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D Kerjaschki

Publications and source records attributed to D Kerjaschki.

At least 91 records · Page 5Linked to original sources
PubMed1989

Expression and modulation of surface antigens in cultured rat glomerular visceral epithelial cells.

This study, using immunocytochemical light and electron microscopy techniques, characterizes the distribution of three antibodies bound to the surface of rat glomerular visceral epithelial cells (GEC) in culture, and tests their ability to redistribute corresponding antigens under conditions appropriate for antigenic modulation (antigen disappearance). At 4 degrees C or after fixation, anti-renal tubular brush border vesicle (BBV) IgG bound diffusely to the surface of GEC and to coated pits. Anti-gp330 IgG had a discrete distribution on the surface of GEC and reacted with coated pits. Anti-podocalyxin IgG was bound diffusely to the surface of GEC but not to coated pits. At 37 degrees C, anti-BBV IgG induced marked redistribution of immune complexes with both shedding and internalization. Anti-gp330 IgG induced weaker redistribution, with internalization of immune complexes predominating. Anti-podocalyxin IgG induced rapid redistribution of immune complexes and antigenic modulation but minimal internalization. Experiments of differential redistribution indicated that anti-BBV IgG modulated the expression of both gp330 and podocalyxin; anti-gp330 IgG had a weaker effect on BBV antigens and podocalyxin; and anti-podocalyxin failed to redistribute BBV antigens or gp330. The relevance of these immunocytochemical studies of antibody-cell surface antigen interaction in cultured GEC to understanding the pathogenesis of Heymann glomerulonephritis (HG) is discussed.

Animals↗
PubMed1989

[The immune complex pathogenesis of experimental membranous glomerulopathy (Heymann nephritis) and its significance in human glomerular disease].

Glomerular subepithelial immune deposits are the hallmark of human membranous glomerulopathy, and of its corresponding experimental rat model, Heymann nephritis (HN). The pathogenesis of HN is now understood in some detail, and recent data on this issue are reviewed in this summary. Immune deposits in HN are caused by the interaction of certain epitopes on the nephritogenic antigen, the glycoprotein gp 330, which is a resident molecule of the clathrin coated pits of glomerular visceral epithelial cells, and specific anti-gp 330 IgG. This step is followed within minutes by detachment of the immune complexes and rapid and firm cross-linking to the glomerular basement membrane, thus preventing its endocytosis by the epithelial cells. Immune complexes accumulate locally to form large immune deposits by repeated cycles of antibody binding to newly synthetized gp 330 antigen, shedding and immobilization in the basement membrane. Also human kidneys contain a gp 330-related (but not identical) protein which appears to be not involved in the formation of immune deposits. However, recent data suggest that also in the human disease similar pathogenic mechanisms as in HN could be involved.

Animals↗
PubMed1989

A beta 1-integrin receptor for fibronectin in human kidney glomeruli.

The fibronectin receptor (FNR) is a transmembrane heterodimeric glycoprotein which shares a common beta 1-chain with several other members of the integrin family of adhesion receptors. The authors have prepared a membrane fraction of isolated human glomeruli, from which two proteins (apparent molecular weights 120 kd and 140 kd) bound to a fibronectin-column, and were selectively released by the synthetic peptide Arg-Gly-Asp-Ser. These molecules were labeled in immune overlays by an antibody raised against the FNR from human placenta that recognizes both the FNR-specific a-chain and the group-specific beta 1-integrin chain. In sections of normal human kidneys this antibody labeled predominately the mesangia and the peripheral capillary walls of glomeruli by an immunoperoxidase procedure. Quantitative immunoelectron microscopy, using an indirect immunogold procedure, revealed a preferential localization along the cell membranes of mesangial, epithelial, and endothelial cells that face the mesangial matrix or the glomerular basement membrane (GBM). In kidney biopsies from patients with various glomerular diseases (membranous and other forms of glomerulonephritis, minimal change disease) the distribution was similar to that in normal glomeruli. These findings indicate that a beta 1-integrin-related FNR is present in normal and diseased human glomeruli.

Adult↗
PubMed1989

Potential pathogenic role of cell-surface polypeptides, cross-reactive with DNA, in systemic lupus erythematosus.

