I will introduce the term "oxidants" and present the evidence available in support of the role of oxidants and iron in chronic kidney disease. The biologic effects of oxidants relevant to chronic kidney disease and their role in experimental glomerular disease1,2,3,4, diabetic nephropathy5,6,7,8, and progression of kidney disease9,10 will be reviewed.
Oxygen normally accepts four electrons and is converted directly to water. However, partial reduction of oxygen can and does occur in biologic systems, leading to the generation of partially reduced and potentially toxic reactive oxygen. Thus, sequential reduction of oxygen along the univalent pathway leads to the generation of superoxide anion, hydrogen peroxide, hydroxyl radical, and water. Superoxide and hydrogen peroxide appear to be the primary species generated Figure 1. These may then play a role in the generation of additional and more reactive oxidants, including the highly reactive hydroxyl radical (or a related highly oxidizing species). Iron salts play a catalytic role in a reaction commonly referred to as the metal-catalyzed Haber-Weiss (Fenton) reaction11. Additional oxidants may be formed as a result of the metabolism of hydrogen peroxide by neutrophil-derived myeloperoxidase (MPO), the enzyme responsible for the green color of pus, to produce highly reactive toxic products, including hypochlorous acid (HOCl, the active ingredient in household bleach). These oxygen metabolites, including the free radical species superoxide and hydroxyl radical, and other metabolites, such as hydrogen peroxide and hypohalous acids, are often collectively referred to as reactive oxygen metabolites (ROM), or oxidants.
BIOLOGIC EFFECTS OF OXIDANTS RELEVANT TO CHRONIC KIDNEY DISEASE
I have divided the biologic effects of oxidants into those that are most relevant to the three major manifestations of glomerular disease2,4: occurrence of proteinuria, altered glomerular filtration rate, and morphologic changes. These effects are relevant both to various proliferative and nonproliferative glomerulonephritides and diabetic nephropathy.
Proteinuria
It is generally accepted that leukocytes cause proteinuria (a hallmark of glomerular diseases) by damaging the glomerular basement membrane (GBM) that serves as the major ultrafiltration barrier to restrict the entry of proteins into the urinary space. The degradation of the GBM by stimulated neutrophils was shown to be due to the activation of a latent metalloenzyme (most likely gelatinase) by hypochlorous acid, or a similar oxidant generated by the myeloperoxidase-hydrogen peroxide-halide system Table 1.
Oxidants may also contribute to glomerular basement membrane damage by increasing its susceptibility to proteolytic damage and by inactivating the alpha-1–proteinase inhibitor (the primary regulator of neutrophil elastase), thus allowing the released elastase to more readily inflict damage to the extracellular matrix. Oxidants also impair the synthesis of glomerular heparan sulfate proteoglycans, which are needed to maintain integrity of the glomerular basement membrane and normal glomerular ultrafiltration.
Several direct in vivo studies indicate that oxidants are capable of inducing glomerular injury, resulting in proteinuria. For example, hydrogen peroxide infused directly into the renal artery causes massive transient proteinuria. Infusion of myeloperoxidase (MPO) followed by hydrogen peroxide in a chloride-containing solution in rats results in significant proteinuria, followed in four to 10 days by the development of a marked proliferative glomerular lesion.
Glomerular filtration rate
The effects of oxidants that potentially contribute to altered GFR are summarized in Table 1. Oxidants reduce the glomerular and mesangial cell planar surface and increase myosin light chain phosphorylation, a biochemical marker of contraction. These effects could modulate the surface area of mesangial cells, thus modifying ultrafiltration coefficient and leading to a decrease in the GFR. In addition, oxidants increase the synthesis of prostaglandins and thromboxane, which have been implicated as important mediators causing proteinuria and/or a fall in the GFR in various experimental models of glomerular disease.
