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Renata Kozyraki - One of the best experts on this subject based on the ideXlab platform.
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loss of Cubilin the intrinsic factor vitamin b12 receptor impairs visceral endoderm endocytosis and endodermal patterning in the mouse
Scientific Reports, 2019Co-Authors: Olivier Cases, Aitana Pereagomez, Vincent Lelievre, Maria V Pulina, Jerome Collignon, Annakaterina Hadjantonakis, Renata KozyrakiAbstract:The visceral endoderm is a polarized epithelial monolayer necessary for early embryonic development in rodents. A key feature of this epithelium is an active endocytosis and degradation of maternal nutrients, in addition to being the source of various signaling molecules or inhibitors required for the differentiation and patterning of adjacent embryonic tissues. Endocytosis across the visceral endoderm epithelium involves specific cell surface receptors and an extensive sub-membrane vesicular system with numerous apical vacuoles/lysosomes. We previously reported that Cubilin, the endocytic receptor for intrinsic factor-vitamin B12, albumin and apolipoproteinA-I/HDL allows maternal nutrient uptake by the visceral endoderm. In the present study, we show that the germline ablation of Cubilin impairs endodermal and mesodermal patterning, and results in developmental arrest at gastrulation. Notably, visceral endoderm dispersal is impeded in Cubilin null embryos. We further confirm the essential role of Cubilin in nutrient internalization by the early visceral endoderm and highlight its involvement in the formation of apical vacuoles. Our results reveal essential roles for Cubilin in early embryonic development, and suggest that in addition to its nutritive function, Cubilin sustains signaling pathways involved in embryonic differentiation and patterning.
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Cubilin the intrinsic factor vitamin b12 receptor in development and disease
Current Medicinal Chemistry, 2018Co-Authors: Renata Kozyraki, Olivier CasesAbstract:: Gp280/Intrinsic factor-vitamin B12 receptor/Cubilin (CUBN) is a large endocytic receptor serving multiple functions in vitamin B12 homeostasis, renal reabsorption of protein or toxic substances including albumin, vitamin D-binding protein or cadmium.Cubilin is a peripheral membrane protein consisting of 8 epidermal growth factor (EGF)-like repeats and 27 CUB (defined as Complement C1r/C1s, Uegf, BMP1) domains. This structurally unique protein interacts with at least two molecular partners, amnionless (AMN) and Lrp2/Megalin. AMN is involved in appropriate plasma membrane transport of Cubilin whereas Lrp2 is essential for efficient internalization of Cubilin and its ligands. Observations gleaned from animal models with Cubn deficiency or human diseases demonstrate the importance of this protein. In this review addressed to basic research and medical scientists we summarize currently available data on Cubilin and its implication in renal and intestinal biology. We also discuss the role of Cubilin as a modulator of Fgf8 signaling during embryonic development and propose that the Cubilin-Fgf8 interaction may be relevant in human pathology, including in cancer progression, heart or neural tube defects. We finally provide experimental elements suggesting that some aspects of Cubilin physiology might be relevant in drug design.
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tubular proteinuria in patients with hnf1α mutations hnf1α drives endocytosis in the proximal tubule
Kidney International, 2016Co-Authors: Sara Terryn, Erik Ilsø Christensen, Renata Kozyraki, Patrick Van Der Smissen, Karo Tanaka, Jeanphilippe Lengele, Eric Olinger, Daniele Duboislaforgue, Serge Garbay, Pierre J CourtoyAbstract:Hepatocyte nuclear factor 1α (HNF1α) is a transcription factor expressed in the liver, pancreas, and proximal tubule of the kidney. Mutations of HNF1α cause an autosomal dominant form of diabetes mellitus (MODY-HNF1A) and tubular dysfunction. To gain insights into the role of HNF1α in the proximal tubule, we analyzed Hnf1a- deficient mice. Compared with wild-type littermates, Hnf1a knockout mice showed low-molecular-weight proteinuria and a 70% decrease in the uptake of β 2 -microglobulin, indicating a major endocytic defect due to decreased expression of megalin/Cubilin receptors. We identified several binding sites for HNF1α in promoters of Lrp2 and Cubn genes encoding megalin and Cubilin, respectively. The functional interaction of HNF1α with these promoters was shown in C33 epithelial cells lacking endogenous HNF1α. Defective receptor-mediated endocytosis was confirmed in proximal tubule cells from these knockout mice and could be rescued by transfection of wild-type but not mutant HNF1α. Transfection of human proximal tubule HK2 cells with HNF1α was able to upregulate megalin and Cubilin expression and to increase endocytosis of albumin. Low-molecular-weight proteinuria was consistently detected in individuals with HNF1A mutations compared with healthy controls and patients with non–MODY-HNF1A diabetes mellitus. Thus, HNF1α plays a key role in the constitutive expression of megalin and Cubilin, hence regulating endocytosis in the proximal tubule of the kidney. These findings provide new insight into the renal phenotype of individuals with mutations of HNF1A .
