Can Ischemia-Modified Albumin Be Used as an Objective Biomarker for Renal Ischemic Damage? An Experimental Study With Wistar Albino Rats H. Kocana, S. Citgeza,*, U. Yucetasb, E. Yucetasc, M. Yazicib, A.S. Amasyalid, E. Unluerb, and A.I. Tascie a Department of Urology, Necmi Ayanoglu Silivri State Hospital, Istanbul, Turkey; bDepartment of Urology, Istanbul Training and Research Hospital, Istanbul, Turkey; cDepartment of Biochemistry, Haseki Training and Research Hospital, Istanbul, Turkey; d Department of Urology, Istanbul Medical Faculty, Istanbul, Turkey; and eDepartment of Urology, Bakırköy Dr. Sadi Konuk Training and Research Hospital, Istanbul, Turkey
ABSTRACT Introduction. This study sought to evaluate the correlation of ischemia-modified albumin (IMA) with time-dependent renal ischemic injury. Methods. We established 5 groups of 8 Wistar albino rats as follows: sham, 10 minutes of renal ischemia, 20 minutes of renal ischemia, 30 minutes of renal ischemia, and 40 minutes of renal ischemia. Renal ischemia was established by occlusion of the right renal pedicle. Blood samples were obtained after exploration of the renal pedicle in the sham group and after thoracotomy and directly from the cardiac chambers at the end of the ischemic period in the other groups. The ischemic kidneys were removed for histopathological evaluation, and the rats were killed. Results. There were significant differences among the IMA levels of the 5 groups (P ¼ .0013). Pathological examination showed that renal ischemic injury corresponded to the duration of ischemia. In the group analysis, the pathological evaluation scores were significantly different among the groups (P < .001). Conclusions. This study shows that IMA levels can be used as a nonselective biomarker for renal ischemic injury. However, further studies are needed to support our findings.
P
ARTIAL nephrectomy has become the standard of care for the management of small renal masses. Although this treatment method has become prevalent, some questions remain to be addressed, such as the warm ischemia time (WIT) and the preservation of renal function. The acceptable limit of WIT before renal injury occurs is unclear [1]. Some investigators have challenged the traditional suggestion of a 30-minute limit for warm ischemia, whereas others have developed techniques to reduce ischemic damage [2]. The WIT also is relevant for living donor kidney transplantation. To date, the influence of WIT on the long-term outcome after living donor kidney transplantation has not been fully addressed. However, prolonged WIT (45 minutes) impairs the long-term graft survival in living donor kidney transplantation [3]. Warm ischemia may contribute to ischemiaereperfusion injury, with increased innate immunogenicity of the allograft, and ultimately may lead to chronic allograft nephropathy [4,5].
Albumin is the most abundant plasma protein, and it has the ability to bind divalent ions, such as cobalt, at its N-terminus. During ischemia and the subsequent production of reactive oxygen species, the N-terminal sequence of albumin is modified, and its ability to bind cobalt is disrupted [6]. This form of albumin is called ischemia-modified albumin (IMA). IMA has been implicated in the detection of acute ischemia before necrosis [7]. Bar-Or et al initially described increased IMA levels in patients with transient myocardial ischemia after elective percutaneous transluminal coronary angioplasty [8]. Subsequently, several studies have shown that IMA levels increase under a variety of ischemic conditions, such as peripheral arterial disease, skeletal muscle ischemia, pulmonary embolism, and stroke [9e11]. *Address correspondence to Sinharib Citgez, MD, Department lu Silivri State Hosof Urology, Floor:1, Prof. Dr. Necmi Ayanog pital, Silivri, Istanbul, Turkey. E-mail: [email protected]
0041-1345/14 http://dx.doi.org/10.1016/j.transproceed.2014.11.001
ª 2014 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
3326
Transplantation Proceedings, 46, 3326e3329 (2014)
ISCHEMIA-MODIFIED ALBUMIN
To our knowledge there is no study investigating serum IMA levels and evaluating their increase over time in an experimental model of renal ischemia in rats in the literature. This study compared the histopathological features and IMA levels to determine whether IMA levels can be used as a predictor of renal ischemia before necrosis. MATERIALS AND METHODS Experiment Schedule A total of 40 female Wistar albino rats with a median weight of 248 g (240 to 260 g) were maintained in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals. The experiments complied with the statement on Animal Experimentation by the National Health and Medical Research Council and were approved by the Istanbul University Experimental Animals Ethics Committee. The rats were weighed daily and housed at a constant temperature with a 12-hour period of lighte dark exposure. The animals were allowed access to standard rat chow and water, with an acclimation period of at least 2 weeks before the procedure. The rats were divided into 5 groups of 8 rats each: sham and 10, 20, 30, and 40 minutes of renal ischemia.
