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1Department of Experimental Pathology, Instituto de Investigaciones Biomédicas, Institut d'Investigacions Biomediques de Barcelona of the Council for Scientific Research, Institut d'Investigacions Biomediques August Pi i Sunyer, and 2Department of Physiology, University of Barcelona, Barcelona, Spain
Submitted 14 September 2006 ; accepted in final form 26 February 2007
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ABSTRACT |
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kidney; nitric oxide; cell death; cytoskeleton derangement
Cytoskeleton alterations have been reported to induce apoptosis in a variety of models, with special attention focused on the disarrangement of the actin cytoskeleton (10, 24), which promotes apoptosis in human airway epithelial cells (40), rat and porcine proximal tubule epithelial cells (35), and also in other renal ischemia-reperfusion (I/R) models (9).
On the other hand, increased expression of nitric oxide synthase 2 (NOS2) and a consequent increase in tissue nitrite and nitrate production occur in cardiac, liver, and kidney I/R (15, 16, 41, 42). In many instances, NOS2 inhibition by arginine analogs or ablation of NOS2 gene expression significantly limits tissue I/R injury (18, 20, 21). Peripheral vascular insufficiency, accompanied by periodic restoration of blood flow, places ischemic organs at risk of additional injury by inducing a proinflammatory state, reflected by enhanced superoxide and hydrogen peroxide (H2O2) generation (11, 27, 43, 44). This setting favors the generation of peroxynitrite (ONOO–), the nitrating and oxidizing species produced by the radical-radical reaction of oxygen and nitric oxide (NO) (3).
In this sense, previous studies performed in our group showed the increase in peroxynitrite generation and its implication in apoptosis development during renal I/R (37).
Peroxynitrite is capable of nitrating tyrosine residues in tissue proteins, conferring to the nitrotyrosine formed over a period of time the ability to be a marker of peroxynitrite production (4, 5). Immunohistochemical studies have revealed that nitration of tyrosine residues takes place in various tissues and organs after ischemia and in other pathological conditions (14, 39).
The identification of actin as a key tissue target for protein nitration and its impaired polymerization properties together with the ability of peroxynitrite to inhibit actin dynamics (8) suggest that in the I/R process the peroxynitrite derived from the reaction between NO and the superoxide radical (O2–) could well have a key role in cytoskeletal breakdown.
Thus the aim of our study was to know the role of peroxynitrite as an inductor of cytoskeleton derangement and apoptosis during renal I/R.
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MATERIALS AND METHODS |
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Animals and anesthesia. The study was performed using male Wistar rats (Ifa Credo, Barcelona, Spain), weighing 250–300 g. The animals were anesthetized by injection of pentobarbital sodium (30 mg/kg ip) and placed in a supine position. Body temperature was maintained between 36 and 37°C. All procedures were conducted under the supervision of our institution's Research Commission and followed European Union guidelines for the handling and care of laboratory animals.
Pharmacological cytoskeleton manipulation. Two different actin cytoskeletal disruptors were used: swinholide A (swin A) is a microfilament-disrupting marine toxin that stabilizes actin dimmers and severs actin filaments (31). As a stabilizing agent, we used jasplakinolide (JP), a compound that stabilizes actin filaments by binding F-actin in vitro (31).
Renal I/R. After a laparotomy and dissection of both renal pedicles, bilateral ischemia was induced by occluding the renal pedicles with an atraumatic microvascular clamp for 45 min. The immediate change in the color of the kidneys signifying the stoppage of blood flow indicates successful occlusion. During reperfusion, clamps were removed and the blood flow to the kidneys was reestablished with visual verification of blood return.
Experimental groups. The following experimental groups (n = 6/group) were studied.
CONTROL.
In the control group, animals were subjected to the identical maneuvers as the I/R group, except that the renal pedicles were not clamped.
I/R.
Animals were subjected to 45 min of bilateral ischemia and 24 h of reperfusion.
I/R+FeTMPyP.
Conditions were similar to those in the I/R group but with the administration of the peroxynitrite scavenger 5,10,15,20-tetrakis-(N-methyl-4-pyridyl) porphinato-iron III (FeTMPyP; 30 mg/kg iv) dissolved in 300 µl of saline 2 min after the beginning of reperfusion by direct puncture in the inferior cava. This dose has been shown to be effective in the rat (30).
