Human and rat mesangial cell receptors for glucose-modified proteins: potential role in kidney tissue remodelling and diabetic nephropathy.

Advanced glycosylation endproducts (AGEs) are derived from the nonenzymatic addition of glucose to proteins. AGEs have been found to accumulate on tissue proteins in patients with diabetes, and their accumulation is thought to play a role in the development of diabetic complications. The finding that macrophages and endothelial cells contain AGE-specific receptors led us to examine whether mesangial cells (MCs) also possess a mechanism for recognizing and processing AGEs. Membrane extracts isolated from rat and human MCs were found to bind AGE-bovine serum albumin (BSA) in a saturable fashion, with a binding affinity of 2.0 +/- 0.4 x 10(6) M-1 (500 nM). The binding was specific for the AGE adduct, since AGE-modified collagen I and ribonuclease both competitively inhibited 125I-AGE-BSA binding to MC membranes, while the unmodified proteins did not compete. Binding of AGE proteins was followed by slow internalization and degradation of the ligand. Ligand blotting of MC membrane extracts demonstrated three distinct AGE-binding membrane proteins of 50, 40, and 30 kD. Growth of MCs on various AGE-modified matrix proteins resulted in alterations in MC function, as demonstrated by enhanced production of fibronectin and decreased proliferation. These results point to the potential role that the interaction of AGE-modified proteins with MCs may play in vivo in promoting diabetic kidney disease.

Radioreceptor assay for advanced glycosylation end products.

Previous assays for nonenzymatic advanced glycosylation end products (AGEs) formed in tissues and/or circulating in blood are unsatisfactory. Based on our earlier identification of AGE-specific receptors on the macrophagelike tumor cell line RAW 264.7, a new assay system for AGEs has been devised. RAW 264.7 cells were used in competitive radioreceptor assays (RRA) after a 3-day culture in 96-well plates with 1 mu CI/ml [3H]glycine. Bovine serum albumin (BSA), modified extensively by incubation with glucose-6-phosphate in vitro to form AGE-BSA, was labeled with 125I and was used as a model ligand at a concn of 10 micrograms/ml. One unit of AGE was defined as the amount of test protein required to inhibit 50% of the specific binding of [125I]-labeled AGE-BSA to the AGE-receptors of intact RAW 264.7 cells. Nonlabeled AGE-BSA was used as a specific competitor to construct standard curves. The reproducibility of the assay was assessed at AGE levels equivalent to mean, maximum, and minimum levels of sensitivity for assays run on a single day and over an extended period, and the RRA had a reproducibility (coefficient of variation) between 5.9 and 14.7%. Protease hydrolysis of in vitro glycosylated proteins before assay increases the competitive ability of these proteins in proportion to their glycosylation. Little or no AGE cross-reactivity was detected in native BSA, Amadori-BSA, maleylated BSA, formaldehyde-treated BSA, palmitic acid-BSA, and acetylated low-density lipoproteins (acetyl-LDL). Polyanions such as heparin or fucoidan strongly interfere with this receptor binding assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of surface binding protein specific for advanced glycosylation end products from mouse macrophage-derived cell line RAW 264.7.

Macrophages internalize and degrade proteins modified by advanced glycosylation end products (AGEs) via a specific receptor (AGE-R). Chemical cross-linking studies with AGE-bovine serum albumin have demonstrated that the molecular weight of this receptor is approximately 90,000. We previously established that the binding constant (Ka) of this receptor site for the chemically synthesized model AGE, 2-(2-furoyl)-4(5)-(2-furanyl)-1H- imidazole-butyric acid (FFI-BA), on cells of the mouse macrophagelike cell line RAW 264.7 is identical to that for AGE proteins. Therefore, FFI was used as an affinity matrix in the first purification step of the AGE-R. The membranes of RAW 264.7 cells were solubilized in octyl-beta-glucoside and subjected to affinity chromatography on FFI-sepharose and gel permeation on Superose 6 fast protein liquid chromatography. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis analysis of this material revealed a high enrichment of a 90,000-Mr protein that had AGE binding activity. Approximately 25% of the protein at this step was the 90,000-Mr protein. The 90,000-Mr membrane protein was purified to homogeneity by rechromatographing the material on Superose 12 in the presence of SDS before and after reduction with 2-mercaptoethanol. After these harsh conditions, the 90,000-Mr protein lost AGE binding activity. Additional cross-linking studies on human peripheral monocytes revealed an AGE-R protein of identical size to that on RAW 264.7 cells, suggesting the relatively highly conserved nature of this molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of glucuronic and iduronic acid units in heparin chains.

