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Macrophages infiltrate injured sites and initiate the inflammatory response (1, 2). It has been reported that infiltrated macrophages have a negative role in ischemia/reperfusion (I/R) injury (3, 4). Although it has recently been demonstrated that infiltrated macrophages are associated with repair and regeneration of damaged tissue, the role that they play after I/R has yet to be defined (5).
I/R in the kidney results in a loss of cellular integrity and fragmentation of DNA (6). I/R also increases the formation of reactive oxygen species (ROS), which in turn continuously induces infiltration of leukocytes, activation of cytokine production, and stimulation of endothelial cells (2). ROS is responsible for apoptotic cell death by regulation of the signal transduction pathways and repair processes through the interruption of normal proliferation in damaged tissue (6–8). Leor et al. reported that macrophage activation accelerates repair of myocardium damaged by infarction (9). Additionally, Yano et al. demonstrated that macrophage colony- stimulating factor (M-CSF) attenuates left ventricular dysfunction by accelerating repair after postmyocardial infarction (10).
Ischemic preconditioning provides kidney resistance to subsequent ischemic injury of the kidney (11). Previously, we reported that this phenomenon is associated with a number of endogenous factors, including heat shock protein-27 and inducible nitrogen oxide synthase (iNOS) (12), which is primarily expressed in macrophages. Therefore, we hypothesized that removal of infiltrated macrophages may affect the repair process of postischemic damaged cells and ischemic preconditioning.
In this study, we investigated whether the removal of infiltrated macrophages affected the recovery of damaged kidneys after I/R injury and the kidney resistance afforded by ischemic preconditioning. We found that removal of infiltrated macrophages retarded repair, but did not contribute to ischemic preconditioning.
MATERIALS AND METHODS
Experiments were performed in 8-week-old BALB/c male mice. Mice were allowed free access to water and standard mouse chow. In all cases, studies were conducted according to animal experimental procedures approved by the Animal Care and Use Committee of the Kyungpook National University School of Medicine. Each animal group consisted of more than four mice.
Animals were anesthetized with pentobarbital sodium (60 mg/kg body weight; intraperitoneally) before surgery. Body temperature was maintained at 36–38°C. Kidneys were exposed through flank incisions and the mice were then subjected to 30 min of bilateral renal ischemia or sham-surgery. Both renal pedicles were clamped with nontraumatic microaneurysm clamps to induce Ischemia (Roboz). The incisions were temporarily closed during ischemia or sham surgery. After the clamps were removed, reperfusion of the kidneys was visually confirmed. Mice were then administered either dichlromethylene bisphosphonate (Cl2MBP) encapsuled by liposome (Lipo-clodronate, i.v.) or PBS-encapsuled by liposome (Lipo-PBS, i.v). Lipoclodronate and Lipo–PBS were prepared as described previously by Van Rooijen et al. (13) Lipoclodronate administration depleted macrophages (14) but did not deplete neutrophils or lymphocytes (13). Mice were then subjected to 30 min of either bilateral ischemia or a sham-surgery on day 8. Kidneys were harvested at the times indicated in the figures and then either snap frozen in liquid nitrogen for biochemical studies or perfusion-fixed in 4% paraformaldehyde for histological analysis.
Plasma Creatinine (PCr) and Blood Urea Nitrogen (BUN) Concentration
Seventy microliters of blood were taken from the retrobulbar vein plexus at the times indicated in the figures. PCr concentrations were measured using a Beckman Creatinine Analyzer II (Beckman). BUN was measured using a BUN assay kit (Asan PHARM Co. LTD, Gyeonggi-Do, Korea) following the manufacturer's protocol.
After perfusion via the left ventricle with 30 mL of PBS for 2 min at 37°C followed by PLP (4% paraformaldehyde, 75 mM L-lysine, 10 mM sodium periodate) fixative for 5 min, kidneys were excised and placed in PLP overnight at 4°C. Kidneys were then washed in PBS and stored at 4°C in PBS containing 0.02% sodium azide. Next, fixed tissue was washed with PBS 3 times for 5 min each, placed overnight in PBS containing 30% sucrose, embedded in oxytetracycline compound (Sakura FineTek, Torrance, CA), and then cut into 5-μm sections using a cryomicrotome (Cryotome). Frozen sections for immunostaining were mounted on Fisher Superfrost Plus (Fisher) microscope slides, air dried, and stored at −20°C. Some kidneys were embedded in paraffin for periodic acid-Schiff (PAS) staining.
