Identification of specific amino acids in diabetic glomerular basement membrane collagen subject to non-enzymatic glucosylation in vivo

Glomerular basement membrane was purified from normal and streptozotocin-diabetic rats, and the insoluble collagenous fraction isolated following reduction and alkylation. Amino acid analysis of diabetic samples revealed three unusual peaks eluting near glucosamine. Two of these peaks had respective elution times identical to those of synthetic glucitol-hydroxylysine and glucitol-lysine. These findings identify lysine-derived amino acids in glomerular basement membrane collagen as sites of excess non-enzymatic glucosylation $\text{in vivo}$ in hyperglycemic diabetes.     

Isolation and characterization of nephritogenic antigen from bovine glomerular basement membrane

A method for isolation of a potent nephritogenic antigen from bovine glomerular basement membrane has been established; the glomerular basement membrane was solubilized by trypsin digestion and fractionated successively by gel filtration on Ultrogel AcA-34, concanavalin A affinity chromatography and affinity chromatography on immobilized antibodies. The antigen thus prepared was found to be highly nephritogenic; it causes glomerulonephritis in rats by a single injection of 0.1 mg per individual. Amino acid and carbohydrate analyses revealed that the antigen is a glycoprotein which contains amino acids and sugars characteristic of collagen, namely, hydroxyproline, hydroxylysine, glycine, glucose and galactose, although the relative amounts of these amino acids and sugars are less than those found in Type IV collagen of glomerular basement membrane.     

Production and immunohistochemical characterization of monoclonal antibodies directed against renal basement membranes of rats

Basement membranes were separated from rat glomeruli and purified by mild procedures, which led to a highly enriched basement membrane fraction. Here, the production and characterization of five monoclonal antibodies against tubular and glomerular basement membranes are described. These antibodies were analyzed immunohistochemically on frozen sections of rat, bovine, and human kidneys as well as on rat embryos. One monoclonal antibody (BM O II) exclusively recognized the glomerular basement membranes, another one (BM O VII) bound to tubular basement membranes and to Bowman's capsule. Three antibodies (BM O IV, BM M II, BM M III) recognized their antigens in both glomerular and tubular basement membranes as well as in mesangial cells. The BM O II antibody showed a stringent species specificity and bound only to glomerular basement membranes of the rat. The other four antibodies cross-reacted with human and bovine glomerular basement membrane and mesangial antigens; they also bound to other tissues in the developing rat embryo. Antibody binding to specific purified components of the basement membranes such as collagen type IV, laminin, heparan sulphate proteoglycan, and fibronectin was investigated by enzyme-linked immunosorbent assay (ELISA). None of these antibodies reacted with any of these known basement membrane components, indicating that the antibodies may serve as useful tools in future investigations of so far unidentified components of basement membranes.     

Isolation, characterization and immunological determination of basement membrane-associated heparan sulfate proteoglycan

Basement membrane-associated heparan sulfate proteoglycan (HSPG) was extracted from isolated porcine glomerular basement membranes and purified by ion-exchange chromatography. The proteogycan was characterized by specific enzymatic digestions, by amino-acid analysis, by SDS-polyacrylamide gel electrophoresis and by density gradient centrifugation. Polyclonal antibodies were raised against the purified HSPG in rabbits. Antibodies were characterized by enzyme immunoassays, immunoprecipitation and immunohistological methods. They were shown to recognize specifically the core protein of HSPG from porcine, human and rat glomerular basement membrane but did not recognize HSPG from guinea pig or rabbit kidney. The affinity-purified antibodies did not cross-react with other basement membrane proteins like laminin, fibronectin or collagen type IV nor with chondroitin sulfate-rich or keratan sulfate-rich proteoglycans from human or bovine tissue. Using these antibodies an enzyme immunoassay was developed for determination of HSPG in the range of 1-100 ng/ml. Studies with cultured porcine endothelial cells showed that subendothelial basement membrane-associated HSPG may be determined with the enzyme immunoassay.     

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.     

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/μg 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.     

