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Journal of the American Society of Nephrology 183

Role of Amadori-Modified Nonenzymatically GlycatedSerum Proteins in the Pathogenesis of DiabeticNephropathy1Margo P. Cohen and Fuad N. Ziyadeh2

M.P. Cohen, Department of Biochemistry, University ofPennsylvania School of Medicine, Philadelphia. PA,and Exocell, Inc. . Philadelphia. PA

F.N. Ziyadeh, Department of Medicine and the PennCenter for Molecular Studies of Kidney Diseases,University of Pennsylvania School of Medicine,Philadelphia, PA

(J. Am. Soc. Nephrol. 199#{243};7:183-190)

ABSTRACTAccelerated nonenzymatic glycation in diabetes, re-

suIting in Amadori-modifled proteins and the later-developing advanced glycation end-products, hasbeen mechanistically linked to the pathogenesis ofdiabetic nephropathy. Recent focus on putative AGE-induced pathophysiology has shifted attention fromthe possible role of Amadori-modified proteins in thedevelopment of diabetic complications. Ample ex-perimental evidence has demonstrated that Ama-don-modified serum proteins adversely affect renalglomerular capillary function, structure, and metabo-lism. Previous studies from the laboratories of thisstudy’s authors have shown that human serum con-taming diabetic concentrations of albumin modified

by Amadori-glucose adducts inhibits the replicationof murine mesangial cells In culture and stimulatesthe production and gene expression of type IV colla-gen. Monoclonal antibodies (A717) specific for Ama-dori-glycated albumin prevent these abnormalities.In other studies, it has also been shown that In vivoadministration of A717 (Fab fragments) retards the

progression of diabetic nephropathy in diabetic

db/db mice. Neutralizing the effects of the elevated

circulating glycated albumin concentration is associ-ated with reduction in proteinuria and mesangialmatrix expansion, and prevention of the overexpres-sion of mRNA encoding type IV collagen and fi-

bronectin in the renal cortex. The renoprotective ef-fects of A717 are independent of any change Inblood glucose concentrations. These studies 1mph-

t Received May 5, 1995. Accepted september 6. 1995.

2 correspondence to Dr. F.N. Zlyadeh, Renal-Electrolyte and Hypertension Dlvi-

slon, 700 clinIcal Research Building, University of Pennsylvania, 422 curIe Boule-

yard, PhiladelphIa, PA 191O4�6144.

1046.6673/0702.0183103.00/0Journal of the American society of Nephrologycopyright © 1996 by the American society of Nephrology

cate Amadori-modified glycated albumin in thepathogenesis of diabetic nephropathy. It Is proposedin this study that abrogating the biologic effects ofincreased glycated albumin in diabetes has noveltherapeutic potential in the management of diabeticrenal complications.

Key Words: Mesangium. albumin, hyperglycemia. glomeru-

lus, glycation

D iabetic nephropathy is a major cause of morbid-ity and mortality. and accounts for most cases of

chronic renal failure that require treatment in anESRD program. One third of patients with Insulin-

dependent diabetes mellitus and at least 1 5% of pa-tients with non-insulin-dependent diabetes will de-velop this complication ( 1-5). In the past two decadesof research, it has become increasingly apparent that

nephropathy, like other chronic complications of dia-betes, has its origin In biochemical disturbances thatmay long antedate the appearance of characteristic

pathologic lesions or of clinical disease manifested byfunctional abnormalities. Experimental evidence link-ing hyperglycemia, the cardinal and pathognomonic

feature of diabetes, to these disturbances has beenextant for many years. Early work demonstrated thathyperglycemia induced in rats by streptozotocin oralloxan injection. or in human patients with pancre-atic insufficiency who develop secondary diabetes,could cause thickening of the glomerular basementmembrane, a characteristic ultrastructural feature ofdiabetic nephropathy (6-1 3). Glomeruli from strepto-zotocin-diabetic rats exhibit increased basement-membrane collagen production and enhanced activityof several enzymes that are intimately involved incollagen biosynthesis, such as lysyl-hydroxylase andglucosyl-transferase (14-18). These changes are pre-vented with correction of the hyperglycemia by insulinadministration ( 19-22). Nevertheless. it remained forthe Diabetes Control and Complications Trial (DCCT)to conclusively establish that intensive regimens for

blood glucose control can lower the risk for develop-ment of diabetic retinopathy, neuropathy. and ne-phropathy. thus linking hyperglycemia to the risk ofcomplications In human diabetes (23).