To immunostain for CD68 and proliferating cell nuclear antigen (PCNA), sections were dried, washed in PBS, incubated in blocking buffer (PBS containing 2% bovine serum albumin) for 20 min at room temperature, incubated in anti-CD68 (Serotec, Oxford, UK) or anti-PCNA (Dako, Glostrup, Denmark) antibody overnight at 4°C, and then washed with PBS. The sections were then incubated in FITC-labeled anti-rat IgG for CD68 staining or rhodamin-labeled anti-mouse IgG for PCNA staining for 40 min at room temperature, washed with PBS 3 times for 10 min each, and then mounted with Prolong Gold anti-fade reagent (Invitrogen, Eugene, OR). For phalloidin staining, sections were incubated in anti-F-actin (phalloidin) antibody conjugated with Cy3 (Sigma) for 2 hr at 37°C, washed with PBS 3 times for 10 min each, and then mounted with Prolong Gold anti-fade reagent (Invitrogen, Eugene, OR). Images were viewed on an Axioplan 2 (Carl Zeiss Vision, Munich, Germany) epifluorescence microscope. Five fields per slide were chosen to count the CD68 or PCNA positive cells.
Immunohistochemical staining was performed using anti-Ly-6G antibody to detect neutrophils (eBioscience Inc. San Diego, CA). To detect the antigens, sections were deparaffinized with xylene and then rehydrated with 100%, 95%, and 80% ethanol. To unmask the antigen epitopes, slides were boiled in 10 mM sodium citrate buffer (pH 6.0) for 5 min, cooled to room temperature for 20 min, and then washed with PBS (2×5 min). To block the activation of endogenous peroxidase, slides were treated with 3% H2O2 in methanol for 30 min at 4°C and then washed with PBS (2×5 min). Sections were then blocked with blocking buffer containing 1% bovine serum albumin (BSA) for 30 min at room temperature. Next, sections were incubated with primary antibodies in 1% BSA overnight at 4°C, washed with PBS 3 times, and then incubated with the respective HRP conjugated secondary antibodies for 60 min at room temperature. The sections were then treated using a DAB substrate kit (Vector Laboratories, Burlingame, CA), and washed with tap water, although some were counter-stained with eosin. Next, the sections were dehydrated and mounted with Permount solution (Fisher) and observed under a light microscope (Nikon).
Western blot analyses were performed as described previously (11) using anti-PCNA (Dako, 1:2000) or GAPDH antibodies (Santacruz, 1:5000).
Sections were mounted on Fisher Superfrost Plus (Fisher Scientific) microscope slides and then stained with Periodic Acid Schiff (PAS) following the standard protocol.
As described previously (15), 50 tubules in the outer medullar of the kidneys were analyzed using the following scoring method: 0, no damage; 1, mild damage with the rounding of epithelial cells and dilated tubular lumen; 2, moderate damage with flattened epithelial cells, dilated lumen, and congestion of lumen; and 3, severe damage with flat epithelial cells lacking nuclear staining and the congestion of lumen. Four kidneys in each experimental animal group were used to determine the histological score.
Nitro Blue Tetrazolium (NBT) Stain
Kidneys were fixed in 4% PLP and sectioned as described previously. Sections were incubated in 1 mg/mL nitro blue tetrazolium (NBT, Biosesang, Gyeonggi-Do, Korea) in phosphate-buffered saline for 2 hr at 37°C and then washed with phosphate-buffered saline. Signals were observed using a light microscope. The area of blue fomarzan was analyzed using LabWorks, an image acquisition and analysis program (Ultra Violet Products Ltd.).
Kidney sections were stained in nitrotyrosine as described above (Cayman, Ann arbor, MI). The area of formation of nitrotyrosine was analyzed using LabWorks, an image acquisition and analysis program (Ultra Violet Products Ltd.).
