THE NATURE OF OXIDATIVE STRESS

In 1989 and in the early 1990s, Daniel Steinberg and colleagues advanced the hypothesis that the atherogenicity of low-density lipoproteins (LDL) was greatly increased by oxidative modification Figure 2⁵. Oxidatively modified LDL is taken up by scavenger receptors, leading to the conversion of monocytes into foam cells, one of the first steps in the atherosclerotic process. Oxidative processes predominantly occur in the mitochondria and the mitochondrial cytochrome oxidase enzyme is responsible for 90% of the oxygen humans metabolize. This enzyme transfers four electrons to oxygen in a concerted reaction that produces two molecules of water as the product. This complex enzyme contains four redox centers, each of which stores a single electron. When all four redox centers are reduced, the simultaneous transfer of four electrons to an oxygen molecule takes place with no detectable intermediate steps, thereby limiting the production of reactive oxygen intermediates. Nevertheless, a small fraction (1 to 2%) of this reaction does proceed via an intermediate step that results in the formation of free radicals⁶. In response to this problem, mammalian systems have evolved numerous intracellular antioxidant systems including enzyme systems, water soluble, and fat-soluble free radical scavengers that can avidly react with and eliminate these intermediate reactive oxygen species before they inflict oxidative damage to vital cellular components and function.

Figure 2.

Potential mechanisms for the role of oxidatively-modified low-density lipoprotein (LDL) in atherogenesis. Endothelial cells, vascular smooth muscle cells, or macrophages may catalyze the oxidative modification of circulating LDL leading to: (I) recruitment of circulating monocytes; (II) macrophage "trapping" in the vessel intima; (III) enhanced uptake of oxidized LDL by resident macrophages leading to foam cell formation; and (IV) endothelial cell destruction as a result of oxidized LDL toxicity (reprinted with permission from Quinn MT et al, Proc Natl Acad Sci USA, 82:5949–5953, 1985).

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While mitochondria are the predominant source of "accidental" oxidative stress, phagocytes "deliberately" utilize high levels of oxygen for host defense against pathogens. The respiratory burst of these phagocytes is known to utilize four enzymes (NADPH oxidase, superoxide dismutase, nitric oxide synthase, and myeloperoxidase) to produce the reactive intermediates superoxide anion, hydrogen peroxide, nitric oxide, and hypochlorous acid (HOCl), respectively, for the destruction of invading microorganisms Figure 3. More recently, an additional novel oxidative pathway has been identified whereby phagocytes convert nitrite to nitryl chloride and nitrogen dioxide via the myeloperoxidase enzyme or by HOCl itself, resulting in nitration of target biomolecules⁷.

Figure 3.

Pathways of oxidant generation by activated leukocytes.

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While each of these phagocyte-derived oxidants can contribute to tissue injury, recent studies have emphasized the importance of myeloperoxidase-catalyzed chlorinating oxidative reactions. Myeloperoxidase (MPO) is one of the most abundant proteins in phagocytes, constituting approximately 5% of neutrophil protein and 1% of monocyte protein. MPO is rapidly secreted upon stimulation and catalyzes the oxidation of halides to their corresponding hypohalous (for example, hypochlorous) acids in the presence of hydrogen peroxide⁸. Quantitative studies have revealed that H₂O₂ generated by activated neutrophils is virtually stochiometrically converted to HOCl. Histological analysis of atherosclerotic tissue have identified catalytically active myeloperoxidase co-localized with foamy macrophages,⁹ and both 3-chlorotyrosine and dityrosine, which are MPO-catalyzed end-products of tyrosine oxidation, have been found in oxidatively modified LDL and in human atherosclerotic lesions^10,11. These phagocyte driven reactions highlight the central role that activated phagocytes and conditions associated with inflammation (and hence phagocyte activation) play in the increased oxidative burden and atherosclerosis. Strong association between elevated white blood cell count, and rapid progression of atherosclerosis and coronary events have been documented in large population studies¹².

Oxidative stress (or oxidant-derived tissue injury) takes place when the production of oxidants exceeds local antioxidant capacity. When this occurs, it results in the oxidation of important macromolecules, including proteins, lipids, carbohydrates, and DNA present in that environment. Because the active oxygen intermediates are produced in vivo in minute quantities and are also highly reactive, direct in vivo detection of the unreacted moities is extremely difficult. Nonetheless, a powerful strategy has evolved for understanding the underlying in vivo mechanisms of oxidative injury by identifying stable end-products of oxidation produced by different reaction pathways. These biomarkers of oxidative pathways increasingly have been used as an in vivo tool to elucidate the importance of oxidative stress as a contributor to many disease states, including uremia^13,14. Table 3 lists the biochemistry of some of the most important of in vivo biomarkers that have been specifically identified at high concentrations in uremia. Figure 4 describes the biochemistry of some of the more recent important biomarker assays of oxidant stress, which are summarized below.

