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Saudi Journal of Kidney Diseases and Transplantation
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Year : 1999  |  Volume : 10  |  Issue : 2  |  Page : 137-143
Free Radicals in Kidney Disease and Transplantation


Department of Medicine, University of Mississippi Medical Center, Jackson, MS 39216, USA

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How to cite this article:
Salahudeen AK. Free Radicals in Kidney Disease and Transplantation. Saudi J Kidney Dis Transpl 1999;10:137-43

How to cite this URL:
Salahudeen AK. Free Radicals in Kidney Disease and Transplantation. Saudi J Kidney Dis Transpl [serial online] 1999 [cited 2020 Sep 27];10:137-43. Available from: http://www.sjkdt.org/text.asp?1999/10/2/137/37218
Reactive oxygen species (ROS) age generally considered as toxic molecules, and are often associated with tissue injury. A large body of accruing evidence also suggests that ROS may play important roles in normal biological process, and consistent with this is the fact that healthy normal cells generate ROS. This article will focus on the pernicious aspects of these molecules in models of renal injury, and will begin with an overview of free radical chemistry.

Free radicals are molecular species possessing one or more unpaired electrons in their outer orbits. Hydrogen peroxide, because of the absence of unpaired electron, is referred to as ROS and not as a free radical. Presence of unpaired electron configuration imparts greater reactivity to the molecule. A handful of reactions comprise the essentials of free radical chemistry. [1],[2],[3]

During the normal cellular metabolism, the generation of ATP from ADP during oxidative phosphorylation is coupled with electron transfer through the mitochondrial electron transport chain. In the final stage, oxygen accepts electrons at the terminal proton pump in the respiratory chain, catalyzed by cytochrome oxidase. The complete reduction of oxygen generates water and no free radicals [(e) in the sequence below).

Unlike complete reduction, incomplete reduction of molecular oxygen leads to free radical formation. It is estimated that 1-3% of oxygen consumed by cells are channeled into the generation of reactive oxygen metabolites. [4] A single electron transfer generates the super oxide anion (a). While two-electron transfer yields hydrogen peroxide (b). The dismutation of the superoxide ion, either spontaneously or also yields hydrogen peroxide (c). Hydrogen peroxide as illustrated by its interaction with the superoxide generates the hydroxyl ion in the presence of transition metal ions (d). Interestingly, superoxide has also been demonstrated to interact with the now widely recognized nitrogen based radical nitric oxide (NO), on one hand, modulating the vascular tone and, on the other, gene­rating the potentially toxic peroxynitrite.

Generation of superoxide may occur at several cellular loci. In addition to mito­chondrial electron transport chain, superoxide is also produced by a variety of enzymatic processes such as the NAD(P)H oxidase, xanthine oxidase/dehydrogenase and aldehyde oxidase and by non-enzymatic processes such as the autoxidation of thiols and catecholamines. The plasma membrane is a rich source of reactive oxygen intermediates. For example, the metabolism of arachidonic acid down either the cyclooxygenase or lipoxygenase pathway generates a variety of reactive oxygen metabolites. Other potential sites include the endoplasmic reticulum and nuclear membrane electron transport system. The hydroxyl ion will react with whatever chemical species or bio-molecules exist in its immediate environs such that this ion is extremely short-lived and dissipates itself within five molecular diameters form its site of generation. While lacking the intense reactivity displayed by the hydroxyl ion, hydrogen peroxide freely diffuses across lipid bilayers, and thus may induce oxidant effect that are quite distant from the site of generation. The chemical reactivity of reactive oxygen species extends to lipids, carbohydrates, protein and nucleic acids. Free radicals such as hydroxyl ion are capable of initiating chain reactions in lipid domains that result in lipid peroxidation. Peroxidation of membrane lipids perturb membrane fluidity, permeability, ion and solute transport. Reactive oxygen species also impair enzymic and structural protein molecules through such mechanisms as oxidation of sulfhydryl group and deamination. Additionally, hydrogen peroxide compro­mises mitochondrial ATP synthesis by inhibiting the ATPase-synthase complex. These changes are followed by the elevation in intracellular calcium, disruption of the cytoskeleton, blebbing of the plasma membrane, and finally cell lysis. Studies from our laboratory have demonstrated a similar role for lipid peroxidation in oxidant­induced early cell injury and two recent studies confirm this observation.[5],[6],[7] In the late phase of oxidant-induced cell injury additional mechanism such as the DNA damage and ATP depletion may determine the reversibility of cell injury.

