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ABB Archives of Biochemistry and Biophysics 412 (2003) 27–33 www.elsevier.com/locate/yabbi
Mitochondrial targets of oxidative stress during renal ischemia/reperfusionq Danielle L. Cruthirds,a Lea Novak,b Kabir M. Akhi,b Paul W. Sanders,c John A. Thompson,b and Lee Ann MacMillan-Crowa,b,* a
c
Department of Pharmacology, UAB School of Medicine, Birmingham, AL 35294, USA b Department of Surgery, UAB School of Medicine, Birmingham, AL 35294, USA Department of Medicine and Department of Physiology and Biophysics, UAB School of Medicine, Birmingham, AL 35294, USA Received 12 December 2002, and in revised form 8 January 2003
Abstract Endogenous tyrosine nitration and inactivation of manganese superoxide dismutase (MnSOD) has previously been shown to occur in both human and rat chronic renal allograft rejection. To elucidate the time course of MnSOD inactivation and mitochondrial dysfunction at earlier times during renal transplantation, we developed a rodent model of renal ischemia/reperfusion (I/R). Renal function was significantly impaired at 16 h reperfusion following 30 min of warm ischemia. Tyrosine nitration of specific mitochondrial proteins, MnSOD and cytochrome c, occurred at the earliest time point examined, an event that preceded significant renal injury. Interestingly, a small percentage of both mitochondrial proteins were also located in the cytosol. This leakage and decreased adenosine 50 -triphosphate levels indicate loss of mitochondrial membrane integrity during renal I/R. Inactivation of MnSOD occurred rapidly in this model of renal I/R, suggesting that loss of MnSOD activity leads to further renal injury and nitration of other mitochondrial targets. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Kidney; Ischemia/reperfusion; Tyrosine nitration; MnSOD; Mitochondria; Transplantation; Cytochrome c; ATP
Kidneys are one of the most successfully transplanted organs today, with excellent (>90%) 1-year organ survival rates. This success can be attributed to the advent of tissue typing protocols and improved immunosuppressive agents. Despite these early successes, many grafts succumb to chronic renal allograft rejection (>1 year posttransplant). The precise cause of chronic rejection of renal allografts remains unclear, but appears to be related to several issues including organ preservation techniques and ischemia/reperfusion (I/R)1 injury q
This work was supported in part by grants from NIH (RO1 DK59872-02, RO1 DK46199), US Department of Transportation, and a Scientist Development Grant from the American Heart Association (L.A.M.C., 9930240N). * Corresponding author. Fax: 1-205-975-7549. E-mail address: [email protected] (L.A. MacMillan-Crow). 1 Abbreviations used: I/R, ischemia/reperfusion; MnSOD, manganese superoxide dismutase; PMSF, phenylmethylsulfonyl fluoride; PAS, periodic acid–Schiff.
prior to and immediately after surgical implantation. The critical role of cold preservation and ischemia to chronic renal rejection is evidenced by the fact that >85% of grafts from living (un)related donor kidneys survive 5 years compared to 50% survival for cadaveric donor kidneys [3–5]. Living (un)related donor kidneys are generally excluded from preservation or cold ischemia in contrast to cadaveric organs, which have undergone both an extended period of cold ischemia and a period of preservation prior to transplantation. Given these statistics, increased interest within the transplantation world has been to investigate the role that I/R plays in transplanted graft function and longterm outcome. It has been demonstrated that reactive oxygen species are generated during the process of I/R [6–14]. Walker et al. [15] nicely demonstrated that in the kidney, ischemia alone causes both oxidant stress and formation of reactive nitrogen species. During this process, there is an
0003-9861/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0003-9861(03)00039-0
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increase in both nitric oxide (NO) and superoxide (O 2 ). These two molecules can react to form the powerful nitrating agent peroxynitrite [16]. Peroxynitrite has been shown to be a potent oxidizing and nitrating agent that leads to a host of injurious events, including lipid peroxidation, depletion of cellular antioxidant defenses, inactivation of enzymes, and nitration of tyrosine residues in proteins that may adversely affect their function and signal transduction processes [17–22]. Walker et al. [15,23] have also shown that nitrotyrosine, a marker of peroxynitrite formation, is present following as little as 10 min of ischemia. Our laboratory has previously demonstrated that during both human and rat chronic renal allograft rejection there is an increase in oxidative stress as evidenced by significant increases in tyrosine nitration and a loss of antioxidant activity [1,2]. In these studies, we demonstrated that manganese superoxide dismutase (MnSOD) was endogenously tyrosine nitrated and inactivated [1,2]. MnSOD is the major antioxidant in the mitochondria. This enzyme catalyzes the dismutation of O 2 generated from misfires in the electron transport chain. Earlier studies have shown that MnSOD is essential for life. Homozygous MnSOD knockout mice survive for only 5–21 days after birth [24–26]. These animals exhibit clinical pathologies including myocardial injury, neurodegeneration, lipid peroxidation, fatty liver, anemia, and mitochondrial damage [24–26]. It has also been shown that MnSOD heterozygous mice (containing only 50% of wild-type MnSOD function) are more susceptible to damage [26,27]. There have been numerous reports that overexpression of MnSOD protects tissues/ organs from I/R-related damage [10,11,28,29]; thus a loss of MnSOD activity during I/R likely contributes to tissue injury. Here we show that MnSOD inactivation occurs following only 30 min of renal ischemia. Mitochondria appear to be especially sensitive to the I/R insult, as has been previously shown [30–32]. More specifically, we show that MnSOD and cytochrome c are specific mitochondrial targets of this oxidative damage.
