Degradation of glomerular basement membrane by human neutrophils in vitro

The glomerular basement membrane is susceptible to immunologic injury when immune complexes or anti-basement-membrane antibodies become lodged in its network. We have studied the digestion of glomerular basement membrane prepared from normal human kidney by isolated neutrophils. In the absence of immunoglobulin aggregates or immune complexes, there was little evidence of neutrophil adherence to the membrane, of release of lysosomal enzymes, or of digestion. However, when the basement membrane contained immunoglobin G (IgG) aggregates generated in situ by heating the membrane impregnated with IgG to 63 degrees C, electron micrographs showed neutrophils adherent to the basement-membrane surface and phagocytosis of smaller fragments. Lysosomal enzymes were detectable in the extracellular medium, and measurements of either total protein or hydroxyproline solubilized showed digestion of 80 micrograms basement membrane/h per 10(7) cells. Hydroxyproline solubilization was almost totally inhibited by phenylmethylsulphonyl fluoride, indicating that the neutrophil serine proteinases, elastase and cathepsin G are responsible for degradation. These findings provide direct evidence for the digestion of extracellular matrix by neutrophils stimulated in situ by deposited immune complexes as a contributor to inflammatory tissue damage.     

Pathogenesis of passive Heymann glomerulonephritis: chlorpromazine inhibits antibody-mediated redistribution of cell surface antigens and prevents development of the disease

Two hypotheses were tested: first, that in LEW rats the interaction of sheep (or rabbit) anti-brush border antibodies with antigens (Heymann antigens) expressed on the plasma membrane of glomerular visceral epithelial cells is characterized by initial redistribution of immune complexes on the cell surface and by subsequent shedding of immune complexes in the subepithelial part of the capillary wall; and secondly, that this interaction is inhibited by chlorpromazine, a drug that displaces calcium ions from binding sites linking the plasma membrane to the cytoskeleton, and which blocks the redistribution of IgG on the surface of B lymphocytes exposed to anti-IgG antibodies. The studies were performed in vitro on cultured LEW glomerular epithelial cells and in vivo in LEW rats. In cultured glomerular epithelial cells exposed at 37 degrees C to anti-brush border IgG, chlorpromazine prevented, in a dose-dependent manner, the redistribution ("capping") of Heymann antigens and the fixation of complement. The renal glomeruli of chlorpromazine-treated LEW rats examined 6 and 48 hr after transfer of anti-brush border antibodies had punctate and, later, punctate and diffuse deposits of sheep (or rabbit) IgG on glomerular epithelial cells, but not similar deposits of rat C3. Moreover, granular subepithelial deposits of sheep (or rabbit) IgG and rat C3, characteristic of passive Heymann glomerulonephritis, did not develop, although deposits of sheep IgG were detected by immunoelectron microscopy on the microvilli of glomerular epithelial cells. Comparative studies on rats with similar reductions in glomerular filtration rates, produced by high doses of chlorpromazine or with renal artery stenosis, showed that the findings were not the consequence of insufficient delivery of antibody to glomerular epithelial cells. The results are consistent with the interpretation that Heymann glomerulonephritis is induced by mechanisms of redistribution of cell surface antigens comparable to those that govern the interaction of surface antigens (or receptors) with appropriate ligands in B lymphocytes and other classical in vitro systems.     

Nephrotic syndrome and subepithelial deposits in a mouse model of immune-mediated anti-podocyte glomerulonephritis

Subepithelial immune complex deposition in glomerular disease causes local inflammation and proteinuria by podocyte disruption. A rat model of membranous nephropathy, the passive Heymann nephritis, suggests that Abs against specific podocyte Ags cause subepithelial deposit formation and podocyte foot process disruption. In this study, we present a mouse model in which a polyclonal sheep anti-mouse podocyte Ab caused subepithelial immune complex formation. Mice developed a nephrotic syndrome with severe edema, proteinuria, hypoalbuminemia, and elevated cholesterol and triglycerides. Development of proteinuria was biphasic: an initial protein loss was followed by a second massive increase of protein loss beginning at approximately day 10. By histology, podocytes were swollen. Electron microscopy revealed 60-80% podocyte foot process effacement and subepithelial deposits, but no disruption of the glomerular basement membrane. Nephrin and synaptopodin staining was severely disrupted, and podocyte number was reduced in anti-podocyte serum-treated mice, indicating severe podocyte damage. Immunohistochemistry detected the injected anti-podocyte Ab exclusively along the glomerular filtration barrier. Immunoelectron microscopy localized the Ab to podocyte foot processes and the glomerular basement membrane. Similarly, immunohistochemistry localized mouse IgG to the subepithelial space. The third complement component (C3) was detected in a linear staining pattern along the glomerular basement membrane and in the mesangial hinge region. However, C3-deficient mice were not protected from podocyte damage, indicating a complement-independent mechanism. Twenty proteins were identified as possible Ags to the sheep anti-podocyte serum by mass spectrometry. Together, these data establish a reproducible model of immune-mediated podocyte injury in mice with subepithelial immune complex formation.     

