This study used male C57BL/6 mice aged 8-10 weeks (Jackson Laboratories, Bar Harbor, Maine, USA).

Mice weighing 20~25 g each were anesthetized with an intraperitoneal injection of Avertin (2,2,2-tribromoethanol: Sigma-Aldrich, Milwaukee, Wisconsin, USA). A midline incision was made and the renal pedicles were bilaterally clamped for 22 min with using microaneurysm clamps. The clamps were then removed and the kidneys were observed to ensure they had regained their original color, which indicated the restoration of blood flow. The abdomen was then closed in two layers. The ischemia time that was chosen provided a reversible model of ischemic ARF and it didn't cause animal mortality. This method resulted in a serum creatinine peak at 24-48 h after reperfusion commenced, and the serum creatinine levels gradually returned to normal within 5-7 d. The control mice underwent sham surgery that consisted of the same surgical procedure except that clamps were not applied. During the 24-h reperfusion period, the animals were kept in an incubator at 29℃. Blood samples were obtained by cardiac puncture at 2, 8, 16 and 24 h after surgery. The BUN and creatinine levels were measured using an Astra Autoanalyser (Beckman Instruments Inc, Fullerton, California, USA). Each time point measurement represents the mean±the standard error of the mean (SEM) from 6 mice.

Kidney tissues were embedded in an optimal cutting temperature (O.C.T.); they were snap-frozen in liquid nitrogen and then stored at -80℃ until sectioning. The cut cryostat sections (5 µm) were fixed in 70% acetone/30% methanol for 5 min, air dried for 5 min, rinsed in phosphate-buffered solution (PBS) for 3 min three times, soaked in 3% paraformaldehyde for 10 min and then rinsed in PBS for 3 min three times. To determine the CD 11b expression, the sections were preincubated with 10% donkey serum for 30 min, and they were next incubated with anti-mouse CD 11b antibody (1:50 dilution in 5% donkey serum in PBS) for 1 h at room temperature; they were rinsed in PBS for 5 min three times and then incubated in anti-donkey horseradish peroxidase (HRP) secondary antibody in 5% donkey serum in PBS. The sections were counterstained by incubating them with 7:1000 Cy3, 1:1,000 wheat germ agglutinin and Alexa Fluor 488 conjugate (green) to identify the cytoplasm and they were incubated with 3:1,000 Hoechst 33,342 (blue) for 60 min at room temperature to identify the nucleus. They were next rinsed in PBS for 5 min three times and then they were rinsed in water for 2 min; they were placed on a slide with mounting medium and a coverslip. The fluorescent images were captured using a digital camera and these were merged with the phase-contrast microscope images. To measure macrophage infiltration, the kidney sections were stained to determine the number of cells expressing CD 11b. Fifteen randomly chosen high-powered fields (×400) of the corticomedullary junction were assessed by a renal pathologist, who was kept "blind: as to how the sections were treated. Tissues from the control (n=4) and ischemia mice (2, 8, 16 and 24 h; n=4 for each time point) were examined.

Clodronate [dichloromethylene bisphosphonate (Cl₂MBP)] was prepared according to the method of van Rooijen et al18). Phosphatidylcholine [86 mg; egg lecithin (20 mg/mL) in chloroform] and cholesterol (8 mg; 8 mg/10 mL chloroform) were evaporated via rotation under reduced pressure (vacuum pump; Rotations evaporator R-114, Buchi Labortechik, Flavil, Switzerland). A suspension of 2.5 g Cl₂MBP in 10 mL PBS was stored under N₂ for 2 h; this was sonicated and then stored overnight at 4℃. The liposomes were centrifuged for 30 min (10,000 g, 4℃) and they were resuspended in 4 mL PBS before use.