To protect the cells against the potentially toxic effects of ROS, several anti-oxidant mechanisms exist. Superoxyde dismutases catalyze the dismutation of superoxide anion and glutathione peroxidase and catalase remove hydrogen peroxide. A number of non-enzymic defense mechanisms also exist. Vitamin E is the major scavenger of free radicals in lipid radicals. Vitamin E inhibits the initiation and propagation of free radical chain reaction in lipid bilayers leading o peroxidation of lipid. Ascorbic acid is a water soluble antioxidant, which not only scavenge a variety of reactive species but also regenerate the reduced form of Vitamin E. Heightened generation of reactive species may overwhelm these antioxidant defense mechanisms. Since reactive oxygen species cannot be easily measured in tissue, increased lipid peroxidation or the glutathione redox ratio provides evidence of oxidative stress. An under-appreciated endogenous scavenger of hydrogen peroxide is the metabolic inter mediary, pyruvate. We have extended the previous observation of its ability to degrade hydrogen peroxide to water into cell culture and animal experiment. [8] Recent studies also underscore an important antioxidant function for the heme-protein degrading enzyme, heme oxygenase I (HO-­I). For example, a priori induction of HO-I protects cells or organs against oxidative injury in a variety of experimental setting. [9] In the remaining part of this article, the potential role of ROS in certain important and well-studied models of renal injury will be discussed.

Renal Ischemia-reperfusion Injury

This model represents such clinical situations, such as the return of blood flow to a kidney rendered ischemic by cross clamping of the aorta, or the transplantation of a donor kidney previously sustained by an artificial preservation solution. Ischemia per se provokes a variety of biochemical insults including the depletion of ATP and the elevation of intra-cellular calcium. In addition, reintroduction of molecular oxygen during reperfusion into the ischemic tissues, by generating free radicals such as super­oxide ion, contributes significantly to injury that accrues in the post-ischemic period. Such generation of superoxide ion is dependent upon stimulation of xanthine oxidase activity in the ischemic and post­ischemic periods. The proposed pathogenic mechanism is based on two essential steps during ischemia: the conversion of xanthine dehydrogenase (type D xanthine oxidase) to xanthine oxidase (type O xanthine oxidase) and the generation of hypoxanthine from the breakdown of ATP during ischemia. Reoxygenation of ischemic tissue, enriched with hypoxanthine and type O xanthine oxidase activity, thereby enhances the generation of superoxide ion. Support for role of ROS in post-ischemic injury in the kidney was provided by a series of studies by Paller and collaborators.[10] Recent studies, including work from our own laboratory in a syngeneic renal transplant model suggest that free radicals beyond tissue injury may participate in the up­regulation of adhesion molecules, cytokines, MHC Class I and II antigens and inducible nitric oxide synthase. This supports the "response-to-injury" hypothesis in the setting of transplantation, i.e. the up-regulation of these pro-inflammatory and immunogenic molecules may trigger or accelerate the immune rejection. [11] Further studies are required to verify the validity of this line of logic.

Cold Storage-associated Injury

Although University of Wisconsin (UW) solution is the most widely used preser­vation solution, the protection it affords against cold storage injury is incomplete,[12],[13] Specifically, lipid peroxidation and cell injuries continue to occur despite storage of organs in UW solution. Evans et al found that UW solution contains traces of catalytically active iron capable of inducing lipid peroxidation.[12] Our findings and the earlier studies suggest that cellular gene­ration of free radicals might be increase at 4 0 C. F2-isoprostances are a family of newly identified vasoconstrictive prostaglandins. These are unique in that they are generated by the peroxidation of arachidonic acid, not by cycloxygenase, but by free radicals. Consistent with the observation that cold storage may cause free radical-catalyzed lipid peroxidation, in a recent study from our laboratory, the levels of F2-isoprostanes rose markedly and time-dependently in the kidneys preserved in cold UW solution.[14] Whether the increase in renal F2-iso­prostanes following cold storage contributes to post-transplant vasoconstriction remains to be determined.