3327
Miscellaneous Measurement All rats were preoperatively anesthetized with 7.5 mg/kg intraperitoneal xylazine (Rhompun, Abdi Ibrahim, Istanbul, Turkey) and 50 mg/kg ketamine hydrochloride (Ketalar, Eczacibasi, Istanbul, Turkey). Under aseptic conditions, a 3-cm midline laparotomy was performed. The right renal pedicle was exposed, and a 3-mL intracardiac blood sample was collected in the sham group (Fig 1). In the ischemic groups, the renal pedicle was ligated with 3-0 silk under 4.5 optical zoom, and blood was sampled at the designated renal ischemia time. During this period, an incision was performed and sterile gauze pads were soaked with saline at room temperature. A nephrectomy was performed in all groups, and the kidneys were stored in 10% formaldehyde for histopathologic evaluation. The animals were killed by drawing the intracardiac blood and decapitation after the procedure.
Biochemical Analysis After the blood samples were collected, the serum and plasma specimens were prepared with 5 minutes of centrifugation at 4000 rpm. The specimens were pipetted into Eppendorf tubes and stored at 80 C until analysis. The serum IMA levels were assessed using the ELISA method (Cusabio Biotech ELISA kit). The results are reported in U/mL.
Fig 1. (A, B) Midline laparotomy under aseptic conditions. (C, D) Exposure of the kidney and the renal pedicle. (E) Ligation of the renal pedicle with 3-0 silk. (F) Intracardiac blood collection.
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KOCAN, CITGEZ, YUCETAS ET AL
Table 1. Mean Serum Ischemia-Modified Albumin Values (U/mL)
Sham 10 min 20 min 30 min 40 min
Mean SD
1.388 1.520 1.783 1.848 5.123
0.3888 0.7683 0.8343 0.5597 4.222
P
.0013
Histopathology The dissected kidneys were fixed in formaldehyde. The specimens were dehydrated, embedded in paraffin blocks, and prepared as 5-mm-thick serial sections. The sections were stained with hematoxylin and eosin by a pathologist who was blinded to the designation of the animal groups. The results were scored according to the percentage of tubule deterioration as follows: grade 0, not damaged; grade 1, 50% [12].
Statistical Analysis The statistical analysis was performed using R scope. The analysis of IMA among groups was performed using the Kruskal-Wallis and Mann-Whitney U tests. Spearman’s correlation analysis was used to assess the relationship between biochemical parameters and histopathologic scores. Statistical significance was assumed at a level of P < .05.
