Prevention of diabetic nephropathy in db/db mice with glycated albumin antagonists. A novel treatment strategy.

Accelerated protein glycation in diabetes has been mechanistically linked to the pathogenesis of diabetic nephropathy. Because glycated albumin induces abnormalities in cultured mesangial cells that resemble those characterizing the glomerular mesangium in diabetes, and monoclonal antibodies (A717) specific for Amadori-modified glycated albumin prevent these abnormalities, we postulated that in vivo administration of A717 could retard the progression of diabetic nephropathy. To test this hypothesis, diabetic db/db mice and their nondiabetic db/m littermates were treated with eight consecutive weekly injections of 150 micrograms of A717 (Fab fragments) to reduce the elevated plasma glycated albumin concentration, or with irrelevant murine IgG (MIg). Relative to nondiabetics, diabetic mice (MIg treated) manifested proteinuria (3.35 +/- 0.15 vs 0.87 +/- 0.1 mg albumin/mg creatinine), 3.8-fold increase in mesangial matrix fraction, and renal cortical overexpression of mRNAs encoding alpha 1(IV) collagen (2.6-fold increase) and fibronectin (3.8-fold increase). Treatment of db/db mice with A717 significantly reduced the proteinuria (1.52 +/- 0.3 mg/mg creatinine), inhibited mesangial matrix expansion, and attenuated overexpression of matrix mRNAs. The nephropathic protective effects of A717 were independent of any change in blood glucose concentrations. Antibodies unreactive with glycated albumin did not duplicate the beneficial effects of A717. Thus, abrogating the biologic effects of increased glycated albumin with A717 has a salutary influence on the pathogenesis of diabetic nephropathy and has novel therapeutic potential in its management.

Production and characterization of monoclonal antibodies against human glycoalbumin.

Hybridomas secreting monoclonal antibodies specific for nonenzymatically glycated albumin were produced by fusion of SP2/0 myeloma cells with spleen cells from BALB/c mice immunized with unreduced nonenzymatically glycated albumin prepared from human plasma. Wells containing hybridomas secreting antibodies against glycoalbumin were identified by binding, in an enzyme-linked immunosorbent assay, to glycoalbumin isolated from human plasma or to albumin that had been glycated in vitro. The colony designated A717, which secreted antibodies discriminating between glycated versus unglycated albumin, was cloned four times by limiting dilution and used for further study, performed with monoclonal antibody purified from mouse ascites fluid. Specificity of A717 was demonstrated by immunoblotting and by ELISA, wherein the monoclonal antibody reacted preferentially with glycated albumin but insignificantly with unglycated albumin. Immunoblotting of human plasma with A717 on nitrocellulose yielded a single band, the electrophoretic mobility of which corresponded with that of authentic glycated albumin, indicating site specificity for glycated epitopes residing in albumin but not in other nonenzymatically glycated serum proteins. A717 differs from other antibodies raised against glycated albumin and other proteins, which recognize glycated residues only after reductive conversion to glucitol-lysine and which do not discriminate between different glycated proteins. Thus, this report describes the establishment of the first hybridoma secreting monoclonal antibody raised against unreduced glycated albumin, which is the physiologic form occurring in vivo, and for the epitope when it resides in albumin but not other proteins.

Measurement of plasma glycoalbumin levels with a monoclonal antibody based ELISA.

We have reported the establishment of hybridomas secreting monoclonal antibody that specifically recognizes glycated albumin. The antibody, raised in mice immunized with nonenzymatically glycated albumin isolated from human plasma, recognizes glycated epitopes residing in albumin but not in other plasma proteins, and does not react with unglycated albumin. We now report utilization of this antibody to measure the amount of nonenzymatically glycated albumin in human plasma. When immobilized onto microtiter wells in ELISA format, A717 yields a linear dose-response relationship upon incubation with authentic purified glycoalbumin. This allows construction of standard curves from which the amount of circulating glycoalbumin can be determined. The glycoalbumin level (mean +/- SEM) in 12 samples from nondiabetic subjects was 2.4 +/- 0.22% of total albumin. In normal plasma spiked with known amounts of purified glycoalbumin, values measured with this assay were close to 100% of expected values. Glycoalbumin levels in samples from 25 diabetic subjects ranged from 1.6 to 11.6%, with a mean +/- SEM of 4.5 +/- 1.2%. Glycoalbumin levels in diabetic samples correlated significantly (r = 0.93) with glycohemoglobin values. The ability of this assay to quantitate glycoalbumin, an index of the prevailing blood glucose concentrations over the preceding 2-3 weeks, makes it a favourable candidate for utilization in the clinical setting to monitor glycemic control in diabetic subjects.

