International Journal of Cardiology 172 (2014) e48–e50
Contents lists available at ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Letter to the Editor
A novel rat model of contrast-induced acute kidney injury Shiqun Sun 1, Tuo Zhang 1, Peng Nie, Liuhua Hu, Ying Yu, Mingli Cui, Zhaohua Cai, Linghong Shen ⁎, Ben He ⁎ Department of Cardiology, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China
a r t i c l e
i n f o
Article history: Received 29 September 2013 Accepted 21 December 2013 Available online 30 December 2013 Keywords: Contrast medium Acute kidney injury Animal model
studies conformed to the guidelines on animal care of Shanghai Jiaotong University School of Medicine. As shown in Fig. 1, twelve rats were randomly assigned to four groups: dehydration for 3 days before contrast medium administration (Group 1); dehydration for 1 day and furosemide injection 20 min before contrast medium administration (Group 2); dehydration for 2 days and furosemide injection 20 min before contrast medium administration (Group 3); dehydration for 3 days and furosemide injection 20 min before contrast medium administration (Group 4). No difference in SCr levels were detected between the baseline value and 24 h after contrast medium administration in Group 1 (21.3 ± 1.3 μmol/L vs.
Contrast-induced acute kidney injury (CI-AKI) is an iatrogenic complication after the administration of contrast medium in diagnostic and interventional procedures. It is a common cause of hospital-acquired renal insufficiency and accounts for about 11% of cases [1,2]. An adequate animal model would be useful for the understanding and prevention of this disorder [3]. In this study, we have developed a new, highly efficient and reproducible model of acute kidney injury following contrast medium infusion in rats. Male Sprague–Dawley rats, weighing approximately 250–300 g, were included in the study. A nonionic low-osmolar contrast medium, Omnipaque (350 mg I/mL, GE Healthcare, Shanghai, China), was injected at 10 mL/kg via tail vein administration over the course of 5 min. Furosemide (Harvest Pharmaceutical Co., Shanghai, China) was injected intramuscularly at 10 mL/kg. Blood samples were taken from the jugular vein in untreated rats and from the abdominal aorta in sacrificed rats. EDTA (1.5 mg/mL) was added to the blood to impair coagulation. Immediately after sacrifice, both kidneys were harvested and embedded in paraffin. Adjacent 4 μm sections were stained with hematoxylin and eosin (H&E). Serum creatine (SCr) levels were measured by a standard spectrophotometric assay (Modular P, Roche Diagnostics GmbH, Penzberg, Germany). Renal tubular necrosis, medullary congestion, and proteinaceous casts were evaluated semi-quantitatively in a blinded manner by the method suggested by Toprak, et al. [4]. The authors of this manuscript certify that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology. All animal
⁎ Corresponding authors at: Department of Cardiology, Renji Hospital, Shanghai Jiaotong University School of Medicine, 1630 Dong Fang Road, Shanghai 200127, China. Tel.: +86 21 58752345; fax: +86 21 68383609. E-mail addresses: [email protected] (L. Shen), [email protected] (B. He). 1 Shiqun Sun and Tuo Zhang equally contributed to this work. 0167-5273/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijcard.2013.12.066
Fig. 1. Preliminary experiment. Male Sprague–Dawley rats were deprived of water as indicated time interval and followed by furosemide injection or not 20 min before Omnipaque administration. Blood samples were drawn before dehydration and 24 h after contrast medium injection (*p b 0.05 vs. baseline SCr value, n = 3). Values are mean ± SEM.
