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.     

Self-assembly of basement membrane collagen

The in vitro self-assembly of murine type IV collagen was examined by using biochemical and morphological techniques. Dimeric collagen undergoes a rapid and reversible thermal gelation at neutral pH without an appreciable lag period. The process is seen to be concentration dependent and inhibited by 2 M urea. The formed complex can be visualized by electron microscopy rotary shadowing as an irregular polygonal lattice network with extensive side by side associations within the collagenous triple-helical part of the molecules, two and three strands thick. Measurements on the matrix suggest a median stagger dimension of 170 nm, one-fifth the length of a dimer. The conversion of pepsin-generated monomers into N-terminally bound tetramers can also be demonstrated in vitro. This process is also concentration dependent and inhibited and reversed by 2 M urea but is thermally irreversible and occurs at a slow rate relative to the lateral associations. These tetramers can be seen by rotary shadowing as four-armed "spider" structures. It is proposed that lateral associations, by virtue of their faster rate of formation, precede 7S bond formation, and several models for the assembly of basement membrane collagen are discussed.     

Reduced synthesis of basement membrane heparan sulfate proteoglycan in streptozotocin-induced diabetic mice

In diabetes, certain basement membranes become thicker yet more porous than normal. To identify possible changes in the basement membrane, we have grown the Engelbreth-Holm-Swarm tumor, a tissue that produces quantities of basement membrane in normal mice and in streptozotocin-treated, insulin-deficient, diabetic mice. The level of laminin, a basement membrane-specific glycoprotein, and the level of total protein were slightly elevated in the diabetic tissue. In contrast, the level of the basement membrane specific heparan sulfate proteoglycan was only 20% of control. The synthesis of this proteoglycan was also reduced in the diabetic animals, while the synthesis of other proteoglycans by tissues such as cartilage was normal. The synthesis of the heparan sulfate proteoglycan in diabetic animals was inversely related to plasma glucose levels showing an abrupt decrease above the normal range of plasma glucose. Insulin restored synthesis to normal but this required doses of insulin that maintained plasma glucose at normal levels for several hours. Since the heparan sulfate proteoglycan in the basement membrane restricts passage of proteins, its absence could account for the increased porosity of basement membrane in diabetes. A compensatory synthesis of other components could lead to their increased deposition and the accumulation of basement membrane in diabetes.     

Increased susceptibility of decay-accelerating factor deficient mice to anti-glomerular basement membrane glomerulonephritis

To prevent complement-mediated autologous tissue damage, host cells express a number of membrane-bound complement inhibitors. Decay-accelerating factor (DAF, CD55) is a GPI-linked membrane complement regulator that is widely expressed in mammalian tissues including the kidney. DAF inhibits the C3 convertase of both the classical and alternative pathways. Although DAF deficiency contributes to the human hematological syndrome paroxysmal nocturnal hemoglobinuria, the relevance of DAF in autoimmune tissue damage such as immune glomerulonephritis remains to be determined. In this study, we have investigated the susceptibility of knockout mice that are deficient in GPI-anchored DAF to nephrotoxic serum nephritis. Injection of a subnephritogenic dose of rabbit anti-mouse glomerular basement membrane serum induced glomerular disease in DAF knockout mice but not in wild-type controls. When examined at 8 days after anti-glomerular basement membrane treatment, DAF knockout mice had a much higher percentage of diseased glomeruli than wild-type mice (68.8 +/- 25.0 vs 10.0 +/- 3.5%; p < 0.01). Morphologically, DAF knockout mice displayed increased glomerular volume (516 +/- 68 vs 325 +/- 18 x 10(3) microm(3) per glomerulus; p < 0.0001) and cellularity (47.1 +/- 8.9 vs 32.0 +/- 3.1 cells per glomerulus; p < 0.01). Although the blood urea nitrogen level showed no difference between the two groups, proteinuria was observed in the knockout mice but not in the wild-type mice (1.4 +/- 0.7 vs 0.02 +/- 0.01 mg/24 h albumin excretion). The morphological and functional abnormalities in the knockout mouse kidney were associated with evidence of increased complement activation in the glomeruli. These results support the conclusion that membrane C3 convertase inhibitors like DAF play a protective role in complement-mediated immune glomerular damage in vivo.     

Cis 3 hydroxyproline reduces glomerular basement membrane thickness and collagen type IV synthesis in diabetic rats

Summary Glomerular basement membrane thickening is thought to be due to increased collagen synthesis and abnormal cross linking. Based upon the observation that the incorporation of distinct proline analogues leads to increased degradation of the newly abnormally formed collagen we administered cis 3 hydroxyproline orally to streptozotocin diabetic rats.Measuring glomerular basement membrane thickness we found in the treated group significantly lower values. The solubility of collagen in the treated group was significantly increased, indicating the mechanism of action of the proline analogue. The collagen content of kidneys in the treated group was reduced as well correlating with the basement membrane thickness. Provided the absence of toxicity of cis 3 hydroxyproline its pharmaceutical use for the inhibition of basement membrane proliferation seems promising.     

