Megalin Expression Research Articles

Receptor-mediated Endocytosis in Renal Tubules and its Dysfunction Induced by Drugs.

In the kidney, various compounds in the plasma are filtrated through glomeruli. Though the glomerular filtration of many plasma proteins is restricted because of their large molecular weights, still significant amount of proteins is filtered. The proteins filtered are reabsorbed in the proximal tubular cells almost completely. The mechanism involved in protein reabsorption is receptor-mediated endocytosis. Not only proteins but also drugs such as aminoglycoside antibiotics are taken up by receptor-mediated endocytosis in renal proximal tubules. Megalin is a large (ca. 600 kDa) endocytic receptor, which is abundantly expressed in renal proximal tubules, and recognizes various compounds such as lipoprotein lipase, aprotinin, transcobalamin-vitamin B12, apolipoprotein J/clusterin, RAP (receptor-associated protein), 25-(OH) vitamin D3-vitamin D binding protein complex as substrates. Albumin, a major plasma protein, was initially reported as a substrate for megalin, but recently the involvement of another protein, cubilin, in albumin endocytosis was indicated. Namely, albumin binds to cubilin, and albumin-cubilin complex binds to megalin for endocytosis; the hypothesis which should be tested further. Megalin is also suggested to be the receptor for endocytosis of aminoglycosides. We have studied the possible role of megalin in in vivo renal accumulation of aminoglycosides. The positive relationship between megalin expression and amikacin accumulation in renal cortex, either under normal or disease condition, strongly suggests that megalin acts as an endocytic receptor for aminoglycosides. There are two important functional proteins for endosomal acidification, which is important for overall endocytic process; these are vacuolar H+-ATPase (V-H+-ATPase) and chloride channel, ClC-5. Cisplatin, an anticancer drug, often induces nephrotoxicity, which is accompanied by albumin(protein) uria. In order to clarify the mechanisms underlying albuminuria by cisplatin, we examined the effect of cisplatin treatment on V-H+-ATPase activity in the kidney. Proton transporting activity as well as ATP hydrolysis activity of V-H+-ATPase was markedly inhibited by cisplatin treatment. Similarly, receptormediated endocytosis of albumin was inhibited by cisplatin in OK cells and in vivo in rats. Reabsorption of a megalin substrate, vitamin D binding protein, was also inhibited and excreted into the urine. These findings suggest that albumin (protein) uria induced by cisplatin would be due to inhibition of V-H+-ATPase. Receptor-mediated endocytosis in the kidney is physiologically and pathophisiologically important process. Also, understanding the receptor-mediated endocytosis would help to consider drug delivery systems, especially those for proteins and genes with large molecular weights.

Cubilin- and megalin-mediated uptake of albumin in cultured proximal tubule cells of opossum kidney

Reabsorption of albumin from the glomerular filtrate occurs via receptor-mediated endocytosis in the proximal tubule. This process is initiated by binding of albumin in apical clathrin-coated pits, followed by endocytosis and degradation in lysosomes. Although binding sites have been characterized by kinetic studies, the receptors responsible for the binding of albumin have not been fully identified. Two giant glycoproteins, cubilin and megalin, constitute important endocytic receptors localized to the kidney proximal tubule. In the present study, we examined the colocalization of cubilin and megalin in the endocytic pathway and the relationship between the uptake of albumin and the expression of cubilin and megalin in opossum kidney (OK) proximal tubule cells by immunocytochemistry and immunoblotting. OK cells expressed both cubilin and megalin. The light microscope labeling patterns for cubilin and megalin were almost identical and were mainly located at the surface area of the cells. Cubilin and megalin were also shown to colocalize on cell surface microvilli, in coated pits, and in endocytic compartments at the electron microscope level. Endocytosed bovine serum albumin (BSA) was identified exclusively in cells expressing megalin and cubilin. Uptake of BSA-FITC was saturable and inhibited by receptor-associated protein (RAP) and by intrinsic factor-vitamin B12 complex (IF-B12) at high concentrations. Significant inhibition was also observed by specific antibodies to cubilin, and megalin and cubilin antisense oligonucleotides likewise significantly reduced albumin uptake. Egg albumin did not affect the uptake of BSA. The present observations suggest that the two receptors cubilin and megalin are both involved in the endocytic uptake of albumin in renal proximal tubule cells.

