defective, indicating that glomerular filtration of TC-B12 is followed by efficient reabsorption in a normal human kidney.


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Fig. 4.   Immunoblotting of TC in the urine of patients with Dent's disease and tubular proteinuria. Lane a shows the very low TC content in the urine of a subject with no known kidney disease, and lanes b-d show the TC content in the urine of 3 different patients with Dent's disease. Twenty microliters of urine with a creatinine concentration of 9.2 mM in control vs. 4.2-6.9 mM in Dent's disease patients were applied to each lane of a 8-16% polyacrylamide SDS gel before immunoblotting.

Internalized B12 accumulates in lysosomes. Immunocytochemistry in normal mouse and rat kidneys using a monoclonal antibody against vitamin B12 showed distinct intracellular labeling concentrated in vesicular structures (Figs. 3a and 5a). No vesicular labeling was observed outside the proximal tubule, although a weak and diffuse cytoplasmic staining of other cell types, including the distal tubules, could be observed at high concentrations of the primary antibody (not shown). Preincubating the antibody with cyano- or hydroxycobalamin at a concentration 50 times higher than that of the primary antibody (~3 pM) totally inhibited labeling (inset, Fig. 5a), whereas attempting to convert all forms of B12 to cyanocobalamin by pretreating sections with 32 µM KCN in acetate buffer did not affect labeling (not shown). Electron microscopy confirmed the immunohistochemical findings, showing a heavy labeling for vitamin B12 in electron-dense structures in selected segment 1 proximal tubules (Fig. 5b). In addition, labeling was seen in the mitochondria of all tubular cells, including distal tubules (not shown). Some gold particles were identified in the endocytic apparatus and cytoplasm of the proximal tubules. Double-labeling immunocytochemistry, showing colocalization of vitamin B12 and the lysosomal membrane protein LAMP-1, confirmed the very intense labeling for the vitamin in some, but not all, lysosomes in the first part of the proximal tubules (Fig. 5b). The vesicular labeling in normal mouse proximal tubules (Fig. 3a) is more apically located than in rat tubules (Fig. 5a), suggesting a more apical localization of B12-containing endosomes/lysosomes in mice compared with rats. This is confirmed by doubling labeling in normal mice and rats (not shown), demonstrating colocalization of the lysosomal enzyme cathepsin B and B12 in vesicular structures located similarly to the B12 labeling in Fig. 3a (mouse) and 5a (rat), respectively.


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Fig. 5.   Immunocytochemistry using a monoclonal anti-B12 antibody (3 pM IgG) on sections of a normal rat kidney cortex. a: The vitamin is identified by a vesicular labeling localized to proximal tubules only. *, The very first part of a proximal tubule attached to the glomerulus. Preincubating the antibody with cyanocobalamin (150 pM) totally inhibits labeling (inset in a). Magnification: ×480; inset, ×180. b and c: Electron-microscopic double-labeling immunocytochemistry using the same anti-B12 antibody (3 pM IgG, 10-nm gold particles) in combination with a polyclonal rabbit anti-LAMP-1 antibody (1:8,000, 5-nm gold particles) on segment 1 rat proximal tubule cell. Vitamin B12 can be localized to lysosomes identified as LAMP-1-positive electron-dense structures (arrowheads pointing to the labeled vesicular membrane). For better identification of gold particles, c shows part of b at a higher magnification. The lower right of b includes part of a LAMP-1-positive vesicular structure with no labeling for vitamin B12, suggesting that not all LAMP-1-positive vesicles contain the vitamin. Also, note the labeling for vitamin B12 in mitochondria (arrows). Magnification: ×45,000 (b), ×100,000 (c).