I/R+JP.
To evaluate the effects of F-actin stabilization, we administered JP (Calbiochem, La Jolla, CA) as a stabilizing agent. Three hundred microliters of saline containing 20 nM JP were administrated by direct puncture in the inferior cava before the beginning of the I/R process. (This dose has proven to be effective in stabilizing actin in previous studies.) (6).
I/R+FeTMPyP+SWIN A (Fet+SW).
To evaluate the effects of direct disruption of the actin cytoskeleton during I/R, together with the inhibition of peroxynitrite formation, the actin cytoskeleton disruptor agent swin A (Sigma-Aldrich, St. Louis, MO) and the peroxynitrite inhibitor FeTMPyP were administered. Three hundred microliters of saline containing 500 nM swin A were administrated by direct puncture in the inferior cava before the beginning of the I/R process. (This dose has proven to be effective in previous studies.) (34).
At the end of the reperfusion period, the kidneys were divided into three parts: one part, obtained from the kidney cortical area, was immediately freeze-clamped to determine caspase-3 activity and Western blotted for nitrotyrosine, cytochrome c, and actin; another was fixed in 4% formalin for terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) and immunohistochemical analysis, and the last part was embedded in optimum cutting temperature compound (OCT; Sakura Finetek, Torrance, CA) and frozen in liquid nitrogen for immunofluorescence procedures.
Biochemical Analysis
Caspase-3 activity. Caspase-3-like activity was determined by measuring proteolytic cleavage of the specific substrate N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (DEVD-AMC; Biomol, Plymouth Meeting, PA). Renal tissue was homogenated and sonicated in assay buffer (50 mM HEPES, 10% sucrose, 0.1% CHAPS, 5 mM GSSG, 5 mM DTT). We used 25 µg of protein of each sample and 12 µM DEVD-AMC to perform the assay. The AMC released was quantified for 1 h at 37°C by fluorospectrophotometry using 380-nm excitation and measurement of 450-nm emission.
Protein concentration. Total protein concentration in homogenates was determined using a commercial kit from Bio-Rad (Munich, Germany).
Western blot analysis. Equal amounts of protein, 30 µg nitrotyrosine, and 10 µg actin were electrophoresed on 10% SDS gels and transferred to nitrocellulose membranes, which were subsequently blocked with 5% nonfat dry milk in 0.06% Tween-TRIS-buffered saline (TTBS) for 1 h. The membranes were incubated overnight with primary antibody (anti-nitrotyrosine 1:1,000 and anti-actin 1:1,000, respectively) at 4°C, washed five times with TTBS, and incubated for 1 h with anti-rabbit IgG for nitrotyrosine (1:2,500) or with horseradish peroxidase-conjugated anti-mouse IgG antibody for cytochrome c and actin (dilution 1:2,000) at room temperature, followed by enhanced chemiluminescence detection.
The sample treatment in the actin Western blot was performed to differentiate the F- and G-actin forms. Briefly, the samples were homogenated in PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, and 2 mM MgCl2, pH 6.9, containing 0.1% Triton X-100, 0.5 mM PMSF, and 0.1 mM DTT). The homogenate was centrifuged at 48,000 g during 5 min at 4°C. The insoluble fraction (cytoskeleton associated) was used for Western blot analysis (25).
Histological Analysis
Samples were embedded in paraffin, cut into 4-µm sections, and stained with hematoxylin-eosin. Evaluation was performed with light microscopy without knowledge of the study groups. Assessment was carried out by expert observers.
Renal Function Test and Blood Pressure Study
Blood urea nitrogen was analyzed using a commercial kit from Sigma. A study of arterial blood pressure was undertaken to test whether the pharmacological compounds administered would alter renal blood flow through systemic influence. Direct measurement of arterial pressure was performed by inserting an aortic catheter (PE-50 tubing, Clay Adams) according to the technique described by Popovic and Popovic (28).
Immunohistological Procedures
Confocal microscopic analyses. One-half of the kidney was embedded in OCT and frozen in liquid nitrogen without prior fixation. Five-micrometer cryosections were fixed in 4% buffered formaldehyde for 10 min and then permeabilized with PBS containing 0.1% Triton X-100 and 1% BSA for 30 min. For actin visualization, the slides were incubated with Alexa Fluor 568-phalloidin (dilution, 1:40, Molecular Probes, Eugene, OR) in PBS with 1% BSA for 30 min. Slides were washed three times for 15 min with PBS and finally mounted using mowiol (Calbiochem). Confocal images were taken with a Leica TCS NT laser microscope (Leica Microsystems, Wetzlar, Germany) equipped with a x100 oil-immersion objective.