S. Sun et al. / International Journal of Cardiology 172 (2014) e48–e50
22.0 ± 3.5 μmol/L, p N 0.05), Group 2 (21.5 ± 2.8 μmol/L vs. 25.1 ± 2.2 μmol/L, p N 0.05) and Group 3 (20.7 ± 0.5 μmol/L vs. 25.7 ± 2.1 μmol/L, p N 0.05). However, SCr elevated more than 50% in Group 4 (23.0 ± 1.5 μmol/L vs. 36.2 ± 7.9 μmol/L, p b 0.05). To confirm the change of SCr in Group 4, another twelve rats were enrolled. Animals were dehydrated for 3 days and underwent furosemide injection 20 min before contrast medium (10 mL/kg, group CM)
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or 0.9% normal saline (10 mL/kg, group NS) administration. As shown in Fig. 2A and B, SCr increased 42.9% after contrast medium injection compared to the baseline value in the group CM and no change was observed in group NS. Since the most commonly used definition of CI-AKI is an increase in SCr ≥25% of the baseline value [1], we calculated the change of SCr and found that 83.3% (5/6) of the group CM reached this threshold. Direct tubular toxicity and tubular obstruction are
Fig. 2. Effect of either normal saline or contrast medium on serum creatinine and kidney histopathological changes. (A and B) Male Sprague–Dawley rats were deprived of water for 3 days and followed by furosemide injection 20 min before normal saline or contrast medium administration (*p b 0.05 vs. baseline value, **p b 0.05 vs. group NS, n = 6). (C) Proteinaceous casts in rat tubular lumens of CM group. (H&E stain, original magnification ×100). (D) Tubular necrosis in rat kidney of group CM (H&E stain, original magnification ×100). (E) Outer medullary congestion in rat kidney of group CM (H&E stain, original magnification ×100). (F) Evaluations of kidney histopathological changes. Tubular necrosis and proteinaceous casts were graded as follows: no damage (0), mild (1, unicellular, patchy isolated damage), moderate (2, damage less than 25%), severe (3, damage between 25 and 50%), and very severe (4, more than 50% damage). The degree of medullary congestion was defined as follows: no congestion (0), mild (1, vascular congestion with identification of erythrocytes by ×400 magnification), moderate (2, vascular congestion with identification of erythrocytes by ×200 magnification), severe (3, vascular congestion with identification of erythrocytes by ×100 magnification) and very severe (4, vascular congestion with identification of erythrocytes by ×40 magnification). *p b 0.05 vs. group NS, n = 6. Values are mean ± SEM.
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S. Sun et al. / International Journal of Cardiology 172 (2014) e48–e50
commonly suggested mechanisms underlying the pathogenesis of CIAKI [1,5]. Therefore, we examined the histopathological changes of the kidneys in these rats. All rats in group NS and group CM developed proteinaceous casts (Fig. 2C, inner zone of the medulla), tubular necrosis (Fig. 2D, outer zone of medulla), and medullary congestion (Fig. 2E, outer zone of medulla). Significantly more tubular necrosis and medullary congestion were seen in group CM compared to group NS (Fig. 2F). Acute kidney injury (AKI) is not easily induced by simply exposing healthy animal to contrast medium alone. Pretreating animals with factors similar to those considered to be risk factors for kidney damage in humans can help induce AKI in animals. Rats are widely used for CIAKI models [3]. Multiple insults, such as uninephrectomy, salt depletion, dehydration, and furosemide or indomethacin injection have been used in rats to predispose them to injury [4,6–8]. Water deprivation is regarded as an appropriate pretreatment method because it is easy to prepare with no pharmacological or surgical procedures. Previous studies have shown that a prolonged dehydration period (3 days) before high-osmolar contrast medium (HOCM) administration promotes renal function deterioration [4]. However, clinical data have demonstrated a significantly lower frequency of CI-AKI in patients with impaired renal function who received low-osmolar contrast medium (LOCM), leading to the adoption of LOCM in clinical practice instead of HOCM [1]. We speculate that water deprivation alone might not be a sufficient pretreatment for the induction of AKI in rats exposed to LOCM. In the present study, we compared multiple pretreatment plans and found that dehydration for 3 days plus furosemide injection 20 min before contrast medium administration is optimal to induced CI-AKI. Under this condition, we obtained adequate and reproducible