The distribution of glucuronic and iduronic acid within the chains of anticoagulantly active and inactive beef lung heparin was investigated. A fraction with an average molecular weight of 19,500 was isolated from the heterodisperse mixture and then separated into active and inactive components by affinity chromatography. Each sample was linked through its reducing terminus to tyramine, reduced with sodium borotritide, and bound covalently to Sepharose via an azo bridge. The bound reduced heparin was treated with a limited amount of HNO2 and the degraded fragments were removed. The sections of the chain contiguous with the original reducing terminus were then detached from the insoluble matrix by reaction with sodium dithionite. The recovered polysaccharide was fractionated according to size on Sephadex G-200 and the amount of each uronic acid in the individual fractions was determined. Inactive heparin showed a constant percentage of glucuronic acid in all fragments, i.e. about 8.9% of the total uronic acid. With active heparin the percentage of glucuronic acid increased with the distance from the reducing terminus of the polysaccharide chain, ranging from 9.5 to 20% of the uronic acids. These results suggest that the biosynthesis of active heparin involves unique reactions or specific processing of the macromolecule.

Location on heparin of the oligosaccharide section essential for anticoagulant activity.

Studies were conducted to define the location of the unique oligosaccharide sequence in heparin that is required for its anticoagulant activity. A heparin fraction with an average molecular weight of 20,000 was linked at its reducing terminus to a Sepharose derivative via an azo bridge. The matrix-linked heparin was subjected to limited degradation with nitrous acid and, after the cleaved segments were removed, the section contiguous with the original reducing terminus was released from the gel by treatment with sodium dithionite. The results of analyses of the products after separation into different molecular weight pools demonstrate that the groups responsible for accelerating the neutralization of thrombin and Factor Xa by antithrombin are located at or near the nonreducing terminus of the heparin chain.

Characterization of a solubilized cell surface binding protein on macrophages specific for proteins modified nonenzymatically by advanced glycosylated end products.

Glucose can react nonenzymatically with free protein amino groups to form Amadori products, 1-amino-1-deoxyketose residues. These adducts can undergo subsequent rearrangements and dehydrations to form a complex group of brown, fluorescent pigments collectively referred to as advanced glycosylation end products (AGE). One AGE has been identified as 2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole (FFI). The AGE-protein adducts accumulate with time and are implicated in irreversible tissue damage. We have previously demonstrated that macrophages bind and degrade AGE-proteins via a specific cell surface binding protein, thus selectively removing senescent macromolecules. In the present communication, we have solubilized this binding protein from the membranes of the murine macrophage cell line RAW 264.7. We have characterized the nature of binding protein-ligand interaction by competition studies using modified ligands. The data indicate that the carbonyl group, the furan ring(s), and the central imidazole structure are all important in the binding protein-ligand interaction. We have established that the binding constant (Ka) of binding protein for the ligand FFI-BA is 3.1 X 10(7) M-1. Chemical crosslinking studies have demonstrated that the molecular weight of the binding protein is 90,000.

The uronic acid composition of anticoagulantly active and inactive heparin.

Bovine heparin was fractionated with respect to chain length and anticoagulant activity. Analysis of each of these fractions for iduronic and glucuronic acids demonstrated that active heparin has a greater amount of glucuronic acid than inactive heparin. The ratio of the uronic acids in the respective fractions was the same for heparin with different molecular weights. Thus, active heparin with longer chain lengths have more additional glucuronate residues than are required for the antithrombin-binding domain. The results indicate that the active and inactive heparin species differ in more than one structural characteristic and suggest a considerable divergence in their respective biosynthesis.

