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At least 19 recordsLinked to original sources
PubMed1990

Isolation and characterization of mouse glomerular basement membrane.

Glomerular basement membrane (GBM) antigens used for immunological studies have until now been isolated from human and rat glomeruli but not from mice. However, given the growing awareness of extracellular matrix species-specificity, the need for a purification method of mouse GBM exists. We now report the purification of GBM from isolated mouse glomeruli. 10 ml 0.01 M phosphate buffered saline (PBS) containing 1.25% Fe3O4 was perfused through the aortae of (C57BL10 x DBA/2)F1 mice over 1 min, kidneys were decapsulated and passed through a 0.075 mm mesh metal screen and collected. After pelletting and washing, the tube was placed against one pole of a magnet and the pelleted glomeruli were washed. This procedure was repeated three times. The glomerular suspension was examined by light microscopy, sonicated, and lyophilized. In suspension no free fragments of tubuli or Bowman's capsule were present as observed by light microscopy. Mouse GBM was prepared from the isolated glomeruli using a modification of earlier described methods for human and rat GBM by enzymatic digestion. Elisa studies showed the presence of laminin, type IV collagen, and fibronectin. Monoclonal antibody directed against tubular epithelial antigen gp160 did not react to either mouse or rat GBM. Sera and glomerular eluates from (C57BL10 x DBA/2)F1 mice suffering from chronic graft-versus-host autoimmune disease gave a higher signal against mouse GBM than against rat GBM. Normal rabbit serum, normal mouse serum and normal mouse eluate did not show significant activity against mouse GBM. Immunoblotting showed the presence of both laminin B1 and B2 chains as well as type IV collagen alpha 1 and alpha 2 subunits. In summary, we describe a method by which it is possible to extract mouse GBM with a high purity.

Animals↗
PubMed1980

Differential solubility and subunit composition of rat glomerular basement membrane.

Glomerular basement membrane (GBM) was prepared from 2- to 3-month-old Sprague-Dawley rats by differential sieving and sonication. 80% of the membrane was soluble in 1% sodium dodecyl sulphate (SDS) and 1% 2-mercaptoethanol. The soluble fraction was resolved into 15 bands in the molecular weight range 30,000 to 300,000 by SDS-polyacrylamide gel electrophoresis. The major bands present had apparent molecular weights of 100,000 and 148,000. Treatment of GBM with SDS alone solubilised mainly low molecular weight components (45,000-150,000) but when the residue was treated with SDS and 2-mercaptoethanol higher molecular weight material was solubilised. Partial solubilisation of GBM was also achieved with pepsin. Digestion for 18 h at 4 degrees C resulted in 20% of the membrane being solubilised but this was increased to 55% at 10 degrees C. The amino-acid composition of pepsin-soluble GBM was more collagen-like than the residue remaining after enzyme digestion. Although the residue was more polar than whole GBM it still contained significant amounts of glycine and hydroxyproline and could be further subfractionated with SDS into a soluble fraction, the amino acid content of which was similar to whole GBM and a collagenous residue containing 317 residues/1,000 of glycine. When pepsin solubilised GBM was subjected to horizontal electrophoresis in SDS-polyacrylamide slab gels the principal bands migrated in the pro-alpha-chain region. This material was heterogeneous and in addition to the principal components, 6 components with apparent molecular weights less than 95,000 were present together with high molecular weight material in the gamma and beta regions of the gel. The band pattern of the pepsin-insoluble material was similar although the intensities of some individual bands varied significantly from that of the pepsin-soluble material. Rat GBM can therefore be fractionated by treatment with SDS alone, SDS together with 2-mercaptoethanol and pepsin digestion. The data reported is compatible with the presence of a mixture of collagen-like and polar regions rather than a major single collagenous component (type IV collagen).

Amino Acids↗
PubMed1983

Intermolecular reducible cross-links in rat glomerular basement membrane.