RESULTS Effect of IMA Levels on the Groups
The mean serum IMA values were 1.388 0.388, 1.520 0.768, 1.783 0.834, 1.848 0.559, 5.123 4.22 U/mL in the sham, 10-, 20-, 30-, and 40-minute ischemia groups, respectively. There was no significant difference between the sham, 10-, 20-, and 30-minute ischemia groups. However, the IMA levels were significantly higher in the 40-minute ischemia group compared with the other groups (P ¼ .0013, KruskalWallis) (Table 1, Fig 2). Furthermore, we determined the 95% confidence interval for the mean IMA values at 30 minutes to determine that time above 30 minutes at which statistical significance began to appear. The upper boundary
of the 95% confidence interval was 2.2345 U/mL, corresponding to a time of 31.18 minutes on the slope from 30 to 40 minutes. Histopathologic Results
During the experiment, no color change was observed macroscopically in the sham, 10-, 20-, or 30-minute ischemia groups. However, the color was dark blue to violet in the 40-minute ischemia group. Based on our microscopic assessment, the histopathologic score for ischemic damage increased in accordance with increased ischemia time (c2 P < .0001). The histopathologic scores are given in Table 2. DISCUSSION
The reversibility of ischemic renal damage depends on the capacity for renal tubular epithelial regeneration, which is negatively correlated with the ischemia time. There is no objective predictor for the duration of ischemia that leads to irreversible renal damage. Biochemical markers, such as creatinine and urea, are late predictors of renal dysfunction because significantly increased levels of these parameters reflect a 50% to 70% reduction of glomerular filtration rate [13]. Additionally, the creatinine level does not give sufficient information in the presence of a normal contralateral kidney. Over the last decade, IMA has been used to measure cobalt binding to albumin and has been suggested as a marker of a variety of ischemic conditions, such as acute myocardial ischemia, peripheral arterial disease, and pulmonary embolism [14]. Subsequently, the US Food and Drug Administration has approved IMA as a biomarker for acute myocardial ischemia [15]. Several experimental studies have evaluated IMA under ischemic conditions, although there are no published data on IMA and renal ischemia in the literature. Ischemia-modified albumin can be measured using the rapid colorimetric test or the ELISA test [16]. Although the majority of studies have used the rapid colorimetric test (absorbance units), we prefer the ELISA test (U/ mL), which is more reliable. The present experimental study was conducted on rats undergoing renal surgery with a contralateral normal kidney to determine whether IMA may be an ideal marker for renal ischemia. A model of acute mesenteric ischemia in rabbits showed that serum IMA levels were significantly higher after 3 and 6 hours of ischemia than in control and sham groups [17]. However, Uygun et al found that serum IMA values were not useful in the early diagnosis of acute mesenteric Table 2. Histopathologic Grading of Groups Grade 0
Fig 2. Distribution of serum ischemia-modified albumin levels according to sham and ischemia groups.
Sham (n ¼ 8) 10 min (n ¼ 8) 20 min (n ¼ 8) 30 min (n ¼ 8) 40 min (n ¼ 8)
3 1 0 0 0
(37.5%) (12.5%) (0%) (0%) (0%)
Values according to c2 test.
Grade 1
5 7 7 2 1
(62.5%) (87.5%) (87.5%) (25%) (12.5%)
Grade 2
0 0 1 6 7
(0%) (0%) (12.5%) (75%) (87.5%)
P
ABSTRACT Introduction. This study sought to evaluate the correlation of ischemia-modified albumin (IMA) with time-dependent renal ischemic injury. Methods. We established 5 groups of 8 Wistar albino rats as follows: sham, 10 minutes of renal ischemia, 20 minutes of renal ischemia, 30 minutes of renal ischemia, and 40 minutes of renal ischemia. Renal ischemia was established by occlusion of the right renal pedicle. Blood samples were obtained after exploration of the renal pedicle in the sham group and after thoracotomy and directly from the cardiac chambers at the end of the ischemic period in the other groups. The ischemic kidneys were removed for histopathological evaluation, and the rats were killed. Results. There were significant differences among the IMA levels of the 5 groups (P ¼ .0013). Pathological examination showed that renal ischemic injury corresponded to the duration of ischemia. In the group analysis, the pathological evaluation scores were significantly different among the groups (P < .001). Conclusions. This study shows that IMA levels can be used as a nonselective biomarker for renal ischemic injury. However, further studies are needed to support our findings.
P
ARTIAL nephrectomy has become the standard of care for the management of small renal masses. Although this treatment method has become prevalent, some questions remain to be addressed, such as the warm ischemia time (WIT) and the preservation of renal function. The acceptable limit of WIT before renal injury occurs is unclear [1]. Some investigators have challenged the traditional suggestion of a 30-minute limit for warm ischemia, whereas others have developed techniques to reduce ischemic damage [2]. The WIT also is relevant for living donor kidney transplantation. To date, the influence of WIT on the long-term outcome after living donor kidney transplantation has not been fully addressed. However, prolonged WIT (45 minutes) impairs the long-term graft survival in living donor kidney transplantation [3]. Warm ischemia may contribute to ischemiaereperfusion injury, with increased innate immunogenicity of the allograft, and ultimately may lead to chronic allograft nephropathy [4,5].