Advanced protein glycosylation induces transendothelial human monocyte chemotaxis and secretion of platelet-derived growth factor: role in vascular disease of diabetes and aging.

Diabetes and aging are commonly accompanied by arterio- and atherosclerosis. Infiltration of the arterial subendothelial intima by macrophages/monocytes is an important early event preceding the development of atheromatous lesions; these macrophages are known to produce mitogenic factors in early atherosclerotic lesions. It has been previously shown that, over time, vascular matrix accumulates proteins nonenzymatically modified by advanced glycosylation end products (AGEs). In view of the fact that macrophages/monocytes have AGE-specific receptors associated with the expression of several growth factors, we investigated the possibility that AGEs mediate initial monocyte-vessel wall interactions that occur before overt formation of vascular lesions. This study demonstrates that (i) in vitro- and in vivo-formed AGEs are chemotactic for human blood monocytes, (ii) sub-endothelial AGEs can selectively induce monocyte migration across an intact endothelial cell monolayer, and (iii) subsequent monocyte interaction with AGE-containing matrix results in the expression of platelet-derived growth factor. These results support the existing hypothesis that in vivo-forming glucose-derived protein adducts can act as signals for the normal turnover of senescent tissue protein by means of the AGE-specific receptor system. Time-dependent glucose-induced deposition of AGEs on matrix proteins may promote monocyte infiltration into the subendothelium. Subsequent AGE-triggered macrophage activation and consequent elaboration of proliferative factors may normally coordinate remodeling but may also lead to the diverse pathogenic changes typical of arterio- and atherosclerosis in diabetic or aging populations.

Advanced glycosylation end products in patients with diabetic nephropathy.

BACKGROUND: Glucose reacts nonenzymatically with proteins in vivo, chemically forming covalently attached glucose-addition products and cross-links between proteins. The excessive accumulation of rearranged late-glucose-addition products, or advanced glycosylation end products (AGEs), is believed to contribute to the chronic complications of diabetes mellitus. METHODS: To elucidate the relation of AGEs to diabetic complications, we used a radioreceptor assay to measure serum and tissue AGEs in diabetic (Types I and Type II) and nondiabetic patients with different levels of renal function. Serum AGEs were measured as a low-molecular-weight (less than or equal to 10 kd) peptide fraction and a high-molecular-weight (greater than 10 kd) protein fraction. RESULTS: The mean (+/- SD) AGE content of samples of arterial-wall collagen from 9 diabetic patients was significantly higher than that of samples from 18 nondiabetic patients (14.5 +/- 5.2 vs. 3.6 +/- 1.5 AGE units per milligram, P less than 0.001). Moreover, diabetic patients with end-stage renal disease had almost twice as much AGE in tissue as diabetic patients without renal disease (21.3 +/- 2.8 vs. 11.5 +/- 1.9 AGE units per milligram, P less than 0.001). The AGE levels in both serum fractions were elevated in the patients with diabetes, and the levels of AGE peptides correlated directly with serum creatinine (P less than 0.001) and inversely with creatinine clearance (P less than 0.005), suggesting that levels of AGE peptides increased with the severity of diabetic nephropathy. In six patients with diabetes who required hemodialysis, the levels of AGE peptides were five times higher than in eight normal subjects (82.8 +/- 9.4 vs. 15.6 +/- 3.4 AGE units per milliliter, P less than 0.001). In another group of diabetic patients the mean serum creatinine level, which decreased by 75 percent during a session of hemodialysis, whereas the level of AGE peptides decreased by only 24 percent. Serum levels of AGE peptides were normal in two patients with normal serum creatinine levels after renal transplantation. CONCLUSIONS: AGEs accumulate at a faster-than-normal rate in arteries and the circulation of patients with diabetes; the increase in circulating AGE peptides parallels the severity of renal functional impairment in diabetic nephropathy.