TUNEL staining. TUNEL assay was performed to detect apoptosis in situ. Renal tissue sections were deparaffinized and rehydrated through three changes between xylene and graded alcohol, later washed in PBS for 5 min, and then incubated in 20 µg/ml proteinase K for 15 min at room temperature. The DNA fragmentation detection Colorimetric-TdT Enzyme Kit (Oncogene Research Products, Boston, MA) was used according to the manufacturer's instructions. Briefly, endogenous peroxidase activity in the kidney sections was blocked by incubation for 5 min with 3% H2O2 in PBS, followed by incubation with equilibration buffer for 10 s. The sections were then incubated for 60 min at 37°C with terminal deoxynucleotidyl transferase (TdT) enzyme in reaction buffer. The reaction was finished by incubation with stopping buffer at room temperature. Sections were then incubated with peroxidase-conjugated anti-digoxigenin antibody for 30 min at room temperature, and the reaction was developed with diaminobenzidine substrate for 4 min at room temperature. Sections were counterstained with methyl green stain, dehydrated through a graded series of alcohol, and mounted in Permount for microscopy.
Nitrotyrosine immunohistochemistry. For nitrotyrosine immunohistochemistry, all specimens were fixed in 10% neutral buffered formalin, paraffin embedded, and processed routinely; 4-µm-thick serial sections were mounted on poly-L-lysine-coated slides. Sections were dewaxed in xylene, dehydrated through graded alcohols and water, and immersed in 0.3% vol/vol H2O2 in methanol for 30 min to block endogenous peroxidases. Antigens were retrieved by microwaving at 750 W for 15 min in 0.01 mol/l trisodium citrate buffer (pH 6.0). Sections were rinsed well in standard PBS (pH 7.2 ± 0.2), and nonspecific binding was blocked with 10% goat serum (Dako, Cambridgeshire, UK) diluted in PBS for 30 min.
Sections were incubated overnight at 4°C with a polyclonal nitrotyrosine antibody at a dilution of 1:75 in PBS for 16 h at 4°C. After being rinsed with PBS, the sections were incubated with biotinylated goat anti-rabbit IgGs (Dako). Sections were rinsed with PBS and incubated with avidin-biotin horseradish peroxidase complex according to the manufacturer's instructions (Vectastain ABC Kit, Vector Laboratories, Peterborough, UK). Peroxides were visualized by incubating the sections in 3,3'-diaminobenzidine (Sigma, Poole, UK) and hydrogen peroxides. Negative control experiments were performed by omitting incubation with the primary antibody.
Statistical Analysis
Data are expressed as means ± SE. Means of different groups were compared using one-way ANOVA. The Student-Newman-Keuls test was used for the evaluation of significant differences between groups. Significant differences were assumed when P < 0.05.
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RESULTS |
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The evaluation of cytoskeletal alterations was performed by the detection of a fluorescent F-actin stain in proximal tubule epithelial cells and F-actin Western blotting.
As shown in Fig. 1, the control group presented an intense stain in the tubular epithelial and in the basal plasma membrane compared with the staining in the I/R group, thus evidencing actin cytoskeletal alterations induced by the process of I/R. By contrast, when the I/R group was treated with the peroxynitrite scavenger FeTMPyP (FeTMPyP group), the F-actin detected was very similar to control animals, indicating the ability of peroxynitrite scavengers to prevent actin cytoskeletal alterations. Administration of the cytoskeletal disruptor swin A to this last group exerted its powerful disruptor effect, generating a potent cytoskeletal damage (Fet+SW group).
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F-actin Western blots showed similar results than thus observed with phalloidin staining. Figure 2 shows the drastic decreases in F-actin levels when the animals were subjected to I/R or when were treated with the cytoskeletal disruptor swin A (Fet+SW group).
