Robert G. Spiro

Studies on Macromolecular Components of Human Glomerular Basement Membrane and Alterations in Diabetes: Decreased Levels of Heparan Sulfate Proteoglycan and Laminin

Treatment of human glomerular basement membrane (GBM) with 4 M guanidine HCI resulted in a preferential extraction of noncollagenous components including laminin, fibronectin, entactin, and heparan sulfate proteoglycan, whereas effective solubilization of type IV collagen required exposure to denaturing solvents in the presence of reducing agents. The guanidine HCI–solubilized constituents were identified by immunochemical procedures after resolution by polyacrylamide gel electrophoresis, CL-6B filtration, and DEAE-cellulose chromatography. Two immunologically related heparan sulfate proteoglycans (Mr ∼350,000 and 210,000) were observed by electrophoresis, with the higher-molecular-weight form being predominant. An examination of the two proteoglycans after heparitinase digestion or chemical deglycosylation indicated that heparan sulfate chains and other carbohydrate units are attached to core proteins with Mr ∼140,000 and 110,000, respectively. Radioimmunoassays indicated that human diabetic GBM contained significantly lower (P < .005) amounts of heparan sulfate proteoglycan and laminin with average values that were 30 and 60%, respectively, of nondiabetic controls; the fibronectin content of the diabetic GBM, however, was not significantly different from the normal. These findings, together with previous studies showing increases in GBM collagen, indicate that an alteration in the macromolecular architecture of this basement membrane occurs in diabetes that may be responsible for the filtration defect and the ultimate glomerular occlusion.

Effect of Diabetes on the Glycosaminoglycan Component of the Human Glomerular Basement Membrane

The glycosaminoglycan (heparan sulfate) component of glomerular basement membranes from human kidneys of diabetic and nondiabetic subjects has been quantitated after isolation from protease digests of the membranes on OEAE-cellulose microcolumns. A significant decrease (P < 0.005) in the glycosaminoglycan content of diabetic membranes was observed. Heparan sulfate was identified as the predominant glycosaminoglycan in both diabetic and control subjects and the extent of its sulfation appeared to be similar. The reduced level of glycosaminoglycan in the diabetic glomerular basement membrane was accompanied by a significant elevation of hexoses, which are primarily associated with the collagen component, suggesting that a redistribution of basement membrane macromolecules occurs in the diabetic state. Since heparan sulfate has been implicated as a major component of the glomerular anionic filtration barrier, its decreased content in diabetic basement membranes may contribute to the proteinuria observed in this disease.

Search for a biochemical basis of diabetic microangiopathy

Diabetic microangiopathy, particularly as seen in the renal glomerulus, is characterized by morphological and biochemical alterations of the capillary basement membrane. Observations from a number of disciplines have indicated that the microvascular disease is not a separately inherited entity but a true consequence or “complication” of insulin deficiency. An evaluation of the biochemical events which could be responsible for the basement membrane lesions of diabetes indicates that the hyperglycemia or plasma somatotropin elevation of this disease alone, or in combination, may play an important role.SummaryDiabetic microangiopathy, particularly as seen in the renal glomerulus, is characterized by morphological and biochemical alterations of the capillary basement membrane. Observations from a number of disciplines have indicated that the microvascular disease is not a separately inherited entity but a true consequence or “complication” of insulin deficiency. An evaluation of the biochemical events which could be responsible for the basement membrane lesions of diabetes indicates that the hyperglycemia or plasma somatotropin elevation of this disease alone, or in combination, may play an important role.

Human Glomerular Basement Membrane: Chemical Alteration in Diabetes Mellitus

The human glomerular basement membrane belongs to the collagen family of proteins. It contains about 7 percent carbohydrate, half of which occurs as glucosylgalactose disaccharide units linked to hydroxylysine. Glomeruli from diabetics contain increased amounts of basement membrane material. In addition, these membranes show a distict chemical alteration c haracterized by a significant decrease in lysine, accoumpanied by an equivalent increase in hydroxylysine and hydroxylysine-linked disaccharide units.

