Heymann Nephritis Antigen Research Articles

Production of heterologous IgG antibody against Heymann nephritis antigen by injections of immune complexes

Heterologous IgG antibody (ab) can be produced against Heymann nephritis (HN) antigen (ag) in rabbits by administering it in Freund's complete adjuvant. The developing abs reacted at high titre with rat kidney brush border (BB) regions of the renal proximal tubules in an indirect fluorescence ab test. A single IV injection of the heterologous ab into a susceptible strain of rat resulted in the localization of IgG ab to glomerular fixed ags, producing immune complex glomerular nephritis. The injected ab also reacted with the BB region of the renal proximal tubules. The aim of this experiment was to find out whether heterologous IgG ab against the HN ag can also be produced in recipient rabbits by injecting immune complexes (ICs) composed of a rat kidney tubular preparation [rat kidney fraction 3 (rKF3)] and donor rabbit-derived rabbit anti-rKF3 IgG ab. We found that anti-rKF3 IgG ab--against the BB region of the renal proximal tubules--could be induced in rabbits injected with ICs, and the resulting ab was able to initiate passive HN in rats. This was the first time a pathogenic IgG ab was produced against HN ag in rabbits without the use of adjuvant. Ab responses in recipient rabbits were achieved by ab information transfer. Recipient rabbits injected with the IC produced the same class of immunoglobulin with the same specificity against the target ag rKF3, as was present in the innoculum, namely rabbit anti-rKF3 IgG ab.

Circulatory antigen of Heymann nephritis. III. Presence of the 70-kD circulatory protein in the immune deposits of Heymann nephritis

An antigen of 70 kD size has been isolated previously from normal rat serum which has immunological cross-reactivity to the Heymann nephritis antigen, F x 1A. Its role in the pathogenesis of Heymann's nephritis was unknown. In this investigation we tested for the presence of 70-kD circulatory antigen in the glomerular immune deposits of Heymann's nephritis. Further, its presence was correlated with severity of disease. It was observed that the presence of the 70-kD antigen strongly correlated with the existence of electron-dense deposits in the lamina rara externa (LRE) of the glomerular capillary wall and with pathologic proteinuria. Temporally, the presence of the 70-kD antigen in the immune deposits was followed by large electron-dense deposits, enhanced complement activity and proteinuria. The data suggest that in the growing immune complex lattice in the LRE, the 70-kD circulatory antigen by virtue of its small size, mobility and antigen cross-reactivity facilitates cross linking and coalescence of immune complexes, resulting in electron-dense immune deposits (EDD) formation which initiates complement activation and consequent proteinuria.

Differentiation-Induced Cultured Podocytes Express Endocytically Active Megalin, a Heymann Nephritis Antigen

Background/Aims: Megalin is a multiligand endocytic receptor expressed in a number of epithelia. In the Lewis rat kidney, podocytes, as well as proximal tubule cells, express megalin that acts as a pathogenic antigen for Heymann nephritis (HN), an experimental model of membranous nephropathy. To obtain a tool to investigate the molecular mechanisms of megalin-mediated endocytosis and the pathogenesis of HN, we examined whether a differentiation-inducible mouse podocyte cell line expressed endocytically active megalin. Methods: Immunofluorescence and immunoprecipitation analyses with an anti-rat megalin antibody were carried out to investigate whether megalin was expressed in the differentiated and undifferentiated podocytes. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was performed to elucidate whether the cells synthesize megalin mRNA. ¹²⁵I-labeled receptor-associated protein (RAP), an endocytic ligand for megalin, was used for cellular internalization and degradation assays. Results: Immunofluorescence, immunoprecipitation and RT-PCR analyses revealed that megalin was synthesized in both differentiated and undifferentiated cells and localized to the cell surfaces. Effective endocytosis of RAP via megalin was shown under the differentiated condition. Conclusion: Endocytically active megalin is expressed in differentiation-induced cultured podocytes. This cell line could be a useful tool for studies on megalin-mediated endocytosis and the pathogenesis of HN.