Glomerular basement membrane (GBM) was purified from adult rats and treated with tritiated borohydride for analysis of the reducible cross-links. After acid hydrolysis, samples were subjected, with and without prior gel filtration on Biogel P-2, to a chromatographic system standardized with known cross-links. The major peak of radioactivity co-eluted with authentic di-hydroxylysinonorleucine (di-OHLNL) standard. Derivation of this cross-link from lysine was corroborated by demonstrating its presence in GBM purified after incubation of isolated glomeruli with [14c]-lysine and after in vivo injection of [14C]-lysine. GBM labeled in vitro with radioactive lysine also contained peaks co-eluting with hydroxylysinonorleucine and lysinonorleucine standards, as well as at unidentified positions in the chromatogram. The findings indicate that rat GBM contains lysine-derived cross-links, of which di-OHLNL is the major reduced form.

Amino Acids↗
PubMed1984

Effect of diabetes on in vivo metabolism of [35S]-labeled glomerular basement membrane.

Glomerular basement membrane (GBM) was labeled in vivo by the injection of tracer amounts of [35S]-sulfate into normal and streptozotocin-diabetic rats. The biosynthesis and turnover of sulfated glycosaminoglycans in the GBM was determined from the specific activity of [35S] after pronase digestion of basement membranes purified from glomeruli isolated 1-7 days after injection. Peak radiolabeling of both normal and diabetic GBM occurred 24 h after injection and, when corrected for differences in serum sulfate specific activities, was less in diabetic than in normal samples. The specific activity of GBM sulfate, expressed as cpm/microgram uronic acid, progressively diminished over the ensuing period of study in both normal and diabetic samples. The rate of decrease in specific activity of [35S]-labeled GBM was not significantly different in diabetic preparations compared with that in normal controls. The findings are compatible with diminished sulfation and/or production but normal turnover of glycosaminoglycans in the renal GBM in experimental diabetes.

Animals↗
PubMed1976

Chemical properties of human glomerular basement membrane in the nephropathy with electron dense deposits inside the basement membrane.

Glomerular basement membranes containing electron dense deposits have been isolated from human kidneys. The presence of electron dense material could not be accounted for by the accumulation of an exogenous substance such as gamma globulin. Chemical analyses of abnormal membranes demonstrated that the electron microscopic findings are the consequence of the accumulation of a basement membrane glycoprotein material slightly different from that normally present.

Adult↗
PubMed1980

Amyloid P-component is a constituent of normal human glomerular basement membrane.

Glomerular and other vascular basement membranes were found to contain an antigen that was immunochemically indistinguishable from serum amyloid P-component. There was no immunological cross-reactivity between antisera to serum amyloid P-component and to collagen types I, III, IV, or V. The amyloid P-component antigen was confined to the endothelial aspect, the lamina rara interna, of glomerular basement membrane. It could not be eluted by high-ionic-strength saline, EDTA, dithiothreitol, or either polar or nonpolar detergents, but was released into solution when isolated glomerular basement membrane was digested by highly purified bacterial collagenase. Most of these P-component molecules and their constituent polypeptide chains were of higher molecular weight and lower isoelectric point than serum amyloid P-component. These findings indicate that, as well as being a normal plasma protein and a universal constituent of amyloid deposits, P-component is also a normal matrix glycoprotein of basement membrane in which it is covalently linked to collagen and/or other matrix proteins. This may be relevant both to the pathogenesis of amyloidosis and to other aspects of physiology and pathology of basement membranes.

Amyloid↗
PubMed2002

Regulation of adhesive interaction between podocytes and glomerular basement membrane.

Glomerular filtration depends on well-orchestrated cell-cell and cell-matrix contacts of glomerular podocytes. Over the last years critical constituents of these contacts have been identified via molecular approaches. Podocyte cell-matrix interactions have been shown to be mediated in part by alpha(3)beta(1)-integrin heterodimers. Disturbances of integrin matrix interaction lead to detachment of podocytes in vitro, corresponding to the critical event of foot process retraction and glomerular basement membrane (GBM) denudation in vivo. Further, dystroglycan-mediated matrix attachment appears to play a critical role for podocyte foot process architecture. Downstream signaling events are currently elucidated concentrating mainly on integrin-dependent cascades and their consequences for podocyte adhesion and proliferation. An activation of the integrin-linked kinase in podocyte damage in vivo and in vitro makes this molecule a particularly interesting candidate for integrin-mediated inside-out and outside-in signaling in podocytes. Podocyte cell-cell interaction has been characterized in a few studies in vitro, indicating the slit diaphragm to be a modified adherens junction. The structural link between the cell-matrix and cell-cell contacts is maintained by the actin cytoskeleton, which may also enable cross-talk between these two cell contact sites. Examining podocyte function in tissue culture, animal models and human expression studies should allow further detailed dissection of the molecular pathways responsible for maintenance and failure of the glomerular filtration barrier.

