Renal uptake of myoglobin is mediated by the endocytic receptors megalin and cub - 《American Journal of Physiology》美国生理学杂志干细胞之家



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作者：Jakub Gburek, Henrik Birn, Pierre J. Verroust, Boguslawa Goj, Christian Jacobsen, Søren K. Moestrup, Thomas E. Willnow,  Erik I. Christensen作者单位：Departments of Cell Biology and Medical Biochemistry, University of Aarhus, DK-8000Aarhus C, Denmark; Department of Biochemistry,Faculty of Pharmacy, Wroclaw Medical University, 50139 Wroclaw, Poland; Institut Nationale de la Santé et de laRecherche Medicalé Unité 53 Centre Hosp
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      【摘要】# Y% e5 [9 N- c6 h; J- @$ r
   Nephrotoxicity of myoglobin is well recognized as playing a part in thedevelopment of acute renal failure in settings of myoglobinuria. However, themolecular mechanism of myoglobin uptake in renal proximal tubules has not beenclarified. Here, we report that the endocytic receptors megalin and cubilin are involved in renal reabsorption of myoglobin. Both receptors were capturedfrom solubilized renal brush-border membranes by affinity chromatography usingmyoglobin-Sepharose. Myoglobin bound to purified megalin and cubilin with K d values of 2.0 and 3 µM, respectively, as evaluatedby surface plasmon resonance analysis. Apomyoglobin bound to megalin with the same affinity, and the affinity of apomyoglobin to cubilin was reduced( K d = 5 µM). Radioiodinated myoglobin could be displaced by apomyoglobin in inhibition studies using isolated renalbrush-border membranes ( K i 2 µM).Receptor-associated protein as well as antibodies directed against megalin and cubilin markedly inhibited the uptake of fluorescent-labeled myoglobin bycultured yolk sac BN-16 cells. The significance of megalin- andcubilin-mediated endocytosis for myoglobin uptake in vivo was demonstrated byuse of kidney-specific megalin knockout mice. Injected myoglobin wasextensively reabsorbed by megalin-expressing proximal tubular cells, whereasthere was very little uptake in the megalin-deficient cells. In conclusion, this study establishes the molecular mechanism of myoglobin uptake in therenal proximal tubule involving the endocytic receptors megalin and cubilin.Identification of the receptors for tubular uptake of myoglobin may beessential for development of new therapeutic strategies for myoglobinuricacute renal failure. 9 U* j2 S# f% V+ R7 d
      【关键词】 endocytosis acute renal failure
   ACUTE RENAL FAILURE (ARF) frequently develops in clinicalsettings associated with increased urinary excretion of myoglobin.Myoglobinuria is a consequence of severe muscle injury, termed rhabdomyolysis, which is usually associated with trauma but may occur in variety of otherclinical conditions such as hyperthermia, muscle ischemia, or exposure tocertain toxins or drugs ( 10, 24 ).

Dependent on etiology myoglobinuria may be accompanied by additional complications but seemingly deposition of myoglobin in the kidney is aprincipal factor contributing to the decline of renal function. At the kidneylevel, the main pathological events in the genesis of myoglobinuric ARF havebeen identified as intense renal vasoconstriction, tubular obstruction byintraluminal pigment casts, and necrosis of proximal tubular epithelium due todirect intra- and/or extracellular toxicity of myoglobin. Because there ismuch evidence for the occurrence of oxidative damage to the kidney inrhabdomyolysis, the toxicity of myoglobin has been attributed to catalysis offree radical reactions. The precise mechanism, whether due to free iron, heme,or heme protein, and which free radical mechanism is involved, as well aswhich cellular organelles are affected, are still debated ( 11, 26 ). It has been convincinglyshown that myoglobin undergoes reabsorption from the glomerular filtrate andis catabolized within proximal tubule cells( 2, 14, 21 ). Thus it is conceivable that particular components of myoglobin can act on different intracellularlevels with the involvement of diverse radical species during reabsorption ofholoprotein and its subsequent decomposition. Consequently, it seems to be ofgreat interest to elucidate the molecular mechanism of myoglobin uptake and degradation in the kidney.