Albumin is the most abundant plasma protein, and it has the ability to bind divalent ions, such as cobalt, at its N-terminus. During ischemia and the subsequent production of reactive oxygen species, the N-terminal sequence of albumin is modified, and its ability to bind cobalt is disrupted [6]. This form of albumin is called ischemia-modified albumin (IMA). IMA has been implicated in the detection of acute ischemia before necrosis [7]. Bar-Or et al initially described increased IMA levels in patients with transient myocardial ischemia after elective percutaneous transluminal coronary angioplasty [8]. Subsequently, several studies have shown that IMA levels increase under a variety of ischemic conditions, such as peripheral arterial disease, skeletal muscle ischemia, pulmonary embolism, and stroke [9e11]. *Address correspondence to Sinharib Citgez, MD, Department lu Silivri State Hosof Urology, Floor:1, Prof. Dr. Necmi Ayanog pital, Silivri, Istanbul, Turkey. E-mail: [email protected]
0041-1345/14 http://dx.doi.org/10.1016/j.transproceed.2014.11.001
ª 2014 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
3326
Transplantation Proceedings, 46, 3326e3329 (2014)
ISCHEMIA-MODIFIED ALBUMIN
To our knowledge there is no study investigating serum IMA levels and evaluating their increase over time in an experimental model of renal ischemia in rats in the literature. This study compared the histopathological features and IMA levels to determine whether IMA levels can be used as a predictor of renal ischemia before necrosis. MATERIALS AND METHODS Experiment Schedule A total of 40 female Wistar albino rats with a median weight of 248 g (240 to 260 g) were maintained in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals. The experiments complied with the statement on Animal Experimentation by the National Health and Medical Research Council and were approved by the Istanbul University Experimental Animals Ethics Committee. The rats were weighed daily and housed at a constant temperature with a 12-hour period of lighte dark exposure. The animals were allowed access to standard rat chow and water, with an acclimation period of at least 2 weeks before the procedure. The rats were divided into 5 groups of 8 rats each: sham and 10, 20, 30, and 40 minutes of renal ischemia.
3327
Miscellaneous Measurement All rats were preoperatively anesthetized with 7.5 mg/kg intraperitoneal xylazine (Rhompun, Abdi Ibrahim, Istanbul, Turkey) and 50 mg/kg ketamine hydrochloride (Ketalar, Eczacibasi, Istanbul, Turkey). Under aseptic conditions, a 3-cm midline laparotomy was performed. The right renal pedicle was exposed, and a 3-mL intracardiac blood sample was collected in the sham group (Fig 1). In the ischemic groups, the renal pedicle was ligated with 3-0 silk under 4.5 optical zoom, and blood was sampled at the designated renal ischemia time. During this period, an incision was performed and sterile gauze pads were soaked with saline at room temperature. A nephrectomy was performed in all groups, and the kidneys were stored in 10% formaldehyde for histopathologic evaluation. The animals were killed by drawing the intracardiac blood and decapitation after the procedure.
Biochemical Analysis After the blood samples were collected, the serum and plasma specimens were prepared with 5 minutes of centrifugation at 4000 rpm. The specimens were pipetted into Eppendorf tubes and stored at 80 C until analysis. The serum IMA levels were assessed using the ELISA method (Cusabio Biotech ELISA kit). The results are reported in U/mL.
Fig 1. (A, B) Midline laparotomy under aseptic conditions. (C, D) Exposure of the kidney and the renal pedicle. (E) Ligation of the renal pedicle with 3-0 silk. (F) Intracardiac blood collection.
