Renal antioxidant enzymes: Their regulation and function

Kidney International, Vol. 45 (1994), pp. 1—9

EDITORIAL REVIEW

Renal antioxidant enzymes: Their regulation and function Over the last decade, experimental evidence has been reactive oxygen metabolites. Although it is currently not possible to give a meaningful estimate of the quantitative importance of each of these enzymes, quantitative assessment of the

amassed to indicate that partially reduced oxygen, more generally termed reactive oxygen metabolites, play a key intermediary role in the pathophysiologic processes of a surprisingly wide variety of clinical and experimental renal diseases. These range from acute to chronic—and from immune to non-immune— injuries, making the kidney unique among other organs as the site in which a spectrum of seemingly unrelated diseases, from minimal change disease to obstructive nephropathy, involves reactive oxygen metabolites (Table 1) [1—17]. In some forms of these injuries, resident renal cells [11, 13, 14], in addition to

role of AOEs in the kidney is possible. The limitations of quantitative efforts arise from both current technological limitations and the specific nature of the reactive oxygen metabolites and AOEs. Because biological membranes are permeable

to oxidants and their metabolites, offending pathogens can reach their targets without hindrance [21], although many of the antioxidants are reasonably compartmentalized. Thus, the site

of oxidant injury may be distant from the site of oxygen polymorphonuclear neutrophils and other circulating cells generation, while the site of antioxidant action may be remote [8—10], can be the source of reactive oxygen metabolites. Thus, from both oxidant generation and injury. Moreover, antioxidant

cultured glomerular mesangial cells have been shown to produce reactive oxygen metabolites when exposed to complement membrane attack complexes C5b-9 [12, 15], which are formed in an experimental model of immune glomerular injury. The current notion of reactive oxygen metabolites as ubiquitous renal pathogenic agents is supported by two lines of experimental evidence, namely, (1) detection of products of oxidant injury in renal tissue or urine, and (2) experimental demonstration of a protective effect of metabolic inhibitors of oxygen radicals in those disease settings. For example, in both hemoglobin- and myoglobin-induced acute renal failure, deferoxamine, an iron chelator, prevented lipid peroxidation and acute renal failure [17]. These results were taken to indicate that hydroxyl radicals are involved in these pigment-induced acute renal failures. Of note, however, many of the metabolites measured and some of the inhibitors administered in many of the above experiments are not highly specific to particular types of oxygen radicals nor to the injuries driven by oxygen radicals. In fact, most recent experimental findings [18-20] are in line with the notion that more restricted types of oxygen radicals are involved in more limited phases/modes of renal injuries than suggested earlier. Thus, tissue injury following renal ischemia represents the injurious effects of both reactive oxygen metabolites [16] and

enzymes of different classes are complementary to each other [22, 23], further obfuscating efforts to quantify the contribution of each enzyme. In this article, we will provide an overview of the current understanding of the antioxidant enzyme system in the kidney. Discussion is limited for some enzymes, and some data are still preliminary. Supplementation of endogenous antioxidant enzymes protects kidneys from a variety of injuries Prototypically, both of the above-discussed types of evidence

