All experiments conformed to the guidelines of and were approved by the Institutional Laboratory Animal Care and the Ethical Committee of the Catholic University of Korea, Seoul, Korea (#2011-0035) on 8 February 2011. All animals were housed in separate cages with a 12 hr light-dark cycle and sufficient water and food. Mice were allowed to acclimatize for 7 days before the experiments. All experiments on mice were performed during the period from March 1 to 31, 2011.

Male C57BL/6 mice (25-30 g) were anesthetized with intraperitoneal pentobarbital, 50 mg/kg or to effect. Mice were placed under a heating lamp and on a 37℃ heating pad. After a midline laparotomy and intraperitoneal application of 20 U of heparin, all mice had a right nephrectomy and dissection of the left renal and hepatic pedicle. The mice were divided into four groups. Group I (sham, n = 8) had a sham operation to assess the effect of laparotomy and dissection of the left renal and hepatic pedicle after right nephrectomy. In group II (IR, n = 8), group III (IPC-IR, n = 8) and group IV (MPG - IPC + IR, n = 8), renal IR injury was created by placing a microvascular clamp on the left renal pedicle for 30 min (1, 7). In group III, hepatic IPC was achieved by brief clamping of the hepatic pedicle for 10 min, followed by 10 min of reperfusion (3), before renal IR injury. In group IV, mice were pretreated with 100 mg/kg N-(2-mercaptopropionyl)-glycine (MPG; a free radical scavenger) 15 min before hepatic IPC and were subjected to renal IR injury (7).

Twenty-four hours after renal IR injury, all mice were re-anesthetized and plasma samples and left kidneys were obtained. For studying the renal effect of hepatic IPC, mice (n = 16) were subjected only to hepatic IPC without renal IR injury, and the left kidney was isolated 15 min (acute, n = 8) or 24 hr (delayed, n = 8) later. The experiments with pretreated MPG mice (n = 16) were performed in the same way.

The harvested kidneys were quickly frozen using liquid nitrogen and stored at -80℃. They were dissected on ice and immediately placed on ice-cold radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 50 mM Tris-HCl, 1 mM EDTA, and 1% Triton-X100 [pH 7.4]) and homogenized for 10 sec on ice. Tissues were centrifuged at 32,500 g and 4℃ under a vacuum for 1 hr. The supernatant was collected and used for immunoblotting as described previously (1, 7). Samples were stabilized with Laemmeli buffer after determining their protein amounts by Bradford assay (BioRad, Hercules, CA, USA) using bovine serum albumin as standard. The proteins, separated by 4 hr 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis at 80 V, were transferred to polyvinylidene fluoride membranes. Membranes were washed with Tris-buffered saline containing 0.1% Tween-20 (TBST) and coated with a blocking solution (5% dry milk in TBST). Measurements of iNOS, phosphorylated Akt (p-Akt), and total Akt were made by immunoblotting. The primary antibodies for p-Akt and total Akt were from Cell Signaling Technologies (Danvers, MA, USA). iNOS antibody was from BD Biosciences Pharmingen (San Jose, CA, USA). The secondary antibody (goat anti-rabbit or anti-mouse IgG conjugated to horseradish peroxidase at 1:5,000 dilution) was detected with enhanced chemiluminescence immunoblotting detection reagents (Amersham, Piscataway, NJ, USA), with subsequent exposure to a CCD camera coupled to a UVP Bio-imaging system (UVP, Upland, CA, USA). The band intensities of the immunoblots were within the linear range of exposure for all experiments.

Semi-quantitative RT-PCR was carried out to analyze the expression of the proinflammatory cytokines (TNF-α) and intercellular adhesion molecule 1 (ICAM-1) (8, 9). A 1 mL aliquot of Trizol reagent (MRC, Cincinnati, OH, USA) was added to the harvested kidneys, which were homogenized and allowed to stand for 5 min. After adding 0.2 mL 1-bromo-3-chloropropane (Sigma-Aldrich, St. Louis, MO, USA), centrifugation was carried out for 15 min at 14,000 g. Subsequently, after separating supernatants and adding an equal amount of phenol: chloroform (5:1; pH 4.7) (Sigma-Aldrich), centrifugation was carried out for 10 min at 14,000 g. After separating supernatants, adding 100% ethanol and allowing the solution react for 1 hr at -20℃, centrifugation was carried out for 10 min at 14,000 g. The solution was kept in at -70℃ after discarding supernatants, washing RNA deposits with 75% ethanol, adding diethylene-pyrocarbonate water, and allowing the solution to react for 5 min at 60℃. RNA concentrations were determined by spectrophotometric absorbance at 260 nm. With extracted RNA (0.5 µg) as the template, cDNA (50 µL) was composed using avian myeloblastosis virus (AMV) reverse transcriptase (Promega, Madison, WI, USA). PCR was done after generating the last reaction liquid 10 µL, including dNTPs (each at 2.5 mM/L), 1 unit I Start Taq DNA polymerase (Intron, Seoul, Korea), and each primer of 20 µM using composed cDNA of 2 µL (Table 1). PCR was done for 35 cycles through 30 sec denaturation at 95℃, 30 sec annealing at 55℃, and 1 min extension at 72℃, and completed after reacting for 10 min at 72℃. β-actin (228 bp) was the internal control gene to determine whether appropriate mRNA had been separated from cells. Extended PCR products were quantified using Gel DocTM XR (BioRad) after 2.0% agarose gel electrophoresis and staining with ethidium bromide dye.

