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AGE, RAGE, and ROS in DiabeticNephropathy

Adeline L. Y. Tan, BBiomedSc, Josephine M. Forbes, PhD, and Mark E. Cooper, PhD

Summary: Diabetic nephropathy is a major cause of morbidity and mortality in diabeticpatients. Two key mechanisms implicated in the development of diabetic nephropathyinclude advanced glycation and oxidative stress. Advanced glycation is the irreversibleattachment of reducing sugars onto amino groups of proteins to form advanced glycation endproducts (AGEs). AGE modification of proteins may lead to alterations in normal function byinducing cross-linking of extracellular matrices. Intracellular formation of AGEs also can causegeneralized cellular dysfunction. Furthermore, AGEs can mediate their effects via specificreceptors, such as the receptor for AGE (RAGE), activating diverse signal transductioncascades and downstream pathways, including generation of reactive oxygen species (ROS).Oxidative stress occurs as a result of the imbalance between ROS production and antioxidantdefenses. Sources of ROS include the mitochondria, auto-oxidation of glucose, and enzymaticpathways including nicotinamide adenine dinucleotide phosphate reduced (NAD[P]H) oxi-dase. Beyond the current treatments to treat diabetic complications such as the optimizationof blood pressure and glycemic control, it is predicted that new therapies designed to targetAGEs, including AGE formation inhibitors and cross-link breakers, as well as targeting ROSusing novel highly specific antioxidants, will become part of the treatment regimen fordiabetic renal disease.Semin Nephrol 27:130-143 © 2007 Elsevier Inc. All rights reserved.Keywords: Diabetes mellitus, diabetic nephropathy/complications, advanced glycationend-products, receptors, oxidative stress

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iabetes mellitus is one of the biggestepidemics affecting human health inthe 21st century. The alarming increase,

articularly of type 2 diabetes, is attributedargely to globalization, along with vast changesn human behavior, lifestyle, and increased lifes-an.1 The complications associated with diabe-es are a major cause of morbidity and mortal-ty, with diabetic nephropathy being one of the

ajor chronic microvascular complications inoth type 1 and 2 diabetic patients. Many fac-ors influence the development of diabetic ne-

lbert Einstein Centre for Diabetes Complications, Baker Heart ResearchInstitute, Melbourne, Victoria, Australia.

upported in part by the Juvenile Diabetes Research Foundation, NationalHealth and Medical Research Council of Australia, Diabetes Australia, andthe National Institutes of Health. J.M.F. is a Juvenile Diabetes ResearchFoundation career development awardee; A.L.Y.T. is supported by a KidneyHealth Australia Medical Research Funding Award.

ddress reprint requests to Mark E. Cooper, Albert Einstein Centre for DiabetesComplications, Baker Heart Research Institute, PO Box 6492, St Kilda Rd, CentralMelbourne, Victoria 8008, Australia. E-mail: Mark.Cooper@baker.edu.au

270-9295/07/$ - see front matter

a2007 Elsevier Inc. All rights reserved. doi:10.1016/j.semnephrol.2007.01.006

Seminars30

hropathy and other complications includingenetic, hemodynamic, environmental, andetabolic factors (Fig. 1). The major contribut-

ng factor is persistent hyperglycemia with theiabetes Control and Complications Trial2 and

he United Kingdom Prospective Diabetestudy3 showing that intensive control of hyper-lycemia can reduce the occurrence and pro-ression of diabetic microvascular complica-ions including nephropathy. The mechanismshereby hyperglycemia leads to diabetic com-lications continue to be under active investi-ation. This review focuses on advanced glyca-ion and oxidative stress, 2 key players in theathogenesis of diabetic nephropathy.

DVANCED GLYCATION END PRODUCTS

dvanced glycation end products (AGEs) areound covalently, reducing sugar modificationsf proteins and lipoproteins.4 AGEs accumulate as

result of natural aging in a time-dependent man-

in Nephrology, Vol 27, No 2, March 2007, pp 130-143

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AGE, RAGE, and ROS 131

er, as supported in experimental models and inuman beings. It is thought that the physiologicole of advanced glycation is to identify senescentroteins for degradation. During aging, AGE for-ation may result from reduced AGE defenses,

ong-term exposure of proteins to reducing sugarsuch as glucose, increased insulin resistance,nd/or deteriorating renal function. In diabetes,GE formation is enhanced by persistent hyper-lycemia and oxidative stress, leading to morextensive modification of long-lived proteins suchs skin collagen, although short-lived proteins alsoecome targets for advanced glycation. Exoge-ous AGEs may be absorbed into the circulationrom reactions between sugars and proteins inoods or from curing of tobacco. Indeed, AGEontent is high in cooked and processed foods,specially those rich in proteins, fat, and sugar.5 Its considered that dietary AGEs are similar to en-ogenous AGEs with regard to their prooxidant,roinflammatory, and signaling properties.6 Thus,he levels of circulating AGE levels in diabeticatients may be a reflection of both endogenously

ormed and exogenously ingested AGEs.Importantly, the kidney is a target for AGE-

igure 1. Potential interactions between metabolic, hemenesis of diabetic nephropathy. ACE, angiotensin-conv

ediated damage and also the main contributor a

o increasing circulating AGE concentrations via aecrease in renal function, by the clearance ofGEs.7

