The elephant in uremia: Oxidant stress as a unifying concept of cardiovascular disease in uremia

Kidney International, Vol. 62 (2002), pp. 1524–1538

PERSPECTIVES IN RENAL MEDICINE

The elephant in uremia: Oxidant stress as a unifying conceptof cardiovascular disease in uremia

JONATHAN HIMMELFARB, PETER STENVINKEL, T. ALP IKIZLER, and RAYMOND M. HAKIM

Division of Nephrology, Department of Medicine, Maine Medical Center, Portland, Maine, USA; Department of RenalMedicine, Huddinge University Hospital, Huddinge, Sweden; Division of Nephrology, Department of Medicine, VanderbiltUniversity Medical Center, and Renal Care Group, Inc., Nashville, Tennessee, USA

pro-atherogenic milieu of uremia. Dialytic therapy, which acts“And so these men of Indostanto reduce the concentration of oxidized substrates, improvesDisputed loud and long,the redox balance. However, processes related to dialytic ther-Each in his own opinionapy, such as the prolonged use of catheters for vascular accessExceeding stiff and strong,and the use of bioincompatible dialysis membranes, can con-Though each was partly in the right,tribute to a pro-inflammatory and pro-oxidative state and thusAnd all were in the wrong!”to a pro-atherogenic state. Anti-oxidative therapeutic strategiesJohn Godfrey Saxefor patients with uremia are in their very early stages; nonethe-less, early studies demonstrate the potential for significant effi-cacy in reducing cardiovascular complications.

The elephant in uremia: Oxidant stress as a unifying concept Cardiovascular disease is the single largest cause ofof cardiovascular disease in uremia. Cardiovascular disease ismortality in the general population, and as such, it notthe leading cause of mortality in uremic patients. In large cross-surprising that it is also the leading cause of mortality insectional studies of dialysis patients, traditional cardiovascular

risk factors such as hypertension and hypercholesterolemia chronic dialysis patients. What is surprising and differenthave been found to have low predictive power, while markers from the general population is the magnitude of cardio-of inflammation and malnutrition are highly correlated with vascular complications and the near equivalence of car-cardiovascular mortality. However, the pathophysiology of the

diac mortality across age groups in uremic patients. Thus,disease process that links uremia, inflammation, and malnutri-cardiac mortality for dialysis patients aged 45 years ortion with increased cardiovascular complications is not well

understood. We hereby propose the hypothesis that increased younger is more than 100-fold greater than in the generaloxidative stress and its sequalae is a major contributor to in- population. Even in the elderly, cardiovascular mortalitycreased atherosclerosis and cardiovascular morbidity and mor- is at least fivefold higher in the end-stage renal diseasetality found in uremia. This hypothesis is based on studies (ESRD) population than in the general population [1].that conclusively demonstrate an increased oxidative burden in

Data are now emerging that strongly suggest that cardio-uremic patients, before and particularly after renal replacementvascular complications accelerate as renal function is pro-therapies, as evidenced by higher concentrations of multiple

biomarkers of oxidative stress. This hypothesis also provides gressively lost, and that a high percentage of incidenta framework to explain the link that activated phagocytes pro- dialysis patients already have a substantial burden ofvide between oxidative stress and inflammation (from infec- cardiovascular disease as they start their lives on dialysistious and non-infections causes) and the synergistic role that

[1, 2]. These findings have led to investigations aboutmalnutrition (as reflected by low concentrations of albuminspecific features of renal insufficiency and loss of kidneyand/or antioxidants) contributes to the increased burden of car-

diovascular disease in uremia. We further propose that retained function that lead to acceleration of atherogenesis.uremic solutes such as beta-2 microglobulin, advanced glycosy- In the past decade, numerous epidemiologic studieslated end products (AGE), cysteine, and homocysteine, which have attempted to determine the causative or associatedare substrates for oxidative injury, further contribute to the factors that contribute to the high cardiovascular mortal-

ity in ESRD patients. Such studies have pointed out thattraditional risk factors for cardiovascular mortality inKey words: chronic dialysis, end-stage renal disease, anti-oxidative ther-

apy, mortality and dialysis, proteinuria, hypertension, blood pressure. the general population such as hypertension and hyper-cholesterolemia do not discriminate well in dialysis pa-Received for publication January 10, 2002tients [3, 4]. More important than these traditional riskand in revised form March 25, 2002

Accepted for publication April 17, 2002 factors are newer “non-traditional” risk factors that areseemingly difficult to reconcile. At the risk of oversimpli- 2002 by the International Society of Nephrology

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Himmelfarb et al: Oxidant stress and cardiovascular disease in uremia 1525

fying these epidemiologic observations, we chose to sum- (Table 2). First, elevated CRP, a marker of the acute phaseresponse, is an indirect marker of increased leukocyte ac-marize them as a starting point for a discussion that

attempts to link these seemingly divergent observations tivation and increased oxidant production. Second, mal-nutrition and more specifically hypoalbuminemia mayto a common and potentially unifying pathophysiological

basis for understanding. contribute to cardiovascular morbidity and mortality byreducing antioxidant defense, thus resulting in a higherlevel of oxidative injury. A third corollary is that renal1. The major causes of mortality in ESRD are cardio-

vascular (50%) and infectious (25%) diseases. replacement therapy, to the extent it can be providedwithout inciting further inflammatory and oxidative stim-2. Inflammation, documented by elevated C-reactive

protein (CRP) concentrations as a measure of acute uli, will relieve the uremic state and improve the oxidant-antioxidant (redox) balance. A final corollary is whenphase response, is associated with increased risk of

cardiovascular mortality. “tissues” have pathologically high concentrations of re-tained uremic substances such as �2-microglobulin (�2m),3. ESRD patients with evidence of protein-calorie mal-

nutrition, documented by lower concentrations of cysteine, and homocysteine, they become substrates foroxidative actions and subsequent tissue injury, propor-albumin and pre-albumin, have a higher relative risk

of mortality that increases with the extent of hypo- tionate to their concentration. This modification may in-crease their pathogenicity.albuminemia.

4. Inflammation and malnutrition are interrelated, but The following discussion attempts to elucidate whatis currently known about the extent to which oxidativeeach contributes independently to mortality risk.