Transmembrane collagen XVII is a novel component of the glomerular filtration barrier

The kidney filtration barrier consists of the capillary endothelium, the glomerular basement membrane and the slit diaphragm localized between foot processes of neighbouring podocytes. We report that collagen XVII, a transmembrane molecule known to be required for epithelial adhesion, is expressed in podocytes of normal human and mouse kidneys and in endothelial cells of the glomerular filtration barrier. Immunoelectron microscopy has revealed that collagen XVII is localized in foot processes of podocytes and in the glomerular basement membrane. Its role in kidney has been analysed in knockout mice, which survive to birth but have high neonatal mortality and skin blistering and structural abnormalities in their glomeruli. Morphometric analysis has shown increases in glomerular volume fraction and surface densities of knockout kidneys, indicating an increased glomerular amount in the cortex. Collagen XVII deficiency causes effacement of podocyte foot processes; however, major slit diaphragm disruptions have not been detected. The glomerular basement membrane is split in areas in which glomerular and endothelial basement membranes meet. Differences in the expression of collagen IV, integrins α3 or β1, laminin α5 and nephrin have not been observed in mutant mice compared with controls. We propose that collagen XVII has a function in the attachment of podocyte foot processes to the glomerular basement membrane. It probably contributes to podocyte maturation and might have a role in glomerular filtration.     

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.     

Degradation of intact basement membranes by human and murine tumor enzymes

Homogenates from malignant tumors, obtained from surgery specimens or from transplants of Walker 256 carcinosarcoma in rats, contained an enzyme activity capable of degrading intact 3H-acetylated basement membranes from bovine lens. The enzyme activity from murine tumor was purified about 7500-fold by (NH4)2SO4 fractionation, ion exchange and gel chromatography. The apparent molecular weight of the purified enzyme was approximately 50,000. The rate of degradation of 3H-labelled basement membrane by the murine tumor enzyme was reduced by addition of excess type IV collagen, but not of excess type I, type III or type V collagen. These results suggested specificity of this enzyme for type IV collagen. Inhibitors of serine proteinases, thiol proteinases and soybean trypsin inhibitor were without effect on the enzyme activity. Chelators such as 1,10-phenanthroline or EDTA reduced the activity to control levels, indicating that the enzyme activity was due to a metalloproteinase. Chromatographic and electrophoretic separation of the enzymatic products from 3H-labelled basement membrane and type IV collagen indicated that the enzyme activity was due to a type IV collagenase. The use of basement membrane in the native physiological state as a substrate for the study of basement membrane-degrading activity by homogenates of solid malignant tumors offers an in vitro model for the investigation of the metastatic potential of these tumors.     

Immunohistochemical distribution of type VI collagen in developing human kidney

Summary The distribution of type VI collagen was investigated immunohistochemically in the developing human kidney from 15 to 32 weeks gestational age and it was compared with that observed in the normal infantile and adult human kidney. In fetal kidney, type VI collagen was widely distributed as a fibrillar network in the subcapsularly undifferentiated mesenchyme and intertubular interstitium, and as a basement membrane-like structure around the ureteral bud branches, tubules, and collecting ducts. During nephrogenesis, type VI collagen disappeared from the induced mesenchyme close to the tips of ureteral branches, while it formed a distinct basement membrane-like structure around the early stages of nephron differentiation (comma-shaped and S-shaped bodies) and later along Bowman's capsule of capillary loop and maturing glomeruli. A strong immureactivity for type VI collagen was also found in the glomerular basement membrane and mesangial areas of capillary loop and maturing glomeruli. In infantile kidney, type VI collagen showed a distribution pattern similar to that observed during the fetal period. In adult human kidney, glomerular basement membrane showed a weak positivity for type VI collagen and the basement membrane-like staining around Bowman's capsule, tubules, and collecting ducts was less evident than in fetal and infantle kidney. Our immunohistochemical findings suggest that type VI collagen is a normal component of the glomerular and extraglomerular extracellular matrix of developing human kidney and that it undergoes changes in the expression during maturation.     

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.     

Type VI collagen in glomeruli of short- and long-term experimental diabetic rats

Type VI collagen was revealed by high-resolution immunocytochemistry in renal glomeruli from short- and long-term streptozotocin-injected hyperglycaemic rats and from their age-matched normoglycaemic controls. The labellings obtained over the glomerular basement membrane and the mesangial matrix were assessed by quantitative evaluations. The labellings over the glomerular basement membrane were low and sparse in the young normoglycaemic animals but became consistent and increased in intensity with age and in both the short- and long-term diabetic animals. For the mesangial matrix, this was labelled more systematically, and its intensity increased with age and in the short-term hyperglycaemic animals. For the long-term hyperglycaemic animals, the intensities of labelling resembled those of their age-matched controls. These results indicate that type VI collagen appears to be a minor constituent of the extracellular matrix of the rat glomeruli, rather concentrated in the mesangial area in the young control animal. Concomitant with the general modifications of the extracellular matrix occurring with age and diabetes, this component increases, but apparently not with the length of the hyperglycaemic state. © Chapman & Hall     