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Cubilin is essential for albumin reabsorption in the renal proximal tubule
Journal of The American Society of Nephrology, 2010Co-Authors: Sabine Amsellem, Jakub Gburek, Erik Ilsø Christensen, Rikke Nielsen, Pierre J Verroust, Ghislaine Hamard, Thomas E Willnow, Olivier Devuyst, Ebba Nexo, Renata KozyrakiAbstract:Receptor-mediated endocytosis is responsible for protein reabsorption in the proximal tubule. This process involves two interacting receptors, megalin and Cubilin, which form a complex with amnionless. Whether these proteins function in parallel or as part of an integrated system is not well understood. Here, we report the renal effects of genetic ablation of Cubilin, with or without concomitant ablation of megalin, using a conditional Cre-loxP system. We observed that proximal tubule cells did not localize amnionless to the plasma membrane in the absence of Cubilin, indicating a mutual dependency of Cubilin and amnionless to form a functional membrane receptor complex. The Cubilin-amnionless complex mediated internalization of intrinsic factor-vitamin B12 complexes, but megalin considerably increased the uptake. Furthermore, Cubilin-deficient mice exhibited markedly decreased uptake of albumin by proximal tubule cells and resultant albuminuria. Inactivation of both megalin and Cubilin did not increase albuminuria, indicating that the main role of megalin in albumin reabsorption is to drive the internalization of Cubilin-albumin complexes. In contrast, cubulin deficiency did not affect urinary tubular uptake or excretion of vitamin D-binding protein (DBP), which binds Cubilin and megalin. In addition, we observed Cubilin-independent reabsorption of the "specific" Cubilin ligands transferrin, CC16, and apoA-I, suggesting a role for megalin and perhaps other receptors in their reabsorption. In summary, with regard to albumin, Cubilin is essential for its reabsorption by proximal tubule cells, and megalin drives internalization of Cubilin-albumin complexes. These genetic models will allow further analysis of protein trafficking in the progression of proteinuric renal diseases.
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multiligand endocytosis and congenital defects roles of Cubilin megalin and amnionless
Current Pharmaceutical Design, 2007Co-Authors: Renata Kozyraki, Francoise GofflotAbstract:Cubilin and megalin are multiligand receptors that mediate uptake of extracellular ligands. Their function has extensively been studied in the kidney where they play a key role in vitamin B12 and vitamin D homeostasis. Amnionless is a plasma membrane protein that binds to Cubilin in various epithelia; the interaction Cubilin-amnionless in the gut is crucial for dietary vitamin B12 uptake. Studies in patients with gene defects in these receptors, and animal models with inactivated Cubilin, megalin or amnionless suggest an important role in embryonic development and normal growth. In this review we will summarize recent data on the biological function of these receptors and focus on their implication in embryonic nutrition and central nervous system malformations.
Soren K Moestrup - One of the best experts on this subject based on the ideXlab platform.
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decreased expression of megalin and Cubilin and altered mitochondrial activity in tenofovir nephrotoxicity
Human Pathology, 2018Co-Authors: Soren K Moestrup, Alexandre Cez, Isabelle Brocheriou, F X Lescure, Clovis Adam, Pierremarie Girard, G Pialoux, Soraya Fellahi, Jeanphilippe BastardAbstract:Summary Tenofovir disoproxil fumarate (TDF) is a commonly used antiretroviral drug for HIV, rarely causing Fanconi syndrome and acute kidney injury. We retrospectively analyzed the clinico pathological presentation of 20 cases of tenofovir-induced tubulopathy, and investigated the renal expression of the megalin and Cubilin proteins, as well as the mitochondrial respiratory chain activity. Estimated glomerular filtration rate (eGFR) before TDF exposure was 92 ml/min/1.73m 2 , decreasing to 27.5 ml/min/1.73m 2 at the time of biopsy, with 30% of patients requiring renal replacement therapy. Proximal tubular expression of megalin and Cubilin was altered in 19 and 18 cases, respectively, whereas it was preserved in patients exposed to TDF without proximal tubular dysfunction and in HIV-negative patients with acute tubular necrosis. Loss of megalin/Cubilin was correlated with low eGFR and high urine retinol binding protein at the time of biopsy, low eGFR at last follow-up, and was more severe in patients with multifactorial toxicity. Patients with additional nephrotoxic conditions promoting tenofovir accumulation showed a lower eGFR at presentation and at last follow-up, and more severe lesions of acute tubular necrosis, than those with isolated tenofovir toxicity. Altered mitochondrial COX activity in proximal tubules was observed and may be an early cellular alteration in tenofovir nephrotoxicity. In conclusion, altered megalin/Cubilin expression represents a distinctive feature in tenofovir-induced tubulopathy, and its severity is correlated with urine retinol binding protein loss and is associated with a poor renal prognosis. Concomitant exposure to other nephrotoxic conditions severely impacts the renal presentation and outcome.