5. Aspects of renal replacement therapy that contrib- stress is the “real elephant” in uremia.ute to inflammation, such as the prolonged use ofcatheters for vascular access or the use of bioincom-

THE NATURE OF OXIDATIVE STRESSpatible membranes, are also associated with increased

In 1989 and in the early 1990s, Daniel Steinberg andcardiovascular and infectious mortality risk.colleagues advanced the hypothesis that the atherogen-6. Hyperhomocysteinemia is highly prevalent in uremiaicity of low-density lipoproteins (LDL) was greatly in-and may be associated with cardiovascular toxicitycreased by oxidative modification (Fig. 2) [5]. Oxida-tively modified LDL is taken up by scavenger receptors,While much has been learned from these studies ofleading to the conversion of monocytes into foam cells,morbidity and mortality predictors in ESRD patients, itone of the first steps in the atherosclerotic process. Oxi-also must be recognized that these correlations are largelydative processes predominantly occur in the mitochon-derived from cross-sectional observations that do notdria and the mitochondrial cytochrome oxidase enzymeclearly define the pathogenesis of the disease process.is responsible for 90% of the oxygen humans metabolize.Thus, these observations that provide pieces of a puzzleThis enzyme transfers four electrons to oxygen in a con-without a unifying hypothesis are akin to the story ofcerted reaction that produces two molecules of water asthe blind men palpating an elephant, set in a poem bythe product. This complex enzyme contains four redoxJohn Godfrey Saxe. These blind men were asked to tellcenters, each of which stores a single electron. When allof their experiences palpating different parts of an ele-four redox centers are reduced, the simultaneous transferphant, without seeing the entire animal (Fig. 1). Eachof four electrons to an oxygen molecule takes place withof the blind men was able to palpate one part of theno detectable intermediate steps, thereby limiting theelephant, and thus was able to describe it in terms thatproduction of reactive oxygen intermediates. Neverthe-were “partly in the right.” However, because none ofless, a small fraction (1 to 2%) of this reaction doesthem was able to encompass the entire elephant in theirproceed via an intermediate step that results in the for-hands, they were also “in the wrong,” in that they failedmation of free radicals [6]. In response to this problem,to identify the whole elephant.mammalian systems have evolved numerous intracellu-The hypothesis we wish to advance for discussion islar antioxidant systems including enzyme systems, waterthat the essence of the “elephant” in uremia is the increasedsoluble, and fat-soluble free radical scavengers that canoxidative burden in the uremic milieu of patients withavidly react with and eliminate these intermediate reac-chronic kidney disease. This oxidative burden, leading totive oxygen species before they inflict oxidative damageincreased oxidation of protein, lipids and carbohydrates,to vital cellular components and function.increases as renal disease progresses and is extensive by

While mitochondria are the predominant source ofthe time ESRD develops. The result is an accelerated“accidental” oxidative stress, phagocytes “deliberately”development of atherogenesis and other manifestationsutilize high levels of oxygen for host defense against patho-of “uremia” such as amyloid bone disease (Table 1).gens. The respiratory burst of these phagocytes is knownThere are several important corollaries of this hypoth-

esis that may link divergent cross-sectional observations to utilize four enzymes (NADPH oxidase, superoxide

Himmelfarb et al: Oxidant stress and cardiovascular disease in uremia1526

Fig. 1. The blind men and the elephant. Poem by John Godfrey Saxe (Cartoon originally copyrighted by the authors; G. Renee Guzlas, artist).


Table 1. The elephant in uremia: A conceptual paradigm

Uremia pathogenesis Additional risk factors Potential mediators Substrates Pathophysiology Pathology

Increased cytokines Acute phase reaction HOCl Cysteine Vascular inflammation AcceleratedIncreased complement Malnutrition H2O2 Homocysteine Endothelial injury atherogenesis

activation RRT characteristics Superoxide anion �2m VSMC proliferation and Coronary restenosisRetained solutes Biocompatibility Peroxynitrite Glucose derivatives migration Accelerated venousNeutrophil priming Catheter use Hydroxyl radical Proteins: Decreased NO effect intimal hyperplasia

? Fe�� exposure Carbonyls Vasoconstriction LVHInfection Thiols Platelet activation �2 amyloidosis

Tyrosine Tissue injuryLipids:

PUFAsLDL

Abbreviations are: RRT, renal replacement therapy; HOCl, hypochlorous acid; H2O2, hydrogen peroxide; �2m, ß2 microglobulin; PUFAs, polyunsaturated fattyacids; LDL, low-density lipoprotein; VSMC, vascular smooth muscle cell; NO, nitric oxide; LVH, left ventricular hypertrophy.

Table 2. Corollaries to the oxidative stress hypothesis light the central role that activated phagocytes and condi-tions associated with inflammation (and hence phagocyteactivation) play in the increased oxidative burden and

� Elevated CRP is a marker for oxidant production� Malnutrition and hypoalbuminemia reduce antioxidant defense,

increasing vulnerability to oxidant injury atherosclerosis. Strong association between elevated white� Renal replacement therapy relieves the uremic state and improvesblood cell count, and rapid progression of atherosclerosisoxidant-antioxidant balance, provided further inflammatory and

oxidative stimuli are avoided and coronary events have been documented in large� Retained uremic solutes, such as �2m, homocysteine, and cysteine,

population studies [12].may become substrates for oxidative injury, thereby contributingto tissue injury, proportionate to their concentration Oxidative stress (or oxidant-derived tissue injury) takes

place when the production of oxidants exceeds local anti-Abbreviations are: CRP, C-reactive protein; �2m, �2 microglobulin.

oxidant capacity. When this occurs, it results in the oxida-tion of important macromolecules, including proteins,lipids, carbohydrates, and DNA present in that environ-

dismutase, nitric oxide synthase, and myeloperoxidase) ment. Because the active oxygen intermediates are pro-to produce the reactive intermediates superoxide anion, duced in vivo in minute quantities and are also highlyhydrogen peroxide, nitric oxide, and hypochlorous acid reactive, direct in vivo detection of the unreacted moities(HOCl), respectively, for the destruction of invading mi- is extremely difficult. Nonetheless, a powerful strategy hascroorganisms (Fig. 3). More recently, an additional novel evolved for understanding the underlying in vivo mecha-oxidative pathway has been identified whereby phago- nisms of oxidative injury by identifying stable end-prod-cytes convert nitrite to nitryl chloride and nitrogen diox- ucts of oxidation produced by different reaction path-ide via the myeloperoxidase enzyme or by HOCl itself,

ways. These biomarkers of oxidative pathways increasinglyresulting in nitration of target biomolecules [7].