Morphologic changes
Several recent studies suggest that oxidants exhibit several effects that may contribute to the morphologic changes observed in a variety of glomerular diseases Table 1. Necrotizing crescentic glomerulonephritis associated with anti-MPO antibodies are part of antineutrophil cytoplasmic autoantibodies (ANCA)-associated glomerulonephritis. It is characterized by segmental fibrinoid necrosis of the GBM, marked infiltration of neutrophils, and mononuclear cells. Similar morphologic changes have been described in rats immunized with MPO and perfused with lysosomal enzyme extract and hydrogen peroxide.
A number of monocyte-specific cytokines have been described and include the monocyte colony-stimulating factor (CSF-1) and the monocyte chemoattractant protein (MCP-1). Generation of reactive oxygen species, possibly by NADPH-dependent oxidase, is involved in the induction of the MCP-1 genes by tumor necrosis factor-
(TNF-) and immunoglobulin G (IgG) complexes in mesangial cells. Local generation of oxidants could represent a factor responsible for the expression of MCP-1 in immune-mediated increased expression of monocyte chemoattractant protein in glomeruli from rats with anti-Thy-1 glomerulonephritis, and in other models where monocyte infiltration is a prominent feature of glomerular disease. Both in vitro and in vivo studies indicate that oxidants are capable of inducing many of the functional and morphologic changes that are observed in glomerular diseases.
ROLE OF OXIDANTS IN LEUKOCYTE-DEPENDENT GLOMERULONEPHRITIS
Several studies strongly support the role of leukocytes in reducing glomerular injury that results in proteinuria Table 2. A wide variety of soluble and particulate stimuli that appears to be relevant to the pathophysiology of glomerulonephritis can activate neutrophils and monocytes to release enormous amounts of oxidants. Thus, stimulated neutrophils or monocytes are potential sources of oxidants in leukocyte-dependent glomerular injury.
Anti-GBM antibody disease
One of the best-characterized models of complement- and neutrophil-dependent glomerular injury is the heterologous phase of anti-GBM antibody disease. Infiltrating neutrophils and macrophages generate large amounts of superoxide anion (hydrogen peroxide) in this model of glomerulonephritis. Treatment with catalase markedly reduces the proteinuria, as do dimethylthiourea (a potent hydroxyl radical scavenger) and deferoxamine. Although the role of iron is not completely understood, the protective effect of iron chelators generally has been taken as evidence for the participation of hydroxyl radical in tissue injury, because iron is critical in the generation of hydroxyl radical.
ROLE OF OXIDANTS IN LEUKOCYTE-INDEPENDENT GLOMERULONEPHRITIS
Puromycin aminonucleoside model of minimal change disease in humans
A single intravenous injection of puromycin aminonucleoside results in marked proteinuria and glomerular morphologic changes which are similar to minimal change disease in humans. It is clear that oxidants play an important role in the development of this model Table 3. Puromycin aminonucleoside enhances the generation of superoxide anion, hydrogen peroxide, and hydroxyl radical when added to freshly isolated glomeruli. Glomerular epithelial cells have been shown to be the target of injury in many of the noninflammatory forms of glomerular disease, including the nephrotic syndrome induced by the injection of puromycin aminonucleoside. In response to puromycin aminonucleoside, cultured glomerular epithelial cells have been shown to enhance the generation of hydrogen peroxide. In vivo evidence for the role of oxidants comes from the ability of several different scavengers of oxidants to prevent or reduce proteinuria in this model of glomerular disease. Bleomycin-detectable iron (iron capable of catalyzing free radical reactions) was markedly increased in glomeruli from nephrotic rats when compared to control. Deferoxamine prevented the increase in bleomycin-detectable iron in glomeruli and provided complete protection against proteinuria, suggesting an important pathogenic role for glomerular catalytic iron in the puromycin aminonucleoside–induced minimal change nephrotic syndrome.
Table 3 - Evidence for the role of oxidants in puromycin aminonucleoside model of minimal change disease2,4,34.