Several glomerular filtrate proteins are reabsorbed as a complex withmegalin and/or cubilin, the multiligand endocytic receptors residing in themembrane of proximal tubular epithelial cells. Megalin is a 600-kDa type Itransmembrane glycoprotein belonging to the LDL receptor family, whereascubilin is a 460-kDa glycoprotein that lacks a classic transmembrane domainand has no homology to any known endocytic receptors. As the vastextracellular domains of these receptors can accommodate a variety of ligands, and the receptors can act both independently or following association as adual-receptor complex, it seems plausible that they facilitate uptake of mostproteins from the primary filtrate( 7 ). This is supported by thefinding of low-molecular-weight proteinuria in megalin knockout mice and indogs that bear an inherited disorder of intracellular cubilin processing( 16, 25 ). Furthermore, low-molecular-weight proteinuria also develops in patients who suffer fromImerslund-Gräsback syndrome, a rare autosomal disorder caused bymutations in the cubilin gene( 1 ).
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We have recently shown that megalin and cubilin are responsible for renalreabsorption of hemoglobin, a heme protein structurally related to myoglobin( 13 ). Thus here we aim atresolving the role of those receptors in myoglobin reabsorption.5 @7 X0 k# I; d; j3 G, i
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MATERIALS AND METHODS
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Proteins. Horse myoglobin was obtained from Serva Electrophoresis (Heidelberg, Germany), and this preparation was used throughout the study. BSAand ovoalbumin were purchased from Sigma (St. Louis, MO). Recombinantreceptor-associated protein (RAP) was prepared as described elsewhere( 20 ). Apomyoglobin wasprepared from myoglobin using the acid acetone procedure( 22 )./ `6 u0 ]+ l5 Z9 H, Q" K0 [

Fluorescence-labeled myoglobin was synthetized by coupling 5(6)-carboxyfluorescein- N -hydroxysuccinimide ester (FLUOS; Roche Diagnostics, Mannheim, Germany) to myoglobin amino groups using afluorescein-labeling kit according to the manufacturer's instructions.Briefly, 5 mg of myoglobin dissolved in PBS, pH 7.4, were incubated with 1.2mg FLUOS for 2 h at room temperature with gentle mixing. Unbound FLUOS wasremoved by Sephadex G-25 gel filtration. The approximatefluorescein-to-protein ratio of the preparation was 10. Aliquots ofFLUOS-myoglobin (1 mg/ml) were stored at -20°C until used.
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125 I-myoglobin was prepared by use of Iodo-gen according to Salacinski et al. ( 23 ).Specific activities of the tracer preparations were in the range of 1.0-1.5µCi/µg protein.4 r$ h3 X/ K- e4 Y
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Protein concentrations were determined using a protein assay reagent(Pierce, Rockford, IL).
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Antibodies. Affinity-isolated IgG to horse myoglobin was purchased from Bethyl Laboratories (Montgomery, TX). Sheep anti-rat megalin and rabbitanti-rat cubilin antisera were obtained as described previously( 15, 19 ). Sera IgG fractions wereprepared by protein A-agarose affinity chromatography according to the manufacturer's instructions (Pierce). A nonimmune sheep serum IgG fraction wasobtained from Sigma. Alexa-conjugated secondary antibodies were purchased fromMolecular Probes (Eugene, OR). All other antibodies used in this study werepurchased from Dako (Glostrup, Denmark)./ R- e! E9 K; L+ t% ~" |

Purification of myoglobin receptors by affinity chromatography. Rat renal brush-border membranes were prepared and solubilized using TritonX-100 as previously described( 12 ). The membrane proteinextract was recirculated at 0.2 ml/min flow through a 1.5-ml ratmyoglobin-Sepharose column equilibrated with PBS, pH 7.4, 0.6 mMCaCl 2, and 0.5%3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). The columnwas washed with 30 ml PBS, pH 7.4, 0.6 mM CaCl 2, 0.5% CHAPS, and 30ml of the same buffer containing 0.5 M NaCl, and again with 10 ml of the firstbuffer. Bound proteins were eluted with PBS, pH 5.0, 10 mM EDTA, and 0.5% CHAPS. Collected 1-ml fractions were concentrated 10 times by ultrafiltrationusing Centricon YM 10 (Millipore, Bedford, MA) and analyzed under nonreducingconditions by 4-16% SDS-PAGE. Protein bands were visualized by Gelcode bluestain reagent (Pierce). For immunochemical analysis, proteins were blotted onto Hybond ECL nitrocellulose membranes (Amersham Pharmacia Biotech, LittleChalfont, Buckingamshire, UK). Nitrocellulose membranes were blocked by 5%skim milk in 80 mM Na 2 HPO 4, 20 mMNaH 2 PO 4, and 0.1% Tween 20, pH 7.5 (PBS-T) for 1 h and incubated with primary antibody in PBS-T overnight at 4°C. After beingwashed with PBS-T, the blots were incubated with horseradishperoxidase-conjugated secondary antibody diluted 1:3,000 in PBS-T. ECL-PLUSreagent (Amersham Pharmacia Biotech) and the Fluor-s imaging system (Bio-RadLaboratories, Hercules, CA) were used for chemiluminescent visualization.. L$ f' l3 a+ W, [6 N+ ~/ j