have been used as an in vivo tool to elucidate the impor-While each of these phagocyte-derived oxidants cantance of oxidative stress as a contributor to many diseasecontribute to tissue injury, recent studies have empha-states, including uremia [13, 14]. Table 3 lists the bio-sized the importance of myeloperoxidase-catalyzed chlo-chemistry of some of the most important of in vivo bio-rinating oxidative reactions. Myeloperoxidase (MPO) ismarkers that have been specifically identified at highone of the most abundant proteins in phagocytes, consti-concentrations in uremia. Figure 4 describes the biochem-tuting approximately 5% of neutrophil protein and 1%istry of some of the more recent important biomarkerof monocyte protein. MPO is rapidly secreted upon stim-assays of oxidant stress, which are summarized below.ulation and catalyzes the oxidation of halides to their

corresponding hypohalous (for example, hypochlorous)acids in the presence of hydrogen peroxide [8]. Quanti- BIOMARKERS OF OXIDATIONtative studies have revealed that H2O2 generated by ac-

Lipid peroxidationtivated neutrophils is virtually stochiometrically con-Several groups of investigators have now used highlyverted to HOCl. Histological analysis of atherosclerotic

sensitive and specific assays to conclusively demonstratetissue have identified catalytically active myeloperoxi-that there are higher levels of plasma lipid oxidation indase co-localized with foamy macrophages, [9] and bothuremic patients compared to healthy subjects. For exam-3-chlorotyrosine and dityrosine, which are MPO-catalyzedple, arachidonic acid has been shown to undergo non-end-products of tyrosine oxidation, have been found inenzymatic, free radical-induced, peroxidation reactionsoxidatively modified LDL and in human atherosclerotic

lesions [10, 11]. These phagocyte driven reactions high- resulting in the formation of a series of prostaglandin


Fig. 2. Potential mechanisms for the role ofoxidatively-modified low-density lipoprotein(LDL) in atherogenesis. Endothelial cells, vas-cular smooth muscle cells, or macrophages maycatalyze the oxidative modification of circulat-ing LDL leading to: (I ) recruitment of circu-lating monocytes; (II ) macrophage “trapping”in the vessel intima; (III ) enhanced uptake ofoxidized LDL by resident macrophages lead-ing to foam cell formation; and (IV ) endothe-lial cell destruction as a result of oxidized LDLtoxicity (reprinted with permission from QuinnMT et al, Proc Natl Acad Sci USA, 82:5949–5953, 1985).

Table 3. In vivo oxidative stress biomarkersuseful in evaluating uremia

LipidsMalondialdehyde and other aldehydesLipid hydroperoxidesOxidized low-density lipoprotein (LDL)Exhaled alkanesAdvanced lipoxidation end products (ALE)

Arachidonic acid derivativesF2 isoprostanesIsolevuglandins

CarbohydratesReactive aldehydesAdvanced glycosylation end products (AGE)

Amino acidsCysteine/cystineHomocysteine/homocystine

Fig. 3. Pathways of oxidant generation by activated leukocytes. Isoaspartate3-chlorotyrosineDityrosine3 nitrotyrosine

ProteinsF2-like compounds known as F2 isoprostanes (F2-IsoPs; Thiol oxidationFig. 4B) [15]. F2-IsoPs are initially formed in situ on Carbonyl formation

Advanced oxidation protein products (AOPP)phospholipids and then subsequently can be released inDNAthe plasma and, therefore, they can be detected either 8 hydroxy 2� deoxyguanine

as free or as phospholipid-bound F2 isoprostanes. Levels OtherSpin traps (electron paramagnetic resonance)of F2-IsoPs in human biologic fluids exceed cyclooxygen-

ase-derived prostanoids by greater than an order of mag-nitude and are a reliable indicator of oxidative stress invivo. Recent findings have shown that plasma F2 isopros-

to plasma proteins because of their extreme reactivity.tane levels are two to four times higher in patients re-Salomon et al have recently demonstrated that patientsceiving chronic hemodialysis therapy as in age- and sex-with ESRD receiving hemodialysis have approximatelymatched controls and correlate closely with levels oftwice the level of isolevuglandin-plasma protein adductsC-reactive proteins [16, 17].as healthy subjects [18]. Handelman et al also demon-Another family of extremely reactive electrophiles,strated that levels of breath ethane (which result from theknown as isolevuglandins, is generated in vivo by free rad-beta scission of lipid hydroperoxides) are approximatelyical-induced lipid oxidation of arachidonic acid. In con-fourfold in hemodialysis patients compared to healthytrast to F2 isoprostanes, isolevuglandins do not circulate

free in the plasma, but are virtually completely adducted subjects (abstract: Handelman et al, J Am Soc Nephrol


Fig. 4. Biochemistry of oxidant stress biomarkers. (A) Oxidation of tyrosine residues. (B) F2-isoprostane generation from arachidonic acid viafree radicals. (C ) Generation of reactive aldehydes (carbonyls). (D) Amino acid thiol group oxidation. (E ) DNA oxidation. Abbreviations are:HOCL, hypochlorous acid; MPO, myeloperoxidase; CML, carboxymethyllysine; HNE, hydroxynonenal.

11:271A, 2000). Thus, a variety of highly sensitive and (HNE), a product of lipid oxidation, with lysine residues[19]. Salomon et al, using an ELISA assay with antibod-specific assay systems all demonstrate that biomarkers

of lipid peroxidation are higher in dialysis patients than ies raised against protein bound 2-pentylpyrrole, havedemonstrated markedly elevated HNE-derived 2-pen-in healthy subjects.

