Detailed investigations of proximal tubular function in Imerslund-Gräsbeck syndrome.

BACKGROUND: Imerslund-Gräsbeck Syndrome (IGS) is a rare genetic disorder characterised by juvenile megaloblastic anaemia. IGS is caused by mutations in either of the genes encoding the intestinal intrinsic factor-vitamin B12 receptor complex, cubam. The cubam receptor proteins cubilin and amnionless are both expressed in the small intestine as well as the proximal tubules of the kidney and exhibit an interdependent relationship for post-translational processing and trafficking. In the proximal tubules cubilin is involved in the reabsorption of several filtered plasma proteins including vitamin carriers and lipoproteins. Consistent with this, low-molecular-weight proteinuria has been observed in most patients with IGS. The aim of this study was to characterise novel disease-causing mutations and correlate novel and previously reported mutations with the presence of low-molecular-weight proteinuria. METHODS: Genetic screening was performed by direct sequencing of the CUBN and AMN genes and novel identified mutations were characterised by in silico and/or in vitro investigations. Urinary protein excretion was analysed by immunoblotting and high-resolution gel electrophoresis of collected urines from patients and healthy controls to determine renal phenotype. RESULTS: Genetic characterisation of nine IGS patients identified two novel AMN frameshift mutations alongside a frequently reported AMN splice site mutation and two CUBN missense mutations; one novel and one previously reported in Finnish patients. The novel AMN mutations were predicted to result in functionally null AMN alleles with no cell-surface expression of cubilin. Also, the novel CUBN missense mutation was predicted to affect structural integrity of the IF-B12 binding site of cubilin and hereby most likely cubilin cell-surface expression. Analysis of urinary protein excretion in the patients and 20 healthy controls revealed increased urinary excretion of cubilin ligands including apolipoprotein A-I, transferrin, vitamin D-binding protein, and albumin. This was, however, only observed in patients where plasma membrane expression of cubilin was predicted to be perturbed. CONCLUSIONS: In the present study, mutational characterisation of nine IGS patients coupled with analyses of urinary protein excretion provide additional evidence for a correlation between mutation type and presence of the characteristic low-molecular-weight proteinuria.

Renal phenotypic investigations of megalin-deficient patients: novel insights into tubular proteinuria and albumin filtration.

BACKGROUND: The reabsorption of filtered plasma proteins, hormones and vitamins by the renal proximal tubules is vital for body homeostasis. Studies of megalin-deficient mice suggest that the large multi-ligand endocytic receptor megalin plays an essential role in this process. In humans, dysfunctional megalin causes the extremely rare Donnai-Barrow/Facio-Oculo-Acustico-Renal (DB/FOAR) syndrome characterized by a characteristic and multifaceted phenotype including low-molecular-weight proteinuria. In this study, we examined the role of megalin for tubular protein reabsorption in humans through analysis of proximal tubular function in megalin-deficient patients. METHODS: Direct sequencing of the megalin-encoding gene (LRP2) was performed in a family in which three children presented with classical DB/FOAR manifestations. Renal consequences of megalin deficiency were investigated through immunohistochemical analyses of renal biopsy material and immunoblotting of urine samples. RESULTS: In the patients, a characteristic urinary protein profile with increased urinary excretion of vitamin D-binding protein, retinol-binding protein and albumin was associated with absence of, or reduced, proximal tubular endocytic uptake as shown by renal immunohistochemistry. In the absence of tubular uptake, urinary albumin excretion was in the micro-albuminuric range suggesting that limited amounts of albumin are filtered in human glomeruli. CONCLUSIONS: This study demonstrated that megalin plays an essential role for human proximal tubular protein reabsorption and suggests that only limited amounts of albumin is normally filtered in the human glomeruli. Finally, we propose that the characteristic urinary protein profile of DB/FOAR patients may be utilized as a diagnostic marker of megalin dysfunction.

Cubilin is expressed in rat and human glomerular podocytes.

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.

Mouse model of proximal tubule endocytic dysfunction.