exist for the experimental model of minimal change nephrotic syndrome [2, 4, 5, 24, 25]. This model, induced by administration of puromycin aminonucleoside, is characterized by massive proteinuria and glomerular lesions that are ultrastructurally and biochemically identical to those of human minimal change disease. Markedly elevated levels of xanthine oxidase activity (localized in the glomerulus) [26, 27] and aldehyde (a nonspecific lipid peroxidation product) were detected in glomeruli isolated from the kidney during the height of massive proteinuria and abnormal epithelial cell morphology, that is "fusion of foot processes" [25]. Concurrent exogenous administration of an antioxidant enzyme, for example, superoxide dismutase (SOD) [2, 3] or catalase [3], was highly effective in attenuating anoxia/hypoxia [19] in varying degrees, depending on the the functional and structural abnormality of this model. The duration and the mode of ischemia. effectiveness of these specific antioxidant enzymes has been Of note, analogous to infectious diseases, systemic or local, demonstrated in many other experimental disease models, the manifestation of antioxidant enzymes in renal diseases is including ischemialreperfusion injury, although some results determined by the dynamic balance between the pathogen and are not entirely consistent. As the nephrotoxicity of oxidants intrinsic defense mechanisms. Although several antioxidant per se is beyond the scope of the present article, the reader is enzymes (AOEs) have been identified, and their molecular referred to excellent recent review articles [4, 7, 28—31] discussstructure, regulation, and reactions extensively studied, some ing in depth these results in other models. are less characterized. In concert with other defense mechanisms, these enzymes protect cells against the toxic effect of Endogenous antioxidant enzymes: Nature's cellular defense mechanism against oxidant injury Of note, reactive oxygen metabolites are constant products of normal aerobic cell metabolism. Thus, auto-oxidations of varReceived for publication March 4, 1993 and in revised form May 7, 1993 ious organic substances, such as hydroquinones, produce suAccepted for publication May 11, 1993 peroxide anion. Membrane-bound cytochromes (P450 and bS) © 1994 by the International Society of Nephrology located in the endoplasmic reticulum generate superoxide anion 1

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Ichikawa et a!: Renal antioxidant enzymes

Table 1. Clinical renal disease entities in which oxidants are considered to be pathogenic Glomerular disease Minimal change disease Membranous glomerulopathy Neutrophil-dependent injurya Acute renal failure Post-ischemic Toxic" Obstructive nephropathy Pyelonephritis Progressive renal failure a Includes

anti-GBM nephritis cis-platinum; gentamicin; contrast media; HUS; myoglobinuria/hemoglobinuria; radiation b Includes:

Thus, transgenic mice carrying the human Mn-SOD gene (in addition to their own) were shown to exert prominent resistance to toxic oxygen exposure, toxicity which would cause pulmonary hemorrhage, hyaline membrane formation and interstitial edema in wild-type mice [39]. Likewise, transgenic mice with excess Cu/Zn-SOD genes were found to be highly resistant to oxidant-induced neuron damage [38]. A most recent human study on the functional role of SODs includes a combination of linkage study and sequence analysis of the Cu/Zn-SOD gene in 13 families afflicted with amyotrophic lateral sclerosis [40]. The

results suggest that this progressive degenerative disease of motor neurons is caused by the toxic effect of reactive oxygen metabolites which are not appropriately metabolized because of

impaired Cu/Zn-SOD. Whereas Mn-SOD is inducible by a variety of biological stimuli, the synthesis of Cu/Zn-SOD is