Effect of Diabetes on the Biosynthesis of the Renal Glomerular Basement Membrane: Studies on the Glucosyltransferase

Kidney glucosyltransferase (UDP-glucose: galactosylhydroxylysine-basement membrane glucosyltransferase), which is involved in the synthesis of the hydroxylsine-linked disaccharide units of glomerular basement membrane, has been measured in the renal cortices of normal and alloxan diabetic rats. The level of this enzyme in diabetic kidneys was found to be significantly elevated over that of age-matched controls, expressed either as specific or total activity, at all times studied (one to five months). However the difference between the normal and diabetic animalsincreased with the duration of the disease. When short-term diabetics were treated with insulin, the level of the glucosyltransferase could be restored to normal. Insulin treatment of long-term diabetic animals, which were more difficult to control, brought the enzyme level close to normal but did not completely restore it. The glucosyltransferase activity in several other tissues of the alloxan diabetic rat, including lung, liver, testes, spleen, and uterus, did not show any significant elevation over that of the normal. Measurement in kidney of a glycosyltransferase (UDP-galactose :N-acetylglucosamine-glycoprotein galactosyltransferase) involved in the synthesis of a different type of carbohydrate unit did not show the marked elevation noted for the glucosyltransferase. It is believed that the insulin-reversible glucosyltransferase elevation of the diabetic renal cortex reflects the increased basement membrane synthesis occurring in this tissue and indicates that this process is directly or indirectly under the control of this hormone.

Studies on the Human Glomerular Basement Membrane: Composition, Nature of the Carbohydrate Units and Chemical Changes in Diabetes Mellitus

The basement membrane has been isolated in purified form from pooled normal human glomeruli and shown by detailed analyses to be composed of glycoprotein material. The peptide portion was characterized by the presence of large amounts of glycine, as well as by the occurrence of a substantial number of hydroxyproline, hydroxylysine, and cystine residues. Approximately 7 per cent of the weight of the membrane was found to be made up of sugar residues. The membrane was extensively solubilized by digestion with bacterial collagenase, and a study of glycopeptides obtained after additional Pronase treatment indicated the occurrence of glucosylgalactose disaccharide units linked to hydroxylysine as well as heteropolysaccharides consisting of N-acetylneuraminic acid, fucose, galactose, mannose and hexosamines similar to those previously found in the bovine glomerular basement membrane. Chemical analyses of glomeruli obtained from diabetic and nondiabetic kidneys indicated that the former contained more basement membrane-like material. Furthermore basement membranes isolated from the diabetic glomeruli had a composition different from those obtained from control subjects. The diabetic membranes were found to have a significant increase (P < 0.01) in their hydroxylysine content and in the number of glucosylgalactose disaccharides linked to this amino acid. The increase in hydroxylysine was accompanied by a decrease in the lysine level so that the sum of these amino acids remained unchanged from the normal. Changes of lesser significance were observed in hydroxyproline, glycine, valine and tyrosine with the former two amino acids showing an increase and the latter two a decrease from the nondiabetic level. No change was found in the number of the heteropolysaccharide units. These results suggest an overproduction in the diabetic state of those subunits of the basement membrane that are rich in hydroxylysmejand its glycosidically linked disaccharide unit. Such alterations in the subunit composition may lead to an abnormal packing of the peptide chains and thereby form the basis for the detective filtration function observed in diabetes.

Effect of High Glucose on Type IV Collagen Production by Cultured Glomerular Epithelial, Endothelial, and Mesangial Cells

Immunochemical and metabolic radiolabeling procedures revealed that homogeneous cultures of calf glomerular epithelial, endothelial, and mesangial cells actively synthesize type IV collagen (primarily as α1(Iv)3) which is secreted into the medium and incorporated into the extracellular matrix. Exposure of confluent cultures of the three cell types to a high glucose concentration (30 mM) for 60 h resulted in a pronounced increase (two- to threefold) in type IV collagen production over that observed at a physiological level (5 mM) of this sugar, as determined by either immunoblotting or fluorography of electrophoretically separated media or cell-matrix components. The elevated glucose did not bring about a change in the rate of cell proliferation or fibronectin production. Moreover, studies with mannitol indicated that the stimulation of type IV collagen synthesis was not a function of hyperosmolarity. In contrast to the glomerular cells, glucose-induced enhancement of formation of this collagen was not observed in 3T3 cells despite a substantial acceleration in the consumption of this sugar. Time studies indicated that the response of the glomerular cells to high glucose occurs over an extended period (maximal at ∼78 h) and, furthermore, that the stimulatory effect on type IV collagen production is only slowly reversed after restoration of the glucose to a normal level. We believe that these findings are relevant to an understanding of the sequence of events that lead to the development of diabetic glomerular lesions.