Despite these dramatic results. the exact mecha-nisms by which hyperglycemia contributes to thedevelopment of nephropathy remain incompletely elu-cidated. Further, it is widely appreciated that imple-mentation and maintenance of intensive regimenssuch as those used in the DCCI are difficult, and thatachievement of requisite glucose homeostasis is prob-lematic In the vast majority of diabetic patients. For

Amadori Glycation in Diabetic Kidney

184 Volume 7 . Number 2 . 1996

these reasons, much effort has been directed at un-derstanding the mechanistic links between hypergly-cemia and nephropathogenesis. which could help inidentifying intervention strategies that address dde-terious factors that act in concert with or independentof glycemic status In the pathogenesis of diabeticcomplications. The use of angiotensin-converting en-zyme inhibitors in diabetic patients with significantproteinuria is one such strategy that has recentlyproved efficacious in arresting decline in renal func-

tion, presumably on the basis of interruption of ad-verse Intraglomerular hemodynamic influences (24).This approach has been heralded as a major thera-peutic advance for forestalling renal failure that re-sults from diabetic nephropathy. Other potential

points of intervention include inhibiting the polyolpathway, blocking the effects of excess nonenzymaticglycation of proteins, and preventing the formation ofadvanced glycation end-products (AGE). Such ap-proaches may hold promise for mitigating the delete-rious metabolic consequences that ensue from hyper-glycemia-induced activation of the polyol pathway(25-30) or increased protein glycation (3 1-38), or thatare related to the modification of proteins by

crosslinks of AGE (39-43). Several excellent reviewsthat discuss the role of polyol pathway activation andmodification by AGE in the pathogenesis of diabeticcomplications have recently been published(25-27,44-49). This review focuses on the role ofAmadori-modified, nonenzymatically-glycated serum

proteins in the development of diabetic nephropathy.

NONENZYMATIC GLYCATION OF PROTEINS

Nonenzymatic glycation is a condensation reactionbetween free glucose and reactive-protein aminogroups, yielding Schiff-base intermediates that un-dergo Amadori rearrangement to form stable protein-glucose adducts (50-54). AGE formation, which isglucose independent. is believed to evolve slowlythrough a series of spontaneous rearrangement. de-hydration, and polymerization reactions (39-43). In

vivo, circulating glycated proteins principally exist asAmadori products, and their concentration, driven by

the ambient glucose concentration to which proteinsare subjected during their residence time in the circu-latlon, is significantly increased in diabetes with ex-posure to a hyperglycemic milieu (50,51,54-60).Amadori-modified matrix proteins also are increased

In diabetes (61-67). This direct relationship between

elevated glucose concentration and increased proteinglycation, together with mounting evidence that glu-cose-derived protein modifications can alter struc-tural and functional properties, has strongly impli-cated accelerated glycation as a potential mechanisticlink between hyperglycemia and the pathogenesis ofdiabetic complications (3 1-43). Recent focus on puta-tive AGE-induced pathophysiology has shifted atten-tion from the possible role of Amadori-modified pro-teins in the development of diabetic complications.

However, it is clear that formation of AGE can only

occur subsequent to nonenzymatic glycation. raisingthe possibility that AGE rearrangement is a “fixative”process, marking the occurrence of glycation-induced

events for which formation of the Amadori glucose-adduct may be the critical event (68). Such consider-ations, coupled with the results of several studies that

demonstrate the adverse effects of glycated serumproteins on glomerular physiology and biochemistry,

has prompted interest in the exploration of the poten-

tial role of Amadori-modified proteins in the develop-ment of diabetic nephropathy.

