Abstract
Objectives
Bilateral lower limb ischemia–reperfusion (I/R) could cause significant oxidative stress, elicit inflammatory response, and subsequently induce kidney injury in animals. We tested the effects of platonin, a potent antioxidant, on mitigating the kidney injury induced by lower limb I/R in rats.
Methods
Adult male rats were allocated to receive I/R or I/R plus platonin (100 μg/kg intravenous injection immediately after reperfusion), and denoted as the I/R or the I/R-P group, respectively (n = 10 in each group). Sham groups were run simultaneously. Bilateral lower limb I/R was achieved by applying rubber-band tourniquets high around each thigh for 3 hours, followed by reperfusion for 6 hours. After sacrifice, the level of kidney injury was assayed.
Results
I/R significantly increased the plasma concentrations of blood urea nitrogen (BUN) and creatinine (Cr). However, this effect could be mitigated by platonin, as the plasma concentrations of BUN and Cr of the I/R-P group were significantly lower than those of the I/R group. Moreover, histological findings revealed moderate injury in kidney tissues of the I/R group and mild injury in those of the I/R-P group. In addition, the leukocyte infiltration and myeloperoxidase activity in kidney tissues as well as the renal concentrations of inflammatory molecules (i.e., cyclooxygenase-2/prostaglandin E2, interleukin-6, and macrophage inflammatory protein-2) and malondialdehyde (i.e., the index of lipid peroxidation) of the I/R group were significantly higher than those of the I/R-P group.
Conclusion
Platonin attenuates kidney injury induced by bilateral lower limb I/R in rats.
Keywords
cytokines; chemokines; ischemia; malondialdehyde; prostaglandins: E2; reperfusion injury;
1. Introduction
Reperfusion of the acutely ischemic limb is a typical ischemia–reperfusion (I/R) injury that may cause injuries to vital organ, including the lungs and kidneys.1 Abundant data indicated that overproduction of reactive oxygen species (ROS) and proinflammatory molecules, and the subsequent inflammatory response induced by I/R is one of the most crucial underlying mechanisms.1, 2, 3, 4 This concept is supported by previous data that the I/R-induced organ injuries could be mitigated by therapies aiming at decreasing the oxidative stress and/or inflammatory response.2, 5, 6, 7
Platonin is a potent antioxidant.8 It is also a potent immunomodulator and is clinically used for rheumatoid arthritis therapy.9 In addition, previous data revealed that platonin could inhibit the endotoxin-induced upregulation of inflammatory molecules.10, 11 It could mitigate circulatory failure and decrease mortality in septic animals.12 Our previous data also revealed that the acute organ injury induced by hemorrhagic shock could be mitigated by platonin.13 Taking together, these data confirmed the potent anti-inflammation capacity of platonin.
Theoretically, platonin may exert protective effects and mitigate acute organ injuries induced by lower limb I/R. This concept was supported by our recent data that the I/R-induced lung injury could be mitigated by platonin.14 However, the question of whether platonin can protect kidney from the insult imposed by I/R remains unanswered. To elucidate further, we thus conducted this study. Our hypothesis was that platonin could mitigate kidney injury induced by lower limb I/R in rats.
2. Materials and methods
A total of 40 adult male Sprague–Dawley rats (200–250 g; BioLASCO Taiwan Co., Ltd, Taipei, Taiwan) were used for the experiment. All rats were fed a standard laboratory chow and were provided water ad libitum until the day of experiment. The animal study was approved by the Institutional Animal Use and Care Committee of Buddhist Tzu Chi General Hospital, Taipei branch, and the care and handling of the animals were in accordance with National Institutes of Health guidelines.
2.1. Animal preparation
All rats were anesthetized with ketamine/xylazine (110/10 mg/kg, intraperitoneal), and then two polyethylene (PE-50) catheters were placed in the right carotid artery (for continuous blood pressure monitoring) and the right external jugular vein (for intravenous injection), ##respectively. After tracheostomy and the tracheostomy tube insertion, all rats were mechanically ventilated with a small animal ventilator (tidal volume: 4 mL of room air; rate: 35 breaths/min; Harvard Apparatus, South Natick, MA, USA). Hemodynamic parameters, including mean arterial pressure (MAP) and heart rate (HR), were continuously monitored (BIOPAC System, Santa Barbara, CA, USA) throughout the experiments. To ensure adequate anesthesia, supplemental dose of ketamine/xylazine mixture (30/3 mg/kg, intravenous) was administered every 1 hour until the end of the experiment.
2.2. Lower limb I/R protocol
Modified from previous reports,1, 5 bilateral lower limb I/R was achieved by applying rubber-band tourniquets high around each thigh for 3 hours, followed by reperfusion for 6 hours.