From the foregoing brief discussion, it is evident that considerable experimental data is available to suggest that administration of antioxidants particularly during cold preser­vation of organs and during reperfusion may mitigate transplant-related tissue injury. It should be noted however that clear clinical evidence for the use of antioxidant is currently lacking, and, hopefully, future studies with more potent and newer antioxidants such as Lazaroid compounds may prove to be efficacious.


   Model of rhabdomyolysis Acute Renal Failure Top


The delivery of heme pigments to the kidney, as occurring after rhabdomyolysis or intravascular hemolysis, is a well­recognized cause of acute renal failure, is a well-recognized cause of acute renal failure. The mechanisms of heme-induced renal injury are tow folds. The oxidative mechanism involves the generation of heme iron-catalyzed free radical production wit consequent lipid peroxidation and tubular injury. The second mechanism involves the widely recognized tubular obstruction from the heme pigments. Recent studies from our laboratory further suggest that these two mechanisms may interact; synergizing the myohemoglobin­induced tubular injury. [15] The commonly used model for this syndrome involves the intramuscular injection of hypertonic glycerol in animals, which induces muscle necrosis with attendant myoglobinemia and myoglobinuria. In this model marked reductions in GFR and renal blood flow rates and prominent histologic damage, occur within hours of the administration of glycerol. Concurrent administration of fluid-mannitol-alkali and antioxidants in this models had the maximum effect in reducing tubular necrosis, cast formation and renal dysfunction. Again, clinical studies are required to verify whether to similar situation in clinical setting.

Toxic Nephropathy Models

ROS have been implicated in the damage arising from a variety of drugs include gentamicin, cyclosporine-A (CSA), cisplatinum, and radiocontrast agents. The potential role of ROS in toxic nephropathy will be discussed using CSA and cisplatin nephro­toxicity as prototypes.

Cyclosporine-A

Although CSA has been popular and has been in use for well over a decade now, the mechanism of its most troublesome side effect of renal toxicity is unclear. Some experimental studies suggest a role for vasoconstrictive prostaglandins, endothelin, angiotensin II or possibly, nitric oxide deficiency. A large body of experimental evidence has also accrued to suggest that excessive production of free radicals might be a potential central mechanism for CSA­nephrotoxicity. CSA had been shown to induce renal microsomal lipid peroxidation in-vitro and reduce antioxidant levels in animal models. In in-vivo experiments from our laboratory, administration of antioxidants such as vitamin E and lazaroid compounds reduced cyclosporine-induced renal functional and structural injury. [16],[17] Conversely, CSA administration in antioxidant defiant rats was accompanied by exacerbation of renal toxicity. Vitamin E administration was also shown to reduce to CSA-induced increase in urinary arachidonic acid metabolites, thromboxane, as well as, free radical-catalyzed vasoconstrictive prostaglandin, F2-iso­prostane. A recent study provides additional evidence for a role for free radical-catalyzed lipid peroxidation in CSA-induced renal vasoconstriction. [18] Blood vessels of hydronephrotic rat kidneys were studied under video microscopy. CSA was applied topically to the kidney in the presence or absence of a lazaroid compound. CSA - induced renal microvascular vasoconstriction and hypoperfusion were significantly reduced by the prior administration of antioxidant lazaroid. Relationship between lipid peroxidation and fibrosis in CSA treated rats is currently being examined. The preliminary findings demonstrate that vitamin E suppresses CSA-induced increase in cyclo-oxygenase II (the induced form) and transforming growth factor-beta (TGF­beta) mRNA expression in the renal tubules. This supports the notion that COX II, through its ability to increase vaso­constrictive prostanoids, and TGF-beta, may contribute to CSA nephrotoxicity. In combination, these studies support the hypothesis that ROS play a role in CSA nephrotoxicity. Again, human studies, for example administering vitamin E in transplant patients receiving CSA, are required to determine whether these observations have relevance in the clinical setting. An additional point is that a free radical mechanism can have an inter­mediate role in controlling other factors such as endothelin, angiotensin II, or possible nitric oxide deficiency. For instance, agiotensin II through NAD(P)H oxidase pathway may increase superoxide, which in turn can reduce the levels of vascular nitric oxide. [19] Recent studies also suggest that free radicals and lipid peroxidation product can stimulate the production of he potent vasoconstrictor, endothelin.