Characterization of a kidney proximal tubule cell line, LLC-PK1, expressing endocytotic active megalin.

Reabsorption and cellular handling of glomerular filtered vitamins, peptides, and hormones in the proximal tubule are essential, but thus far, poorly elucidated processes. The multiligand receptor megalin, initially described as a Heymann nephritis antigen and later identified as a member of the LDL receptor gene family, mediates reabsorption of several molecules, such as transcobalamin-vitamin B12 and albumin, in the proximal tubule. Consequently, a differentiated cell line of proximal tubular origin expressing megalin is an important requisite for examination of the above-mentioned processes. This study shows, using electron microscopy, that the cell line LLC-PK1, originating from the proximal tubule, maintained differentiated morphology and had a well developed endocytotic apparatus. Furthermore, by immunoblotting and immunohisto- and cytochemistry, megalin was identified in the endocytotic compartments of these cells. Megalin was situated mainly in the endosomes and in the dense apical tubules, but it was also identified in coated pits and in the brush border. The ability of megalin to mediate internalization and degradation of labeled receptor-associated protein (RAP) in a RAP-inhibitable manner was demonstrated. By autoradiography, the endocytosed, iodinated RAP was located in endosomes and lysosomes in the apical part of the cells. Moreover, the LLC-PK1 cells assembled in a monolayer with a hindrance toward diffusion of labeled mannitol, inulin, and dextran at a satisfactory level for the study of proximal tubule handling of smaller proteins. This study reveals a proximal tubule cell line expressing megalin in a functional manner well suited for binding, uptake, and transcellular transport studies.

Nested sets of protein fragments and their use in epitope mapping: characterization of the epitope for the S4D5 monoclonal antibody binding to receptor associated protein

The present report describes a new general procedure by which linear and some structure-dependent epitopes may be mapped in a protein antigen using a nested set of protein fragments prepared from partial proteolysis products of a recombinant protein. Briefly, the antigen, fused to an affinity tag, is partially fragmented and affinity sorted under denaturing conditions to produce a nested set of polypeptides, consisting of N- (or C-)terminal fragments. Immunoblots of SDS-PAGE fractionated sets of fragments are therefore directly readable in terms of molecular mass — i.e., approximate sequence positions — that identify sequence segments harbouring an epitope and any additional structural elements, required to maintain epitope conformation. Blots of N- and C-terminal nested sets of polypeptide fragments representing the human receptor associated protein (RAP) were prepared and probed with mAb S4D5 (Moestrup and Gliemann, 1991). Fragments 1–177 and 94–323 were the shortest fragments detected by the antibody, suggesting the presence of an epitope within the 94–177 segment. Independent mapping based on recombinant fragments of the RAP homologue, rat Heymann nephritis antigen, confirmed that the epitope resides in the Pro 115-Asp 177 segment. The model study demonstrates the utility of nested sets of protein fragments as fast and inexpensive tools for epitope mapping.

A Protein Involved in Calcium Sensing of the Human Parathyroid and Placental Cytotrophoblast Cells Belongs to the LDL-Receptor Protein Superfamily

Monoclonal anti-parathyroid antibodies have been utilized to isolate a single-chain glycoprotein of 500 kDa, which apparently acts as a sensor of the extracellular calcium concentration and is expressed on the surface of human parathyroid, placental, and kidney tubule cells. The present contribution reports the isolation of a cDNA clone encoding this protein in human placenta and subsequent Northern blots confirming the mRNA expression also in human parathyroid and kidney cells. Close similarity in sequence as well as in tissue distribution is demonstrated with the rat Heymann nephritis antigen, a kidney tubule glycoprotein with calcium-binding ability. The 500-kDa protein belongs to the LDL-receptor superfamily of glycoproteins, claimed to function primarily as protein receptors and characterized by functionally important calcium-binding capacity. It is proposed that the currently identified protein constitutes part of a common structure for the sensing of extracellular calcium concentrations and influences calcium homeostasis in different organs.

