title: Megalin and cubilin are endocytic receptors involved in renal clearance of hemoglobin.
abstract: The kidney is the main site of hemoglobin clearance and degradation in conditions of severe hemolysis. Herein it is reported that megalin and cubilin, two epithelial endocytic receptors, mediate the uptake of hemoglobin in renal proximal tubules. Both receptors were purified by use of hemoglobin-Sepharose affinity chromatography of solubilized renal brush-border membranes. Apparent dissociation constants of 1.7 microM for megalin and 4.1 microM for cubilin were determined by surface plasmon resonance analysis. The binding was calcium dependent in both cases. Uptake of fluorescence-labeled hemoglobin by BN-16 cells was inhibited by anti-megalin and anti-cubilin antibodies as well as by receptor-associated protein, a chaperone for LDL-receptor family proteins. Partial inhibition by myoglobin was observed, whereas bovine serum albumin, intrinsic factor-cobalamin complexes, and beta2-microglobulin did not affect the uptake. By use of immunohistochemistry, it was demonstrated that uptake of hemoglobin in proximal tubules of rat, mouse, and dog kidneys occurs under physiologic conditions. Studies on normal and megalin knockout mouse kidney sections showed that megalin is responsible for physiologic clearance of hemoglobin. Labeling intensities in kidneys from normal and cubilin-malexpressing dogs were similar, which suggests that, in the normal state, the role of cubilin in uptake of hemoglobin is rather limited. However, cubilin is likely to assist hemoglobin endocytosis in settings of hemoglobinuria. In conclusion, the study provides a molecular explanation for long-standing observations of hemoglobin uptake in renal proximal tubules that involve the endocytic receptors megalin and cubilin. The findings may prove to be essential for further research on the pathophysiology of hemoglobinuric acute renal failure and proteinuria-associated tubulointerstitial nephritis.
authors.affiliation: *Department of Biochemistry, Faculty of Pharmacy, Wrocław Medical University, Wrocław, 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, Wrocław Agricultural University, Wrocław, Poland; Department of Medical Biochemistry, University of Aarhus, Aarhus, Denmark.
authors.forename: Jakub
authors.initials: J
authors.lastname: Gburek
authors.affiliation: *Department of Biochemistry, Faculty of Pharmacy, Wrocław Medical University, Wrocław, 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, Wrocław Agricultural University, Wrocław, Poland; Department of Medical Biochemistry, University of Aarhus, Aarhus, Denmark.
authors.forename: Pierre J
authors.initials: PJ
authors.lastname: Verroust
authors.affiliation: *Department of Biochemistry, Faculty of Pharmacy, Wrocław Medical University, Wrocław, 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, Wrocław Agricultural University, Wrocław, Poland; Department of Medical Biochemistry, University of Aarhus, Aarhus, Denmark.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Willnow
authors.affiliation: *Department of Biochemistry, Faculty of Pharmacy, Wrocław Medical University, Wrocław, 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, Wrocław Agricultural University, Wrocław, Poland; Department of Medical Biochemistry, University of Aarhus, Aarhus, Denmark.
authors.forename: John C
authors.initials: JC
authors.lastname: Fyfe
authors.affiliation: *Department of Biochemistry, Faculty of Pharmacy, Wrocław Medical University, Wrocław, 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, Wrocław Agricultural University, Wrocław, Poland; Department of Medical Biochemistry, University of Aarhus, Aarhus, Denmark.
authors.forename: Wojciech
authors.initials: W
authors.lastname: Nowacki
authors.affiliation: *Department of Biochemistry, Faculty of Pharmacy, Wrocław Medical University, Wrocław, 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, Wrocław Agricultural University, Wrocław, Poland; Department of Medical Biochemistry, University of Aarhus, Aarhus, Denmark.
authors.forename: Christian
authors.initials: C
authors.lastname: Jacobsen
authors.affiliation: *Department of Biochemistry, Faculty of Pharmacy, Wrocław Medical University, Wrocław, 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, Wrocław Agricultural University, Wrocław, Poland; Department of Medical Biochemistry, University of Aarhus, Aarhus, Denmark.
authors.forename: Søren K
authors.initials: SK
authors.lastname: Moestrup
authors.affiliation: *Department of Biochemistry, Faculty of Pharmacy, Wrocław Medical University, Wrocław, 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, Wrocław Agricultural University, Wrocław, Poland; Department of Medical Biochemistry, University of Aarhus, Aarhus, Denmark.
authors.forename: Erik I
authors.initials: EI
authors.lastname: Christensen
mesh_terms: D000042:Absorption; D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity; D004285:Dogs; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006454:Hemoglobins / Q000378:metabolism*; D007150:Immunohistochemistry; D007668:Kidney / Q000378:metabolism*; D026561:Low Density Lipoprotein Receptor-Related Protein-2 / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout / Q000235:genetics; D008871:Microvilli / Q000378:metabolism; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism*; D012016:Reference Values; D022161:Transport Vesicles / Q000378:metabolism*
chemical_list: D005452:Fluoresceins; D005456:Fluorescent Dyes; D006454:Hemoglobins; D026561:Low Density Lipoprotein Receptor-Related Protein-2; D011956:Receptors, Cell Surface; C097499:fluorescein carboxylate; C029486:intrinsic factor-cobalamin receptor