Attachment of cells to basement membrane collagen type IV

Of ten different cell lines examined, three showed distinct attachment and spreading on collagen IV substrates, and neither attachment nor spreading was enhanced by adding soluble laminin or fibronectin. This reaction was not inhibited by cycloheximide or antibodies to laminin, indicating a direct attachment to collagen IV without the need of mediator proteins. Cell-binding sites were localized to the major triple-helical domain of collagen IV and required an intact triple helical conformation for activity. Fibronectin showed preferential binding to denatured collagen IV necessary to mediate cell binding to the substrate. Fibronectin binding sites of collagen IV were mapped to unfolded structures of the major triple-helical domain and show a similar specificity to fibronectin-binding sites of collagen I. The data extend previous observations on biologically potential binding sites located in the triple helix of basement membrane collagen IV.     

Deletion of the basement membrane heparan sulfate proteoglycan type XVIII collagen causes hypertriglyceridemia in mice and humans

Background

Lipoprotein lipase (Lpl) acts on triglyceride-rich lipoproteins in the peripheral circulation, liberating free fatty acids for energy metabolism or storage. This essential enzyme is synthesized in parenchymal cells of adipose tissue, heart, and skeletal muscle and migrates to the luminal side of the vascular endothelium where it acts upon circulating lipoproteins. Prior studies suggested that Lpl is immobilized by way of heparan sulfate proteoglycans on the endothelium, but genetically altering endothelial cell heparan sulfate had no effect on Lpl localization or lipolysis. The objective of this study was to determine if extracellular matrix proteoglycans affect Lpl distribution and triglyceride metabolism.

Methods and Findings

We examined mutant mice defective in collagen XVIII (Col18), a heparan sulfate proteoglycan present in vascular basement membranes. Loss of Col18 reduces plasma levels of Lpl enzyme and activity, which results in mild fasting hypertriglyceridemia and diet-induced hyperchylomicronemia. Humans with Knobloch Syndrome caused by a null mutation in the vascular form of Col18 also present lower than normal plasma Lpl mass and activity and exhibit fasting hypertriglyceridemia.

Conclusions

This is the first report demonstrating that Lpl presentation on the lumenal side of the endothelium depends on a basement membrane proteoglycan and demonstrates a previously unrecognized phenotype in patients lacking Col18.     

Increased degradation of dermal collagen in diabetic rats.

The effect of alloxan induced diabetes on the dermal collagen content of albino rats was studied in relation to few lysosomal enzymes. Diabetes decreased the dermal collagen content. The specific activities of the lysosomal enzymes studied in the diabetic rat skin were elevated. It has been established that lysosomal enzymes degrade the connective tissue components. Thus, it may be suggested that the increase in the lysosomal enzymes studied should have facilitated the decrease in dermal collagen content of diabetic rats by increasing the degradation of dermal collagen.     

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.     

Structural heterogeneity of the noncollagenous domain of basement membrane collagen

The noncollagenous domain of collagen from three different basement membranes of bovine origin (glomerular, lens capsule, and placental) was excised with bacterial collagenase, purified under nondenaturing conditions, and characterized. In each case the domain existed as a hexamer comprised of four distinct subunits (alpha 1 (IV) NC1, alpha 2 (IV) NC1, M2*, and M3). Each subunit exists in both monomeric and dimeric (disulfide-cross-linked) forms. Certain dimers also exist which contain nonreducible cross-links. The hexamers from the three membranes differ with respect to stoichiometry of subunits and subunit isoforms and to the degree of cross-linking of monomers into dimers. The minor subunits, M2* and M3, vary in quantity over a 20-fold range relative to the major ones among the three hexamers. The results indicate that: 1) at least two populations of triple-helical collagen molecules, differing in chain composition, exist in each membrane and that their relative proportions are tissue-specific; and 2) the chemical nature of the noncollagenous domain of these populations is tissue-specific with regard to subunit isoforms and relative proportion of reducible and nonreducible cross-links in dimers. A novel structural feature of the noncollagenous domain of basement membrane collagen was also evinced from these studies. Namely, that each of the four monomeric subunits exists in charge isoforms.     