Materials and methods Rat I/R model Animals were treated according to strict UAB IACUC guidelines. Male inbred Fisher 344 rats weighing 250– 300 g were used in this study. Animals were maintained on a formulated diet (Dyets, Inc., Bethlehem, PA, USA) containing 1% (w/w) NaCl for 2 weeks before initiation of the study. The model is one of bilateral ischemia in which a right nephrectomy is performed followed by immediate occlusion of the left renal artery and vein for 30 min. The
left kidney is then allowed to reperfuse for a period of up to 16 h. At specific time points, the animals were anesthetized with ketamine/xylazine, the left kidney was removed, and blood was collected via intracardiac injection. Animals that underwent identical surgery but without the I/R episode served as sham animals. Serum analysis Blood was collected at time of sacrifice via intracardiac injection. Serum was made from whole blood using Super Serum according to manufacturer directions (Allegiance Healthcare, McGaw Park, IL, USA). Serum creatinine levels were determined using a Roche Serum Analyzer. Immunohistology Renal tissue was formalin-fixed, paraffin-embedded, and processed as described [33]. Nitrotyrosine staining was performed using the polyclonal anti-nitrotyrosine antibody (1:500) as described [2]. TUNEL staining was performed using the DeadEnd colorimetric TUNEL system kit (Promega, Madison, WI, USA). Renal tissue injury was assessed in tissue sections stained using the periodic acid–Schiff (PAS) reaction. Renal extract preparation Renal extracts were made from frozen tissue by homogenizing (0.1 g/ml) using a Polytron homogenizer in buffer containing 50 mM potassium phosphate, pH 7.4, and 1 mM PMSF. Solubilized extracts were sonicated and centrifuged at 10,000 rpm (5 min, 4 °C) to remove tissue debris. Protein concentrations were determined by Bradford Assay (Pierce, Rockford, IL, USA). Immunoprecipitation and western blot analysis Renal extracts (500 lg) were incubated (4 °C, 2 h) with 10 ll of the monoclonal anti-nitrotyrosine agarose conjugate (Upstate Biotechnology, Lake Placid, NY, USA). MnSOD Western analysis was performed using the polyclonal anti-MnSOD antibody (Upstate Biotechnology; 1:1000). Cytochrome c Western analysis was performed using the monoclonal anti-cytochrome c antibody (Pharmingen, San Diego, CA, USA; 1:500). Probed membranes were washed three times and immunoreactive proteins were detected using horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence. MnSOD activity assays Enzymatic activity of MnSOD was determined in renal extracts by the cytochrome c reduction method
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using 1 mM KCN as described by McCord and Fridovitch [34]. Mitochondria/cytosol fractionation Separation of mitochondria and cytosol from each kidney was carried out by centrifugation in mitochondrial lysis buffer (250 mM sucrose, 20 mM Hepes, pH 7.5, 10 mM KCl, 1.5 mM MgCl2 , 1 mM EDTA, 1 mM PMSF, 1 mM dithiothreitol) as follows. Kidneys were homogenized in a glass homogenizer in a small volume of lysis buffer. Samples were centrifuged at 750g for 10 min. The supernatant was centrifuged at 10,000g for 10 min. The resulting mitochondrial pellet was resuspended in 300 ll of lysis buffer. The supernatant was further centrifuged at 100,000g for 25 min to obtain the pure cytosolic fraction. Proper mitochondrial fractionation was assessed via Western blot analysis of the mitochondrial encoded protein, cytochrome c oxidase subunit I (1:300; Molecular Probes), which should be present only in the mitochondrial fractions. ATP measurements Total ATP measurements were made using an ATP bioluminescent assay kit (Sigma–Aldrich, St. Louis, MO, USA). Measurements were made on a TD 20/20 luminometer (Turner Designs, Sunnyvale, CA, USA). Quantification of ATP was made by creating an ATP standard curve.
Results and discussion Our laboratory has previously utilized a rodent model of chronic renal allograft rejection and demonstrated that the mitochondrial antioxidant protein, MnSOD, was tyrosine nitrated and inactivated at the earliest time point measured (2 weeks posttransplant) [1]. To further elucidate the time course of MnSOD inactivation and mitochondrial dysfunction at earlier times during renal transplantation we developed a rodent model of I/R. In this model of I/R, where only the left kidney was reperfused, significant renal dysfunction was observed at 16 h of reperfusion, as evidenced by increased serum creatinine levels (Fig. 1). Although there was noted elevation in serum creatinine by 3 h, it was not significant due to the already elevated levels in the corresponding shams. We attribute this basal increase in serum creatinine to trauma associated with completion of the right nephrectomy. Increased oxidant production, as measured by nitrotyrosine immunostaining, appeared as early as 30 min of ischemia of the sole left kidney (data not shown). As early as 3 h of reperfusion, nitrotyrosine stain was present in proximal and distal tubules and at the surface of glomerular tufts (Fig. 2B). Nitration ap-
Fig. 1. Serum creatinine measurements of samples from rats undergoing renal I/R. *Significantly different from corresponding shamtreated animals (p < 0:05).