Full table There is other supporting evidence for a role for oxidants in puromycin aminonucleoside–induced nephrotic syndrome Table 3. Glutathione peroxidase is a seleno enzyme that catalyzes the reduction of hydrogen peroxide to water. Feeding rats a selenium-deficient diet results in marked diminution of glutathione peroxidase and is accompanied by a marked increase in urinary protein following puromycin aminonucleoside injection, suggesting an important role of glutathione peroxidase in this model of glomerular disease. Similarly, inhibition of superoxide dismutase augments puromycin aminonucleoside-induced proteinuria. These studies not only demonstrate the importance of endogenous antioxidant defenses, but also provide additional support for the role of oxidants in these models of glomerular injury.
Passive Heymann nephritis model of membranous nephropathy
Passive Heymann nephritis, induced by a single intravenous injection of anti-Fx1A, is a complement-dependent and neutrophil-independent model of glomerular disease that resembles membranous nephropathy in humans Table 4. In in vivo studies, enhanced generation of hydrogen peroxide has been demonstrated in this model of membranous nephropathy.
Table 4 - Evidence for the role of oxidants in an animal model of membranous nephropathy2,4,34.
Full table Administration of scavengers of hydroxyl radical or deferoxamine markedly reduces proteinuria, which suggests a role of the hydroxyl radical in passive Heymann nephritis. Similarly, hydroxyl radical scavengers significantly reduce proteinuria in the cationized gamma globulin–induced immune complex glomerulonephritis, a complement-[dependent?] and neutrophil-independent model of membranous nephropathy. Glutathione peroxidase is also an important defense enzyme in this model. Rats on a selenium-deficient diet have decreased glutathione peroxidase activity and a worsening of proteinuria. Malondialdehyde adducts (a marker of lipid peroxidation) are localized to the GBM, and type IV collagen is modified by malondialdehyde adducts. Taken together, these studies suggest an important role for hydroxyl radical in animal models of membranous nephropathy.
DIABETIC NEPHROPATHY
There is a large body of evidence indicating that diabetes is a state of increased oxidative stress12,13,14. I will summarize only the information directly pertinent to diabetic nephropathy. It was reported that a hydroxyl radical–like species was responsible for the changes in the arterial wall proteins in a model of diabetes in monkeys15. In an excellent review, Nishikawa and Brownlee argue and provide convincing evidence that oxidants are the causative link for all the major pathways that have been implicated in diabetic complications including activation of aldose reductase pathway, induction of the diacylglycerol pathway, activation of protein kinase C, and induction of advanced glycation end-products in an article entitled, "The missing link: A single unifying mechanism for diabetic complications"5.
In addition to these effects on the vascular bed5,15,16,17, which are likely to be important in the glomerular vascular bed, there is more direct evidence for oxidants in diabetic nephropathy6,7,8,18,19. In in vitro studies, it has been shown that high glucose results in increased generation of reactive oxygen species by mesangial cells. Similarly, it has been shown that high glucose results in lipid peroxidation in isolated glomeruli, which is prevented by hydroxyl radical scavengers6. High glucose has also been shown to affect several biologic processes in glomeruli that have been implicated in diabetic nephropathy, and antioxidants have been shown to inhibit these processes. For example, antioxidants prevented glucose-induced activation of protein kinase C (PKC) and nuclear factor-
B (NK-B) up-regulation of tumor growth factor-
1 (TGF-1), up-regulation of fibronectin7, and up-regulation of endothelin-18. In in vivo studies, glomeruli isolated from diabetic rats have increased production of superoxide and hydrogen peroxide7,8, and kidneys from diabetic rats exhibit lipid peroxides and 8-OhdG6. Diabetic nodular lesions in humans stained positive for MDA, an index of lipid peroxidation19.
Iron content in the kidney has been shown to be increased in an animal model of diabetes20. It has been shown that the urinary iron excretion is increased early in the course of diabetic renal disease in humans21. Howard et al demonstrated that diabetic patients with microalbuminuria and overt proteinuria have a marked increase in urinary iron.
