Oxidative Stress and Reactive Nitrogen Species Generation during Renal Ischemia

Lisa M. Walker*, J. Lyndal York{dagger}, Syed Z. Imam{ddagger}, Syed F. Ali*,{dagger},{ddagger}, Kenneth L. Muldrew* and Philip R. Mayeux*,1

* Departments of Pharmacology and Toxicology, and {dagger} Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205; and {ddagger} Neurochemistry Laboratory, Division of Neurotoxicology, National Center for Toxicological Research/FDA, Jefferson, Arkansas 72079

Received March 23, 2001; accepted June 4, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previous evidence suggests that both oxygen radicals and nitric oxide (NO) are important mediators of injury during renal ischemia-reperfusion (I-R) injury. However, the generation of reactive nitrogen species (RNS) has not been evaluated in this model at early time points. The purpose of these studies was to examine the development of oxidant stress and the formation of RNS during I-R injury. Male Sprague-Dawley rats were anesthetized and subjected to 40 min of bilateral renal ischemia followed by 0, 3, or 6 h of reperfusion. Control animals received a sham operation. Plasma urea nitrogen and creatinine levels were monitored as markers of renal injury. Glutathione (GSH) oxidation and 4-hydroxynonenal (4-HNE)-protein adducts were used as markers of oxidant stress. 3-Nitrotyrosine (3-NT) was used as a biomarker of RNS formation. Significant increases in plasma creatinine concentrations and urea nitrogen levels were found following both 3 and 6 h of reperfusion. Increases in GSH oxidation, 4-HNE-protein adduct levels, and 3-NT levels were observed following 40 min of ischemia with no reperfusion. Since these results suggested RNS generation during the 40 min of ischemia, a time course of RNS generation following 0, 5, 10, 20, and 40 min of ischemia was evaluated. Significant increases in 3-NT generation was detected as early as 10 min of ischemia and rose to values nearly 10-fold higher than Control at 40 min of ischemia. No additional increase was observed following reperfusion. The data clearly demonstrate that oxidative stress and RNS generation occur in the kidney during ischemia.

Key Words: 3-nitrotyrosine; peroxynitrite; superoxide; 4-hydroxynonenal; renal ischemia; acute renal failure; glutathione.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Renal ischemia-reperfusion injury (I-R) may occur as a result of transient severe systemic hypotension or aortic occlusion during surgery. Molecular mechanisms underlying renal I-R injury are poorly understood. It is critical to understand how events initiated during the ischemic period lead to injury of renal proximal tubule epithelial cells and result in cortical necrosis and renal failure (Weight et al., 1996Go). The ability of superoxide dismutase (SOD), allopurinol, deferoxamine, and other antioxidants to attenuate renal I-R injury suggests that reactive oxygen species contribute to the development of renal injury (De Vecchi et al., 1998Go; Paller and Hedlund, 1988Go; Paller et al., 1984Go). However, these compounds also scavenge or inhibit the formation of peroxynitrite (ONOO), a highly reactive species derived from nitric oxide (NO) and superoxide (Arteel et al., 1999Go; Crow and Beckman, 1995Go; Denicola et al., 1995Go; Whiteman and Halliwell, 1997Go). Indeed, several lines of evidence now implicate reactive nitrogen species (RNS) as contributors to renal I-R injury (Chiao et al., 1997Go; Edelstein et al., 1997Go; Noiri et al., 1996Go; Shoskes et al., 1997Go).

There are a number of RNS derived from NO (Patel et al., 1999Go). Of these, peroxynitrite (ONOO) is the best characterized and appears have the most biological activity (Crow and Beckman, 1996Go). ONOO- is formed by the biradical reaction of NO and O2-. The reaction is extremely fast and will occur at a near diffusion-limited rate (Huie and Padmaja, 1993Go). A number of oxidation and nitration products are produced from the reaction of ONOO- with cellular macromolecules. One such product, 3-nitrotyrosine (3-NT), can be used as a marker of ONOO formation in vivo (Crow and Beckman, 1995Go).