peared to be contained within the cytosol of tubular cells. Interestingly, by 16 h of reperfusion, there was an apparent increase in tyrosine nitration appearing at the tubular lumen (Fig. 2C). Sham-operated animals (16 h) presented with very low levels of nitrotyrosine immunostaining (Fig. 2A). Preincubation (30 min, 25 °C) of the polyclonal anti-nitrotyrosine antibody with 10 mM 3-nitrotyrosine completely blocked immunostaining (data not shown). Others have shown that I/R injury leads to tubular dilatation and loss of brush border integrity [23,35,36]. To examine tubular histopathologic changes associated with loss of membrane integrity due to ischemic injury, PAS histochemical staining was performed (Fig. 2). In the sham animals, the tubules showed normal thickness of the brush border (Fig. 2D). By 3 h of reperfusion, several tubules were dilated and the brush border appeared thinner, indicating a loss of membrane integrity (Fig. 2E). Consistent with progressive damage, there was much more tubular dilation and thinning of tubular brush borders by the 16-h time point (Fig. 2F, arrow). Another consequence of increased oxidative stress is the induction of apoptosis; thus, we used TUNEL staining as a marker of apoptotic cell death during renal I/R. In this model of I/R, there was a small degree of TUNEL staining apparent in the cortex and medulla by 16 h of reperfusion (while not present at 3 h) (Figs. 2G and H). The staining was mainly localized to the tubules with more intense staining of the cells within the lumen of the tubules. Based on the fact that we had previously shown MnSOD to be tyrosine nitrated and inactivated during both human and rat renal allograft rejection, we sought to more specifically look at the time course of MnSOD nitration and inactivation during renal I/R. Nitrotyrosine immunoprecipitation followed by MnSOD Western analysis was carried out on renal extracts to assess nitration of MnSOD. Tyrosine-nitrated MnSOD was seen
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Fig. 2. Nitrotyrosine, periodic acid–Schiff (PAS), and TUNEL staining of renal tissue following I/R. Nitrotyrosine staining: I/R model (A–C); 16-h Sham, 3- and 16-h reperfusion; 100. PAS staining: I/R model (D–F); 16-h Sham, 3- and 16-h reperfusion; arrow shows thinning of brush border; 400. TUNEL staining: I/R model (G, H); 3- and 16-h reperfusion; 100.
as early as 30 min of ischemia (Fig. 3A, isc) and appeared to increase with time of reperfusion. A small degree of MnSOD nitration was observed in sham animals, which is likely due to the surgical trauma of the right nephrectomy. No band with the apparent molecular weight of MnSOD was noted with the antinitrotyrosine conjugate alone (Fig. 3A, NT, negative control). Significant inactivation of MnSOD (40% of sham activity) was seen in kidneys subjected to ischemia
alone and it remained lowered throughout the reperfusion period (Fig. 3B, 3- and 16-h time points). It was important to document that this decrease in activity was not due to a reduction in MnSOD protein levels. Thus, MnSOD Western analysis of these same renal extracts was performed and showed no decrease in MnSOD protein levels following I/R (Fig. 3B, inset). It was also interesting that the sham animals had normal renal MnSOD activity despite the small amount of tyrosine
Fig. 3. (A) MnSOD Western analysis of nitrotyrosine immunoprecipitation (NT IP). Std, recombinant MnSOD; isc, ischemia only; sh, sham-operated rats (16 h); NT, nitrotyrosine agarose beads only. (B) MnSOD activity of renal extracts using the cytochrome c reduction method. *Significantly different from sham-operated rats (p < 0:05). Inset: MnSOD Western analysis of kidney extracts from rats sacrificed at 3–16 h reperfusion. (C) Cytochrome c Western analysis of nitrotyrosine immunoprecipitation from the blot shown in A (after stripping of primary antibody).
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nitration observed (Fig. 3A, sh). This likely suggests that the degree of nitration was not sufficient to cause inactivation. The Western blot in Fig. 3A shows that the apparent levels of nitrated MnSOD increase with time of reperfusion; however, the extent of MnSOD inactivation does not change. The biochemical reasoning for this discrepancy remains unknown. We have previously shown that both tyrosine nitration and tyrosine oxidation (dityrosine production) were required to completely inactivate MnSOD by peroxynitrite in vitro [52]. Interestingly, we have been unable to detect dityrosine crosslinked MnSOD in this model of renal I/R. The other possibility is that nitration of different tyrosine residues within MnSOD may lead to different degrees of inactivation. Current studies are underway to determine the precise tyrosine residue(s) within MnSOD that are nitrated in vivo. Loss of MnSOD activity leads to increased oxidant production within the mitochondria, which could lead to nitration of other mitochondrial proteins. Previous studies demonstrated that cytochrome c was nitrated during rat chronic renal allograft rejection [1]; therefore, the same blot shown in Fig. 3A was stripped of primary antibody and reprobed with the cytochrome c antibody to assess whether it was also nitrated during I/R. Cytochrome c Western analysis following nitrotyrosine immunoprecipitation showed that this protein was also endogenously nitrated (Fig. 3C). The nitration of cytochrome c followed a time pattern similar to that of the nitration of MnSOD. Cytochrome c was nitrated early in the process (ischemia alone) and remained nitrated throughout the reperfusion period. In our previous studies using a rodent model of renal transplantation, we showed that nitration of MnSOD preceded cytochrome c nitration [1]. We speculated that this was due to progressive mitochondrial injury, which lead to increased production of nitrating agent and subsequent nitration of cytochrome c. One possible explanation for the change in the nitration time course in the current I/R model is that the rat undergoes a rapid loss of both kidneys, which could produce a large flux of nitrating species leading to nitration of both MnSOD and cytochrome c. In our previous studies dealing with renal transplantation, there was loss of one kidney followed by a 2-week delay before the loss of the second kidney; this may in fact produce a much lower level of nitrating species over a longer time course. Like MnSOD, cytochrome c nitration was observed in sham animals, which was not unexpected since serum creatinine was also elevated in sham animals. One indicator of mitochondrial dysfunction is the loss of mitochondrial membrane integrity leading to alterations in the electron transport chain, most notably ATP production and protein leakage from the mitochondria. Previous studies have shown that there is a severe decline in ATP levels following renal I/R [41–43]. To fur-
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Fig. 4. ATP levels of renal extracts at all timepoints following renal I/ R. *Significantly different from sham-operated rats (p < 0:05).
ther examine the extent of mitochondrial dysfunction, ATP measurements were made. By 3 h of reperfusion, and at all later time points, there was a significant decrease in renal ATP levels (Fig. 4). Release of cytochrome c into the cytosol has been implicated in the apoptotic signaling pathway [37–40]. Therefore, isolation of mitochondrial/cytosolic fractions
Fig. 5. Cytochrome c Western analysis of mitochondrial (A) and cytosolic (B) fractions isolated from rat renal tissue at specified times following renal I/R. (C and D) MnSOD Western analysis of blots shown in A and B (after stripping of primary antibody). (E and F) Cytochrome c oxidase subunit 1 Western analysis (Anti-COX) of blots shown in A and B. Isc, ischemia only; Std, recombinant MnSOD.