Preservation of fixed anionic sites in the GBM in the acute proteinuric phase of cationic antigen mediated in-situ immune complex glomerulonephritis in the rat

Cationic antigens have been observed to bind with the negatively charged glomerular basement membrane (GBM). Using the cationic reagent polyethyleneimine (PEI), the distribution of glomerular anionic sites was evaluated ultrastructurally in the early stage (2 h-day 7) of cationic antigen mediated in-situ immune complex formation type glomerulonephritis (GN) in the rat. Renal perfusion via the renal artery with 100 micrograms of cationized human IgG(pI greater than 9.5), followed by the i.v. injection of specific antibodies, led to an initial increase in urinary albumin excretion, subsequent massive globulinuria and the formation of numerous subepithelial deposits on day 7. The most striking alteration in glomerular anionic sites was observed on the epithelial cell surface coat; the PEI deposition on the epithelial cell surface was almost identical to that in control glomeruli at 2 and 4 h after the induction of GN; thereafter, on day 7, a broad loss of anionic sites was observed on flattened epithelial foot processes. In contrast, fixed anionic sites of the laminae rarae of the GBM showed no apparent alterations in the distribution and number from 2 h to day 7 and did not disappear even in the lamina rara externa adjacent to subepithelial deposits. These findings not only show that fixed anionic sites of the GBM, in contrast to the rapid decrease in those of the epithelial cell surface, are not completely neutralized or destroyed even in GN, in which cationic antigen participates in the in-situ formation of GBM-deposits.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alteration of glomerular anionic sites by the development of subepithelial deposits in experimental glomerulonephritis in the rat

Using highly cationic polyethleneimine, alteration of glomerular anionic sites were evaluated ultrastructurally in two types of rat glomerulonephritis (GN); chronic serum sickness GN and heterologous (passive) or autologous (active) Heymann's GN. Daily i.v. injections of egg white lysozyme in physiologic saline into presensitized rats led to the formation of numerous mesangial and subepithelial deposits. In the non-proteinuric period in which immune deposits were localized predominantly in the mesangium, anionic sites of the laminae rarae and the epithelial cell coat were clearly observed. In the subsequent proteinuric period in which numerous subepithelial deposits were superimposed, a broad loss of anionic sites in the epithelial cell coat was seen. Splitting and focal loss of anionic sites on the lamina rara externa adjacent to the subepithelial deposits were commonly observed both in passive and active Heymann's GN and in lysozyme GN. These findings indicate that the subepithelial deposits are closely involved in the development of proteinuria by injuring the anionic sites, especially those on lamina rare externa of the glomerular basement membrane.     

Ultrastructural distribution of endogenous IgGs in the glomerular wall of control and diabetic rats

Summary Endogenous IgG molecules were revealed with high resolutionem over the glomerular wall in renal tissues sampled from short and longterm control and streptozotocin induced diabetic rats by applying the protein A-gold immunocytochemical approach. In tissues from control animals, IgG antigenic sites were revealed on the subendothelial side of the basement membrane, the epithelial side being only weakly labelled. In contrast, in longterm diabetic animals IgG antigenic sites were present throughout the entire thickness of the basement membrane, and in patches closely associated with the plasma membrane of the epithelial cells. Deposits of basement membrane-like material present in the mesangial area were also highly labelled for IgG. Numerous intensely labelled lysosome-like structures were present in the epithelial cells. Morphometrical evaluation of the distribution of the labelling over the basement membrane confirmed these observations. In control animals a peak of labelling was found at 30 nm from the endothelial cell region corresponding to the subendothelial side of the lamina densa. In longterm diabetic animals the labelling was more uniformly distributed throughout the entire thickness of the basement membrane. These data were correlated to biochemical determinations of proteinuria and IgG excretion in urine samples from the control and the diabetic animals. These results suggest that in normal conditions the lamina densa may represent the main barrier for the restriction of the passage of IgGs through the glomerular wall. Modifications at that level occur during diabetes leading to or participating in the loss of the selective permeability of the basement membrane.     