ENESIS AND STRUCTURE OF AGES

dvanced glycation (advanced glucosylation orlycosylation) is the nonenzymatic processhereby the carbonyl (aldehyde or ketone) of

educing sugars such as glucose react nonenzy-atically with lysine and N-terminal amino

roups in a variety of proteins, lipoproteins, anducleic acids, leading to the formation of earlylycation products via the Maillard reaction.4

hese go through further rearrangements leadingo the formation of various reactive intermediateroducts including �-dicarbonyls or oxoalde-ydes. Examples of �-dicarbonyls include 3-de-xyglucosone, glyoxal, and methylglyoxal. �-di-arbonyls react with amino groups of intracellularnd extracellular proteins to form AGEs, a heter-geneous class of stable and irreversible covalentdducts. Many AGEs such as pentosidine haventrinsic florescence and hence tissue and plasmauorescence may be used as markers of AGE

amic, genetic, and environmental factors in the patho-enzyme; BP, blood pressure.

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ccumulation. Other AGEs such as carboxymeth-

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132 A.L.Y. Tan, J.M. Forbes, and M.E. Cooper

llysine (CML) are not fluorescent. In vivo, theaillard reaction is slow under homeostatic con-itions because glucose, the predominant sugarsed for fuel, is the least reactive of the biologicalugars to the Maillard reaction.4

Glycation may be accompanied by oxidationnd induction of intracellular metabolic path-ays such as the polyol pathway, to form gly-

oxidation products including CML, carboxy-thyllysine, and pentosidine. AGEs also can beenerated through nonoxidative pathways, forxample, pyralline, a methylglyoxal lysine dimer

igure 2. Possible pathways for AGE formation. Intermlycation leads to formation of �-dicarbonyl intermediatean occur through oxidative or nonoxidative pathways. Mthyllysine; GOLD, glyoxal lysine dimer; MOLD, methylg

igure 3. AGEs and their receptor-independent or -deetabolic defects, and structural abnormalities. TCA,

eceptor types A, B.

erived from methylglyoxal during nonoxidativenaerobic glycolysis, or deoxyglucosone lysineimer derived from 3-deoxyglucosone releaseduring Amadori rearrangements (Fig. 2).4

The effects of AGEs can be classified as re-eptor-independent or -dependent (Fig. 3).irst, AGEs modify long-lived structural compo-ents of the basement membrane or extracellu-

ar matrix (ECM). This may occur by increasinghe expression of protein components such asype IV collagen in the kidney or via abnormalnteractions of AGEs with other matrix compo-

lipid metabolism, the polyol pathway, and advancedEs, and advanced lipoxidation end-products (ALEs). Thismethylglyoxal; 3-DG, 3-deoxyglucosone; CEL, carboxy-l lysine dimer; DOLD, deoxyglucosone lysine dimer.

nt effects leading to renal clearance or inflammation,oxylic acid cycle; MSR-A, B, macrophage scavenger

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AGE, RAGE, and ROS 133

ents and cellular matrix receptors. In addition,ovalent intermolecular and intramolecularross-links can form between glycated ECMroteins leading to structural alterations includ-

ng changes in packing density, surface charge,embrane permeability, resistance to proteo-

ytic digestion, and thermal stability. AGEs dis-upt normal cell-matrix contact or preventhysiologic cellular growth and intercellularontact, thus preventing maintenance of tissuentegrity and normal function.4

The formation of AGEs is not exclusive to gly-ation of extracellular proteins. AGEs and �-dicar-onyl intermediates also can form from intracel-

ular components and this can occur after onlyays of hyperglycemia or via increases in reduc-

ng sugars owing to altered metabolism.8

ECEPTOR-MEDIATED EFFECTS OF AGES

GEs also mediate their effects via receptorsFig. 3) including the receptor for AGE (RAGE),acrophage scavenger receptor types I and II

types A and B1/CD36), oligosaccharyl trans-erase-48 (AGE-R1), 80K-H phosphoproteinAGE-R2), galectin-3 (AGE-R3), CD-36,4 and theecently identified ezrin, radixin, and moesinroteins.9 Other multiligand receptors includ-

ng megalin also bind AGEs. AGE receptors arexpressed on various cell types such as mono-ytes, macrophages, endothelial cells, mesan-ial cells, podocytes, tubular epithelial cells,strocytes, microglia, and smooth muscleells.10 Many AGE-receptors have multiple li-ands and can be activated by non-AGE moi-ties, as well as a range of structurally distinctGEs. It remains unknown which AGEs have

he greatest affinity and activating potential forGE receptors. Ultimately, the total empiricinding capacity rather than the specific AGEoiety that is binding may be more important.