Mouse heparin binding protein-44 (HBP-44) associates with brushin, a high-molecular-weight glycoprotein antigen common to the kidney and teratocarcinomas.

Heparin binding protein-44 (HBP-44) is a heparin binding protein of 44 kDa, found by cDNA cloning using antibodies against teratocarcinoma glycoproteins [Furukawa, T. et al. (1990) J. Biochem. 108, 297-302]. The N-terminal sequence analysis reported in this publication establishes the structure of its mature form. Immunohistochemical staining revealed that HBP-44 was located in the tubular brush border of the kidney. HBP-44 formed a complex with brushin, a high molecular weight (450 kDa) glycoprotein antigen common to the kidney and teratocarcinoma, but not with OR8 antigen, another antigen (350 kDa) of the same category. Brushin was shown to be the mouse counterpart of rat Heymann nephritis antigen, called gp330. The association between HBP-44 and brushin was revealed not only by co-precipitation upon indirect immunoprecipitation, but also by ligand blotting with HBP-44-maltose binding protein fusion protein. Calcium ion stabilized the association. Disulfide bonds in brushin seemed to be necessary for the complex formation, since reductive cleavage of the bonds resulted in failure of the protein to associate with HBP-44 in a ligand blotting experiment. Association of HBP-44 with brushin occurred both in teratocarcinoma cells, in which these molecules are mainly located in extraembryonic endoderm cells, and in the kidney, suggesting that the complex has an unknown common function in the renal tubular brush border and the extraembryonic endoderm.

Identification of a cell line that expresses a cell surface and a soluble form of the gp330/receptor-associated protein (RAP) Heymann nephritis antigenic complex.

gp330 is a large glycoprotein located in clathrin-coated pits at the surface of the glomerular and proximal tubule epithelia in the rat kidney. It was originally identified as the target of autoimmune antibodies in Heymann nephritis (HN) and has since been shown to be a member of the low density lipoprotein receptor gene family and to form a stable association with receptor-associated protein (RAP), which together constitute the HN antigen complex (HNAC). Progress in defining the normal functions of gp330 as well as the molecular mechanisms of HN has been hampered by the lack of an available kidney cell line that expresses this protein. We here report the identification of a rat yolk sac carcinoma cell line (L2) that synthesizes HNAC and expresses it in coated pits at the cell surface. gp330 and RAP from L2 cells are immunologically identical to their kidney counterparts, and peptide maps of gp330 yielded identical peptide fragments. Characterization of the cell line revealed that there are 3.3 x 10(4) gp330 molecules per L2 cell and that the cells produce a soluble form of gp330 that is released into the medium. Heparin ligand blot analysis demonstrated that RAP but not gp330 binds heparin. By heparin affinity chromatography, gp330 and RAP copurify, indicating that the glycosaminoglycan binding site within RAP is accessible when the subunit is complexed with gp330. These results indicate that the L2 cell line provides a valid and useful model for studies on the function of HNAC and the pathogenesis of HN.

Apical expression of an antigen common to rabbit yolk sac endoderm and kidney proximal tubule epithelium

The tissue distribution, molecular weight, and biochemical nature of an antigen detected by a mouse monoclonal antibody designated 283D3 and raised against rabbit visceral yolk sac endodermal cells, has been investigated. The antigen is located on the luminal side of apical tubules and large sub-apical vesicles in rabbit yolk sac endoderm and proximal kidney tubule epithelial cells. It is expressed in a similar polarised fashion in epithelial cells lining the epididymis. Western blotting showed the antigen to comprise proteins of molecular weight 330–380 kDa. The antigen has been affinity purified from yolk sac and kidney and is predominantly protein in nature with a small percentage of N-linked carbohydrate. In terms of tissue distribution and molecular weight it has close similarity to Heymann nephritis antigen but differs in not being confined to coated pits. Its function is not known, but the association with endocytic elements implies a possible role in non-specific protein absorption.