AMADORI-GLYCATED PROTEINS ANDMODULATION OF MESANGIAL CELL BIOLOGY

Diabetic nephropathy originates in the glomerularmesangium, which is bathed with serum that con-

tans increased concentrations of glycated proteins in

diabetes. Amadori-glycated serum proteins are prefer-

entially transported across the filtration barrier and

can induce adverse cellular effects (69-73). Glomeruli

isolated from normal rats exhibit preferential uptake

of glycated serum albumin relative to nonglycated

albumin, which is accompanied by an increase In cell

hydrogen peroxide production (72). Epithelial and

mesangial cells in glomeruli from streptozotocin-dia-betic rats show increased hydrogen peroxide produc-

tion, and this abnormality can be duplicated in gb-merular cells from normal rats by injecting Amadori-

glycated albumin and raising its circulating

concentration (73). Nondiabetic mice injected with

glycated plasma proteins have been reported to de-velop glomerular basement membrane thickening and“pseudodiabetic” glomerular lesions (74). A moststriking effect of Amadori-glycated serum proteins isthe induction of glomerular hyperfiltration, an earlyfunctional abnormality implicated in the development

of diabetic nephropathy. Sabbatini and colleagues

showed that normal rats manifest hyperfiltration

when they are transfused with plasma subjected toshort-term incubation with glucose to achieve concen-

trations ofAmadori-modified glycated proteins similar

to those found in streptozotocin-diabetic rats (75).

This glycated serum-induced hyperfiltration was de-

monstrable under normoglycemic conditions, with animpressive exaggeration observed under conditions ofacute hyperglycemia.

The gbomerular lesion in diabetic nephropathy ischaracterized by expansion of the mesangial matrix,leading to encroachment of cellular elements and lossof filtration area, and thickening of the peripheral

capillary basement membrane (76-80). The increasedmesangial cell elaboration of matrix macromolecules,

an early and consistent feature of diabetic gbomeru-

lopathy, has been exploited in cell culture studies to

investigate potential nephropathic factors by exam!-nation of the response to diabetes-related manipula-

tions of the culture environment (81-88). For exam-ple, high glucose concentration induces increased

Cohen and Ziyadeh


mesangial cell production and gene expression of typelv collagen, laminin. and fibronectin (83,84,87). Thesechanges are accompanied by activation of proteinkinase C, transient elevation of mRNA levels of c-fos

and c-Jun oncogenes, and corresponding Increases Inc-fos and c-Jun proteins, and delayed growth inhibi-tion in association with bioactivation of transforminggrowth factor-(31 (TGF-�1) (82,89-92). Given that themesangium in diabetes is exposed to serum contain-Ing Increased concentrations of nonenzymatically gly-

cated proteins. it appeared appropriate to examinemesangial cell growth and matrix biosynthesis underconditions that simulate this in vivo diabetic milieu.Serum that contains diabetic concentrations of Ama-

don-modified glycated proteins inhibits the prolifera-tion of mesangial cells in culture and stimulates theelaboration and gene expression of type IV collagen(93). These effects are demonstrable in physiologicglucose concentration and are accentuated in mediathat contain elevated glucose concentration, indicat-ing that glycated serum proteins influence mesangialcell biology both independent of and additive to theprevailing glucose concentration. Thus, under condi-tions approximating the in vivo situation in whichthere are periods of normoglycemia and hyperglyce-mia, glycated serum proteins induce mesangial cellabnormalities resembling those associated with theglomerulopathic lesion in diabetes.

The observed increase in type IV collagen mRNAinduced by Amadori-modified serum proteins isgreater than that previously seen when mesangialcells or proximal tubule epithelial cells, another cell

line that overproduces type N collagen in a hypergly-cemic milieu, were incubated with elevated glucoseconcentration in serum-free media (94-97). The tubu-lointerstitium and gbomeruli exhibit parallel changes

In diabetes. with overproduction of extracellular ma-trix and increased basement membrane thickness

(92,98-100). High media glucose concentrationcauses an approximately twofold increment in the

relative type N collagen:GAPDH mRNA ratio in me-

sangial or proximal tubule epithelial cells, whereas

Amadori-glycated serum in high glucose media in-

duces a ninefold increase in the type N collagen:GADPH mRNA ratio relative to the ratio observed incells cultured with low (5.5 mM) glucose and no serumsupplement. It is thus clear that Amadori-glycatedserum proteins influence mesangial cell mRNA levelsof type N collagen independent of glucose concentra-tion, and can markedly augment the modest effect ofhigh glucose.