Animals↗
PubMed1979

Presence of heparan sulfate in the glomerular basement membrane.

The glomerular basement membrane was subjected to digestion with specific enzymes to determine the chemical nature (sialoglycoproteins, collagenous peptides, or glycosaminoglycans) of the anionic sites previously demonstrated in the laminae rarae. Enzyme digestion was carried out both in situ and in vitro. Kidneys were perfused in situ with enzyme solutions followed by perfusion with fixative containing the cationic dye, ruthenium red, to detect the anionic sites. Glomerular basement membranes were isolated by detergent treatment of glomeruli and incubated with enzyme solutions, followed by incubation with cationized ferritin (pI 7.3-7.5) to label the anionic sites. Only highly purified enzymes free of proteolytic activity were used. The findings were the same both in situ and in vitro. The anionic sites were unaffected by treatment with neuraminidase, chondroitinase ABC, and testicular or leech hyaluronidase. However, they could no longer be demonstrated after digestion with crude heparinase, purified heparitinase, or Pronase or after nitrous acid oxidation. The results demonstrate that the sites contain heparan sulfate since they are removed by treatment with heparitinase and by nitrous acid oxidation-procedures specific for heparan sulfate; and that sialoglycoproteins or other glycosaminoglycans do not represent major components of these sites since the latter are not affected by digestion with neuraminidase and other glycosaminoglycan-specific enzymes. Identical findings were obtained on basement membranes in other locations (Bowman's capsule, tubule epithelium, and endothelium of peritubular capillaries). The presence of heparan sulfate in the glomerular basement membrane is discussed in relation to the charge-selective properties of the glomerular filter and in relation to its potential involvement in various types of glomerular injury.

Animals↗
PubMed1992

Studies of the permeation properties of glomerular basement membrane: cross-linking renders glomerular basement membrane permeable to protein.

Cross-linking glomerular basement membrane (GBM) has been shown to render it more permeable to protein. Isolated pig GBM was cross-linked with dimethylmalonimidate which reacts selectively with lysine epsilon-NH2 groups or with glutaraldehyde, a less selective cross-linking agent. Studies of the ultrafiltration properties of these materials in vitro using cytochrome c, myoglobin, bovine serum albumin and immunoglobulin showed that cross-linking had markedly increased solvent and protein fluxes as compared with native membranes particularly at higher pressures. Filtration studies with serum demonstrated that the cross-linked membranes were more permeable to serum proteins. Thickness measurements under pressure indicated that cross-linked membrane was less compressed than native membrane as pressure was increased. Pore theory did not provide a suitable model for analysis of the results, but analysis of the results using the fibre-matrix hypothesis indicated that cross-linking had the effect of bundling together the fibres (type IV collagen) in the GBM matrix. The effect of cross-linking on filtration could be explained by a combination of contraction of the membrane, fibre bundling and increased rigidity compared with native membrane. Cross-linking of GBM might lead to long-term damage of the glomerular capillary wall in nephritis, so promoting proteinuria.

Animals↗
PubMed1982

In vivo biosynthesis and turnover of 35S-labeled glomerular basement membrane.

Renal glomerular basement membrane was labeled in vivo by the injection of tracer amounts of radioactive sulfate into normal adult rats. The biosynthesis and turnover of [35S]glycosaminoglycans in purified basement membrane was determined from the specific activity of 35S in pronase digests of basement membranes isolated 1-7 days after injection. Peak radioactive labeling occurred 24 h after injection following which the specific activity of basement membrane sulfate, expressed as cpm/microgram uronic acid, progressively declined over the ensuing period of study. The biologic half-life of radioactive sulfate in basement membrane was estimated at about 7 days, which is within the range previously reported for [35S]glycosaminoglycans in whole renal cortex. The findings indicate that 35S-labeled components of glomerular basement membrane have a relatively rapid turnover.

