1. Cloning of Megalin
As a result of a search for an etiologic antigen of Heymann nephritis, which is a model for experimental membranous nephropathy, Kerjaschki, D. and Farquhar, M. G. identified a cell membrane protein, gp330, in 1982 (Kerjaschki D., Farquhar M. G., 1982, Proc. Natl. Acad. Sci. U.S.A., 79, 5557-5561). In 1994, Saito, A. et al. determined the complete primary structure of a rat gp330 and designated it as megalin, because it was the largest cloned cell membrane protein of a vertebrate (Saito A. et al., 1994, Proc. Natl. Acad. Sci. U.S.A., 91, 9725-9729).
2. Megalin-Expressing Site
Megalin is also known as glycoprotein 330 (gp330) or low-density lipoprotein (LDL) receptor-related protein 2 (LRP-2). It is a glycoprotein having a molecular weight of about 600 kDa, which is expressed in kidney proximal tubule epithelial cells, other tissues and cells, such as type II alveolar cells, spermary, uterine endometrium, placenta, or inner ear epithelium, renal epithelium, germo-vitellarium, and neural ectoderm (see Christensen E. I., Willnow, T. E., 1999, J. Am. Soc. Nephrol. 10, 2224-2236; Juhlin C., Klareskog L. et al., 1990, J. Biol. Chem. 265, 8275-8279; and Zheng G, McCluskey R. T. et al., 1994, J. Histochem. Cytochem. 42, 531-542). In the kidney, megalin functions as an endocytosis receptor associated with endocytosis and reabsorption of proteins and the like in the proximal tubule prior to urinary excretion. The reabsorbed proteins and the like are then degraded by lysosomes (see Mausbach A. B., Christensen E. I., 1992, Handbook of Physiology: Renal Physiology, Windhager, editor, New York, Oxford University Press, 42-207).
3. Nucleotide Sequence of Megalin
Megalin is a glycoprotein that is the most frequently expressed on the kidney proximal tubule epithelial membrane of a mammalian animal. The cDNA-encoding sequence thereof has nucleotide identity with the human megalin cDNA sequence having gene accession number U04441 disclosed in Korenberg, J. R. et al. (1994) or the human megalin cDNA sequence having gene accession number U33837 disclosed in Hjacln, G., et al. (1996) (see Korenberg J. R. et al., 1994, Genomics 22, 88-93; and Hjalm G. et al., 1996, Eur. J. Biochem. 239, 132-137).
Also, rat megalin having homology with human megalin has been discovered by Saito et al. (1994), and the cDNA sequence thereof having gene accession number L34049 has already been disclosed (see Saito A. et al., 1994, Proc. Natl. Acad. Sci. U.S.A., 91, 9725-9729).
4. Amino Acid Sequence and Protein Structure of Megalin
Megalin is a gigantic cell membrane protein consisting of 4,655 amino acids (in the case of human megalin) and 4,660 amino acids (in the case of rat megalin). The molecular weight deduced based on the amino acid sequence is about 520 kDa, and it can be as great as about 600 kDa, when including a sugar chain (see Saito A. et al., 1994, Proc. Natl. Acad. Sci. U.S.A., 91, 9725-9729). Megalin belongs to the LDL receptor gene family, a gigantic extracellular region thereof has four functional domains, and the extracellular region is connected to a thin intracellular region through a single transmembrane region. Megalin is mainly present in a clathrin-coated pit on the glomerulus (rat) or the epithelial luminal membrane (luminal and basal membrane in the glomerular epithelial cell) of the proximal tubule, type II alveolar cell, epididymal glands, thyroid glands, accessory thyroid glands, yolk sac membrane, inner ear, small intestine, or chorioidea, and it is associated with intake of various ligands into the cells and metabolism thereof (see Farquhar M. G. et al., 1995, J. Am. Soc. Nephrol. 6, 35-47; and Christensen E. I. et al., 2002, Nat. Rev. Mol. Cell. Biol. 3, 256-266). Low-molecular-weight proteinuria, bone metabolism disorders, respiratory failure, malformation of brain, and other disorders occur in megalin-knockout mice (see Willnow T. E. et al., 1996, Proc. Natl. Acad. Sci. U.S.A., 93, 8460-8464). A megalin homolog is also present in nematodes (C. elegans), and the biological importance thereof has been suggested (see Yochem J. et al., 1993, Proc. Natl. Acad. Sci. U.S.A., 90, 4572-4576).