Macromolecular organization of collagen fibres in natural and tanned basement membrane

The elastic constants and ultrastructure of natural and tanned basement membrane of the crystalline lens of the adult rat have been investigated. Sonicated and negatively stained specimens of both membranes show parallel filaments that have similar spacing of 3.5(+/- 0.1) nm and a different periodicity. In natural membrane the periodicity is 3.7(+/- 0.13) nm, whilst in tanned basement membrane the periodicity is 3.2(+/- 0.15) nm. The periodicity ratio of tanned membrane to natural membrane was 0.86 +/- 0.04, whilst the elongation ratio of tanned membrane compared with natural membrane was 0.88 +/- 0.05. In contrast to this, the thickness ratio of tanned to natural membrane was 1.098 +/- 0.045. Tanned basement membrane showed a shrinkage of 12% in length but an increase in thickness of about 10%. These data suggest, firstly, that the degree of extension of the superhelices of the filaments follows closely the degree of extension of the intact membrane and, secondly, that the coiled superhelices of tanned membrane have an angle of tilt of about 42 degrees compared with those of natural membrane, where the angle is about 50 degrees. The Young's modulus of elasticity and ultimate stress of tanned basement membrane are, respectively, eight times greater and one-third as great as natural membrane. The entropy change in basement membrane was calculated from the external work necessary to extend the tanned membrane, and was estimated to be -13.5(+/- 2.4) J K-1 mol-1. An estimate of the change in entropy from thermodynamic measurements made on a suspension of collagen tanned with glutaraldehyde was found to be -30.1(+/- 9.5) J K-1 mol-1. The two different estimates of the change in entropy of collagen following tanning suggest that in basement membrane only about 45% of the collagenous protein has an extensile helical structure.     

Modifications of glomerular basement membrane cross-links in experimental diabetic rats

Glomerular basement membranes were isolated from normal and streptozotocin-diabetic rats. after elimination of non collagenous components with 8M urea the extent of ketoamine-linked hexoses and cross-links in the preparation was determined using NaB³H₄ reduction and radiochromatography. The observed increase in the level of N-glycosylation appeared to be closely related to a lowered amount of intermolecular cross-links in diabetic rats as compared to normal animals. Such a glycosylation may affect the stability and physicochemical properties of G.B.M. and thereby contribute to the vascular sequelae of diabetes mellitus.     

Isolation and partial characterization of a novel basement membrane collagen

A guanidine-HCl extraction of lens capsule basement membrane dissolves collagenous material. This material was fractionated on an Agarose A-5M column. Fractions 1, 2 and 3 were further purified and partially characterized immunochemically and by amino acid analysis. Fraction 3 has a molecular weight of 55,000 when compared with collagen type I standard. The CNBr peptide pattern and composition of fraction 3 are different from those of alpha 1 (IV) 95K and alpha 2 (IV) 95K chains. The results described suggest the presence of a new chain in lens capsule basement membrane.     

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 anttigen 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.     

Calcium-dependent binding of basement membrane protein BM-40 (osteonectin, SPARC) to basement membrane collagen type IV

Basement membrane protein BM-40, prepared from the mouse Engelbreth-Holm-Swarm tumor, was used in native, denatured and proteolytically processed form for binding to various extracellular matrix proteins. BM-40 and its derivatives were also characterized by CD spectroscopy, calcium binding and epitope analysis. Of several basement membrane proteins tested only collagen IV showed a distinct and calcium-dependent binding of BM-40 in an immobilized ligand assay. This interaction was specific as shown by a low activity of other collagen types (I, III, V, VI) in direct binding and competition assays. The binding was reduced or abolished by metal-ion-chelating or chaotropic agents, high salt and reduction of disulfide bonds in BM-40. Fragment studies indicated that domains III (alpha-helix) and/or IV (EF hand) of BM-40 possess the binding site(s) for collagen IV, while the N-terminal domains I and II provide the major antigenic determinants. A major BM-40-binding site on collagen IV was dependent on a triple-helical conformation and could be localized to a pepsin fragment from the central portion of the triple-helical domain, in agreement with electron microscopic visualization of BM-40--collagen-IV complexes.     

Biosynthesis of basement membrane collagen by rabbit corneal endothelium in vitro.

The synthesis of collagen has been demonstrated in endothelial cells of Descemet's membrane isolated from rabbit cornea. Incorporation of [14C]proline and [14C]lysine into nondialyzable protein was measured in the medium and cell fraction after incubating Descemet's membrane for up to 5 hours. In the [14C]collagen synthesized by the endothelium, 15% of the hydroxy[14C]proline was present as the 3-isomer. About 98% of the hydroxy[14C]lysine in the 14C-labeled-protein found in the medium was glycosylated; 95% of the glycosylated hydroxy[14C]lysine was in the form of the disaccharide glucosyl-galactosyl-hydroxy[14C]lysine. Time course experiments with [14C]proline indicated that there was a delay of about 60 min before significant amounts of [14C]collagen were secreted into the medium. The initial polypeptides of [14C]collagen synthesized by the corneal endothelium had an apparent molecular weight of 155,000. The chemical and physical properties of the [14C]collagen synthesized by rabbit corneal endothelium are consistent with those of basement membrane collagen synthesized by other cell types.