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*Department of Biochemistry, Faculty of Pharmacy, Wroc
aw Medical University, Wrocaw, Poland; Department of Cell Biology, Institut of Anatomy, University of Aarhus, Aarhus, Denmark; Institut Nationale de la Santé et de la Recherche St.-Antoine, Paris, France; Max Delbrueck Centre for Molecular Medicine, Berlin, Germany; ¶Department of Microbiology, Michigan State University, East Lansing, Michigan; ||Department of Veterinary Prevention and Immunology, Wrocaw Agricultural University, Wrocaw, Poland; |Department of Medical Biochemistry, University of Aarhus, Aarhus, Denmark.
Correspondence to Dr. Jakub Gburek, Department of Cell Biology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C, Denmark. Phone: 45-8942-3030; Fax: 45-8619-8664; E-mail: jgbu{at}ana.au.dk
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However, several studies have clarified the molecular events responsible for tubular uptake of a subset of glomerular filtrate proteins by two endocytic receptors expressed by tubular epithelium: megalin, a 600-kD transmembrane glycoprotein that belongs to the LDL-receptor family, and cubilin, a 460-kD glycoprotein that lacks a classical transmembrane domain and is internalized via association with megalin. A variety of glomerular filtrate proteins have been demonstrated to be megalin ligands. The list is composed of vitamin-binding proteins such as retinol and vitamin D–binding proteins, enzymes (e.g., lipoprotein lipase), lipoproteins, the hormone carrier transthyretin, and a number of other low-molecular-mass proteins (9,10). Severe abnormalities, including proteinuria, in surviving megalin knockout mice underscores the physiologic relevance of this receptor (11).
The cubilin ligands identified to date include intrinsic factor-cobalamin complexes (IF-B12), albumin, Ig light chains, apolipoprotein A-I, HDL, and Clara cell secretory protein, but it is likely that the 27 CUB repeats that constitute the majority of cubilin structure can also bind other filtrate proteins (10,12). The significance of cubilin as a protein scavenger in normal proximal tubules is supported by the finding of proteinuria in patients who have megaloblastic anemia type 1, a disorder caused by cubilin mutations in some families (13,14) and in dogs affected with an inherited abnormality of cubilin trafficking to the apical membrane (15,16).
We recently demonstrated the presence of specific binding sites for hemoglobin on renal brush-border membranes (17). Because their characteristics shared similarities with those of megalin and cubilin, we examined the possibility that renal uptake of hemoglobin is mediated by these receptors.
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Materials and Methods |
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Fluorescence-labeled rat hemoglobin was synthesized by coupling of 5 (6)-carboxyfluorescein-N-hydroxysuccinimide ester (FLUOS) to hemoglobin amino groups by use of the Fluorescein Labeling Kit, according to the manufacturer’s instructions (Roche Diagnostics GmbH, Mannheim, Germany). Briefly, 4 mg of rat hemoglobin dissolved in phosphate-buffered saline (PBS; pH 7.4) was incubated with 0.4 mg FLUOS for 2 h at room temperature with gentle mixing. Unbound FLUOS was removed by Sephadex G-25 gel filtration. The approximate fluorescein to protein ratio of the preparation was 10. Aliquots of FLUOS-hemoglobin (1 mg/ml) were stored at -20°C until used. Protein concentrations were determined by use of Protein Assay Reagent (Pierce Chemical Co., Rockford, IL).
Antibodies
Antisera to rat, mouse, and dog hemoglobins were raised in rabbits by repeated immunization with 0.2 mg of the antigen intravenously and subcutaneously. Sheep anti-rat megalin and rabbit anti-rat cubilin antisera were obtained as described elsewhere (19,20). Sera IgG fractions were prepared by Protein A–agarose affinity chromatography according to the manufacturer’s instructions (Pierce Chemical Co.). Specific anti-hemoglobin IgG was affinity-selected by hemoglobin-Sepharose chromatography as follows: IgG fraction (5 ml) was passed through 0.5 ml hemoglobin-Sepharose, and the bed was washed with 20 ml PBS (pH 7.4). Specific IgG was eluted with 3 ml of 0.1 M glycine buffer (pH 2.5). Immediately after adjustment of pH to 7.4 with 1 N NaOH, 200 µl of haptoglobin-Sepharose was added to remove minute amounts of hemoglobin released from the column during acidic elution. After 5 min of end-over-end mixing, haptoglobin-Sepharose was removed by centrifugation at 3000 x g for 5 min, and the supernatant that contained the specific IgG was collected. The antibodies were stored at 4°C in the presence of 0.1% BSA and 0.02 M NaN3. The beds were prepared by coupling of proteins to CNBr-activated Sepharose 4B by use of a protocol provided by the manufacturer (Pharmacia Biotech AB, Uppsala, Sweden).
