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Ann Thorac Surg 1998;66:351-355
© 1998 The Society of Thoracic Surgeons

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Original articles: general thoracic

Recombinant kunitz protease inhibitor ameliorates reperfusion injury in rat lung transplantation

^a Division of Cardiothoracic Surgery, Department of Surgery, Washington University School of Medicine, St. Louis, Missouri, USA
^b Scios Inc, Sunnyvale, California, USA

Address reprint requests to Dr Patterson, Division of Cardiothoracic Surgery, Washington University School of Medicine, 3108 Queeny Tower, One Barnes Hospital Plaza, St. Louis, MO 63110

Presented at the Forty-fourth Annual Meeting of the Southern Thoracic Surgical Association, Naples, FL, Nov 6–8, 1997.

	Abstract

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Background. Recombinant Kunitz protease inhibitor (rKPI-BG022) is more homologous to human Kunitz protease inhibitor than is aprotinin. Because aprotinin has been reported to inhibit free radicals, we hypothesized that rKPI would ameliorate reperfusion injury caused by free radicals. We examined its effect and the timing of administration in an in vivo rat lung transplantation model.

Methods. All lungs were flushed with low-potassium dextran–1% glucose solution and stored for 24 hours at 4°C, then orthotopic left lung transplantations were performed. Rats were divided into 4 groups (n = 6) as follows: group 1 served as control; in Group 2, rKPI was added to the flush solution (10 µmol/L); in group 3, rKPI (5 mg/kg) was administered intravenously to the recipient just after reperfusion; and in group 4, rKPI was added to the flush solution (10 µmol/L) and rKPI (5 mg/kg) was administered intravenously to the recipient just after reperfusion. Twenty-four hours after transplantation, the right main pulmonary artery and right main bronchus were ligated, and the rats were ventilated with 100% O₂ for 5 minutes. Peak airway pressure, blood gas analysis, serum lipid peroxide level, tissue myeloperoxidase activity, and wet-dry weight ratio were measured.

Results. The partial oxygen tension values of group 2 were higher than those of groups 1 and 4 (groups 1, 2, and 4: 104.8 ± 15.8, 245.1 ± 49.0, 101.4 ± 4.5 mm Hg, respectively; p < 0.01). The partial carbon dioxide tension values of groups 3 and 4 were lower than those of group 1 (groups 1, 3, and 4: 74.5 ± 5.7, 42.0 ± 11.0, 46.0 ± 8.4 mm Hg, respectively; p < 0.05). Peak airway pressures were lower in groups 2 and 3 than in groups 1 and 4 (groups 1, 2, 3, and 4: 22.5 ± 0.5, 18.2 ± 0.5, 19.2 ± 0.8, 22.5 ± 1.1 mm Hg; p < 0.01). Serum lipid peroxide levels in groups 2 and 3 were lower than those of groups 1 and 4 (groups 1, 2, 3, and 4: 0.793 ± 0.037, 0.577 ± 0.069, 0.560 ± 0.029, and 0.785 ± 0.053 nmol/mL, respectively; groups 2 and 3 vs group 1, and group 3 vs group 4: p < 0.01; group 2 vs group 4: p < 0.05). There were no differences in wet-dry weight ratio and tissue myeloperoxidase activity between the groups.

Conclusion. Recombinant Kunitz protease inhibitor ameliorates reperfusion injury caused by free radicals in an in vivo rat lung transplantation model. Administration of rKPI through the flush solution and intravenous injection after reperfusion were effective separately, but the combination of the two administrations was not effective.

	Introduction

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Aprotinin belongs to a group of proteins known as the Kunitz protease inhibitors (KPIs) that share a Kunitz domain in their polypeptide structure which confers an ability to inhibit serine protease. Aprotinin is extracted from bovine lung tissue and is only 45% homologous to human KPI [1]. The recombinant Kunitz protease inhibitor KPI-BG022 (rKPI) has been produced (Scios Inc, Sunnyvale, CA) from a transformed strain of the yeast Pichia pastoris and is a 61-amino acid polypeptide more than 95% homologous to human KPI at the Kunitz domain. We hypothesized that rKPI ameliorates reperfusion injury because it is more homologous than aprotinin, which has been reported to have an advantageous effect in organ preservation and reperfusion injury [2–5]. In this study, we determined the effect of rKPI in reperfusion injury after a period of prolonged ischemia in an orthotopic rat lung transplantation model.

	Material and methods

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Male inbred specific-pathogen-free Fisher rats weighing 250 to 350 g were purchased from Harlan Sprague Dawley (Indianapolis, IN). The animals were given humane care in compliance with the "Guide for the Care and the Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 86-23, revised 1985).