Figure 4.

Biochemistry of oxidant stress biomarkers. (A) Oxidation of tyrosine residues. (B) F2-isoprostane generation from arachidonic acid via free 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.

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Table 3 - In vivo oxidative stress biomarkers useful in evaluating uremia.

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BIOMARKERS OF OXIDATION

Lipid peroxidation

Several groups of investigators have now used highly sensitive and specific assays to conclusively demonstrate that there are higher levels of plasma lipid oxidation in uremic patients compared to healthy subjects. For example, arachidonic acid has been shown to undergo non-enzymatic, free radical-induced, peroxidation reactions resulting in the formation of a series of prostaglandin F₂-like compounds known as F₂ isoprostanes (F₂-IsoPs; Figure 4b)¹⁵. F₂-IsoPs are initially formed in situ on phospholipids and then subsequently can be released in the plasma and, therefore, they can be detected either as free or as phospholipid-bound F₂ isoprostanes. Levels of F₂-IsoPs in human biologic fluids exceed cyclooxygenase-derived prostanoids by greater than an order of magnitude and are a reliable indicator of oxidative stress in vivo. Recent findings have shown that plasma F₂ isoprostane levels are two to four times higher in patients receiving chronic hemodialysis therapy as in age- and sex-matched controls and correlate closely with levels of C-reactive proteins^16,17.

Another family of extremely reactive electrophiles, known as isolevuglandins, is generated in vivo by free radical-induced lipid oxidation of arachidonic acid. In contrast to F₂ isoprostanes, isolevuglandins do not circulate free in the plasma, but are virtually completely adducted to plasma proteins because of their extreme reactivity. Salomon et al have recently demonstrated that patients with ESRD receiving hemodialysis have approximately twice the level of isolevuglandin-plasma protein adducts as healthy subjects¹⁸. Handelman et al also demonstrated that levels of breath ethane (which result from the beta scission of lipid hydroperoxides) are approximately fourfold in hemodialysis patients compared to healthy subjects (abstract: Handelman et al, J Am Soc Nephrol 11:271A, 2000). Thus, a variety of highly sensitive and specific assay systems all demonstrate that biomarkers of lipid peroxidation are higher in dialysis patients than in healthy subjects.

Several investigators also have re-examined the issue of whether plasma LDL is more oxidized in patients with uremia compared to healthy subjects, using more sensitive and specific assays than in the past. Free plasma LDL produces detectable 2-pentylpyrrole epitopes that are generated by the reaction of 4-hydroxy-2-nonenal (HNE), a product of lipid oxidation, with lysine residues¹⁹. Salomon et al, using an ELISA assay with antibodies raised against protein bound 2-pentylpyrrole, have demonstrated markedly elevated HNE-derived 2-pentylpyrrole levels in LDL in patients on both hemo- and peritoneal dialysis compared to healthy subjects²⁰. Using different techniques, Ziouzenkova et al measured the amino group oxidation in APO B100 protein to detect minimal oxidative modifications in LDL²¹. They demonstrated that minimally oxidatively modified LDL levels (which are associated with atherosclerosis) are higher in hemodialysis patients compared to healthy subjects, suggesting that this may be free radical-mediated by the interaction of dityrosine with LDL-associated protein. Taken together, these studies provide compelling evidence that increased free radical-mediated oxidative processes lead to increased lipid, apolipoprotein, and lipoprotein oxidation in patients with ESRD receiving dialysis therapy.

Protein and amino acid oxidation

Oxidatively modified plasma proteins and amino acids also can serve as important in vivo biomarkers of oxidative stress. The ready accessibility of plasma proteins and amino acids for sampling, the relatively long plasma half-lives of many proteins, and the well-defined biochemical pathways that lead to protein and amino acid oxidation can be used to detect more specific pathways of oxidative stress than plasma lipids. For example, oxidation of amino acyl side chains of amino acids in proteins is an attractive biomarker of oxidative reactions because of the high specificity of the end products for specific oxidation pathways. Detection of tyrosine residue oxidation currently constitutes the most sensitive and specific means available for detecting end products of specific oxidative pathways Figure 4a²². In particular, oxidation of tyrosine residues leads to the formation of 3-chlorotyrosine, 3-nitrotyrosine, or dityrosine, depending on whether the predominant oxidizing species is hypochlorous acid, a reactive nitrogen species, or a free radical (such as hydroxyl ion), respectively²². Our group has recently demonstrated that plasma proteins from hemodialysis patients contain elevated levels of 3-chlorotyrosine, but not 3-nitrotyrosine or dityrosine Figure 5. Since this product (3-chlorotyrosine) is a specific product of myeloperoxidase catalyzed reaction, it further suggests a specific and important role for phagocyte-initiated oxidative reactions as the cause of excess oxidative stress in uremia²³.