5. Importance of Megalin as a Cause of Nephritis
Megalin, which is a major etiologic antigen of experimental membranous nephropathy (Heymann nephritis), is an epithelial scavenger receptor, and biological and pathological roles thereof have been elucidated. Animal models have been used for a long time in order to elucidate the mechanism of human membranous nephropathy development, and rat Heymann nephritis is a model of membranous nephropathy. The analysis of Heymann nephritis has been more advanced than that of any other model. Saito A. et al. disclosed the results of analysis of the pathological epitope and the ligand-binding domain of Heymann nephritis, and they have also demonstrated the major antigen region of megalin and a functional domain of megalin that mainly contribute to binding to a ligand (see Kerjaschki D. et al., 1992, Proc. Natl. Acad. Sci. U.S.A., 89, 11179-11183; Saito A., Farquhar M. G. et al., 1996, Proc. Natl. Acad. Sci. U.S.A., 93, 8601-8605; Yamazaki H., Farquhar M. G. et al., 1998, J. Am. Soc. Nephrol. 9, 1638-1644; and Orlando R. A., Farquhar M. G. et al., 1997, Proc. Natl. Acad. Sci. U.S.A., 94, 2368-2373).
6. Various Ligands of Megalin
Megalin is expressed most abundantly on the luminal side of the proximal tubule epithelial cells in vivo. In human kidney, megalin expression is not observed at sites other than the proximal tubule epithelial cells, including at glomeruli. Megalin incorporates various ligands (e.g., a low-molecular-weight protein or drugs) that are filtered by glomeruli into cells via endocytosis, megalin transports them to lysosomes, and they reappear on the cell surface via recycling (see Farquhar M. G. et al., 1995, J. Am. Soc. Nephrol. 6, 35-47; and Christensen E. I. et al., 2002, Nat. Rev. Mol. Cell. Biol. 3, 256-266). Also, megalin is associated with transcytosis from the luminal side to the basal membrane side. Megalin is also associated with intake and metabolism of binding proteins, such as vitamins A, B12, and D (see Christensen E. I. et al., 2002, Nat. Rev. Mol. Cell. Biol. 3, 256-266). Christensen and Willnow demonstrated that megalin mediates reabsorption of three vitamin carrier proteins, vitamin D binding proteins (DBP), retinol binding protein (RBP), and transcobalamin (TC) and vitamins associated therewith; i.e., (OH) vitamin 25D3, vitamin A (retinol), and vitamin B12 (see Christensen E. I., Willnow T. E., 1999, J. Am. Soc. Nephrol. 10, 2224-2236). Saito A. et al. demonstrated that leptin, which is secreted from adipocytes and increase in the blood of obese patients, is incorporated into and metabolized by the proximal tubule epithelial cells as the megalin ligand (see Saito A., Gejyo F. et al., 2004, Endocrinology. 145, 3935-3940). The adipocytes, that is, accumulated visceral fats, result in combined pathological conditions, i.e., metabolic syndrome. Leptin, which is an adipocytokine secreted from adipocytes, increases in the blood of a metabolic syndrome patient. It is suggested that the kidney is the organ in which leptin in the blood is most likely to accumulate and that leptin plays a nephropathic role (see Tarzi R. M. Lord G. M. et al., 2004, Am. J. Pathol. 164, 385-390). A so-called leptin receptor is also found in a region between the proximal tubule and the collecting tubule located downstream of the megalin functioning region.
The term “metabolic syndrome” is defined as a disease complication of visceral obesity, elevated blood pressure, hyperlipidemia, impaired glucose tolerance, and other symptoms, the primary risk factor of which is insulin resistance. Such conditions are highly likely to lead to development of arteriosclerotic diseases and proteinuria, and may result in the development of nephropathy with glomerulus and renal tubular hypertrophy as histological features. When such a case is combined with apparent diabetes, the feature of hyperglycemia is further developed, diabetic nephropathy is manifested, and the disease conditions may further become serious. Type II diabetes is basically preceded by or simultaneously develops with metabolic syndrome. Accordingly, the feature of nephropathy could be included as nephropathy associated with metabolic syndrome.