   Cisplatinum Top


Cisplatin is a widely used anticancer drug. One of the proposed mechanisms for its nephrotoxicity is that, in the low intracellular chloride milieu, chloride ions of cisplatin may exchange for cellular SH moieties resulting in glutathione depletion, hydrogen peroxide accumulation and lipid peroxidation. A recent cell culture work from our laboratory has provided some support for this potential mechanism. [20] Furthermore, this study demonstrated that Cisplatin-mediated renal cell injury was also accompanied by a thiol donor N-acetyl cysteine, suppressible prod8ction of F2­isoprostance. Cisplatin causes renal vasoconstriction and isoprostanes have been speculated as mediators of vasoconstriction in this setting. A number of in-vivo studies utilizing the administration of thiol compounds and other forms of antioxidants have verified the role of free radicals, and these include clinical studies which indicate that thiol donors administered to patient receiving cisplatin may mitigate the cisplatin-toxicity including renal damage. Currently a thiol formulation is available for routine clinical use.


   Models of Glomerulonephritis Top


Glomeruli are often infiltrated with leukocytes and macrophages, and a large body of experimental evidence suggests that they, through in part free radical mechanism, may play a role in glomeruli injury. [21] Neutrophil occupy a central role in glome­rular injury in certain models as demon­strated by the marked reduction in the rates of protein excretion and attenuation in endothelial and epithelial injury with neutrophil depletion. Key mechanisms, through which neutrophils inflict glomerular damage, involve the generation of reactive oxygen species particularly hydrogen peroxide. Another pathway for glomerular damage is the neutrophil-dependent hypochlorus/ myeloperoxidase system. [22] Activated neutrophils have been shown to release copious amounts of the highly injurious acid from hydrogen peroxide and halides such as chloride. Platelet activation, which itself may be triggered by the myelo­peroxidase-hydrogen peroxide system, represents an additional source of inflam­matory and mitogenic agents including platelet derived growth factor, platelet co­factor 4, platelet activating factor (PAF) and thromboxane. Platelets in turn enhance the free radical response of activated neutrophils possibly through their stores of adenine nucleotides. Thus by recruiting additional pathways for tissue injury such as platelet activation, as well as generation hypochlorus acid, the myelo-peroxidase­hydrogen peroxide system promotes glomerular injury. Yet another mechanism for hydrogen peroxide-facilitated injury is based on the degradation of glomerular basement mem­brane by metalloproteinases derived from neutrophils. [23] Metalloenzymes such as collagenase and gelatinase reside within neutrophils in a dormant state. Activation is necessary before these enzymes can degrade basement membrane. These investigators noted that catalase but not superoxide dis­mutase inhibited degradation of glomerular basement membrane by activated neutrophils. Metal chelators, inhibitors of myeloperoxidase and scavengers of hypochlorous acid also inhibited degradation of basement membrane by supernatant extracts for activated neutro­phils. These findings suggest that oxidants derived from the myeloperoxidase-hydrogen peroxide-halide system activate the proteolytic capacity of metallo-proteinases towards the glomerular basement membrane.

The aminonucleoside of puromycin administration model of glomerulonephritis mimics the minimal change disease. Puromycin administration impairs glomerular permselectivity in rats such that protein excretory rates are markedly increased.

Foot processes fusion of the glomerular epithelial cells and a paucity of prolix­ferative or infiltrative changes, findings that are similar to human minimal change disease, characterize the histologic lesion. The aminonucleoside of puromycin is metabolized to hypoxanthine and the availability of hypoxanthine would serve as substrate for xanthine oxidase there by generating superoxide. [24] In this model, marked attenuation in rates of protein excretion was observed with allopurinol (xanthine oxidase inhibitor) and superoxide dismutase. Generation of hydrogen peroxide is also implicated in the loss of glomerular permselectivity in this model. In addition to glomerular damage, puromycin also induces an acute tubulo-interstitial nephritis. It is possible that the generation of hydroxyl and other reactive species may also contribute to acute tubulo-interstitial injury.