Interestingly, the effects of glycated serum on cellreplication and collagen production are prevented inthe presence of the monocbonal antibody A7 1 7 thathas been shown to specifically recognize albumin

modified by Amadori glucose adducts (94), Indicatingthat the observed changes are principally the result ofincreased Amadori-glycated albumin relative to otherglycated serum proteins. The influence of Amadori-glycated albumin on mesangial cell biology has been

confirmed by direct examination of the effects of thepurified protein on mesangial cell growth and collagensecretion. Again, the glycated albumin-induced de-crease In 3H-thymidlne incorporation and increase incollagen IV secretion are demonstrable In physiologicglucose concentration, and are exaggerated in highglucose media ( 10 1 ). The changes In cell replicationand collagen N production are also prevented by theantiglycated albumin monoclonal antibodies A7 17,whereas immunogbobulin G not reactive with glycatedalbumin has no effect on 3H-thymidlne incorporationor collagen IV secretion ( 1 0 1 ). These data havestrengthened the notion that increased levels of Ama-dori-glycated albumin in diabetes is an importantcontributor to mesangial cell pathobiology. and thatthe deleterious effects of glycated albumin are mani-fest regardless of glycemic fluctuations but are accen-tuated with hyperglycemic excursions.

The increased collagen N mRNA in mesangial cells

exposed to glycated serum coincides temporally (72 hofculture) with the increased TGF-13l gene expressionand bioactivity that have been observed in proximaltubule cells and mesangial cells grown in serum-freemedia that contains an elevated glucose concentra-tion (82.92.97). It has been postulated that TGF-�1plays a primary causative role in diabetic renal dis-

ease ( 102-1 10). According to this hypothesis, in-creased TGF-�1 expression leads to diabetic renalhypertrophy and Increased matrix production by tu-bular and gbomerular mesangial cells. This raises thepossibility that glycated serum proteins, like elevated

ambient glucose alone, can exert deleterious effects oncells through the modulation of TGF-�1 activity orgene expression. We are currently examinIng the in-terrelations that may exist between the effects ofAmadori-glycated serum proteins on collagen and fi-

bronectin production In mesangial cells and theirpossible mediation by increased TGF-131 expression.

GLYCATED PROTEIN RECEPTORS

Recent work has established that mesangial cellsexpress receptors that recognize albumin modified byAmadori glucose adducts, and suggests that a ligand-

receptor system for glycated albumin promotes tran-scriptional activation of the al(IV) collagen gene( 1 1 1-1 13). Ligand-blnding studies conducted with

phenotypically stable nontransformed mesangial cells

have demonstrated selective binding of glycated albu-mm in a dose-dependent and saturable manner, andare consistent with the existence of more than oneaffinity class ofglycated albumin receptors. In compe-

tition experiments. specific binding of glycated albu-mm to mesangial cell receptors is inhibited by the

antiglycated albumin monoclonal antibodies. The an-tibody does not interfere with the binding of nongly-cated albumin. In parallel studies, we have demon-strated that Amadori-glycated albumin stimulatesal(IV) collagen mRNA levels in mesangial cells, in partbecause of transcriptional activation of the a 1(IV)


186 Volume 7 . Number 2 . 1996

collagen gene as assayed by transfection of a lucif-erase reporter ( 1 14, 1 15); these effects are completelyprevented by the antiglycated albumin monoclonalantibodies. Because glycated albumin effectuateschanges in mesangial cell biology that are not ob-served with nonglycated albumin. it is reasonable toposit that blocking biologically active sites with themonocbonal antibody prevents an interaction withcellular binding sites that mediate the effects of gly-cated albumin on collagen al(N) gene expression.

AMADORI-GLYCATED PROTEINS ANDGLOMERULAR PATHOLOGY IN VIVO

The data from in vitro studies with mesangial cells in

culture strongly suggest that Increased levels of Ama-

dori-glycated albumin in diabetes is pathophysiologi-cally Important in the development of diabetic ne-

phropathy. With due caution in extrapolating datafrom tissue-culture studies to the in vivo situation, we

reasoned that abrogation of the influence of excessglycated albumin could have a salutary influence onthe development of gbomerular pathology in diabetes.To test this hypothesis, the antiglycated albumin

monocbonal antibodies A7 1 7 were administered todiabetic db/db mutant mice, a rodent model of genetictype II diabetes mellitus that develops glomerular

mesangial expansion resembling that found in human

diabetes ( 1 16-1 18). Plasma glycated albumin concen-trations were elevated twofold to threefold fold indb/db mice compared with their nondiabetic db/m

littermates, and declined after monocbonal antibodyadministration. Eight to 10 wk after the onset ofhyperglycemia, the steady-state levels of mRNA thatencode the a 1(N) collagen relative to 1 85 rRNA wereincreased 2.6-fold in kidney cortex of db/db mice;similarly, levels of mRNA that encode fibronectin rel-