Kinetics of myoglobin and apomyoglobin binding to cubilin and megalin. The binding of myoglobin and apomyoglobin to megalin and cubilinwas studied by surface plasmon resonance analysis on a BiaCore 2000 instrument(BiaCore, Uppsala, Sweden). The procedure was essentially as describedpreviously ( 5 ). Briefly, BiaCore type CM5 sensor chips 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 recommendations. Megalin and cubilin were purified by RAP or IF-B12 affinitychromatography, respectively. The preparations produced single bands inSDS-PAGE followed by Coomassie brilliant blue staining. No cross-contaminationof the two proteins could be detected. The proteins were immobilized atconcentrations 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. Theresulting receptor densities were in the range of 23-40 fmolreceptor/mm 2. A control flow cell was made by performing theactivation and blocking procedures only. Immobilized receptor proteins werereduced by injection of 0.5% dithiothreitol in 6 M guanidine hydrochloride, 5mM EDTA, and 50 mM Tris, pH 8.0, into the flow cell. Samples were dissolved in10 mM HEPES, 150 mM NaCl, 2 mM CaCl 2, and 0.005% Tween 20, pH 7.4, or 10 mM HEPES, 150 mM NaCl, 20 mM EGTA, and 0.005% Tween 20, pH 7.4. Sampleand running buffers were identical. The regeneration of sensor chips aftereach analysis cycle was performed with 1.6 M glycine-HCl buffer, pH 3.0. TheBiaCore response is expressed in relative response units, i.e., the differencein response between proteins and the control flow channel. Kinetic parameters were determined by using BIAevaluation 3.1 software.
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Competition between myoglobin and apomyoglobin for binding to receptorsites was studied by the rapid filtration technique using isolated ratbrush-border membranes as previously described ( 12 ). In brief, membranes (100µg protein) were incubated in binding buffer (Tris-buffered saline) withtracer amounts of 125 I-myoglobin ( 10 4 counts/min)and increasing concentrations of apomyoglobin in the range of 0.3-5 µM in afinal volume of 0.2 ml for 60 min. After incubation, 0.15-ml samples were applied onto GVWP 0.22-µm membrane filters (Millipore) and washed with 5 mlof binding buffer to remove unbound ligands. The radioactivity of the filterscorresponding to the amount of bound 125 I-myoglobin was measured ina gamma counter (Polon). K i was evaluated by computerizednonlinear regression analysis using Prizm software (GrapPad Software)." U. C4 V. {" F, ]
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Immunohistochemistry. Immunohistochemical studies were performed in kidneys excised from control mice (C57BL) or kidney-specific megalinknockout mice ( 15 ) injectedwith myoglobin (35 mg/kg body wt in PBS, pH 7.4) into the femoral vein at 15and 30 min after injection, respectively.  e4 J& X3 }  |8 R9 _, g! W; D
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Cortical tissue specimens were prepared from kidneys after fixation with 2%paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4, by retrograde perfusionthrough the abdominal aorta. Blocks of tissue were further fixed by immersionin 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 (0.8µm) were cut using a Reichert Ultracut S microtome (Richert-Jung, Vienna,Austria) and placed onto glass slides. Endogenous peroxidase activity wasquenched with PBS, pH 7.4, 10% methanol, and 3% H 2 O 2,and nonspecific binding was blocked with PBS, pH 7.4, 1% BSA, and 0.05 Mglycine.
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For the immunoperoxidase reaction, the sections were subsequently incubatedwith goat anti-myoglobin affinitypurified IgGs diluted 1:400-1:800 andperoxidase-conjugated rabbit IgG anti-goat IgGs diluted 1:300. The reactionwas visualized with diaminobenzidine. The sections were counterstained withMeyer's hematoxilin.