elevation of SCr and histopathologic changes of kidney tissue consistent with the changes observed in human CI-AKI. Therefore, we believe that the model described here is a suitable model of experimental nephropathy and is comparable to the human CI-AKI. Acknowledgments This work was supported by the Grant Numbers 30800453, 30971185, 81370399, 81070239, 81100200, 81100199, 81270374 and 81170192 from the National Natural Science Foundation and the Grant Numbers 10JC1409400, 11JC1407100, 11ZR1421600 and 12140902600 from the Shanghai Municipal Natural Science Foundation. References [1] McCullough PA. Contrast-induced acute kidney injury. J Am Coll Cardiol 2008;51: 1419–28. [2] Zhang T, Shen LH, Hu LH, et al. Statins for the prevention of contrast-induced nephropathy: a systematic review and meta-analysis. Am J Nephrol 2011;33:344–51. [3] Idee JM, Bonnemain B. Reliability of experimental models of iodinated contrast media-induced acute renal failure. From methodological considerations to pathophysiology. Invest Radiol 1996;31:230–41. [4] Toprak O, Cirit M, Tanrisev M, et al. Preventive effect of nebivolol on contrast-induced nephropathy in rats. Nephrol Dial Transplant 2008;23:853–9. [5] Schrier RW, Wang W, Poole B, et al. Acute renal failure: definitions, diagnosis, pathogenesis, and therapy. J Clin Invest 2004;114:5–14. [6] Heyman SN, Brezis M, Reubinoff CA, et al. Acute renal failure with selective medullary injury in the rat. J Clin Invest 1988;82:401–12. [7] Agmon Y, Peleg H, Greenfield Z, et al. Nitric oxide and prostanoids protect the renal outer medulla from radiocontrast toxicity in the rat. J Clin Invest 1994;94:1069–75. [8] Gazi S, Altun A, Erdogan O. Contrast induced nephropathy: preventive and protective effects of melatonin. J Pineal Res 2006;41:53–7.
Contents lists available at ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Letter to the Editor
A novel rat model of contrast-induced acute kidney injury Shiqun Sun 1, Tuo Zhang 1, Peng Nie, Liuhua Hu, Ying Yu, Mingli Cui, Zhaohua Cai, Linghong Shen ⁎, Ben He ⁎ Department of Cardiology, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China
a r t i c l e
i n f o
Article history: Received 29 September 2013 Accepted 21 December 2013 Available online 30 December 2013 Keywords: Contrast medium Acute kidney injury Animal model
studies conformed to the guidelines on animal care of Shanghai Jiaotong University School of Medicine. As shown in Fig. 1, twelve rats were randomly assigned to four groups: dehydration for 3 days before contrast medium administration (Group 1); dehydration for 1 day and furosemide injection 20 min before contrast medium administration (Group 2); dehydration for 2 days and furosemide injection 20 min before contrast medium administration (Group 3); dehydration for 3 days and furosemide injection 20 min before contrast medium administration (Group 4). No difference in SCr levels were detected between the baseline value and 24 h after contrast medium administration in Group 1 (21.3 ± 1.3 μmol/L vs.
Contrast-induced acute kidney injury (CI-AKI) is an iatrogenic complication after the administration of contrast medium in diagnostic and interventional procedures. It is a common cause of hospital-acquired renal insufficiency and accounts for about 11% of cases [1,2]. An adequate animal model would be useful for the understanding and prevention of this disorder [3]. In this study, we have developed a new, highly efficient and reproducible model of acute kidney injury following contrast medium infusion in rats. Male Sprague–Dawley rats, weighing approximately 250–300 g, were included in the study. A nonionic low-osmolar contrast medium, Omnipaque (350 mg I/mL, GE Healthcare, Shanghai, China), was injected at 10 mL/kg via tail vein administration over the course of 5 min. Furosemide (Harvest Pharmaceutical Co., Shanghai, China) was injected intramuscularly at 10 mL/kg. Blood samples were taken from the jugular vein in untreated rats and from the abdominal aorta in sacrificed rats. EDTA (1.5 mg/mL) was added to the blood to impair coagulation. Immediately after sacrifice, both kidneys were harvested and embedded in paraffin. Adjacent 4 μm sections were stained with hematoxylin and eosin (H&E). Serum creatine (SCr) levels were measured by a standard spectrophotometric assay (Modular P, Roche Diagnostics GmbH, Penzberg, Germany). Renal tubular necrosis, medullary congestion, and proteinaceous casts were evaluated semi-quantitatively in a blinded manner by the method suggested by Toprak, et al. [4]. The authors of this manuscript certify that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology. All animal
⁎ Corresponding authors at: Department of Cardiology, Renji Hospital, Shanghai Jiaotong University School of Medicine, 1630 Dong Fang Road, Shanghai 200127, China. Tel.: +86 21 58752345; fax: +86 21 68383609. E-mail addresses: [email protected] (L. Shen), [email protected] (B. He). 1 Shiqun Sun and Tuo Zhang equally contributed to this work. 0167-5273/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijcard.2013.12.066
Fig. 1. Preliminary experiment. Male Sprague–Dawley rats were deprived of water as indicated time interval and followed by furosemide injection or not 20 min before Omnipaque administration. Blood samples were drawn before dehydration and 24 h after contrast medium injection (*p b 0.05 vs. baseline SCr value, n = 3). Values are mean ± SEM.