Although NO, generated from L-arginine by nitric oxide synthase (NOS), participates in numerous physiological processes in kidney (Kone and Baylis, 1997Go), NO also appears to contribute to renal I-R injury (Edelstein et al., 1997Go; Noiri et al., 1996Go; Shoskes et al., 1997Go). The purpose of the present study was to examine the development of oxidative stress and the generation of RNS as early events during renal I-R injury.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Induction of renal I-R injury.
All experiments and procedures were conducted with the approval of the University of Arkansas for Medical Sciences Institutional Animal Care and Use Committee, and all animals were housed in accordance with the Guide for the Care and Use of Laboratory Animals. Renal I-R surgery was performed on male Sprague-Dawley rats (225–250 g) as described previously (Walker et al., 2000Go). Rats were randomly assigned one of four groups. The Control group received a sham operation. The I-R group received 40 min of bilateral renal ischemia. The 3-h I-R and 6-h I-R groups received 40 min of renal ischemia followed by 3 h or 6 h of reperfusion, respectively. Briefly, rats were placed on a warming pad and anesthetized with pentobarbital sodium (50 mg/kg). Using aseptic technique, bilateral flank incisions were made to expose the kidneys, and the renal pedicles were isolated and occluded for 40 min with microvascular clamps (except Control group). Reperfusion was for 0, 3, or 6 h. At the end of the ischemic or reperfusion periods, rats were reanesthetized if necessary, blood was drawn, and renal tissue was harvested and frozen in liquid nitrogen. In a separate set of experiments, rats were subjected to 5, 10, 20, or 40 min of ischemia without reperfusion.

Quantitation of GSH and GSSG.
Tissue samples, prepared from whole kidneys snap-frozen in liquid nitrogen, were prepared and analyzed by HPLC using the procedures of Richie and Lang, (Richie and Lang, 1987Go) with slight modification, in that the buffer concentration was lowered to 0.02 M and eluants were measured at 214 nm. GSH and GSSG eluted as clearly resolved peaks at 5.25 and 16.75 min, respectively, and showed a linear relationship between peak area and amount of sample applied. The column needed to be cleaned with acetonitrile and re-equilibrated after every dozen samples. Total amounts of GSH equivalents were calculated from GSH and GSSG levels (total equivalents = GSH + [2 x GSSG]). The percentage of oxidized equivalents was determined from GSSG levels per total equivalents (oxidized equivalents [%] = [(2 x GSSG)/total equivalents] x 100).

Detection of 4-HNE-protein adducts by Western blot analysis.
Homogenates of whole kidney were subjected to Western blot analysis using a rabbit anti-4-HNE antiserum purchased from Calbiochem (San Diego, CA), as described previously (Zhang et al., 2000aGo). Briefly, kidneys were homogenized in homogenization buffer (125 mM sucrose, 10 mM HEPES, 1 mM EDTA, 100 µM PMSF, 2 µM leupeptin, and 1.5 µM pepstatin, pH 7.4) using a Dounce glass homogenizer. Protein concentrations were determined using Coomassie Plus® protein assay reagent. Samples (100 µg protein per lane) were separated by SDS–PAGE on 4–20% polyacrylamide gels and were transferred to polyvinylidene difluoride (PVDF) membranes. Nonspecific protein binding was blocked by incubation of the membranes with blocking buffer (10 mM Tris, 100 mM NaCl, 0.1% Tween 20, 5% nonfat milk, pH 7.4) overnight at 4°C. Rabbit anti-4-HNE-modified antibody (1:500; Calbiochem, San Diego, CA) was incubated with the membrane for 3 h at room temperature in blocking buffer. The membranes were washed and subsequently incubated with donkey anti-rabbit IgG peroxidase conjugate (1:3000) for 60 min at room temperature. Membranes were then washed and developed using the Enhanced Chemiluminescent Substrate (Amersham Pharmacia Biotech, Inc., Piscataway, NJ) according to the manufacturer's directions. Between each step, membranes were washed four times (10 min each) with washing buffer (10 mM Tris, 100 mM NaCl, 0.1% Tween 20, pH 7.4).