2.3. Experimental protocols
Rats were randomly allocated to one of the four groups (n = 10 in each group), i.e., the sham instrumentation (Sham), Sham instrumentation plus platonin (Sham-P), I/R, and I/R plus platonin (I/R-P) groups. Rats of the I/R-P and Sham-P groups received 100 μg/kg platonin dissolved in 0.5 mL normal saline intravenously immediately after reperfusion, while those in the Sham group received sham instrumentation at comparable time points. In addition, rats of the Sham and I/R groups also received 0.5 mL normal saline intravenously at comparable time points to offset the falling-short volume of the vehicle. The dosage of platonin (i.e., 100 μg/kg) was determined according to previous data that platonin could protect septic rats from circulatory failure and mortality at the dosage of 100 μg/kg.12 After reperfusion for 6 hours, all rats were sacrificed with a high-dose pentobarbital injection.
2.4. Tissue sample collection
At the end of each experiment, 5 mL of blood was drawn and centrifuged to separate the plasma. Then, both kidneys were removed. The left kidney was snap frozen in liquid nitrogen and stored at −80°C for subsequent analysis. The right kidney was immersed in 10% formaldehyde for histological analysis.
2.5. Biochemical analysis
Rises in plasma concentrations of blood urea nitrogen (BUN) and creatinine (Cr) were established indices of kidney injury.15 To determine the plasma concentrations of BUN and Cr, the collected plasma samples were analyzed by a chemistry analyzer (Roche Reflotron 1 Chemistry Analyzer; Roche Diagnostic Corp., Indianapolis, IN, USA).
2.6. Histological analysis
The formaldehyde-immersed kidney tissues were then embedded in paraffin wax, serially sectioned, and stained with hematoxylin and eosin. Histological characteristics, including polymorphonuclear leukocyte (PMN) infiltration and fibrin thrombosis in glomeruli, interstitial edema, and vacuolar change of proximal tubular cells, were assessed with a light microscope to evaluate kidney injury, as we had previously reported.16 Each histological characteristic was scored with a scale of 0 (normal) to 5 (severe) by a pathologist who was blinded to the experiment. The overall kidney injury was categorized according to the sum of the score (0–5: normal to minimal injury; 6–10: mild injury; 11–15: moderate injury; 16–20: severe injury). In addition, the number of PMN per high-power field (HPF, 400×) in 10 randomly selected areas of each sample was counted to quantify leukocyte infiltration, which was one of the indices of kidney injury.16
2.7. Myeloperoxidase activity assay
We quantified kidney injury by measuring renal myeloperoxidase (MPO) activity, i.e., the activity of infiltrated leukocytes.16 A commercial MPO activity assay kit (MPO fluorometric detection kit; Enzo Life Sciences, Plymouth Meeting, PA, USA) was employed, and the procedures were performed according to the manufacture's protocol. In brief, snap-frozen tissue samples were weighed and added to 1× assay buffer, and then homogenized at 4°C followed by centrifugation at 10,000g for 20 minutes at 4°C. The pellets were resuspended, sonicated, and then centrifuged again. The supernatant was collected, and kidney homogenate samples of equal-volume (50 μL in triplicates) were then analyzed to determine the MPO activity of each sample.
2.8. Inflammatory molecules
Snap-frozen kidney tissues were weighed (approximately 200 mg) and then added into prechilled CelLytic MT reagent (Sigma-Aldrich, St. Louis, MO, USA) with a 1% protease inhibitor (Sigma-Aldrich) (2.5 mL per gram tissues). After incubation at 4°C for 30 minutes, the samples were homogenized and then centrifuged at 10,000g for 20 minutes at 4°C. The supernatants were then collected, and the protein concentrations of each sample were measured using a BCA assay (Pierce Biotechnology Inc., Rockfold, IL, USA). Kidney homogenate samples of equal volume (50 μL in triplicates) were then analyzed using enzyme-linked immunosorbent assay (ELISA) to determine the renal concentrations of the inflammatory molecules, including prostaglandin E2 (PGE2), cytokine [e.g., interleukin-6 (IL-6)], and chemokine [e.g., macrophage inflammatory protein-2 (MIP-2)]. Commercial ELISA kits were employed (ELISA Kits for PGE2 and IL-6; Pierce; MIP-2 ELISA kit; R&D Systems, Inc., Minneapolis, MN, USA), and the procedures were performed according to the manufacturers' protocols. Protein levels of inflammatory molecules were corrected for the total amounts of protein, and the results were expressed as pg/mL.