Sections were washed with PBS and permeated 0.1% Triton X-100 in 0.1% sodium citrate for 2 min on ice, reacted with TUNEL reaction mixture (Roche, Indianapolis, IN) for 30min at 37°C, rinsed with PBS, then reacted with 4′-6-diamidino-2-phenylindole (DAPI) for 1 min, and then mounted with anti-fade reagent (Invitrogen, Eugene, OR).
Myeoloperoxidase (MPO) Activity in Kidney Tissues
MPO activity in kidney tissues was measured as described previously (16).
Results are expressed as the mean±the SE. Statistical differences among groups were calculated using the paired Student's t test. Differences between groups were considered statistically significant at a p value of <0.05.
I/R Induces Infiltration of Macrophages
Ischemia-induced severe tubular epithelial cell disruption in the outer stripe of the outer medulla that was coupled with functional impairments (Fig. 1B). Plasma creatinine levels were not distinguishable from base line levels 8 days after ischemia, indicating that renal function had recovered (Fig. 1A). Numerous infiltrated cells, including macrophages, were observed in the interstitium of the kidneys exposed to I/R (Fig. 1B).
Clodronate Administration Reduces the Levels of Infiltrated Macrophages
To deplete tissue macrophages, we administered either Lipo-clodronate or Lipo-PBS into the tail vein 6 days after either ischemia or sham-operation. Three days after treatment, the kidneys and spleens were harvested and the number of CD68-positive cells was determined. Although CD68 antibody is commonly used to detect macrophages, CD68 is also expressed in dendrite cells (17). The number of CD68- positive cells in kidneys harvested from mice treated with Lipo-clodronate was significantly lower than the number of CD68-positive cells in kidneys harvested from mice treated with Lipo-PBS (Fig. 2A and 2B). Similar to the results observed in the kidneys, the number of CD68-positive cells in spleens harvested from mice treated with Lipo-clodronate was significantly lower than the number of CD68-positive cells observed in spleens harvested from mice treated with Lipo-PBS (Fig. 2C and 2D). These results indicate that, despite the possibility that Lipo-clodronate-administration could affect dendritic cells, it effectively removed macrophages from the kidney and spleen. It should be noted that although the removal of macrophages was effective, it was not complete, which is similar to the results reported by Day and colleagues (14).
High Levels of Postischemic Apoptotic Cells and Cell Debris Are Present in the Kidneys of Mice Treated With Lipo-Clodronate
Macrophages are responsible for phagocytosis in dying cells. We treated mice with either Lipo-clodronate or Lipo-PBS 6 days after they had been subjected to ischemia and then used a TUNEL assay to determine the number of apoptotic cells 3 days after the Lipo-clodronate or Lipo-PBS treatment. There were no significant differences in the levels of PCr between Lipo-clodronate and Lipo-PBS treated mice at 2 days or 3 days after treatment (data not shown); however, there were twofold more TUNEL-positive cells in kidneys harvested from mice treated with Lipo-clondronate than in those harvested from mice treated with Lipo-PBS (Fig. 3A and 3B). Additionally, kidneys from mice treated with Lipo-clodronate exhibited excessive debris composed of disrupted tubules when compared wth kidneys from mice treated with Lipo-PBS (Fig. 3C and 3D), which suggests that macrophages that infiltrate the kidney after I/R eliminate dying cells and cell debris (18, 19). In addition, kidney damage was significantly more severe in Lipo-clodronate-treated mice than in Lipo-PBS-treated mice (Fig. 3D and 3E).
Removal of Infiltrated Macrophages Increases the Formation of ROS and RNS
I/R produces excessive amounts of ROS, which induce oxidative stress in cells. To examine whether infiltrated macrophages affected ROS and reactive nitrogen species (RNS) formation, we conducted NBT staining and immunostaining with anti-nitrotyrosine antibody to evaluate superoxide levels and peroxinitrate levels (20), respectively. Superoxide produces blue formazan by reacting with nitro blue tetrazolium (NBT). Formation of blue formazan was 8-fold higher in kidneys harvested from mice treated with Lipo-clodronate than in those harvested from mice treated with Lipo-PBS (Fig. 4A, B). Similarly, peroxynitrite levels in kidneys treated with Lipo-clodronate were significantly greater than those in kidneys treated with Lipo-PBS (Fig. 4C and 4D).