ROLE OF OXIDANTS AND IRON IN PROGRESSIVE KIDNEY DISEASE
Although there is evidence for both oxidants and iron in progressive renal disease, in this section we emphasize the role of iron because of the possibility of using iron chelators in preventing progression. The data supporting the role of iron in models of progressive renal disease consists of demonstration of increased iron in the kidney in these models of progressive kidney disease; enhanced oxidant generation, which provides a mechanism by which iron can be mobilized; and, more directly, the beneficial effect of iron-deficient diets and iron chelators. Rats with proteinuria have increased iron content in proximal tubular cells, and iron accumulation was the only independent predictor of both functional and structural damage22. Similarly, it has been shown there is a substantial iron accumulation associated with increased cortical malondialdehyde in proximal tubular cells in the remnant kidney, suggesting reactive oxygen species generation. Iron accumulation has also been studied in human chronic renal disease23.
The sources of increased iron in the kidney have not been well delineated, but Alfey et al have suggested that urinary transferrin provides a potential source of iron. Glomerular injury of any type impairs glomerular permselectivity, thereby leading to the leakage of proteins, including transferrin, into the urinary space.
At a pH below 6.5, iron has been shown to dissociate from transferrin in urine. As pH decreases as urine courses along the nephron, iron is released from transferrin24,25, thus providing a source of iron that could act on renal tubular epithelial cells or be absorbed into the tubules. Information about other potential sources, including intracellular sources of iron in the kidney, is available for only acute models of renal injury. Thus, mitochondria have been suggested as sources of iron in gentamicin nephrotoxicity, whereas cytochrome C has been shown to be an important source of catalytic iron in ischemic and toxic injury26.
For iron to be important in causing renal injury, it is also important to demonstrate increased generation of oxidants. The oxidants would then play a role in mobilizing iron, as well as interacting with the mobilized iron to generate highly reactive metabolites. Nath et al have carried out a series of studies that have provided compelling evidence for a role for oxidants in progressive renal disease27,28. Increased rates of oxygen consumption, which occur in surviving nephrons, is linked to ammoniagenesis and increased generation of reactive oxygen species, both of which have been incriminated in progressive renal injury27. It has been shown also that diets deficient in selenium and vitamin E weanling rats enhances ammoniagenesis, compromises renal function in the intact kidney, and induces tubulointerstitial injury29.
Several studies have examined the effect of iron-deficient diet or iron chelators on progression of chronic kidney disease. Alfrey et al have shown a marked effect of iron-deficient diet or an iron chelator on preventing the development of tubulointerstitial disease and renal functional deterioration in nephrotoxic serum nephritis24,30. Remuzzi et al have shown that rats fed an iron-deficient diet had significant reduction in proteinuria, and developed less glomerulosclerosis31. Iron chelator significantly reduced iron accumulation and tubular damage32 in rat remnant kidneys (a model for progressive renal disease).
The precise cellular mechanisms by which oxidants and iron participate in progression are not known. However, oxidants and iron have many cellular effects that are potentially important in tubulointerstial damage, fibrosis, and matrix accumulation. For example, it is well established that oxidants and iron play an important role in cellular injury and cell death, including apoptosis. Thus, conceivably they could play a role in tubular atrophy and loss of cells, which is a common feature of progressive renal disease. In addition, lipid peroxidation has been shown to be important in induction of collagen gene expression33, and may thus contribute to renal fibrosis.
CONCLUSION
A sufficient body of in vitro and in vivo information exists to postulate that oxidants appear to be important mediators in glomerular pathophysiology and in progressive kidney disease. Nonetheless, the multifaceted nature of tissue injury makes it almost a certainty that the cooperative and sometimes complex interactions between different injurious mechanisms are important in the final expression of injury.
While the collective information on the role of oxidants and iron derived both from models of glomerular disease, as well as progressive renal failure, is impressive, there is virtually no information on the potential role of these mechanisms in human disease. There are many differences between animal models of glomerular disease and glomerular disease in humans. For example, the animal model of minimal change disease is a toxic model, whereas the mechanism of minimal change disease in humans is not known. Similarly, the anti-Fx1A antibody that is used for the animal model of membranous nephropathy has been difficult to demonstrate in human membranous nephropathy.