Non-immune sheep serum IgG fraction was provided by Sigma Chemical Co. Non-immune rabbit IgG serum fraction, peroxidase-conjugated goat anti-rabbit IgG, and peroxidase conjugated rabbit anti-sheep IgG were purchased from DAKO (Glostrup, Denmark).
Purification of Hemoglobin Receptors by Affinity Chromatography
Rat renal brush-border membranes were prepared from 20 2-mo-old Wistar rats, each weighing 250 g. Before excision, kidneys were perfused via the abdominal aorta with saline that contained heparin (15 U/ml) at 120 mmHg for 5 min. Cortical tissue was excised and stored frozen at -20°C. Brush-border membranes were isolated from frozen cortices by a magnesium precipitation and differential centrifugation technique, as described by Biber et al. (21). All steps were performed at 4°C. The tissue was thawed in 5 volumes of 2 mM Tris-HCl (pH 7.0) that contained 10 mM mannitol and protease inhibitors (100 µg/ml Pefabloc, 5 µg/ml leupeptin, 1 µg/ml pepstatin, and 2 µg/ml E-64; Roche Diagnostics GmbH) and homogenized 3 x 30 s in a Waring blender. The homogenate was filtered through two layers of surgical gaze, and 0.01 volume of 1 M MgCl2 was added to the filtrate. The mixture was stirred for 15 min. Precipitated material was removed by centrifugation at 3000 x g for 12 min and discarded. The supernatant was centrifuged at 50,000 x g for 1 h. The pellet containing brush-border membranes was washed twice with 20 volumes of 50 mM phosphate buffer (pH 7.4) by dispersion and centrifugation at 50,000 x g for 1 h. The final preparation was diluted with the washing buffer to 10 mg/ml protein concentration and stored frozen at -20°C. Specific activity of alkaline phosphatase, a brush-border membrane marker, in the final membrane fraction was 7.2-fold to 8.5-fold greater than in the starting homogenate (22).
Membranes (30 ml) were solubilized by addition of Triton X-100 to the final concentration of 1% and mixing end-over-end for 4 h. Nondissolved material was removed by centrifugation at 50,000 x g for 1 h. The supernatant was recirculated at 0.2 ml/min flow through a 1.5 ml rat hemoglobin-Sepharose column equilibrated with PBS (pH 7.4), 0.6 mM CaCl2, and 0.5% CHAPS. The column was washed with 30 ml PBS (pH 7.4), 0.6 mM CaCl2, 0.5% CHAPS, 30 ml of the same buffer containing 0.5 M NaCl, and again with 10 ml of the first buffer. Bound proteins were eluted with PBS (pH 5.0), 10 mM ethylenediaminetetraacetic acid (EDTA), and 0.5% CHAPS. Collected 1-ml fractions were concentrated 10 times by ultrafiltration by use of Centricons YM 10 (Millipore, Bedford, MD) and analyzed under nonreducing conditions by 4% to 16% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Protein bands were visualized by GELCODE blue stain reagent (Pierce Chemical Co.). For immunochemical analysis, proteins were blotted onto Hybond ECL nitrocellulose membrane (Amersham Pharmacia Biotech, Little Chalfont, Buckingamshire, UK). Nitrocellulose membranes were blocked by 5% skimmed milk in PBS-T (80 mM Na2HPO4, 20 mM NaH2PO4, and 0.1% Tween 20 [pH 7.5]) for 1 h and incubated with primary antibody in PBS-T overnight at 4°C. After washing with PBS-T, the blots were incubated with horseradish peroxidase–conjugated secondary antibody diluted 1:3000 in PBS-T. ECL-PLUS reagent (Amersham Pharmacia Biotech) and Fluor-s imaging system (Bio-Rad Laboratories Inc., Hercules, CA) was used for chemiluminescent visualization.