The authors report their studies on the cross-reactivity of certain anti-DNA antibodies with polypeptides expressed on the membrane of different cell types. These lupus associated membrane proteins (LAMPs) could play an important role in the pathophysiology of systemic lupus erythematosus (SLE) since anti-LAMP antibodies have been found in renal eluates of MRL/lpr/lpr mice and in the serum of patients with active SLE.

Animals↗
PubMed1987

Initial events in the formation of immune deposits in passive Heymann nephritis. gp330-anti-gp330 immune complexes form in epithelial coated pits and rapidly become attached to the glomerular basement membrane.

The nephritogenic antigen of Heymann's nephritis (HN), gp330, was previously demonstrated (4-9) to be a resident glycoprotein of coated pits in the glomerular and proximal tubule epithelium of rats, and anti-gp330 IgG given intravenously was found to form IDs in glomeruli (passive HN). The purpose of this study was to investigate the detailed events that occur in the formation of IDs in passive HN. HN was induced by the injection of either 125I-labeled or unlabeled anti-gp330 IgG. At various times after injection (15 min to 8 d) the kidneys of some of the injected rats were fixed by perfusion, and the distribution of the rabbit IgG was determined by immunofluorescence and by immunoelectron microscopy. Glomeruli were isolated from the kidneys of injected rats and used for isolation of GBM fractions or for elution of the bound IgG. At 15 min to 1 h after injection, the rabbit IgG was localized by immunocytochemistry exclusively in coated pits along the podocyte plasmalemma facing the GBM. By 1-8 d, anti-gp330 IgG was detected in larger electron-dense IDs often located under the slit diaphragms. Serial sectioning revealed that each of the IDs maintained contact with a coated pit at some level. When GBMs isolated from rats given radiolabeled anti-gp330 IgG were examined by electron microscopy, the IDs were found to remain attached to the GBMs as early as 15 min after injection and coisolated with them at all time points. By double-immunolabeling of the isolated GBMs with two sizes of gold particles, both the antigen (gp330) and the anti-gp330 IgG could be demonstrated in IDs at all time points. When the amount of radiolabeled anti-gp330 bound to GBM fractions was compared with that of isolated glomeruli, it was found that 20% of the radiolabel remained bound to the purified GBMs at 15 min after injection, and 90% at 3 d. The bound IgG was released only by treatments that disrupt antibody-antigen complexes (high and low pH), but not by the other treatments we tried (detergent, high salt, heparinase, or collagenase digestion). When the IgG bound to glomeruli was eluted with acid citrate buffer 3 d after injection, it was found to specifically immunoprecipitate only gp330 from detergent-solubilized 125I-labeled kidney microvillar vesicles. By isoelectric focusing the eluate was found to be enriched in IgGs with acidic isoelectric points.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Identification of a 400-kd protein in the brush borders of human kidney tubules that is similar to gp330, the nephritogenic antigen of rat Heymann nephritis.

The nephritogenic antigen of Heymann nephritis (HN)--a well-studied experimental rat model disease of human membranous glomerulonephritis (MGN)--was recently shown to be a 330-kd glycoprotein (gp330) which is present in the membranes of both the rat tubular brush borders and of podocytes. Because the pathogenic antigen(s) of MGN are unknown, the authors have searched for a gp330-like molecule in human kidney and for its role in MGN. The authors here report that a membrane protein (apparent molecular weight 400 kd) is present in human kidney which is immunologically cross-reactive with rat gp330. By immunoelectron microscopy (using rabbit anti-rat gp330 IgG or a monoclonal anti-400-kd IgG) this molecule is similarly localized in human proximal tubules, but it is absent from the podocytes of human glomeruli. The 400-kd molecule is not detected in the glomerular immune deposits of 30 biopsies of MGN. It is proposed that this is due to the lack of the 400-kd protein in human glomeruli which prevents the formation of initial 400-kd anti-400-kd IgG immune complexes in situ.

Animals↗
PubMed1986

Endothelial cell membranes contain podocalyxin--the major sialoprotein of visceral glomerular epithelial cells.