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of the renal I/R tissues was performed to determine whether cytochrome c was being released into the cytosol. As shown in Fig. 5B, a small fraction of cytochrome c was being released into the cytosol during ischemia alone and following reperfusion (3–16 h). No apparent change in the levels of mitochondrial-associated cytochrome c was noted, indicating that the relative amount of cytochrome c being released is small (Fig. 5A). Interestingly, when membranes identical to those used in Figs. 5A and B were stripped of antibodies and reprobed with the polyclonal anti-MnSOD antibody, a small percentage of MnSOD protein also appeared in the cytosolic fraction following reperfusion, but not after ischemia alone (Fig. 5D). These results suggest that the release of MnSOD into the cytosol follows the release of cytochrome c. Western blot analysis of the mitochondrial-encoded protein, cytochrome c oxidase subunit I (1:300; Molecular Probes), was performed to ensure that the mitochondrial fractionation procedure itself was not responsible for the release of these mitochondrial proteins into the cytosol (Figs. 5E and F). Results from this experiment strongly suggested that the fractionation procedure was reliable, since the majority of cytochrome c oxidase was present in the mitochondrial fraction where it is made. These data showing release of cytochrome c and the data showing increased TUNEL staining and decrease in ATP levels lend further support to an apoptotic-mediated pathway following renal I/R. To our knowledge, this is the first report of MnSOD being localized in the cytosolic compartment. MnSOD is synthesized in the nucleus as a precursor protein and is transported to the mitochondria via a mitochondrialtargeting sequence, where it is thought that the monomeric protein is folded into the active, tetrameric 96-kDa protein. Current in vitro studies are underway to determine the mechanism of MnSOD release, e.g., whether release is dependent on the opening of the mitochondrial pore, which may be the mechanism responsible for release of mitochondrial cytochrome c. Alternatively, it is possible that newly synthesized MnSOD is unable to enter the mitochondria, due to a disruption in the mitochondrial targeting domain of MnSOD. Regardless of the mechanism, the appearance of MnSOD in the cytosolic fraction clearly indicates a loss of mitochondrial membrane integrity. Preliminary studies failed to detect nitration of either cytosolic cytochrome c or cytosolic MnSOD (data not shown). The relative amount of these proteins within the cytosolic compartment appears to be quite low; thus, it is possible that the levels of tyrosine-nitrated cytochrome c or MnSOD were below the level of detection in these experiments. An important aspect of the I/R model used in these studies is that the period of ischemia was restricted to 30 min. In our experience, experiments with the rodent
model of renal transplantation demonstrated that 30 min of warm ischemia appears to be the longest time that a kidney can remain ischemic before it is no longer viable for transplant. Many other studies have used as long as 90 min of ischemia [11,43–45]; however, it seems that these results are not applicable in a transplant setting. Collectively, this study shows that reactive oxygen species are generated during renal I/R, which results in increased oxidative stress contributing to renal dysfunction [31,32,46,47]. Previous studies have shown that reactive oxygen species, particularly nitric oxide and superoxide, are generated during I/R [11,23,28]. Mitochondria play a critical role in I/R injury. Even short periods of ischemia increase the electronegativity of the electron transport chain complexes and leakage of electrons. When the ischemic period is prolonged, electron transport complexes are altered. There is a reduction in all activities accompanied by structural damage. This damage can increase electron leakage, which is sustained during reperfusion. Due to the fact that mitochondrial antioxidants are depleted during ischemia, cells become more susceptible to oxidative stress. Upon reperfusion, there is a burst of O 2 when oxygen is reintroduced to the organ. This may damage the electron transport chain complexes, resulting in leakage of electrons that react with oxygen to generate even more superoxide in a continuous cycle [31]. Despite the fact that data presented here do not reveal the functional status of isolated cytochrome c during renal I/R injury, as pointed about by Cassina et al. [48], nitration of even a small fraction of cytochrome c may alter the apoptotic-related signaling properties of this important protein. Numerous studies have shown that the overexpression and supplementation with MnSOD is protective during I/R [27,28]. In a model of myocardial I/R, MnSOD transgenic mice showed better functional recovery and limited infarct size. Both results were attributed to the increased MnSOD activity in the transgenic mice [27]. In another study of gastric I/R, SOD was added at the onset of reperfusion. This administration caused a decrease in nitrotyrosine staining, indicating a decrease in peroxynitrite formation [28]. Another option to using SOD is to use SOD mimetics. One successful mimetic has been MnTBAP. MnTBAP has been shown to be a potent inhibitor of peroxynitrite-induced oxidative reactions, but has no effect on NO levels [49]. MnTBAP has been shown to decrease superoxide in a dose-dependent manner and is cell permeable [50]. Studies have shown this compound to be protective in rat models of ischemia and shock [51]. In summary, the process of renal I/R alone leads to increased oxidant production and mitochondrial protein nitration. The two mitochondrial proteins, MnSOD and
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cytochrome c, appear to be specific targets of endogenous nitration. Loss of MnSOD activity likely occurs early during I/R and may contribute to poor graft function and long-term graft survival.
