Van-Yu Wu

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.

Disturbances in Glomerular Basement Membrane Glycosaminoglycans in Experimental Diabetes

Glycosaminoglycans (GAGs) were purified from basement membranes isolated from glomeruli of control and streptozocin-induced diabetic rats and were quantitatively analyzed with a recently described competitive binding assay that is specific for and sensitive to microgram amounts of chondroitin and heparan sulfate. Total GAG content in glomeruli from diabetic rats and in the basement membranes prepared from these samples (17.22 ± 1.45 and 6.56 ± 0.49 μg/105 glomeruli, respectively) was significantly less than that found in comparable control preparations (43.71 ± 3.35 and 16.05 ± 1.41 μg/105 glomeruli, respectively). The portion of total GAG in the water-soluble fraction recovered after osmotic lysis of isolated glomeruli was also markedly decreased in diabetic samples (26.11 ± 4.55 vs. 3.30 ± 0.32 μg/105 glomeruli, control vs. diabetic). Treatment of lysed glomeruli with the ionic detergent deoxycholate, required for liberation of the extracellular matrix from plasma membrane lipoproteins and purification of the insoluble glomerular basement membrane (GBM), resulted in solubilization of ∼10% of the waterinsoluble GAG in control samples but >50% in diabetic membranes. Heparan sulfate comprised >90% of the GAGs in both control and diabetic GBM, defined as the water- and detergent-insoluble matrix. The findings clearly demonstrate that the GAG content of GBM is diminished in experimental diabetes and provide evidence that the reduction in GBM anionic sites associated with diabetes derives from a decrease in the constituent GAGs of this extracellular matrix. The results further suggest that the interaction between GBM and populations of GAG associated with the surface of plasma membranes of adjacent cells is disturbed in diabetes.

Undersulfation of Glomerular Basement Membrane Heparan Sulfate in Experimental Diabetes and Lack of Correction With Aldose Reductase Inhibition

In this study we examined the effect of experimental diabetes and of treatment with an aldose reductase inhibitor on the level of sulfation of glomerular basement membrane (GBM) heparan sulfate, the principal glycosaminoglycan in this extracellular matrix. Glycosaminoglycans were isolated from GBM purified from control, streptozocin-induced diabetic, and sorbinil-treated diabetic rats and analyzed for sulfate and uronate content. Glomerular yields from diabetic kidneys were greater than those from control animals, but the amount of sulfate per glomerulus in diabetic samples, both untreated and sorbinil treated, did not differ significantly from that in control samples. However, the sulfate-to-uronate ratio in heparan sulfate isolated from diabetic GBM (0.34 ± 0.08) was significantly less than in control samples (0.69 ± 0.11), and treatment of diabetic rats with an aldose reductase inhibitor did not correct this reduced ratio (0.36 ± 0.06). The results indicate that there is an undersulfation of heparan sulfate of GBM in experimental diabetes, an abnormality that may contribute to loss of anionie sites and decreased charge selectivity of the glomerular filtration barrier. The findings further suggest that this abnormality results from disturbances in glycosaminoglycan synthesis and/or metabolism in diabetes that are independent of polyol-pathway activation in the renal glomerulus.

Purification of glycated hemoglobin.

Publisher Summary The term glycohemoglobin (glycated hemoglobin) refers to hemoglobin that has been modified postribosomally by the attachment of glucose to the polypeptide chain. This modification, generically designated nonenzymatic glycation, is a condensation reaction between the carbohydrate and a free amino group at the amino terminus or ɛ-amino group of a lysine residue. The study of hemoglobin glycation has served as a model system; for it is now clear that many proteins undergo nonenzymatic glycation. The reaction is initiated with attachment of acyclic glucose to a protein amino group via nucleophilic addition, forming an aldimine, also known as a Schiff base. This intermediate product subsequently undergoes an Amadori rearrangement to form a 1-amino-1-deoxyfructose derivative in stable ketoamine linkage. The reaction is slow and continuous, and the extent of glycation is largely dependent on the glucose concentration to which the protein is exposed during its lifetime in the circulation. Other factors that influence the sites and amount of glycation include the accessibility and pK a of the amino groups within the structure of the protein.

Amelioration of Diabetes-Associated Abnormalities in the Vitreous Fluid by an Inhibitor of Albumin Glycation

PURPOSE Albumin modified by Amadori glucose adducts is a plasma-borne factor that activates cell signaling pathways, modulates the expression of growth factors and cytokines, and participates in the pathogenesis of microvascular complications of diabetes. In the present study, streptozotocin diabetic rats were treated with an orally administered compound that inhibits the nonenzymatic glycation of albumin to evaluate whether increased glycated albumin contributes to diabetes-associated abnormalities in the vitreous fluid. METHODS Vitreous obtained from age-matched nondiabetic and streptozotocin-diabetic rats, half of which received the test compound 2-(3-chlorophenylamino) phenylacetic acid (23CPPA) by oral gavage for 26 weeks, was analyzed by immunoassay for pigment epithelium-derived factor (PEDF), vascular endothelial growth factor (VEGF) and glycated albumin content, by measurement of thiobarbituric acid reactive substances (TBARs) for lipid peroxide products and by colorimetric assay for hyaluronan content. RESULTS Compared with that of nondiabetic controls, vitreous of diabetic rats contained decreased PEDF, increased VEGF, higher VEGF/ PEDF ratio, and elevated levels of TBARs, glycated albumin, and hyaluronan. These changes were significantly attenuated in rats treated with test compound despite the presence of marked hyperglycemia. CONCLUSIONS Results indicate that inhibiting the formation of glycated albumin, which is increased in diabetes, ameliorates vitreous changes in angiogenic and metabolic factors associated with the development of diabetic retinopathy. The observed improvement in vitreous alterations associated with reductions in glycated albumin suggests that elevated levels of glycated albumin play a retinopathogenic role in diabetes that is operative and that can be therapeutically addressed independently of glycemic status.