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contribution of Cubilin and amnionless to processing and membrane targeting of Cubilin amnionless complex
Journal of The American Society of Nephrology, 2005Co-Authors: Gwenaelle Coudroy, Jakub Gburek, Mette Madsen, Renata Kozyraki, Soren K Moestrup, Germain Trugnan, P Verroust, Michele MauriceAbstract:Cubilin is a peripheral apical membrane receptor for multiple ligands that are taken up in several absorptive epithelia. Recently, amnionless (AMN) was identified to form a functional receptor complex with Cubilin. By expression in transfected polarized MDCK cells of AMN and several Cubilin fragments, including a functional “mini” version of Cubilin, the processing, sorting, and membrane anchoring of the complex to the apical membrane were investigated. The results show that truncation mutants, including the N-terminal domain of Cubilin, did not appear at the plasma membrane but instead were retained in the endoplasmic reticulum or partially secreted into the medium. Coexpression with AMN led to efficient transport to the apical cell surface of the Cubilin constructs, which included the EGF domains, and prevented release into the medium. AMN co-precipitated with Cubilin and co-localized with Cubilin at the apical cell surface. Apical sorting was observed for a broad set of nonoverlapping Cubilin fragments without the N-terminal region, in the absence of AMN. The preference for apical sorting disappeared when glycosylation was inhibited by tunicamycin. In conclusion, it is shown that both units contribute to the processing of the Cubilin–AMN complex to the apical membrane: AMN interacts with the EGF domains of Cubilin and is responsible for membrane attachment and export of the complex from the endoplasmic reticulum, whereas the extracellular Cubilin molecule is responsible for apical sorting of the complex in a carbohydrate-dependent manner.
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Contribution of Cubilin and Amnionless to Processing and Membrane Targeting of Cubilin–Amnionless Complex
Journal of The American Society of Nephrology, 2005Co-Authors: Gwenaelle Coudroy, Jakub Gburek, Mette Madsen, Pierre J Verroust, Renata Kozyraki, Soren K Moestrup, Germain Trugnan, Michele MauriceAbstract:Cubilin is a peripheral apical membrane receptor for multiple ligands that are taken up in several absorptive epithelia. Recently, amnionless (AMN) was identified to form a functional receptor complex with Cubilin. By expression in transfected polarized MDCK cells of AMN and several Cubilin fragments, including a functional “mini” version of Cubilin, the processing, sorting, and membrane anchoring of the complex to the apical membrane were investigated. The results show that truncation mutants, including the N-terminal domain of Cubilin, did not appear at the plasma membrane but instead were retained in the endoplasmic reticulum or partially secreted into the medium. Coexpression with AMN led to efficient transport to the apical cell surface of the Cubilin constructs, which included the EGF domains, and prevented release into the medium. AMN co-precipitated with Cubilin and co-localized with Cubilin at the apical cell surface. Apical sorting was observed for a broad set of nonoverlapping Cubilin fragments without the N-terminal region, in the absence of AMN. The preference for apical sorting disappeared when glycosylation was inhibited by tunicamycin. In conclusion, it is shown that both units contribute to the processing of the Cubilin–AMN complex to the apical membrane: AMN interacts with the EGF domains of Cubilin and is responsible for membrane attachment and export of the complex from the endoplasmic reticulum, whereas the extracellular Cubilin molecule is responsible for apical sorting of the complex in a carbohydrate-dependent manner.