Removal of Infiltrated Macrophages Affects Cell Proliferation
To examine whether infiltrated macrophages affected cell proliferation after I/R, we used immunoblot and immunohistochemical analysis to evaluate PCNA expression. After 30 min of bilateral renal ischemia, PCNA expression peaked within 2–3 days, then gradually returned to baseline. The number of PCNA-positive cells in kidneys harvested from mice treated with Lipo-clodronate was approximately 2-fold greater than that of kidneys harvested from mice treated with Lipo-PBS (Fig. 5A and 5B). Similarly, the level of PCNA protein expression was greater in kidneys harvested from mice treated with Lipo-clodronate than in those harvested from mice treated with Lipo-PBS (Fig. 5C and 5D). PCNA is a cofactor for DNA polymerase that is associated not only with DNA repair of damaged cells but also with the proliferation of cells. Therefore, the increased PCNA expression that was observed in clodronate-treated animals when compared with animals that were treated with PBS may indicate greater proliferation of cells and less removal of damaged cells. Overall, these results indicate that infiltrated macrophages are associated with postischemic repair processes and that removal of infiltrated macrophages may delay recovery after I/R.
Depletion of Infiltrated Macrophages With Clodronate-Administration Does Not Abolish Kidney Resistance Afforded by Ischemic Preconditioning
To determine whether the protection afforded by ischemic preconditioning was associated with infiltrated macrophages, we treated mice that had been subjected to either 30 min of bilateral renal ischemia (preconditioned) or sham-operation (nonpreconditioned) 6 days earlier with Lipo-clodronate or Lipo-PBS. The mice were then subjected to 30 min of bilateral renal ischemia 2 days after treatment, and the plasma creatinine and BUN levels were then determined 24 hr after the second ischemia. In nonpreconditioned mice, administration of Lipo-clodronate significantly inhibited postischemic increases of PCr (Fig. 6B) and BUN (Fig. 6A), indicating that removal of macrophages reduces kidney susceptibility to I/R injury (Fig. 6A an 6B). These results are consistent with those reported by Day et al., which showed that macrophage depletion by Lipo-clodronate prevented postischemic increases in plasma creatinine (14). As previously reported (11), ischemic preconditioning protected the kidney from 30 min of bilateral renal ischemia 8 days later (Fig. 6). In preconditioned mice, PCr and BUN levels were unchanged in Lipo-clodronate treated mice (Fig. 6), indicating that removal of infiltrated macrophages did not affect the kidney resistance afforded by ischemic preconditioning. Interestingly, when nonpreconditioned mice were treated with Lipo-clodronate 2 days before exposure to a second ischemia, the postischemic increase of plasma creatinine was lower than that of the Lipo-PBS-treated mice (Fig. 6A). To determine whether the protective effect of Lipo-clodronate is associated with inflammatory responses, we determined the activity of MPO by biochemically measuring changes in the MPO activity and by evaluating neutrophil infiltration by immunohistochemical staining using anti-Ly-6G antibody. Both MPO activity (Fig. 6C) and neutrophil infiltration (Fig. 6D and 6E) were significantly lower in the kidneys of clodronate-treated mice than in the kidneys of PBS-treated mice. These results suggest that the lower increases in BUN and PCr levels in the clodronate-treated mice that were not preconditioned may be associated with lower inflammatory responses, which subsequently result in reduced production of cytokines, oxygen-free radicals, and proteases.
I/R results in apoptosis and necrosis (21). Dying cells and dead cell debris stimulate cytokine and ROS production from normal or modestly damaged cells (19, 22), leading to increased stress in the injured sites, which induces infiltration of macrophages into the damaged sites. Macrophage infiltration into the injured tissues exacerbates the cell damage by the production of harmful factors, including cytokines, chemokines, and ROS (1, 2). It has been reported that excessive oxidative stress delays the recovery of the ischemic kidney and results in chronic inflammation, which can cause chronic renal failure (23). Interestingly, there is accumulating evidence that macrophages that have infiltrated injured tissues reduce inflammatory responses through the ingestion of dying cells or dead cell debris, thereby mitigating tissue damage (24, 25). In the present study, the amount of apoptotic cells and cell debris in the kidneys of mice that had been treated with Lipo-clodronate was greater than in kidneys from control mice. In addition, levels of superoxide and peroxinitrite were greater in kidneys from mice that had been treated with Lipo-clodronate. Overall, these results suggest that infiltrated macrophages may mitigate oxidative stress through the ingestion of damaged cell debris and dying cells, which in turn accelerates the cell repair process.