Detection of 3-nitrotyrosine (3-NT).
3-NT and tyrosine content in whole kidney tissue snap-frozen in liquid nitrogen were determined by CoulArray-HPLC (ESA, Cambridge, MA) using electrochemical detection as described previously (Imam and Ali, 2000Go), with slight modifications (Walker et al., 2000Go). After treatment of kidney homogenates with pronase (4 U per mg of homogenate protein for 18 h at 50°C), undigested protein was precipitated with 10% trichloroacetic acid. 3-NT and tyrosine were then quantified and the data expressed as moles of 3-NT per 100 moles of tyrosine.

Statistical analysis.
Data are expressed as mean ± SEM. Statistical differences were detected by analysis of variance (ANOVA). A one-way ANOVA was performed followed by a Newman-Keuls post hoc test. p < 0.05 was considered significantly different for all tests performed.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To confirm that our model of 40 min of bilateral renal ischemia produced renal failure, plasma creatinine and urea nitrogen levels were used to assess renal function (Fig. 1Go). Plasma creatinine concentrations in the 3-h I-R and 6-h I-R groups were significantly different from Control and each other (p < 0.05). Likewise, plasma urea nitrogen concentrations in the 3-h I-R, and 6-h I-R groups were significantly different from Control and each other (p < 0.05). The progressive decline in renal function was also accompanied by a progressive increase in morphological changes assessed by light microscopy (data not shown).



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FIG. 1. Effect of ischemia on renal function. Animals were subjected to 40 min of bilateral ischemia followed by reperfusion for the indicated time. Data are mean ± SE; n = four animals per group. (A) Plasma creatinine levels were increased at 3 h and again at 6h (*p < 0.05 compared to Control and 6-h I-R groups; **p < 0.05 compared to Control and 3-h I-R groups). (B) Plasma urea nitrogen levels were increased at 3 h and again at 6 h (*p < 0.05 compared to Control and 6-h I-R groups; **p < 0.05 compared to Control and 3-h I-R groups).

 
Oxidant stress in renal tissue was evaluated by measuring renal GSH and GSSG levels in tissues using HPLC. Lower GSH levels were detected in both the 40-min I and 3-h I-R groups compared to the Control group (Fig. 2AGo). Levels of GSH in Control, 40-min I, and 3-h I-R groups were 1.60 ± 0.2, 0.80 ± 0.08, and 1.00 ± 0.06 nmol GSH/mg wet weight, respectively. In the 6-h I-R group, GSH levels (1.78 ± 0.30 nmol GSH/mg wet weight) recovered to levels similar to the Control group. GSSG levels also were altered during renal I-R injury (Fig. 2BGo). Ischemia caused a significant increase in GSSG levels (0.14 ± 0.03 nmol GSSG/mg wet weight) compared to the Control group (0.03 ± 0.01 nmol GSSG/mg wet weight). GSSG levels in the 3-h I-R and 6-h I-R groups were 0.02 ± 0.01 and 0.08 ± 0.01 nmol GSSG/mg wet weight, respectively. When expressed as a percentage, the percent oxidized GSH equivalents was elevated only in the 40-min I group (Fig. 2CGo). Total amounts of GSH equivalents (Fig. 2DGo), calculated from the GSH and GSSG values, were 1.68 ± 0.20, 1.07 ± 0.12, 1.05 ± 0.07, and 1.95 ± 0.24 nmol total GSH equivalents/mg wet weight for the Control, 40-min I, 3-h I-R, and 6-h I-R groups, respectively. Although a decrease in total GSH equivalents was observed in both the 40-min I and 3-h I-R groups when compared to the 6-h I-R group, levels were not significantly different from the Control group.



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FIG. 2. Reduced and oxidized levels of GSH in the kidney during I-R injury. Data are presented as mean ± SE; n = four animals per group. (A) GSH levels (*p < 0.05 compared to Control group). (B) GSSG levels (*p < 0.05 compared all other groups). (C) Percent oxidized GSH equivalents (*p < 0.05 compared to all other groups). (Panel D) Total GSH equivalents (*p < 0.05 compared to 6-h I-R group).