Megalin-Mediated Transcytosis of Thyroglobulin by Thyroid Cells is a Calmodulin-Dependent Process

Megalin, a multiligand receptor expressed on the apical surface of thyroid cells, mediates transepithelial transport (transcytosis) of thyroglobulin (Tg) across thyrocytes, resulting in diversion of Tg from the lysosomal pathway and reduction of the extent of thyroid hormone release from internalized Tg molecules. The calcium regulatory protein calmodulin facilitates some forms of transcytosis. Here we investigated the role of calmodulin in megalin-mediated transcytosis of Tg by thyroid cells. For this purpose, we studied the effect of calmodulin antagonists on Tg transcytosis by Fisher rat thyroid cells (FRTL-5), an established, differentiated thyroid cell line. FRTL-5 cells were cultured on permeable filters in the upper chamber of dual chambered devices, with megalin expression exclusively on the upper surface. Unlabeled Tg was added to the upper chamber at 37 degrees C, and transcytosed Tg was detected by enzyme-linked immunosorbent assay (ELISA) in fluids collected 1 hour later from the lower chamber. To study the role of calmodulin in Tg transcytosis, cells were preincubated with one of two calmodulin antagonists, either trifluoperazine or W7. Both antagonists markedly reduced transcytosis of Tg by FRTL-5 cells. These inhibitory effects and those of a monoclonal antimegalin antibody were not additive, indicating that calmodulin acts on the megalin-mediated pathway. Furthermore, trifluoperazine increased the extent of triiodothyronine (T3) release from exogenously added Tg by FRTL-5 cells, indicating that Tg transported in the calmodulin-dependent, megalin-mediated pathway, bypasses the lysosomal pathway.

Megalin Acts in Concert with Cubilin to Mediate Endocytosis of High Density Lipoproteins

Cubilin has recently been shown to function as an endocytic receptor for high density lipoproteins (HDL). The lack of apparent transmembrane and cytoplasmic domains in cubilin raises questions as to the means by which it can mediate endocytosis. Since cubilin has been reported to bind the endocytic receptor megalin, we explored the possibility that megalin acts in conjunction with cubilin to mediate HDL endocytosis. While megalin did not bind to HDL, delipidated HDL, or apoA-I, it was found to copurify with cubilin isolated by HDL-Sepharose affinity chromatography. Cubilin and megalin exhibited coincident patterns of mRNA expression in mouse tissues including the kidney, ileum, thymus, placenta, and yolk sac endoderm. The expression of both receptors in yolk sac endoderm-like cells was inducible by retinoic acid treatment but not by conditions of sterol depletion. Suppression of megalin activity or expression by treatment with either megalin antibodies or megalin antisense oligodeoxynucleotides resulted in inhibition of cubilin-mediated endocytosis of HDL. Furthermore, megalin antisense oligodeoxynucleotide treatment resulted in reduced cell surface expression of cubilin. These data demonstrate that megalin acts together with cubilin to mediate HDL endocytosis and further suggest that megalin may play a role in the intracellular trafficking of cubilin.