BACKGROUND: Several studies have indicated the central role of the megalin/cubilin multiligand endocytic receptor complex in protein reabsorption in the kidney proximal tubule. However, the poor viability of the existing megalin-deficient mice precludes further studies and comparison of homogeneous groups of mice. METHODS: Megalin- and/or cubilin-deficient mice were generated using a conditional Cre-loxP system, where the Cre gene is driven by the Wnt4 promoter. Kidney tissues from the mice were analysed for megalin and cubilin expression by quantitative reverse transcription-polymerase chain reaction, western blotting and immunohistochemistry. Renal albumin uptake was visualized by immunohistochemistry. Twenty-four-hour urine samples were collected in metabolic cages and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and western blotting. Urinary albumin/creatinine ratios were measured by ELISA and the alkaline picrate method. RESULTS: The Meg(lox/lox);Cre(+), Cubn(lox/lox);Cre(+) and Meg(lox/lox), Cubn(lox/lox);Cre(+) mice were all viable, fertile and developed normal kidneys. Megalin and/or cubilin expression, assessed by immunohistology and western blotting, was reduced by >89%. Consistent with this observation, the mice excreted megalin and cubilin ligands such as transferrin and albumin in addition to low-molecular weight proteins. We further show that megalin/cubilin double-deficient mice excrete albumin with an average of 1.45 ± 0.54 mg/day, suggesting a very low albumin concentration in the glomerular ultrafiltrate. CONCLUSIONS: We report here the efficient genetic ablation of megalin, cubilin or both, using a Cre transgene driven by the Wnt4 promoter. The viable megalin/cubilin double-deficient mice now allow for detailed large-scale group analysis, and we anticipate that the mice will be of great value as an animal model for proximal tubulopathies with disrupted endocytosis.

Cubilin is essential for albumin reabsorption in the renal proximal tubule.

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.

Renal proximal tubular dysfunction is a major determinant of urinary connective tissue growth factor excretion.

Connective tissue growth factor (CTGF) plays a key role in renal fibrosis. Urinary CTGF is elevated in various renal diseases and may have biomarker potential. However, it is unknown which processes contribute to elevated urinary CTGF levels. Thus far, urinary CTGF was considered to reflect renal expression. We investigated how tubular dysfunction affects urinary CTGF levels. To study this, we administered recombinant CTGF intravenously to rodents. We used both full-length CTGF and the NH(2)-terminal fragment, since the NH(2)-fragment is the predominant form detected in urine. Renal CTGF extraction, determined by simultaneous arterial and renal vein sampling, was 18 +/- 3% for full-length CTGF and 21 +/- 1% for the NH(2)-fragment. Fractional excretion was very low for both CTGFs (0.02 +/- 0.006% and 0.10 +/- 0.02%, respectively), indicating that >99% of the extracted CTGF was metabolized by the kidney. Immunohistochemistry revealed extensive proximal tubular uptake of CTGF in apical endocytic vesicles and colocalization with megalin. Urinary CTGF was elevated in megalin- and cubilin-deficient mice but not in cubilin-deficient mice. Inhibition of tubular reabsorption by Gelofusine reduced renal uptake of CTGF and increased urinary CTGF. In healthy volunteers, Gelofusine also induced an increase of urinary CTGF excretion, comparable to the increase of beta(2)-microglobulin excretion (r = 0.99). Furthermore, urinary CTGF correlated with beta(2)-microglobulin (r = 0.85) in renal disease patients (n = 108), and only beta(2)-microglobulin emerged as an independent determinant of urinary CTGF. Thus filtered CTGF is normally reabsorbed almost completely in proximal tubules via megalin, and elevated urinary CTGF may largely reflect proximal tubular dysfunction.

Receptor-mediated endocytosis in renal proximal tubule.

Proteins filtered in renal glomeruli are removed from the tubular fluid by endocytosis in the proximal tubule mediated by the two receptors megalin and cubilin. After endocytic uptake, the proteins are transferred to lysosomes for degradation, while the receptors are returned to the apical cell membrane by receptor recycling in dense apical tubules. In the renal proximal tubule, there is no significant transcellular transport of protein. The reabsorptive process is extremely efficient as evidenced by the virtual protein free urine in humans. The two receptors bind a variety of ligands. The process serves not only to remove the proteins from the ultrafiltrate but also to conserve a variety of essential substances such as vitamins and trace elements carried by plasma proteins. The endocytic apparatus is highly developed in the proximal tubule demonstrating the high capacity of the cells; however, under certain circumstances like diseases affecting the glomeruli, the system is overloaded resulting in proteinuria.

