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.     

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 potential role of human kidney cortex cysteine proteinases in glomerular basement membrane degradation

(1) The degradation of glomerular basement membrane and some of its constituent macromolecules by human kidney lysosomal cysteine proteinases has been investigated. Three cysteine proteinases were extracted from human renal cortex and purified to apparent homogeneity. These proteinases were identified as cathepsins B, H and L principally by their specific activities towards Z-Arg-Arg-NHMec, Leu-NNap and Z-Phe-Arg-NHMec, respectively, and their Mr on SDS-polyacrylamide gel electrophoresis under reducing conditions. (2) Cathepsins B and L, at acid pH, readily hydrolysed azocasein and degraded both soluble and basement membrane type IV and V collagen, laminin and proteoglycans. Their action on the collagens was temperature-dependent, suggesting that they are only active towards denatured collagen. Cathepsin L was more active in degrading basement membrane collagens than was cathepsin B but qualitatively the action of both proteinases were similar, i.e., at below 32 degrees C the release of an Mr 400,000 hydroxyproline product which at 37 degrees C was readily hydrolysed to small peptides. (3) In contrast, cathepsin H had no action on soluble or insoluble collagens or laminin but did, however, hydrolyse the protein core of 35S-labelled glomerular heparan sulphate-rich proteoglycan. (4) Thus renal cysteine proteinases form a family of enzymes which together are capable of degrading the major macromolecules of the glomerular extracellular matrix.     

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.     

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.     

Increased glycosylation of glomerular basement membrane collagen in diabetes

Basement membrane was purified from glomeruli isolated from normal and streptozotocin-diabetic rats. After extraction of non-collagen protein with 8M urea, the extent of glycosylation in glomerular basement membrane collagen was determined with a specific colorimetric reaction that detects carbohydrate in ketoamine linkage with proteins. The level of glycosylation of glomerular basement membrane collagen purified from diabetic rats was significantly greater than that in non-diabetic animals. Increased basement membrane glycosylation may alter structure-function relationships of the capillary filtration barrier.     

Metal-binding properties of the isolated glomerular basement membrane

The nature of binding of metal cations to the glomerular basement membrane has been investigated using isolated bovine glomerular basement membrane. Highest-affinity binding for a number of ions is attributable to the glycosaminoglycans (mostly heparan sulfate) of the membrane. Some ions, such as divalent Mn, Ca and Ni, have specific binding sites on these polymers, while for others the ion-polyelectrolyte interaction is of a non-specific nature. Both structural and binding data indicate a linear charge density of close to unity for the heparan sulfate of the glomerular basement membrane, which at the ionic composition of the plasma filtrate corresponds to a polymer surface potential of about -45 mV. Several independent observations are better explained by a model of counter-ion condensation about the glycosaminoglycans than by conventional double layer theories. These include the valence dependence of ion binding, the sharp ejection of divalent ions at a critical concentration of La3+, and the relative insensitivity of 63Ni2+ binding to NaCl concentration in the neighbourhood of physiological ionic strength. In its interactions with metal ions, the glomerular basement membrane behaves like a dilute solution of polyelectrolytes. This conclusion has important consequences for the extent of charge reduction of the filtration barrier of the kidney, bathed as it is in an electrolyte solution of mainly monovalent salts.     

Synthesis of the glomerular basement membrane in the rat kidney

To study the origin and the formation of the glomerular basement membrane, autoradiographic investigations with³H-proline and³H-leucine have been performed in ultrathin and semithin sections of the glomeruli of 42 male rats. The results of this study indicate that, of the three cell types of the glomerulus, the epithelial cells (=podocytes) synthesize the proline-rich scleroproteins of the glomerular basement membrane. Our autoradiographic studies have yielded no evidence for participation of the endothelial or mesangial cells in the formation of the basement membrane. The mesangial cells appear to be responsible for the synthesis of the mesangial matrix only.     

Ultrastructural architecture of proteoglycans in the glomerular basement membrane. A cytochemical approach

Rat kidneys were perfused with fixative solutions containing either a) a polycationic dye (Alcian blue 8 GX, Astra blue 6 GLL, cuprolinic blue, ruthenium red), b) a monocationic dye (safranine 0), or c) Alcian blue in the presence of a 0.3 M MgCl2 concentration. Whereas solutions of a revealed the glomerular basement membrane proteoglycans as particles or threads 60 nm apart and arranged in a reticular pattern, solutions of b and c demonstrated new morphological aspects of these molecules. They appeared as tiny filamentous structures, about 100 to 160 nm long, ordered in a network-like pattern with a mesh of about 60-nm width. The filaments displayed lateral branches about 20 nm apart and about 25 nm long, projecting within the meshes. We suggest that the filamentous structures are the protein core, and the branches are the glycosaminoglycans of proteoglycan molecules. Because of this arrangement the negatively charged sites of the glomerular basement membrane would lie closer to each other than previously assumed.     

Ontogenesis of glomerular basement membrane: structural and functional properties

Protein A-gold immunocytochemistry was applied in combination with morphometrical approaches to reveal the alpha 1(IV), alpha 2(IV), and alpha 3(IV) chains of type IV collagen as well as entactin on renal basement membranes, particularly on the glomerular one, during maturation. The results have indicated that a heterogeneity between renal basement membranes appears during the maturation process. In the glomerulus at the capillary loop stage, both the epithelial and endothelial cell basement membranes were labeled for the alpha 1(IV) and alpha 2(IV) chains of type IV collagen and entactin. After fusion, both proteins were present on the entire thickness of the typical glomerular basement membrane. At later stages, the labeling for alpha 1(IV) and alpha 2(IV) chains of type IV collagen decreased and drifted towards the endothelial side, whereas the labeling for the alpha 3(IV) chain increased and remained centrally located. Entactin remained on the entire thickness of the basement membrane during maturation and in adult stage. The distribution of endogenous serum albumin in the glomerular wall was studied during maturation, as a reference for the functional properties of the glomerular basement membrane. This distribution, dispersed through the entire thickness of the basement membrane at early stages, shifted towards the endothelial side of the lamina densa with maturation, demonstrating a progressive acquisition of the permselectivity. These results demonstrate that modifications in the content and organization of the different constituents of basement membranes occur with maturation and are required for the establishment of the filtration properties of the glomerular basement membrane.     

Differential solubility of subcomponents of rat glomerular basement membrane

Marked differences were found in the electrophoretic profiles and amino-acid compositions of components prepared from rat glomerular basement membrane (GBM) by a number of different solubilization procedures. Treatment with reducing agent resulted in a simplified electrophoretic pattern which was characterized by the presence of a major collagenous component with a mol.wt, of 150 000. In contrast, detergent solubilized mainly lower-mol.-wt, material which had a more polar amino-acid composition. When both reagents were used together the majority of the basement-membrane material was soJubilized within 2 h and components with mol.wts, of 170 000 and 135 000 were predominant i n the pro- region of the gel. Treatment for a further 16 h was required to solubilize higher-mol.-wt, material and to achieve maximum solubility of components in the pro- region with mol.wts, of 185 000 and 150 000. These methods provide a means of separating subcomponents of rat GBM while avoiding the problems of degradation inherent in enzymatic procedures.