In immunofluorescence studies, the following antibodies were used: goatanti-horse myoglobin affinity-purified IgGs diluted 1:400-1:800; sheepanti-rat megalin antiserum diluted 1:25,000-1:50,000; rabbit anti-rat cubilinantiserum diluted 1:2,000-1:4,000; Alexa 568-labeled donkey IgG anti-goat IgGsdiluted 1:300; Alexa 488-labeled donkey IgG anti-sheep IgGs diluted 1:300; andFITC-conjugated swine IgG anti-rabbit IgGs diluted 1:40. The sections wereanalyzed using Leica SP2 confocal microscope.) A; F# i/ a3 u7 ]7 h) X( N5 g

Uptake studies in cell culture. Rat yolk sac carcinoma BN-16 cells( 18 ) were routinely grown in25-cm 2 plastic culture flasks (Corning Costar, Badhoevedrop,Holland) in Eagle's MEM (Bio-Whittaker, Walkersville, MD) supplemented with10% fetal calf serum (Biological Industries, Fredensborg, Denmark), 2 mM L -glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin (Bio-Whittaker) in a humidified atmosphere containing 5% CO 2 at37°C. Cells were passaged every fourth day at a split ratio of 1:5 bytrypsinization with 500 mg/l trypsin and 200 mg/l EDTA (Bio-Whittaker).5 C, b0 R6 v- Q4 f
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For uptake experiments, cells were cultured in eight-chamber glass slides(Nalge Nunc International, Naperville, IL). One day before the cells reachedconfluence, the medium was replaced with medium supplemented with 0.5%ovoalbumin instead of 10% serum (serum-free medium). Cell monolayers wereincubated with 5 µg/ml FLUOS-myoglobin in serum-free medium for 10 min andfixed with 1% paraformaldehyde in PBS, pH 7.4, for 3 min. For inhibitionstudies, the following proteins were added to the incubation mixture: 1 µMrecombinant receptor-associated protein, a 200-mg/l sheep anti-rat megalin IgGserum fraction and a sheep nonimmune IgG serum fraction, or 400-mg/l rabbit anti-rat cubilin IgG serum fraction and rabbit nonimmune IgG serum fraction.The slides were mounted with 50% glycerol, 2% N -propyl-gallat, and2.4% Tris and examined using a fluorescence microscope (Leica DMR) equippedwith a color video camera (Sony 3CCD).- x8 G' m" q- W0 ~1 U

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Myoglobin affinity chromatography. To identify potential endocytic receptors for myoglobin in the kidney proximal tubule, we performed affinitychromatography on a myoglobin-Sepharose column. Solubilized rat renalbrush-border membranes were passed over the affinity matrix, and, after anextensive washing, the retained proteins were eluted at pH 5.0. Analysis offinal acid eluate in SDS-PAGE revealed the presence of two proteins, whichcomigrated with cubilin and megalin, seen as the two predominant bands at the top area of the brush-border membrane fraction lane( Fig. 1 ). We further confirmedthe identity of the proteins by Western blot analysis of the peak fractionusing specific antibodies to megalin and cubilin( Fig. 1 ).7 P/ z+ X& h- u9 L8 Y) M1 T
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Fig. 1. Analysis of affinity-purified myoglobin-binding proteins by SDS-PAGE andWestern blotting. Rat renal brush-border membranes solubilized in 1% TritonX-100 were passed over myoglobin-Sepharose. After being washed, the boundfraction was eluted with PBS, pH 5.0, 10 mM EDTA, and 0.5%3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Left :SDS-PAGE of consecutive eluate fractions ( lanes 1-7 ) with Coomassiebrilliant blue (CBB) staining. The myoglobin-binding fraction contains similaramounts of cubilin and megalin. Mb, 1 µg myoglobin; BB, brush-bordermembranes (10 µg protein); M, molecular mass standards. Right :Western blotting of peak fraction ( lane 4 ) from myoglobin-Sepharoseaffinity chromatography using anti-cubilin or anti-megalin antibodies forimmunoperoxidase chemiluminescent visualization.