Interstitial fibrosis: tubular hypothesis versus glomerular hypothesis.

The pathogenesis of renal interstitial fibrosis leading eventually to renal failure is highly debatable. Whereas the so-called tubular hypothesis, involving an increased tubular uptake of potentially toxic substances that induce a variety of cytokines, growth factors, and profibrogenic factors, is based to a large extent on cell-culture studies, the glomerular hypothesis is based mainly on careful morphological observations. Unraveling the pathways appears to be extremely complex, but in vivo studies appear to offer the most reliable results.

Characterization of a plasma membrane-resident albumin-binding protein associated with the proliferation of estrogen-target, serum-sensitive cells.

Estrogens control the proliferation of their target cells through a receptor-mediated pathway. Recently presented evidence suggests that estradiol cancels the proliferative inhibition exerted by human albumin (HA) and recombinant human albumin (rHA) on estrogen-target serum-sensitive cells (indirect-negative hypothesis). We postulate that this mechanism requires the presence of a plasma membrane estrogen receptor (mER) and a plasma membrane albumin-binding protein (mABP). Direct evidence confirming the presence of mERalpha in MCF7 cells has recently been presented. Herein, we now show that Western blot analysis of purified T47D membrane proteins with the C542 ERalpha specific monoclonal antibody also revealed specific, multiple M(r) mERs (67, 110, and 130k M(r)). In addition, Western blot analysis with an ABP antiserum revealed a potential 60k M(r) ABP in both MCF7 and T47D plasma membrane extracts. No such evidence was observed in similar extracts from ER-negative, serum-insensitive MDA-MB231 cells. Ligand blot analysis of similar plasma membrane extracts with bovine serum albumin confirmed the presence of a 60k M(r) ABP in MCF7 and T47D cells; again, no such evidence was observed in comparable extracts from MDA-MB231 cells. Fluorescence and confocal microscopy of MCF7 cells fixed in 2.0% paraformaldehyde/0.1% glutaraldehyde identified specific membrane ABP antigenic sites by immunocytochemistry. Serum-insensitive MDA-MB231 cells fixed and labeled similarly did not exhibit this mABP. These results suggest that the proposed mABP is expressed only in serum-sensitive estrogen-target cells and is not expressed in cells insensitive to the proliferative inhibition of HA and rHA. Also, the present data suggest that the proposed mABP may be the recognition mechanism by which both HA and rHA inhibit MCF7 and T47D cell proliferation.

[Cubilin: physiopathologic role and relationship with megalin]. / Cubiline: rôle physiopathologique et relations avec la mégaline.

Epithelia which line the renal proximal convoluted tubule, the visceral layer of the yolk sac and the ileum have the ability to internalize a variety of substances which not only serve as nutrients, but may also be transported from one compartment to another. Cubilin, a multiligand receptor, in association with megalin, also a multiligand receptor, appears to be important under both normal and pathological conditions.

Receptor-mediated endocytosis of α-galactosidase A in human podocytes in Fabry disease.

Injury to the glomerular podocyte is a key mechanism in human glomerular disease and podocyte repair is an important therapeutic target. In Fabry disease, podocyte injury is caused by the intracellular accumulation of globotriaosylceramide. This study identifies in the human podocyte three endocytic receptors, mannose 6-phosphate/insulin-like growth II receptor, megalin, and sortilin and demonstrates their drug delivery capabilities for enzyme replacement therapy. Sortilin, a novel α-galactosidase A binding protein, reveals a predominant intracellular expression but also surface expression in the podocyte. The present study provides the rationale for the renal effect of treatment with α-galactosidase A and identifies potential pathways for future non-carbohydrate based drug delivery to the kidney podocyte and other potential affected organs.