considered largely constitutive [32]. Of interest, a recent study by auto-oxidation (Fig. 1). The cytochrome-catalyzed genera- by Yim, Chock and Stadtman [41] suggests the possibility that tion of superoxide anion from oxygen is particularly active in an Cu/Zn-SOD can also act as an oxygen radical-generating enenvironment in which the concentrations of electron donors, zyme. such as NADPH and NADH, are relatively high (Fig. 1). Catalase is present in subcellular peroxisomes and catalyzes Arachidonic acid metabolisms of cyclooxygenase and 5-lipoxy- the decomposition of hydrogen peroxide to yield oxygen and genase pathways, which are involved in a variety of fundamen- water. Glutathione peroxidase catalyzes the decomposition of tal cell functions, also produce partially reduced oxygen mole- hydrogen peroxide or organic peroxides and reduces glutathicules. However, cells which are under constant threat of the one (GSH) which forms oxidized glutathione (GSSG). GSSG is toxic effect of reactive oxygen species are furnished with highly again reduced to GSH by glutathione reductase, thus forming effective devices to eliminate these toxic oxygen species. A the redox cycle. These enzymes are found in both cytosol and major mechanism of this elimination is the antioxidant enzyme mitochondria. cascade [27]. Superoxide dismutases (SODs), catalase and A new class of antioxidant enzyme has recently become a glutathione peroxidase, serve to eliminate primary products of focus of intense investigation [42, 43], namely, heme oxygenpartially reduced oxygen (Fig. 1). SODs are found mainly in the ase, a rate-limiting microsomal enzyme for heme degradation, intracellular compartment. They exist as Mn-containing pro- that is, utilizing heme as its substrate. Iron is a critical compoteins primarily in the mitochondrial matrix but are found in nent involved in the generation of reactive oxygen metabolites cytosol, as well [32], and as CulZn-containing enzymes primar- and lipid peroxidation (Fig. 1). Since not only iron but also ily in the cytoplasm; they also exist in lesser quantities in certain hemoproteins (such as cytochrome P-450 and hemoglolysosomes [33] and the mitochondrial intermembrane space bin) can promote the formation of hydroxyl radical and lipid [34]. In addition, SODs are present at low concentrations in peroxidation [44], this enzyme can serve as an oxidant scavenextracellular fluids [35]. By binding to heparan sulfate proteo- ger by degrading hemoproteins to bile pigments. Moreover, bile glycans on endothelial cells, these extracellular SODs are pigments per se can act as important intracellular and extracelspeculated to function as a "protective coat" [36]. These lular antioxidants [45]. different forms of superoxide dismutase promote the same In view of many successful experimental demonstrations of biochemical reaction, that is, the dismutation of the superoxide the therapeutic effectiveness of exogenous antioxidant enanion, which is an immediate oxygen metabolite with a high zymes, clinical applications of AOEs are seriously considered biologic activity, to yield hydrogen peroxide and oxygen. as important therapeutic measures in many diseases, including Hydrogen peroxide is removed by hydroperoxidases (such as, respiratory distress syndrome (both adult and neonatal forms), glutathione peroxidase), but this can give rise to the extremely acute inflammation, myocardial infarction, stroke, and organ toxic hydroxyl radical in the presence of reduced iron, which is viability during transplantation [46]. also produced by superoxide anion from ferric iron (Fig. 1). Thus, too little SOD can lead to increased availability of both Antioxidant enzymes are regulated to meet biological need superoxide anion and reduced iron in favor of hydroxyl radical formation, whereas excessive SOD can produce hydrogen Among antioxidant enzymes, superoxide dismutase has been peroxide (a precursor of the hydroxyl radical) at a rate in excess studied extensively, particularly in prokaryotes. It intercepts of that at which hydroperoxidases remove it. A delicate balance the first product of the univalent reduction of oxygen molecules must, therefore, be maintained among the availability of super- (Fig. 1). Mn-SOD has been shown to be modulated by several oxide, hydrogen peroxide, and reduced iron to minimize the key stimuli, including reactive oxygen metabolites and iron highly toxic hydroxyl radical formation. In E. coli, the species chelators [47—5 1]. Studies indicate that superoxide and iron studied extensively thus far, the level of SOD is known to be regulate positively and negatively, respectively, transcription of tightly regulated under a wide range of oxidant stresses to meet the Mn-SOD gene. As the bacteriocidal activities of the host's this need [37]. Unequivocal evidence for the functional impor- phagocytic cells (neutrophils, eosinophils and mononuclear tance of SOD in mammalian cells has been provided by studies phagocytes) rely on microbial killing by reactive oxygen metabusing transgenic mice carrying excess SOD gene(s) [38, 39]. olites, induction of SOD in the microorganism is considered an

Ichikawa et a!: Renal antioxidant enzymes

3

Water

Oxygen 02 Xanthine oxidase Lipoxygenase Cyclooxygenase NAD(P)H oxidase Cytochrome P450

H20

Catalase glutathione peroxidase Superoxide dismutase

Superoxide anion

Hydrogen peroxide H202

Fe2

\
Table 2. Some of the biological stimulants for superoxide dismutase in mammalian tissues/cells Stimulant

Tissue/cell

High oxygen tension Ozone Paraquat Hydrogen peroxide Exercise Endotoxin Chiorpromazine Glucocorticoid Heat TNFfIL.l/IL-6

Lung Lung Fibroblast Kidney/trachea Lung Lung/kidney/liver Brain Lung/iris/kidney/liver Lung Lung/kidney/liver/pancreas/intestine

Hypochlorite OCI-

Fig. 1. Key metabolic pathways of reactive oxygen metabolites in the cell. Although not discussed in the text, myeloperoxidase from neutrophils can metabolize hydrogen peroxide to produce highly reactive oxidants, most notably hypochlorite [1]. Other more specific antioxidant enzymes, although conceivably important for certain cells, are not included in this diagram.