Non-collagen protein and proteoglycan in renal glomerular basement membrane

Extraction of rat glomerular basement membrane, purified by osmotic lysis and sequential detergent treatment, with 8 M urea containing protease inhibitors solubilizes protein that is devoid of hydroxyproline and hydroxylysine. This material represents 8-12% of total membrane protein, elutes mainly as two high molecular weight peaks on agarose gel filtration, and is associated with glycosaminoglycans. Isolated rat renal glomeruli incorporate [35S]sulfate into basement membrane from which this non-collagenous 35S-labeled fraction can be subsequently solubilized. The radioactivity incorporated into urea-soluble glomerular basement membrane eluted primarily with the higher molecular weight peak (Mr greater than 250 000). Cellulose acetate electrophoresis after pronase digestion of the urea-soluble fraction revealed glycosaminoglycan that was resistant to digestion with Streptomyces hyaluronidase and chondroitinase ABC, sensitive to nitrous acid treatment, and contained [35S]sulfate. The findings indicate that one of the non-collagenous components of glomerular basement membrane is a proteoglycan containing heparan sulfate.

Evaluation of the effect of aldose reductase inhibition on increased basement membrane collagen fluorescence in diabetic rats

1. It has been proposed that increased fructose contributes to the formation of fluorescent pigments in diabetic tissues. 2. Since the aldose reductase inhibitor sorbinil lowers glomerular fructose concentrations, we examined the effect of sorbinil on the formation of advanced glycation end products in glomerular basement membrane of streptozotocin diabetic rats. 3. Treatment with sorbinil for 30 days after induction of diabetes did not influence the increased fluorescence observed in collagen from glomerular basement membrane of untreated diabetic rats. 4. The results suggest that nonenzymatic glycation by fructose is not a major contributor to the formation of fluorescent advanced glycation end products in basement membrane in experimental diabetes.

Production and characterization of monoclonal antibodies against non-A1c glycated hemoglobin

Hybridomas secreting monoclonal antibodies specific for hemoglobin nonenzymatically glycated in the non-A1c position were produced by fusion of SP 2/0 myeloma cells with spleen cells from BALB/c mice immunized with nonenzymatically glycated hemoglobin prepared from human erythrocytes. Wells containing hydridomas secreting antibodies against glycohemoglobin were identified by binding, in an enzyme-linked immunosorbent assay, to purified glycated hemoglobin. The colony designated E85, which secreted antibodies discriminating between glycated versus unglycated hemoglobin, was cloned at least four times by limiting dilution and used for further study, performed with purified monoclonal antibody. Specificity of E85 was demonstrated by immunoblotting and by ELISA, wherein the monoclonal antibody reacted with glycated hemoglobin but not with hemoglobin A1c or with unglycated hemoglobin. Immunoblotting of human plasma with E85 on nitrocellulose yielded no reactive proteins, indicating site specificity for glycated epitopes residing in hemoglobin but not in other nonenzymatically glycated proteins present in plasma. E85 differs from other antibodies raised against glycated hemoglobin and other glycated proteins, which recognize hemoglobin glycated at the N terminal valine of the beta chain (HbA1c) or which recognize glycated residues only after reductive conversion to glucitollysine and which do not discriminate between different glycated proteins. Thus, this report describes the establishment of the first hybridoma secreting monoclonal antibody raised against a physiologic (unreduced) form of non-A1c glycohemoglobin, and for the glycated epitope when it resides in glycohemoglobin but not in other proteins or in hemoglobin A1c.

Platelet factor 3: Quantitation and characterization

Abstract Platelet factor 3, isolated according to the method of Alkjaersig et al. (1), was heterogenous on 4% SDS-polyacrylamide gels. Procoagulant activity was lost after five days at 4°C. Optimal stability was at pH 7.2–8.3. The product contained 38% protein, 49% lipid and 8% carbohydrate. Activity was destroyed by phospholipases A, C and D, and trypsin. Delipidation of platelet factor 3 with sec-butyl alcohol or ethanol resulted in loss of all procoagulant activity. Recombination of the protein residue with the lipid extract restored 80% of the procoagulant activity if sec-butyl alcohol was used and 44% using absolute ethanol. Platelet factor 3 activated human prothrombin complex more rapidly than crude cephalin, which was inactive in the platelet factor 3 assay. Attempts to purify the protein components and maintain procoagulant activity have been limited by instability in deoxycholate, SDS and Triton.

Nonenzymatic Glycosylation of Glomerular Basement Membrane

To test the hypothesis that increased nonenzymaticc glycosylation contributes to the microangiopathic sequelae of diabetes, we examined the interaction of glucose with basement membranes in vivo and in vitro. The level of nonenzymatic glycosylation, determined by hydroxymethylfurfuraldehyde (HMF) generation, in glomerular basement membrane (GMB) collagen purified from rats with streptozotocin diabetes was significantly greater than that of nondiabetic GMB. Incubation of purified rat GBM and lens capsule basement membrane with [14C]-glucose in vitro resulted in the incorporation of [14C] into acid precipitable radioactivity in a reaction that was dependent on time, temperature, and glucose concentration. Glucose-dependent nonenzymatic glycosylation of GBM in vitro was documented by measurement of HMF generation after incubation with unlabeled glucose. These results provide direct experimental evidence that hyperglycemia, via nonenzymatic glycosylation, modifies the chemistry of proteins specifically involved in diabetic glomerulosclerosis.