Gene expression analysis defines the proximal tubule as the compartment for endocytic receptor-mediated uptake in the Xenopus pronephric kidney.

Endocytic receptors in the proximal tubule of the mammalian kidney are responsible for the reuptake of numerous ligands, including lipoproteins, sterols, vitamin-binding proteins, and hormones, and they can mediate drug-induced nephrotoxicity. In this paper, we report the first evidence indicating that the pronephric kidneys of Xenopus tadpoles are capable of endocytic transport. We establish that the Xenopus genome harbors genes for the known three endocytic receptors megalin/LRP2, cubilin, and amnionless. The Xenopus endocytic receptor genes share extensive synteny with their mammalian counterparts. In situ hybridizations demonstrated that endocytic receptor expression is highly tissue specific, primarily in the pronephric kidney, and did not occur prior to neurulation. Expression was strictly confined to proximal tubules of the pronephric kidney, which closely resembles the situation reported in mammalian kidneys. By immunohistochemistry, we demonstrated that Xenopus pronephric tubule epithelia express high amounts of the endocytic receptors megalin/lrp2 and cubilin in the apical plasma membrane. Furthermore, functional aspects of the endocytic receptors were revealed by the vesicular localization of retinol-binding protein in the proximal tubules, probably representing endocytosed protein. In summary, we provide here the first comprehensive report of endocytic receptor expression, including amnionless, in a nonmammalian species. Remarkably, renal endocytic receptor expression and function in the Xenopus pronephric kidney closely mirrors the situation in the mammalian kidney. The Xenopus pronephric kidney therefore represents a novel, simple model for physiological studies on the molecular mechanisms underlying renal tubular endocytosis.

Megalin-dependent internalization of cadmium-metallothionein and cytotoxicity in cultured renal proximal tubule cells.

Chronic cadmium (Cd2+) exposure results in renal proximal tubular cell damage. Delivery of Cd2+ to the kidney occurs mainly as complexes with metallothionein-1 (molecular mass approximately 7 kDa), freely filtered at the glomerulus. For Cd2+ to gain access to the proximal tubule cells, these complexes are thought to be internalized via receptors for small protein ligands, such as megalin and cubilin, followed by release of Cd2+ from metallothionein-1 in endosomal/lysosomal compartments. To investigate the role of megalin in renal cadmium-metallothionein-1 reabsorption, megalin expression and dependence of cadmium-metallothionein-1 internalization and cytotoxicity on megalin were studied in a renal proximal tubular cell model (WKPT-0293 Cl.2 cells). Expression of megalin was detected by reverse transcriptase-polymerase chain reaction and visualized by immunofluorescence both at the cell surface (live staining) and intracellularly (permeabilized cells). Internalization of Alexa Fluor 488-coupled metallothionein-1 was concentration-dependent, saturating at approximately 15 microM. At 14.3 microM, metallothionein-1 uptake could be significantly attenuated by 30.9 +/- 6.6% (n = 4) by 1 muM of the receptor-associated protein (RAP) used as a competitive inhibitor of cadmium-metallothionein-1 binding to megalin and cubilin. Consistently, cytotoxicity of a 24-h treatment with 7.14 muM cadmium-metallothionein-1 was significantly reduced by 41.0 +/- 7.6%, 61.6 +/- 3.4%, and 26.2 +/- 1.8% (n = 4-5 each) by the presence of 1 microM RAP, 400 microg/ml anti-megalin antibody, or 5 microM of the cubilin-specific ligand, apo-transferrin, respectively. Cubilin expression in proximal tubule cells was also confirmed at the mRNA and protein level. The data indicate that renal proximal tubular cadmium-metallothionein-1 uptake and cell death are mediated at least in part by megalin.

Contribution of cubilin and amnionless to processing and membrane targeting of cubilin-amnionless complex.

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.

Pathophysiology of cubilin: of rats, dogs and men.

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.

Megalin and cubilin, role in proximal tubule function and during development.