Lysosomal accumulation of B12 depends on vitamin intake. Feeding rats with a low-vitamin B12 diet caused an ~30-fold reduction in renal vitamin content as well as a reduction in serum B12, in combination with increased unsaturated serum B12 binding capacity (Table 2). Conversely, daily injections of cyanocobalamin increased renal vitamin concentration approximately twofold in combination with increased serum B12 and decreased unsaturated serum B12 binding capacity (Table 2). Thus changes in vitamin intake are reflected in changes in renal B12 accumulation. Immunocytochemistry revealed dose-dependent labeling for vitamin B12 in kidney proximal tubules (Fig. 6). In vitamin-deprived rats, hardly any vitamin labeling could be observed (Fig. 6a), whereas, in rats fed a normal diet, vesicular labeling is evident in proximal tubules (Fig. 6b). In vitamin-loaded rats, more intense labeling is seen (Fig. 6c). Double-labeling immunocytochemistry identifying vitamin B12 and the lysosomal enzyme cathepsin B showed that the B12 labeling in rats loaded by vitamin injection colocalized with lysosomal labeling (Fig. 6d), suggesting that the vitamin under conditions of vitamin surplus accumulates in proximal tubule lysosomes. No vitamin B12 labeling was observed at the light-microscopic level in the ileal epithelial cells or in the liver cells of vitamin-loaded or -deprived rats (not shown).

                              
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Table 2.   Serum vitamin B12 concentration and unsaturated B12-binding capacity as well as kidney vitamin B12 concentration in rats



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Fig. 6.   Immunocytochemistry using a monoclonal anti-B12 antibody (0.6 pM IgG) on cryostat sections (a-c) of kidney cortex from vitamin-deprived rats (a), rats fed a normal diet (b), and rats loaded with the vitamin by subcutaneous injection (c). Little labeling is observed in the kidneys of vitamin-deprived rats, whereas proximal tubules of rats fed a normal diet reveal vesicular labeling. Labeling appears most strongly in the very first part of the proximal tubule, as identified by its connection with the glomerulus (arrow in b). In vitamin-loaded rats, more intense labeling is seen. Both the number of immunolabeled tubular cross sections and the labeling intensity within each tubule are increased. d: Immunoflorescent, double-labeling immunocytochemistry identifying both vitamin B12 (3 pM IgG, red fluorescence) and lysosomal enzyme cathepsin B (1:2,000, green fluorescence) in a semithin cryosection of vitamin-loaded rat kidney cortex. Colocalization of almost all vitamin B12 labeling with cathepsin B is reflected by abundant yellow fluorescence (arrows) and little red fluorescence. Green fluorescence (arrowheads) represents lysosomal structures with no immunodetectable vitamin B12. The tubular cross section outlined in the upper right-hand corner shows no vitamin B12 labeling and may represent part of a proximal tubule located downstream to the point where all the filtered vitamin is reabsorbed. Magnification: ×100 (a-c), ×550 (d).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The data presented show that megalin is important for the renal tubular uptake and accumulation of vitamin B12. The present gene-knockout data are substantiated by previous studies showing that megalin binds TC-B12 with high affinity and is able to mediate the endocytosis and cellular uptake of this complex (25). Whereas the previous study did not examine the physiological significance of this mechanism, the present study demonstrates that megalin deficiency is associated with increased urinary excretion of vitamin B12 and its carrier protein TC and with a decreased accumulation of vitamin in the renal proximal tubules. The present data, combined with the strong apical expression of megalin in the proximal tubule, support an essential function of megalin to prevent urinary loss of this highly conserved vitamin.

Vitamin B12 is absorbed in the intestines bound to intrinsic factor. The intrinsic factor-vitamin B12 receptor cubilin is a 460-kDa peripheral membrane protein with no obvious transmembrane domain (26). Cubilin may be internalized after binding to megalin (26). Thus, although cubilin is expressed in megalin-deficient mice, it may be functionally impaired, affecting the intestinal absorption of vitamin B12. Although this may contribute to the observed reduction in serum B12 levels, it does not affect renal handling of B12. The finding of an approximately fourfold increase in urinary excretion of B12 in megalin-deficient mice, reflecting a >20-fold increase in B12 clearance, is important. This establishes defective tubular uptake and shows that the decrease in renal accumulation of TC and B12 is not due to the reduced plasma vitamin levels or decreased filtration of the vitamin, as these would both result in a decrease in urinary vitamin excretion. Very little, if any, intrinsic factor is present in serum or urine and, whereas megalin binds TC-B12 strongly [dissociation constant (Kd), ~180 nM; 25], the binding of this complex to cubilin is very weak and not strong enough to allow any estimation of Kd when analyzed by surface plasmon analysis (data not shown).

Megalin is also expressed in other absorptive epithelia, including enterocytes (1), ependyma (45), yolk sac (11, 22, 35), and possibly placental syncytiotrophoblast cells (45) and thus may be involved in the transport of vitamin B12 to other, especially fetal, tissues.