Acknowledgments The authors thank Shirin Ahki, Dodie Madden, and Qi Wu for excellent technical support.
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Mitochondrial targets of oxidative stress during renal ischemia/reperfusionq Danielle L. Cruthirds,a Lea Novak,b Kabir M. Akhi,b Paul W. Sanders,c John A. Thompson,b and Lee Ann MacMillan-Crowa,b,* a
c
Department of Pharmacology, UAB School of Medicine, Birmingham, AL 35294, USA b Department of Surgery, UAB School of Medicine, Birmingham, AL 35294, USA Department of Medicine and Department of Physiology and Biophysics, UAB School of Medicine, Birmingham, AL 35294, USA Received 12 December 2002, and in revised form 8 January 2003
Abstract Endogenous tyrosine nitration and inactivation of manganese superoxide dismutase (MnSOD) has previously been shown to occur in both human and rat chronic renal allograft rejection. To elucidate the time course of MnSOD inactivation and mitochondrial dysfunction at earlier times during renal transplantation, we developed a rodent model of renal ischemia/reperfusion (I/R). Renal function was significantly impaired at 16 h reperfusion following 30 min of warm ischemia. Tyrosine nitration of specific mitochondrial proteins, MnSOD and cytochrome c, occurred at the earliest time point examined, an event that preceded significant renal injury. Interestingly, a small percentage of both mitochondrial proteins were also located in the cytosol. This leakage and decreased adenosine 50 -triphosphate levels indicate loss of mitochondrial membrane integrity during renal I/R. Inactivation of MnSOD occurred rapidly in this model of renal I/R, suggesting that loss of MnSOD activity leads to further renal injury and nitration of other mitochondrial targets. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Kidney; Ischemia/reperfusion; Tyrosine nitration; MnSOD; Mitochondria; Transplantation; Cytochrome c; ATP
Kidneys are one of the most successfully transplanted organs today, with excellent (>90%) 1-year organ survival rates. This success can be attributed to the advent of tissue typing protocols and improved immunosuppressive agents. Despite these early successes, many grafts succumb to chronic renal allograft rejection (>1 year posttransplant). The precise cause of chronic rejection of renal allografts remains unclear, but appears to be related to several issues including organ preservation techniques and ischemia/reperfusion (I/R)1 injury q
This work was supported in part by grants from NIH (RO1 DK59872-02, RO1 DK46199), US Department of Transportation, and a Scientist Development Grant from the American Heart Association (L.A.M.C., 9930240N). * Corresponding author. Fax: 1-205-975-7549. E-mail address: [email protected] (L.A. MacMillan-Crow). 1 Abbreviations used: I/R, ischemia/reperfusion; MnSOD, manganese superoxide dismutase; PMSF, phenylmethylsulfonyl fluoride; PAS, periodic acid–Schiff.
prior to and immediately after surgical implantation. The critical role of cold preservation and ischemia to chronic renal rejection is evidenced by the fact that >85% of grafts from living (un)related donor kidneys survive 5 years compared to 50% survival for cadaveric donor kidneys [3–5]. Living (un)related donor kidneys are generally excluded from preservation or cold ischemia in contrast to cadaveric organs, which have undergone both an extended period of cold ischemia and a period of preservation prior to transplantation. Given these statistics, increased interest within the transplantation world has been to investigate the role that I/R plays in transplanted graft function and longterm outcome. It has been demonstrated that reactive oxygen species are generated during the process of I/R [6–14]. Walker et al. [15] nicely demonstrated that in the kidney, ischemia alone causes both oxidant stress and formation of reactive nitrogen species. During this process, there is an
0003-9861/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0003-9861(03)00039-0
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increase in both nitric oxide (NO) and superoxide (O 2 ). These two molecules can react to form the powerful nitrating agent peroxynitrite [16]. Peroxynitrite has been shown to be a potent oxidizing and nitrating agent that leads to a host of injurious events, including lipid peroxidation, depletion of cellular antioxidant defenses, inactivation of enzymes, and nitration of tyrosine residues in proteins that may adversely affect their function and signal transduction processes [17–22]. Walker et al. [15,23] have also shown that nitrotyrosine, a marker of peroxynitrite formation, is present following as little as 10 min of ischemia. Our laboratory has previously demonstrated that during both human and rat chronic renal allograft rejection there is an increase in oxidative stress as evidenced by significant increases in tyrosine nitration and a loss of antioxidant activity [1,2]. In these studies, we demonstrated that manganese superoxide dismutase (MnSOD) was endogenously tyrosine nitrated and inactivated [1,2]. MnSOD is the major antioxidant in the mitochondria. This enzyme catalyzes the dismutation of O 2 generated from misfires in the electron transport chain. Earlier studies have shown that MnSOD is essential for life. Homozygous MnSOD knockout mice survive for only 5–21 days after birth [24–26]. These animals exhibit clinical pathologies including myocardial injury, neurodegeneration, lipid peroxidation, fatty liver, anemia, and mitochondrial damage [24–26]. It has also been shown that MnSOD heterozygous mice (containing only 50% of wild-type MnSOD function) are more susceptible to damage [26,27]. There have been numerous reports that overexpression of MnSOD protects tissues/ organs from I/R-related damage [10,11,28,29]; thus a loss of MnSOD activity during I/R likely contributes to tissue injury. Here we show that MnSOD inactivation occurs following only 30 min of renal ischemia. Mitochondria appear to be especially sensitive to the I/R insult, as has been previously shown [30–32]. More specifically, we show that MnSOD and cytochrome c are specific mitochondrial targets of this oxidative damage.