Saito A. et al. have conducted an experiment using rat yolk sac epithelium-derived cells (L2 cells) in which megalin is expressed at high levels and found that incorporation of 125I-labelled AGE (advanced glycation end products) (derived from glucose) into L2 cells would be significantly inhibited by an anti-megalin antibody. Thus, they demonstrated that megalin is associated with a pathway for such incorporation (see Saito A. Gejyo F. et al., 2003, J. Am. Soc. Nephrol. 14, 1123-1131). As a mechanism of diabetic nephropathy development, association of advanced glycation end products (AGE) with glycated and modified proteins by the Maillard reaction has been pointed out. A low-molecular-weight AGE in the blood is filtered by glomeruli, and it is reabsorbed and metabolized by the proximal tubule epithelial cells. If nephropathy further advances, a higher-molecular weight AGE also is filtered by glomeruli, accumulates in the proximal tubule epithelial cells, and imposes excessive metabolic loads. Further, Saito A. et al. also demonstrate that megalin is also associated with incorporation of AGE derived from methylglyoxal, glyceraldehyde, or glycolaldehyde into cells, in addition to glucose. Also, metabolic syndrome is often complicated with hepatopathy, such as fatty liver. Liver type fatty acid binding proteins (L-FABP) that are abundantly present in the liver are released into the blood of a healthy person. In case of hepatopathy, more L-FABP is released into and increased in the blood. Saito A. et al. have also demonstrated that L-FABP in the blood is rapidly filtered by glomeruli and it is reabsorbed by the proximal tubule epithelial cells via megalin (see Takeda T., Gejyo F., Saito A. et al., 2005, Lab. Invest. 85, 522-531).
7. Functional Protein that Interacts with Megalin
In order to elucidate the mechanism of megalin transportation in cells, adaptor molecules that bind to megalin intracellular domains are searched for, and various proteins, such as Dab2, ANKRA, MAGI-1, GAIP, GIPC, Galphai3, MegBP, and ARH, have been identified (see Oleinikov A. V. et al., 2000, Biochem. J. 347, 613-621; Rader K., Farquhar M. G. et al., 2000, J. Am. Soc. Nephrol. 11, 2167-2178; Patrie K. M., Margolis B. et al., 2001, J. Am. Soc. Nephrol. 12, 667-677; Lou X., Farquhar M. G. et al., 2002, J. Am, Soc. Nephrol. 13, 918-927; Petersen H. H., Willnow T. E., 2003, J. Cell. Sci. 116, 453-461; and Takeda T., Farquhar M. G. et al., 2003, Mol. Biol. Cell. 14, 4984-4996). Through such molecules, megalin is associated with endocytosis or transcytosis, and megalin is also associated with signal transmission related thereto. Also, megalin functions conjugatively with a cell membrane receptor, i.e., cubilin, in the proximal tubule epithelial cells, so as to be further involved with incorporation of various ligands into cells (see Saito A. et al., 1994, Proc. Natl. Acad. Sci. U.S.A., 91, 9725-9729). For example, cubilin is a receptor that directly binds to transferrin, albumin, endogenous vitamin B12, or the like, and megalin is indirectly involved with endocytosis thereof. Also, megalin is known to interact with the Na+—H+ exchanger isoform 3 (NHE3) in the proximal tubule epithelial cells (see Biemesderfer D. et al., 1999, J. Biol. Chem. 274, 17518-17524). NHE3 is an antiporter that plays an important role in reabsorption of Na+, and NHE3 also influences incorporation of a ligand by megalin (see Hryciw D. H. et al., 2004, Clin. Exp. Pharmacol. Physiol. 31, 372-379). Also, megalin may be involved with inactivation and metabolism of NHE3. At an early stage of diabetic nephropathy or metabolic syndrome-related nephropathy, glomerular filtration becomes excessive. Enhanced reabsorption of Na+ of the proximal tubule is deduced to be a primary cause (see Vallon V. et al., 2003, J. Am. Soc. Nephrol. 14, 530-537), NHE3 plays a key role in such a case, and inactivation and metabolism of MHE3 by megalin is considered to be involved therewith (see Hryciw D. H. et al., 2004, Clin. Exp. Pharmacol. Physiol. 31, 372-379).
8. Correlation of Urinary Excretion of Megalin and Urinary Excretion of Ligand by Megalin
Leheste et al. disclosed that megalin-knockout mice and Fanconi syndrome patients with weakened proximal tubule functions would experience increased excretion of proteins and retinol in the urine (see Leheste J. et al., 1999, Am. J. Pathol. 155, 1361-1370). Further, Moestrup S. K. et al. demonstrated that the amount of megalin excreted in urine of patients of Fanconi syndrome is significantly lower than that excreted by healthy individuals. This causes deterioration of megalin functions and expression in the proximal tubule and consequently increases the amount of glomerular-filtered proteins containing retinol-binding proteins excreted in urine (see Anthony G. W., Moestrup S. K. et al., 2002, J. Am. Soc. Nephrol. 13, 125-133).