Several investigators also have re-examined the issue tylpyrrole levels in LDL in patients on both hemo- andperitoneal dialysis compared to healthy subjects [20].of whether plasma LDL is more oxidized in patients with

uremia compared to healthy subjects, using more sensi- Using different techniques, Ziouzenkova et al measuredthe amino group oxidation in APO B100 protein to de-tive and specific assays than in the past. Free plasma

LDL produces detectable 2-pentylpyrrole epitopes that tect minimal oxidative modifications in LDL [21]. Theydemonstrated that minimally oxidatively modified LDLare generated by the reaction of 4-hydroxy-2-nonenal


levels (which are associated with atherosclerosis) arehigher in hemodialysis patients compared to healthy sub-jects, suggesting that this may be free radical-mediatedby the interaction of dityrosine with LDL-associated pro-tein. Taken together, these studies provide compellingevidence that increased free radical-mediated oxidativeprocesses lead to increased lipid, apolipoprotein, andlipoprotein oxidation in patients with ESRD receivingdialysis therapy.

Protein and amino acid oxidation

Oxidatively modified plasma proteins and amino acidsalso can serve as important in vivo biomarkers of oxida- Fig. 5. Elevated plasma protein 3-chlorotyrosine, a specific biomarker

of myeloperoxidase-catalyzed oxidation, in hemodialysis patients.tive stress. The ready accessibility of plasma proteins(Adapted with permission from Himmelfarb et al, Free Radical Biol-and amino acids for sampling, the relatively long plasmaogy & Medicine 31:1163–1169, 2001.)

half-lives of many proteins, and the well-defined bio-chemical pathways that lead to protein and amino acidoxidation can be used to detect more specific pathwaysof oxidative stress than plasma lipids. For example, oxi- oxidative protein products (AOPP) in the plasma of ure-dation of amino acyl side chains of amino acids in pro- mic patients [26–28]. These investigators also demon-teins is an attractive biomarker of oxidative reactions strated that hypochlorous acid derived from activatedbecause of the high specificity of the end products for spe- phagocytes in vitro could replicate the production ofcific oxidation pathways. Detection of tyrosine residue plasma AOPP to levels seen in uremic patients [28]. Fi-oxidation currently constitutes the most sensitive and nally, l-isoaspartyl residues in plasma proteins fromspecific means available for detecting end products of uremic patients are also increased as a manifestation ofspecific oxidative pathways (Fig. 4A) [22]. In particular, oxidative injury [29]. Collectively, these studies usingoxidation of tyrosine residues leads to the formation of protein and amino acids as biomarkers strongly suggest3-chlorotyrosine, 3-nitrotyrosine, or dityrosine, depend- that myeloperoxidase-catalyzed hypochlorous acid de-ing on whether the predominant oxidizing species is hy- rived from activated phagocytes is an important oxidantpochlorous acid, a reactive nitrogen species, or a free in uremic patients.radical (such as hydroxyl ion), respectively [22]. Our The previous discussion has served to highlight thegroup has recently demonstrated that plasma proteins

specific pathways of oxidative stress and their perva-from hemodialysis patients contain elevated levels of

siveness in uremia. In order to highlight the clinical im-3-chlorotyrosine, but not 3-nitrotyrosine or dityrosineportance of these oxidation by-products, it is important(Fig. 5). Since this product (3-chlorotyrosine) is a specificto link these pathways to specific and common diseaseproduct of myeloperoxidase catalyzed reaction, it furthermanifestations in patients with uremia. For the sake ofsuggests a specific and important role for phagocyte-brevity, we will attempt to demonstrate the clinical rele-initiated oxidative reactions as the cause of excess oxida-vance of two specific pathways: one related to the forma-tive stress in uremia [23].tion and accumulation of aldehydes and one to the deple-In addition to oxidized tyrosine residues, other impor-tion of reduced thiols.tant amino acyl groups subject to oxidation include sulfur

groups (cysteine, methionine), amino groups (such as ly-sine), and alcohol groups (including threonine). Recent UREMIA, OXIDATIVE STRESS, ANDstudies from our group and other investigators have clearly ALDEHYDE FORMATIONdemonstrated high levels of thiol group oxidation as well

Reactive aldehydes can be formed as the end productas carbonyl formation in plasma proteins from patientsof a variety of oxidative reactions including oxidation ofwith uremia (Fig. 6) [24]. While not entirely specific foralcohol groups, amino groups, and via the addition ofa single oxidative pathway, levels of plasma protein oxi-oxygen to unsaturated carbon double bonds in carbohy-dation and carbonyl formation seen in uremic patientsdrates, lipids, or amino acids (Fig. 4C) [30]. Biochemicalin vivo are similar to patterns of protein thiol oxidationassays designed to detect accumulation of aldehydes relyobserved in vitro after oxidation with hypochlorous acid,on identification of carbonyl (C � O) groups, leading tobut not with hydrogen peroxide, again suggesting an im-the use of the term “carbonyl stress.” Myeloperoxidase-portant role for myeloperoxidase in generating oxidantcatalyzed oxidative modification of amino acids also canstress [24, 25]. A series of studies by Witko-Sarsat and col-

leagues have demonstrated elevated levels of advanced lead to the formation of a variety of reactive aldehyde


sclerosis [30, 35]. AGEs may promote atherosclerosisthrough interactions with specific receptors (RAGE),causing increased expression of adhesion molecules andenhanced attraction of circulating monocytes to the ves-sel wall. It is important to note that the interaction ofAGE with its RAGE receptor also leads to the increasedproduction of interleukin-6 (IL-6) by monocytes and in-directly to the excess formation by CRP in the liver, thusparticipating in the genesis of inflammation.

Reactive aldehydes have been shown to be involvedin the oxidative modification of LDL cholesterol, bothin vitro and in vivo, thereby making this modified LDLa target for monocyte phagocytosis and the developmentof foam cells, a precursor of the atherosclerotic process[36]. The participation of reactive aldehydes in the ath-erosclerotic process also is suggested by immunohisto-chemical analyses of atherosclerotic lesions from humanaorta, in which intense positivity of a variety of aldehydeadducts including HNE-histadine, MDA lysine, andacrolein-lysine, can be demonstrated in association withmacrophages and foam cells [37].