Primary structure of alpha 2-macroglobulin receptor-associated protein. Human homologue of a Heymann nephritis antigen

The alpha 2-macroglobulin (alpha 2M) receptor complex as purified by affinity chromatography contains three polypeptides: a 515-kDa heavy chain, an 85-kDa light chain, and a 39-kDa associated protein. Previous studies have established that the 515/85-kDa components are derived from a 600-kDa precursor whose complete sequence has been determined by cDNA cloning (Herz, J., Hamann, U., Rogne, S., Myklebost, O., Gassepohl, H., and Stanley, K. (1988) EMBO J. 7,4119-4127). We have now determined the primary structure of the human 39-kDa polypeptide, termed alpha 2M receptor-associated protein, by cDNA cloning. The deduced amino acid sequence contains a putative signal sequence that precedes the 323-residue mature protein. Comparative sequence analysis revealed that alpha 2M receptor-associated protein has 73% identity with a rat protein reported to be a pathogenic domain of Heymann nephritis antigen gp 330 and 77% identity to a mouse heparin-binding protein termed HBP-44. The high overall identity suggests that these molecules are interspecies homologues and indicates that the pathogenic domain, previously thought to be a portion of gp 330, is in fact a distinct protein. Further, the 120-residue carboxyl-terminal region of alpha 2M receptor-associated protein has 26% identity with a region of apolipoprotein E containing the low density lipoprotein receptor binding domain. Pulse-chase experiments revealed that the newly formed alpha 2M receptor-associated protein remains cell-associated, while surface labeling experiments followed by immunoprecipitation suggest that this protein is present on the cell surface forming a complex with the alpha 2M receptor heavy and light chains.

Luminal surfaces of fetal rat alveolar type II and clara lung cells react with antibody to heymann nephritis antigen

We have used affinity purified antibody, which reacts with the renal antigen gp600, to examine the immunoreactivity of pulmonary cells in the quick frozen lung tissue of fetal rats. On immunoelectron microscopy, we found that the reaction was specifically localized to the luminal surfaces of Clara cell and, as previously reported, to alveolar type II cell (ATII) membranes. No immunoreaction was seen on or in type I cells, endothelial cells or any other lung or blood cell. These studies suggest that the anti-gp600 immunoselection can be used as a marker to identify and quantify Clara and ATII cells in rat lung tissue.

Nephritogenicity and immunocytochemical localization of the 70-kilodalton glycoprotein subunit (gp70) of heymann antigen

Heymann nephritis (HN) is an experimentally induced glomerulonephropathy of the rat characterized by subepithelial immune deposits and proteinuria. Immunization with a complex multimeric glycoprotein, gp600, comprising four subunits gp330, gp140, gp110, and gp70 has been shown to induce the complete form of the disease including proteinuria. Examination of three different batches of heterologous anti-gp600 antisera by immunoblot technique showed that the reactivity toward gp70 was dominant and common to all three antisera. gp70 was isolated from Triton X-100-solubilized Fx1A by lectin Lens culinaris affinity chromatography, and the purity was confirmed by SDS-PAGE. Ten rats were actively immunized with 200 μg of gp70. All 10 animals developed circulating brush border antibody and typical granular IgG deposits in the glomerulus but only 1 10 animals developed abnormal proteinuria. A potent antiserum against gp70 was prepared in the rabbit. It reacted strongly to the glomerular capillary wall and the proximal tubular brush border by immunofluorescence. By Protein A immunogold technique using anti-gp70, gold particles were found associated with the glomerular basement membrane (GBM)-endothelial region. By immunoblot analysis of rat GBM using the same anti-gp70 antiserum, a 70-kDa cross-reactive antigen was demonstrated in GBM preparations. These results show that the smallest subunit, gp70 of the complete HN antigen, gp600 Fx1A can independently induce the lesion of HN, but without proteinuria. The presence of gp70 on the endothelial side of the GBM is consistent with a role for in situ antigen-antibody reactions at sites other than the subepithelial region in the pathogenesis of HN.