Ultrastructural studies in passive in situ immune complex glomerulonephritis. A rat model for membranous glomerulonephritis

Morphologic studies were performed in a passive model of in situ immune complex glomerulonephritis in rats. The formation and fate of subepithelial immune complexes as well as the role of glomerular polyanion in the induction of disease were examined. Unilateral in situ immune complex glomerulonephritis was induced in rats by perfusion of cationised horse spleen ferritin (pI greater than 9.5) (400 micrograms/rat) into the left kidney followed by systemic injection of 0.2 ml (= 400 micrograms precipitating antibody) of sheep anti-ferritin antiserum 2 h later. This schedule induced glomerulonephritis with proteinuria (mean maximum 100 mg/24 h between the 5th and the 12th day). Rats were sacrificed at intervals between 1 h and 42 days after induction of glomerulonephritis, samples of renal tissue were examined by light, immunofluorescence and electron microscopy (including staining of anionic sites by polyethyleneimine). The lesion induced closely resembled that of membranous glomerulonephritis in man as massive subepithelial deposits were seen with very little cellular infiltration or proliferation. The antigen (ferritin) deposits were initially located subepithelially; from 2 weeks onwards intramembranous deposits in the thickened basement membrane were present, the apparent translocation being due to excessive newly synthesised basement membrane material which encloses the deposits. A loss of anionic sites in the lamina rara interna, lamina rara externa and on the epithelial cell surface coat preceded the development of proteinuria.     

Antibody localization in the glomerular basement membrane may precede in situ immune deposit formation in rat glomeruli

The administration of cationized antibodies, specific to human serum albumin, into the renal artery of rats caused transient presence of IgG in glomeruli by immunofluorescence microscopy. Intravenous infusion of appropriate doses of antigen after the injection of cationized antibodies resulted in immune deposit formation in glomeruli that persisted through 96 hr. By electron microscopy, these deposits were located in the subepithelial area. The injection of large doses of antigen produced immune deposits which were present in glomeruli for only a few hours, presumably due to formation of only small-latticed immune complexes. The presented data indicate that cationic antibodies bound to the fixed negative charges of the glomerular basement membrane can interact with circulating antigen to form immune deposits in glomeruli. This mechanism may be important because anionic antigens have been shown to induce the synthesis of cationic antibodies.     

Ultramicroscopic localization of cationized antigen in the glomerular basement membrane in the course of active, in situ immune complex glomerulonephritis

The sequence of antigen localization and the interaction of immune deposits with the anionic sites of the glomerular basement membrane (GBM) were investigated in an active model of in situ immune complex glomerulonephritis using a cationized ferritin. Three weeks after immunization with native horse spleen ferritin, the left kidneys of rats were perfused with 500 micrograms of cationized ferritin through the left renal artery. One h after renal perfusion, most of ferritin particles localized subendothelially, corresponding to the anionic sites of the lamina rara interna. In the glomerular capillary loops, infiltrating polymorphonuclear leukocytes and monocytes were seen. Some of these monocytes were in direct contact with immune complexes containing ferritin aggregates associated with anionic sites of the lamina rara interna. At 24 h, numerous ferritin aggregates were present subepithelially, preferentially beneath the slit membrane. The subepithelial location of ferritin did not always correspond to the anionic sites of the lamina rara externa. From days 3 to 7, there was remarkable endocapillary cell proliferation in some loops and pronounced effacement of epithelial foot processes. Focal detachment of epithelium from the GBM was observed occasionally. From days 14 to 28, most of ferritin aggregates were located intramembranously and subepithelially. Membranous transformation has already begun around the subepithelial deposits. This morphological study provides insight into the fate of immune deposits and injury to the GBM in the glomerulonephritis.     

Developmental expression and cellular origin of the laminin alpha2, alpha4, and alpha5 chains in the intestine.

