Male C57BL/6 mice weighing 23–25 g were used (Damul Science, Daejeon, Korea). They were kept at room temperature (23℃–25℃), 40%–60% relative humidity on a 12/12-hour light/dark cycle and were fed a standard pellet diet with water ad libitum. Mice (including those in the sham group) were anesthetized by intraperitoneal administration of a mixture of xylazine (10 mg/kg) and ketamine (100 mg/kg) and were placed on a heating pad under a warming light to maintain their body temperature between 36℃ and 38℃. Bilateral flank incisions were made and a right nephrectomy was performed. The left renal pedicle was occluded with a nontraumatic microaneurysm clamp to induce renal ischemia. Reperfusion was initiated by removing the clamp. Mice were randomly divided into five experimental groups (n = 5 for each group, Table 1). In the I/R group, the renal pedicle was clamped for 30 minutes and then reperfused for 24 hours. In the IP groups, just before the sustained ischemia, kidneys were subjected to 3, 5, or 7 minutes of ischemia followed by 10 minutes of reperfusion. Soon after, the renal pedicle was clamped for 30 minutes then reperfused. The data from each of these groups were compared to those of a sham group in which animals underwent only right nephrectomy. Twenty-four hours after reperfusion the mice were sacrificed and serum and tissue samples were collected and immediately fixed in 10% formalin or stored at −80℃ until further analysis. All animal procedures and experiments were performed according to a protocol approved by the Institutional Animal Care and Use Committee of the Clinical Research Institute at Daejeon St. Mary's Hospital at the Catholic University of Korea (institutional review board #CMCDJ-AP-2012-018).

Blood samples were collected 24 hours after reperfusion. BUN and serum Cr were measured using IDEXX VetTest Chemistry Analyzer (IDEXX Laboratories Inc., Westbrook, ME, USA).

Kidney tissues were fixed in 10% neutral buffered formalin and then embedded in paraffin. Tissue was sectioned at 0.5-µm and then stained with hematoxylin and eosin (H&E) and periodic acid-Schiff reagent (PAS) for light microscopic analyses. Histopathologic damage was defined as tubular epithelial swelling, loss of brush border, vacuolar degeneration, necrotic tubules, cast formation, and desquamation. The degree of tubular injury was estimated at ×400 magnification using 5 randomly selected fields for each kidney using the following standard: 0, normal; 1, damage involving <25% of tubules; 2, damage involving 25%–50% of tubules; 3, damage involving 50%–75% of tubules; and 4, damage involving 75%–100% of tubules.

Apoptotic nuclei were identified with deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining using the ApopTag Plus Peroxidase in situ Apoptosis Kit (Millipore, Billerica, MA, USA). TUNEL-positive areas from 2 to 3 fields (×400)/animal were quantified with Image J. Results are expressed as total area percent and were quantified from 5 animals per group.

Kidney tissues were homogenized using TissueLyser II (Qiagen, Hilden, Germany) in radioimmunoprecipitation assay (RIPA) buffer (Elpis Biotech., Daejeon, Korea) containing protease inhibitor cocktail tablets (Roche Diagnostics GmbH, Mannheim, Germany). Homogenates were centrifuged at 13,000 rpm for 15 minutes at 4℃, and the supernatants were collected. Nuclear proteins were extracted using a Nuclear/Cytosol Fractionation kit (BioVision, Mountain View, CA, USA) according to the manufacturer's instructions. After measuring the protein concentration (BCA Protein Assay Kit; Pierce, Rockford, IL, USA), equal amounts of protein were separated by electrophoresis on a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred electrophoretically onto a nitrocellulose membrane (Pall Corporation, Ann Arbor, MI, USA). After blocking with 5% nonfat powdered milk, the membrane was incubated overnight at 4℃ with primary antibodies directed against Cleaved caspase-3 (Cell Signaling Technology, Beverly, MA, USA), Bax (Cell Signaling Technology), Bcl-2 (Cell Signaling Technology), and TLR4 (Abcam, Cambridge, MA, USA) or NF-κB (Abcam). They were then incubated with horseradish peroxidase-conjugated anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-mouse IgG (Invitrogen, Carlsbad, CA, USA). Positive bands were detected and analyzed by chemiluminescence technology using the ChemiDoc XRS+ (Bio-Rad Laboratories, Hercules, CA, USA). As loading controls, each membrane was probed with GAPDH (Cell Signaling Technology) or Histone H3.1 (Santa Cruz Biotechnology).