In addition to megalin, a 62-kDa (normally present as a 124-kDa dimer) TC-B12 binding protein has been identified in the kidney, placenta, intestines, and liver (3-5). However, in contrast to megalin, this 62-kDa protein of still unknown structure is preferentially expressed in basolateral membranes of renal cells when analyzed by membrane fractionation (4). On the basis of the present data, we suggest that megalin is important for the luminal uptake of filtered TC-B12 and for the renal accumulation of B12. Other proximal tubule TC-B12 binding proteins may possibly be involved in basolateral uptake or secretion as well as in intracellular transport.

Dent's disease is a Fanconi-like renal tubular defect caused by mutations in the renal chloride channel ClC-5 (41). It is characterized by hypercalciuric nephrolithiasis and low-molecular-weight proteinuria similar to that observed in megalin-deficient mice (21). The increased excretion of TC in urine from patients suffering from this tubular defect compared with normal human subjects indicates that TC-B12 is filtered in the human glomeruli but normally reabsorbed, suggesting that renal tubular luminal uptake in humans may prevent loss of the vitamin in urine. This is also consistent with previous suggestions that an endocytic dysfunction may underlie the tubular proteinuria of this disease (10, 16, 43). Recently, it has been shown that ClC-5 disruption impairs proximal tubule endocytosis and causes a reduction in proximal tubule luminal expression of megalin (34), indicating a functional relationship between ClC-5 and megalin-mediated endocytosis. Also, in a rat model of autosomal-dominant polycystic kidney disease, tubular proteinuria is associated with decreased endocytic activity in cyst-lining epithelial cells colocalizing with defective expression of both ClC-5 and megalin (31). Traces of TC were identified by immunoblotting in urine from normal human subjects but not in normal mouse urine. This is most likely due to the use of two different anti-TC antibodies. A rabbit anti-recombinant human TC antibody is used to identify human TC, whereas a guinea pig anti-rabbit TC is used to identify mouse TC. The affinity of anti-human TC for human TC is likely to be higher than the affinity of anti-rabbit TC for mouse TC, explaining why TC is detected in human urine only.

Most megalin-deficient mice die perinatally (44); however, ~1 of 50 survive to adulthood, forming the basis for the present study. The kidneys of these mice are generally normal. Ultrastructurally, the glomeruli are normal, whereas the proximal tubule cells reveal a loss of endocytic invaginations, vesicles, and dense apical tubules, which supports decreased endocytic activity (21). An ~40% reduction in urea clearance is observed in megalin-deficient mice, which, for the first time, estimates renal function in these animals. The reason for this is unknown. Although megalin has been localized in the glomeruli of rats, it has not been identified in other species, including mice. There is no evidence of changes in the glomerular ultrastructure or in the filtration of proteins, and, in particular, there is no suggestion of glomerular proteinuria in megalin-deficient mice (8, 21, 30). Megalin is a multiligand receptor that is important for the tubular reabsorption of other vitamin-carrier protein complexes, including retinol-binding protein (8) and vitamin D-binding protein-vitamin D (30). Thus megalin appears to serve a specific and important vitamin-scavenger-receptor function that prevents excessive urinary loss of filtered, protein-bound vitamins.

Vitamin B12 identified by immunohistochemistry accumulates in the lysosomes of rat kidney proximal tubules, and this accumulation is dependent on the vitamin status of the animal. The lysosomal accumulation in cryostat sections appears to be concentrated in the first part of the proximal tubule, indicating an efficient tubular uptake of filtered TC-B12. This is in accordance with the high-affinity binding of TC-B12 to megalin (25). Lysosomal accumulation of B12 in the rat kidney supports previous studies based on tissue fractionation by gradient centrifugation (27, 28).These studies showed that the concentration of endogenous cobalamin in kidney lysosomes is five to six times that of mitochondria (27). Variations in cobalamin supply are associated with extensive changes in rat kidney vitamin accumulation, confirming the results of previous studies (15, 37, 39). Animals loaded with vitamin B12 in the present study exhibited a 60-fold increase in renal vitamin accumulation compared with vitamin-depleted animals. This is associated with a similar change in the immunolabeling for vitamin B12 in the renal proximal tubules. When animals are loaded with cobalamin, increased labeling of lysosomes in the proximal tubule is observed. In contrast, rats fed on a low-vitamin diet revealed no immunodetectable vitamin in the proximal tubule lysosomes by light microscopy. Interestingly, no labeling was observed at the light-microscopic level in the intestinal epithelial cells or in the liver of vitamin-loaded rats, suggesting the immunodetectable lysosomal accumulation to be characteristic of the kidney proximal tubule cells.