Generation of free radicals and initiation of radical reactions in nitrones - Fe2+ - phosphate buffer systems

Three antigenic fractions were isolated from collagenase-solubilised human glomerular basement membrane utilising affinity columns containing purified human anti-glomerular basement membrane autoantibodies. The three fractions were able to inhibit the binding of sera containing anti-glomerular basement membrane autoantibodies when measured by solid phase radio-immunoassay. Amino acid and carbohydrate analyses revealed differences in the composition of the three fractions and were consistent with them being derived from the non-collagenous components of glomerular basement membrane.     

Glomerular basement membrane: evidence for collagenous domain of the alpha 3 and alpha 4 chains of collagen IV

A collagenous component(s) of Mr = 60K was extracted from glomerular basement membrane with urea and was purified. Upon digestion, it yielded a collagenase-resistant fragment(s) of Mr = 23.5K. Both component and fragment showed immunochemical identity with the noncollagenous domains of the new alpha 3 & alpha 4 chains of collagen IV. The component is characterized by a collagenous domain of about 280 residues and a noncollagenous domain of about 250 residues. These findings further establish these new chains as distinct entities of collagen IV.     

Characterization of a novel glycoprotein isolated from the basement membrane matrix of the Engelbreth-Holm-Swarm tumor

A previously undescribed protein has been isolated and purified from the extracellular matrix of the Engelbreth-Holm-Swarm (EHS) tumor, a murine tumor that synthesizes an extensive matrix composed of basement membrane molecules. Molecular characterization of the molecule determined that it is a glycoprotein with internal disulfide bonds and an isoelectric point of 6.0. Electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the glycoprotein migrated as a diffuse band with a molecular weight of approximately 72,000-80,000. The amino acid composition was significantly different from known basement membrane components. Polyclonal antibodies that specifically recognize the glycoprotein localized it to the kidney glomerular basement membrane. These antibodies did not cross-react with either known basement membrane components (laminin, type IV collagen, and heparan sulfate proteoglycan), with 70K "culture shock" protein or with components of normal mouse serum (including mouse transferrin, albumin, or alpha-fetoprotein), when analyzed by "Western" immunoblots. Our data indicate that the glycoprotein is synthesized by the EHS tumor cells and is present at relatively high levels in the EHS tumor matrix.     

Studies on the rat glomerular basement membrane: Age-related changes in composition

To examine the effect of age on the glomerular basement membrane, compositional analyses were performed on membranes isolated in highly purified form from rats at different stages of their growth (35 to 200 days old). Substantial age-related changes were observed in the amino acid composition of the basement membranes. A significant correlation with age (P < 0.01) was evident in the contents of 3- and 4-hydroxyproline, threonine, serine, alanine, valine, half-cystine, hydroxylysine, and lysine. Of these amino acids, hydroxylysine and both isomers of hydroxyproline demonstrated a progressive increase with age, while the others were found to decline. The direct relationship of hydroxylysine content with age (P < 0.001) was associated with an inverse correlation of lysine with age (P < 0.001) so that the ratio of hydroxylysine to lysine increased in a highly significant manner from 0.92 at 35 days to 1.33 at 200 days. This elevation in the hydroxylysine content was accompanied by an augmentation in the number of saccharide units linked to it so that the percentage glycosylation of this amino acid was not significantly affected by age. The relative differences in the hydroxylysine and lysine levels between young and older rats were maintained in sodium dodecyl sulfateextracted membranes. These results suggest that the compositional changes observed during the aging process reflect an alteration in the subunit makeup of the basement membrane, possibly due to an increased synthesis or decreased degradation of the more collagen-like polypeptide components.     

Collagen synthesis and secretion by isolated rat renal glomeruli.