AGE

lthough RAGE first was described as a receptoror AGEs, it later was discovered to be a multili-and receptor that recognizes a pattern or com-on motif. Its primary function is in the innate

mmune response in which it plays a major role in t

ost pathogen defense. Other ligands of RAGEther than AGEs include amphoterin, amyloid-peptide, S100/calgranulins, and Mac-1.11

RAGE is a multiligand member of the immuno-lobulin superfamily with 394 amino acids, a sin-le hydrophobic transmembrane domain (19mino acids), and a highly charged COOH-termi-al cytosolic tail (43 amino acids) that mediate

ntracellular signaling pathways. Extracellularly,AGE has a terminal V-type ligand binding do-ain and 2 C-type domains (V-C-C’) (Fig. 4).11

In addition to full-length RAGE, there are otheressenger RNA (mRNA) splice variants for RAGE

hat encode truncated proteins with various bio-ogical properties (Fig. 4).12 Endogenous secre-ory or soluble RAGE (sRAGE) lacks the COOHerminal and transmembrane domains. It is se-reted in a paracrine way and can bind extracel-ular ligands independently of direct cell contact.xcess sRAGE may competitively bind RAGE li-ands, preventing their interaction with the cellurface RAGE receptor and hence preventing cel-ular signaling. Hence, the balance between syn-

igure 4. RAGE, dominant-negative (DN)-RAGE, andhe major splice variants of RAGE - endogenous secretoryES)-RAGE and N-truncated (NT)-RAGE. RAGE has 3 ex-racellular domains: 1 terminal V-type domain and 2-type domains (V-C-C’), a transmembrane (TMD) do-ain, and a cytosolic tail (ct).

hesis of sRAGE and full-length RAGE may be a

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134 A.L.Y. Tan, J.M. Forbes, and M.E. Cooper

ey determinant of AGE-induced pathology. Lowpicomolar) levels of sRAGE have been found inhe plasma of animals and human beings, pro-uced by native expression of the truncated formf RAGE. This suggests that therapeutically, exog-nous administration of sRAGE of the same spe-ies may not trigger an immunologic response.ndeed, infusion of sRAGE of the same speciesnto animals such as mice is not immunogenic,ven up to 6 months.13 The potential therapeuticalue of sRAGE has been observed both in exper-mental diabetic atherosclerosis14 and nephropa-hy.15 Other RAGE isoforms include the secretedAGE isoform that lacks the transmembrane do-ain only, and N-truncated RAGE, which lacks

he terminal V-type domain. N-truncated RAGE isnchored in the cell membrane but does not bindigands and its role remains unknown.

In the human kidney, RAGE protein is foundn tubular epithelial cells,16 mesangial cells,17

odocytes,15,18 and within vascular and neuralompartments. In diabetes, RAGE expression isncreased at sites of macrovascular and micro-ascular injury. This is supported by AGE andAGE colocalization in susceptible organs iniabetes.19

RAGE binding by AGEs or other ligands acti-ates diverse signal transduction cascades in-luding p21ras, p38, p44/p42 (erk1/2, extracel-ular signal-related kinase), and stress-activatedrotein kinase/c-Jun N-terminal kinase mitogen-ctivated protein (MAP) kinases, the Janus ki-ase/signal transducers and activators of tran-cription pathway, and protein kinase C (PKC)athway. Signal transduction leads to down-tream consequences including generation ofeactive oxygen species (ROS) and activation ofranscription factors such as nuclear factorappa B (NF-�B).11 One important consequencef NF-�B translocation is the up-regulation ofAGE itself because the promoter region ofAGE contains functional binding elements forF-�B.20 AGE-RAGE induction of NF-�B or otherathways contributes to the release of proin-ammatory cytokines, and the expression ofdhesion molecules and growth factors that aremplicated in the pathogenesis of diabetic com-lications. These include transforming growth

actor-�1 (TGF-�1), vascular endothelial growth R

actor, connective tissue growth factor, inter-eukin-1� and -6, insulin-like growth factor-1,latelet-derived growth factor, tumor necrosis

actor (TNF)-�, and vascular cell adhesion mol-cule (VCAM)-1.11

GE, RAGE, AND DIABETICEPHROPATHY: ANIMAL STUDIES

nimal studies support the role of AGEs andAGE in the pathogenesis of diabetic nephrop-thy. First, diabetic animals have significant in-reases in renal AGEs assayed by a range ofechniques.21 Second, pathologic changes inhe diabetic kidney are reduced with AGE for-ation inhibitors such as aminoguanidine,22