Glomerular Basement Membrane Metabolism in the Diabetic Rat: In Vivo Studies

The effect of diabetes on the metabolism of the renal glomerular basement membrane has been studied in the rat with the aid of injected tracer doses of tritiated proline. At various times after administration of the labeled amino acid, the specific radioactivities of the proline and hydroxyproline of the basement membranes from alloxan diabetic rats were determined and compared with those of age-matched normal rats. In both normal and diabetic animals the incorporation of radioactivity into the basement membrane was slow and, after a maximum was reached, an extended period of almost constant specific activity of proline and hydroxyproline was observed. The diabetic basement membrane, however, differed from the normal by attaining specific activities of the amino acids which were about twice as high as normal (P < 0.001 at 42 h after injection of radioisotope). Although the proline concentration of serum and renal cortical fluid was the same in normal and diabetic rats, there were substantial differences in the specific activity of this precursor amino acid in these pools that had to be taken into account to compare the two types of animals. The results of the present study are consistent with an accelerated rate of glomerular basement membrane polypeptide synthesis and proline hydroxylation in diabetes.

Glucose residues as key determinants in the biosynthesis and quality control of glycoproteins with N-linked oligosaccharides.

Although glucose residues do not occur as constituents of mature N-linked oligosaccharides in eukaryotic cells, it has been appreciated for some time that they are integral components of the polymannose oligosaccharides of newly synthesized glycoproteins and their lipid-linked precursors (1). Indeed it has been shown that they play an essential role in the cotranslational transfer of a preassembled triglucosylated oligosaccharide (Glc3Man9GlcNAc2) from a dolichyl pyrophosphoryl carrier to asparagine in Asn-XSer(Thr) sequences on the polypeptide chain (2, 3). Moreover, it has recently become apparent that the most internal of the three glucose residues, after being brought to a terminal position through the action of ER-situated glucosidases, interacts with lectin-like chaperones to mediate proper folding and/or oligomerization during protein quality control (4, 5). From these observations it has become evident that the presence of transient glucose residues on the polymannose oligosaccharides provides ideal recognition signals for crucial biological events, which have implications for a number of disease states as well as for viral replication. A rather complicated enzymatic machinery occurs in eukaryotic cells to achieve glucose attachment (6) and removal (7, 8), and this has been studied effectively with the help of mutants and inhibitors. It is the purpose of this article to provide a succinct overview of this distinctive area of glycobiology.

Fetuin: Immunochemistry and quantitative estimation in serum

Abstract Immune serum was prepared in rabbits by injection of purified fetuin. Since the antiserum was found to be immunochemically homogeneous and was shown to be directed against fetuin, it was used to estimate quantitatively fetuin in fetal and adult bovine sera. The ratio of fetuin to total serum proteins was found to be 0.4–0.5 and did not vary significantly during development of the fetus. Fetuin was found in adult sera but at markedly lower levels. Cross reactions of fetuin antiserum with fetal and adult goat and sheep sera have been found. By various criteria, these heterologous antigens appeared to be quite similar, but not identical, to the protein derived from fetal calf serum. No cross reactions were observed with the sera of several other species tested. Fetuin from which the terminal sialic acid residues were removed reacted to a greater extent than the original antigen with fetuin antibody in the quantitative complement fixation assay. Glycopeptides derived from fetuin by proteolytic digestion did not react with fetuin antiserum, nor did they inhibit to any significant extent complement fixation of native fetuin with its homologous antibody.