Rat Heymann nephritis antigen is closely related to brushin, a glycoprotein present in early mouse embryo epithelia

Heymann nephritis is a glomerulonephritis induced in rat by injecting kidney extracts. The responsible antigen is known to consist of two high molecular weight glycoproteins, gp330 and gp300, located in renal tubular brush border cells. We show that rabbit antibodies made against purified rat gp330 react in immunofluorescence tests with several polarized epithelia present in pre- and post-implantation mouse embryos, as well as with murine trophoblastoma cells. Immunoprecipitation experiments demonstrate that anti-gp330 antibodies also recognize two components with relative molecular weights of 330 and 300 Kd in mouse embryonic cells. Furthermore, sequential immunoprecipitation experiments and limited proteolysis tests suggest that these two components are identical with brushin, a glycoprotein set known to be present in a mouse early embryonic epithelium and in adult kidney cells. Finally, ultrastructural studies demonstrate that anti-gp330 antibodies react with molecules preferentially located in coated pits of murine early epithelial cells. These results show that Heymann nephritis antigen has an embryonic counterpart closely related to, if not identical with, previously identified brushin glycoproteins. These molecules could be a receptor to an unknown ligand or a structural component of coated pit membrane.

The membrane composition of coated pits, microvilli, endosomes, and lysosomes is distinctive in the rat kidney proximal tubule cell.

The distribution of a number of membrane proteins on plasmalemmal microdomains (microvilli, coated pits) and in endosomes and lysosomes of the proximal tubule epithelial cell was determined in normal rat kidneys by immunofluorescence and immunoelectron microscopy. Two major brush border proteins, 130 and 94 kD, and gamma-glutamyl transpeptidase were detected on the membranes of the microvilli but were not found on membranes of coated pits. Gp330, the Heymann nephritis antigen, and clathrin were localized in coated pits. The lysosomal membrane glycoprotein, lgp120 (Lewis, V., S. A. Green, M. Marsh, P. Vihko, A. Helenius, and I. Mellman, 1985, J. Cell Biol., 100: 1839-1847) was restricted to lysosomes where it co-localized with beta-glucuronidase. Endosomes, identified by preloading with HRP injected 5-15 min before rats were killed, did not contain detectable amounts of any antigen tested. The distribution of the same proteins was also determined in rats given sodium maleate, which is known to slow or reduce protein absorption by the proximal tubule and to cause vacuolation of the endocytic apparatus. After maleate treatment the distribution of microvillar and lysosomal markers was unchanged, but the coated pit markers were redistributed--gp330 was concentrated in newly formed apical vacuoles, and clathrin was diffusely distributed in the apical cytoplasm or on apical coated vesicles. These findings indicate that the membrane composition of microvilli, coated pits, endosomes, and lysosomes is distinctive in the proximal tubule cell; and that gp330, unlike other known coated pit membrane components, is not transferred to endosomes during endocytosis. After maleate treatment, the coated pits lose their clathrin coats, and the corresponding membrane is internalized.

Microdomains of distinctive glycoprotein composition in the kidney proximal tubule brush border.