LT-946,22 OPB-9195,23 EXO-226,24 and A717,25

r other approaches to reduce AGE accumula-ion such as the cross-link breaker ALT-711 (al-gebrium-chloride; Alteon Inc., Ramsey, NY).26

hese renal pathologic changes also can beiminished by treating the diabetic animalsith soluble RAGE15 or a RAGE-specific neutral-

zing antibody.27 Third, genetic manipulation ofAGE expression influences the renal pheno-

ype in the setting of diabetes. For example,iabetic transgenic mice that overexpress hu-an RAGE have more advanced renal diseasehen compared with diabetic wild-type mice.hese changes included increases in albumin-ria and serum creatinine levels, mesangial ex-ansion, and advanced glomerulosclerosis.28

onsistent with these findings, RAGE knockoutice made diabetic by using streptozotocinave less renal injury in comparison with dia-etic wild-type mice. In particular, these RAGEnockout mice do not have significant mesan-ial expansion or glomerular basement mem-rane thickening.15 Finally, normal rats or micedministered with AGE-albumin develop renalhanges reminiscent of those seen in diabeticephropathy including increased renal AGEontent and glomerular volume, glomerularasement membrane thickening, mesangial ma-rix expansion, NF-�B activation, and increasedollagen IV and TGF-� mRNA expression.29

hese changes are reduced with administrationf the AGE inhibitor aminoguanidine29 or a

AGE-specific neutralizing antibody.30

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AGE, RAGE, and ROS 135

GE, RAGE AND DIABETICEPHROPATHY: HUMAN DIABETES

linical studies in both type 1 and 2 diabetestrongly implicate AGEs in the development ofiabetic complications. Type 1 diabetic patientshat advance from normal renal function to sub-equent microalbuminuria, clinical nephropathy,nd hemodialysis have significantly increased se-um levels of fluorescent non-CML AGEs, but notML or pentosidine.31 Other investigators alsoave shown that CML in type 1 diabetic patientsorrelates with the presence and severity of ne-hropathy and retinopathy.32,33 A lower meanlycated hemoglobin value in type 1 diabetic pa-ients with intensified insulin treatment, as ob-erved in the Diabetes Control and Complicationsrial, also is associated with less carotid intima-edia thickening, and this has been postulated to

e linked to less AGE accumulation.34 It should beoted, however, that associations between AGEccumulation and the development of diabeticomplications remained significant even after ad-ustment for the glycated hemoglobin level.33

Similarly, in type 2 diabetic patients therelso are significant increases in serum AGE con-entrations, including increased CML–humanerum protein35 and hydroimidazolone levels.36

ML–human serum protein levels were highern those patients with retinopathy or microalbu-

inuria.35 In addition, increases in circulatingGE peptides correlated with the severity ofenal impairment in diabetic subjects.37 The se-erity of diabetic nephropathy in human beingsorrelated to the extent of AGE formation inlomerular and tubulointerstitial compart-ents.18 Furthermore, these patients had in-

reased podocyte RAGE expression.18

THER AGE RECEPTORS

t has been proposed that AGE-R1, -R2, and -R3nteract closely in the AGE receptor complex, a

olecular aggregate on cell surfaces involved inGE catabolism.38 AGE-R1 or P60 is a 48-kd,

ype I integral membrane protein originally dis-overed in the lumen of the endoplasmic retic-lum and was thought to act as a stabilizingomponent of the oligosaccharyltransferase sys-em. Later, it was identified on cell surfaces

here it bound AGEs significantly. It has been g

uggested that AGE-R1 may have a protectiveffect against AGE-induced injury. In diabeticidney disease, AGE-R1 expression is sup-ressed in both human beings39 and nonobeseiabetic mice.40 Moreover, in mesangial cells,p-regulation of AGE-R1 enhances AGE removalnd down-regulates RAGE and downstream sig-aling pathways such as NF-�B activity andAP kinase phosphorylation, whereas down-

egulation of AGE-R1 increases AGE-inducedAP kinase activation.10 Furthermore, mice

ransgenic for AGE-R1 are protected against theevelopment of diabetic nephropathy.41

AGE-R2 or P90 is an 80- to 90-kd protein,ound to be identical to an 80- to 87-kd AGE-nducible tyrosine-phosphorylated protein. Itnitially was thought to act as a substrate forinase C, but later was found to be involved inhe intracellular signaling of various receptors,ncluding the fibroblast growth factor receptor.he P90 protein is located in the plasma mem-rane and can bind to other adaptor moleculesuch as Shc. Because P90 is phosphorylatedhen exposed to AGEs, it was suggested that itas a role in the early stages of AGE signaling,nd hence was termed AGE-R2.