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the functional cobalamin vitamin b12 intrinsic factor receptor is a novel complex of Cubilin and amnionless
Blood, 2004Co-Authors: John C Fyfe, Erik Ilsø Christensen, Mette Madsen, Peter Hojrup, Stephan M Tanner, Albert De La Chapelle, Quianchuan He, Soren K MoestrupAbstract:Imerslund-Grasbeck syndrome (I-GS, megaloblastic anemia 1) is an autosomal recessive disorder characterized by intestinal cobalamin (vitamin B 12 ) malabsorption and proteinuria. I-GS–causing mutations are found in either of 2 genes encoding the epithelial proteins: Cubilin and amnionless (AMN). Cubilin recognizes intrinsic factor (IF)–cobalamin and various other proteins to be endocytosed in the intestine and kidney, respectively, whereas the function of AMN is unknown. Here we show that Cubilin and AMN colocalize in the endocytic apparatus of polarized epithelial cells and copurify as a tight complex during IF-cobalamin affinity and nondenaturing gel filtration chromatography. In transfected cells expressing either AMN or a truncated IF-cobalamin–binding Cubilin construct, neither protein alone conferred ligand endocytosis. In Cubilin transfectants, Cubilin accumulated in early biosynthetic compartments. However, in cells cotransfected with AMN and the Cubilin construct, Cubilin trafficked to the cell surface and endosomes, and the cells exhibited IF-cobalamin endocytosis and lysosomal degradation of IF. These data indicate that Cubilin and AMN are subunits of a novel Cubilin/AMN (cubam) complex, where AMN binds to the amino-terminal third of Cubilin and directs subcellular localization and endocytosis of Cubilin with its ligand. Therefore, mutations affecting either of the 2 proteins may abrogate function of the cubam complex and cause IG-S.
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megalin and Cubilin are endocytic receptors involved in renal clearance of hemoglobin
Journal of The American Society of Nephrology, 2002Co-Authors: Jakub Gburek, Thomas E Willnow, John C Fyfe, Soren K Moestrup, P Verroust, Christian Jacobsen, Wojciech Nowacki, Erik Ilsø ChristensenAbstract: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 μM for megalin and 4.1 μM 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 β 2 -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.
P Verroust - One of the best experts on this subject based on the ideXlab platform.
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Cubilin is expressed in rat and human glomerular podocytes
Nephrology Dialysis Transplantation, 2012Co-Authors: Thaneas Prabakaran, Erik Ilsø Christensen, Rikke Nielsen, P VerroustAbstract:Background. The bulk of proteins filtered in the glomeruli are reabsorbed in the proximal tubule by endocytosis mediated by two multiligand receptors operating in concert, megalin and Cubilin. Podocytes can also internalize protein and megalin; this was initially reported in rat proximal tubular and glomerular epithelial cells and has recently also been demonstrated in human podocytes. Cubilin, crucial for albumin reabsorption in the proximal tubule, has not been identified in glomerular epithelial cells. Methods. In the present study, we used immunocytochemistry and reverse transcription-polymerase chain reaction on laser-captured glomeruli to demonstrate synthesis and expression of Cubilin in rat and human glomeruli. In parallel experiments, the expression of Cubilin was studied in cultured podocytes. Results. This study identifies Cubilin in rat and human glomeruli according to a pattern similar to that reported for megalin. Cubilin revealed a surface expression but also intracellular expression in the podocytes. Conclusion. Our findings show that the podocytes display the two endocytic receptors which are responsible for the only documented process for protein reabsorption in proximal tubule cells.
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overlapping expression patterns of the multiligand endocytic receptors Cubilin and megalin in the cns sensory organs and developing epithelia of the rodent embryo
Gene Expression Patterns, 2005Co-Authors: Emeline Assemat, P Verroust, Francois Chatelet, Olivier Cases, Jacqueline Chandellier, Frederic Commo, Renata KozyrakiAbstract:Abstract Cubilin and megalin are multiligand epithelial endocytic receptors well characterized in the adult kidney and ileum where they form a complex essential for protein, lipid and vitamin uptake. Although inactivation of the megalin gene leads to holoprosencephaly and administration of anti-Cubilin antibodies induces fetal resorptions or cranio-facial malformations their function in the developing embryo remains unclear. We recently showed that both proteins are strongly expressed by the maternal–fetal interfaces and the neuroepithelium of the early rodent embryo where they co-localize and form a complex important for nutrient uptake. The aim of the present study was the further investigation of Cubilin expression at later developmental stages of the rodent embryo and its correlation to that of megalin. Immunohistochemical and in situ hybridization analysis showed striking similarities in the spatial and temporal expression patterns of Cubilin and megalin. The electrophoretic mobility of both proteins was identical to that of the adult as revealed by Western blot analysis. Cubilin and megalin were strongly expressed in the sensory organs, the central nervous system, the respiratory and urogenital tracts as well as in the thymus, parathyroids and thyroid. In each site, the expression mainly concerned epithelial structures and correlated with the onset of epithelial induction. Depending on the site, a decreased or restricted expression was observed by the end of the gestation for both proteins.