Evaluation and performance characteristics of a novel ELISA using monoclonal antibody to glycated albumin.

To evaluate the clinical utility of a highly specific monoclonal antibody directed against the glycated epitopes residing in human albumin, we developed an ELISA using this antibody to measure glycated albumin in plasma samples from nondiabetic and diabetic individuals. The assay is predicated on the ability of immobilized monoclonal antibody to distinguish glycated albumin from all other plasma proteins, followed by detection and quantitation of the bound glycoalbumin with an enzyme-conjugated second antibody directed against human albumin. The relative percent concentration of glycated albumin in a sample is then determined by dividing the microgram glycated albumin in the sample by the total microgram albumin in the sample. The mean glycoalbumin level in samples from 12 non-diabetic subjects was 2.4% +/- 0.22% (mean +/- SD). In samples from 30 diabetic subjects, glycated albumin levels ranged from 1.6% to 14.9%, and individual values correlated positively and significantly with glycohemoglobin levels measured in simultaneously collected red cells. The intra- and inter-assay coefficients of variation of the method were 6.8 and 7.4%, respectively. The findings indicate that this ELISA allows sensitive, specific and reproducible measurement of glycoalbumin, and provides a clinically relevant objective parameter of integrated glycemic control.

Glycated albumin: a marker of glycaemic status in rats with experimental diabetes.

Monoclonal antibodies that specifically recognize non-enzymatically glycated epitopes residing in albumin were used to measure levels of glycated albumin in the plasma of control and streptozotocin diabetic rats. Standard or sample was incubated with monoclonal antibody immobilized onto preactivated tubes, and binding was detected with enzyme-conjugated polyclonal antibody to rat albumin and substrate. Rat glycated albumin exhibited a linear dose response in the assay, and plasma samples contained between 30-100 micrograms/50 microliters. Levels were significantly increased in diabetic compared with control rats, and correlated positively and significantly with mean blood glucose concentrations measured in the fasting and fed states. The results indicate that glycated albumin levels measured by immunoassay reflect recent integrated glycaemia and provide and objective index of glycaemic status in the rat experimental model of diabetes.

Amelioration of diabetic nephropathy by treatment with monoclonal antibodies against glycated albumin.

The pathogenesis of diabetic nephropathy is incompletely understood, but increased nonenzymatic glycation of proteins is considered an important contributory factor. Glycated albumin, which is increased in diabetic sera and is preferentially transported into the renal glomerulus, induces an increase in Type IV collagen production and a decrease in proliferative capacity by mesangial cells in culture. These effects resemble the abnormalities that characterize the glomerular mesangium in diabetes and are prevented by monoclonal antibodies that specifically react with Amadori adducts in glycated albumin. To explore the possibility that the in vitro effects of glycated albumin on mesangial cell biology pertain to the in vivo situation, we examined the effect of treatment with the A717 monoclonal antibodies on glomerular functional and structural changes in a rodent model of genetic diabetes, the db/db mouse. Weekly parenteral antibody administration reduced the elevated albumin excretion and attenuated the mesangial expansion that were observed in the untreated db/db mice that served as controls. Monoclonal antibody treatment also was shown to lower plasma concentrations of glycated albumin in diabetic mice. Thus, reducing glycated albumin concentrations and/or blocking its biologically active epitopes has a salutary influence on the pathogenesis of diabetic nephropathy. The findings indicate that glycated albumin participates in the development of the glomerular lesion in the db/db mouse, and suggest a new approach to the therapy of this complication of diabetes.

Evolution of renal function abnormalities in the db/db mouse that parallels the development of human diabetic nephropathy.

The db/db mutant mouse is a rodent model of genetic diabetes that develops renal glomerular lesions with striking mesangial matrix accumulation by the age of 16 weeks, after 8-10 weeks of sustained hyperglycemia. However, abnormalities in renal function that antedate or accompany the appearance of these pathologic changes, which resemble those found in human diabetes, have not been delineated. We therefore examined renal function in young db/ db mice and their nondiabetic db/m littermates from the age of 8 through 15 weeks. Serum creatinine and blood urea nitrogen concentrations at the onset of diabetes in db/db mice did not differ significantly from mean concentrations in db/m controls. An elevated creatinine clearance, due in large part to increased body weight, and increased urinary albumin excretion were observed in db/db compared with db/m mice soon after establishment of sustained hyperglycemia. A relative reduction in creatinine clearance was demonstrable in db/db mice at the age of 15 weeks, coincident with the appearance of overt compromise in renal function manifested by frank increases in the serum creatinine and blood urea nitrogen. The findings indicate that the well-documented glomerular pathology in db/db mice is accompanied by definable alterations in renal function, which are similar in chronology and nature to those found in human diabetes.