onstrated to alter tissue/cell levels of AOEs [7 1—75]. It has been

shown that treatment of pregnant rats in late gestation with glucocorticoids raises AOE activities in fetal lungs and protects them against oxygen toxicity [721. In addition, in vitro studies by Randhawa et al [73, 74] have suggested that glucocorticoids can act directly on fetal lung explants or fibroblasts in culture to increase AOE activities. An increase in SOD gene expression, determined by mRNA, has been shown to occur in response to several stimuli [76, 77]. Thus, lipopolysaccharide (endotoxin), interleukin- 1, and tumor necrosis factor have been shown to raise Mn-SOD mRNA levels in rat pulmonary epithelial cells [77].

Induction of antioxidant enzymes is considered in the context ide dismutase; Its role in xenobiotic detoxification. Pharmac Ther of tumor cell malignancy; the increase in AOEs has been Adapted by permission from CANADA AT, CALABRESE EJ: Superox-

44:288, 1989

important determinant of their virulence [52]. Catalase in microorganisms is also known to be multi-regulated in an elaborate fashion [53, 54]. AOEs in eukaryotes are also regulated by multiple factors [55—60] (Table 2). Following exposure to a variety of nonlethal oxidant stresses, tissue SOD activity often increases along with acquisition of resistance to otherwise lethal doses of the same oxidant stress, a phenomenon seen in both prokaryotes and eukaryotes [55]. When rats were exposed to 85% oxygen for seven days, Cu/Zn-SOD and Mn-SOD activities in the lung increased by 67% and 140%, respectively. When rats were exposed to 10% oxygen, only Mn-SOD activity increased. Of interest, when these preexposed rats were subsequently exposed to 100% oxygen, which is otherwise lethal, both of these groups survived [611. Similar findings have since been obtained by a number of investigators in their in vivo and in vitro studies [62—65]. While ozone, another form of oxidant stress, induces

SOD [66], rats exposed to a high concentration of ozone develop cross-tolerance to a variety of lethal oxidant stresses, that is, not only to ozone [67] but also to oxygen [68], nitrogen dioxide [69] and radiation [70]. Of interest to nephrologists, treatment with glucocorticoids is one of several experimental maneuvers which has been dem-

detected in aggressive tumor cell populations [78], suggesting that this adaptive response may allow the tumor cell to survive in the environment of high self-generated oxidants [127]. Human small-cell lung cancer cells exhibit progressive increase in resistance to oxidant injury [79]. This phenomenon is thought to underlie the radioresistance seen in many malignant tumors. Although experimental evidence is at best indirect, antioxidant enzyme levels are postulated by some investigators to be an important determinant of a species' life span. By examining the tissues from 13 different primate species including the human, Cutler [80] found an impressively linear relationship between the SOD level and the species' maximum life span potential. In conjunction with the speculation that aging involves impairment of the continuous repair of DNA molecules damaged by reactive oxygen metabolites, these investigators were intrigued by the possibility that the availability of AOEs may be an important determinant of a species' life span. A variety of agents and biological conditions which directly or indirectly imposes oxidant stress on tissues has been demonstrated to activate heme oxygenase, as well [22, 81—83]. As heme oxygenase is a heat-shock protein [84], it is expected to respond to the spectrum of stimuli to which heat-shock proteins in general are sensitive. Glucocorticoids have also been shown to induce heme oxygenase in certain settings [81]. Of interest, when endogenous glutathione peroxidase was experimentally suppressed by feeding animals with a selenium deficient diet,