Materials and methods Rat I/R model Animals were treated according to strict UAB IACUC guidelines. Male inbred Fisher 344 rats weighing 250– 300 g were used in this study. Animals were maintained on a formulated diet (Dyets, Inc., Bethlehem, PA, USA) containing 1% (w/w) NaCl for 2 weeks before initiation of the study. The model is one of bilateral ischemia in which a right nephrectomy is performed followed by immediate occlusion of the left renal artery and vein for 30 min. The
left kidney is then allowed to reperfuse for a period of up to 16 h. At specific time points, the animals were anesthetized with ketamine/xylazine, the left kidney was removed, and blood was collected via intracardiac injection. Animals that underwent identical surgery but without the I/R episode served as sham animals. Serum analysis Blood was collected at time of sacrifice via intracardiac injection. Serum was made from whole blood using Super Serum according to manufacturer directions (Allegiance Healthcare, McGaw Park, IL, USA). Serum creatinine levels were determined using a Roche Serum Analyzer. Immunohistology Renal tissue was formalin-fixed, paraffin-embedded, and processed as described [33]. Nitrotyrosine staining was performed using the polyclonal anti-nitrotyrosine antibody (1:500) as described [2]. TUNEL staining was performed using the DeadEnd colorimetric TUNEL system kit (Promega, Madison, WI, USA). Renal tissue injury was assessed in tissue sections stained using the periodic acid–Schiff (PAS) reaction. Renal extract preparation Renal extracts were made from frozen tissue by homogenizing (0.1 g/ml) using a Polytron homogenizer in buffer containing 50 mM potassium phosphate, pH 7.4, and 1 mM PMSF. Solubilized extracts were sonicated and centrifuged at 10,000 rpm (5 min, 4 °C) to remove tissue debris. Protein concentrations were determined by Bradford Assay (Pierce, Rockford, IL, USA). Immunoprecipitation and western blot analysis Renal extracts (500 lg) were incubated (4 °C, 2 h) with 10 ll of the monoclonal anti-nitrotyrosine agarose conjugate (Upstate Biotechnology, Lake Placid, NY, USA). MnSOD Western analysis was performed using the polyclonal anti-MnSOD antibody (Upstate Biotechnology; 1:1000). Cytochrome c Western analysis was performed using the monoclonal anti-cytochrome c antibody (Pharmingen, San Diego, CA, USA; 1:500). Probed membranes were washed three times and immunoreactive proteins were detected using horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence. MnSOD activity assays Enzymatic activity of MnSOD was determined in renal extracts by the cytochrome c reduction method
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using 1 mM KCN as described by McCord and Fridovitch [34]. Mitochondria/cytosol fractionation Separation of mitochondria and cytosol from each kidney was carried out by centrifugation in mitochondrial lysis buffer (250 mM sucrose, 20 mM Hepes, pH 7.5, 10 mM KCl, 1.5 mM MgCl2 , 1 mM EDTA, 1 mM PMSF, 1 mM dithiothreitol) as follows. Kidneys were homogenized in a glass homogenizer in a small volume of lysis buffer. Samples were centrifuged at 750g for 10 min. The supernatant was centrifuged at 10,000g for 10 min. The resulting mitochondrial pellet was resuspended in 300 ll of lysis buffer. The supernatant was further centrifuged at 100,000g for 25 min to obtain the pure cytosolic fraction. Proper mitochondrial fractionation was assessed via Western blot analysis of the mitochondrial encoded protein, cytochrome c oxidase subunit I (1:300; Molecular Probes), which should be present only in the mitochondrial fractions. ATP measurements Total ATP measurements were made using an ATP bioluminescent assay kit (Sigma–Aldrich, St. Louis, MO, USA). Measurements were made on a TD 20/20 luminometer (Turner Designs, Sunnyvale, CA, USA). Quantification of ATP was made by creating an ATP standard curve.
Results and discussion Our laboratory has previously utilized a rodent model of chronic renal allograft rejection and demonstrated that the mitochondrial antioxidant protein, MnSOD, was tyrosine nitrated and inactivated at the earliest time point measured (2 weeks posttransplant) [1]. To further elucidate the time course of MnSOD inactivation and mitochondrial dysfunction at earlier times during renal transplantation we developed a rodent model of I/R. In this model of I/R, where only the left kidney was reperfused, significant renal dysfunction was observed at 16 h of reperfusion, as evidenced by increased serum creatinine levels (Fig. 1). Although there was noted elevation in serum creatinine by 3 h, it was not significant due to the already elevated levels in the corresponding shams. We attribute this basal increase in serum creatinine to trauma associated with completion of the right nephrectomy. Increased oxidant production, as measured by nitrotyrosine immunostaining, appeared as early as 30 min of ischemia of the sole left kidney (data not shown). As early as 3 h of reperfusion, nitrotyrosine stain was present in proximal and distal tubules and at the surface of glomerular tufts (Fig. 2B). Nitration ap-
Fig. 1. Serum creatinine measurements of samples from rats undergoing renal I/R. *Significantly different from corresponding shamtreated animals (p < 0:05).