Glomeruli were isolated from rat renal cortex and incubated with radioactive lysine to study in vitro collagen synthesis in these preparations. Glomerular basement membrane was obtained by sonication, and the appearance of [-14C]lysine and hydroxylysine in medium, membrane and intracellular proteins was determined. Total glomerular incorporation of [-14C]lysine into protein linearly increased for up to 2-h period, and membrane hydroxylysine content gradually rose during this time. Hydroxy[-14C]lysine was recovered in the 105 000 times g pellet, reaching a hydroxylysine content of 22 percent in this intracellular fraction after 90 min of incubation. 60 percent of the protein secreted into the medium, and about 75 percent of newly synthesized sonicated basement membrane was acetic acid soluble. Hydroxylysine content was 33 percent in the acetic acid-insoluble fraction of sonicated membrane, suggesting that basement-membrane collagen was a significant component of total collagen synthesized by these preparation, The ability of isolated glomeruli to synthesize and secrete basement-membrane protein will be useful for studies concerning control of glomerular collagen and basement-membrane synthesis.     

Glomerular lysozyme binding in protein-overload proteinuria

Binding of the cationic molecule lysozyme to the glomerular basement membrane and to the glomerular epithelial cell coat was investigated in the glomerulus of normal female Wistar rats and in rats in which heavy proteinuria was induced by the daily administration of 1 g of bovine serum albumin. In normal rats the binding of lysozyme to the anionic groups in the glomerular basement membrane and the cell coat had no effect on the ultrastructure of the glomerular epithelial cell, in particular the foot processes were unchanged. In the proteinuric rats the lysozyme-binding to the glomerular basement membrane and the epithelial cell coat was completely lost in the damaged glomeruli. In the apparently normal glomeruli present in these proteinuric animals binding was similar to that seen in normal rats. These results suggest that in protein-overload proteinuria there is a loss of glomerular anion and hence a reduction in the glomerular charge barrier. This may account, at least in part, for the increased glomerular leak of negatively charged serum albumin in this experimental model of proteinuria.     

Histochemical localization of protein-polysaccharides in renal tissue

The purpose of this study was to investigate the distribution of protein-polysaccharides in the glomerular and non-glomerular regions of the nephron. The techniques used include the digestion of kidney slices with specific polysaccharidases: neuraminidase, hyaluronidase, chondroitinase ABC, and collagenase followed by several cytochemical techniques to identify the glycosaminoglycans and glycoproteins at the light and electron microscope levels. Differential staining of hyaluronic acid and sulphated glycosaminoglycans was accomplished with Alcian Blue at pH 2.5 and pH 0.5, respectively. Sialoproteins were stained with Alcian Blue at pH 2.5. The periodic acid Schiff’s reaction technique was employed for the visualization of collagen. At the electron microscope level the polysaccharides were identified with the periodic acid-chromic acid-silver methenamine reaction. Our results indicated that the major polysaccharide components of the glomerular basement membrane were sialoproteins and collagen, with smaller amounts of hyaluronic acid and various sulphated glycosaminoglycans. Hyaluronidase digestion resulted in partial detachment of epithelial processes from the glomerular basement membrane indicating the hyaluronic acid may have a role in the stability of the attachment of these processes. Tubular basement membranes also contain sialoproteins and sulphated glycosaminoglycans but in considerably lower concentrations than the glomerular basement membrane. Bowman’s capsule appears to contain mostly sulphated glycosaminoglycans and has a lower concentration of sialoproteins and hyaluronic acid.     

Metabolism of glomerular basement membrane in short- and long-term streptozotocin diabetic rats

The synthesis of glomerular basement membrane (GBM) total protein and collagen was assessed by two methods in vivo in normal and streptozotocin diabetic rats 4-6 weeks and 42-44 weeks after onset of hyperglycaemia, using L-[2, 3, 3H] proline as a radioactive precursor. The incorporation of tritiated proline into GBM hydroxyproline was used as a measure of collagen synthesis and that into proline as total protein synthesis. The basement membrane fractions from both short- and long-term diabetic rats attained much higher proline and hydroxyproline specific activities compared to normal GBM proline and hydroxyproline specific activities. Early insulin therapy with normalization of blood sugar levels in short-term (4-6 weeks) diabetic rats returned the abnormal increases in GBM total protein and collagen synthesis to normal. By contrast, poor glycaemic control with insulin did not prevent the increases in GBM protein synthesis. The results of the present study suggest that overall enhancement of GBM protein synthesis occurs in both short- and long-term streptozotocin diabetes. Early insulin therapy with normalization of blood sugar levels prevents this increase in GBM protein synthesis. Poor glycaemic control had no effect on abnormal GBM protein synthesis. This may be of potential significance in view of preventing chronic diabetic microvascular complications such as nephropathy.