Laminins are extracellular matrix glycoproteins that are involved in various cellular functions, including adhesion, proliferation, and differentiation. In this study, we examine the expression patterns and the cellular origins of the laminin alpha2, alpha4, and alpha5 chains in the developing mouse intestine and in in vitro mouse/chick or chick/mouse interspecies hybrid intestines. In situ hybridization and Northern blot analysis revealed that mRNA levels for all three laminin alpha chains are highest in the fetal intestine undergoing intense morphogenetic movements. Laminin alpha4 mRNA and polypeptide are associated with mesenchyme-derived cell populations such as endothelium and smooth muscle. In contrast, laminin alpha2 and alpha5 chains participate in the structural organization of the subepithelial basement membrane and, in the mature intestine, show a complementary pattern of expression. All three laminin alpha chains occur in the smooth muscle basement membrane, with a differential expression of laminin alpha5 chain in the circular and longitudinal smooth muscle layers. The cellular origin of laminin alpha2 and alpha5 chains found in the subepithelial cell basement membrane was studied by immunocytochemical analysis of mouse/chick or chick/mouse interspecies hybrid intestines at various stages of development using mouse-specific antibodies. Laminin alpha2 was found to be deposited into the basement membrane exclusively by mesenchymal cells, while the laminin alpha5 chain was deposited by both epithelial and mesenchymal cells in an apparently developmentally regulated pattern. We conclude that (1) multiple laminin alpha chains are expressed in the intestine, implying specific roles for individual laminin isoforms during intestinal development, and (2) reciprocal epithelial/mesenchymal interactions are essential for the formation of a structured subepithelial basement membrane.     

The relation between connective tissue cells and intercellular substances, including basement membranes.

Basement membranes are distributed widely in the body forming an extracellular matrix for epithelial and endothelial cells. The collagenous and glycoprotein constituents of basement membranes are synthesized by these two cell types. Disturbance of the interactions between basement membranes and their associated epithelial and endothelial cells can lead to the pathological changes seen in diseases involving basement membranes. These changes are illustrated here by reference to glomerulonephritis induced by the deposition of immune complexes in the glomerulus of the kidney, and chronic inflammatory changes occurring in the lung after inhalation of asbestos. In these diseases basement membrane changes can occur in several ways. Hydrolytic enzymes released from inflammatory cells degrade basement membranes while other constituents by epithelial and endothelial cells. Alternatively the physical separation of epithelial and endothelial cells from their basement membrances by space-occupying substances such as immune complexes can interfere with feedback mechanisms leading to synthesis of basement membrane constituents and cell proliferation. Studies of these pathological changes at a cellular level should shed new light on the ways in which cells interact with their pericellular environment.     

The effect of oxidants on neutrophil-mediated degradation of glomerular basement membrane collagen

The contribution of activated oxygen species to neutrophil-mediated degradation of basement membrane collagen was investigated. In preliminary experiments, pre-exposure of either albumin or glomerular basement membrane to neutrophil myeloperoxidase with H2O2 and chloride increased their susceptibility to proteolysis 2-3-fold. In the basement membrane model, neutrophils are stimulated by trapped immune complexes to adhere, produce oxidants and degranulate. Degradation, measured as the amount of hydroxyproline solubilised, was due to neutral proteinases, particularly elastase, and depended on cell number and the amount of proteinase released. Experiments with oxidant scavengers and inhibitors and with neutrophils from donors with chronic granulomatous disease or myeloperoxidase deficiency showed that oxidants did not affect degradation of the basement membrane when this was measured on a per cell basis. However, oxidative inactivation of the released granule enzymes occurred. Activities of elastase, beta-glucuronidase and lysozyme were 1.5-2-times higher in the presence of catalase, but were unaffected by superoxide dismutase or hydroxyl radical scavengers. Inactivation did not occur with chronic granulomatous disease or myeloperoxidase deficient neutrophils. When related to the activity of released elastase, or to other degranulation markers, collagen degradation was decreased in the presence of catalase, or with chronic granulomatous disease or myeloperoxidase deficient cells. This implies that the basement membrane was made more digestible by myeloperoxidase-derived oxidants, as occurred in the cell-free experiments. Taken together, the results indicate that neutrophil oxidants have two opposing effects. They increase the susceptibility of the collagen to proteolysis and inactivate the proteinases responsible.     