Podocalyxin is the major sialoprotein in the glycocalyx of glomerular podocytes. Here we report on its extraglomerular localization, using a monospecific antibody which was obtained by affinity purification of IgG on nitrocellulose transfers of glomerular podocalyxin. By indirect immunofluorescence, podocalyxin was found in the blood vessels of several organs (lung, heart, kidney, small intestine, brain, pancreas, aorta, the periportal blood vessels in liver, and the central arteries of follicles of the spleen, but not in the endothelia that line the sinusoids of the latter organs). By immunoelectron microscopy--using immunogold conjugates in diffusion ("pre-embedding") and surface ("postembedding") procedures--podocalyxin was localized on the luminal membrane domain of endothelial cells, in a patchy distribution. The presence of podocalyxin was confirmed in SDS extracts of lung tissue by immunoblotting. We conclude that (a) podocalyxin is a widespread component of endothelial plasma membranes, (b) it is restricted to the luminal membrane domain, and (c) it is distributed unevenly on the endothelial cell surface.

Animals↗
PubMed1986

Identification of a major sialoprotein in the glycocalyx of human visceral glomerular epithelial cells.

Glomerular visceral epithelial cells are endowed with a sialic acid-rich surface coat (the "glomerular epithelial polyanion"), which in rat tissue contains the sialoprotein podocalyxin. We have identified a major membrane sialoprotein in human glomeruli that is similar to rat podocalyxin in its sialic acid-dependent binding of wheat germ agglutinin and in its localization on the surface of glomerular epithelial and endothelial cells, as shown by immunoelectron microscopy, using the monoclonal antibody PHM5. Differences in the sialoproteins of the two species are indicated by the discrepancy of their apparent molecular weights in sodium dodecyl sulfate gels, by the lack of cross reactivity of their specific antibodies, and by the lack of homology of their proteolytic peptide maps. It is therefore possible that the human glomerular sialoprotein and rat podocalyxin are evolutionarily distinct, but have similar functions.

Animals↗
PubMed1986

Epithelial polyanion (podocalyxin) is found on the sides but not the soles of the foot processes of the glomerular epithelium.

The distribution of podocalyxin was determined by several different immunoelectron microscopic methods, which included so-called diffusion methods carried out on cryostat sections and surface methods done on ultrathin sections. When localization was carried out by three different indirect immunogold methods (using ultrathin frozen sections, ultrathin sections cut from tissues embedded in Lowicryl K4M or LR White, or 20- mu cryostat sections for incubations), gold particles were found exclusively on the urinary surfaces of glomerular epithelial cells and, at a lower concentration, on the luminal surface of glomerular endothelial cells. No gold was bound to the basal surfaces (soles) of the epithelial foot processes, the filtration slit diaphragms, the glomerular basement membrane (GBM), or those regions of the endothelial plasmalemma that face the GBM. When podocalyxin was localized by an indirect immunoperoxidase method on cryostat sections, heavy deposits of reaction product were seen on the same cell surfaces as with the immunogold methods. Each of the methods used had certain advantages as well as limitations, but the collective results obtained were convergent and complimentary. It is concluded that podocalyxin is restricted in its distribution to the urinary surfaces of epithelial cells and the luminal surface of endothelial cells; it is missing or present in very low concentrations on the soles (basal surface) of the epithelial foot processes, the slit diaphragms and the basement membrane surface of the endothelium; and the podocalyxin-containing regions of the epithelial and endothelial plasmalemmae constitute microdomains of distinctive membrane protein composition on the corresponding cell surfaces.

Animals↗
PubMed1985

[Molecular pathology of the glomerular sialoglycoprotein podocalyxin, a major component of the glomerular polyanion in experimental and human minimal glomerular change].

Visceral glomerular epithelial cells (or podocytes) are endowed with a highly polyanionic glycokalyx which is rich in sialic acid. We have identified a single sialoprotein - MW 140 kD - which we have called "Podocalyxin", and which is the major carrier of glomerular sialic acid. We have shown that podocalyxin of normal rat glomeruli contains 20 sialic acid residues per molecule, whereas in aminonucleoside nephrotic glomeruli only 4-5 sialic acid per molecule are present. We conclude that the loss of histochemical staining for the "glomerular polyanion" is due both to the reduction of sialic acid per podocalyxin molecule, and to the reduction of the surface of the podocyte's plasmalemma caused by spreading of footprocesses. We furtheron report that also in human glomeruli there is a sialoprotein which is distinct from rat podocalyxin in its electrophoretic mobility and immunological structure, but otherwise shows all features which are characteristic for rat podocalyxin.

Animals↗
PubMed1985

[Cellular pathology of Heyman nephritis: monoclonal antibodies against the pathogenic antigen Gp 330].