 
Generation of 4-HNE was used as a second marker of oxidant stress. Immunoblot analysis was used to monitor 4-HNE-protein adducts in the kidney (Fig. 3Go). Several immunoreactive bands were observed in all groups, but the intensity of staining was greatest in animals subjected to ischemia alone. The immunoreactivity of these bands was decreased in both the 3-h I-R and 6-h I-R groups. Protein staining of the immunoblot confirmed equal loading of protein in all lanes.



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FIG. 3. 4-HNE-protein adducts during renal I-R injury. Kidney proteins (100 µg) were separated by SDS-PAGE, transferred to a membrane, and probed with antiserum directed against 4-HNE-modified proteins. Each lane represents a sample from a different animal. Equal loading of protein was confirmed by staining the membrane for total protein. Treatments are listed below each lane.

 
The appearance of 3-NT containing protein was the third biomarker of oxidative stress monitored and indicates the development of RNS. The levels of 3-NT were significantly increased as early as 10 min of ischemia and increased again at 40 min to levels approximately 10-fold higher than Control (Fig. 4AGo). In a separate set of experiments, 3-NT levels were determined following reperfusion (Fig. 4BGo). There was no change in the levels of 3-NT detected up to 6 h of reperfusion compared to 40 min of ischemia alone.



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FIG. 4. Analysis of 3-NT. 3-NT was determined from total kidney protein following enzymatic hydrolysis. Data are expressed as mean ± SE; n = four to seven animals per group. (A) Time course of ischemia (*p < 0.05 compared to Control, 5 min, and 40 min; **p < 0.05 compared to all other groups). (B) Time course of reperfusion (*p < 0.05 compared to Control group).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Despite the fact that numerous antioxidants afford some degree of protection against renal I-R injury (De Vecchi et al., 1998Go; Paller and Hedlund, 1988Go; Paller et al., 1984Go), the role of oxidants in the development of acute renal failure due to I-R remains controversial because direct evidence of hydroxyl radial formation is lacking (Zager et al., 1992Go). Collectively, our data show that in the kidney, ischemia alone causes both oxidant stress and RNS formation.

GSH depletion and altered GSH/GSSG ratios can signal the development of oxidant-mediated tissue injury (De Vecchi et al., 1998Go; White et al., 1986Go) and have been observed in several models of oxidant-mediated acute renal failure (Abul-Ezz et al., 1991Go; Scaduto et al., 1988Go). GSH peroxidase can scavenge a number of oxidants and RNS such as ONOO (Karoui et al., 1996Go; Patel et al., 1999Go; Stamler et al., 1992Go) directly or through the actions of GSH. The increases in GSSG content and percent oxidized GSH equivalents show that GSH oxidation occurs during ischemia and indicates the development of oxidative stress prior to reperfusion. This is in contrast to what is reported in the isolated rat liver during ischemia. In a study using the isolated perfused rat liver, GSH and GSSG levels did not change during a 30- or 120-min period of ischemia indicating the absence of oxidant stress in this model (Jaeschke et al., 1988Go).

Lipid peroxidation is an autocatalytic pathway that causes oxidative damage to cell membranes and results in the release of reactive lipid aldehydes. These cytotoxic metabolites such as 4-HNE (Esterbauer et al., 1991Go; Uchida and Stadtman, 1992Go) diffuse from the site of production and react with cellular macromolecules. The appearance of 4-HNE-protein adducts during ischemia is additional evidence that oxidative stress occurs during ischemia. However, we cannot rule out the possibility that the increase in oxidant stress products during ischemia may be, at least in part, a result of accumulation due to reduced clearance.