Figure 5.

Elevated plasma protein 3-chlorotyrosine, a specific biomarker of myeloperoxidase-catalyzed oxidation, in hemodialysis patients. (Adapted with permission from Himmelfarb et al, Free Radical Biology & Medicine 31:1163–1169, 2001.)

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In addition to oxidized tyrosine residues, other important amino acyl groups subject to oxidation include sulfur groups (cysteine, methionine), amino groups (such as lysine), and alcohol groups (including threonine). Recent studies from our group and other investigators have clearly demonstrated high levels of thiol group oxidation as well as carbonyl formation in plasma proteins from patients with uremia Figure 6²⁴. While not entirely specific for a single oxidative pathway, levels of plasma protein oxidation and carbonyl formation seen in uremic patients in vivo are similar to patterns of protein thiol oxidation observed in vitro after oxidation with hypochlorous acid, but not with hydrogen peroxide, again suggesting an important role for myeloperoxidase in generating oxidant stress^24,25. A series of studies by Witko-Sarsat and colleagues have demonstrated elevated levels of advanced oxidative protein products (AOPP) in the plasma of uremic patients^26,27,28. These investigators also demonstrated that hypochlorous acid derived from activated phagocytes in vitro could replicate the production of plasma AOPP to levels seen in uremic patients²⁸. Finally, L-isoaspartyl residues in plasma proteins from uremic patients are also increased as a manifestation of oxidative injury²⁹. Collectively, these studies using protein and amino acids as biomarkers strongly suggest that myeloperoxidase-catalyzed hypochlorous acid derived from activated phagocytes is an important oxidant in uremic patients.

Figure 6.

Plasma protein thiol group oxidation (A) and carbonyl formation (B) in patients with chronic kidney disease and hemodialysis patients, demonstrating that plasma protein oxidation develops in renal disease well before ESRD. (Adapted with permission from Himmelfarb et al, Kidney Int 58:2571–2578, 2000.)

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The previous discussion has served to highlight the specific pathways of oxidative stress and their pervasiveness in uremia. In order to highlight the clinical importance of these oxidation by-products, it is important to link these pathways to specific and common disease manifestations in patients with uremia. For the sake of brevity, we will attempt to demonstrate the clinical relevance of two specific pathways: one related to the formation and accumulation of aldehydes and one to the depletion of reduced thiols.

UREMIA, OXIDATIVE STRESS, AND ALDEHYDE FORMATION

Reactive aldehydes can be formed as the end product of a variety of oxidative reactions including oxidation of alcohol groups, amino groups, and via the addition of oxygen to unsaturated carbon double bonds in carbohydrates, lipids, or amino acids Figure 4c³⁰. Biochemical assays designed to detect accumulation of aldehydes rely on identification of carbonyl (C = O) groups, leading to the use of the term "carbonyl stress." Myeloperoxidase-catalyzed oxidative modification of amino acids also can lead to the formation of a variety of reactive aldehyde compounds including glyoxal, methylglyoxal, acrolein, glycoaldehyde, and parahydroxy phenacetaldehyde³¹.

Several groups have demonstrated that reactive carbonyl compounds can be detected in uremia in concentrations far in excess of normal healthy subjects^{24,25,31,32,33}. Assays measuring total reactive carbonyl compounds (by measuring hydrazone formation after reaction with 2,4-dinitrophenylhydrazine) have shown that total carbonyl compounds are found at a concentration up to tenfold higher in uremic plasma compared to normal plasma^24,31.

The significance of elevated concentrations of aldehydes, measured as reactive carbonyl compounds in uremia is most clearly demonstrated by their role in the formation of advanced glycosylation end products (AGEs). AGEs are formed nonenzymatically by irreversible reaction of reactive carbonyl compounds with various amine groups in proteins³⁴.