Animals↗
PubMed1980

Characterization of the collagenous domain of bovine glomerular basement membrane.

Bovine glomerular basement membrane was subjected to limited pepsin digestion and the solubilized collagenous polypeptides were characterized. Several electrophoresis systems were used which enabled an examination over a molecular weight range from 20,000 to greater than 10(6). A 0.1% SDS-5% polyacrylamide gel system resolves the reduced digestion product into 17 polypeptides ranging in molecular weight from 78,000 to 340,000. The larger collagenous components were resolved on a 0.1% SDS-2.5% agarose gel system. The nonreduced digestion product resolves into 13 components which vary in molecular weight from 85,000 to 5 million. Upon reduction, the majority of this material is converted to a 165,000 molecular weight component(s) and cross-linked (aldehyde derived) multimers of this component(s) containing as many as 6 cross-linked monomers. The digestion product was subjected to a second pepsin digestion after reduction and alkylation under nondenaturing conditions. This results in a conversion of a larger polypeptides to three lower molecular weight peptides, two of which exhibit an electrophoretic migration identical to alpha 1- and alpha 2-chains of collagen. The results indicate that the collagenous domain of glomerular basement membrane consists of various size collagen molecules connected by disulfide bonds and aldehyde-derived cross-links to form high molecular weight aggregates containing as many as 30 of these polypeptides, and that the larger collagenous polypeptides contain alpha-size segments within their structure.

Animals↗
PubMed1980

Biological thixotropy of glomerular basement membrane and the implications of thixotropic in explaining basement membrane permeability.

Glomerular basement membranes (GBMs) are considered to exhibit the characteristics of biological thixotropic systems, and because of this GBM permeability is determined by intravascular pressure. The significance of pressure as the major determinant of GBM permeability is discussed. Factors leading to GBM permeability to albumin following interruption of renal blood flow are examined, and a possible explanation for the phenomenon is proposed.

Animals↗
PubMed1991

Electron microscopic histochemical and immunochemical analyses of heparan sulfate proteoglycan distribution in renal glomerular basement membranes.

Renal glomerular basement membranes (GBMs) exhibit a charge-selective barrier, comprised of anionic sites, that restrict the passage of anionic molecules into the urine. These sites are located primarily in the laminae rarae interna (LRI) and externa (LRE) of the GBM and consist of heparan sulfate proteoglycan (HSPG). Previous efforts to localize HSPG core protein within various layers of the GBM have been contradictory. In the present study when rat renal cortex blocks were treated by immersion with the cationic probe, polyethyleneimine (PEI), GBMs exhibited anionic sites concentrated primarily in the LRE and more irregularly within the LRI and lamina densa. All sites were heparitinase sensitive indicating that PEI positive sites represent negatively charged groups associated with heparan sulfate. In order to gain information on the distribution of the HSPG protein core, antibodies to HSPG from the EHS tumor matrix [anti-(EHS) HSPG] and GBMs [anti-(GBM) HSPG] were used together with immunogold to label thin sections of Lowicryl embedded kidney cortex. Depending upon the antisera used, markedly different distributions of HSPG were obtained. Immunolabelling with anti-(GBM) HSPG suggested a distribution of HSPG which was restricted to the laminae rarae, whereas labelling with anti-(EHS) HSPG indicated that the protein core penetrates through all layers of the GBM.

Animals↗
PubMed1989

[Structure and antigenicity of the glomerular basement membrane].