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Ichikawa et a!: Renal antioxidant enzymes

heme oxygenase was induced in a compensatory manner [221, suggesting the existence of adaptive responses among different antioxidant enzymes. More detailed biological implications of antioxidant enzyme regulations for non-renal tissues are discussed elsewhere [42, 43, 55, 85]. Endogenous antioxidant enzymes are an essential defense system against oxidant injury for the kidney Direct experimental evidence of the importance of AOEs in

renal disease has recently been shown in a study by Nath and Salahudeen [86] using a diet deficient in selenium, a metal essential for glutathione peroxidase and other intra- and extracellular AOEs. When animals were raised with a diet deficient in selenium and vitamin E, they developed renal injury characterized (functionally) by proteinuria and reduced GFR and (structurally) by interstitial cellular infiltrate. When endogenous glutathione peroxidase was experimentally depressed in a similar manner by a selenium deficient diet [871 or administration of diethyl maleate [88] in rats treated with puromycin aminonucle-

oside, the characteristic proteinuria was even more markedly augmented. Of interest, experimental depletion of SOD by administration of diethyldithiocarbamate was also shown to aggravate the same purotnycin aminonucleoside nephrosis in rats [89]. Antioxidant enzymes may operate as a renal protective mechanism through yet another channel. Given that the kidney under oxidant stress is characterized by a profound

vasoconstriction [90] and that nitric oxide acts as a potent vasodilator [91] readily degraded by superoxide anion [921, antioxidants, particularly SODs (through their ability to scavenge superoxide anion), can maximize the renal protective action of nitric oxide [931. Convincing evidence for the protective role of SODs of endogenous origin was provided in a study with transgenic mice carrying additional SOD gene(s). Morphologic analysis of the kidney following experimental transient ischemialreperfusion in these mice revealed a distinctive pattern of tissue protection from injury [941. Acute renal failure secondary to severe rhabdomyolysis is another clinical setting in which oxidant stress plays a major pathogenic role since, in the experimental rhabdomyolysis induced by administration of glycerol, the characteristic depression of GFR can be ameliorated by exogenous administration of dimethyithiourea, a hydroxyl radical scavenger, or deferoxamme [17, 95]. The importance of heme oxygenase in this setting was convincingly demonstrated in a recent study using tinprotoporphyrin, a competitive inhibitor of heme oxygenase [96]. In this study, animals with mild glycerol-induced rhabdomyolysis developed severe azotemia only when they had been pretreated with tin-protoporphyrin [96]. Collectively, studies point to the highly significant role of endogenous AOEs as an effective defense mechanism against oxidative renal injury. Renal antioxidant enzymes are also regulated

Similar to other cells, renal cells respond to many biological stimuli, including reactive oxygen metabolites, with increased AOE activities [97]. Cultured rat glomerular mesangial cells incubated with hydrogen peroxide were shown, in a preliminary study, to have Mn-SOD activity significantly elevated over the

level of untreated cells, while there was no difference in Cu/Zn-SOD activity between hydrogen peroxide-treated and untreated cells [98]. These phenomena were identified in vivo; by assessing specific lipid hydroperoxides with a sensitive method, a 30-minute complete renal ischemia followed by reperfusion was shown to impose significant oxidant stress on the kidney [18]. Glomeruli isolated from rats subjected to the above ischemialreperfusion protocol had elevated levels of antioxidant enzyme activities [99]. Thus, the activities of total SOD, Mn-SOD, glutathione peroxidase, and catalase were uniformly increased over control levels. These effects appeared somewhat selective, as the LDH (cytosolic enzyme) and fumarase (mitochondrial enzyme)—both non-AOE enzymes—measured in the experiments were unaffected [99]. In the absence of data on renal tubules, it is speculated, without evidence, that AOEs were also induced in the renal tubules, the major target of renal ischemiaJreperfusion injury. As in the lung, renal AOEs are also up-regulated by glucocorticoids. Of interest, glucocorticoid pretreatment of rat gbmerular mesangial cells cultured in vitro was shown, after a lag period, to decrease the hydrogen peroxide released when cells were exposed to zymosan [13]. Glomeruli isolated from rats given a daily injection of methylprednisobone for three to nine days were found to have significantly higher activities of total SOD, Mn-SOD, glutathione peroxidase, and catalase than those from untreated rats [251. In a subsequent study, glomerular endothelial cells were incubated in vitro with or without methylprednisolone, and antioxidant enzyme activities were determined 24 hours later. Mn-SOD and catalase activities were significantly elevated in methylpreclnisolone-treated cells, although Cu/Zn-SOD or glutathione peroxidase activity was not appreciably affected in these experiments [1001, Renal antioxidant enzymes can be down-regulated, as well, by certain biological stimuli. When antioxidant enzyme activi-

ties in renal cortex were compared between acutely water deprived and non-water deprived rats, water deprived rats were found to have catalase and superoxide dismutase activities that were reduced to —50% of baseline levels [101]. Renal antioxidant enzymes undergo postnatal development. Oberley et at demonstrated immunohistochemical localization of SODs and catalase in the developing hamster kidney [102].