Orthotopic left lung transplantations were performed by using a modification of the cuff technique as described by Mizuta and associates [6]. A reproducible reperfusion injury model using rat lung transplantation, which has been developed in our laboratory [7, 8], was used in the present study. All transplantations were performed by a single surgeon (I.N.) using an operating microscope (Wild Heerbrugg M-691; Wild Leitz, Rockleigh, NJ).

Donor harvest
Anesthesia of donor animals was induced with inhaled halothane and maintained with intraperitoneal pentobarbital (20 mg/kg) administration. The animals were intubated by a tracheostomy and ventilated with room air at a respiratory rate of 60 breaths per minute, tidal volume of 3.0 mL, and positive end-expiratory pressure of 0.5 cm H₂O. After heparinization (1,000 unit/kg intravenously), the left atrium was incised, and the lungs were flushed with 20 mL of cold (4°C) low-potassium dextran–1% glucose solution [9] delivered from a height of 25 cm H₂O. The heart-lung block was excised and immersed in low-potassium dextran–1% glucose solution and stored at 4°C until implantation.

Just before implantation, the left main pulmonary artery, left main pulmonary vein, and left main bronchus of the lung graft were divided and cuffed with segments of a 14-gauge angiocatheter.

Recipient operation
Recipient animals were anesthetized with halothane, premedicated with intraperitoneal administration of ketamine (40 mg/kg) and atropine (0.25 mg/kg), and intubated orotracheally. The animals were maintained under general anesthesia with a mixture of halothane and oxygen. The left hilum was dissected through a left third intercostal space thoracotomy, and the left main pulmonary artery, the left main pulmonary vein, and the left main bronchus were occluded with vascular clamps (Yasagil mini aneurysm clip; Aesculap AG, Tuttlingen, Germany). A longitudinal arteriotomy and venotomy, and horizontal bronchotomy were created, and the donor cuffs were inserted and held in place with 5-0 silk ties. The chest was closed, and a chest tube was left in place until after removal of the tracheal tube. The animals were placed in a cage with supplemental oxygen (2 L/min) and food and water ad libitum.

Assessment
At 24 hours after reperfusion, all animals were anesthetized with halothane and pentobarbital (10 mg/kg intraperitoneally), intubated, and ventilated. Access was gained to the right pulmonary hilum by laparotomy and median sternotomy. The ventilator was set at a 100% fraction of inspired oxygen, tidal volume of 1.5 mL, respiratory rate of 100 breaths per minute, and positive end-expiratory pressure of 0.5 cm H₂O. The right main pulmonary artery and mainstem bronchus were occluded with a 4-0 silk tie. Five minutes thereafter, 4 mL of arterial blood was obtained from the ascending aorta for measurements of blood gas, hematocrit, hemoglobin, and thiobarbituric acid reactive substances assay. Peak airway pressure was measured and monitored continuously by means of a tracheal tube. After exsanguination, the graft was flushed with 10 mL of saline delivered from a height of 25 cm, and the graft was extracted from the thorax for myeloperoxidase activity assay. For wet-dry weight ratio measurement, the graft was extracted without flushing with saline solution.

Serum thiobarbituric acid reactive substances assay
The blood samples were centrifuged at 3,000 g for 10 minutes at 4°C, and the serum was stored at -80°C until measurement. Serum thiobarbituric acid reactive substances assay was performed by the method of Fleming [10].

Myeloperoxidase assay
To measure myeloperoxidase the graft was placed into a cryotube, snap frozen in liquid nitrogen, and stored at -80°C until measurement. Quantitative lung homogenate myeloperoxidase activity was determined as previously described and modified in our laboratory [11].

Wet-dry weight ratio
To obtain wet-dry weight ratio, the graft was placed in an oven at 180°C for 48 hours, and the original weight was divided by the weight after drying.

Experimental groups
Experimental groups were designed as follows: group 1 (n = 6) served as control; in group 2 (n = 6), rKPI was added to the flush solution (10 µmol/L); in group 3 (n = 6), rKPI (5 mg/kg) was administered intravenously to the recipient just after reperfusion; and in group 4 (n = 6), rKPI was added to the flush solution (10 µmol/L) and rKPI (5 mg/kg) was administered intravenously to the recipient just after reperfusion. The rKPI was provided by Tyler White, PhD (Scios Inc, Sunnyvale, CA).

Statistical analysis
Data are expressed as the mean ± standard error of the mean. Groups were compared using analysis of variance with the Fisher multiple comparison method. Differences are considered significant when p is less than 0.05.

