Albumin And Megalin You Need To Know

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Part Ⅱ：Renal Handling of Albumin—From Early Findings to Current Concepts

Jakub Gburek, Bogusława Konopska and Krzysztof Goł ˛ab

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3.3. Cubilin

Cubilin was identified as the albumin receptor by Birn et al. in 2000 [64]. The authors isolated cubilin from the membranes of rat kidney brush-border using affinity chromatography with immobilized albumin. They also determined the dissociation constant of the complex by surface plasmon resonance(Kd=0.63 uM) and demonstrated that inbred dogs with a functional defect of cubilin and mice with inhibited megalin expression excrete significant amounts of albumin in the urine. Moreover, they also indicated the mutual interaction of both receptors in albumin endocytosis.

Cubilin is an extracellular glycoprotein with a molecular weight of about 460 kDa. Initially, it was identified as the antigen of teratogenic antibodies produced in rabbits after injection of renal brush border. Originally known as glycoprotein 280 (gp280), it was located in the proximal renal tubules and the yolk sac epithelium. Its expression was also found in the ileum, uterus, and placenta. Initially, no ligands were attributed to this protein. Later studies showed that cubilin was identical to the receptor of the intrinsic factor-vitamin B12 (IF-B12)complex present in the small intestine. The function of a receptor protein was assigned to it [65]. The human cubilin gene was located on the 10p12.33-p13 chromosome. This protein is composed of a 110 amino acid N-terminal part, followed by 8 epidermal growth-factor-like(EGF-like) domains and 27 domains similar to complement C1r/C1s, epidermal growth-factor-related sea urchin protein (Uegf), and bone morphogenic protein1(CUB). Cubilin does not have a transmembrane domain. The N-terminus is responsible for protein embedding in the membrane. Each CUB domain consists of 110 amino acid residues. The structure of CUB domains consists of two layers of five antiparallel β-sheets also connected by β-structures. They constitute the most conservative area of the molecule and are responsible for ligand binding. In addition to albumin and IF-B12, iron-containing proteins(transferrin, hemoglobin, myoglobin), apolipoprotein Al and vitamin D binding protein may also be bound 56,59,66,67I. Analysis of albumin binding to in vitro expressed cubilin fragments made it possible to identify the binding sites within CUB domains 6-8 [68]. These sites are likely to partially overlap with the binding sites of hemoglobin as demonstrated in a competition study using HK-2 cells (human kidney proximal tubule, adult male, papilloma immortalized) [69].

Cubilin is not a fully independent endocytic receptor. For its proper membrane localization and stability, it must interact with the AMN protein. In addition, this protein is responsible for cubilin internalization. Recently, it was demonstrated that the FXNPXF sequence in the AMN cytoplasmic domain is functionally active and mediates the sequestration of the complex by interacting with adaptor proteins: the disabled protein-2 (Dab2) and autosomal recessive cholesterolemia protein(ARH). CUBAM internalization may also occur through association with megalin [55,70]. Megalin seems to be responsible for cubilin maintaining the cell membrane. Simultaneous shedding of cubilin from the membrane and its loss with urine was found in a patient with the Donnai-Barrow/FOAR syndrome (Facio-Oculo-Acustico-Renal Syndrome) due to a functional mutation in this receptor gene leading to its cytoplasmic retention [71].

The main role of CUBAM in tubular albumin uptake was demonstrated in recent studies in mice with renal-specific knockout of cubilin and megalin, in which moderate tubular albuminuria was observed [54,72]. This observation was also confirmed by the genetic and phenotypic analysis of patients with Imerslund-Gräsbeck syndrome， caused by a mutation in the cubilin or AMN gene. In both cases, albumin and other ligands of cubilin were lost in the urine [58].

Interestingly, cubilin and megalin have been also detected in rat and human glomeruli according to a pattern similar to that reported for megalin. Cubilin revealed both surface and intracellular expression in the podocytes [73]. Moreover, cubilin mRNA expression was detected in mesangial cells, but the absence of amnion implied that cubilin is not fully functional in these cells. Nevertheless, albumin endocytosis was significantly impaired under inducible megalin knockdown conditions in stably transduced mesangial cells |74]. These findings may be important for a better understanding of glomeruli pathies.