9. Importance of Megalin Function Found by Experiments Using Models For Uremia and Models for Organ Regeneration
As described above, megalin is involved with intake of various low-molecular-weight proteins into the proximal tubule epithelial cells and metabolism thereof. When the pathological condition advances to kidney failure, the mechanism of the metabolism is disturbed, and low-molecular-weight proteins are consequently accumulated in the blood and tissues as uremic proteins. A representative example thereof is β2-microglobulin (β2-m), which may cause dialysis-related amyloidosis in a long-term dialysis patient (see Gejyo F., Schmid K. et al., 1985, Biochem. Biophys. Res. Commun. 129, 701-706). The aforementioned AGE is also suggested as a cause of arteriosclerosis or organ failure due to its accumulation in the blood of patients with kidney failure or dialysis, and AGE is considered as a type of uremic protein (see Henle T., Miyata T., 2003, Adv. Ren. Replace Ther. 10, 321-331). Further, leptin accumulates in the blood of a dialysis patient and thus is considered to be involved with malnutrition or immunity compromise. Tabata Y. and Gejyo F. et al. disclosed the effects and effectiveness of models for metabolizing uremic protein using megalin functions (see Saito A., Tabata Y., Gejyo F. et al., 2003, J. Am. Soc. Nephrol. 14, 2025-2032 and WO 02/091955). That is, megalin-expressing cells are transplanted as scaffold proteins in vivo, and low-molecular-weight proteins leaked from peripheral blood vessels (newborn blood vessels) are incorporated into the cells with the aid of megalin for metabolization. The megalin-expressing cells used for transplantation (i.e., yolk sac epithelium-derived L2 cells) incorporate and metabolize β2-m with the aid of megalin (see Saito A., Tabata Y., Gejyo F. et al., 2003, J. Am. Soc. Nephrol. 14, 2025-2032). Both kidneys of a nude mouse into which L2 cells had been subcutaneously transplanted were removed, the condition of kidney failure was induced, and cell incorporation in the tissue mass into which 125I-labeled β2-m had been transplanted and in organs via intraperitoneal injection was measured. As a result, the cell mass into which L2 cells had been transplanted was found to more significantly incorporate 125I-labeled β2-m compared with other organs, and the 125I-labeled β2-m clearance was found to significantly advance in a group to which L2 cells had been transplanted, compared with a control group into which L2 cells had not been transplanted (see Saito A., Tabata Y., Gejyo F. et al., 2003, J. Am. Soc. Nephrol. 14, 2025-2032).
10. Proteolysis and Urinary Excretion of Megalin
In recent years, the possibility of megalin being subjected to proteolysis in a Notch-like signaling pathway has been suggested (see Zou Z., Biemesderfer D. et al., 2004, J. Biol. Chem. 279, 34302-34310; and Grigorenko A. P. et al., 2004. Proc. Natl. Acad. Sci. U.S.A., 101, 14955-14960). This also includes a two-step cleavage system of shedding of an ectodomain mediated by metalloprotease and intramembrane proteolysis mediated by gamma-secretase.
Also, megalin is known to express in the type II alveolar cell.
Thus, megalin has been extensively studied in respect of its correlation with the metabolism in organs such as the kidney. However, the correlation between diseases of organs, including the kidney, and megalin has not yet been elucidated, and expression of megalin or excretion thereof to the body fluid in connection with a variety of organ diseases has not yet been studied.
To date, a method involving tissue staining or Western blotting using a polyclonal antibody obtained by immunizing an immune animal, such as a rabbit, has been known as a method for detecting megalin.
This technique, however, involves staining of a cell or a protein separated via electrophoresis, and this necessitates very complicated procedures and a long period of time for immobilizing tissues, preparing tissue slices, electrophoresis, and transfer onto the membrane. Thus, it is difficult to quantify megalin.
From the viewpoint of diagnosis of the degrees of functional disorders of tissues or organs, particularly in the case of kidney disorders, there is no effective means for diagnosing kidney tubule failure in a specific and simple manner. At present, many methods of diagnosis that detect albumin, creatinine. β2-microglobulin, L-FABP, or the like in urine or blood as a diagnosis marker for renal diseases have been employed. Such diagnosis markers, however, are not derived from kidney tissue, and they merely result from all phenomenon and functions during filtration in kidney glomeruli and reabsorption in kidney tubules. That is, it is difficult to identify glomerulus failure and failure of kidney tubule failure in the kidney even with the use of such marker. Also, such marker is an indirect marker derived from organs other than the kidney. Thus, effectiveness is poor for early diagnosis of a disease. The same applies to KL-6 (markers of acute inflammation), which is an existing diagnosis marker for lung diseases, and particularly for inflammation.
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