peared to be contained within the cytosol of tubular cells. Interestingly, by 16 h of reperfusion, there was an apparent increase in tyrosine nitration appearing at the tubular lumen (Fig. 2C). Sham-operated animals (16 h) presented with very low levels of nitrotyrosine immunostaining (Fig. 2A). Preincubation (30 min, 25 °C) of the polyclonal anti-nitrotyrosine antibody with 10 mM 3-nitrotyrosine completely blocked immunostaining (data not shown). Others have shown that I/R injury leads to tubular dilatation and loss of brush border integrity [23,35,36]. To examine tubular histopathologic changes associated with loss of membrane integrity due to ischemic injury, PAS histochemical staining was performed (Fig. 2). In the sham animals, the tubules showed normal thickness of the brush border (Fig. 2D). By 3 h of reperfusion, several tubules were dilated and the brush border appeared thinner, indicating a loss of membrane integrity (Fig. 2E). Consistent with progressive damage, there was much more tubular dilation and thinning of tubular brush borders by the 16-h time point (Fig. 2F, arrow). Another consequence of increased oxidative stress is the induction of apoptosis; thus, we used TUNEL staining as a marker of apoptotic cell death during renal I/R. In this model of I/R, there was a small degree of TUNEL staining apparent in the cortex and medulla by 16 h of reperfusion (while not present at 3 h) (Figs. 2G and H). The staining was mainly localized to the tubules with more intense staining of the cells within the lumen of the tubules. Based on the fact that we had previously shown MnSOD to be tyrosine nitrated and inactivated during both human and rat renal allograft rejection, we sought to more specifically look at the time course of MnSOD nitration and inactivation during renal I/R. Nitrotyrosine immunoprecipitation followed by MnSOD Western analysis was carried out on renal extracts to assess nitration of MnSOD. Tyrosine-nitrated MnSOD was seen
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Fig. 2. Nitrotyrosine, periodic acid–Schiff (PAS), and TUNEL staining of renal tissue following I/R. Nitrotyrosine staining: I/R model (A–C); 16-h Sham, 3- and 16-h reperfusion; 100. PAS staining: I/R model (D–F); 16-h Sham, 3- and 16-h reperfusion; arrow shows thinning of brush border; 400. TUNEL staining: I/R model (G, H); 3- and 16-h reperfusion; 100.
as early as 30 min of ischemia (Fig. 3A, isc) and appeared to increase with time of reperfusion. A small degree of MnSOD nitration was observed in sham animals, which is likely due to the surgical trauma of the right nephrectomy. No band with the apparent molecular weight of MnSOD was noted with the antinitrotyrosine conjugate alone (Fig. 3A, NT, negative control). Significant inactivation of MnSOD (40% of sham activity) was seen in kidneys subjected to ischemia
alone and it remained lowered throughout the reperfusion period (Fig. 3B, 3- and 16-h time points). It was important to document that this decrease in activity was not due to a reduction in MnSOD protein levels. Thus, MnSOD Western analysis of these same renal extracts was performed and showed no decrease in MnSOD protein levels following I/R (Fig. 3B, inset). It was also interesting that the sham animals had normal renal MnSOD activity despite the small amount of tyrosine
Fig. 3. (A) MnSOD Western analysis of nitrotyrosine immunoprecipitation (NT IP). Std, recombinant MnSOD; isc, ischemia only; sh, sham-operated rats (16 h); NT, nitrotyrosine agarose beads only. (B) MnSOD activity of renal extracts using the cytochrome c reduction method. *Significantly different from sham-operated rats (p < 0:05). Inset: MnSOD Western analysis of kidney extracts from rats sacrificed at 3–16 h reperfusion. (C) Cytochrome c Western analysis of nitrotyrosine immunoprecipitation from the blot shown in A (after stripping of primary antibody).
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nitration observed (Fig. 3A, sh). This likely suggests that the degree of nitration was not sufficient to cause inactivation. The Western blot in Fig. 3A shows that the apparent levels of nitrated MnSOD increase with time of reperfusion; however, the extent of MnSOD inactivation does not change. The biochemical reasoning for this discrepancy remains unknown. We have previously shown that both tyrosine nitration and tyrosine oxidation (dityrosine production) were required to completely inactivate MnSOD by peroxynitrite in vitro [52]. Interestingly, we have been unable to detect dityrosine crosslinked MnSOD in this model of renal I/R. The other possibility is that nitration of different tyrosine residues within MnSOD may lead to different degrees of inactivation. Current studies are underway to determine the precise tyrosine residue(s) within MnSOD that are nitrated in vivo. Loss of MnSOD activity leads to increased oxidant production within the mitochondria, which could lead to nitration of other mitochondrial proteins. Previous studies demonstrated that cytochrome c was nitrated during rat chronic renal allograft rejection [1]; therefore, the same blot shown in Fig. 3A was stripped of primary antibody and reprobed with the cytochrome c antibody to assess whether it was also nitrated during I/R. Cytochrome c Western analysis following nitrotyrosine immunoprecipitation showed that this protein was also endogenously nitrated (Fig. 3C). The nitration of cytochrome c followed a time pattern similar to that of the nitration of MnSOD. Cytochrome c was nitrated early in the process (ischemia alone) and remained nitrated throughout the reperfusion period. In our previous studies using a rodent model of renal transplantation, we showed that nitration of MnSOD preceded cytochrome c nitration [1]. We speculated that this was due to progressive mitochondrial injury, which lead to increased production of nitrating agent and subsequent nitration of cytochrome c. One possible explanation for the change in the nitration time course in the current I/R model is that the rat undergoes a rapid loss of both kidneys, which could produce a large flux of nitrating species leading to nitration of both MnSOD and cytochrome c. In our previous studies dealing with renal transplantation, there was loss of one kidney followed by a 2-week delay before the loss of the second kidney; this may in fact produce a much lower level of nitrating species over a longer time course. Like MnSOD, cytochrome c nitration was observed in sham animals, which was not unexpected since serum creatinine was also elevated in sham animals. One indicator of mitochondrial dysfunction is the loss of mitochondrial membrane integrity leading to alterations in the electron transport chain, most notably ATP production and protein leakage from the mitochondria. Previous studies have shown that there is a severe decline in ATP levels following renal I/R [41–43]. To fur-
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Fig. 4. ATP levels of renal extracts at all timepoints following renal I/ R. *Significantly different from sham-operated rats (p < 0:05).
ther examine the extent of mitochondrial dysfunction, ATP measurements were made. By 3 h of reperfusion, and at all later time points, there was a significant decrease in renal ATP levels (Fig. 4). Release of cytochrome c into the cytosol has been implicated in the apoptotic signaling pathway [37–40]. Therefore, isolation of mitochondrial/cytosolic fractions
Fig. 5. Cytochrome c Western analysis of mitochondrial (A) and cytosolic (B) fractions isolated from rat renal tissue at specified times following renal I/R. (C and D) MnSOD Western analysis of blots shown in A and B (after stripping of primary antibody). (E and F) Cytochrome c oxidase subunit 1 Western analysis (Anti-COX) of blots shown in A and B. Isc, ischemia only; Std, recombinant MnSOD.