In summary, carbonyl concentration as an index ofreactive aldehyde formation represents the end productof oxidative and other pathways. Carbonyl concentra-tion is important both in the pathogenesis of atheroscle-rosis in uremia and as a potential biomarker to monitorantioxidative therapeutic strategies. To the extent thatcarbonyl formation is a byproduct of phagocyte-derived

Fig. 6. Plasma protein thiol group oxidation (A) and carbonyl forma- myeloperoxidase reactions, carbonyl concentration is alsotion (B) in patients with chronic kidney disease and hemodialysis pa-a marker of phagocyte activation and the inflammatorytients, demonstrating that plasma protein oxidation develops in renal

disease well before ESRD. (Adapted with permission from Himmelfarb burden.et al, Kidney Int 58:2571–2578, 2000.)

UREMIA, OXIDATIVE STRESS, ANDDEPLETION OF REDUCED THIOLcompounds including glyoxal, methylglyoxal, acrolein,

In addition to the generalized metabolic disturbanceglycoaldehyde, and parahydroxy phenacetaldehyde [31].in uremia resulting in excess accumulation of reactiveSeveral groups have demonstrated that reactive car-aldehydes, it is becoming increasingly clear that there isbonyl compounds can be detected in uremia in concen-a concomitant depletion of reduced thiol groups. Thiolstrations far in excess of normal healthy subjects [24, 25,are a class of organic sulfur derivatives that are character-31–33]. Assays measuring total reactive carbonyl com-ized by the presence of sulfhydryl residues at their activepounds (by measuring hydrazone formation after reac-site. Thiols have critical intracellular and extracellulartion with 2,4-dinitrophenylhydrazine) have shown thatfunction as redox buffers, via protein thiol (S-H)/disul-total carbonyl compounds are found at a concentrationfide (S-S) concentration equilibrium (Fig. 4D) [38]. Be-up to tenfold higher in uremic plasma compared to nor-cause the formation and cessation of disulfide bonds aremal plasma [24, 31].involved in many enzymatic and transport processes, thiolThe significance of elevated concentrations of alde-oxidation has significant effects on protein structure andhydes, measured as reactive carbonyl compounds in ure-function [39]. For example, intracellular thiols such asmia is most clearly demonstrated by their role in the for-glutathione and thioredoxin play an important role inmation of advanced glycosylation end products (AGEs).maintaining the highly reduced environment inside theAGEs are formed nonenzymatically by irreversible reac-cell [38]. Extracellular thiols also constitute an importanttion of reactive carbonyl compounds with various aminecomponent in antioxidant defense relevant to cardiovascu-groups in proteins [34].lar disease [40–46]. The extracellular fluids of the humanThe importance of increased reactive aldehyde andbody, including plasma, contain little or no catalase activ-AGE formation is evidenced by their prominent role in

the pathogenesis of vascular diseases including athero- ity and only low levels of common antioxidants such as


superoxide dismutase, glutathione and selenium-contain- INFLAMMATION AND OXIDATIVE STRESSing glutathione peroxidase [47]. Thus, traditional anti- Inflammation is a common feature of ESRD and itoxidant enzymes that are crucial in intracellular defense has been recognized that about 30 to 50% of predialysis,are unavailable and play a minimal role in protection hemodialysis (HD), and peritoneal dialysis (PD) patientsagainst oxidative injury in extracellular fluids. Halliwell have serologic evidence of an activated inflammatoryand others have demonstrated that the protein-associ- response [54]. Ward and McLeish have reported thatated thiols and ascorbate constitute the major extra- phagocyte cells that are “primed” in uremia may contrib-cellular defense against oxidant stress in plasma [47–49]. ute to increased production of both reactive oxygen spe-The importance of plasma protein-associated free thiols cies as well as cytokines [55]. An elevation of plasmain scavenging both free radicals and myeloperoxidase- CRP is one indication of a cytokine-driven (especially

IL-6) acute phase inflammatory response. The clinicalgenerated oxidants has been demonstrated in severalsignificance of CRP in dialysis patients has been wellstudies [50].documented in a series of recent studies in which anGiven the importance of the antioxidant actions ofelevated CRP has been shown to be a strong predictorthiols both intracellularly and particularly extracellularly,of adverse clinical outcomes and increased cardiovas-it is instructive to examine the relative concentrations ofcular mortality [reviewed in Arici and Walls, 56]. Thereduced and oxidized thiols in uremia. Ceballos-Picot etinteraction of AGE with its RAGE receptor leading toal investigated the glutathione antioxidant system in theincreased production of IL-6 and CRP also was pre-plasma of a large cohort of patients with chronic renalviously discussed [56]. Finally, the highly skewed distri-failure and demonstrated diminished plasma glutathionebution of CRP and IL-6 elevations in dialysis patientslevels and a profound drop in glutathione peroxidase func-and the episodic nature of CRP increases suggest thattion [51]. Our group has demonstrated that plasma pro-patient-specific processes (such as clotted access grafts,tein thiols are extensively oxidized in uremic patientsdialysis membrane bioincompatibility, or persistent clini-compared to healthy subjects [24]. We and others alsocal or sub-clinical infections) may be important causeshave examined the redox status of the low molecularof chronic inflammation in ESRD patients.weight amino thiols cysteine, homocysteine, and cys-

Although the association between atherosclerosis andteinyl-glycine, which are well-known accumulate in ure-chronic inflammation in the dialysis patient popula-mia [52, 53]. In each of these aminothiols, the ratio oftion is well documented, we do not know if the inflam-oxidized to reduced thiols is considerably increased inmatory response merely reflects an epiphenomenon ac-hemodialysis patients compared to healthy subjects [53].companying established atherosclerotic disease or whetherThus, these studies demonstrate a generalized increaseinflammation and various acute-phase reactants are in-in thiol oxidation and a concomitant decrease in bothvolved in the initiation and/or progression of athero-

protein-associated and low molecular weight reducedsclerosis. Clearly, systemic infection and other causes of

plasma thiols, which are quantitatively the major redoxchronic inflammation may contribute directly to mortal-

change in uremic plasma. ity independent of the atherosclerotic process. Nonethe-It should be noted that the increase in reactive alde- less, data are beginning to emerge linking inflammation

hyde formation and decrease in reduced thiol, which are (as manifested by an increase in CRP) and oxidative stressconcomitantly present in uremia, might be interrelated in dialysis patients. Nguyen-Khoa et al recently demon-and synergistic in their biologic effects. Reactive al- strated a positive correlation between elevated plasmadehyde groups preferentially bind to thiols, which can CRP levels and plasma thiobarbituric acid reaction sub-thereby function to detoxify or scavenge reactive alde- stance (TBARS) as a measure of lipid peroxidation inhydes. Thus, the presence of depleted thiols in the setting chronic hemodialysis patients [57]. These investigatorsof increased reactive aldehyde production in uremia may additionally demonstrated a negative correlation betweensynergistically tip the redox balance in favor of increased plasma CRP levels and plasma alpha tocopherol levels,injury from oxidative stress. consistent with the hypothesis that inflammation depletes