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contribution of Cubilin and amnionless to processing and membrane targeting of Cubilin amnionless complex
Journal of The American Society of Nephrology, 2005Co-Authors: Gwenaelle Coudroy, Jakub Gburek, Mette Madsen, Renata Kozyraki, Soren K Moestrup, Germain Trugnan, P Verroust, Michele MauriceAbstract:Cubilin is a peripheral apical membrane receptor for multiple ligands that are taken up in several absorptive epithelia. Recently, amnionless (AMN) was identified to form a functional receptor complex with Cubilin. By expression in transfected polarized MDCK cells of AMN and several Cubilin fragments, including a functional “mini” version of Cubilin, the processing, sorting, and membrane anchoring of the complex to the apical membrane were investigated. The results show that truncation mutants, including the N-terminal domain of Cubilin, did not appear at the plasma membrane but instead were retained in the endoplasmic reticulum or partially secreted into the medium. Coexpression with AMN led to efficient transport to the apical cell surface of the Cubilin constructs, which included the EGF domains, and prevented release into the medium. AMN co-precipitated with Cubilin and co-localized with Cubilin at the apical cell surface. Apical sorting was observed for a broad set of nonoverlapping Cubilin fragments without the N-terminal region, in the absence of AMN. The preference for apical sorting disappeared when glycosylation was inhibited by tunicamycin. In conclusion, it is shown that both units contribute to the processing of the Cubilin–AMN complex to the apical membrane: AMN interacts with the EGF domains of Cubilin and is responsible for membrane attachment and export of the complex from the endoplasmic reticulum, whereas the extracellular Cubilin molecule is responsible for apical sorting of the complex in a carbohydrate-dependent manner.
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expression and role of Cubilin in the internalization of nutrients during the peri implantation development of the rodent embryo
Biology of Reproduction, 2005Co-Authors: Emeline Assemat, P Verroust, Stephanie Vinot, Francoise Gofflot, Patrick Linselnitschke, Francoise Illien, Francois Chatelet, Sophie Louvetvallee, Franz Rinninger, Renata KozyrakiAbstract:Histiotrophic nutrition is essential during the peri-implantation development in rodents, but little is known about receptors involved in protein and lipid endocytosis derived from the endometrium and the uterine glands. Previous studies suggested that Cubilin, a multiligand receptor for vitamin, iron, and protein uptake in the adult, might be important in this process, but the onset of its expression and function is not known. In this study, we analyzed the expression of Cubilin in the pre- and early post-implantation rodent embryo and tested its potential function in protein and cholesterol uptake. Using morphological and Western blot analysis, we showed that Cubilin first appeared at the eight-cell stage. It was expressed by the maternal-fetal interfaces, trophectoderm and visceral endoderm, but also by the future neuroepithelial cells and the developing neural tube. At all these sites, Cubilin was localized at the apical pole of the cells exposed to the maternal environment or to the amniotic and neural tube cavities, and had a very similar distribution to megalin, a member of the LDLR gene family and a coreceptor for Cubilin in adult tissues. To analyze Cubilin function, we followed endocytosis of apolipoprotein A-I and HDL cholesterol, nutrients normally present in the uterine glands and essential for embryonic growth. We showed that internalization of both ligands was Cubilin dependent during the early rodent gestation. In conclusion, the early Cubilin expression and its function in protein and cholesterol uptake suggest an important role for Cubilin in the development of the peri-implantation embryo.
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pathophysiology of Cubilin of rats dogs and men
Nephrology Dialysis Transplantation, 2002Co-Authors: P VerroustAbstract:At variance from small solutes which are reabsorbed by transporters and channels, proteins present in the proximal convoluted tubule lumen are internalized by endocytosis via a vesicular system, transferred to the lysosomes and degraded. We summarize here recent data on Cubilin, a 460 kDa peripheral protein which, together with megalin, serves as a multiligand receptor providing for the reabsorption of most proteins present in the tubule lumen. Interestingly, Cubilin is also expressed by the yolk sac, which has endocytic and degradative properties reminiscent of the proximal tubule and serves as the receptor for intrinsic factor-vitamin B12 complexes.
Erik Ilsø Christensen - One of the best experts on this subject based on the ideXlab platform.