Heymann nephritis is an experimental auto-immune glomerulonephritis, closely resembling human epimembranous nephritis, which is induced by identified antigens in the brush border membranes of kidney proximal tubules. The hallmark of the disease is the accumulation of immune deposits in a granular pattern in the lamina rara externa of the glomerular basement membrane. We have established that a large membrane glycoprotein with an apparent molecular weight of 330 kDal (pg 330) is the pathogenic antigen. By means of immunohistochemistry, using mono- and polyclonal anti-pg 330 IgG we found that gp 330 is present in the cell membranes of glomerular epithelial cells and that it is concentrated there in coated pits (specialized areas off the cell membrane which play a key role in receptor mediated endocytosis for ligands such as insulin and others). On the basis of these findings we propose the following mechanism of formation of an immune deposit: (1) "In-situ" formation of immune complexes of gp 330 and anti-gp 330 IgG; (2) shedding of the immune complexes into the basement membrane and (3) crosslinking of the immune complexes into the basement membrane. This scheme could be a general mechanism by which immune deposits are formed also in various other auto-immune diseases.

Animals↗
PubMed1985

Antibody-induced redistribution of Heymann antigen on the surface of cultured glomerular visceral epithelial cells: possible role in the pathogenesis of Heymann glomerulonephritis.

In this study we analyze the ability of antibodies that produce passive Heymann glomerulonephritis to induce antigen redistribution on the surface of cultured glomerular visceral epithelial cells (GEC). Polyclonal antibodies produced by immunization with membrane vesicles prepared from proximal tubule brush borders (BB) and polyclonal (rabbit) and monoclonal (mouse) antibodies to a membrane glycoprotein (gp 330) purified from epithelial cells of rat proximal tubule were used. The study by immunofluorescence of GEC kept at 4 degrees C or fixed with paraformaldehyde showed that the three antibody preparations reacted with the plasma membrane in a punctate pattern known to be due to staining of coated pits or coated vesicles on the cell surface. At 37 degrees C and, at a slower rate, at 22 degrees C, the two polyclonal antibodies induced a rapid clustering of antigen-antibody complexes on the nonadherent surface of living cultured GEC with subsequent formation of patches and caps and, after prolonged incubation, with temporary disappearance of Heymann antigen from the cell surface, so-called antigenic modulation. Antigen redistribution and modulation were inhibited by sodium azide, indicating that these processes are energy dependent. Monovalent Fab fragments of antibodies to BB vesicles did not alter the distribution of Heymann antigen unless they were subsequently cross-linked. Monoclonal anti-gp330 induced a modest degree of antigen redistribution, which was increased by subsequent cross-linking. Exposure of glomerular epithelial cells to cytochalasin B, colchicine, or ionophore A23187 prevented or altered antigen redistribution at 37 degrees C. Furthermore, the antibody-induced antigen redistribution was associated with changes in distribution of cytoplasmic actin, myosin, and tubulin, indicating that it is related to the contractile activity GEC. LEW rats, given i.v. injection of IgG directed against BB membrane vesicles, developed passive Heymann glomerulonephritis (i.e., immune deposits in the lamina rara externa of the glomerular basement membrane). In contrast, the glomeruli of rats exposed for longer periods to larger amounts of Fab fragments of the same antibodies failed to develop immune deposits. These studies show that the antibodies to the nephritogenic antigen of Heymann glomerulonephritis may induce a redistribution of immune complexes (IC) in the membrane of glomerular epithelial cells that is similar to that produced by other plasma membrane antigen-ligand interactions.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Reduced sialylation of podocalyxin--the major sialoprotein of the rat kidney glomerulus--in aminonucleoside nephrosis.

In this study the sugar composition of podocalyxin was determined in puromycin aminonucleoside-treated (PAN) rats and controls. Podocalyxin from both control and PAN rats bound 125I-WGA and 125I-peanut lectin (the latter only after neuraminidase treatment) on nitrocellulose transfers. Purified podocalyxin from both control and PAN rats was found to contain sialic acid, Gal, GlcNac, and Man but lacked Fuc and GalNac by gas-liquid chromatography. In PAN rats the sialic acid content of podocalyxin was reduced from 4.5% to 1.5%, whereas the concentration of the other sugars (with the possible exception of Gal) was similar to that of controls. The density of podocalyxin on the epithelial cell surface was estimated after immunogold labeling with anti-podocalyxin IgG, and no differences were found between PAN rats and controls. These data indicate that the reduced total glomerular sialic acid content found in PAN is due to the combined effects of the decreased podocyte plasmalemmal surface area and the reduced sialic acid content of podocalyxin.