The importance of increased reactive aldehyde and AGE formation is evidenced by their prominent role in the pathogenesis of vascular diseases including atherosclerosis^30,35. AGEs may promote atherosclerosis through interactions with specific receptors (RAGE), causing increased expression of adhesion molecules and enhanced attraction of circulating monocytes to the vessel wall. It is important to note that the interaction of AGE with its RAGE receptor also leads to the increased production of interleukin-6 (IL-6) by monocytes and indirectly to the excess formation by CRP in the liver, thus participating in the genesis of inflammation.

Reactive aldehydes have been shown to be involved in the oxidative modification of LDL cholesterol, both in vitro and in vivo, thereby making this modified LDL a target for monocyte phagocytosis and the development of foam cells, a precursor of the atherosclerotic process³⁶. The participation of reactive aldehydes in the atherosclerotic process also is suggested by immunohistochemical analyses of atherosclerotic lesions from human aorta, in which intense positivity of a variety of aldehyde adducts including HNE-histadine, MDA lysine, and acrolein-lysine, can be demonstrated in association with macrophages and foam cells³⁷.

In summary, carbonyl concentration as an index of reactive aldehyde formation represents the end product of oxidative and other pathways. Carbonyl concentration is important both in the pathogenesis of atherosclerosis in uremia and as a potential biomarker to monitor antioxidative therapeutic strategies. To the extent that carbonyl formation is a byproduct of phagocyte-derived myeloperoxidase reactions, carbonyl concentration is also a marker of phagocyte activation and the inflammatory burden.

UREMIA, OXIDATIVE STRESS, AND DEPLETION OF REDUCED THIOL

In addition to the generalized metabolic disturbance in uremia resulting in excess accumulation of reactive aldehydes, it is becoming increasingly clear that there is a concomitant depletion of reduced thiol groups. Thiols are a class of organic sulfur derivatives that are characterized by the presence of sulfhydryl residues at their active site. Thiols have critical intracellular and extracellular function as redox buffers, via protein thiol (S-H)/disulfide (S-S) concentration equilibrium Figure 4d³⁸. Because the formation and cessation of disulfide bonds are involved in many enzymatic and transport processes, thiol oxidation has significant effects on protein structure and function³⁹. For example, intracellular thiols such as glutathione and thioredoxin play an important role in maintaining the highly reduced environment inside the cell³⁸. Extracellular thiols also constitute an important component in antioxidant defense relevant to cardiovascular disease^{40,41,42,43,44,45,46}. The extracellular fluids of the human body, including plasma, contain little or no catalase activity and only low levels of common antioxidants such as superoxide dismutase, glutathione and selenium-containing glutathione peroxidase⁴⁷. Thus, traditional antioxidant enzymes that are crucial in intracellular defense are unavailable and play a minimal role in protection against oxidative injury in extracellular fluids. Halliwell and others have demonstrated that the protein-associated thiols and ascorbate constitute the major extracellular defense against oxidant stress in plasma^47,48,49. The importance of plasma protein-associated free thiols in scavenging both free radicals and myeloperoxidase-generated oxidants has been demonstrated in several studies⁵⁰.

Given the importance of the antioxidant actions of thiols both intracellularly and particularly extracellularly, it is instructive to examine the relative concentrations of reduced and oxidized thiols in uremia. Ceballos-Picot et al investigated the glutathione antioxidant system in the plasma of a large cohort of patients with chronic renal failure and demonstrated diminished plasma glutathione levels and a profound drop in glutathione peroxidase function⁵¹. Our group has demonstrated that plasma protein thiols are extensively oxidized in uremic patients compared to healthy subjects²⁴. We and others also have examined the redox status of the low molecular weight amino thiols cysteine, homocysteine, and cysteinyl-glycine, which are well-known accumulate in uremia^52,53. In each of these aminothiols, the ratio of oxidized to reduced thiols is considerably increased in hemodialysis patients compared to healthy subjects⁵³. Thus, these studies demonstrate a generalized increase in thiol oxidation and a concomitant decrease in both protein-associated and low molecular weight reduced plasma thiols, which are quantitatively the major redox change in uremic plasma.

It should be noted that the increase in reactive aldehyde formation and decrease in reduced thiol, which are concomitantly present in uremia, might be interrelated and synergistic in their biologic effects. Reactive aldehyde groups preferentially bind to thiols, which can thereby function to detoxify or scavenge reactive aldehydes. Thus, the presence of depleted thiols in the setting of increased reactive aldehyde production in uremia may synergistically tip the redox balance in favor of increased injury from oxidative stress.