Kidneys damaged after I/R recover structurally and functionally within several days of the recovery of renal blood flow (11, 16). Although the precise mechanisms by which the regenerative process occurs are unknown, it has been reported that regeneration is achieved via the migration and proliferation of normal cells (26, 27). Recently, it has been demonstrated that macrophages accelerate the repair of injured tissues (5, 9). Macrophage colony-stimulation factor, which induces infiltration of macrophages, accelerates the repair of damaged heart tissue (10). The repair of damaged tissue is related to growth factors, including hepatic growth factor and vascular endothelial growth factor, which are secreted by macrophages (28). In postmyocardial injury, infiltrating macrophages promote wound healing through secretion of transforming growth factor-β and vascular endothelial growth factor-α (25). After 30 min of bilateral renal ischemia in mice, cell proliferation peaks within 3 days, then gradually decreases over time to nearly base line levels within 8 days of the ischemia (12, 29). Witzgal et al. reported that postischemic regeneration peaked within 3 days in rat kidneys (30). In this study, plasma creatinine levels were not distinguishable from baseline levels 9 days after ischemia. Further, after 9 days, the amount of PCNA-positive cells in kidneys from mice that had been treated with Lipo- clodronate was higher than those of kidneys from mice treated with Lipo-PBS. Overall, these results suggest that removal of infiltrated macrophages may delay repair of damaged tissues. Interestingly, although treatment with clodronate on day 6 after ischemia delayed the repair of tissue damage, renal function and the level of inflammation were the same in Lipo-clodronate and Lipo-PBS treated mice. This dissociation may be caused by a lower sensitivity of plasma creatinine and BUN to tissue damage when compared with the use of biochemical and histological evaluation for tissue damage. We previously reported that, although plasma creatinine levels returned to normal several days after ischemia, postischemic histological changes did not (12). In that study we also observed that the kidney injury molecule-1, a sensitive marker of tubular cell injury (16, 31–33), was still expressed after the PCr levels had returned to normal (12).
Recently, we reported that ischemic preconditioned kidneys are resistant to I/R induced several days to weeks later and that this resistance is associated with various factors, including mitogen activated protein kinases and heat shock proteins (11). In addition, we found that iNOS gene-deletion mitigates the kidney resistance afforded by ischemic preconditioning (12). Because macrophages are accumulated in the kidneys after ischemic preconditioning and express iNOS (12), we examined whether infiltrated macrophages are associated with the ischemic preconditioning phenomenon. In the present study, clodronate-administration did not mitigate the resistance afforded by ischemic preconditioning. In contrast, clodronate-administration into non-preconditioned mice reduced postischemic kidney functional impairment, suggesting that macrophages and neutrophils recruited soon after I/R may accelerate cell damage. Day et al. and Jo et al. conducted experiments similar to ours and found that macrophage depletion reduced I/R-induced kidney injury (1, 14). In addition, Day et al. and others have demonstrated that a reduction in macrophages reduced the production of cytokines and the adhesion of leukocytes (14, 34, 35). Therefore, protection induced by clodronate- administration in non-preconditioned mice may be associated with a reduction in inflammatory responses, resulting in reduced production of cytokines, oxygen free radicals and proteases.
In conclusion, our results demonstrated that macrophages that have infiltrated injured tissues may accelerate the repair process by clearing dying cells and dead cell debris, and that infiltrated tissue macrophages may not be a critical contributor to ischemic preconditioning.
This work was supported by the KOSEF (F01-2006-000-10034-0 to K.M. Park) and the Stem cell research program of the Ministry of Science & Technology (M10641450001-06N4145-00110 to K.M. Park).
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