AGE-R3 or galectin-3 is a 32-kd protein thatinds to carbohydrates, laminin, and immuno-lobulin E and is associated with several cellularunctions including activation, inflammation,umor growth activity, and apoptosis. Galec-in-3 binds to AGE ligands with high affinity andncreases in surface expression of AGE-R3 leadso an increase in AGE-ligand binding and endo-ytosis by macrophages. It is proposed thatGE-R3 is involved in the regulation of AGE

urnover and hence maintenance of tissue in-egrity,42 a compensatory event to combat in-reasing circulating and tissue AGE concentra-ions in diabetes. In diabetic rats, increases inlomerular AGE-R3 mRNA and protein expres-ion were observed 2 months after induction ofiabetes and continued to increase as com-ared with undetectable levels in nondiabeticats until 12 months of age.42 The importance ofGE-R3 in AGE turnover is best shown in defi-ient mice that have accelerated diabetic glo-erulopathy, increased proteinuria, and mesan-

ial expansion.43

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XIDATIVE STRESS

xidative Stress and Antioxidantefense

xidative stress is defined as the excess formationr insufficient removal by antioxidant defenses ofighly reactive molecules including ROS and re-ctive nitrogen species (RNS) (Fig. 5A). Examplesf ROS include free radicals such as superoxideO2

·�), hydroxyl (HO·), peroxyl (O2·), hydroper-

xyl (HO2·), and nonradical species such as hy-

rogen peroxide (H2O2) and hydrochlorous acidHOCl). Examples of RNS include free radicalsuch as nitric oxide (NO·) and nitrogen dioxideNO2

·), and nonradicals such as peroxynitriteONOO�), alkyl peroxynitrates (RONOO), anditrous oxide (HNO2). The major free radical im-licated in diabetic complications is O2

·�, whichan be produced by various sources including theitochondrial electron transport chain (ETC) dur-

ng normal oxidative phosphorylation, by nicotin-mide adenine dinucleotide phosphate reduced

igure 5. Oxidative stress and the generation of reactolecules including ROS and RNS are not sufficiently r

pecies may occur when oxygen is converted to O2·�,

onverted to H2O by catalase or glutathione peroxidase, o2

·� also can react rapidly with NO· to form ONOO�.

NAD[P]H) oxidase, xanthine oxidase, cyclooxy- c

enase, lipoxygenase, cytochrome P-450, and ni-ric oxide synthase in certain contexts (Fig. 5B).44

In normal conditions, O2·� is eliminated rap-

dly by antioxidant defense mechanisms. O2·�

an dismutate spontaneously to form H2O2. Al-ernatively, superoxide dismutase (SOD) canatalyze the dismutation of O2

·� to H2O2. SODas 3 major isoforms: cytosolic CuZnSODSOD1), mitochondrial MnSOD (SOD2), and ex-racellular SOD (SOD3). H2O2 is converted to

2O and O2 via catalase in lysosomes or gluta-hione peroxidase (GPx) in the mitochondriand cytosol. In the presence of transition metalsuch as iron and copper, H2O2 can be con-erted to the highly reactive HO· radical via theenton reaction. Excess O2

·� also can reactith NO· to form ONOO� (Fig. 5B).44

Common effects of the various ROS de-cribed earlier such as O2

·�, H2O2, HO·, andNOO� include oxidation of important macro-olecules including lipids, DNA, proteins, and

ecies. (A) Oxidative stress results when highly reactiveed by antioxidant defences. (B) Generation of reactiveh then is dismutated to H2O2 by SOD. H2O2 may beO· by reaction with copper (Cu) or iron (Fe). In addition,

ive spemovwhicr to H

arbohydrates. ROS can induce peroxidation of

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AGE, RAGE, and ROS 137

embrane lipids that may alter membranetructure and fluidity and hence function. Thisay result in the production of toxic lipid per-

xides. DNA damage may result in the modifi-ation of transcription factors, thus modulatinghe expression of a range of proteins includingytokines and enzymes involved in glucose res-iration. Oxidants also can increase signalingolecules such as p38 or c-Jun N-terminal ki-

ase MAP kinases.44

xidative Stress in Diabetes

onenzymatic sources

t is widely recognized that oxidative stress is aey component in the development of diabeticomplications. Nonenzymatic sources of oxida-ive stress induced by diabetes include glucoseuto-oxidation, advanced glycation, the polyolathway, and the mitochondrial ETC.45 It haseen suggested that the primary initiating event