ative to 18S rRNA were 3.8-fold greater in the db/db

mice compared with the db/m littermates. Histomor-phometric measurements demonstrated 3.8-fold in-crease in the mesangial matrix fraction in db/db

relative to db/m mice. Treatment with the monocbonalantiglycated albumin antibody for 8 consecutive wksignificantly reduced urinary protein excretion, renaltype N collagen. and fibronectin mRNA levels, andprevented mesangial matrix accumulation without

any change in glycemic status. Administration of an-

tibodies unreactive with glycated albumin was unableto duplicate these effects (1 19-120). Thus, treatmentof diabetic db/db mice with a murine monocbonalantibody directed against Amadori-modified albuminprevented abnormalities in renal gbomerular function,histopathology, and cell biology that are characteristic

of the nephropathic lesion in diabetes. Notably, the

db/db mice in these experiments were not treatedwith insulin or other glucose-lowering agents, andblood glucose concentrations at the conclusion of thestudy were as elevated as they were at the initiation ofthe study period. Thus. although hyperglycemia is thedriving force for increased nonenzymatic glycation.

the results of these studies indicate that the nephro-pathogenic effects of increased Amadori-glycated al-bumin in vivo can be corrected independent of antihy-

perglycemic therapy.Treatment with an antiglycated albumin monoclo-

nal antibody blocks the biologically active glycated

epitope and/or accelerates its removal from the circu-latlon, diminishing the putative interaction of glycatedalbumin at the cellular level. The glycated albuminepitope is readily accessible in the circulation, pro-moting rapid binding to the complementarity-deter-

mining region of the monocbonal antibody Fab frag-

ments, and presumably is followed by clearance of the

complex by the reticuloendothelial system.There are no human studies that evaluate the role of

Amadori-modified proteins in diabetic complications,

nor are there studies that directly correlate glycoalbu-niln levels with long-term clinical outcome. The DCCThas shown that long-term lowering of the mean glyco-

hemoglobin level diminishes the incidence of diabeticnephropathy, retinopathy, and neuropathy (23). Near-

normalization of the glycohemogbobin over extendedperiods of time correlates with a lessened risk for

complications of diabetes. The Amadori form of gly-

cated albumin (glycoalbumin) is a measure of inter-mediate-term Integrated glycemia, and correlates withglycohemoglobin when ambient glycemia is main-tamed in the same range during the preceding 2 to 10

wk. The corollary that near normalization of gly-

coalbumin over extended periods of time would simi-larly correlate with reduced risk for complications is

reasonable.

DISTINCTION BETWEEN EFFECTS THAT RESULTFROM AMADORI-GLUCOSE ADDUCTS VERSUSTHOSE RESULT FROM AGE

It should be noted that the cellular effects we havedescribed in vitro and in vivo (93, 1 1 1-1 13, 1 15, 1 19-

123) are the result of Amadori-modified rather thanAGE-modified proteins. The glycated albumin for in

vitro experiments was purified from the humanplasma of normal subjects or was prepared underconditions known to yield Amadori and not AGE mod-ification (4-day Incubation with 28 mM glucose atroom temperature). Although it might be argued thatglycated albumin contains an AGE-modified residue,

it is clear that the observed biologic effects depended

on the presence of an Amadori modification becausethe A7 1 7 monoclonal antibody inhibited these effects.The A7 1 7 antibody was raised against glycated albu-mm that was purified from normal plasma and did notcontain AGE-modified albumin (94). The antibodyspecifically reacts with glycated lysine epitopes inalbumin, does not recognize nonglycated albumin,and does not immunoreact with albumin In the pass-

through fraction after affinity-chromatography with aphenylboronate column. Even if AGE products ofserum glycoalbumin are formed in vivo (or in vitro aftershort-term glucose incubation), they would be present

Cohen and Ziyadeh


in the phenylboronate pass-through fraction because

coplanar cis-hydroxyl groups are required for bindingto phenylboronate. AGE products, which are irrevers-

ibly formed, do not have this configuration.

AGE-modified proteins are predominantly identified

by their characteristic fluorescence and their capacity

to react with antisera raised against AGE-modifiedRNase or BSA. Such antibodies recognize AGE-mod!-fled proteins independent of the nature of the protein

itself, and react with epitopes common to many pro-

teins that have been subjected to extensive modifica-

tion in vitro by incubation for prolonged periods with

extremely high concentrations of reducing sugars.