They found no positive staining before birth and a postnatal increase in immunoreactive enzymes which developed in a centrifugal pattern; positive staining first appeared in the juxtamedullary region and then extended to superficial nephrons. In contrast, the brain and heart demonstrated constant staining of the enzymes. Similar findings were described in the fetal rat kidney using radioimmunoassay for SODs [103]. Exposure of the kidney to exogenous hemoglobin or, more indirectly, endogenous myogbobin induced by glycerol administration was shown to increase local heme oxygenase activity and mRNA expression of this antioxidant enzyme, while this

adaptive response was not seen in catalase or glutathione peroxidase [96]. Using hydrogen peroxide, reactive oxygen metabolites have been shown to induce heme oxygenase in proximal tubule cells in vitro [1041.

Recent studies by several investigators have been directed toward evaluating the functional significance of these renal antioxidant enzyme regulations. The experimental evidence heretofore collected, although still limited, is suggestive of a

Ichikawa et a!: Renal antioxidant enzymes

broad pathophysiologic and therapeutic significance of AOE regulation, as discussed below.

5

animals acquired resistance to the toxic effect of a direct intrarenal arterial injection of hydrogen peroxide, which otherwise would have induced massive proteinuria in normal un-

treated animals [25]. Indeed, the above-discussed rats with PAN nephrosis, which had been treated with glucocorticoids, showed less impairment in renal function, lacked the typical Some forms of acute renal insult which involve generation of morphologic feature of fusion of glomerular epithelial foot reactive oxygen metabolites in kidneys induce a resistance to processes, and had lower glomerular lipid peroxidation product Pathophysiologic and therapeutic implications of renal antioxidant enzyme regulation

recurrent insults of a similar nature [1051. Given that reactive

levels, an index of oxidant stress [25]. Overall, the mechanism

oxygen metabolites induce activation of AOEs, not only in for therapeutic effect of glucocorticoids on minimal change prokaryotic but also mammalian cells (both renal and non- nephrotic syndrome appears to include an enhancement of renal), as discussed above, it is conceivable that this acquisition by the kidney of resistance to reactive oxygen metabolites may involve enhancement of local levels of AOEs. This possibility has been tested recently in some models of renal oxidant injury.

endogenous glomerular AOEs, which attenuates the production

of reactive oxygen metabolites within the glomerular tissue. Given that polymorphonuclear leukocytes are involved in some inflammatory processes of renal parenchymal and microvascu-

Rats, in which glomerular AOE activities were raised by a lar lesions, the capacity of glucocorticoids to induce MnSOD in 30-minute complete renal ischemia followed by reperfusion, these circulating cells [108] may also contribute to minimizing were rechallenged six days later with either 30 minutes of the oxidative injuries caused by the oxygen radicals released ischemia plus 60 minutes of reperfusion or unilateral intrarenal from these cells [109]. arterial infusion of hydrogen peroxide [99]. Whereas in normal control rats these experimental manipulations induced severe Speculated molecular mechanisms for renal antioxidant enzyme regulation reductions in GFR and massive proteinuria, respectively, those The mechanism regulating antioxidant enzyme levels in mamwith elevated AOEs were protected from these functional deteriorations. Thus, enhanced local AOE activities induced by malian cells is the focus of current intense investigation. In this exposure to reactive oxygen metabolites provided kidneys with regard, transcriptional regulation appears to be at least a part of an effective defense system against the toxic effect of reactive the mechanism of local antioxidant enzyme regulation [77, 110, 111]. Thus, in addition to the experimental evidence for up! oxygen metabolites. As discussed earlier, heme oxygenase, which has been shown down-regulation of AOE activities and protein contents, there to act as an important endogenous antioxidant in acute renal are several experimental studies reporting regulated expression failure secondary to rhabdomyolysis, can be induced by hemo- of AOE genes. In general, regulation of protein (that is, globin [961. The functional significance of this induction was enzyme) synthesis can occur at one of several steps: transcripverified by the observation that animals with enhanced heme tion; processing (of RNA transcripts); stabilization (of mRNA); oxygenase by hemoglobin pretreatment became resistant to the translation and post-translation [112]. Among these, the rate of development of severe azotemia and tubular necrosis charac- transcription is the most common step for regulating specific teristic of an experimental rhabdomyolysis, although the heme gene expression. In fact, the gene expressions of several