Albumin modified by Amadori glucose adducts activates mesangial cell type IV collagen gene transcription.

The direct relationship between elevated glucose concentrations and accelerated protein glycation has implicated increased glycation as a potential mechanistic link between hyperglycemia and the pathogenesis of diabetic nephropathy. Albumin modified by Amadori glucose adducts has been shown to stimulate collagen secretion by mesangial cells in vitro, and to contribute to the overproduction of glomerular mesangial matrix in vivo. To delineate mechanisms responsible for these effects, we examined the influence of glycated albumin on transcriptional activation of the alpha 1 (IV) collagen gene in renal glomerular mesangial cells. These experiments used a stably transfected reporter mesangial cell line that exhibits responses to media manipulations that are directionally parallel with those of non-transformed mesangial cells, and that expresses luciferase driven by 5'-flanking and first intron regions of the alpha 1 (IV) collagen gene. In these cells, purified glycated albumin stimulated collagen IV gene transcription, whereas glucose-free albumin did not. Further, glycated albumin induced a significant increase in mesangial cell collagen IV mRNA, assessed by Northern blot analysis and quantified by calculation of the ratio of collagen IV mRNA to 18S ribosomal RNA after densitometric scanning. The stimulation of collagen gene transcription and mRNA expression were both prevented by monoclonal antibodies known to specifically recognize Amadori-modified albumin. The findings indicate that glycated albumin promotes mesangial cell transcriptional activation and mRNA expression of the alpha 1 (IV) collagen gene and further implicate increased glycated albumin in diabetes in the pathogenesis of diabetic nephropathy.

Glycated albumin modified by Amadori adducts modulates aortic endothelial cell biology.

Increased protein glycation has been mechanistically linked to accelerated vascular pathobiology in diabetes. To test the influence of protein modified by Amadori glucose adducts on vascular cell biology, we examined the effect of glycated albumin on replicative capacity and basement membrane collagen production by aortic endothelial cells in culture. Relative to carbohydrate-free albumin, which supported cell proliferation and Type IV collagen synthesis, glycated albumin significantly inhibited 3H-thymidine incorporation and Type IV collagen production. The glycated albumin-induced effects were prevented by monoclonal antibodies (A717) that specifically react with Amadori-modified albumin, but not by IgG that was unreactive with glycated albumin. A717 had no effect on thymidine incorporation or collagen synthesis by cells cultured in the presence of nonglycated albumin. The findings indicate that the interaction of glycated albumin with endothelial cells, which have been shown to display dose-responsive, saturable receptors, limits cell replication and triggers maladaptive biosynthetic programs, which may contribute to degenerative macrovascular disease in diabetes.

Inhibiting albumin glycation ameliorates diabetic nephropathy in the db/db mouse.

Albumin modified by Amadori glucose adducts stimulates the expression of extracellular matrix proteins by glomerular mesangial and endothelial cells, and has been mechanistically linked to the pathogenesis of diabetic nephropathy. To test the hypothesis that inhibiting the formation of glycated albumin might beneficially influence the development of kidney disease in diabetes, we treated diabetic db/db mice for 12 weeks with a low-molecular-weight compound (EXO-226) that impedes the condensation of free glucose with lysine epsilon-amino groups in albumin. Administration of EXO-226 (3 mg/kg) twice daily by gavage normalized the plasma concentration of glycated albumin within days after initiation of treatment and maintained glycated albumin within the normal range throughout the study, despite persistent and severe hyperglycemia. Urine albumin excretion, which was markedly increased at the start of the study (age 12 weeks), was significantly reduced in treated diabetic animals compared with their untreated diabetic littermates. The fall in creatinine clearance that was observed in untreated diabetic animals was prevented in diabetic littermates that received treatment. Compared with the nondiabetic controls, the amount of glomerular mesangial matrix was threefold greater in untreated diabetic mice; in contrast, the mesangial matrix fraction was only 1. 5 times that of nondiabetic controls in the treated diabetic animals, representing a reduction in mesangial matrix accumulation of more than 50%. EXO-226 also reduced the overexpression of mRNA encoding for alpha1 (IV) collagen in renal cortex of db/db mice. We conclude that normalization of plasma glycated albumin concentrations with the glycation inhibitor EXO-226 ameliorates the glomerular structural and functional abnormalities associated with diabetic nephropathy in the db/db mouse.