The administration of an antibody to the tubular epithelial antigen, Fx1A, induces proteinuria in rats and a glomerular lesion characterized by thickening of the capillary loops and the subepithelial immune deposits, thus resembling clinical membranous nephropathy. In this model glomerular leak of macromolecules is dependent on comple­ment but not on neutrophils. Iron chelators and scavengers of hydroxyl ion reduced proteinuria in this disease model. [25] Such beneficial effects were exerted in the absence of alterations in the deposition of antibody an complement in the glomeruli. These data suggest that the in-situ binding of the anti-Fx1 A antibody trigger the generation of hydroxyl ion leading to glomerular injury. The findings in these models indicate that Immune reactants may evoke the generation of reactive oxygen species by intrinsic glomerular constituents. The role of free radicals in biology and medicine continue to evolve and their role in kidney disease and transplantation are no exception. The newer findings that cellular redox states influence signals transduction and that oxidant by activating transcription factors induce several pathologically relevant genes provide basis to explain how free radical-involvement can account for a wide variety of renal disease processes, including diabetic nephropathy. [26]


   Acknowledgments Top


The author's basic research which forms the basis for this article has or had been supported by grants from American Heart Association, Baxter Extramural Grant Program, Kidney Care Inc., and US National Institute of Environmental Health Services. The secretarial assistance of Ms. J. Ginn is appreciated.

