Human glomerular basement membrane. Antigenic determinants in human urine

Antisera against particulate human glomerular basement membrane prepared from cadaver kidneys were raised in rabbits. It was shown that both normal individuals and patients with glomerular and tubular diseases excrete in their urine several antigens reactive with these antibodies. One antigen crossreacted immunologically with an antigen from human glomerular basement membrane while several others did not. One of the urinary antigens and the antigen crossreacting with the basement membrane were separated from the others by ion exchange chromatography and gel filtration, respectively.The pattern of antigen excretion differed depending on the underlying renal disease but the multitude of different antigens detected complicates the interpretation of the patterns of excretion in different diseases.     

Human glomerular basement membrane. Heterogeneity of antigenic determinants

Human glomerular basement membrane was solubilized by digestion with proteolytic enzymes and immunoreactive components were quantitated and characterized by using rabbit antibodies raised against the particulate membrane. A number of antigens were demonstrated but they did not separate on gel filtration. However, two antigenic components in a collagenase digest of the membrane could be separated and isolated by Sepharose 6B chromatography. Chemical characterization suggests that both fragments are noncollagenous glycopeptides (molecular weights approx. 1 000 000 and 60 000–200 000, respectively).     

Human glomerular basement membrane. Heterogeneity of antigenic determinants.

Human glomerular basement membrane was solubilized by digestion with proteolytic enzymes and immunoreactive components were quantitated and characterized by using rabbit antibodies raised against the particulate membrane. A number of antigens were demonstrated but they did not separate on gel filtration. However, two antigenic components in a collagenase digest of the membrane could be separated and isolated by Sepharose 6B chromatography. Chemical characterization suggests that both fragments are noncollagenous glycopeptides (molecular weights approx. 1,000,000 and 60,000--200,000, respectively).     

Reducible cross-links in human glomerular basement membrane

Reducible cross-links in purified human glomerular basement membrane (GBM) were examined with an ion exchange chromatographic system that provided complete separation of cross-link standards and glucosylamines. After hydration in phosphate buffer, lyophilized GBM was reduced with tritiated borohydride. Chromatographic separation revealed two major radioactive peaks, identified as di-hydroxylysinonorleucine (di-OHLNL) and hydroxyaldolhistidine (HAH) by coelution with authentic di-OHLNL and HAH standards. Radioactive glucitol-lysine and glucitol-hydroxylysine were also identified on the basis of their co-elution with synthetic standards. The findings document the existence and establish the nature of the major reducible cross-links in adult human GBM.     

The glomerular basement membrane

The kidney's glomerular filtration barrier consists of two cells-podocytes and endothelial cells-and the glomerular basement membrane (GBM), a specialized extracellular matrix that lies between them. Like all basement membranes, the GBM consists mainly of laminin, type IV collagen, nidogen, and heparan sulfate proteoglycan. However, the GBM is unusually thick and contains particular members of these general protein families, including laminin-521, collagen α3α4α5(IV), and agrin. Knockout studies in mice and genetic findings in humans show that the laminin and type IV collagen components are particularly important for GBM structure and function, as laminin or collagen IV gene mutations cause filtration defects and renal disease of varying severities, depending on the nature of the mutations. These studies suggest that the GBM plays a crucial role in establishing and maintaining the glomerular filtration barrier.     

Heparan sulfate proteoglycan from human tubular basement membrane. Comparison with this component from the glomerular basement membrane

Heparan sulfate proteoglycan (HSPG) was extracted from human tubular basement membrane (TBM) with guanidine and purified by ion-exchange chromatography and gel filtration. The glycoconjugate was sensitive to heparitinase and resistant to chondroitinase ABC, had an apparent molecular mass of 200-400 kDa and consisted of 70% protein and 30% glycosaminoglycan. The amino acid composition was characterized by its high content of glycine, proline, alanine and glutamic acid. Hydrolysis with trifluoromethanesulfonic acid yielded core proteins of 160 and 110 kDa. The heparan sulfate (HS) chains obtained after alkaline NaBH4 treatment had a molecular mass of about 18 kDa. Results of heparitinase digestion and HNO2 treatment suggest a clustering of sulfate groups in the distal portion of the HS side chains. These chemical data are comparable to those obtained previously on glomerular basement membrane (GBM) HSPG (Van den Heuvel et al. (1989) Biochem. J. 264, 457-465). Peptide patterns obtained after trypsin, clostripain or V8 protease digestion of TBM and GBM HSPG preparations showed a large similarity. Polyclonal antisera and a panel of monoclonal antibodies raised against both HSPG preparations and directed against the core protein showed complete cross-reactivity in ELISA and on Western blots. They stained all basement membranes in an intense linear fashion in indirect immunofluorescence studies on human kidneys. Based on these biochemical and immunological data we conclude that HSPGs from human GBM and TBM are identical, or at least very closely related, proteins.     

Ultrastructural development features of the human glomerular basement membrane

In the human embryo, the basement glomerular membrane appears early, at about ten weeks of age. The study of 36 human embryos aged 8-20 weeks revealed many arguments suggesting the epithelial origin of the basement membrane of the visceral capsula.     

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.     

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.     