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megalin dependent urinary cystatin c excretion in ischemic kidney injury in rats
PLOS ONE, 2017Co-Authors: Erik Ilsø Christensen, Henrik Birn, Rikke Nielsen, Danny Jensen, Casper Kierulflassen, Marie Louise Vindvad Kristensen, Rikke Norregaard, Kathrin WeyerAbstract:Background Cystatin C, a marker of kidney injury, is freely filtered in the glomeruli and reabsorbed by the proximal tubules. Megalin and Cubilin are endocytic receptors essential for reabsorption of most filtered proteins. This study examines the role of these receptors for the uptake and excretion of cystatin C and explores the effect of renal ischemia/reperfusion injury on renal cystatin C uptake and excretion in a rat model. Methods Binding of cystatin C to megalin and Cubilin was analyzed by surface plasmon resonance analysis. ELISA and/or immunoblotting and immunohistochemistry were used to study the urinary excretion and tubular uptake of endogenous cystatin C in mice. Furthermore, renal uptake and urinary excretion of cystatin C was investigated in rats exposed to ischemia/reperfusion injury. Results A high affinity binding of cystatin C to megalin and Cubilin was identified. Megalin deficient mice revealed an increased urinary excretion of cystatin C associated with defective uptake by endocytosis. In rats exposed to ischemia/reperfusion injury urinary cystatin C excretion was increased and associated with a focal decrease in proximal tubule endocytosis with no apparent change in megalin expression. Conclusions Megalin is essential for the normal tubular recovery of endogenous cystatin C. The increase in urinary cystatin C excretion after ischemia/reperfusion injury is associated with decreased tubular uptake but not with reduced megalin expression.
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tubular proteinuria in patients with hnf1α mutations hnf1α drives endocytosis in the proximal tubule
Kidney International, 2016Co-Authors: Sara Terryn, Erik Ilsø Christensen, Renata Kozyraki, Patrick Van Der Smissen, Karo Tanaka, Jeanphilippe Lengele, Eric Olinger, Daniele Duboislaforgue, Serge Garbay, Pierre J CourtoyAbstract:Hepatocyte nuclear factor 1α (HNF1α) is a transcription factor expressed in the liver, pancreas, and proximal tubule of the kidney. Mutations of HNF1α cause an autosomal dominant form of diabetes mellitus (MODY-HNF1A) and tubular dysfunction. To gain insights into the role of HNF1α in the proximal tubule, we analyzed Hnf1a- deficient mice. Compared with wild-type littermates, Hnf1a knockout mice showed low-molecular-weight proteinuria and a 70% decrease in the uptake of β 2 -microglobulin, indicating a major endocytic defect due to decreased expression of megalin/Cubilin receptors. We identified several binding sites for HNF1α in promoters of Lrp2 and Cubn genes encoding megalin and Cubilin, respectively. The functional interaction of HNF1α with these promoters was shown in C33 epithelial cells lacking endogenous HNF1α. Defective receptor-mediated endocytosis was confirmed in proximal tubule cells from these knockout mice and could be rescued by transfection of wild-type but not mutant HNF1α. Transfection of human proximal tubule HK2 cells with HNF1α was able to upregulate megalin and Cubilin expression and to increase endocytosis of albumin. Low-molecular-weight proteinuria was consistently detected in individuals with HNF1A mutations compared with healthy controls and patients with non–MODY-HNF1A diabetes mellitus. Thus, HNF1α plays a key role in the constitutive expression of megalin and Cubilin, hence regulating endocytosis in the proximal tubule of the kidney. These findings provide new insight into the renal phenotype of individuals with mutations of HNF1A .
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from bowel to kidneys the role of Cubilin in physiology and disease
Nephrology Dialysis Transplantation, 2013Co-Authors: Erik Ilsø Christensen, Rikke Nielsen, Henrik BirnAbstract:: Cubilin is a large endocytic receptor serving such diverse functions as the intestinal absorption of the intrinsic factor-B(12) complex and the renal proximal tubule reabsorption of filtered proteins including albumin, transferrin, vitamin D-binding protein and other important plasma carriers. Cubilin is a structurally unique, peripheral membrane protein, which depends on the membrane protein amnionless (AMN) for correct apical translocation. In addition, AMN appears important for efficient internalization of intrinsic factor-B(12) in the intestine, whereas in the proximal tubule Cubilin interacts with another endocytic receptor, megalin, for effective reabsorption. The importance of Cubilin has been demonstrated in several animal models of Cubilin deficiency as well as in a variety of human diseases. Recent demonstration of Cubilin in podocytes from various species awaits further clarification with respect to the functional role as well as its role in pathology.