Indeed, the lessons from animal models of acute renal failure have been disappointing when attempting to translate to human disease. In the future, control studies in a large number of patients will be required to demonstrate the efficacy of antioxidants and/or iron chelators in retarding progression of renal failure, and may offer important therapeutic modality to patients with chronic kidney disease.
References
| 1. | DIAMOND JR. The role of reactive oxygen species in animal models of glomerular disease. Am J Kidney Dis 1992; 19: 292–300. | PubMed | ISI | ChemPort | |
| 2. | SHAH SV. The role of reactive oxygen metabolites in glomerular disease. Annu Rev Physiol 1995; 57: 245–262 10.1146/annurev.ph.57.030195.001333. | Article | PubMed | ChemPort | |
| 3. | JOHNSON RJ, LOVETT D & LEHRER RI et al. Role of oxidants and proteases in glomerular injury. Kidney Int 1994; 45: 352–359. | PubMed | ISI | ChemPort | |
| 4. | KAUSHAL GP, MAYEUX PR & SHAH SV. Proteases and oxidants in glomerular injury,. inImmunologic Renal Diseases 2001; 2nd ed., edited by Neilson EG, Couser WB Philadelphia, Lippincott Williams & Wilkins pp 395–414. |
| 5. | NISHIKAWA T, EDELSTEIN D & BROWNLEE M. The missing link: A single unifying mechanism for diabetic complications. Kidney Int 2000; 58: S26–S30 10.1046/j.1523-1755.2000.07705.x. |
| 6. | HA H & KIM KH. Role of oxidative stress in the development of diabetic nephropathy. Kidney Int 1995; 48: S18–S21. |
| 7. | KITCHING AR, RUGER BM & DAVIS PF. Oxidant stress in the kidney in experimental diabetes mellitus. Kidney Int 1997; 51: 1314. |
| 8. | CHEN H-C, GUH J-Y & SHIN S-J et al. Reactive oxygen species enhances endothelin-1 production of diabetic rat glomeruli in vitro and in vivo. J Lab Clin Med 2000; 135: 309–315. | PubMed | |
| 9. | SHAH SV. Role of iron in progressive renal disease. Am J Kidney Dis 2001; 37: S30–S33. | PubMed | ISI | ChemPort | |
| 10. | HAUGEN E & NATH KA. The involvement of oxidative stress in the progression of renal injury. Blood Purif 1999; 17: 58–65 10.1159/000014377. | Article | PubMed | ISI | ChemPort | |
| 11. | HALLIWELL B & GUTTERIDGE JMC. Role of free radicals and catalytic metal ions in human disease: An overview. Meth Enzymol 1990; 186: 1–85. | PubMed | ChemPort | |
| 12. | HARTNETT ME, STRATTON RD & BROWNE RW et al. Serum markers of oxidative stress and severity of diabetic retinopathy. Diabetes Care 2000; 23: 234–240. | PubMed | |
| 13. | MATTEUCCI E & GIAMPIETRO O. Oxidative stress in families of type I diabetic patients. Diabetes Care 2001; 24: 167–168. | PubMed | |
| 14. | SHIN CS, MOON BS & PARK KS et al. Serum 8-hydroxy-guanine levels are increased in diabetic patients. Diabetes Care 2001; 24: 733–737. | PubMed | |
| 15. | PENNATHUR S, WAGNER JD & LEEUWENBURGH C et al. A hydroxyl radical-like species oxidizes cynomolgus monkey artery wall proteins in early diabetic vascular disease. J Clin Invest 2001; 107: 853–860. | PubMed | ChemPort | |
| 16. | SHARPE PC, LIU W-H & YUE KKM et al. Glucose-induced oxidative stress in vascular contractile cells. Diabetes 1998; 47: 801–809. | PubMed | ISI | ChemPort | |
| 17. | ORANJE WA & WOLFFENBUTTEL BHR. Lipid peroxidation and atherosclerosis in type II diabetes. J Lab Clin Med 1999; 34: 19–32 10.1016/S0022-2143(99)90050-1. |