In summary, recent evidence accumulated by multiple antioxidants. Elevated plasma F2 isoprostane levels in theinvestigators incontrovertibly demonstrate that the ure- hemodialysis patient population are positively correlatedmic milieu is a milieu of increased oxidant stress, affect- also with CRP, a finding our group has corroborateding lipid, carbohydrate, protein, amino acid, and DNA [16, 17]. These observations suggest an important patho-structure. With this background, we now wish to examine physiologic link between markers of inflammation andthe important and somewhat more speculative relation- biomarkers of oxidant stress in dialysis patients. How-ships between increased oxidative stress and inflamma- ever, more research is required to firmly establish the path-tion, malnutrition, renal replacement therapy, and solute ophysiologic relationships between inflammation and

oxidant stress in uremia, and to determine if oxidantretention in patients with uremia.


injury is the direct mediator of accelerated cardiovascu-lar complications observed in dialysis patients with ele-vated markers of acute phase reactants.

MALNUTRITION AND OXIDATIVE STRESS:ROLE OF HYPOALBUMINEMIA

Oxidative injury generally takes place only when localoxidant production exceeds antioxidant defense. As dis-cussed previously, the intracellular milieu is maintainedin a highly reduced state, and cells have evolved complexenzymatic defense against oxidant injury. In contrast, ex-tracellular fluids generally contain much lower concen-trations of similar antioxidants [47]. In plasma, the mostimportant antioxidant is provided by thiol groups, whichare largely located on the albumin molecule. The concen-tration of these thiol moieties has been estimated to be Fig. 7. The malnutrition inflammation syndrome tips the redox balance

toward oxidative stress and cardiovascular disease.as high as 500 �mol/L. These thiol moieties act as scav-engers of hypochlorous acid and other oxidants [58].While ascorbate is also an important extracellular anti-

gistic effect on the risk for cardiovascular toxicity, sinceoxidant, albumin, via its thiol groups, provides quantita-

inflammation would result in increased production of oxi-tively almost tenfold greater antioxidant protection [49].

dants by leukocytes and hypoalbuminemia results in re-The albumin molecule has been demonstrated to inhibit

duced scavenging capacity for these oxidants (Fig. 7). Us-copper ion-dependent generation of hydroxyl radicals

ing plasmalogen as an index of oxidative stress, our groupand lipid peroxidation. In other studies, in addition to

has demonstrated that malnourished patients with ad-its active antioxidant moieties, albumin has been shown

vanced chronic kidney disease have increased oxidativeto be selectively oxidized by a variety of oxidants, thereby stress compared to well-nourished patients [66].functioning as a “suicide scavenger” preventing oxida- When hypoalbuminemia arises, in part or in whole,tive injury to both lipoproteins and the vascular wall because of poor nutritional intake, low albumin concen-[47, 58]. Our group has recently demonstrated that oxida- tration may be a surrogate for the lowered concentrationtion of free thiol groups on plasma albumin is one of of other important antioxidants as well. For example,the hallmarks of uremic oxidant stress, quantitatively the major source of reducing equivalents to maintainaccounting for most of the measurable oxidation-induced intracellular redox health comes from nutrients that arealterations in plasma [24]. Another recent study from substrates for the pentose phosphate pathway. It isour group also demonstrated that albumin is the major tempting to speculate that circumstances associated withplasma protein target for carbonyl formation in patients hypoalbuminemia resulting from decreased dietary pro-with uremia [59]. Albumin can act as a binding protein tein intake also are associated with caloric deprivation,for products of oxidation of carbohydrates, lipids, and thereby diminishing the activity of the pentose phos-proteins. As an example, AGEs such as pentosidine and phate pathway and the ability to maintain the intracellu-carboxymethyllysine are more than 90% albumin-bound lar milieu in a highly reduced state. Similarly, when hypo-when circulating in the plasma [60, 61]. albuminemia occurs due to diminished nutritional intake,

These observations provide the framework for a path- because of either illness or anorexia, the burden of oxida-ophysiologic link between the clinical observations in tive injury may be increased by diminished intake ofcross-sectional studies that hypoalbuminemia correlates exogenous antioxidants such as ascorbate and tocopher-with cardiovascular mortality in ESRD patients [62–64]. ols. Ascorbate in particular is dialyzed through mostAccording to this hypothesis, patients with malnutrition high-flux dialysis membranes, and its concentration inand a low plasma albumin concentration will have a dialysis patients is significantly lower.significantly diminished plasma antioxidant capacity dueto the diminished availability of thiol groups. A direct

RETAINED UREMIC SOLUTES MAY BECOMEcorrelation between antioxidant capacity of plasma andSUBSTRATES FOR OXIDATIVE INJURYserum albumin concentrations in patients with nephrotic

syndrome supports this hypothesis [65]. The oxidative As renal failure progresses, compounds begin to accu-stress hypothesis of vascular injury also suggests that the mulate in uremic blood and tissues directly or indirectlycombination of increased inflammation (as manifested by due to declining renal clearance. Vanholder et al recently

pointed to a number of retention solutes that may con-elevated CRP) and hypoalbuminemia will have a syner-


tribute to vascular damage in uremia, including comple- Hyperhomocysteinemia in renal failure has been postu-lated to contribute to atherosclerosis development, al-ment peptides, cytokines, phosphate, oxalate, and di-

methyl arginine [67]. Vanholder et al suggested that urea, though at present, the pathogenetic linkage betweenhyperhomocysteinemia and the development of athero-oxalic acid, parathyroid hormone, and �2 strictly meet

the criteria of uremic toxins. These uremic retention sol- sclerosis is controversial in this patient population [re-viewed in 76].utes lead to a deterioration in biochemical and physio-

logic function, resulting in the uremic syndrome [68]. In In addition to being present in the plasma in higherquantities, both homocysteine and cysteine are morethe past decade, a number of additional solutes including

homocysteine, indoles, and para-cresol also have been oxidized in uremic patients than healthy subjects. Similarto �2m, the extent of homocysteine and cysteine oxida-suggested to function as uremic toxins.