Role of Megalin (gp330) in Transcytosis of Thyroglobulin by Thyroid Cells: A NOVEL FUNCTION IN THE CONTROL OF THYROID HORMONE RELEASE

When thyroglobulin (Tg) is endocytosed by thyrocytes and transported to lysosomes, thyroid hormones (T4 and T3) are released. However, some internalized Tg is transcytosed intact into the bloodstream, thereby avoiding proteolytic cleavage. Here we show that megalin (gp330), a Tg receptor on thyroid cells, plays a role in Tg transcytosis. Following incubation with exogenous rat Tg at 37 degrees C, Fisher rat thyroid (FRTL-5) cells, a differentiated thyroid cell line, released T3 into the medium. However, when cells were incubated with Tg plus either of two megalin competitors, T3 release was increased, suggesting that Tg internalized by megalin bypassed the lysosomal pathway, possibly with release of undegraded Tg from cells. To assess this possibility, we performed experiments in which FRTL-5 cells were incubated with either unlabeled or (125)I-labeled Tg at 37 degrees C to allow internalization, treated with heparin to remove cell surface-bound Tg, and further incubated at 37 degrees C to allow Tg release. Intact 330-kDa Tg was released into the medium, and the amount released was markedly reduced by megalin competitors. To investigate whether Tg release resulted from transcytosis, we studied FRTL-5 cells cultured as polarized layers with tight junctions on permeable filters in the upper chamber of dual chambered devices. Following the addition of Tg to the upper chamber and incubation at 37 degrees C, intact 330-kDa Tg was found in fluids collected from the lower chamber. The amount recovered was markedly reduced by megalin competitors, indicating that megalin mediates Tg transcytosis. We also studied Tg transcytosis in vivo, using a rat model of goiter induced by aminotriazole, in which increased release of thyrotropin induces massive colloid endocytosis. This was associated with increased megalin expression on thyrocytes and increased serum Tg levels, with reduced serum T3 levels, supporting the conclusion that megalin mediates Tg transcytosis. Tg transcytosis is a novel function of megalin, which usually transports ligands to lysosomes. Megalin-mediated transcytosis may regulate the extent of thyroid hormone release.

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.

Expression of oligo/polyalpha2,8-linked deaminoneuraminic acid and megalin during kidney development and maturation: mutually exclusive distribution with polyalpha2,8-linked N-acetylneuraminic acid of N-CAM.

The expression of homopolymers of alpha2,8-linked deaminoneuraminic acid (oligo/polyalpha2,8-KDN) and of megalin during rat kidney development was investigated using immunocytochemistry and immunoblotting, and compared to homopolymers of alpha2,8-linked N-acetylneuraminic acid (polyalpha2,8-Neu5Ac) of the neural cell adhesion molecule (N-CAM). Both, oligo/polyalpha2,8-KDN and megalin were found in early proximal tubules of embryonic day 18 kidneys. In addition, megalin, but not oligo/polyalpha2,8-KDN, was detectable in late S-shaped bodies and early capillary loop stages. Until postnatal day 7, oligo/polyalpha2,8-KDN and megalin immunoreactivity was present, not only in convoluted but also in straight proximal tubules, and then restricted to the convoluted part as in adult kidney. Immunoblotting revealed increasing megalin expression until postnatal week 3 of kidney development, when the level corresponded to adult kidney. Combined immunoprecipitation/immunoblot analyses showed a steady level of oligo/polyalpha2,8-KDN on megalin throughout development. This was in striking contrast to the expression of polyalpha2,8-Neu5Ac and N-CAM, which was highest in early embryonic kidney, undetectable in kidneys of 3-week-old rats, and mutually exclusive with oligo/polyalpha2,8-KDN in its distribution. These findings demonstrated the coincidence of oligo/polyalpha2,8-KDN and megalin expression and the first appearance of proximal tubules, and revealed the high degree of specialization of the biosynthetic machinery for protein polysialylation in kidney.

Megalin-mediated endocytosis of transcobalamin-vitamin-B12 complexes suggests a role of the receptor in vitamin-B12 homeostasis.