Kinetics of Hemoglobin Binding to Cubilin and Megalin
The binding of human hemoglobin to megalin and cubilin was studied by surface plasmon-resonance (SPR) analysis on a BiaCore 2000 instrument (BiaCore AB, Uppsala, Sweden). The procedure was essentially as described elsewhere (23). Briefly, BiaCore sensor chips type CM5 were activated with a 1:1 mixture of 0.2 M N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide and 0.05 M N-hydroxysuccimide in water, according to the manufacturer’s instructions. Megalin and cubilin were purified by RAP or IF-B12 affinity chromatography, respectively. The preparations gave single bands in sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by coomassie brilliant blue staining. No cross-contamination of the two proteins could be detected. The proteins were immobilized at concentrations up to 50 µg/ml in 10 mM sodium acetate (pH 4.5), and the remaining binding sites were blocked with 1 M ethanolamine (pH 8.5). The resulting receptor densities were 23 fmol megalin/mm2 and 38 fmol cubilin/mm2, respectively. A control flow cell was made by performing the activation and blocking procedures only. In some experiments, immobilized receptor proteins were reduced by injection of 0.5% dithiothreitol in 6 M guanidine hydrochloride, 5 mM EDTA, and 50 mM Tris (pH 8.0) into the flow cell. Samples were dissolved in 10 mM HEPES, 150 mM NaCl, 2 mM CaCl2, and 0.005% Tween-20 (pH 7.4) or in 10 mM HEPES, 150 mM NaCl, 20 mM ethyleneglycol-bis(ß-aminoethyl ether)-N-N-N'-N'-tetraacetic acid, and 0.005% Tween-20 (pH 7.4). Sample and running buffer were identical. Regeneration of sensor chips after each analysis cycle was performed with 1.6 M glycine-HCl buffer (pH 3.0). The BiaCore response is expressed in relative response units, i.e., the difference in response between protein and control flow channel. Kinetic parameters were determined by use of BIAevaluation 3.1 software.
Immunohistochemistry
Immunohistochemical studies were performed on kidneys excised from normal rats or rats injected with rat hemoglobin (200 mg/kg body wt in PBS [pH 7.4]) into the femoral vein at 5, 10, 20, and 30 min after injection, respectively. Kidneys from normal and megalin knockout mice (24) as well as normal and cubilin-malexpressing dogs (25) were also studied but without hemoglobin infusion. Cortex tissue specimens were prepared from kidneys after fixation with 1% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4) by retrograde perfusion through abdominal aorta or directly through the renal arteries in dogs. Blocks of tissue were further fixed by immersion in the same solution for 1 h and transferred to 2.3 M sucrose in PBS (pH 7.4) for 0.5 h before being frozen in liquid nitrogen. Semithin cryosections (0.8 µm) were cut by use of a Reichert Ultracut S microtome (Richert-Jung, Vienna, Austria) and placed onto glass slides. Endogenous peroxidase activity was quenched with PBS (pH 7.4), 10% methanol, and 3% H2O2, and nonspecific binding was blocked with PBS (pH 7.4), 1% BSA, and 0.05 M glycine. The sections were subsequently incubated with the appropriate anti-hemoglobin affinity-selected IgGs diluted 1:20 to 1:200 and peroxidase-conjugated goat IgG anti-rabbit immunoglobulins diluted 1:100. The reaction was visualized with diaminobenzidine. The sections were counterstained with Mayer’s hematoxilin.
Uptake Studies on Cell Culture
Rat yolk sac carcinoma cells BN-16 (20) were routinely grown in 25 cm2 plastic culture flasks (Corning Costar, Badhoevedrop, Holland), in Eagle’s Minimal Essential Medium (Bio-Whittaker, Welkersville, MD) supplemented with 10% fetal calf serum (Biological Industries, Fredensborg, Denmark), 2 mM L-glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin (Bio-Whittaker) in humidified atmosphere that contained 5% CO2 at 37°C. Cells were passaged every fourth day with a split ratio of 1:5 by trypsinization with 500 mg/L Trypsin and 200 mg/L EDTA (Bio-Whittaker).
For uptake experiments, cells were cultured in eight-chamber glass slides (Nalge Nunc International, Naperville, IL). One day before the cells reached confluence, the medium was replaced with medium supplemented with 0.5% ovoalbumin instead of 10% serum (serum-free medium). Cell monolayers were incubated with 10 µg/ml FLUOS-hemoglobin in serum-free medium for 10 min and fixed with 1% paraformaldehyde in PBS (pH 7.4) for 3 min. For inhibition studies, the following proteins were added to the incubation mixture: 20 µM unlabeled hemoglobin, 20 µM BSA, 20 µM IF-B12, 1 µM RAP, 200 to 400 mg/L sheep anti-rat megalin IgG serum fraction, sheep nonimmune IgG serum fraction, or rabbit anti-rat cubilin IgG serum fraction, rabbit nonimmune IgG serum fraction. The slides were mounted with 50% glycerol, 2% N-propylgallat, 2.4% Tris, and examined by use of a fluorescence microscope (Leica DMR) equipped with a color video camera (Sony 3CCD).
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85% and 80% decrease of binding to megalin and cubilin, respectively. The binding to megalin was affected to the same extent also by reductive conditions. Reduction of cubilin did not influence its interaction with hemoglobin (data not shown).