n the development of diabetic complications is

2·� formation by mitochondria.46 One theory

uggests that hyperglycemia induces changes inhe mitochondrial voltage gradient by increas-ng electron donors of the ETC or via uncou-ling protein-1.46 Another hypothesis is thatyperglycemia may inhibit F0F1-adenosineriphosphate (ATP) synthase, slowing electronransfer and ATP synthesis, leading to an excessf electrons that would combine with molecu-

ar O2 to form O2·�. Indeed, diabetic rats have

itochondrial enlargement in renal proximalubules associated with disturbed ATP metabo-ism.47 Alternatively, production of excess O2

lso may result when NAD� cannot be regener-ted during electron transfer and NADH oxi-ase is activated, generating O2

·� as a byprod-ct. Moreover, mitochondrial swelling inducedy permeability transition pore opening in iso-

ated rat liver mitochondria inhibits the activityf ETC complex I.48 Furthermore, diabetic ratsave altered mitochondrial permeability transi-ion evident in kidney49 mitochondria. In addi-ion to induction of mitochondrial permeabilityransition, oxidative damage also can affect mi-ochondrial function by altering oxidative phos-horylation, calcium homeostasis, and protein

urnover.50 t

nzymatic sources

t has been recognized that there are a numberf enzymatic sources of ROS, however, thesere not discussed within this section but haveeen comprehensively reviewed previous-

y.45,51 NAD(P)H oxidase is a membranous en-yme consisting of 5 subunits: 2 membrane-ssociated subunits, p22phox and gp91phox,nd 3 major cytosolic subunits, p47phox,40phox, and p67phox. Gp91phox has otheromologues including nox-1 and nox-4.52

AD(P)H oxidase is a major source of cellular

2·� and is an important source of vascular O2

·�

n both nondiabetic and diabetic patients. Dia-etic rats also show significantly increasedAD(P)H oxidase activity and subunit expres-

ion within the kidney.53-55 In support of this,revention of NAD(P)H oxidase assembly by

nitiation of membranous translocation of47phox and p67phox from the cytosol usinguboxistaurin reduces ROS generation in glo-eruli of diabetic rats.53 Cultured endothelial

ells exposed to high glucose levels activateAD(P)H oxidase via PKC, leading to ROS gen-ration.56 AGE treatment of human endothelialells also leads to oxidative stress that is atten-ated with the NAD(P)H oxidase inhibitor di-henyliodonium.57

Changes in the antioxidants enzymes GPx,atalase, CuZnSOD, and MnSOD also may con-ribute to oxidative stress in diabetes. In dia-etic rats, GPx activity is decreased in the liver,rain, kidney, and heart, whereas catalase activ-

ty is increased in heart and kidney, but not livernd brain. CuZnSOD activity in diabetes is de-reased in heart, but not liver, brain, or kid-ey.58 Interestingly, overexpression of MnSOD

n diabetic mice attenuates diabetic renal inju-y.59 Furthermore, MnSOD overexpression inultured glomerular mesangial cells preventshe increase in hyperglycemia-induced cellular

2·� and collagen synthesis.59

NTERPLAY BETWEEN AGES AND ROS

his review has focused on advanced glycationccumulation and oxidative stress as mechanismsnvolved in the pathogenesis of diabetic compli-ations, particularly nephropathy. However,

here is increasing evidence to suggest that there

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138 A.L.Y. Tan, J.M. Forbes, and M.E. Cooper

s interplay between these and other pathwaysesponsible for diabetic complications.46 For ex-mple, oxidative stress may facilitate both theormation of intracellular AGEs and cross-linkingn diabetes.60 Human diabetic glomerular lesionshow colocalization of oxidative stress and AGE-odified proteins.61 Moreover, in spontaneously

iabetic rats, good glycemic control prevents thencrease in both glycation and oxidation endroducts in collagen.62 Indeed, studies using aange of antioxidants have been successful in re-ucing AGE formation. For example, the use ofntioxidants such as butylated hydroxytoluenend probucol leads to decreased renal AGE con-entrations in diabetic rats.21

Although these studies show that ROS canrigger AGE generation, there also is evidenceo suggest that the converse occurs with AGEormation triggering ROS production. For ex-mple, AGEs induce decreases in the activitiesf antioxidant enzymes such as SOD and cata-

ase,63 decreases glutathione stores, or can di-ectly stimulate ROS production.64 In addition,iological effects of AGEs may be modulated byhanges in oxidative stress. Antioxidant treat-ent of cultured cells prevents the AGE-in-

uced activation of NF-�B, TGF-�1, and celleath.65 Furthermore, depletion of the intracel-

ular antioxidant glutathione in cultured ratesangial cells decreases the AGE concentra-

ions required to activate downstream signalingathways including NF-�B and PKC-�1.65 In ad-ition, antioxidant administration to mice in-used with AGE albumin prevents the increasen endothelial cell oxidant stress as measuredy thiobarbituric acid reactive substance gener-tion and NF-�B translocation.30 Indeed, it haseen suggested that glycation of antioxidativenzymes also may enhance ROS production andellular oxidative damage.66