As our understanding of uremic toxicity grows, it is tion is correlated with their respective plasma concentra-tions in the uremic patient population [53]. These find-becoming increasingly clear that many retained uremic

solutes can become substrates for further biochemical ings suggest that homocysteine and cysteine functionas uremic retention solutes that are further subject tomodification in the uremic milieu and may thus further

contribute to toxicity. Indeed, there is evidence to sug- oxidative modification by the uremic environment. Invitro studies have shown that cystine, the oxidized formgest that they may be toxic only after oxidative modifica-

tion. For example, while urea may or may not be directly of cysteine, activates vascular smooth muscle cells tofurther produce reactive oxygen species such as superox-toxic [69], many molecules can be carbamylated in the

presence of sustained high concentrations of urea. Car- ide [79]. Thus, the oxidation or auto-oxidation of theseuremic solutes may contribute to vascular wall toxicitybamylated LDL is another modification of LDL that

can be taken up by scavenger receptors on monocytes, and may further increase their atherogenicity.While these findings are provocative, it must be recog-thereby initiating the atherosclerotic plaque and contrib-

uting to the pathogenesis of atherosclerosis [70]. nized that our understanding of the uremic syndromeSeveral lines of evidence are beginning to suggest that on a biochemical level, although progressing, is still in

oxidative modification of retained uremic solutes may its infancy. The interactions between retained uremicpotentiate their pathogenicity. It is now well known that solutes and further biochemical modifications in the ure-�2m amyloidosis is a serious complication that occurs in mic milieu that contribute to increased toxicity is anthe majority of patients undergoing long-term hemodial- emerging concept and more research in this area willysis [71]. While the pathogenesis of �2m amyloidosis is likely develop in the future.still not completely understood, recent studies have em-phasized that in addition to substrate retention, biochem-

RENAL REPLACEMENT THERAPYical modification of the �2m molecule in the uremic milieuIMPROVES REDOX BALANCEcontributes to its pathogenicity. For example, an isoform

If the uremic milieu is characterized by an alterationof the �2m molecule with a more acidic isoelectric pointin redox chemistry favoring oxidant-mediated injury, itis now recognized as a component of amyloid depositsis logical to assume that amelioration of the uremic statefrom patients undergoing dialysis [72, 73]. Acidificationby renal replacement therapy would correspondingly im-of the �2m molecule is due to progressive glycation andprove the redox balance toward the normal state. Thisoxidation through the non-enzymatic Maillard reactionissue has recently been investigated by examining the re-[74]. The resulting AGE-modified �2m can bind to AGEdox state of plasma thiols, an important marker of plasmareceptors (RAGE) on monocytes leading to monocyteantioxidant capacity, before and after the hemodialysisactivation, cytokine production, and further reactive ox-procedure in patients with chronic renal failure [24, 53].ygen species formation [75]. Monocyte infiltration is nowIn an initial study, our group demonstrated that plasmarecognized as an important component of the tissue de-protein thiol groups are markedly oxidized prior to thestruction that occurs in �2m amyloidosis. Our group hasdialysis procedure but are restored to the levels seen inshown that polymerization of �2m is greatly facilitatedhealthy subjects by the end of the dialysis procedurein the presence of an oxidative stress, reproduced in vitro[24]. In a subsequent investigation, we demonstrated thatby complement-activated neutrophils.each of the major plasma amino thiols (cysteine, homo-Our recent work suggested that homocysteine and cys-cysteine, cysteinyl-glycine, and glutathione) is more oxi-teine also function as retained uremic solutes that be-dized in uremic patients prior to the hemodialysis proce-come substrates for oxidative modification [53]. Bothdure than in healthy subjects. For each of these fourhomocysteine and cysteine plasma levels are elevatedplasma amino thiols (which collectively constitute anseveral-fold in uremic patients compared to healthy sub-important extracellular redox buffering system), redoxjects [44, 52, 53, 76, 77]. Epidemiologic studies have cor-status improved toward the level seen in healthy subjectsrelated hyperhomocysteinemia and hypercysteinemia with

atherosclerotic disease in the general population [78]. by the end of the dialysis procedure [53]. These results


strongly suggest that a major beneficial effect of the genic properties and may result in oxalate accumulation[86]. Furthermore, vitamin C can contribute to the libera-hemodialysis procedure is to restore redox balance to-

ward normal. In other words, alleviation of the uremic tion of ferrous iron from stored iron, which may be aparticular problem in intravenous iron-treated patientsstate exceeds the potential pro-inflammatory effects of

the dialysis procedure itself in improving redox balance. [87]. The reducing potential of vitamin C potentiallyleads to the metal catalyzed production of hydroxyl radi-Support for this concept also comes from earlier work

by Roselaar et al, who demonstrated the presence of a cals with deleterious consequences [87]. In the absence ofovert ascorbate deficiency, risks and benefits of ascorbatestable low molecular weight dialyzable oxidant in the

plasma of chronic hemodialysis patients using electron administration in the uremic population will have to beweighed carefully.spin resonance spectroscopy [80]. These investigators

also demonstrated a redox improvement toward normal The administration of alpha tocopherol (vitamin E) isa much more promising strategy to alleviate oxidativefrom pre- to post-dialysis. Our group demonstrated that

twelve months of dialysis treatment improves plasmalo- complications in uremic patients. Studies to date havereported conflicting results as to whether vitamin E levelsgen levels (a marker of oxidative stress) toward levels

seen in healthy subjects [66]. are low, normal, or even high in uremic patients com-pared to healthy subjects. However, the administrationHowever, since it is well established that extracorpo-

real dialytic therapies can lead to complement activation, of alpha tocopherol to uremic patients almost universallyhas been associated with improvements in biochemicalleukocyte activation, and other pro-inflammatory changes