Epithelia lining the proximal convoluted epithelium and the visceral yolk sac in rodents are characterized by their ability to internalize and degrade many proteins facing their apical pole. It has become apparent over the last decade that this property is mediated through a very high endocytic activity and the expression of at least two distinct multiligand receptors, cubilin and megalin, characterized by the accumulation of ligand-binding units, LDL receptor class A repeats and CUB domains, respectively. We review here evidence that both receptors are involved in proximal tubule function and embryonic development.

Receptor-associated protein is important for normal processing of megalin in kidney proximal tubules.

The receptor-associated protein (RAP) has been identified as a chaperone regulating the expression and processing of the LDL receptor-related protein. RAP also binds to the related 600-kD multiligand endocytic receptor megalin expressed in many absorptive epithelia including renal proximal tubule. The present study examines the effect of RAP gene disruption on megalin expression and subcellular distribution in the proximal tubule as well as the effect on tubular protein reabsorption. It is shown that RAP is important for the normal expression and function of megalin. Megalin expression was reduced to approximately 23% estimated by immunoblotting and supported by immunocytochemistry and by the amount of megalin recovered by RAP affinity chromatography. Light- and electron microscope immunocytochemistry as well as analyses on separated membrane fractions showed significant changes in the subcellular distribution of megalin. A significant reduction in the normal brush border labeling was observed in association with increased labeling of rough endoplasmic reticulum and the smooth paramembranous endoplasmic reticulum along the basolateral membranes. RAP deficiency was associated with changes in urinary protein composition, enabling the identification of alpha-amylase as a new ligand for megalin. In addition, an increased excretion of vitamin D-binding protein, a recently identified ligand to megalin, was observed supporting changes in tubular protein reabsorption. The present data show that RAP is of crucial importance for normal processing and function of megalin, suggesting a chaperone-like function of this protein in the kidney proximal tubule.

The tandem endocytic receptors megalin and cubilin are important proteins in renal pathology.

The molecular mechanisms controlling proximal tubule reabsorption of proteins have been much elucidated in recent years. Megalin and cubilin constitute two important endocytic receptor proteins involved in this process. Although structurally very different the two receptor proteins interact to mediate the reabsorption of a large number of filtered proteins, including carrier proteins important for transport and cellular uptake of several vitamins, lipids and other nutrients. Dysfunction of either protein results in tubular proteinuria and is associated with specific changes in vitamin metabolism due to the defective proximal tubular reabsorption of carrier proteins. Additional focus on the two receptors is attracted by the possible pathogenic role of excessive tubular protein uptake during conditions of increased filtration of proteins, and by recent findings implicating members of the low density lipoprotein-receptor family, which includes megalin, in the transduction of signals by association with cytoplasmic proteins.

Megalin mediates renal uptake of heavy metal metallothionein complexes.

Although several heavy metal toxins are delivered to the kidney on the carrier protein metallothionein (MT), uncertainty as to how MT enters proximal tubular cells limits treatment strategies. Prompted by reports that MT-I interferes with renal uptake of the megalin ligand beta(2)-microglobulin in conscious rats, we tested the hypothesis that megalin binds MT and mediates its uptake. Three lines of evidence suggest that binding of MT to megalin is critical in renal proximal tubular uptake of MT-bound heavy metals. First, MT binds megalin, but not cubilin, in direct surface plasmon resonance studies. Binding of MT occurs at a single site with a K(d) approximately 10(-4) and, as with other megalin ligands, depends on divalent cations. Second, antisera and various known megalin ligands inhibit the uptake of fluorescently labeled MT in model cell systems. Anti-megalin antisera, but not control sera, displace >90% bound MT from rat renal brush-border membranes. Megalin ligands including beta(2)-microglobulin and also recombinant MT fragments compete for uptake by megalin-expressing rat yolk sac BN-16 cells. Third, megalin and fluorescently labeled MT colocalize in BN-16 cells, as shown by fluorescent microscopic techniques. Follow-up surface plasmon resonance and flow cytometry studies using overlapping MT peptides and recombinant MT fragments identify the hinge SCKKSCC region of MT as a critical site for megalin binding. These findings suggest that disruption of the SCKKSCC motif can inhibit proximal tubular MT uptake and thereby eliminate much of the renal accumulation and toxicity of heavy metals such as cadmium, gold, copper, and cisplatinum.