	Results

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Flushing times and total ischemic times were not different between any of the groups (Table 1). Significant differences between groups were noted in arterial oxygen tension (p < 0.05), arterial carbon dioxide tension (p < 0.05), peak airway pressure (p < 0.01) and serum thiobarbituric acid reactive substances levels (p < 0.01). Group 2 arterial oxygen tension levels were significantly higher than those of groups 1 and 4 (Fig 1). Group 3 arterial carbon dioxide tension levels were significantly lower than those of group 1. Peak airway pressures were also significantly lower in groups 2 and 3, but the values in group 4 were higher and showed no difference from the control group (Fig 2). Serum thiobarbituric acid reactive substances levels in groups 2 and 3 were significantly lower than those of groups 1 and 4 (Fig 3).

View larger version (18K): [in this window] [in a new window]   Fig 1. Arterial blood gas analysis. The data are shown as the mean ± standard error of the mean. (Pa CO2 = partial carbon dioxide tension; Pa O2 = partial oxygen tension; ** = p < 0.01; * = p < 0.05.)

View larger version (16K): [in this window] [in a new window]   Fig 2. Peak airway pressure. The data are shown as the mean ± standard error of the mean. (** = p < 0.01.)

View larger version (17K): [in this window] [in a new window]   Fig 3. Serum thiobarbituric acid reactive substances levels. The data are shown as the mean ± standard error of the mean. (* = p < 0.05; ** = p < 0.01.)

	Comment

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Lung allograft reperfusion injury remains a significant and often unpredictable problem in clinical lung transplantation. Although several mechanisms have been proposed to explain the pathogenesis of ischemia-reperfusion injury, most attention has been focused on the role of reactive oxygen metabolites and inflammatory leukocytes. This work has led to the proposition that free radical ablation or inhibition of postischemic neutrophil infiltration may prove useful for therapeutic intervention in ischemia-reperfusion injury [12].

Aprotinin has been reported to have a beneficial effect on neuron preservation [2] and ischemia-reperfusion injury in various organs [3–5]. Aprotinin has been also reported to inhibit the production of superoxide [13] and hydrogen peroxide [14] by human polymorphonuclear leukocytes and chemiluminescence induced by proteases from rat glomeruli [15]. On the basis of those reports, we hypothesized that rKPI would be as efficacious as aprotinin in ameliorating reperfusion injury by suppressing free radical generation. In the present study, we showed that rKPI administered either in the flush solution or intravenously after reperfusion (groups 2 and 3) was effective against reperfusion injury after a prolonged ischemic period. In addition to functional measurements of the grafts, serum lipid peroxide levels, which reflect the membrane destruction by free radicals, were significantly lower in groups 2 and 3 than in the control group. These results indicate that rKPI ameliorates reperfusion injury caused by free radicals.

Myeloperoxidase activity, which reflects polymorphonuclear infiltration to interstitial tissue of the graft, and wet-dry weight ratios did not differ between the groups. We cannot explain these results but speculate that the 24-hour preservation period was too long and deleterious to the grafts to allow differences between study and control groups to occur.

The grafts that received rKPI both before and after preservation (group 4) showed poorer function than the grafts that received rKPI only before or after preservation (groups 2 and 3). The most probable cause for this difference in function would be the dosage of rKPI. We determined the dosage from studies conducted by other groups in which rKPI was administered during cardiopulmonary bypass and resulted in decreased bypass-associated bleeding and improved lung function. The present study was performed in a totally different setting, and systematic determination of the appropriate dosage of rKPI is needed in a lung transplantation model.

The effect of aprotinin is complex, but it is evidently an inhibitor of the kallikrein-kinin system. Plasma kallikrein releases bradykinin from kininogens [16], and it has been reported that bradykinin increases during the ischemia-reperfusion period. Aprotinin ameliorates reperfusion injury by suppressing bradykinin [17, 18]. Recombinant Kunitz protease inhibitor is a more potent kallikrein inhibitor than aprotinin (Table 2), which suggests that rKPI may ameliorate ischemia-reperfusion injury by virtue of its inhibitory effect. Another important effect of aprotinin is inhibition of plasmin. Much work has been done with regard to the hemostatic effect of aprotinin, particularly during or after cardiopulmonary bypass [19]. This effect is also expected for rKPI, and we tried to demonstrate it by measuring recipient hemoglobin and hematocrit levels 24 hours after operation but did not find any differences between the control and study groups. Higher dosage levels would be needed to derive the hemostatic effect of rKPI in this model, because rKPI’s inhibitory effect against plasmin is one-twenty fifth that of aprotinin (Table 2).

In conclusion, we showed that rKPI ameliorated ischemia-reperfusion injury and suppressed serum lipid peroxide levels after reperfusion. The genetic engineering of the protein structure would provide a more desirable agent by the alterability of its actions, and this approach holds promise for the future.

	Acknowledgments

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
We thank Jill Manchester for assisting with myeloperoxidase and thiobarbituric acid reactive substances assay, and Dawn Schuessler and Mary Ann Kelly for secretarial support.

	References

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 

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