Surface plasmon resonance analysis. Kinetic parameters ofmyoglobin binding to megalin or cubilin were examined by means of surface plasmon resonance analysis. K d values for the interactions of myoglobin with megalin or cubilin were 2.0 and 3.0 µM, respectively( Fig. 2, A and B ). Apomyoglobin bound to megalin with the same affinityas myoglobin ( K d = 2 µM), whereas the affinity ofapomyoglobin for cubilin was lower ( K d = 5 µM). Thebinding of both myoglobin and apomyoglobin was completely abolished in thepresence of EDTA ( Fig. 2, C and D ). Competition of both myoglobin forms for binding siteson the receptors was tested in an inhibition study using isolated brush-bordermembranes. Radioiodinated myoglobin could be displaced by apomyoglobin with a K i of 2 µM ( Fig.3 ).5 j- V: p; I$ i

Fig. 2. Surface plasmon resonance analysis of myoglobin binding to cubilin andmegalin. Megalin and cubilin were immobilized onto sensor chips, and myoglobinsamples (40 µl) were passed over the flow cells at 5 µl/min. A and B : sensorgrams with myoglobin concentrations in the range of0.2-5 µM were recorded for evaluation of kinetic parameters. The affinityof myoglobin for megalin ( A ) was determined as K d = 2.0 µM and the affinity of myoglobin for cubilin as K d = 3.0 µM, assuming 1 class of binding sites for eachreceptor. C and D : sensorgrams with myoglobin (solid lines)and apomyoglobin (dotted lines) in a concentration of 5 µM. The binding ofmyoglobin as well as apomyoglobin was almost abolished (below 5 responseunits) when 20 mM EDTA was included in sample and running buffer, showingcalcium dependency of the interactions.

Fig. 3. Competition of myoglobin and apomyoglobin for receptor-binding sites.Isolated brush-border membranes were incubated with tracer amount ofradioiodinated myoglobin and increasing concentrations of apomyoglobin in therange of 0.3-5.0 µM for 1 h. The total amount of bound (B) radioligand wasdetermined by the rapid filtration technique. Apomyoglobin inhibited myoglobinbinding to receptor sites with a K i of 2 µM.

Uptake of myoglobin in vitro. The role of megalin and cubilin inthe endocytic uptake of myoglobin was investigated by an inhibition study incultured BN-16 cells, a cell line derived from the rat yolk sac. The cellscould intensively internalize FLUOS-myoglobin from the incubation medium,which appeared as green fluorescence accumulating within theendosomal/lysosomal compartment of the cell. The uptake was site limitedbecause an excess of unlabeled myoglobin (20 µM) virtually prevented fluorescent labeling (not shown). We observed a marked inhibition ofFLUOS-myoglobin uptake with anti-megalin or anti-cubilin antibodies at aconcentration of 200 and 400 µg/ml, respectively, or with 1 µM RAP( Fig. 4 ).
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Fig. 4. Characterization of myoglobin uptake by BN-16 cells using fluorescencemicroscopy. Cells were grown to confluence on glass slides and incubated with5 µg/ml 5(6)-carboxyfluorescein- N -hydroxysuccinimide estermyoglobin in Eagle's MEM medium containing 0.5% ovoalbumin for 10 min alone( A ) or in the presence of 1 µM receptor-associated protein (RAP; B ), 200 mg/l nonimmune sheep IgG ( C ), 200 mg/l sheepanti-megalin IgG ( D ), 400 mg/l nonimmune rabbit IgG ( E ), or400 mg/l rabbit anti-cubilin IgG ( F ). The uptake of myoglobin ismarkedly inhibited by RAP and anti-megalin or cubilin antibodies (compare A and B, C and D, E and F ). Magnification: x 700.

Uptake of myoglobin in vivo. The significance of megalin- and cubilin-mediated endocytosis for myoglobin reabsorption in vivo wasinvestigated by immunocytochemistry in kidney sections from normal andkidney-specific megalin knockout mice( 15 ). Because endogenousmyoglobin could be detected in neither normal mouse nor knockout mouseproximal tubule (data not shown), we performed our experiments using miceinjected with exogenous myoglobin at a dose of 35 mg/kg body wt. Thirtyminutes after injection, a strong immunoperoxidase reaction withanti-myoglobin antibodies was identified mainly along the brush border and within the apical endosomal/lysosomal compartment in virtually all cells ofthe proximal tubule from normal mice. There was also additionalimmunoreactivity in the extratubular space ( Fig. 5 A ). In themegalin knockout mouse, some proximal tubular cells were devoid ofimmunoreactivity, whereas in others the distribution of the immunoreaction wasanalogous to that in the normal mouse ( Fig.5 B ). To demonstrate a relationship between the expressionof megalin or cubilin and reabsorption of myoglobin in those two subsets ofcells, we employed the double-immunofluorescence technique. Normal anddeficient cells could be easily distinguished by anti-megalin antibodies.There was a pronounced reduction of cubilin expression in the cells lackingmegalin. Both receptors colocalized mainly in the apical membrane area( Fig. 6 ).Megalin-immuno-reactive cells exhibited substantial deposition of myoglobin atthe brush border and in the apical endosomal/lysosomal apparatus as well as invesicular structures located deeper in the cell. Myoglobin and megalin orcubilin, respectively, colocalized at the brush border and in vesiclesadjacent to the apical plasma membrane. Uptake of myoglobin by the cellsdevoid of megalin was either very low or not seen at all.