2.9. Reverse transcription and polymerase chain reaction
Production of PGE2 is mediated by cyclooxygenase 2 (COX-2). Transcriptional expression of COX-2 of the harvested lung tissues was measured using reverse transcription and polymerase chain reaction (RT–PCR). The primer sequences and amplification protocols for COX-2 and β-actin (as an internal standard) were adapted from the previously published studies.17, 18 After separation, PCR-amplified cDNA band densities were quantified using densitometric techniques (Scion Image for Windows, Scion Corp., Frederic, MD, USA).
2.10. Malondialdehyde assay
We quantified kidney oxidative status by measuring renal malondialdehyde (MDA) activity, i.e., the status of lipid peroxidation.16 A commercial MDA assay kit (MDA assay kit; BioVision, Mountain View, CA, USA) was employed, and the procedures were performed according to the manufacturer's protocol. In brief, snap-frozen kidney tissues (10 mg) were homogenized in 300 μL MDA lysis buffer and then centrifuged (13,000g, 10 minutes at 4°C). Then, phosphoric acid and thiobarbituric acid solution were added to 0.2 mL of homogenates. The mixture was heated in boiling water for 45 minutes. After cooling, the absorbance at 532 nm was measured and the amounts of lipid peroxides were calculated.
2.11. Statistical analysis
One-way analysis of variance with the post hoc Tukey test was used for multiple comparisons. Data were presented as means ± standard deviations. The significance level was set at 0.05. A commercial software package (SigmaStat for Windows; SPSS Science, Chicago, IL, USA) was used for data analysis.
3. Results
3.1. Hemodynamics
Comparable baseline HR and MAP of these four groups were noted (data not shown). In the Sham and Sham-P groups, HR and MAP remained stable throughout the experiment, and the end HR and MAP (i.e., measured at the end of the experiment) of the experiment of these two groups were comparable. In contrast, the end HR of the I/R group was significantly higher than that of the Sham group (p = 0.028; Table 1), whereas the end MAP of the I/R group was significantly lower than that of the Sham group (p = 0.015; Table 1). Moreover, the end HR and MAP of the I/R-P group were comparable to those of the I/R group (Table 1).
3.2. Biochemical analysis
The plasma concentrations of BUN and Cr of the Sham and Sham-P groups were comparable (Table 1). In contrast, the plasma BUN and Cr of the I/R group were significantly higher than those of the Sham group (p = 0.007 and 0.011, respectively; Table 1). However, the plasma BUN and Cr concentrations of the I/R-P group were significantly lower than those of the I/R group (p = 0.025 and 0.032, respectively; Table 1).
3.3. Histology analysis and injury scores
The kidney tissues of the Sham and Sham-P groups revealed normal to minimal kidney injury histological characteristics, as evaluated under light microscope (Figs. 1A and 1B). In contrast, the kidney tissues of the I/R group revealed moderate kidney injury histological characteristics (Fig. 1C). Moreover, the kidney tissues of the I/R-P group revealed mild kidney injury histological characteristics (Fig. 1D). Findings of the kidney injury score (Fig. 2A) paralleled the findings of the histological analysis.
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3.4. Leukocyte infiltration and MPO activity
The leukocyte infiltration (as indexed by PMN per 10 HPF) and MPO activity in kidney tissues of the Sham and Sham-P groups were low (Figs. 2B and 2C). The leukocyte infiltration and MPO activity of the I/R group were significantly higher than those of the Sham group (p = 0.012 and 0.018, respectively; Figs. 2B and 2C). In contrast, the leukocyte infiltration and MPO activity of the I/R-P group were significantly lower than those of the I/R group (p = 0.029 and 0.035, respectively; Figs. 3C and 3D).
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3.5. Concentrations of inflammatory molecules
The concentrations of COX-2 mRNA and PGE2 in kidney tissues of the Sham and Sham-P groups were low (Fig. 3A). In contrast, the concentrations of COX-2 mRNA and PGE2 of the I/R group were significantly higher than those of the Sham group (p = 0.001 and 0.013, respectively; Fig. 3A). Moreover, the concentrations of COX-2 mRNA and PGE2 of the I/R-P group were significantly lower than those of the I/R group (p = 0.023 and 0.030, respectively; Fig. 3A). Our data further revealed that findings of the IL-6 and MIP-2 (Figs. 3B and 3C) paralleled those of the COX-2 and PGE2 (Fig. 3A).
3.6. MDA assay
The MDA concentrations in kidney tissues of the Sham and Sham-P groups were low (Fig. 4). In contrast, the MDA concentration of the I/R group was significantly higher than that of the Sham group (p = 0.021; Fig. 4). Moreover, the MDA concentration of the I/R-P group was significantly lower than that of the I/R group (p = 0.024; Fig. 4).