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renal uptake of 99mtc dimercaptosuccinic acid is dependent on normal proximal tubule receptor mediated endocytosis
The Journal of Nuclear Medicine, 2013Co-Authors: Kathrin Weyer, Erik Ilsø Christensen, Henrik Birn, Rikke Nielsen, Steen V Petersen, Michael RehlingAbstract:UNLABELLED: (99m)Tc-labeled dimercaptosuccinic acid ((99m)Tc-DMSA) accumulates in the kidney cortex and is widely used for imaging of the renal parenchyma. Despite its extensive clinical use, the mechanism for renal targeting of the tracer is unresolved. Megalin and Cubilin are cooperating receptors essential to the proximal tubule endocytic uptake of proteins from the glomerular ultrafiltrate. We have used megalin/Cubilin-deficient mice produced by gene knockout to determine whether receptor-mediated endocytosis is responsible for the renal uptake of (99m)Tc-DMSA. METHODS: Control or megalin/Cubilin-deficient mice were injected intravenously with 0.5 MBq of (99m)Tc-DMSA or (99m)Tc-mercaptoacetyltriglycine (MAG3). Whole-body scintigrams and the activity in plasma, urine, and the kidneys were examined 6 h after injection. The size and identity of (99m)Tc-DMSA-bound proteins in urine were analyzed by fractionation by centrifugation and separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by autoradiography and mass spectrometry. RESULTS: No renal accumulation of (99m)Tc-DMSA was identified in scintigrams of megalin/Cubilin-deficient mice. The renal accumulated activity of the tracer was reduced to 11.4% (± 2.5%, n = 7) of the normal uptake in control mice, correlating with a reduction in renal megalin/Cubilin expression in knockout mice to about 10% of normal. The reduced renal uptake in megalin/Cubilin-deficient mice was accompanied by an increase in the urinary excretion of (99m)Tc-DMSA. Size separation of the urine by ultracentrifugation and sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated that in megalin/Cubilin-deficient mice an increased amount of (99m)Tc-DMSA was excreted in an approximately 27-kDa form, which by mass spectrometry was identified as the plasma protein α1-microglobulin, an established megalin/Cubilin ligand. CONCLUSION: (99m)Tc-DMSA is filtered bound to α1-microglobulin and accumulates in the kidneys by megalin/Cubilin-mediated endocytosis of the (99m)Tc-DMSA protein complex. Renal accumulation of (99m)Tc-DMSA is thus critically dependent on megalin/Cubilin receptor function and therefore is a marker of proximal tubule endocytic activity.
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Cubilin is expressed in rat and human glomerular podocytes
Nephrology Dialysis Transplantation, 2012Co-Authors: Thaneas Prabakaran, Erik Ilsø Christensen, Rikke Nielsen, P VerroustAbstract:Background. The bulk of proteins filtered in the glomeruli are reabsorbed in the proximal tubule by endocytosis mediated by two multiligand receptors operating in concert, megalin and Cubilin. Podocytes can also internalize protein and megalin; this was initially reported in rat proximal tubular and glomerular epithelial cells and has recently also been demonstrated in human podocytes. Cubilin, crucial for albumin reabsorption in the proximal tubule, has not been identified in glomerular epithelial cells. Methods. In the present study, we used immunocytochemistry and reverse transcription-polymerase chain reaction on laser-captured glomeruli to demonstrate synthesis and expression of Cubilin in rat and human glomeruli. In parallel experiments, the expression of Cubilin was studied in cultured podocytes. Results. This study identifies Cubilin in rat and human glomeruli according to a pattern similar to that reported for megalin. Cubilin revealed a surface expression but also intracellular expression in the podocytes. Conclusion. Our findings show that the podocytes display the two endocytic receptors which are responsible for the only documented process for protein reabsorption in proximal tubule cells.
Christian Jacobsen - One of the best experts on this subject based on the ideXlab platform.
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Cubilin and megalin co localize in the neonatal inner ear
Audiology and Neuro-otology, 2009Co-Authors: Jacob Tauris, Erik Ilsø Christensen, Christian Jacobsen, Anders Nykjaer, Claus Munck Petersen, Therese OvesenAbstract:Cubilin and megalin are multifunctional endocytotic receptors expressed in many absorptive epithelia. The receptors have separate functions but may act in concert in several tissues including the smal
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megalin and Cubilin are endocytic receptors involved in renal clearance of hemoglobin
Journal of The American Society of Nephrology, 2002Co-Authors: Jakub Gburek, Thomas E Willnow, John C Fyfe, Soren K Moestrup, P Verroust, Christian Jacobsen, Wojciech Nowacki, Erik Ilsø ChristensenAbstract: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 μM for megalin and 4.1 μM 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 β 2 -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.