In summary, recent evidence accumulated by multiple investigators incontrovertibly demonstrate that the uremic milieu is a milieu of increased oxidant stress, affecting lipid, carbohydrate, protein, amino acid, and DNA structure. With this background, we now wish to examine the important and somewhat more speculative relationships between increased oxidative stress and inflammation, malnutrition, renal replacement therapy, and solute retention in patients with uremia.

INFLAMMATION AND OXIDATIVE STRESS

Inflammation is a common feature of ESRD and it has been recognized that about 30 to 50% of predialysis, hemodialysis (HD), and peritoneal dialysis (PD) patients have serologic evidence of an activated inflammatory response⁵⁴. Ward and McLeish have reported that phagocyte cells that are "primed" in uremia may contribute to increased production of both reactive oxygen species as well as cytokines⁵⁵. An elevation of plasma CRP is one indication of a cytokine-driven (especially IL-6) acute phase inflammatory response. The clinical significance of CRP in dialysis patients has been well documented in a series of recent studies in which an elevated CRP has been shown to be a strong predictor of adverse clinical outcomes and increased cardiovascular mortality [reviewed in Arici and Walls, 56]. The interaction of AGE with its RAGE receptor leading to increased production of IL-6 and CRP also was previously discussed⁵⁶. Finally, the highly skewed distribution of CRP and IL-6 elevations in dialysis patients and the episodic nature of CRP increases suggest that patient-specific processes (such as clotted access grafts, dialysis membrane bioincompatibility, or persistent clinical or sub-clinical infections) may be important causes of chronic inflammation in ESRD patients.

Although the association between atherosclerosis and chronic inflammation in the dialysis patient population is well documented, we do not know if the inflammatory response merely reflects an epiphenomenon accompanying established atherosclerotic disease or whether inflammation and various acute-phase reactants are involved in the initiation and/or progression of atherosclerosis. Clearly, systemic infection and other causes of chronic inflammation may contribute directly to mortality independent of the atherosclerotic process. Nonetheless, data are beginning to emerge linking inflammation (as manifested by an increase in CRP) and oxidative stress in dialysis patients. Nguyen-Khoa et al recently demonstrated a positive correlation between elevated plasma CRP levels and plasma thiobarbituric acid reaction substance (TBARS) as a measure of lipid peroxidation in chronic hemodialysis patients⁵⁷. These investigators additionally demonstrated a negative correlation between plasma CRP levels and plasma alpha tocopherol levels, consistent with the hypothesis that inflammation depletes antioxidants. Elevated plasma F₂ isoprostane levels in the hemodialysis patient population are positively correlated also with CRP, a finding our group has corroborated^16,17. These observations suggest an important pathophysiologic link between markers of inflammation and biomarkers of oxidant stress in dialysis patients. However, more research is required to firmly establish the pathophysiologic relationships between inflammation and oxidant stress in uremia, and to determine if oxidant injury is the direct mediator of accelerated cardiovascular complications observed in dialysis patients with elevated markers of acute phase reactants.

MALNUTRITION AND OXIDATIVE STRESS: ROLE OF HYPOALBUMINEMIA

Oxidative injury generally takes place only when local oxidant production exceeds antioxidant defense. As discussed previously, the intracellular milieu is maintained in a highly reduced state, and cells have evolved complex enzymatic defense against oxidant injury. In contrast, extracellular fluids generally contain much lower concentrations of similar antioxidants⁴⁷. In plasma, the most important antioxidant is provided by thiol groups, which are largely located on the albumin molecule. The concentration of these thiol moieties has been estimated to be as high as 500 mol/L. These thiol moieties act as scavengers of hypochlorous acid and other oxidants⁵⁸. While ascorbate is also an important extracellular antioxidant, albumin, via its thiol groups, provides quantitatively almost tenfold greater antioxidant protection⁴⁹. The albumin molecule has been demonstrated to inhibit copper ion-dependent generation of hydroxyl radicals and lipid peroxidation. In other studies, in addition to its active antioxidant moieties, albumin has been shown to be selectively oxidized by a variety of oxidants, thereby functioning as a "suicide scavenger" preventing oxidative injury to both lipoproteins and the vascular wall^47,58. Our group has recently demonstrated that oxidation of free thiol groups on plasma albumin is one of the hallmarks of uremic oxidant stress, quantitatively accounting for most of the measurable oxidation-induced alterations in plasma²⁴. Another recent study from our group also demonstrated that albumin is the major plasma protein target for carbonyl formation in patients with uremia⁵⁹. Albumin can act as a binding protein for products of oxidation of carbohydrates, lipids, and proteins. As an example, AGEs such as pentosidine and carboxymethyllysine are more than 90% albumin-bound when circulating in the plasma^60,61.