[81], it is also conceivable that the dialysis procedure measures of oxidant stress and interestingly has frequentlybeen associated with improvements in the anemia ofitself can lead to pro-inflammatory and pro-oxidative

changes. Thus, for example, it is well known that dialy- chronic renal disease, likely as a result of decreased oxi-dant-mediated erythrocyte destruction (Table 4).sis using unmodified cellulosic (bioincompatible) mem-

branes vigorously activate the alternative pathway of Recently, Boaz et al published the results of an impor-tant study (Secondary Prevention with Antioxidants ofcomplement and lead to both neutrophil and monocyte

activation, with increased production of cytokines and Cardiovascular Disease in End Stage Renal Disease –SPACE) in which 196 patients on chronic hemodialysisoxidants [81]. While it is noteworthy that our group and

others have not been able to demonstrate an increase therapy were randomized to receive either placebo or800 IU of alpha-tocopherol daily, with a median follow-in either protein-associated 3-chlorotyrosine or plasma

F2 isoprostane content (biomarkers of different oxidative up of 519 days [88]. This study demonstrated a substantialreduction in myocardial infarction and other cardiovas-pathways) during a single dialysis session with cellulosic

membranes, it is also true that the long-term repetitive cular events in the vitamin E-treated group comparedto patients receiving placebo (Fig. 8). However, despiteuse of bioincompatible membranes has been associated

with worsening nutritional status [82, 83], increased car- the marked improvement in cardiovascular morbidity,there was no difference in overall survival between thediovascular and infectious mortality [84], and an increase

in �2m amyloidosis and higher CRP levels. Given these two treatment groups, suggesting the need to investigateadditional strategies to improve overall survival. Further,countervailing proclivities, it is difficult to predict a priori

what the effect of the dialysis procedure will be on the studies of vitamin E administration for secondary cardio-vascular prevention in non-uremic patients have gener-redox balance in uremic patients. However, pro-inflam-

matory processes such as the use of bioincompatible mem- ally not been found to be beneficial [89].branes or catheters should be avoided whenever possiblein order to favor the restoration of the anti-oxidant po- WHITHER THE ELEPHANT?tential of dialysis.

In the foregoing, we have attempted to provide mul-tiple lines of evidence that increased oxidative stress,

EVIDENCE FROM CLINICAL TRIALS primarily (but not exclusively) resulting from activatedphagocytes via the myeloperoxidase pathway, leads toThe emerging evidence linking uremia to an increase

in oxidative stress and cardiovascular injury will of neces- oxidation of several types of macromolecules in uremia.The consequences of excess oxidation include lipid andsity lead to new therapeutic approaches designed to ame-

liorate the devastating consequences of vascular disease LDL peroxidation, excess oxidation of atherogenic thiolssuch as homocysteine and cysteine, excess generation ofin this patient population. Logical candidates for therapy

include free radical chain-breaking antioxidants such as atherogenic reactive carbonyl groups, and depletion ofimportant plasma antioxidants. The sequela of this pro-vitamin C and vitamin E. Caution must be exercised,

however, in considering the administration of large doses cess includes, among others, accelerated development ofthe atherosclerotic plaque. We also attempted to provideof vitamin C, as vitamin C can function as both an antioxi-

dant and a pro-oxidant [85], and may have procarcino- a pathophysiologic link between inflammation, malnu-


Table 4. Effect of alpha-tocopherol (vitamin E) in dialysis patients

Duration NStudy Dose days uremic patients Effect

Giardini et al [91]Clin Nephrol, 1984 300 mg IM 15 19 Increased Hct

Ono et al [92]Nephron, 1985 600 mg 30 30 Increased Hct

Yukawa et al [93]Nephrol Dial Transplant, 1985 600 mg 14 5 Improved MDA-LDL metabolism

Lubrano et al [94]Artif Organs, 1986 300 mg IM 15 10 Decreased PBMC MDA

Lubrano et al [95] Decreased RBC, MDANutrition, 1992 300 mg IM 15 9 Decreased hemolysis

Sanaka et al [96] No treatment effects onClin Nephrol, 1995 500 mg 11 9 phospholipid hydroperoxidase

Cristol et al [97]Nephrol Dial Transplant, 1997 500 mg 180 7 Reduced EPO dose

Roob et al [98] Attenuate iron-induced lipidJ Am Soc Nephrol, 2000 800 IU 1 22 peroxidation

Islam et al [99] Prolonged lag phase LDLAtherosclerosis, 2000 800 IU 84 33 oxidation

Boaz et al [88] Decreased MILancet, 2000 800 IU 519 97 Decreased CV event

Abbreviations are: Hct, hematocrit; LDL, low-density lipoprotein; PBMC, peripheral blood mononuclear cells; MDA, malodialdehyde; RBC, red blood cells;EPO, erythropoietin; MI, myocardial infarction; CV, cardiovascular.

process, which hopefully will lead to the way to larger,more comprehensive clinical trials.

The SPACE trial may be analogous to early studiesexamining the hypothesis that lowering serum choles-terol levels could reduce cardiovascular morbidity andmortality. In studies by the Lipid Research Clinics andother groups, utilizing lipid-lowering agents that by to-day’s standards would be considered relatively ineffec-tive, cardiovascular morbidity was lowered without a cor-responding improvement in cardiovascular mortality [90].Only with the development of combination anti-lipi-demic therapy, and then subsequently with the develop-ment of statins, was a profound reduction in cardiovascu-Fig. 8. Major end points of the SPACE Study. Symbols are: ( ) pla-

cebo; (�) vitamin E. (Adapted with permission from Boaz et al, Lancet lar mortality demonstrated. Only if and when similar356:1213–1218, 2000.) results can be demonstrated with antioxidant therapy in

uremia will the true identity of the elephant be fullyrecognized.trition, and an acceleration of both the oxidative injury

process and atherosclerosis. Oxidative modification of Reprint requests to Jonathan Himmelfarb, M.D., Division of Ne-phrology, Maine Medical Center, 22 Bramhall Street, Portland, Maineother substances (for example, �2m) leads to other se-04102, USA.quelae of uremia.E-mail: [email protected]

By advocating for this integrated pathophysiology thatlinks inflammation, chronic infection, malnutrition, and

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