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Cubilin dysfunction causes abnormal metabolism of the steroid hormone 25 oh vitamin d 3
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Anders Nykjaer, Renata Kozyraki, John C Fyfe, Albert De La Chapelle, P Verroust, Christian Jacobsen, Jorg Robert Leheste, Morten Nielsen, Maria Aminoff, Soren K MoestrupAbstract:Steroid hormones are central regulators of a variety of biological processes. According to the free hormone hypothesis, steroids enter target cells by passive diffusion. However, recently we demonstrated that 25(OH) vitamin D3 complexed to its plasma carrier, the vitamin D-binding protein, enters renal proximal tubules by receptor-mediated endocytosis. Knockout mice lacking the endocytic receptor megalin lose 25(OH) vitamin D3 in the urine and develop bone disease. Here, we report that Cubilin, a membrane-associated protein colocalizing with megalin, facilitates the endocytic process by sequestering steroid–carrier complexes on the cellular surface before megalin-mediated internalization of the Cubilin-bound ligand. Dogs with an inherited disorder affecting Cubilin biosynthesis exhibit abnormal vitamin D metabolism. Similarly, human patients with mutations causing Cubilin dysfunction exhibit urinary excretion of 25(OH) vitamin D3. This observation identifies spontaneous mutations in an endocytic receptor pathway affecting cellular uptake and metabolism of a steroid hormone.
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megalin dependent Cubilin mediated endocytosis is a major pathway for the apical uptake of transferrin in polarized epithelia
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Renata Kozyraki, Jakub Gburek, Erik Ilsø Christensen, Thomas E Willnow, John C Fyfe, P Verroust, Christian Jacobsen, Alice Dautryvarsat, Soren K MoestrupAbstract:Cubilin is a 460-kDa protein functioning as an endocytic receptor for intrinsic factor vitamin B12 complex in the intestine and as a receptor for apolipoprotein A1 and albumin reabsorption in the kidney proximal tubules and the yolk sac. In the present study, we report the identification of Cubilin as a novel transferrin (Tf) receptor involved in catabolism of Tf. Consistent with a Cubilin-mediated endocytosis of Tf in the kidney, lysosomes of human, dog, and mouse renal proximal tubules strongly accumulate Tf, whereas no Tf is detectable in the endocytic apparatus of the renal tubule epithelium of dogs with deficient surface expression of Cubilin. As a consequence, these dogs excrete increased amounts of Tf in the urine. Mice with deficient synthesis of megalin, the putative coreceptor colocalizing with Cubilin, also excrete high amounts of Tf and fail to internalize Tf in their proximal tubules. However, in contrast to the dogs with the defective Cubilin expression, the megalin-deficient mice accumulate Tf on the luminal Cubilin-expressing surface of the proximal tubule epithelium. This observation indicates that megalin deficiency causes failure in internalization of the Cubilin–ligand complex. The megalin-dependent, Cubilin-mediated endocytosis of Tf and the potential of the receptors thereby to facilitate iron uptake were further confirmed by analyzing the uptake of 125I- and 59Fe-labeled Tf in cultured yolk sac cells.
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Cubilin p1297l mutation associated with hereditary megaloblastic anemia 1 causes impaired recognition of intrinsic factor vitamin b12 by Cubilin
Blood, 2000Co-Authors: Mette Kristiansen, Albert De La Chapelle, P Verroust, Christian Jacobsen, Maria Aminoff, Ralf Krahe, Soren K MoestrupAbstract:Megaloblastic anemia 1 (MGA1) is an autosomal recessive disorder caused by the selective intestinal malabsorption of intrinsic factor (IF) and vitamin B12/cobalamin (Cbl) in complex. Most Finnish patients with MGA1 carry the disease-specific P1297L mutation (FM1) in the IF-B12 receptor, Cubilin. By site-directed mutagenesis, mammalian expression, and functional comparison of the purified wild-type and FM1 mutant forms of the IF–Cbl-binding Cubilin region (CUB domains 5-8, amino acid 928-1386), we have investigated the functional implications of the P1297L mutation. Surface plasmon resonance analysis revealed that the P1297L substitution specifically increases the K d for IF–Cbl binding several-fold, largely by decreasing the association rate constant. In agreement with the binding data, the wild-type protein, but not the FM1 mutant protein, potently inhibits 37°C uptake of iodine 125–IF–Cbl in Cubilin-expressing epithelial cells. In conclusion, the data presented show a substantial loss in affinity of the FM1 mutant form of the IF–Cbl binding region of Cubilin. This now explains the malabsorption of Cbl and Cbl-dependent anemia in MGA1 patients with the FM1 mutation.