OTENTIAL INTERVENTIONS FORIABETIC COMPLICATIONS

argeting AGEs

ietary reduction of AGEsotential interventions for diabetic complicationsre summarized in Fig. 6. The first approach toonsider is a dietary reduction in exogenously

erived AGEs. Patients with diabetic nephropathy A

ave been reported to have decreased renal ex-retion of exogenously derived AGEs and diabeticatients on a high AGE diet may have an in-reased risk of renal and vascular injury.67 Thus,ecreasing the AGE content in the diet may be an

mportant adjunct therapy in the treatment ofiabetic nephropathy.

Low dietary AGE intake in animal models,ncluding those with diabetes, is associated notnly with decreased atherosclerosis,68 but alsoith decreased nephropathy.69 Diabetic pa-

ients on a low-AGE diet have decreased serumGE levels and a reduction in the inflammatoryediators TNF-� and VCAM-1.70 Furthermore, a

eduction in dietary AGE intake by nondiabetic,hronic renal failure patients with increasederum AGE levels leads not only to a decrease inerum AGE levels, but also reduced TNF-�,CAM-1, and vascular endothelial growth factor

evels.71 Moreover, long-term dialysis patientsave significant correlations between dietaryGE intake and serum AGE levels that appear toe independent of dietary constituents such asat, protein, and carbohydrate.72

GE formation inhibitors

ne of the earliest strategies used to reduce AGEccumulation was the use of AGE formation in-ibitors. These agents act in a variety of ways

ncluding trapping of reactive carbonyl and dicar-onyl compounds, chelation of transition metal

ons, and direct inhibition of the conversion ofmadori intermediates to AGEs.73 A number ofGE formation inhibitors have been described

ncluding aminoguanidine, ALT-946, pyridoxam-ne, and OPB-9195.74 The first agent to be inves-igated extensively was aminoguanidine, a nucleo-hilic hydrazine compound that inhibits in vitrond in vivo formation of AGEs via binding to earlylycation and glycoxidation products, dicarbonylntermediates, and aldehyde products.75 Amino-uanidine is a nonspecific inhibitor because it alsonhibits inducible nitric oxide synthase and dia-

ine oxidase.76 Aminoguanidine has been showno slow the development of diabetic complica-ions including nephropathy.22 Diabetic ratsreated with aminoguanidine and other AGE for-ation inhibitors including OPB-9195, ALT-946,

nd pyridoxamine have shown reduced renal

GE accumulation, less mesangial expansion, and

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AGE, RAGE, and ROS 139

lower progression of glomerulosclerosis andlbuminuria.22,77 Furthermore, the renoprotec-ive effects of these agents appear to be relatedo the duration of the treatment.78 In humanlinical studies, type 1 diabetic patients withephropathy treated with pimagedine (amino-uanidine hydrochloride) were shown to havelower decreases in glomerular filtration rate,ut overall there was no significant beneficialffect on the progression of overt nephropa-hy.79 Unfortunately, aminoguanidine interferesith several important regulatory systems76 and

oxic side effects were observed with use ofhis agent in clinical trials. Thus, it has beeniscontinued for further clinical develop-ent.80 Interestingly, one of the current clinical

herapies for diabetic nephropathy, angioten-in-converting enzyme inhibitors, have beendentified as potent inhibitors of AGE forma-ion81 and it is postulated that at least some ofhe nonhemodynamic renoprotection con-erred by angiotensin-converting enzyme inhib-

igure 6. Therapeutic intervention to reduce AGE-indu

tors may involve effects on AGE accumulation. s

GE cross-link breakersGE cross-link breakers are compounds thateduce AGE accumulation by cleavage of pre-ormed AGE-mediated cross-links.4 Examples ofGE cross-link breakers include N-phenacylthia-olium bromide (PTB) and alagebrium chloride,,5-Dimethyl-3-(2-oxo2-phenylethyl)-thiazoliumhloride (ALT-711).26,82 Indeed, ALT-711 haseen reported to attenuate renal injury in ex-erimental diabetes26 and is deemed safe inuman clinical trials in other nondiabetic dis-ases (www.alteon.org). Clinical trials are nowarranted to confirm that renoprotective ef-

ects of ALT-711 (alagebrium chloride; Alteonnc.) also are seen in human beings.

nhibitors of AGE bindingnhibitors of AGE receptor ligand binding includeoluble RAGE and RAGE-specific neutralizing an-ibodies, which have been used in both in vivond in vitro studies to block the biological effectsf RAGE. Indeed, diabetic mice treated with

amage.