These observations provide the framework for a pathophysiologic link between the clinical observations in cross-sectional studies that hypoalbuminemia correlates with cardiovascular mortality in ESRD patients^62,63,64. According to this hypothesis, patients with malnutrition and a low plasma albumin concentration will have a significantly diminished plasma antioxidant capacity due to the diminished availability of thiol groups. A direct correlation between antioxidant capacity of plasma and serum albumin concentrations in patients with nephrotic syndrome supports this hypothesis⁶⁵. The oxidative stress hypothesis of vascular injury also suggests that the combination of increased inflammation (as manifested by elevated CRP) and hypoalbuminemia will have a synergistic effect on the risk for cardiovascular toxicity, since inflammation would result in increased production of oxidants by leukocytes and hypoalbuminemia results in reduced scavenging capacity for these oxidants Figure 7. Using plasmalogen as an index of oxidative stress, our group has demonstrated that malnourished patients with advanced chronic kidney disease have increased oxidative stress compared to well-nourished patients⁶⁶.

Figure 7.

The malnutrition inflammation syndrome tips the redox balance toward oxidative stress and cardiovascular disease.

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When hypoalbuminemia arises, in part or in whole, because of poor nutritional intake, low albumin concentration may be a surrogate for the lowered concentration of other important antioxidants as well. For example, the major source of reducing equivalents to maintain intracellular redox health comes from nutrients that are substrates for the pentose phosphate pathway. It is tempting to speculate that circumstances associated with hypoalbuminemia resulting from decreased dietary protein intake also are associated with caloric deprivation, thereby diminishing the activity of the pentose phosphate pathway and the ability to maintain the intracellular milieu in a highly reduced state. Similarly, when hypoalbuminemia occurs due to diminished nutritional intake, because of either illness or anorexia, the burden of oxidative injury may be increased by diminished intake of exogenous antioxidants such as ascorbate and tocopherols. Ascorbate in particular is dialyzed through most high-flux dialysis membranes, and its concentration in dialysis patients is significantly lower.

RETAINED UREMIC SOLUTES MAY BECOME SUBSTRATES FOR OXIDATIVE INJURY

As renal failure progresses, compounds begin to accumulate in uremic blood and tissues directly or indirectly due to declining renal clearance. Vanholder et al recently pointed to a number of retention solutes that may contribute to vascular damage in uremia, including complement peptides, cytokines, phosphate, oxalate, and dimethyl arginine⁶⁷. Vanholder et al suggested that urea, oxalic acid, parathyroid hormone, and ₂ strictly meet the criteria of uremic toxins. These uremic retention solutes lead to a deterioration in biochemical and physiologic function, resulting in the uremic syndrome⁶⁸. In the past decade, a number of additional solutes including homocysteine, indoles, and para-cresol also have been suggested to function as uremic toxins.

As our understanding of uremic toxicity grows, it is becoming increasingly clear that many retained uremic solutes can become substrates for further biochemical modification in the uremic milieu and may thus further contribute to toxicity. Indeed, there is evidence to suggest that they may be toxic only after oxidative modification. For example, while urea may or may not be directly toxic⁶⁹, many molecules can be carbamylated in the presence of sustained high concentrations of urea. Carbamylated LDL is another modification of LDL that can be taken up by scavenger receptors on monocytes, thereby initiating the atherosclerotic plaque and contributing to the pathogenesis of atherosclerosis⁷⁰.

Several lines of evidence are beginning to suggest that oxidative modification of retained uremic solutes may potentiate their pathogenicity. It is now well known that ₂m amyloidosis is a serious complication that occurs in the majority of patients undergoing long-term hemodialysis⁷¹. While the pathogenesis of ₂m amyloidosis is still not completely understood, recent studies have emphasized that in addition to substrate retention, biochemical modification of the ₂m molecule in the uremic milieu contributes to its pathogenicity. For example, an isoform of the ₂m molecule with a more acidic isoelectric point is now recognized as a component of amyloid deposits from patients undergoing dialysis^72,73. Acidification of the ₂m molecule is due to progressive glycation and oxidation through the non-enzymatic Maillard reaction⁷⁴. The resulting AGE-modified ₂m can bind to AGE receptors (RAGE) on monocytes leading to monocyte activation, cytokine production, and further reactive oxygen species formation⁷⁵. Monocyte infiltration is now recognized as an important component of the tissue destruction that occurs in ₂m amyloidosis. Our group has shown that polymerization of ₂m is greatly facilitated in the presence of an oxidative stress, reproduced in vitro by complement-activated neutrophils.