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4. Discussion
Data from this study confirmed that bilateral lower limb I/R could cause significant kidney injury in rats. Previous data indicated that bilateral lower limb ischemia for 3 hours followed by reperfusion for 3 hours could induce significant injuries to vital organs, including the lungs and the kidney.1, 5 This concept was partially confirmed by our recent data that significant lung injury could be observed in rats that underwent the above-mentioned process of bilateral lower I/R.14 However, our preliminary data revealed that the above-mentioned I/R protocol induced only mild kidney injury in rats (data not shown). The mechanism(s) underlying this discrepancy remains to be studied. Nevertheless, previous data also indicated a positive correlation between reperfusion time and the level of kidney injury.1 Those data seemed to suggest that increasing the reperfusion time could allow the I/R-induced kidney injury to develop. Judging from those data, we thus chose to modify the I/R protocol, i.e., ischemia for 3 hours followed by reperfusion for 6 hours, and our data confirmed that this protocol could induce significant kidney injury in rats.
Our recent data demonstrated that platonin could mitigate the lung injury induced by lower limb I/R.14 As data from this study, in concert with those previous ones,1, 5, 14 indicated that the kidney tissues may react differently from the lung tissues in situations associated with lower limb I/R, it was thus possible that platonin might not be able to exert protective effects against kidney injury induced by lower limb I/R. To elucidate further in this regard, we thus conducted this study and our data confirmed that platonin could mitigate kidney injury induced by lower limb I/R in rats. Judging from these data, we thus speculate that incorporating platonin as part of the therapies should be beneficial in clinical situations associated with lower limb I/R.
It is well established that the I/R-induced overproduction of ROS is one of the most crucial mechanisms that underlie the adverse effects of I/R on inducing vital organ injuries, as the I/R-induced burst of ROS could imbalance the cellular redox condition and damage the vital pathways that are related to cellular survival, including energy metabolism, survival/stress responses, apoptosis, etc.1, 2, 3, 4, 19 Platonin possesses potent ROS scavenging capacity.8, 20 In line with this notion, we thus speculated that platonin could reduce the oxidative stress and attenuate the kidney injury induced by I/R. Data from this study confirmed our speculation, as our data revealed that the I/R-induced oxidative stress (evidenced by the increase in MDA concentration) in harvested kidney tissues could be significantly mitigated by platonin.
In addition, the I/R-induced burst of ROS could activate leukocytes and induce cytokine production and eventually lead to the induction of inflammatory response,21 i.e., another crucial mechanism that underlies the adverse effects of I/R on inducing vital organ injuries.1, 2, 3, 4 This concept was supported by our data that rats of the I/R group had significant increases in leukocyte infiltration (evidenced by histology, PMN per 10 HPF, and MPO assays) and upregulation of inflammatory molecules (including COX-2/PGE2, cytokine, and chemokine) in harvested kidney tissues. In addition to its antioxidation capacity, data from our group as well as those from the other groups revealed that platonin possesses potent anti-inflammation capacity.10, 11, 12, 13 Judging from these data, we thus further speculated that the protective effects of platonin on attenuating kidney injury induced by lower limb I/R may also involve its anti-inflammation capacity. This speculation was confirmed by our data, as we observed that the I/R-induced increases in leukocyte infiltration and upregulation of inflammatory molecules in harvested kidney tissues could be attenuated by platonin.
Although data from this study confirmed the protective effects of platonin on attenuating the I/R-induced kidney injury, certain study limitations do exist. Firstly, platonin was administered immediately after reperfusion in this study. Whether platonin could exert similar protective effects if administered in the later phase of reperfusion remains unstudied. Secondly, only one dosage of platonin was tested in this study. Whether the protective effects of platonin in this regard are dose dependent also remains unstudied. Thirdly, in addition to the lungs and kidney, liver has been reported to be subject to the influence of lower limb I/R.1 Whether platonin could protect liver from the I/R-induced adverse effects remains unstudied. To elucidate further, a follow-up study is currently being conducted in our laboratory.
In conclusions, platonin significantly attenuated kidney injury induced by bilateral lower limb I/R in rats. Moreover, the mechanisms may very likely involve mitigating the oxidative stress and the subsequent inflammatory response induced by lower limb I/R.
Acknowledgments
This work was performed mainly in Buddhist Tzu Chi General Hospital, Taipei branch. It was supported by grants from Buddhist Tzu Chi General Hospital, Taipei branch (TCRD-TPE-100-42), awarded to KYH and from National Science Council, Taiwan (NSC 98-2314-B-303-012-MY3), awarded to CJH.