Therefore, what these assays really measure is not well

established, but is likely to encompass a host of end-

products, some of which are inert, that accumulate inrenal failure even without diabetes (43). Such measure-ments may therefore be interpreted as markers of glyca-

tion-related processes or damage, more than being es-pecially causative of diabetic nephropathy.

CONCLUSIONS

Many abnormalities of the diabetic milieu contrib-

ute to glomerular injury. The important role of hyper-glycemia in the genesis of diabetic renal disease has

been strengthened by the application of tissue-culture

techniques. Likely mediators of the effects of highambient glucose ( 1 25) include the following: adaptivechanges in glomerular hemodynamics (increasedtranscapillary hydraulic pressure); activation of the

polyol pathway; altered cellular myo-Inos!tol metabo-

lism; biochemical abnormalities related to intracellu-

bar signaling, such as increased protein kinase C

activity: alterations in hormonal or cytokine balance,

especially activation of the TGF-�3 system (126);and / or nonenzymatic glycatlon of various serum or

matrix proteins (Table 1 ). A unifying hypothesis en-

compassing the contribution of all these factors in the

pathogenesis of diabetic nephropathy remains specu-

lative. One postulate implicates the hyperglycemia-induced increase In the redox state (e.g. , high NADH/NAD� ratio) to manifestations of diabetes in targettissues, such as increased polyol pathway activity,

increased de novo synthesis of diacylglycerol, and

stimulation of PKC activity. The oxidative stress pro-

duced by nonenzymatic glycation may also be linkedto the cellular dysfunction associated with the hyper-

glycemia-induced “pseudohypoxic” state ( 1 27). In thisreview, we examined several lines of evidence thatpointed to a causal relation between increased nonen-zymatic glycation of serum proteins and the develop-

ment of diabetic nephropathy. The cell biologychanges that underlie the structural abnormalities

that characterize the diabetic renal glomerulus in vivo,

including accumulation of extracellular matrix pro-

tein at the expense of cellular replicative capacity,have been reproduced by the incubation of mesangial

cells with Amadori-modified serum proteins (93), in

particular, glycated albumin ( 10 1 ). Blocking the bio-

TABLE 1. Mediators of diabetic renal disease

Genetic/Familial PredispositionAltered Intrarenal HemodynamicsHumoral Imbalance

Metabolic consequences of insulin deficiencyActivation of intrarenal cyfokines or growth factors

Angiotensin IIThromboxaneNitric oxideInsulin-like growth factor 1Platelet-derived growth factorTransforming growth factor-13

Activation of Pathways for Glucose MetabolismAldose reductase-dependent polyol pathway

(increased sorbitol)

Pentosephosphate shunt (increased UDPglucose)De novo synthesis of diacylglycerol and stimulation of

protein kinase CDisordered cellular myo-inositol metabolismAltered cellular redox state (increased �,

NADH/NAD�)Altered glycosphingolipid metabolismRenal tubular hypermetabolism and oxidant injury

Nonenzymatic Glycation of Circulating or Matrix ProteinsAmadori-modified glucose adductsAdvanced glycosylation end-products (AGE)

logically active epitope in glycated albumin with highly

specific monoclonal antibodies prevents the induction

of these changes in cultured mesangial cells. Glycated

serum transfused into normal animals provokes hy-

perfiltration (75), the prognostically Important gbomer-ular hemodynamic dysfunction in early diabetes. In-jection of diabetic mice with antibodies that are

capable of neutralizing excess glycated albumin pre-vents the overproduction of matrix proteins, accumu-

lation of extracellular matrix, and excessive urineprotein excretion ( 1 1 1 . 1 19, 120). The mechanism bywhich glycated serum proteins induce pathobiologicevents in the renal gbomerulus is incompletely de-fined, but may relate to receptor-induced events ( 1 1 1-

1 13, 121-124). Increased glycated albumin in diabetes

appears to be a potent stimulus in vivo of renal

extracellular matrix production and gene expressionand, consequently, a target for intervention therapy In

diabetic nephropathy.

ACKNOWLEDGMENTS

Work performed in the authors’ laboratories was supported in part by

National Institutes of Health Grants DK-38308, DK-45 19 1 . and DK-44513. and Training Grant DK-07006.

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