| 18. | REDDI AS & BOLLINENI JS. Selenium-deficient diet induces renal oxidative stress and injury via TGF- 1 in normal and diabetic rats. Kidney Int 2001; 59: 1342–1353 10.1046/j.1523-1755.2001.0590041342.x. | PubMed | |
| 19. | HORIE K, MIYATA T & MAEDA K et al. Immunohistochemical colocalization of glycoxidation products and lipid peroxidation products in diabetic renal glomerular lesions. J Clin Invest 1997; 100: 2995–3004. | PubMed | ISI | ChemPort | |
| 20. | JOHNSON WT & EVANS GW. Effects of the interrelationship between dietary protein and minerals on tissue content of trace metals in streptozotocin-diabetic rats. J Nutrit 1984; 114: 180–190. | PubMed | |
| 21. | HOWARD RL, BUDDINGTON B & ALFREY AC. Urinary albumin, transferrin and iron excretion in diabetic patients. Kidney Intl 1991; 40: 923–926. |
| 22. | HARRIS DC, TAY C & NANKIVELL BJ. Lysosomal iron accumulation and tubular damage in rat puromycin nephrosis and ageing. Clin Exp Pharmacol Physiol 1994; 21: 73–81. | PubMed | |
| 23. | NANKIVELL BJ, BOADLE RA & HARRIS DCH. Iron accumulation in human chronic renal disease. Am J Kidney Dis 1992; 20: 580–584. | PubMed | ISI | ChemPort | |
| 24. | ALFREY AC. Toxicity of tubule fluid iron in the nephrotic syndrome. Am J Physiol 1992; 263: F637–F641. | PubMed | |
| 25. | COOPER MA, BUDDINGTON B & MILLER NL et al. Urinary iron speciation in nephrotic syndrome. Am J Kidney Dis 1995; 25: 314–319. | PubMed | ISI | ChemPort | |
| 26. | BALIGA R, UEDA N & WALKER PD et al. Oxidant mechanisms in toxic acute renal failure. Am J Kidney Dis 1997; 29: 465–477. | PubMed | ISI | ChemPort | |
| 27. | NATH KA, FISCHEREDER M & HOSTETTER TH. The role of oxidants in progressive renal injury. Kidney Int 1994; 45: S111–S115. | ISI | ChemPort | |
| 28. | NATH KA, CROATT AJ & HOSTETTER TH. Oxygen consumption and oxidant stress in surviving nephrons. Am J Physiol 1990; 258: F1354–F1362. | PubMed | ISI | ChemPort | |
| 29. | NATH KA & SALAHUDEEN AK. Induction of renal growth and injury in the intact rat kidney by dietary deficiency of antioxidants. J Clin Invest 1990; 86: 1179–1192. | PubMed | ISI | ChemPort | |
| 30. | ALFREY AC, FROMENT DH & HAMMOND WS. Role of iron in the tubulointerstitial injury in nephrotoxic serum nephritis. Kidney Int 1989; 36: 753–759. | PubMed | ISI | ChemPort | |
| 31. | REMUZZI A, PUNTORIERI S & BRUGNETTI B et al. Renoprotective effect of low iron diet and its consequence on glomerular hemodynamics. Kidney Int 1991; 39: 647–652. | PubMed | |
| 32. | NANKIVELL BJ, CHEN J & BOADLE RA et al. The role of tubular iron accumulation in the remnant kidney. J Am Soc Nephrol 1994; 4: 1598–1607. | PubMed | ISI | ChemPort | |
| 33. | HOUGLUM K, BRENNER DA & CHOJKIER M. D- -tocopherol inhibits collagen a1 (I) gene expression in cultured human fibroblasts. Modulation of constitutive collagen gene expression by lipid peroxidation. J Clin Invest 1991; 87: 2230–2235. | PubMed | |
| 34. | SHAH SV. Role of reactive oxygen metabolites in experimental glomerular disease. Kidney Int 1989; 35: 1093–1106. | PubMed | ISI | ChemPort | |
Acknowledgments
The author would like to thank Cindy Reid for her assistance in editing this manuscript.
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