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7 nM) (27) in comparison to other ligands, one could speculate that the presence of both receptors in the eluate might be due to their association. However, in other studies, only trace amounts of megalin, as detected by immunoblotting, copurified with cubilin in affinity chromatography that used sole cubilin ligands such as IF-B12 or HDL (23,28). Apparently, in solubilized membranes the interaction between cubilin and megalin is largely abolished. Thus the results obtained by affinity chromatography, at least in the case of cubilin, are not considerably influenced by a copurification phenomenon. We verified the binding of hemoglobin to both receptors using SPR. On the basis of dissociation constants, 1.7 µM for megalin and 4.1 µM for cubilin, the interactions can be classified as "low-affinity binding," which applies to many ligands of these receptors. Both the affinity chromatography results and the SPR data indicate that hemoglobin binds to cubilin and megalin in a calcium-dependent manner, as do all other of their characterized ligands. We further examined the significance of those interaction for cellular uptake of hemoglobin using a cell-culture model. Because in cultured cells of proximal tubule origin, like LLC-PK1 or OK cells, the expression of megalin and cubilin is much lower in comparison to those in vivo, we chose BN-16 cells for these experiments. BN-16 cells, which originate from yolk sac epithelium, are structurally and functionally similar to renal proximal tubule cells but exhibit high rates of cubilin- and megalin-mediated endocytosis. These cells have been used elsewhere as an in vitro model of proximal tubular endocytosis (20,29). Indeed, we observed a rapid accumulation of fluorescence-labeled hemoglobin in the cells. Involvement of cubilin and megalin in the uptake was further demonstrated by the inhibitory action of antisera raised against purified receptors. Moreover, the uptake was virtually abolished by RAP, a chaperone that affects binding of most megalin and cubilin ligands (30). We also observed inhibition of hemoglobin uptake by myoglobin. These proteins share structural similarity, so it is plausible that they can occupy common binding sites on these receptors. Elucidation of myoglobin catabolic pathways in the kidney would be of particular clinical importance, because myoglobinuria underlies as many as 15% of all acute renal failure cases (3).
Renal uptake of hemoglobin has been demonstrated repeatedly in different experimental models of hemoglobinuria (5–8). Our immunohistochemical study documents that hemoglobin uptake also occurs under physiologic conditions. Comparison of kidneys of normal and megalin knockout mice confirmed that megalin is responsible for physiologic clearance of hemoglobin. The physiologic significance of hemoglobin uptake in the kidney is open to question and may be a mechanism of iron retrieval. One can calculate, taking free plasma hemoglobin concentration of 0.03 g/L (31) and the fractional filtration coefficient for hemoglobin of 0.03 (4), that lack of hemoglobin reabsorption in humans would result in a loss of 0.6 mg iron daily, which is rather negligible in comparison to 4 g overall iron body pool (32). However, extensive withdrawal of hemoglobin from the primary filtrate may protect the lower urinary tract from bacterial invasion. Endogenous host hemoproteins are commonly used by pathogenic bacteria as an iron source. Inaccessibility of this element, as well as heme in bacteria unable to synthesize protoporphyrin IX, is the factor most limiting for their growth and virulence (33,34). Recent findings that Neisseria gonorrhoeae and Gardnerella vaginalis, pathogenic bacteria that provoke urinary tract infections, can acquire iron from human hemoglobin would support this hypothesis (35,36).
We found no difference in renal hemoglobin uptake between normal dogs and those that do not express cubilin in the apical membrane of proximal tubule cells. It is conceivable that, under physiologic conditions, megalin itself is sufficient to scavenge the small amounts of filtered hemoglobin. Generally, hemoglobin released into blood circulation is irreversibly captured by the plasma protein haptoglobin and the complex, being too large to be filtered in the glomeruli, is catabolized by the mononuclear phagocyte system in liver and spleen (37). Recently, the receptor for the hemoglobin-haptoglobin complex has been identified as the acute phase–regulated and signal-inducing monocyte/macrophage protein CD163 (38). The role of cubilin may also be limited by its lower affinity for hemoglobin. However, cubilin may become an essential scavenger in hemolytic disorders when glomerular filtrate hemoglobin concentration is greatly elevated. The involvement of two receptors would serve as a high-capacity mechanism for hemoglobin uptake in such circumstances. The existence of an effective reabsorption mechanism for hemoglobin suggests that it may have a role in the development of hemoglobinuria/proteinuria-induced interstitial tubular nephritis. To date, investigations of other potentially nephrotoxic plasma proteins have focused mainly on albumin and transferrin (39).
In conclusion, this study reveals a molecular mechanism of hemoglobin uptake in the renal proximal tubule that involves the endocytic receptors megalin and cubilin. Identification of the receptors for tubular hemoglobin uptake may be essential for development of new therapeutic agents to block hemoglobin entry into tubular epithelial cells and prevent its cytotoxicity in hemoglobinuric states. To our knowledge, this is the first report demonstrating the uptake of endogenous hemoglobin under physiologic conditions, the pathophysiologic role of which remains to be elucidated.
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) apical membrane staining; (
) endosomal/lysosomal labeling. Magnifications: x800 in A, C, and D; x1050 in B, E, and F.