Glomerular permselection: shape and flow

The selective permeability of the glomerular basement membrane to macromolecules is a function of the size and charge of the macromolecule. Evidence suggests that shape may also be a factor. The orientation of macromolecules in solution is dependent on their size, shape, and frictional interactions with moving solvent molecules. The spaces between the glomerular visceral epithelial cells (slit pores) may produce a non-uniform distribution of fluid flow within the basement membrane, and this non-uniformity may increase during disease. This report is of a model that relates the filtration of rigid prolate ellipsoidal (cigar) shaped macromolecules to the size and shape of the filter and to the velocity of solvent flow. The calculations, using published macromolecular and glomerular parameters correspond well to published data. The glomerular visceral epithelial cell, by altering the number, size and distribution of the intercellular spaces, may regulate the passage of ellipsoidal shaped macromolecules, such as albumin and IgG, into and through glomerular structures.     

Permeation of endogenous IgG with an anionic subpopulation into glomerular basement membrane in rat

Conflicting results of previous electron microscopy studies and concerns about the validity of immunoperoxidase technique employed in those studies to accurately localize endogenous IgG in rat glomerular basement membrane (GBM) prompted us to use other techniques to answer the following question: Does endogenous IgG permeate the matrix of GBM? Immunofluorescence, radioimmunoassay (RIA), isoelectric focusing, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and immunodetection on Western blots were used to detect endogenous IgG in GBM. Direct immunofluorescence of normal frozen rat kidney sections prepared from in vivo perfused kidney showed endogenous IgG in a linear pattern of staining in the GBM. RIA for rat IgG found the IgG content of collagenase-solubilized GBM to be 0.48% of the dry weight. Immunodetection for rat IgG on Western blots of SDS-PAGE-separated GBM demonstrated endogenous IgG in purified collagenase-solubilized GBM. IgG was detected as an intact molecule with covalently linked light and heavy chains and not as small immunoreactive catabolic fragments. Isoelectric focusing followed by immunodetection on Western blot showed that part of the endogenous IgG in GBM was anionic. The results clearly show that under normal conditions, endogenous IgG can permeate into the collagen matrix of GBM in rat and that some of this IgG is more anionic than the IgG in serum. These findings may assist in understanding the transit of autoantibodies to subepithelial glomerular antigens located beneath the matrix of GBM in membranous glomerulonephropathy.     

Schistosoma japonicum: Immunopathology of nephritis in Macaca fascicularis

Macaca monkeys experimentally infected with Schistosoma japonicum developed a chronic progressive kidney lesion characterized by an increase of mesangial matrix, local glomerular hypercellularity, and local thickening of glomerular basement membrane. Immunofluorescence studies revealed the localization of IgG, IgM, IgA, and IgE immunoglobulins mostly in the mesangial area of the glomeruli accompanied by the deposition of Schistosoma antigens. By electron microscopy, in addition to the local thickening of the glomerular basement membrane, dense homogeneous deposits and those with moth-eaten appearance were detected in the mesangial matrix. These findings suggest that worms in the bloodstream continuously release antigenic materials that stimulate host's antibody response belonging to various immunoglobulin classes including IgE. The produced antibodies and antigens would form immune complexes that deposited in the glomeruli. The increased vascular permeability caused by antigen-IgE antibody interaction may play an important role in the deposition of immune complexes and in the rapid development of kidney injury.     

Extralysosomal localisation of acid phosphatase in the rat kidney

 There is strong evidence that acid phosphatase (AcPase) plays an important role in the catabolism of the glomerular basement membrane (GBM) and the removal of macromolecular debris resulting from ultrafiltration. Recent enzyme histochemical investigations provide new evidence of the antithrombotic and anti-inflammatory function of ADPase and on the distribution of AcPase in mouse kidney tubule cells. By means of 3 mM cerium as the trapping agent and 1 mM p-nitrophenyl phosphate as the substrate, extralysosomal AcPase could be demonstrated at the ultrastructural level. Following a mild perfusion fixation (2% formaldehyde + 0.07% glutaraldehyde), an effective postfixation and short enzyme incubations (20 min) with microwave irradiation, highly specific enzyme histochemical reaction product and reasonable structural preservation were obtained. Extralysosomal, membrane-bound AcPase was observed along the endoplasmic reticulum, the trans-Golgi cisternae, the nuclear envelope, basal infoldings of the proximal and distal tubular cells and on glomerular profiles, e.g. cell membranes of podocytes, endothelium and basement membrane. Large amounts of extralysosomal AcPase were observed in the basement membrane of glomeruli, in contrast to no AcPase activity in the tubular and mesangial basement membrane. The observed difference in AcPase activity in the tubular epithelial basement membrane and the GBM supports the idea that AcPase in GBM specifically serves in the clearance of macromolecular debris to facilitate ultrafiltration. In the GBM a laminar distribution is observed, suggesting that both epithelial and endothelial cells are involved in the production of AcPase. Accepted: 16 September 1997     