Our recent work suggested that homocysteine and cysteine also function as retained uremic solutes that become substrates for oxidative modification⁵³. Both homocysteine and cysteine plasma levels are elevated several-fold in uremic patients compared to healthy subjects^{44,52,53,76,77}. Epidemiologic studies have correlated hyperhomocysteinemia and hypercysteinemia with atherosclerotic disease in the general population⁷⁸. Hyperhomocysteinemia in renal failure has been postulated to contribute to atherosclerosis development, although at present, the pathogenetic linkage between hyperhomocysteinemia and the development of atherosclerosis is controversial in this patient population [reviewed in⁷⁶.

In addition to being present in the plasma in higher quantities, both homocysteine and cysteine are more oxidized in uremic patients than healthy subjects. Similar to ₂m, the extent of homocysteine and cysteine oxidation is correlated with their respective plasma concentrations in the uremic patient population⁵³. These findings suggest that homocysteine and cysteine function as uremic retention solutes that are further subject to oxidative modification by the uremic environment. In vitro studies have shown that cystine, the oxidized form of cysteine, activates vascular smooth muscle cells to further produce reactive oxygen species such as superoxide⁷⁹. Thus, the oxidation or auto-oxidation of these uremic solutes may contribute to vascular wall toxicity and may further increase their atherogenicity.

While these findings are provocative, it must be recognized that our understanding of the uremic syndrome on a biochemical level, although progressing, is still in its infancy. The interactions between retained uremic solutes and further biochemical modifications in the uremic milieu that contribute to increased toxicity is an emerging concept and more research in this area will likely develop in the future.

RENAL REPLACEMENT THERAPY IMPROVES REDOX BALANCE

If the uremic milieu is characterized by an alteration in redox chemistry favoring oxidant-mediated injury, it is logical to assume that amelioration of the uremic state by renal replacement therapy would correspondingly improve the redox balance toward the normal state. This issue has recently been investigated by examining the redox state of plasma thiols, an important marker of plasma antioxidant capacity, before and after the hemodialysis procedure in patients with chronic renal failure^24,53. In an initial study, our group demonstrated that plasma protein thiol groups are markedly oxidized prior to the dialysis procedure but are restored to the levels seen in healthy subjects by the end of the dialysis procedure²⁴. In a subsequent investigation, we demonstrated that each of the major plasma amino thiols (cysteine, homocysteine, cysteinyl-glycine, and glutathione) is more oxidized in uremic patients prior to the hemodialysis procedure than in healthy subjects. For each of these four plasma amino thiols (which collectively constitute an important extracellular redox buffering system), redox status improved toward the level seen in healthy subjects by the end of the dialysis procedure⁵³. These results strongly suggest that a major beneficial effect of the hemodialysis procedure is to restore redox balance toward normal. In other words, alleviation of the uremic state exceeds the potential pro-inflammatory effects of the dialysis procedure itself in improving redox balance. Support for this concept also comes from earlier work by Roselaar et al, who demonstrated the presence of a stable low molecular weight dialyzable oxidant in the plasma of chronic hemodialysis patients using electron spin resonance spectroscopy⁸⁰. These investigators also demonstrated a redox improvement toward normal from pre- to post-dialysis. Our group demonstrated that twelve months of dialysis treatment improves plasmalogen levels (a marker of oxidative stress) toward levels seen in healthy subjects⁶⁶.

However, since it is well established that extracorporeal dialytic therapies can lead to complement activation, leukocyte activation, and other pro-inflammatory changes⁸¹, it is also conceivable that the dialysis procedure itself can lead to pro-inflammatory and pro-oxidative changes. Thus, for example, it is well known that dialysis using unmodified cellulosic (bioincompatible) membranes vigorously activate the alternative pathway of complement and lead to both neutrophil and monocyte activation, with increased production of cytokines and oxidants⁸¹. While it is noteworthy that our group and others have not been able to demonstrate an increase in either protein-associated 3-chlorotyrosine or plasma F₂ isoprostane content (biomarkers of different oxidative pathways) during a single dialysis session with cellulosic membranes, it is also true that the long-term repetitive use of bioincompatible membranes has been associated with worsening nutritional status^82,83, increased cardiovascular and infectious mortality⁸⁴, and an increase in ₂m amyloidosis and higher CRP levels. Given these countervailing proclivities, it is difficult to predict a priori what the effect of the dialysis procedure will be on the redox balance in uremic patients. However, pro-inflammatory processes such as the use of bioincompatible membranes or catheters should be avoided whenever possible in order to favor the restoration of the anti-oxidant potential of dialysis.