The glomerular basement membrane is a complex extracellular matrix formed of various molecules which build a supramolecular network. The major structural components are collagen IV, laminin, heparan sulfate proteoglycan, and nidogen/entactin. Cross-reacting antibodies against laminin, nidogen, and collagen IV may occur after several infectious diseases. They are however of doubtful pathogenetic significance. The pathogenetic relevant autoantibodies in Goodpasture's syndrome and rapidly progressive glomerulonephritis with linear immunofluorescence pattern are directed against epitopes which are located on the collagenase resistant C-terminal globule NC1 of collagen IV. The human NC1 globule appears as a hexamer which dissociates into monomers and dimers under various experimental conditions. Dissociation is paralleled by a significant increase in available epitopes. Immunisation with the dissociated NC1 globule initiates a pulmo-renal syndrome in rabbits similar to the human Goodpasture's syndrome. In hereditary nephritis one of the alpha-chains which form the triple-helix of collagen IV seems to be altered within the NC1 region. This may possibly explain the typical morphologic findings in this disease as well as the reduced binding of antiglomerular basement membrane antibodies to basement membranes of kidneys in Alport's syndrome.

Animals↗
PubMed1980

Reduced susceptibility of nonenzymatically glucosylated glomerular basement membrane to proteases: is thickening of diabetic glomerular basement membranes due to reduced proteolytic degradation?

Glomerular basement membrane (GBM) preparations were enzymatically glucosylated and applied to proteolytic degradation by several enzymes. The split products were then characterized and quantitatively estimated by high pressure liquid chromatography. For this purpose, GBMs were isolated by a sieving and sonication method, incubated with glucose and digested with the proteases trypsin, chymotrypsin, papain, pepsin and a lysosomal preparation. Comparison of the concentrations of split products obtained by proteolytic degradation of normal and nonenzymatically glucosylated membranes showed a remarkable reduced susceptibility of the nonenzymatically glucosylated membranes, possibly due to steric hindrance or altered electrical charge of the glucosylated membrane proteins. This could be interpreted as an additional factor for accumulation of basement membrane material in the diabetic state, that not only increased basement membrane synthesis may occur but also reduced catabolism could possibly contribute to the diabetic changes.

Animals↗
PubMed1978

Cellular reactivity to altered glomerular basement membrane in glomerulonephritis.

Glomerular basement membrane may be altered during glomerulonephritis, exposing antigens that are recognized as foreign. Immunochemical studies suggest that removal of peripheral glycopeptides from the basement membrane with glycosidase mimics this pathogenetic event. To examine these hypotheses, we studied 24 patients with biopsy-proved glomerulonephritis by means of the lymphocyte-blast-transformation assay. Three preparations of normal glomerular basement membrane were used: two mimicked the native state for the peripheral glycopeptides, and one was altered by glycosidases. Results showed minimal differences in responses to native glomerular basement-membrane preparations among patients with glomerulonephritis and control groups. However, patients with glomerulonephritis had a significant blastogenic response to the glycosidase-treated glomerular basement membrane as compared to patients with nonglomerular renal disease and normal controls (P less than 0.0005). These studies suggest that cellular reactivity to altered glomerular basement-membrane antigens can be detected in certain forms of progressive glomerulonephritis.

Antigens↗
PubMed1980

Comparison of the chemical and polypeptide composition of tubular and glomerular basement membranes.

Renal tubular and glomerular basement membranes were isolated from rabbit, rat and mouse kidneys. Methods were developed to obtain the basement membrane from limited numbers of animals. Rabbit and mouse tubular basement membrane as well as rabbit and rat glomerular basement membrane were obtained from single animals in quantities sufficient for electrophoresis and chemical analysis. The chemical composition of all the basement membranes were compared and sodium dodecylsulfate-polyacrylamide gel electrophoresis was performed on rabbit tubular and glomerular basement membranes in order to compare their polypeptide composition. The chemical composition of the tubular basement membranes were similar, each species having nearly equal concentrations of glycine, hydroxyproline and hydroxylysine, respectively. Glomerular basement membranes were also similar to each other and close to tubular basement membranes in chemical composition. Within each species, the glycine, hydroxyproline and hydroxylysine values for tubular basement membrane were about 10% higher than for glomerular basement membrane. The polypeptide composition of a reduced, sodium dodecylsulfate-soluble fraction of the rabbit basement membranes appeared to be alike with two exceptions. A prominent band of Mr = 160,000 seen in gels of tubular basement membrane was present as a lightly staining band in glomerular basement membrane samples and a prominent band of Mr = 140,000 in gels of glomerular basement membrane was seen as a light band in samples of tubular basement membrane.

Animals↗