oxygenase induction did not bring about a cross tolerance to hormones or enzymes have been shown to be regulated by other forms of oxidant stress, that is, ischemiaireperfusion [96]. oxidants or local oxygen tension in non-renal tissues, including Together with the concurrent up-regulation of ferritin [96], an the lung, cardiomyocytes [113], and erythropoetin-producing iron binding protein which serves to minimize the toxic effect of

the irons freed from heme, heme oxygenase induction by iron pigments confers a significant protection against a specific form of renal oxidant injury. Glucocorticoids constitute the first-line treatment of nephrotic syndrome and are remarkably effective in treating minimal change nephrotic syndrome [106]. The mechanism by which glucocorticoids bring minimal change nephrotic syndrome into remission has not been clarified. While glucocorticoids can affect the function of infiltrating cells (such as, neutrophils) by

hepatoma cells [11]. Many regulatory elements for transcription may be located adjacent to the promoter region and may act as

activators to turn on (or off) the functional promoters. It is within these regulatory elements that the response elements for a variety of steroid hormones and other regulators reside [11, 115—122]. Along with the nucleotide sequence, several impor-

tant functional features of this regulatory element have been clarified for the Mn-SOD gene in prokaryotes [37]. Regulation of gene expression by steroid hormones is thought to proceed as follows [112, 115]: (1) ligand-receptor binding; (2)

suppressing the generation of reactive oxygen metabolites activation of the steroid receptor and transformation; (3) bindproduced during phagocytosis [13], minimal change disease ing of the hormone-receptor complex with a specific DNA lacks clear evidence for local cell infiltration or inflammatory processes. Of interest, glucocorticoids have also been shown to be protective in an animal model of nephrotic syndrome, that is, puromycin aminonucleoside nephrosis (PAN) although, unlike the human counterpart, this animal model lacks evidence of T cell activation through which glucocorticoids may potentially work [107]. As discussed earlier, glucocorticoids can raise glomerular antioxidant enzyme activity in vivo [25] and in vitro [100]. Importantly, when glomerular antioxidant enzyme activity was raised by daily administration of methylprednisolone,

sequence (in the regulatory region); and (4) changes in the rate of transcription. While much of the published data have been consistent with a primary effect of steroid hormones at the level of gene transcription, it is possible that, in some circumstances, the rate of transcription remains relatively constant during protein induction and that the accumulation of mRNA is due simply to a steroid-induced suppression of mRNA degradation [112, 115] or activation of translation. The latter has recently been reported for the regulation of Na-K-ATPase subunit by glucocorticoids [123].

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Ichikawa et al: Renal antioxidant enzymes

Putative NF-KB binding site Putative GRE GGGGCTTTCC

100 bp

Putative GRE

Putative ORE

TGTCCT GAACGGCCGTGTTCT TGACGCGGC

Exon I

The binding sites for the glucocorticoids-receptor complex, or glucocorticoid response element (GRE), have been studied on various genes in various cells [116—122]. The binding sites are usually located between 40 to 2,700 bp upstream of the transcription initiation site [116—120, 1221. Several nucleotide sequences have been identified for GRE [112, 116, 122, 124]: GRE contains an imperfect palindromic consensus sequence

Exon II

Fig. 2. Putative nuclear factor ,cB (NF-KB) binding site, glucocorticoid response elements (GRE), and oxygen radical response element (ORE) on the rat man ganese-superoxide dismutase (Mn-SOD) gene.