EVIDENCE FROM CLINICAL TRIALS

The emerging evidence linking uremia to an increase in oxidative stress and cardiovascular injury will of necessity lead to new therapeutic approaches designed to ameliorate the devastating consequences of vascular disease in this patient population. Logical candidates for therapy include free radical chain-breaking antioxidants such as vitamin C and vitamin E. Caution must be exercised, however, in considering the administration of large doses of vitamin C, as vitamin C can function as both an antioxidant and a pro-oxidant⁸⁵, and may have procarcinogenic properties and may result in oxalate accumulation⁸⁶. Furthermore, vitamin C can contribute to the liberation of ferrous iron from stored iron, which may be a particular problem in intravenous iron-treated patients⁸⁷. The reducing potential of vitamin C potentially leads to the metal catalyzed production of hydroxyl radicals with deleterious consequences⁸⁷. In the absence of overt ascorbate deficiency, risks and benefits of ascorbate administration in the uremic population will have to be weighed carefully.

The administration of alpha tocopherol (vitamin E) is a much more promising strategy to alleviate oxidative complications in uremic patients. Studies to date have reported conflicting results as to whether vitamin E levels are low, normal, or even high in uremic patients compared to healthy subjects. However, the administration of alpha tocopherol to uremic patients almost universally has been associated with improvements in biochemical measures of oxidant stress and interestingly has frequently been associated with improvements in the anemia of chronic renal disease, likely as a result of decreased oxidant-mediated erythrocyte destruction Table 4.

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

Full table

Recently, Boaz et al published the results of an important study (Secondary Prevention with Antioxidants of Cardiovascular Disease in End Stage Renal Disease – SPACE) in which 196 patients on chronic hemodialysis therapy were randomized to receive either placebo or 800 IU of alpha-tocopherol daily, with a median follow-up of 519 days⁸⁸. This study demonstrated a substantial reduction in myocardial infarction and other cardiovascular events in the vitamin E-treated group compared to patients receiving placebo Figure 8. However, despite the marked improvement in cardiovascular morbidity, there was no difference in overall survival between the two treatment groups, suggesting the need to investigate additional strategies to improve overall survival. Further, studies of vitamin E administration for secondary cardiovascular prevention in non-uremic patients have generally not been found to be beneficial⁸⁹.

Figure 8.

Major end points of the SPACE Study. Symbols are: () placebo; () vitamin E. (Adapted with permission from Boaz et al, Lancet 356:1213–1218, 2000.)

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WHITHER THE ELEPHANT?

In the foregoing, we have attempted to provide multiple lines of evidence that increased oxidative stress, primarily (but not exclusively) resulting from activated phagocytes via the myeloperoxidase pathway, leads to oxidation of several types of macromolecules in uremia. The consequences of excess oxidation include lipid and LDL peroxidation, excess oxidation of atherogenic thiols such as homocysteine and cysteine, excess generation of atherogenic reactive carbonyl groups, and depletion of important plasma antioxidants. The sequela of this process includes, among others, accelerated development of the atherosclerotic plaque. We also attempted to provide a pathophysiologic link between inflammation, malnutrition, and an acceleration of both the oxidative injury process and atherosclerosis. Oxidative modification of other substances (for example, ₂m) leads to other sequelae of uremia.

By advocating for this integrated pathophysiology that links inflammation, chronic infection, malnutrition, and accelerated atherosclerosis via oxidative stress from activated phagocytes, we run the risk of being another group of "blind men palpating the elephant." Nevertheless, we believe that this hypothesis has several testable elements that if confirmed could lead to major advances in the treatment of patients with uremia. Indeed, we believe that the time is right for clinical trials to test the critical elements of this hypothesis by providing study patients with appropriate antioxidants, perhaps in combination with nutritional repletion and anti-inflammatory therapy. The SPACE trial is an important first step in this process, which hopefully will lead to the way to larger, more comprehensive clinical trials.

The SPACE trial may be analogous to early studies examining the hypothesis that lowering serum cholesterol levels could reduce cardiovascular morbidity and mortality. In studies by the Lipid Research Clinics and other groups, utilizing lipid-lowering agents that by today's standards would be considered relatively ineffective, cardiovascular morbidity was lowered without a corresponding improvement in cardiovascular mortality⁹⁰. Only with the development of combination anti-lipidemic therapy, and then subsequently with the development of statins, was a profound reduction in cardiovascular mortality demonstrated. Only if and when similar results can be demonstrated with antioxidant therapy in uremia will the true identity of the elephant be fully recognized.

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