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Apoptosis
As shown in Fig. 3, caspase-3 activity was increased as a consequence of I/R (I/R group). By contrast, when the I/R group was treated with the peroxynitrite scavenger FeTMPyP (FeTMPyP group), caspase-3 activity decreased significantly, indicating the ability of peroxynitrite scavengers to prevent apoptosis alterations. The administration of the cytoskeletal disruptor swin A to the FeTMPyP-treated rats reversed the effects of the peroxynitrite scavenger by increasing apoptosis (Fet+SW group), indicating the role of cytoskeleton derangement as an inductor of apoptosis. On the other hand, the cytoskeleton stabilizer (JP group) prevented the apoptosis alterations in a similar way to that with the peroxynitrite scavenger.
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Nitrotyrosine Western blotting. Nitrotyrosine Western blotting has been used as a marker of peroxynitrite formation; peroxynitrite reacts with a variety of cellular targets, among them, L-tyrosine, which is converted to 3-nitro-L-tyrosine. Figure 5 shows the nitration of the proteins in kidneys from the different groups; the I/R group presents an increased nitration pattern with respect to the control group. This nitration increase was reversed in all the other groups studied.
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Histology showed acute tubular necrosis incidence in the I/R group (Fig. 7). Tubular necrosis was also detected in the swin A-treated group. By contrast, FeTMPyP- or JP-treated groups showed the reverse of the injury, and renal structure were almost normal and well preserved.
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Pharmacological compounds administration produced just small and short transitory drops of mean arterial pressure (MAP) similar to those induced by surgical manipulation by venous puncture without drug injection. After intravenous JP administration, mean blood pressure diminished by a maximum of 16% compared with the control period, and swin A injection decreased MAP by a maximum of only 12%. In both cases, normal preadministration values of MAP were reestablished rapidly within a period of 1.5 min. Therefore, in terms of the systemic parameters the arterial blood pressure showed no significant change.
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DISCUSSION |
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Between the possible mechanisms able to induce cytoskeletal alterations, the nitration of actin has been pointed out in other models (1). Actin nitration provokes G-actin depolymerization, which leads to the subsequent cytoskeletal disruption (2, 12, 13).
On the other hand, F-actin could be easily nitrated in conditions of oxidative stress such as I/R (1). As observed in Figs. 4 and 5, nitrotyrosine Western blot and immunohistochemical analysis showed that protein nitration was increased after kidney I/R. The increase in protein nitration was parallel with cytoskeletal alterations (Fig. 1) and apoptotic variations (Fig. 3) at the end of the I/R period.
Increased production of reactive oxygen species and NO as a consequence of the I/R process promotes peroxynitrite formation, which could induce protein nitration, apart from cell damage and renal function alterations. One of the consequences of protein nitration could be the nitration of actin tyrosine residues in the cytoskeleton, thus possibly inducing cytoskeletal alterations. The fact that the peroxynitrite scavenger FeTMPyP was able to revert the cytoskeletal derangement provoked by I/R indicates the direct relationship between peroxynitrite and cytoskeletal alterations. Thus this study confirms the role of peroxynitrite as an inductor of cytoskeletal derangement.
In addition, the peroxynitrite scavenger FeTMPyP is able to reduce apoptosis and renal damage derived from the I/R process, indicating the role of peroxynitrite in apoptosis development. In this sense, an interesting finding was shown when rats previously treated with the peroxynitrite scavenger were treated with cytoskeletal disruptors; in this case, the cytoskeleton disruptor accomplishes a reversal of the effect of the peroxynitrite scavenger in preventing apoptosis, indicating that cytoskeletal alteration is an intermediate step between peroxynitrite production and apoptosis.
The role of actin depolymerization and apoptosis has been previously studied; on one hand, actin depolymerization could induce apoptosis-related events (22, 29, 32), and on the other hand caspase-3 could directly act on actin, which is a direct substrate for caspase-3, indicating the possible role of caspase-3 as an inductor of cytoskeletal alterations (23). In addition, active caspase-3, by acting on its substrate gelsoline, could activate it, thus inducing F-actin depolymerization (17). Moreover, the fact that cytoskeleton stabilizers such as JP were able to reduce the apoptotic parameters and to improve renal function indicates that cytoskeletal alterations induce apoptosis and, subsequently, renal damage.
In summary, our results indicate that in the I/R process, peroxynitrite acts on the actin cytoskeleton to induce apoptosis.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* G. Hotter and A. Sola contributed equally to and codirected this work. 
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