Total RNA was extracted from kidney tissues with TRIzol reagent (Invitrogen) according to the manufacturer's instructions. cDNA was synthesized from 1 µg RNA using Reverse Transcriptase Premix (Elpis Biotech). The expression levels were quantified by Power SYBR Green PCR Master Mix (Applied Biosystems, Warrington, UK) according to the manufacturer's instructions. The following primer sets (sense sequence and antisense sequence, respectively) were used: Tumor necrosis factor-α (TNF-α) forward, 5'-ACG GCA TGG ATC TCA AAG AC-3', reverse, 5'-GTG GGT GAG GAG CAC GTA GT-3'; Interleukin-1β (IL-1) forward, 5'-GCC CAT CCT CTG TGA CTC AT-3', reverse, 5'-AGG CCA CAG GTA TTT TGT CG-3'; Interleukin-6 (IL-6) forward, 5'-GAT GCT ACC AAA CTG GAT ATA ATC-3', reverse, 5'-GGT CCT TAG CCA CTC CTT CTG TG-3'; monocyte chemoattractant protein-1 (MCP-1) forward, 5'-CCA GCA AGA TGA TCC CAA TGA G-3', reverse, 5'-CTC TCT CTT GAG CTT GGT GAC AAA A-3'; glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward, 5'-TGC AGT GGC AAA GTG GAG ATT-3', reverse, 5'-CGT GAG TGG AGT CAT ACT GGA ACA-3'. Each reaction was performed by ABI 7500 FAST (Applied Biosystems, Foster City, CA, USA) in triplicate. The relative levels of mRNA were normalized to GAPDH.

Graph Pad Prism 5 (Graph Pad Inc., La Jolla, CA, USA) was used to analyze and present data. Differences between groups were analyzed using a paired parametric t-test or a one-way analysis of variance. Values are expressed as the mean ± standard error of the mean. A P-value of <0.05 was considered statistically significant.

The levels of serum BUN and Cr were significantly increased in the I/R group and were decreased in the IP-5 group (P < 0.01) (Fig. 1A). Histopathologic analysis revealed swelling in the renal tubular epithelial cells, vacuole degeneration, disappearance of the brush border, coagulation necrosis, and massive inflammatory cell infiltration in the I/R group compared with the sham group. However, IP reduced this severe renal damage (Fig. 1C). Quantitative analysis revealed a markedly decreased tubular injury score in the IP groups compared with the I/R group (P < 0.05) (Fig. 1B).

Although the major cause of cell death during renal I/R injury is necrosis, apoptotic cell death is also observed during the reperfusion process. The extent of apoptosis was evaluated by TUNEL staining (Fig. 2A). The number of TUNEL-positive apoptotic cells was markedly decreased in IP-injured mice compared to that in the I/R group (P < 0.05) (Fig. 2B). Increased protein levels of anti-apoptotic Bcl-2 and decreased protein levels of pro-apoptotic cleaved caspase-3 and Bax were observed in the IP groups (Fig. 2C, D).

The inflammatory response following I/R injury is related to renal dysfunction. To investigate whether IP mitigated renal dysfunction, we investigated the mRNA expression of TNF-α, IL-1β, IL-6, and MCP-1, which are key inflammatory cytokines produced by infiltrated inflammatory cells. Real-time RT-PCR analysis confirmed decreased accumulation of macrophages and suppressed inflammation in IP groups compared to the I/R group (P < 0.05) (Fig. 3).

Kidney TLR4 protein expression was significantly elevated after I/R, but not in the IP groups (Fig. 4A, B). IP groups had significantly decreased NF-κB expression induced by IR (Fig. 4C, D).