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of the renal I/R tissues was performed to determine whether cytochrome c was being released into the cytosol. As shown in Fig. 5B, a small fraction of cytochrome c was being released into the cytosol during ischemia alone and following reperfusion (3–16 h). No apparent change in the levels of mitochondrial-associated cytochrome c was noted, indicating that the relative amount of cytochrome c being released is small (Fig. 5A). Interestingly, when membranes identical to those used in Figs. 5A and B were stripped of antibodies and reprobed with the polyclonal anti-MnSOD antibody, a small percentage of MnSOD protein also appeared in the cytosolic fraction following reperfusion, but not after ischemia alone (Fig. 5D). These results suggest that the release of MnSOD into the cytosol follows the release of cytochrome c. Western blot analysis of the mitochondrial-encoded protein, cytochrome c oxidase subunit I (1:300; Molecular Probes), was performed to ensure that the mitochondrial fractionation procedure itself was not responsible for the release of these mitochondrial proteins into the cytosol (Figs. 5E and F). Results from this experiment strongly suggested that the fractionation procedure was reliable, since the majority of cytochrome c oxidase was present in the mitochondrial fraction where it is made. These data showing release of cytochrome c and the data showing increased TUNEL staining and decrease in ATP levels lend further support to an apoptotic-mediated pathway following renal I/R. To our knowledge, this is the first report of MnSOD being localized in the cytosolic compartment. MnSOD is synthesized in the nucleus as a precursor protein and is transported to the mitochondria via a mitochondrialtargeting sequence, where it is thought that the monomeric protein is folded into the active, tetrameric 96-kDa protein. Current in vitro studies are underway to determine the mechanism of MnSOD release, e.g., whether release is dependent on the opening of the mitochondrial pore, which may be the mechanism responsible for release of mitochondrial cytochrome c. Alternatively, it is possible that newly synthesized MnSOD is unable to enter the mitochondria, due to a disruption in the mitochondrial targeting domain of MnSOD. Regardless of the mechanism, the appearance of MnSOD in the cytosolic fraction clearly indicates a loss of mitochondrial membrane integrity. Preliminary studies failed to detect nitration of either cytosolic cytochrome c or cytosolic MnSOD (data not shown). The relative amount of these proteins within the cytosolic compartment appears to be quite low; thus, it is possible that the levels of tyrosine-nitrated cytochrome c or MnSOD were below the level of detection in these experiments. An important aspect of the I/R model used in these studies is that the period of ischemia was restricted to 30 min. In our experience, experiments with the rodent
model of renal transplantation demonstrated that 30 min of warm ischemia appears to be the longest time that a kidney can remain ischemic before it is no longer viable for transplant. Many other studies have used as long as 90 min of ischemia [11,43–45]; however, it seems that these results are not applicable in a transplant setting. Collectively, this study shows that reactive oxygen species are generated during renal I/R, which results in increased oxidative stress contributing to renal dysfunction [31,32,46,47]. Previous studies have shown that reactive oxygen species, particularly nitric oxide and superoxide, are generated during I/R [11,23,28]. Mitochondria play a critical role in I/R injury. Even short periods of ischemia increase the electronegativity of the electron transport chain complexes and leakage of electrons. When the ischemic period is prolonged, electron transport complexes are altered. There is a reduction in all activities accompanied by structural damage. This damage can increase electron leakage, which is sustained during reperfusion. Due to the fact that mitochondrial antioxidants are depleted during ischemia, cells become more susceptible to oxidative stress. Upon reperfusion, there is a burst of O 2 when oxygen is reintroduced to the organ. This may damage the electron transport chain complexes, resulting in leakage of electrons that react with oxygen to generate even more superoxide in a continuous cycle [31]. Despite the fact that data presented here do not reveal the functional status of isolated cytochrome c during renal I/R injury, as pointed about by Cassina et al. [48], nitration of even a small fraction of cytochrome c may alter the apoptotic-related signaling properties of this important protein. Numerous studies have shown that the overexpression and supplementation with MnSOD is protective during I/R [27,28]. In a model of myocardial I/R, MnSOD transgenic mice showed better functional recovery and limited infarct size. Both results were attributed to the increased MnSOD activity in the transgenic mice [27]. In another study of gastric I/R, SOD was added at the onset of reperfusion. This administration caused a decrease in nitrotyrosine staining, indicating a decrease in peroxynitrite formation [28]. Another option to using SOD is to use SOD mimetics. One successful mimetic has been MnTBAP. MnTBAP has been shown to be a potent inhibitor of peroxynitrite-induced oxidative reactions, but has no effect on NO levels [49]. MnTBAP has been shown to decrease superoxide in a dose-dependent manner and is cell permeable [50]. Studies have shown this compound to be protective in rat models of ischemia and shock [51]. In summary, the process of renal I/R alone leads to increased oxidant production and mitochondrial protein nitration. The two mitochondrial proteins, MnSOD and
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cytochrome c, appear to be specific targets of endogenous nitration. Loss of MnSOD activity likely occurs early during I/R and may contribute to poor graft function and long-term graft survival.
Acknowledgments The authors thank Shirin Ahki, Dodie Madden, and Qi Wu for excellent technical support.
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