[131]. The possibility exists, therefore, that activation of NF-icB, followed by its binding to its recognition site, is

involved in the transcriptional up-regulation of the Mn-SOD gene by oxygen radicals. Alternatively, activation of the MnSOD gene may involve a separate set of unknown binding protein and nucleotide elements responsive to oxygen radical species. Of interest, in this regard, recent studies have identified a with a highly conserved core hexanucleotide (such as 5'TGTTCT-3' or 5'-TGTCCT-3'). Within the rat Mn-SOD gene, nucleotide consensus sequence (GTACNNNGC) linked to an there are at least two sequences relevant to the core hexanu- enhancement in transcription of the rat glutathione-S-transcleotide of GRE in the upstream region, one of which has a ferase Ya subunit gene in response to hydrogen peroxide [130]. palindromic structure (Fig. 2) [1241. It is, therefore, possible This consensus sequence is present in a non-coding region of rat that glucocorticoids activate the Mn-SOD gene by involving Mn-SOD genomic DNA (Fig. 2). Collectively, recent studies point to the notion that the these GREs. A recent preliminary study demonstrated enhancement of Mn-SOD activity and mRNA content in methyl- kidney inherits and acquires a powerful defense system against prednisolone-treated cultured glomerular cells [100]. Therefore, oxidant injuries which responds to a variety of natural and within the glomerulus, glucocorticoids are likely to regulate therapeutic stimuli. Insight into such alterations of the antioxantioxidant enzyme levels at the gene transcriptional step, as idant system may unwind the mechanism and therapeutic approach to a variety of renal injuries. they do for other proteins [115, 1251. The acquisition by the kidney of functionally important elevated antioxidant enzyme levels following exposure to oxiIEKUNI ICHUCAWA, SHIGERU KIYAMA, AND dant stress (discussed earlier) may also involve activation of TOSHIMASA YosHIoIcA local antioxidant enzyme genes. LLC-PK1 (proximal tubule Vanderbilt University cell-like cells derived from the porcine kidney) cells exposed to hydrogen peroxide have been shown to have enhanced mRNA expression of heme oxygenase [104]. Renal epithelial cell lines

Nashville, Tennessee, USA and Tokyo Women's College Tokyo, Japan

exposed to hydrogen peroxide chronically acquire elevated

Acknowledgments

catalase and glutathione peroxidase levels [1261. The Mn-SOD The publication of this review article was supported in part by mRNA level in rat mesangial cells was significantly elevated National Institutes of Health Grants DK 44757 and DK 40527. The after exposure to hydrogen peroxide [98]. As described earlier, authors are greatly indebted to Dr. Karl A. Nath of the University of

in addition to oxygen radicals per se, some cytokines, for Minnesota and Dr. Sudhir V. Shah of the University of Arkansas for example, TNF-a, IL-i and IL-6, have been shown to transcriptionally up-regulate SOD in a manner somewhat specific to the cell type (Table 2). As these cytokines play key roles in tissue inflammatory and immune responses, as well as multiple other biological phenomena, the mechanism of this cytokine-induced transcriptional activation has been a focus of a great interest. Thanks to recent research endeavors several intermediary steps have been identified for a few genes. Studies have identified NF-icB, a multisubunit transcription factor protein which, in collaboration with cell-specific factors, can activate the expression of the cytokine genes described above [1271. Since TNF-a fails to activate NF-KB when cells are concurrently treated with antioxidants [128], reactive oxygen species appear to be the secondary messenger for cytokine activation of NF-KB and to act as its direct activator. Of note, TNF-a activates NF-icB and the Mn-SOD gene in human papillary thyroid carcinoma cells [129]. Moreover, the Mn-SOD gene contains, in its upstream region (Fig. 2), a concensus decanucleotide sequence which has been determined in other genes to be the binding site for NF-KB

their valuable suggestions during the preparation of this article. The authors are also grateful to Ms. Mary Beehan for her editorial assistance. Reprint requests to lekuni Ichikawa, M.D., C-4204 Medical Center North, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2584, USA.

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