RAGE have less albuminuria and glomeruloscle-

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140 A.L.Y. Tan, J.M. Forbes, and M.E. Cooper

osis.15 RAGE-specific neutralizing antibodies ad-inistered to diabetic mice prevent diabetes-in-

uced renal changes including mesangialxpansion and albuminuria.27 RAGE is consideredn attractive target for developing new treatmentsor diabetic complications with an active programurrently in development to target this receptorpecifically (http://www.lifesciencesworld.com/ews/view/8312).

ARGETING ROS

large number of experimental studies haveeen performed using a range of antioxidants tossess their potential actions as renoprotectivegents. This has included the use of vitamins Cnd E and �-lipoic acid. Rat mesangial cellsreated with vitamin E and a ROS scavengeritecapone have less AGE-dependent NF-�B ac-ivation and normalization of PKC activity.65

iabetic rats treated with the ROS scavengeritecapone normalized urinary sodium excre-ion and oxidative stress parameters, preventedyperfiltration, focal glomerulosclerosis, and al-uminuria, and inhibited activation of glomer-lar PKC activity.83 The potential beneficial ef-ects of antioxidant therapy in human beingsemain controversial. Type 2 diabetic patientsdministered vitamin C have improved endo-helial dysfunction in their forearm resistanceessels.84 Type 1 diabetic patients with high-ose vitamin E supplementation have normal-

zed baseline retinal blood flow and creatininelearance, suggesting a role in improving retinalemodynamics and renal function in diabeticatients.85 In the Cambridge Heart Antioxidanttudy, vitamin E administration to patients withoronary atherosclerosis decreased the primaryrial end point of cardiovascular death and myo-ardial infarction.86 However, in the Heart Out-omes Prevention Evaluation trial, administra-ion of vitamin E to older patients with anncreased risk of cardiovascular events includ-ng a significant proportion with diabetes, there

as no significant effect on primary and sec-ndary cardiovascular outcomes.87 Idebenone,short analogue of CoQ10 that acts as a potent

ree radical scavenger, protects Friedriechtaxia patients from iron/ROS injury in theeart muscle and reduces cardiac hypertrophy

n these patients.88 It remains to be determined l

f such a strategy, potentially targeting mito-hondrial ROS generation, may be useful inatients with diabetic nephropathy.It remains unexplained as to why no clear-cut

eneficial effects of antioxidants that have under-one trials to date have been observed. A numberf explanations have been raised including lowbsorption rates and the likelihood that conven-ional antioxidants such as various vitamins mayot effectively reduce O2

·� levels and indeed maye prooxidants in certain contexts. It has beenuggested that more effective reductions in O2

·�

oncentrations may be seen with a catalytic anti-xidant, such as a SOD/catalase mimetic, thatould continuously scavenge ROS. Indeed, thenSOD mimetic, MnTBAP, prevented hypergly-

emia-induced ROS injury in endothelial cells.89

he role of such an approach in diabetic compli-ations remains to be elucidated.

ONCLUSIONS

here is strong evidence to support the pathoge-icity of excess AGE accumulation and ROS gen-ration in diabetic nephropathy. It is essential tourther understand these pathways and other met-bolic and hemodynamic factors that may interacto contribute to the pathogenesis of diabetic com-lications. Such knowledge would aid in design-

ng therapeutic interventions to add to the cur-ent treatment regimens that focus on bloodlucose and blood pressure control. Combinationherapies targeting multiple pathways are likely toe more successful than targeting single path-ays alone. A variety of therapies including AGE

ormation inhibitors, AGE cross-link breakers,RAGE, and free-radical scavengers ultimately maye proven to be the appropriate adjunct therapieso optimize the prevention of diabetes-associatedenal injury.

A range of issues still need to be resolved inhe field of advanced glycation. In particular, its critical to further understand the relative im-ortance of RAGE and the other AGE receptorshat have been identified recently. With respecto oxidative stress in diabetic complications,urther elucidation of the relative importance ofhe various sources of ROS generation is re-uired. Furthermore, current pharmacologictrategies to target ROS generation and its bio-

ogical effects do not appear to be particularly

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AGE, RAGE, and ROS 141

otent and are relatively nonselective. Finally, its critical that we understand in more detailow AGE and ROS interact, not only with eachther, but also with other relevant pathways iniabetic complications such as the renin-angio-ensin system. This would allow us to designherapies that target these pathways appropri-tely and in the long term provide better treat-ent strategies for diabetic patients at risk of orith established renal disease.

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