Co-culture of glomerular epithelial cells and mesangial cells on collagen-gauze-fiber gel

A novel collagen-gauze-fiber gel was created as a scaffold for the co-culture of renal glomerular epithelial cells and mesangial cells at its opposite sides. This gauze-fiber-gel provides a mimic environment like that of renal glomeruli in vivo. The cell morphology, cell growth and cell viability were investigated and the results showed that this novel scaffold maintains cell growth and cell viability without changing cell morphology for more than 3 weeks. Interestingly, glomerular epithelial cells co-cultured with mesangial cells on the gauze-fiber gel resulted in the polarity formation which usually appears on the normal epithelial cells existing at glomerular basement membrane in vivo, but seldom appears on the epithelial cells when cultured in vitro.     

A morphologic and immunofluorescent analysis of primary guinea pig glomerular cell types grown in chemically defined media. Evidence for clonal growth and cell differentiation

Primary kidney guinea pig glomerular cells were successfully grown in chemically defined media containing insulin, transferrin, and fibronectin or glycylhistidyllysine and fibronectin. Morphologic analysis of glomerular cells grown in either of these chemically defined media provided identical results with respect to cell growth properties and cell types involved. Electron microscopic studies of glomeruli early after they had been placed in culture showed definite evidence of "dedifferentiation" of some glomerular cells. Most glomerular cells in later cultures were undifferentiated. However, since electron microscopic analyses of glomeruli in confluent cultures demonstrated that the majority of cells in culture grow from the epithelial side of the glomerular basement membrane, we suggest that these cells were some form of epithelial cell. This conclusion was further strengthened by the fact that cells resembling well differentiated glomerular epithelial cells were seen in cultures of glomeruli grown in chemically defined media; these cells have never been observed in glomeruli grown in calf serum. Fluorescent microscopy of cell stained with the mitochondrial stain rhodamine 123 allowed identification of several glomerular cell types according to distribution, number, and morphology of mitochondria. Similarly, indirect immunofluorescent microscopy studies using antibodies to fibronectin or laminin provided evidence that glomerular cells separated into cell types according to mitochondrial staining properties were unique biochemically. Using these histochemical criteria it was possible to demonstrate that certain of the glomerular cell types could be selectively grown by addition of the enzyme galactose oxidase to the media. Analysis of our morphologic and histochemical results suggests the possibility that clonal growth and differentiation of glomerular epithelial cells occurs when glomeruli are placed in chemically defined media, and our results are compatible with the hypothesis that either "stem cells" or "dedifferentiated" cells are the primary cells dividing in culture.     

A possible ligand of serum origin for the kidney autoantigen of Heymann nephritis

Earlier studies have localized the Heymann nephritis (HN) autoantigen (gp330) in the coated pits of the plasma membrane and multivesicular bodies of the glomerular epithelial cell. Because of these locations in the glomerular epithelial cells, it has been suggested that the HN Ag may be a receptor. The aim of our study was to search for a ligand which can bind the HN autoantigen. Normal rat serum was subjected to SDS-PAGE under reducing and non-reducing conditions followed by Western analysis of the separated polypeptides. A reaction was revealed directly by autoradiography using 125I labeled HN autoantigen as a probe and indirectly by enzyme immunodetection using unlabeled nephritogenic autoantibody (anti-gp330) eluted from glomeruli of diseased rats followed by biotinylated rabbit anti-rat IgG avidin-peroxidase complex. A polypeptide of 76 kDa Mr was identified under non-reducing conditions as a serum protein reacting with the HN autoantigen. Reactivity of the 76-kDa polypeptide was lost when serum was electrophoresed under reducing conditions. Direct binding of the 76-kDa polypeptide obtained from serum to the HN autoantigen obtained from kidney suggests that the 76-kDa polypeptide may be a ligand for the autoantigen. This is the first documentation of a possible ligand for the HN autoantigen. Not only does this polypeptide bind to the HN autoantigen but it also shows direct binding with the nephritogenic autoantibody eluted from glomerular deposits. This characteristic of the 76-kDa polypeptide indicates that this serum protein may potentially play a role in the development of the glomerular lesion of active HN. Further analysis of this serum component should assist in understanding the normal function of the HN autoantigen.