Fig. 5. Immunoperoxidase demonstration of myoglobin uptake in normal andkidney-specific megalin knockout mouse. The animals were injected withmyoglobin (35 mg/kg body wt), perfusion fixed after 30 min, and the kidneyswere processed for cryoimmunocytochemistry. A : cortex of normalmouse. B : cortex of kidney-specific megalin knockout mouse. Note thatall proximal tubular cells are labeled in the normal mouse, whereas in megalinknockout mouse a number of cells remined unlabeled ( * ). Arrows,brush-border staining; arrowheads, vacuolar staining. Magnification: x 1,200.5 U3 X/ e; @  v9 h* t

Fig. 6. Identification of myoglobin uptake in kidney-specific megalin knockout miceby double immunofluorescence. The animals were injected with myoglobin (35mg/kg body wt), perfusion fixed after 30 min, and the kidneys were processedfor cryoimmunocytochemistry. A - C : expression of megalin andcubilin in the proximal tubule. D - F : colocalization ofmegalin and injected myoglobin in the proximal tubule. G - I :colocalization of cubilin and injected myoglobin in the proximal tubule. Notethat in megalin-deficient cells, expression of cubilin is markedly reduced andthe reabsorption of myoglobin is negligible compared with megalin-expressingcells. Endocytosis of myoglobin by megalin-expressing cells is reflected byits colocalization with megalin and cubilin at the brush border and in theapical endosomal compartment. Magnification: x 1,200.3 s, ]: p* o- @. F' R

DISCUSSION
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Megalin and cubilin are the major endocytic receptors responsible forreabsorption of glomerular ultrafiltrate proteins in the proximal tubule.Here, we report that the receptors also facilitate epithelial uptake ofmyoglobin, a highly nephrotoxic protein released into the circulation duringrhabdomyolysis. This is supported by several lines of evidence. Affinitychromatography of solubilized renal brush-border membranes on a myoglobin matrix yielded principally megalin and cubilin in approximately equal amounts.Lack of other proteins in the affinity preparation suggested that thereceptors are solely responsible for myoglobin reabsorption in the proximaltubule. Because purified megalin and cubilin retained myoglobin-bindingactivity, we could characterize the kinetics of the binding (interactions)using surface plasmon resonance analysis. Based on K d values, 2 µM for megalin and 3 µM for cubilin, the interactions can beclassified as "low-affinity binding." Low affinity might raise thequestion of the efficiency of scavenging and thereby the pathophysiological significance of such interactions. However, in the case of the kidney proximaltubule, the low affinity is likely to be compensated for by copious amounts ofthe receptors at the brush border and increased glomerular filtrate massconvection facilitated by the microvillar apparatus. Indeed, megalinandcubilin-ligand interactions of the above strengths turned out to be essential for the gross uptake of other ligands such as albumin or hemoglobin ( 4, 13 ). Furthermore, the plasmaconcentration of myoglobin in most common rhabdomyolytic states is ratherhigh, exceeding the estimated K d values severalfold( 3 ).
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To clarify whether these relatively weak interactions can genuinely promotethe uptake of myoglobin, we employed a cell culture model in our studies.Because in cultured cells of proximal tubule origin like LLCPK 1 oropossum kidney cells the expression of megalin and cubilin is much lowercompared with those in vivo, we chose BN-16 cells for these experiments. BN-16cells, originating from yolk sac epithelium, are structurally and functionally similar to renal proximal tubule cells but exhibit high rates of cubilin- andmegalin-mediated endocytosis. These cells have previously been used as an invitro model of proximal tubular endocytosis( 17, 18 ). Significantly,accumulation of fluorescence-labeled myoglobin in the cells was inhibited byantibodies raised against purified receptors and by RAP, a chaperone thataffects binding of most megalin and cubilin ligands( 6 ).