3328
KOCAN, CITGEZ, YUCETAS ET AL
Table 1. Mean Serum Ischemia-Modified Albumin Values (U/mL)
Sham 10 min 20 min 30 min 40 min
Mean SD
1.388 1.520 1.783 1.848 5.123
0.3888 0.7683 0.8343 0.5597 4.222
P
.0013
Histopathology The dissected kidneys were fixed in formaldehyde. The specimens were dehydrated, embedded in paraffin blocks, and prepared as 5-mm-thick serial sections. The sections were stained with hematoxylin and eosin by a pathologist who was blinded to the designation of the animal groups. The results were scored according to the percentage of tubule deterioration as follows: grade 0, not damaged; grade 1, 50% [12].
Statistical Analysis The statistical analysis was performed using R scope. The analysis of IMA among groups was performed using the Kruskal-Wallis and Mann-Whitney U tests. Spearman’s correlation analysis was used to assess the relationship between biochemical parameters and histopathologic scores. Statistical significance was assumed at a level of P < .05.
RESULTS Effect of IMA Levels on the Groups
The mean serum IMA values were 1.388 0.388, 1.520 0.768, 1.783 0.834, 1.848 0.559, 5.123 4.22 U/mL in the sham, 10-, 20-, 30-, and 40-minute ischemia groups, respectively. There was no significant difference between the sham, 10-, 20-, and 30-minute ischemia groups. However, the IMA levels were significantly higher in the 40-minute ischemia group compared with the other groups (P ¼ .0013, KruskalWallis) (Table 1, Fig 2). Furthermore, we determined the 95% confidence interval for the mean IMA values at 30 minutes to determine that time above 30 minutes at which statistical significance began to appear. The upper boundary
of the 95% confidence interval was 2.2345 U/mL, corresponding to a time of 31.18 minutes on the slope from 30 to 40 minutes. Histopathologic Results
During the experiment, no color change was observed macroscopically in the sham, 10-, 20-, or 30-minute ischemia groups. However, the color was dark blue to violet in the 40-minute ischemia group. Based on our microscopic assessment, the histopathologic score for ischemic damage increased in accordance with increased ischemia time (c2 P < .0001). The histopathologic scores are given in Table 2. DISCUSSION
The reversibility of ischemic renal damage depends on the capacity for renal tubular epithelial regeneration, which is negatively correlated with the ischemia time. There is no objective predictor for the duration of ischemia that leads to irreversible renal damage. Biochemical markers, such as creatinine and urea, are late predictors of renal dysfunction because significantly increased levels of these parameters reflect a 50% to 70% reduction of glomerular filtration rate [13]. Additionally, the creatinine level does not give sufficient information in the presence of a normal contralateral kidney. Over the last decade, IMA has been used to measure cobalt binding to albumin and has been suggested as a marker of a variety of ischemic conditions, such as acute myocardial ischemia, peripheral arterial disease, and pulmonary embolism [14]. Subsequently, the US Food and Drug Administration has approved IMA as a biomarker for acute myocardial ischemia [15]. Several experimental studies have evaluated IMA under ischemic conditions, although there are no published data on IMA and renal ischemia in the literature. Ischemia-modified albumin can be measured using the rapid colorimetric test or the ELISA test [16]. Although the majority of studies have used the rapid colorimetric test (absorbance units), we prefer the ELISA test (U/ mL), which is more reliable. The present experimental study was conducted on rats undergoing renal surgery with a contralateral normal kidney to determine whether IMA may be an ideal marker for renal ischemia. A model of acute mesenteric ischemia in rabbits showed that serum IMA levels were significantly higher after 3 and 6 hours of ischemia than in control and sham groups [17]. However, Uygun et al found that serum IMA values were not useful in the early diagnosis of acute mesenteric Table 2. Histopathologic Grading of Groups Grade 0
Fig 2. Distribution of serum ischemia-modified albumin levels according to sham and ischemia groups.
Sham (n ¼ 8) 10 min (n ¼ 8) 20 min (n ¼ 8) 30 min (n ¼ 8) 40 min (n ¼ 8)
3 1 0 0 0
(37.5%) (12.5%) (0%) (0%) (0%)
Values according to c2 test.
Grade 1
5 7 7 2 1
(62.5%) (87.5%) (87.5%) (25%) (12.5%)
Grade 2
0 0 1 6 7
(0%) (0%) (12.5%) (75%) (87.5%)
P