 
   References Top

1.Grisham MB, McCord J. Chemistry and cytotoxicity of reactive oxygen metabnloites. In"Physiology of oxygen radical" Am. Physiol Soc, (Ed AE Taylor, S Matalon, P Wrd), Williams and Wilkins Co. Baltimore, MD 1986;pp1-18.  Back to cited text no. 1    
2.Halliwel B, Gutteridge JM, Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem J 1984;219:1-14.  Back to cited text no. 2    
3.Shah SV. Role of reactive oxygen metabolities in experimental glomerular disease. Kid Int 1989;35:1093-106.  Back to cited text no. 3    
4.Chance B, Sies H, Boveris A. Hydroperoxide metabolism in mamma,ian organs. Physiol Rev 1979;59:527-605.  Back to cited text no. 4  [PUBMED]  [FULLTEXT]
5.Salahydden AK. Role of lipid peroxidation in H2O2-induced renal pithelial (LLC-PK1) cell injury. AM J Physiol 1995;268:F30-8.  Back to cited text no. 5    
6.Andreoli SP, Mallett CP. Disassociation of oxidant-induced ATP depletion and DNA damage from early cytotocicity I LLC-PK1 cells. Am J Physiol 1997;272:F729-35.  Back to cited text no. 6  [PUBMED]  [FULLTEXT]
7.Sheridan AM, Fitzpatrick S, Wang C, Wheeler DCm Lieberthal W. Lipid peroxidation contributes to hydrogen peroxide induced cytotoxicity in renal epithelial cells. Kidney Int 1996;49:88-93.  Back to cited text no. 7    
8.Salahudden AK, Clark EC, Nath KA. Hydrogen peroxide-induced renal injury. A protective role of pyruvate in vitro and in vivo. J Clin Invest1991;88:1886-93.  Back to cited text no. 8    
9.Nath KA, Balla G, Vercellotti GM, et al. Induction of heme oxygenase is a rapid, protective response in rhabdomyolysis in the rat. J Clin Invest 1992;90:267-70.  Back to cited text no. 9  [PUBMED]  [FULLTEXT]
10.Paller MS, Hoidal JR, Ferris TF. Oxygen free radicals in ischemic acute renal failure in the rat. J Clin Invest 1984;74:1156-64.  Back to cited text no. 10  [PUBMED]  [FULLTEXT]
11.Salahudeen A, Wang C, McDaniel O, Lagoo Denadyalan S, Bigler S, Bigler S, Barber H. Anti-oxidant lazaroid U-74006F improves renal function and reduces the expression of cytokines, inducible nitric oxide synthase, and MHC antigens in a syngeneic renal transplant model. Partial support for the response-to-injury hypo­theses. Transplantation 1996;62:1628-33.  Back to cited text no. 11    
12.Evans PJ, Tredger JM, Dunne JB, Halliwell B, Catalytic metal ions and the loss of reduced glutathione from University of Wisconsin preservation solution. Transplantation 1996;62:1628-33.  Back to cited text no. 12    
13.Peters SM, Rauen U, Tijsen MJ, et al. Cold preservation of isolated rabbit proximal tubules induced radical-mediated-cell injury Transplantation 1998;65:625-32.  Back to cited text no. 13    
14.Salahudeen AK, Navaz M, Poovala V, et al. cold induces time-dependent F2-isoprostane formation in renal tubular cells and rat kidneys stored in university of Wisconsin solution: potential implication for immediate post-transplant renal vasoconstriction. Kidney Int (In press).  Back to cited text no. 14    
15.Salahudeen AK, Wang C, Bigler SA, Dai Z, Tachikawa H. Synergistic renal protection by combining alkaline-diuresis with lipid peroxidation inhibitors in rhabdomyolysis possible interaction between oxidant and non-oxidant mechanisms. Nephrol Dial Transplant 1996;11:635-42.  Back to cited text no. 15  [PUBMED]  [FULLTEXT]
16.Wang C, Salahudeen AK. Lipid peroxidation accompanies cyclosporine nephrotoxicity: effects of vitamin E. Kidney Int 1995;47:927-34.  Back to cited text no. 16  [PUBMED]  
17.Wang C, Salahudeen AK. Cyclosporine­nephrotoxicity: attenuation by an antioxidant -inhibitor of lipid peroxidation in-vitro and in-vivo. Transplantation 1994;58:940-6.  Back to cited text no. 17  [PUBMED]  
18.Krysztopik RJ, Bentley FR, Spain DA, Wilson MA, Garrison RN. Lazaroids prevent acute cyclosporine-induced renal vasoconstriction. Transplantation 1997;63:1215-20.  Back to cited text no. 18  [PUBMED]  [FULLTEXT]
19.Gryglewski RJ, Palmer RM, Moncada S. Superoxide anion is involved in the break­down of endothelium derived relaxing factor Nature 1986;320:454-6.  Back to cited text no. 19    
20.Salahudeen A, Poovala V, Parry W, et al. Cisplatin induces N-accetyl cysteine suppressible F2-isoprostane production and injury in renal tubular epithelial cells. J Am Soc Nephrol 1998;9:1448-55.  Back to cited text no. 20  [PUBMED]  
21.Weiss SJ. Tissue destruction by neutrophils. N Engl J Med 1989;320:365-76.  Back to cited text no. 21  [PUBMED]  
22.Johnson RJ, Couser WG, chi Ey, Adler S, Klebanoff SJ. New mechanism for glomerular injury: my eloperoxidase­hydrogen peroxide-halide system. J Clin Invest 1987;79:1379-87.  Back to cited text no. 22  [PUBMED]  [FULLTEXT]
23.Shah SV, Baricos Wh, Basci A. Degradation of human glomerular basement membrane by stimulated neutrophils. Acivation of a metalloproteinase (s) by reactive oxygen metabokites. J clin Invest 1987;79:25-31.  Back to cited text no. 23    
24.Diamond JR, bonvertre JV, Karnovsky MJ. A role for oxygen free radicals in aminonucleoside nephrosis. Kidney Int 1986;29:478-83.  Back to cited text no. 24    
25.Shah SV. Evidence suggesting a role for hydroxyl radical in passive Heymann nephritis in rats. Am J Physiol 1988;254:F337-44.  Back to cited text no. 25  [PUBMED]  [FULLTEXT]
26.Salahudeen AK, Kanji V, Rechelhoff JK, Schmidt AM. Pathogenesis of diabetic nephropathy: a radical approach. Nephrol Dial Transplant 1997;12:664-4.  Back to cited text no. 26    

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Correspondence Address:
Abdulla K Salahudeen
Associated Professor in Medicine, Physiology and Biophysics, University of Mississippi Medical Center, 2500 North State Street, Jackson, Ms 39216-4504
USA
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PMID: 18212421

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