Clodronate (100 µL/10 g mouse) was injected via a tail vein 5 days before ischemia induction and again 2 days before ischemia induction. At 24 h post-ischemic reperfusion, the mice' blood samples were analyzed for monocytes, and the spleens were prepared for flow cytometry as we have previously described15). Empty liposomes, which did not containing clodronate, were prepared in the same manner as the LECs, and the empty liposomes were used as the vehicle (V). Staining was detected by a Beckton-Dickinson FACsCalibur (BD Immunocytometry Systems, San Jose, CA) flow cytometer. CellQuest Software (BD Immunocytometry Systems) was used to analyze the flow cytometry data of the mice that were treated with LECs. Following ischemia induction, the serum creatinine levels were determined.

The values are expressed as means±SEMs. The non-normally distributed data was analyzed by the nonparametric unpaired Mann-Whitney test. Multiple-group comparisons were performed by the Kruskal-Wallis test and using post-tests. A p value < 0.05 was considered to indicate a significant difference.

The serum creatinine level in the control mice (n=6) was 0.17±0.03 mg/dL at 24 h after sham surgery. The serum creatinine levels at 2, 8, 16 and 24 h from the ischemia mice were 0.30±0.06, 0.37±0.1, 0.63±0.03 (p<0.05 vs. the control and at 2 h and 8 h post-ischemia) and 2.07±0.12 mg/dL (p<0.05 vs. the control and at 2 h, 8 h and 16 h post-ischemia) (n=6 for each time point), respectively. The BUN level in control mice (n=6) was 25.0±2.0 mg/dL at 24 hr after sham surgery. The BUN levels at 2, 8, 16 and 24 h from the ischemia mice were 48.0±2.8, 45.6±4.2, 72.0±3.0 (p<0.05 vs. control and 2 h and 8 h post-ischemia) and 156.0±8.5 mg/dL (p<0.05 vs. the control and at 2 h, 8 h and 16 h post-ischemia) (n=6 for each time point), respectively (Figure 1A and 1B).

The number of CD 11b-postive cells in the sections from the control and at 2, 8, 16 and 24 h from the ischemia mice was 0.2±0.1, 3.5±0.3, 2.9±0.4, 13.8±1.5 and 11.3±0.7, respectively (n=4 for each time point) (Figure 2A). This data showed there was a significant increase in the number of CD 11b-positive cells in the 2 h ischemia mice compared to the controls (p<0.01), and that macrophage infiltration continued to increase to 16-24 h after ischemia was induced. The representative sections are shown in Figures 2B-F.

We determined the number of CD 11b-positive cells in various kidney regions. This analysis showed there were 1.1±0.3, 13.2±0.9 and 1.6±0.3 CD 11b-positive cells in the 24 hr post-ischemia sections from the cortex, outer medulla and inner medulla, respectively (p<0.01, n=4 for each group, Figure 3A). The representative images are shown in Figures 3B-D.

The numbers of blood monocytes (10⁶/L) were 55±18 in the sham-operated mice and 145±38 in the ischemic ARF with vehicle (ARF-V) mice and 82±24 in the ischemic ARF with clodronate (ARF-LEC) mice. There was a significant decrease of the blood monocytes in the ARF-LEC mice compared to the ARF-V mice. Administering LECs resulted in a 70% reduction of the macrophages in the spleen (data not shown) and a significant decrease of macrophage infiltration in the kidney of the ischemic ARF mice (^*p<0.01 vs. the sham operated, ARF-V mice, ^**p<0.05 vs. the sham operated mice, n=4 for each group) (Figure 4A). The representative images are shown in Figures 4B-D. The ARF-V mice had higher serum creatinine levels compared to the sham operated mice and the ARF-LEC mice (0.20±0.01 vs 2.10±0.79 vs 1.09±0.67 mg/dL, respectively, ^*p<0.05, n=8). The ARF-LEC mice had lower serum creatinine levels compared to the ARF-V mice (1.09±0.67 vs. 2.10±0.79 mg/dL, respectively, ^**p<0.05, n=8 for each group) (Figure 4E).