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3.4. Megalin

Megalin was identified as the albumin receptor by Cui et al. in 1996 [75]. The study was performed in vivo by injecting proximal tubules of rats with albumin solutions labeled with colloidal gold or 125Iiodine isotope. In the experiment, the influence of many compounds on the tubular uptake of these proteins was determined. Administration of receptor-associated protein(RAP), cytochrome C reduced and gentamicin completely inhibited tubular reabsorption of labeled albumin. These ligands are characterized by a high affinity for megalin, and their presence effectively hampers the binding of albumin through this receptor. The same applied to EDTA, a chelator of Ca2+ ions which also inhibited the uptake. This indicated that megalin contributes to the tubular reabsorption of this protein. These conclusions were confirmed by affinity chromatography on a column with immobilized megalin.

Megalin was originally discovered as an antigen in Heymann-type membranous nephropathy and called glycoprotein 330(gp 330). In 1994, after a successful cloning attempt, its structure was better known, and the name was changed to the currently used one [76. The human megalin gene was located on chromosome 2g24-g31. Megalin is a large transmembrane protein that belongs to the family of low-density lipoprotein receptors. In proximal tubules, megalin is found in the apical membrane and clathrin-coated endocytic vesicles. This protein has a molecular weight of 600 kDa and is made up of about 4600 amino acid residues. The megalin molecule contains a large N-terminal extracellular domain, a single transmembrane domain and a short C-terminal cytoplasmic fragment. The extracellular domain has four areas rich in cysteine and is responsible for ligands binding. Several repeats of sequence similar to epidermal growth factor(EGF)and a cysteine-poor fragment containing YWTD motif separate the ligand-binding region from the embedding portion. The signaling sequence is responsible for the dissociation of the ligand in the acidic endosome environment. The cytoplasmic part contains three NPXY motifs that initiate the process of albumin endocytosis by concentrating receptors in coated pits via the disabled protein-2(Dab2) adapter protein [77]. Recruitment of this adapter is in turn dependent on phosphorylation catalyzed by protein kinases B(Aktl and Akt2) [78,79].

Megalin also has the ability to bind many other proteins. Besides albumin, its ligands include inter alia: apolipoproteins(B, E, HD), vitamin transporting proteins (A, D, B12), hormones (angiotensin I, epithelial growth factor, leptin), enzymes and their inhibitors (lysozyme, plasminogen) and others [80]. In the case of albumin, the binding sites on the receptor molecule have not been mapped yet. It also internalizes the CUBAM complex and its ligands, including albumin. The complexes formed are internalized with the formation of early endosomes. After acidification of the endosome environment, the complex dissociates and a free receptor is recycled to the cell surface via dense apical tubules [59,66,67,81].

It is worth mentioning that high glucose regulates megalin-mediated albumin endocytosis in renal proximal tubule cells. Elevated levels of plasma glucose inhibited the megalin expression and consequently albumin endocytosis [61].

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3.5. Neonatal Fc Receptor of Immunoglobulin Fragment

Recent studies on renal albumin absorption indicate the important role of neonatal Fc receptor(FcRn) in the process of albumin recovering from the filtrate and protecting this protein against intracellular degradation. FcRn is a heterodimeric transmembrane protein similar in its structure to major histocompatibility complex(MHC) class I molecules. It is common in endothelial and epithelial cells. Its presence was confirmed in glomerular podocytes and in proximal tubules. It has the ability to independently bind albumin and immunoglobulins IgG in an acidic environment (pH 5-6). An increase in the pH value above7 results in the release of ligands from the receptor [82]. FcRn in podocytes is present in vesicles belonging to the acidic endosomal compartment and is responsible for the binding of immunoglobulins and albumin that passed through the glomerular capillary endothelium to the filtration space. The filtered proteins are absorbed by the podocyte through endocytosis and/or pinocytosis and then bound by FcRn at the acidic pH of the endosomes. Endocytic vesicles laden with proteins undergo transcytosis toward the lumen of the Bowman's capsule. In conditions of increasing pH, the protein-receptor complex is dissociated, and the intact protein is released from the cell to the primary filtrate. This process is supposed to protect narrow filtration slits from clogging with large proteins [83,84]. Part of the albumin, filtered and reabsorbed in the proximal tubules, is not a subject of degradation in the lysosomes of the tubular epithelium and is most likely recovered in the transcytosis process. In this theoretical model, FcRn binds albumin, which is released from its receptors, presumably the megalin/cubilin complex in the acidic endosome environment, and then is directed through transcytosis to the basal site of tubular epithelial cells, where it is released into the interstitial tissue as a consequence of increasing pH [85]. Although in the light of current research results, the recovery of albumin by transcytosis involving FcRn in proximal tubules takes place, this is a marginal phenomenon for normal albumin homeostasis in the body [56].

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