Lipid peroxidation is frequently used as an indicator of oxidative damage in the kidney (Paller et al., 1984Go; Ramsammy et al., 1986Go; Zhang et al., 2000bGo) and provides additional support for oxidant-mediated injury in rat I-R model. 4-HNE-protein adducts have also been detected in the kidney following treatment with iron (Zainal et al., 1999Go) and during ischemia (Eschwege et al., 1999Go) using immunohistochemistry. Eschwege et al. (Eschwege et al., 1999Go) found that 4-HNE-protein adduct levels were increased in the rat kidney cortex following 30, 40, and 60 min of ischemia. However, it was not determined whether 4-HNE-protein adducts were detectable after reperfusion (Eschwege et al., 1999Go). In the isolated perfused hearts (Eaton et al., 1999Go), 4-HNE-protein adducts were found after 10 min of ischemia. Moreover, adduct accumulation increased steadily through 30 min of ischemia, but no further increases were found beyond this time. When rat hearts were subjected to 20 min of ischemia and 5–30 min of reperfusion, no additional increases in 4-HNE-protein adducts were detected following reperfusion. Thus, in both heart and kidney, oxidant generation occurs during periods of ischemia. Furthermore, the generation of 4-HNE is also likely to have contributed to the observed decreases in GSH levels. GSH scavenges lipid peroxidation products such as 4-HNE (Grune et al., 1997Go; Hu and Tappel, 1992Go) as well as RNS (Briviba et al., 1998Go; Patel et al., 1999Go; Sies et al., 1997Go).

We recently showed immunohistochemical detection of 3-NT-protein adducts 6 h following reperfusion (Walker et al., 2000Go), and others have shown the appearance of immunoreactive 3-NT containing protein 24 h following reperfusion (Chiao et al., 1997Go). In the present study a highly specific and quantifiable method was used to detect 3-NT. These data provide the first evidence that the generation of ONOO precedes the development of renal injury and failure. Most striking is how early ONOO is generated during ischemia. It has been assumed that inducible nitric oxide synthase (iNOS) is responsible for NO-mediated injury in this model (Noiri et al., 1996Go). It is doubtful that the rapid appearance of 3-NT within 10 min of ischemia is due to such a rapid induction of iNOS. The source of NO is most likely constitutively expressed NOS found in the renal tubule (Mayeux et al., 1995Go) or a latent form of iNOS. Importantly, these findings offer one explanation for the lack of complete protection against renal I-R injury by the iNOS inhibitor L-iminoethyl lysine (Walker et al., 2000Go).

ONOO is a potent and versatile oxidant that can react with cellular lipids, proteins, and DNA (Pryor and Squadrito, 1995Go). Although the contribution of early generation of RNS to the development of renal failure has yet to be determined, it is tempting to speculate that the generation of RNS, rather than hydroxyl radical, is most responsible for the injury associated with renal I-R. By 3 h of reperfusion, oxidant stress appears to begin to subside, and by 6 h GSH levels have returned to Control levels. Yet, 3-NT levels are still elevated at 6 h. The turnover rate of 3-NT-modified proteins in the kidney is not known. Therefore, the presence of 3-NT at 6 h could reflect the persistence of 3-NT-protein adducts or a steady state production of ONOO. Nevertheless, 3-NT-modified proteins can affect protein function (MacMillan-Crow and Thompson, 1999Go; Padmaja et al., 1998Go; Roberts et al., 1998Go) and should be considered as potential contributors to renal cell injury.

In summary, the present studies show that both oxidative stress and RNS generation occur during renal ischemia. These conclusions are supported by evidence of GSH oxidation, 4-HNE-protein adduct formation, and the appearance of 3-NT, a biomarker of ONOO generation. Most significantly, 3-NT formation occurs very rapidly during the ischemic period. This suggests that a constitutive NOS isoform is responsible for rapid generation of NO leading to ONOO generation. Furthermore, the ability of scavengers of ONOO or inhibitors of its synthesis (De Vecchi et al., 1998Go; Paller and Hedlund, 1988Go; Paller et al., 1984Go; Walker et al., 2000Go) to attenuate renal I-R injury provides compelling evidence to support an important role of RNS in this cause of acute renal failure.


    ACKNOWLEDGMENTS
 
The authors thank Mr. John Skinner for his technical assistance. This work was supported by an American Heart Association Heartland Affiliate Predoctoral Fellowship to L.M.W. and by National Institutes of Health grant DK44716 to P.R.M.


    NOTES
 
1 To whom correspondence should be addressed at University of Arkansas for Medical Sciences, Department of Pharmacology and Toxicology, Mail Slot 611, 4301 W. Markham St., Little Rock, AR 72205. Fax: (501) 686-5521. E-mail: mayeuxphilipr{at}uams.edu. Back


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