Animals↗
PubMed1984

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.

Animals↗
PubMed1984

Identification and characterization of podocalyxin--the major sialoprotein of the renal glomerular epithelial cell.

The glomerular epithelial polyanion is a specialized cell surface component found on renal glomerular epithelial cells (podocytes) that is rich in sialoprotein(s), as detected by staining with cationic dyes (colloidal iron, alcian blue) and wheat germ agglutinin (WGA). We have isolated rat glomeruli and analyzed their protein composition by SDS PAGE in 5-10% gradient gels. When the gels were stained with alcian blue or "Stains All," a single band with an apparent Mr of 140,000 was detected that also stained very prominently with silver, but not with Coomassie Blue. This band predominated in fluorograms of gels of isolated glomeruli that had been labeled in their sialic acid residues by periodate-[3H]borohydride. In lectin overlays, the 140-kilodalton (kd) band was virtually the only one that bound [125I]wheat germ agglutinin, and this binding could be prevented by predigestion with neuraminidase. [125I]Peanut lectin bound exclusively to the 140-kd band after neuraminidase treatment. An antibody was prepared that specifically recognizes only the 140-kd band by immunoprecipitation and immuneoverlay. By immunoperoxidase and immunogold techniques, it was localized to the surface coat of the glomerular epithelium and, less extensively, to that of endothelial cells. When analyzed (after electroelution from preparative SDS gels), the 140-kd band was found to contain approximately 20% hexose and approximately 4.5% sialic acid. These findings indicate that the 140-kd protein is the major sialoprotein of the glomerulus, and it is the only component of glomerular lysates with an affinity for cationic dyes and lectins identical to that defined histochemically for the epithelial polyanion in situ. Since this molecule is a major component of the cell coat or glycocalyx of the podocytes, we have called it "podocalyxin."

Animals↗
PubMed1984

Presence of an extensive clathrin coat on the apical plasmalemma of the rat kidney proximal tubule cell.

The nature of the cytoplasmic coat present on the apical invaginations of the kidney proximal tubule cell was investigated by immuneoverlay and immunocytochemistry of renal brush borders with anticlathrin antibodies. When kidney cortex was prepared for electron microscopy using methods that enhance visualization of clathrin coats, the apical invaginations at the base of the brush border microvilli were seen to be backed by a nearly continuous coating which resembles but is more extensive than the lattice-like clathrin coats found around brain coated vesicles. When isolated brush border fractions were prepared under conditions that preserve the coats, separated by SDS PAGE, and transferred to nitrocellulose, the presence of clathrin heavy and light chains was detected by immuneoverlay using two different affinity-purified anticlathrin IgGs--one that we prepared, which detects only the clathrin light chains, and the other, prepared by Louvard et al. ( Louvard , D., C. Morris, G. Warren, K. Stanley, F. Winkler , and H. Reggio , 1983, EMBO [Eur. Mol. Biol. Organ.] J., 2:1655-1664), which detects both the heavy and light chains. As viewed by light microscopy (immunofluorescence or immunoperoxidase), staining with both anticlathrins was concentrated at the base of the proximal tubule microvilli. Immunoelectron microscopic localizations carried out on brush border fractions (using peroxidase and gold conjugates) demonstrated specific binding of anticlathrin IgGs to the lattice-like cytoplasmic coat. When brush border fractions were reacted with monoclonal antibodies prepared against gp330 and maltase, proteins that serve as markers for the membrane of the apical invaginations and microvilli, respectively ( Kerjaschki , D., L. Noronha - Blob , B. Sacktor , and M. G. Farquhar , 1984, J. Cell Biol., 98:1505-1513), the two proteins retained their restrictive distribution in the brush border. The findings demonstrate (a) that the cytoplasmic coat of the proximal tubule intermicrovillar apical invaginations is composed of clathrin heavy and light chains, and (b) that the differential distribution of proteins in these two brush border microdomains is maintained in appropriately prepared brush border fractions.

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