Margo P. Cohen

Increased glycosylation of glomerular basement membrane collagen in diabetes

Abstract 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.

Renal glomerular collagen synthesis in streptozotocin diabetes Reversal of increased basement membrane synthesis with insulin therapy

The effect of diabetes and insulin on basement membrane synthesis in vitro be renal glomeruli obtained from normal and diabetic rats was determined. Four groups of experimental animals were used: age-matched controls; streptozotocin-diabetic; and streptozotocin-diabetic treated with insulin for half or all of the duration of diabetes. Isolated glomeruli were incubated with [14C]-lysine and the radioactive lysine and hydroxylysine in glomerular proteins were measured. [14C]Lysine incorporation and hydroxy[14H]lysine synthesis were elevated in diabetic glomeruli. Progressive diminution in 14C-labelled protein and hydroxy[14C]lysine formation was observed in incubations containing glomeruli from insulin-treated diabetic rats, with greater reversal toward normal following longer periods of exogenous insulin administration. Basement membrane synthesis, determined by the appearance of labelled hydroxylysine in membranes obtained from sonicated glomeruli, was increased in diabetic preparations. Reversal of these changes toward normal values was observed in glomeruli from rats treated with insulin immediately following induction of diabetes. The results indicate that basement membrane synthesis is increased in renal glomeruli from streptozotocin-diabetic rats, and that this process is restored toward normal with continuous insulin therapy.

Effect of diabetes andinsulin on rat renal glomerular protocollagen hydroxylase activities

The effect of diabetes and insulin on the activities of both prolyl hydroxylase (trivial name; proline,2-oxoglutarate dioxygenase, EC 1.14.11.2) and lysyl hydroxylase (trivial name; lysine,2-oxoglutarate dioxygenase, EC 1.14.11.4) in isolated rat renal glomeruli was determined. Three groups of experimental animals were used: age-matched controls, streptozotocin-diabetic, and insulin-treated streptozotocin-diabetic. Using 14C-labeled lysine or proline hydroxylase substrate prepared from chick embryo tibiae, glomerular 17 000 X g supernatant enzyme was incubated in a complete hydroxylating system for 60 and 120 min Lysyl hydroxylase activity was significantly increased in diabetic preparations, but prolyl hydroxylase activity did not differ from control. Administration of insulin to streptozotocin-injected animals completely restored glomerular lysyl hydroxylase to normal levels. The results suggest that the specific elevation of lysyl hydroxylase relates to the biochemical changes contributory to diabetic nephropathy, and that insulin may reverse this process.

Glomerular protocollagen lysyl-hydroxylase activity in streptozotocin diabetes

Using [14-C]lysine protocollagen substrate prepared from chick embryo tibiae, lysyl hydroxylase activity was found in the 17 000 times g supernatant and particulate fractions obtained from homogenates of isolated rat renal glomeruli. Specific activities using the latter as an enzyme source were about 20-30% that of the supernatant. [14-C]Hydroxylysine formation was proportional to substrate and enzyme concentration, and to time for up to 120 min of incubation. Omission of alpha-ketoglutarate and ascorbate in the incubational assay markedly depressed activity. Hydroxylation of substrate by supernatant enzyme from streptozotocin diabetic rats was significantly increased over that of normal. In contrast, the activity of supernatant fractions from glomeruli of pancreatectomized, normoglycemic animals did not differ from that of non-operated controls. It is concluded that elevated glomerular lysine hydroxylase activity accompanies the increased glomerular collagen synthesis found in streptozotocin diabetes, and that chronic hyperglycemia may be implicated in these changes.

Glycosaminoglycans are integral constituents of renal glomerular basement membrane.

Abstract Basement membranes from canine renal glomeruli were isolated following osmotic lysis and sequential detergent treatment. Substantial amounts of uronic acid in unfractionated membranes were demonstrated with the carbazole and orcinol reactions. About 10–15% of basement membrane uronic acid was solubilized with neutral salt solutions. Denaturation in 8M urea solubilized ⋍70% of the uronic acid but only ⋍10% of basement membrane hydroxyproline; the latter was solubilized after reduction and alkylation. Uronic acid containing glycoprotein isolated by denaturation did not bind to carboxymethylcellulose and migrated as a high molecular weight band on SDS-gel electrophoresis. The ability of isolated rat glomeruli to incorporate radioactive sulfate in vitro was demonstrated. These findings indicate that sulfated glycosaminoglycans are integral components of glomerular basement membrane.

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.

Evidence for enhanced basement membrane synthesis and lysine hydroxylation in renal glomerulus in experimental diabetes.

Abstract Isolated glomeruli prepared from the renal cortex of normal and pancreatectomized diabetic rats were incubated in the presence of radioactive lysine, and the appearance of labeled lysine and hydroxylysine in non-dialyzable protein of cellular fractions was determined. Lysine incorporation and percent hydroxylysine content were higher than normal in the membrane fraction and lower than normal in the cytosol fraction in diabetes. The results are consistent with enhanced glomerular basement membrane secretion and increased hydroxylation of lysine residues in released collagen peptides in diabetes.

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

Abstract 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 in vivo in hyperglycemic diabetes.

Clinical, pathophysiological and structure/function consequences of modification of albumin by Amadori-glucose adducts.

BACKGROUND The nonenzymatic condensation of glucose with albumin results in the formation of albumin modified by Amadori glucose adducts, the principal form in which glycated albumin exists in vivo. SCOPE OF REVIEW This review focuses on (a) the utility of measurement of Amadori-modified glycated albumin (AGA) as a biomarker in diabetes, where elevated levels attend the hyperglycemic state; (b) the role of AGA as a causal factor in the pathogenesis of complications of diabetes; (c) effects on transport properties; and (d) structural and functional consequences of the modification of albumin by Amadori glucose adducts. It does not discuss counterparts with respect to Advanced Glycation Endproducts (AGE), which may be found in other publications. MAJOR CONCLUSIONS Nonenzymatic glycation of albumin, which is increased in diabetes, has clinical relevance and pathophysiologic importance, with ramifications for the management of this disease, the development of its complications, and the transport of endogenous and exogenous ligands. GENERAL SIGNIFICANCE Appreciation of the manifold consequences of AGA has afforded new avenues for assessing clinical management of diabetes, awareness of the impact of nonenzymatic glycation on albumin biology, insights into the pathogenesis of vascular complications of diabetes, and avenues of investigation of and intervention strategies for these complications. This article is part of a Special Issue on albumin. This article is part of a Special Issue entitled Serum Albumin.

Presence of glycosaminoglycans in retinal capillary basement membrane

Retinal microvessels were isolated from bovine eyes and the basement membranes were purified either directly or after incubation with [35S]sulfate and [14C]glucosamine. The basement membranes, which were purified by osmotic lysis and sequential treatment with detergents, had the general compositional features associated with basement membrane collagens, including high levels of hydroxyproline and hydroxylysine and the presence of 3-hydroxyproline and cystine. After pronase digestion, cellulose acetate electrophoresis of glycosaminoglycans from retinal microvessel basement membrane revealed material comigrating with heparan sulfate that was insensitive to digestion with Streptomyces hyaluronidase ad chondroitinase ABC. Retinal microvessels also incorporated [35S]- and [14C]glucosamine into glycosaminoglycans that were isolated following pronase digestion of the retinal microvessel basement membrane purified from these incubations. The findings provide the first demonstration that glycosaminoglycans are integral components of the retinal microvascular basement membrane and suggest that heparan sulfate is the major glycosaminoglycan species in this basement membrane.