The conceivable vitamin-conserving function of the proximal tubule implies that the reabsorbed and stored vitamin B12 eventually is returned to the circulation. The mechanism for this process is not resolved; however, in light of our present data, this must involve release of the vitamin from the lysosomal compartment. A specific transport system for vitamin B12 has been identified in rat liver lysosomal membrane vesicles, suggesting that the vitamin initially is transported into the cytoplasm (18). Also, an unusually large lysosomal accumulation of vitamin B12 has been demonstrated in fibroblasts from patients suffering from cblF disease. This is an inborn error of cobalamin metabolism, characterized by a defective transfer of the vitamin to target enzymes (42). These data indicate the presence of a specific mechanism for the release of vitamin B12 from lysosomes. Further cellular processing of vitamin B12 requires secretion at the basolateral membranes in complex with carrier proteins or as free B12, which is then complexed extracellularly. Whether the vitamin is metabolized by transformation into different cobalamin derivatives within the proximal tubule cell has not been established.

At the electron-microscopic level, vitamin B12 labeling was observed both in the mitochondria and, to a lesser extent, in the cytoplasm of all cell types analyzed, including intestinal epithelial cells and liver cells. This labeling corresponds to the known localization of the vitamin B12-dependent enzymes. Binding competition analysis of the monoclonal anti-B12 antibody used in this study suggested that the antibody reacted with equal affinity for cyanocobalamin, hydroxycobalamin, and methylcobalamin but with significantly less affinity for 5'-deoxyadenosylcobalamin. All forms of vitamin B12 are found in tissue extracts, but the major part of 5'-deoxyadenosylcobalamin is localized in the mitochondria. Both methyl- and 5'-deoxyadenosylcobalamin are converted to hydroxycobalamin on exposure to light and, with addition of cyanide, hydroxycobalamin is converted to cyanocobalamin. It is unlikely that any free 5'-deoxyadenosylcobalamin remains stable in thin cryosections heavily exposed to light for 1-2 h before immunolabeling. This implies that any possible in vivo changes in the relative distribution of various cobalamin forms would not influence labeling intensity. This hypothesis is supported by the finding that preincubation of sections with KCN did not affect labeling intensity of kidney proximal tubule cells.

In conclusion, the present study establishes the importance of megalin in renal tubular luminal reabsorption of TC-B12 and vitamin accumulation, emphasizing its role as a vitamin-carrier protein-scavenger receptor. Our studies in Dent's disease implicate proximal tubular function in humans as necessary for physiological TC-B12 reabsorption from the glomerular filtrate. Also, in rat proximal tubules, a vitamin intake-dependent lysosomal accumulation of vitamin is observed, suggesting that this organelle may serve a reserve function in the rat kidney. Whether this function is quantitatively important and actively regulated, possibly by regulated release from the kidney, remains to be elucidated.


    ACKNOWLEDGEMENTS

The authors thank Dr. Raj Thakker, Molecular Endocrinology Group, Imperial College School of Medicine, The Hammersmith Hospital, London, United Kingdom, for performing the genetic characterization of the Dent's disease patients. Also, the skillful technical assistance by Ann Vad Steffensen, Pia Nielsen, Hanne Sidelmann, Inger Kristoffersen, and Anna Lisa Christensen is fully appreciated.


    FOOTNOTES

This work was supported in part by the Danish Medical Research Council, the University of Aarhus, the Birn Foundation, the Novo-Nordisk Foundation, the Biomembrane Research Center, and the Sir Jules Thorn Charitable Trust. The study was in part presented at the American Society of Nephrology's 32nd Annual Meeting, Miami, FL, November 5-8, 1999, and has appeared in abstract form.

Address for reprint requests and other correspondence: H. Birn, Dept. of Cell Biology, Inst. of Anatomy, Bldg. 234, DK-8000 Aarhus C, Denmark (E-mail: hb{at}ana.au.dk).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajprenal.00206.2000

Received 13 July 2000; accepted in final form 14 October 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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