Clogging of the glomerular basement membrane

The negative charges of the sulfated glycosaminoglycans (GAGs) of the glomerular basement membrane (GBM) were differentially neutralized by perfusin with high molarity buffers in order to determine whether or not these charges protect the GBM from being clogged by circulating plasma macromolecules. Progressive elimination of the negative charges resulted in clogging of the GBM by perfused native ferritin (NF) and bovine serum albumin as evidenced ultrastructurally by the increase in accumulation of NF in the GBM. In addition, the permeability of the GBM to 125I-insulin, a macromolecule which is normally freely permeable, and the glomerular filtration rate (as determined by [3H]inulin clearance) were markedly reduced after the GBM had been clogged with NF in the presence of high molarity buffer, thereby indicating that clogging severely reduces the ability of the GMB to act as a selective filter. These findings are consistent with the idea that the sulfated GAGs of the GBM serve as anticlogging agents.     

Human kidney cathepsins B and L. Characterization and potential role in degradation of glomerular basement membrane.

Cathepsins B and L were purified from human kidney. SDS/polyacrylamide-gel electrophoresis demonstrated that cathepsins B and L, Mr 27000-30000, consist of disulphide-linked dimers, subunit Mr values 22000-25000 and 5000-7000. The pH optimum for the hydrolysis of methylcoumarylamide (-NHMec) substrates (see below) is approx. 6.0 for each enzyme. Km and kcat. are 252 microM and 364s-1 and 2.2 microM and 25.8 s-1 for the hydrolysis of Z-Phe-Arg-NHMec (where Z- represents benzyloxycarbonyl-) by cathepsins B and L respectively, and 184 microM and 158 s-1 for the hydrolysis of Z-Arg-Arg-NHMec by cathepsin B. A 10 min preincubation of cathepsin B (40 degrees C) or cathepsin L (30 degrees C) with E-64 (2.5 microM) results in complete inhibition. Under identical conditions Z-Phe-Phe-CHN2 (0.56 microM) completely inhibits cathepsin L but has little effect on cathepsin B. Incubation of glomerular basement membrane (GBM) with purified human kidney cathepsin L resulted in dose-dependent (10-40 nM) GBM degradation. In contrast, little degradation of GBM (less than 4.0%) was observed with cathepsin B. The pH optimum for GBM degradation by cathepsin L was 3.5. Cathepsin L was significantly more active in degrading GBM than was pancreatic elastase, trypsin or bacterial collagenase. These data suggest that cathepsin L may participate in the lysosomal degradation of GBM associated with normal GBM turnover in vivo.     

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 ?-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.     

Analysis of nephritogenic antigens in human glomerular basement membrane by two-dimensional gel electrophoresis

Collagenase digests of GBM were partially purified by column chromatography and analyzed by 2-D gel electrophoresis. Silver staining of 2-D gels showed charge- and size-related heterogeneity of proteins in the 45 to 50 kDa and 25 to 27 kDa regions. These components were transferred to nitrocellulose sheets and reacted with 10 human anti-GBM autoantibodies. Detection of bound anti-GBM autoantibodies to blotted proteins was carried out with peroxidase-labeled goat anti-human IgG and revealed binding predominantly to the cationic (pI 8 to 9.0) 45 to 50 kDa and 25 to 27 kDa components. Positive-staining patterns of blotted proteins were similar with all anti-GBM autoantibodies except that three sera additionally identified neutral (pI 5.5 to 6.5) protein components. One anti-GBM autoantibody, which developed following renal transplantation, lacked reactivity with the most cationic components in the 25 to 27 kDa region. These findings suggest heterogeneity of nephritogenic GBM antigens. The cationic 45 to 50 kDa components were sensitive to reduction, while one neutral 45 to 50 kDa component was resistant; a complex array of 25 to 30 kDa proteins (pI 5.5 to 7.5) were observed by silver staining postreduction. None of the reduced protein components reacted with anti-GBM antibodies, suggesting that epitopes on nephritogenic GBM antigens may be related to disulfide-bonded regions. Although there is variable immunohistochemical reactivity of anti-GBM autoantibodies with the GBM of infant kidneys, 2-D gels of collagenase-digested human infant GBM blotted and reacted with anti-GBM autoantibodies and showed staining patterns similar to that of adult GBM. These studies demonstrate the presence of nephritogenic antigens in the GBM of immature human kidney which are not detectable by immunohistochemical analysis.     

Isolation and partial characterization of heparan sulphate proteoglycan from the human glomerular basement membrane.

Heparan sulphate proteoglycan was solubilized from human glomerular basement membranes by guanidine extraction and purified by ion-exchange chromatography and gel filtration. The yield of proteoglycan was approx. 2 mg/g of basement membrane. The glycoconjugate had an apparent molecular mass of 200-400 kDa and consisted of about 75% protein and 25% heparan sulphate. The amino acid composition was characterized by a high content of glycine, proline, alanine and glutamic acid. Hydrolysis with trifluoromethanesulphonic acid yielded core proteins of 160 and 110 kDa (and minor bands of 90 and 60 kDa). Alkaline NaBH4 treatment of the proteoglycan released heparan sulphate chains with an average molecular mass of 18 kDa. HNO2 oxidation of these chains yielded oligosaccharides of about 5 kDa, whereas heparitinase digestion resulted in a more complete degradation. The data suggest a clustering of N-sulphate groups in the peripheral regions of the glycosaminoglycan chains. A polyclonal antiserum raised against the intact proteoglycan showed reactivity against the core protein. It stained all basement membranes in an intense linear fashion in immunohistochemical studies on frozen kidney sections from man and various mammalian species.