Two membrane proteins, maltase and gp330 (the pathogenic antigen of Heymann nephritis), present in the proximal tubule brush border have recently been independently purified and found to be large glycoproteins of similar molecular weight (Mr = approximately 300,000) by SDS PAGE. To determine the relationship between the two, monoclonal antibodies raised against the purified proteins were used for comparative immunochemical analyses and immunocytochemical localization. When a detergent extract of [35S]methionine-labeled rat renal cortex was used for immunoprecipitation with monoclonal antimaltase IgG, a single band of approximately 300 kdaltons was precipitated, whereas a single 330-kdalton band was precipitated with monoclonal anti-gp330 IgG. Monoclonal antimaltase (gp300) IgG also immunoprecipitated maltase activity from solubilized renal maltase preparations, whereas monoclonal anti-gp330 IgG failed to do so. When cyanogen bromide-generated peptide maps of the two proteins were compared, there were many similar peptides, but some differences. When maltase and gp330 were localized by indirect immunofluorescence and by indirect immunoperoxidase and immunogold techniques at the electron microscope level, they were found to be differently distributed in the brush border of the initial (S1 and S2) segments of the proximal tubule: maltase was concentrated (approximately 90%) on the microvilli, and gp330 was concentrated (approximately 90%) in the clathrin-coated apical invaginations located at the base of the microvilli. We conclude that maltase (gp300) and the Heymann nephritis antigen (gp330) are structurally related membrane glycoproteins with a distinctive distribution in the proximal tubule brush border which may serve as markers for the microvillar and coated microdomains, respectively, of the apical plasmalemma.

Immunocytochemical localization of the Heymann nephritis antigen (GP330) in glomerular epithelial cells of normal Lewis rats.

The nephritogenic antigen of Heymann's nephritis (HN) was previously purified from tubular brush-border fractions of rat kidney and found to be a 330,000- mol-wt glycoprotein (gp330). This study was conducted to determine whether gp330 is also present in the rat glomerulus, and, if so, to establish where in the glomerulus it is located. Rabbit polyclonal and mouse monoclonal antibodies were raised against purified gp330, which specifically immunoprecipitated gp330 from solubilized brush-border fractions and specifically stained microvilli and coated invaginations (located at the base of the microvilli) of proximal tubule cells. Accordingly, they were used to localize gp330 by immunoprecipitation and immunocytochemistry in glomeruli of normal Lewis rats. For immunoprecipitation, purified glomerular fractions were prepared from [(35)S]-methionine-labeled kidneys, extracted with Triton X-100, and the extract was used for immunoprecipitation with affinity-purified rabbit polyclonal, or mouse monoclonal, anti-gp330 IgG. Analysis of immunoprecipitates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis fluorography indicated that a band corresponding in mobility to gp330 was specifically precipitated. When unfixed cryostat sections were incubated for indirect immunofluorescence with monoclonal or affinity-purified polyclonal IgG, a fine granular fluorescent staining was seen throughout the glomerulus. When aldehyde-fixed cryostat sections were incubated for indirect immunoperoxidase, reaction product was detected only in the epithelial cells and was not seen in the GBM, endothelium, or mesangium. Within the epithelium it was localized to the endoplasmic reticulum, occasional Golgi elements, multivesicular bodies, and coated pits at the cell surface. The reactive coated pits were distributed all along the cell membrane, including the sides and base of the foot processes. Reaction product was detected in the latter location only in sections that had been digested with neuraminidase before antibody incubation. When rats were given rabbit anti-gp330 IgG by intravenous injection and their kidneys stained for direct immunoperoxidase 3 d later, rabbit IgG was seen to be deposited beneath the slit diaphragms and in the coated pits at the base of the foot processes. The immunocytochemical and immunoprecipitation data indicate, in confirmation of the results of others, that the nephritogenic HN antigen is present in renal glomeruli as well as in proximal tubular brush borders. The immunocytochemical results further demonstrate that gp330 is an epithelial, rather than a glomerular basement membrane, antigen. It appears to be synthesized by glomerular epithelial cells and subsequently becomes concentrated in coated pits. As both the endogenous antigen (gp330) and exogenously administered anti-gp330 antibody were localized to coated pits, it seems likely that coated pits located at the base of the foot processes are the sites where the HN antigen (gp330) and circulating antibodies directed against gp330 meet and where immune complexes are formed.