Kidney cortex is a main target for circulating vitamin B12 (cobalamin) in complex with transcobalamin (TC). Ligand blotting of rabbit kidney cortex with rabbit 125I-TC-B12 and human TC-57Co-B12 revealed an exclusive binding to megalin, a 600-kDa endocytic receptor present in renal proximal tubule epithelium and other absorptive epithelia. The binding was Ca2+ dependent and inhibited by receptor-associated protein (RAP). Surface plasmon resonance analysis demonstrated a high-affinity interaction between purified rabbit megalin and rabbit TC-B12 but no measurable affinity of the vitamin complex for the homologous alpha 2-macroglobulin receptor (alpha 2MR)/low density lipoprotein receptor related protein (LRP). 125I-TC-B12 was efficiently endocytosed in a RAP-inhibitable manner in megalin-expressing rat yolk sac carcinoma cells and in vivo microperfused rat proximal tubules. The radioactivity in the tubules localized to the endocytic compartments and a similar apical distribution in the proximal tubules was demonstrated after intravenous injection of 125I-TC-B12. The TC-B12 binding sites in the proximal tubule epithelium colocalized with megalin as shown by ligand binding to cryosections of rat kidney cortex, and the binding was inhibited by anti-megalin polyclonal antibody, EDTA, and RAP. These data show a novel nutritional dimension of megalin as a receptor involved in the cellular uptake of vitamin B12. The expression of megalin in absorptive epithelia in the kidney and other tissues including yolk sac and placenta suggests a role of the receptor in vitamin B12 homeostasis and fetal vitamin B12 supply.

The expression of megalin (gp330) and LRP diverges during F9 cell differentiation.

The receptor-associated protein, RAP, is a chaperonin-like molecule that binds to two members of the low density lipoprotein receptor (LDLR) superfamily-megalin (gp330) and the LDL receptor-related protein (LRP). In F9 embryonal carcinoma cells, expression of RAP mRNA increases when differentiation is induced with retinoic acid and dibutyryl-cyclic AMP. We have investigated the expression of megalin and LRP and their interaction with RAP in F9 cells using biochemical and immunocytochemical methods. Both receptors are expressed in uninduced F9 cells, but only megalin co-precipitates with RAP. When F9 cells were induced to differentiate into parietal endoderm, the expression of megalin was dramatically increased. The expression of megalin exceeded that of LRP and RAP by an order of magnitude and both receptors co-precipitated with RAP. By immunoelectron microscopy, megalin and LRP were localized to clathrin-coated pits at the cell surface in both undifferentiated and differentiated F9 cells, whereas RAP was found mainly in the ER. A sizeable pool of LRP was also detected in the ER. When F9 cells were grown in suspension in the presence of RA and induced to develop into embryoid bodies, the expression of megalin and LRP segregated into different cell types: megalin was found in the outer epithelial layer and LRP in the stem cells of the inner core. Our results demonstrate that F9 cells induced to differentiate in monolayer culture express megalin, LRP and RAP, and RAP is capable of interacting simultaneously with both receptors. In embryoid bodies the expression of megalin and LRP diverges, and only megalin is expressed in the outer epithelial layer.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterisation of immunoglobulins bound to the placenta of sheep

Aristolochic acid impedes endocytosis and induces DNA adducts in proximal tubule cells.Aristolochic acid (AA), present in Aristolochia plants, appears to be the toxin responsible for Chinese herbs nephropathy (CHN), a rapidly progressive tubulointerstitial nephritis. One of the earliest sign of CHN is the urinary excretion of low-molecular-weight proteins (LMWP), suggesting that AA is toxic to proximal tubules (PT).The effects of AA on PT functions including reabsorption of LMWP were investigated on the well-established opossum kidney (OK) cell line, a model for PT, and compared with those of the classical PT toxin cadmium chloride (CdCl2).OK cell monolayers internalized albumin and β2-microglobulin by receptor-mediated endocytosis, both proteins apparently competing for the same receptor, a complex of megalin and cubulin. The process was significantly impaired by 24-hour preincubation with AA (10 or 20 μmol/L) or CdCl2 (15 μmol/L). Furthermore, 24-hour exposure to AA followed by its removal during one to six days led to a persistent inhibition of the uptake of albumin, in contrast to the substantial recovery observed after CdCl2 removal. Neither AA nor CdCl2 affected cell viability, Na+-glucose cotransport or total rate of protein synthesis. AA significantly decreased megalin expression and formed specific DNA adducts in OK cells, similar to those found in kidneys from CHN patients.The present data support the involvement of AA in the early PT dysfunction found in CHN; furthermore, they suggest a causal relationship between DNA adduct formation, decreased megalin expression, and inhibition of receptor-mediated endocytosis of LMWP.