To assess the role of the receptors in the clearance of myoglobin in vivo,we performed experiments using kidney-specific megalin knockout mice. The micewere designed specifically to conquer the problems of high perinatal lethalityand severe complex phenotype associated with full megalin gene knockout, sofar limiting the usefulness of the model. Renal specific knockout mice presenta severe tubular reabsorption deficiency associated with up to a 90% reductionin the number of proximal tubular cells expressing megalin but exhibit normaldevelopment and viability( 15 ). Mice were injectedintravenously with myoglobin at a dose of 35 mg/kg, which is compatible withthe concentration range observed in rhabdomyolysis( 3 ). A comparison of myoglobin deposition in megalin-deficient and normal cells after intravenous administration of the protein clearly implied that megalin-mediated endocytosis is a predominant route of myoglobin entry into proximal tubulecells. In normal cells, myoglobin colocalized with the receptors at the brushborder and within the apical vacuolar apparatus, which reflected its bindingto the receptors and subsequent endocytosis. Part of the endocytosed myoglobincould be detected in vacuolar structures devoid of the receptors, which isconsistent with early sorting of the receptors into the recycling compartment( 8 ). Deposition of injectedmyoglobin in megalin-deficient cells was strikingly reduced and limited torelatively small vacuolar structures, which resembled uptake by fluid-phaseendocytosis. This process has been previously characterized in the proximaltubule as a minor nonreceptor endocytic transport system accounting for uptake ( 9 ).Besides the epithelium, there was also deposition of myoglobin in theinterstitium, indicating a substantial leak of the protein through thecapillary walls. The finding possibly discloses one more potential target ofmyoglobin toxicity in the kidney.$ Z" ?8 W& b) D4 [+ w- }& R; _
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Necrosis of proximal tubular epithelium is a trait/histopathological phenomenon found in the myoglobinuric kidney and thus is believed to be animportant factor in the development of the disease. Substantial evidence haslinked this lesion to iron-driven oxidative stress in connection withintracellular breakdown of the hemoprotein( 26 ). In light of ourfindings, one could envisage that restriction of megalin-mediated endocytosisof myoglobin, especially early after rhabdomyolytic insult, could have abeneficial effect on myoglobinnuric ARF. A potential competitor of myoglobinendocytosis that emerged during this study was apomyoglobin. Lacking theneprotoxic heme, apomyoglobin can displace myoglobin from the receptor-bindingsites. However, it is not known to what extent such competition can occur in vivo and whether administration of the doses required for inhibition wouldhave its own adverse effects.0 U8 \+ q2 B. G! J8 ~8 C2 k
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In conclusion, our study establishes a molecular mechanism of myoglobinuptake in the renal proximal tubule involving the endocytic receptors megalinand cubilin. Our findings may offer a perspective for experimental therapiespreventing development of myoglobinuric ARF. We suggest that organ-specificmegalin knockout mice may be employed in further studies on thepathophysiology of myoglobinuric ARF. Such investigations may in particular clarify the significance of tubular myoglobin reabsorption for the developmentof ARF as well as its role during the maintainance and recovery phase of thisdisease.
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DISCLOSURES+ w3 @. R% R( v( \' m" Z: X$ e1 z+ V; a
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This work has been supported by the Danish Medical Research Council, theUniversity of Aarhus Research Foundation, the Marie Curie Fellowship of theEuropean Community's Improving Human Potential Program (contract no.HPMF-CT-2001-01129), the NOVO-Nordisk Foundation, and the Biomembrane ResearchCentre of Aarhus University.

ACKNOWLEDGMENTS
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The technical assistance of Hanne Sidelmann, Inger Kristoffersen, and PiaKamuk Nielsen is greatly appreciated.* F7 q% ^4 }- W) G; Z, \( K
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Aminoff M,Carter JE, Chadwick RB, Johnson C, Gräsbeck R, Abdelaal MA, Broch H,Jenner LB, Verroust PJ, Moestrup SK, de la Chapelle A CA, and Krahe R. Mutations in CUBN, encoding the intrinsic factor-vitamin B 12 receptor, cubilin, cause hereditary megaloblastic anaemia 1. NatGenet 21:309-313, 1999.
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