Renal function was assessed by measurement of serum creatinine 24 hr after renal IR injury. Serum creatinine levels were measured spectrophotometrically using a commercially available quantitative colorimetric assay (Sigma-Aldrich) (10).

The harvested kidneys were fixed in 10% formalin solution, embedded in paraffin, sectioned at 5 µm thickness, and stained with hematoxylin-eosin. Morphological assessment was performed by an experienced renal pathologist who was unaware of the treatment that the animal had received. A previously described grading scale of 0-4 (11) was used for the histopathological assessment of ischemia and reperfusion-induced damage of the proximal tubules.

Data are mean ± SEM. Statistical processing was done using SPSS 12.0 (SPSS, Chicago, IL, USA). Data were analyzed using t test or the analysis of variance (ANOVA). The ordinal values of the Jablonski scale were analyzed by the Kruskal-Wallis non-parametric test with Dunnett's test comparison among groups. A P value of < 0.05 was considered statistically significant.

Kidneys collected 15 min after hepatic IPC displayed elevation of p-Akt by 85% without affecting iNOS or Akt expression (P = 0.011) (Fig. 1). Kidneys collected 24 hr after hepatic IPC displayed a significant elevation (44%) in the expression of iNOS (P = 0.038), but elevated expressions of p-Akt at the acute phase were abolished. On the other hand, elevations of protective proteins caused by hepatic IPC did not appear in acute and delayed phase of pretreated MPG mice (Fig. 1).

Kidney inflammation was determined by the measurement of mRNA-encoding markers of inflammation, including TNF-α and ICAM-1 at 24 hr after renal IR injury. Although there were significant elevations in TNF-α and ICAM-1 level in group II (66.7 ± 8.7 and 91.9 ± 13.7; P < 0.001, respectively) vs group I (44.6 ± 7.5 and 61.7 ± 8.9, respectively), these elevations were markedly attenuated in group III (52.4 ± 7.4 and 67.3 ± 10.1, respectively; P = 0.003 and P = 0.001) vs group II (Fig. 2). Proinflammatory genes were significantly re-elevated in MPG pretreated group IV (62.6 ± 8.3 and 89.7 ± 12.8, respectively; P = 0.022 and P = 0.002) vs group III (Fig. 2).

As expected, serum creatinine at 24 hr after reperfusion was significantly elevated in group II (IR, 2.7 ± 0.6 mg/dL; n = 8; P < 0.001) and group IV (MPG - IPC + IR, 2.2 ± 0.5 mg/dL; n = 8; P < 0.001) compared with group I (sham operation, 0.4 ± 0.1 mg/dL; n = 8) (Fig. 3). Hepatic IPC in group III (IPC + IR, 0.6 ± 0.1 mg/dL; n = 8; P < 0.001) significantly improved renal function compared with group II. However, renal protective effects of hepatic IPC were lost by MPG treatment in group IV (P < 0.001) vs group III (Fig. 3).

Histopathologic findings at 24 hr after reperfusion were nearly normal in hepatic IPC treated animals (group III) (Fig. 4). In contrast, several areas of tubular dilation and necrosis, medullary luminal congestion, and hemorrhage were present in the kidneys of mice subjected to IR (group II) and pretreated MPG before hepatic IPC and renal IR (group IV) (Fig. 4). Jablonski histology grading scores in the outer medullary of kidneys are shown in Fig. 5. Groups II and IV resulted in severe acute tubular necrosis (grade 3.5 ± 0.5 and 3.3 ± 0.5, respectively; P < 0.001) vs sham grade (0.2 ± 0.3). Group III (grade 1.1 ± 0.6; P < 0.001) showed significantly improved necrosis scores compared with groups II and IV.