Fetal IGF-1 and IGFBP-2 response to betamethasone and thyroxine

Aristolochic acid impedes endocytosis and induces DNA adducts in proximal tubule cells.Aristolochic acid (AA), present in Aristolochia plants, appears to be the toxin responsible for Chinese herbs nephropathy (CHN), a rapidly progressive tubulointerstitial nephritis. One of the earliest sign of CHN is the urinary excretion of low-molecular-weight proteins (LMWP), suggesting that AA is toxic to proximal tubules (PT).The effects of AA on PT functions including reabsorption of LMWP were investigated on the well-established opossum kidney (OK) cell line, a model for PT, and compared with those of the classical PT toxin cadmium chloride (CdCl2).OK cell monolayers internalized albumin and β2-microglobulin by receptor-mediated endocytosis, both proteins apparently competing for the same receptor, a complex of megalin and cubulin. The process was significantly impaired by 24-hour preincubation with AA (10 or 20 μmol/L) or CdCl2 (15 μmol/L). Furthermore, 24-hour exposure to AA followed by its removal during one to six days led to a persistent inhibition of the uptake of albumin, in contrast to the substantial recovery observed after CdCl2 removal. Neither AA nor CdCl2 affected cell viability, Na+-glucose cotransport or total rate of protein synthesis. AA significantly decreased megalin expression and formed specific DNA adducts in OK cells, similar to those found in kidneys from CHN patients.The present data support the involvement of AA in the early PT dysfunction found in CHN; furthermore, they suggest a causal relationship between DNA adduct formation, decreased megalin expression, and inhibition of receptor-mediated endocytosis of LMWP.

291 Growth hormone receptors in human breast cancer: Correlation with oestrogen receptor status

Cubilin- and megalin-mediated uptake of albumin in cultured proximal tubule cells of opossum kidney.Reabsorption of albumin from the glomerular filtrate occurs via receptor-mediated endocytosis in the proximal tubule. This process is initiated by binding of albumin in apical clathrin-coated pits, followed by endocytosis and degradation in lysosomes. Although binding sites have been characterized by kinetic studies, the receptors responsible for the binding of albumin have not been fully identified. Two giant glycoproteins, cubilin and megalin, constitute important endocytic receptors localized to the kidney proximal tubule.In the present study, we examined the colocalization of cubilin and megalin in the endocytic pathway and the relationship between the uptake of albumin and the expression of cubilin and megalin in opossum kidney (OK) proximal tubule cells by immunocytochemistry and immunoblotting.OK cells expressed both cubilin and megalin. The light microscope labeling patterns for cubilin and megalin were almost identical and were mainly located at the surface area of the cells. Cubilin and megalin were also shown to colocalize on cell surface microvilli, in coated pits, and in endocytic compartments at the electron microscope level. Endocytosed bovine serum albumin (BSA) was identified exclusively in cells expressing megalin and cubilin. Uptake of BSA-FITC was saturable and inhibited by receptor-associated protein (RAP) and by intrinsic factor-vitamin B12 complex (IF-B12) at high concentrations. Significant inhibition was also observed by specific antibodies to cubilin, and megalin and cubilin antisense oligonucleotides likewise significantly reduced albumin uptake. Egg albumin did not affect the uptake of BSA.The present observations suggest that the two receptors cubilin and megalin are both involved in the endocytic uptake of albumin in renal proximal tubule cells.