S. Sun et al. / International Journal of Cardiology 172 (2014) e48–e50
22.0 ± 3.5 μmol/L, p N 0.05), Group 2 (21.5 ± 2.8 μmol/L vs. 25.1 ± 2.2 μmol/L, p N 0.05) and Group 3 (20.7 ± 0.5 μmol/L vs. 25.7 ± 2.1 μmol/L, p N 0.05). However, SCr elevated more than 50% in Group 4 (23.0 ± 1.5 μmol/L vs. 36.2 ± 7.9 μmol/L, p b 0.05). To confirm the change of SCr in Group 4, another twelve rats were enrolled. Animals were dehydrated for 3 days and underwent furosemide injection 20 min before contrast medium (10 mL/kg, group CM)
e49
or 0.9% normal saline (10 mL/kg, group NS) administration. As shown in Fig. 2A and B, SCr increased 42.9% after contrast medium injection compared to the baseline value in the group CM and no change was observed in group NS. Since the most commonly used definition of CI-AKI is an increase in SCr ≥25% of the baseline value [1], we calculated the change of SCr and found that 83.3% (5/6) of the group CM reached this threshold. Direct tubular toxicity and tubular obstruction are
Fig. 2. Effect of either normal saline or contrast medium on serum creatinine and kidney histopathological changes. (A and B) Male Sprague–Dawley rats were deprived of water for 3 days and followed by furosemide injection 20 min before normal saline or contrast medium administration (*p b 0.05 vs. baseline value, **p b 0.05 vs. group NS, n = 6). (C) Proteinaceous casts in rat tubular lumens of CM group. (H&E stain, original magnification ×100). (D) Tubular necrosis in rat kidney of group CM (H&E stain, original magnification ×100). (E) Outer medullary congestion in rat kidney of group CM (H&E stain, original magnification ×100). (F) Evaluations of kidney histopathological changes. Tubular necrosis and proteinaceous casts were graded as follows: no damage (0), mild (1, unicellular, patchy isolated damage), moderate (2, damage less than 25%), severe (3, damage between 25 and 50%), and very severe (4, more than 50% damage). The degree of medullary congestion was defined as follows: no congestion (0), mild (1, vascular congestion with identification of erythrocytes by ×400 magnification), moderate (2, vascular congestion with identification of erythrocytes by ×200 magnification), severe (3, vascular congestion with identification of erythrocytes by ×100 magnification) and very severe (4, vascular congestion with identification of erythrocytes by ×40 magnification). *p b 0.05 vs. group NS, n = 6. Values are mean ± SEM.
e50
S. Sun et al. / International Journal of Cardiology 172 (2014) e48–e50
commonly suggested mechanisms underlying the pathogenesis of CIAKI [1,5]. Therefore, we examined the histopathological changes of the kidneys in these rats. All rats in group NS and group CM developed proteinaceous casts (Fig. 2C, inner zone of the medulla), tubular necrosis (Fig. 2D, outer zone of medulla), and medullary congestion (Fig. 2E, outer zone of medulla). Significantly more tubular necrosis and medullary congestion were seen in group CM compared to group NS (Fig. 2F). Acute kidney injury (AKI) is not easily induced by simply exposing healthy animal to contrast medium alone. Pretreating animals with factors similar to those considered to be risk factors for kidney damage in humans can help induce AKI in animals. Rats are widely used for CIAKI models [3]. Multiple insults, such as uninephrectomy, salt depletion, dehydration, and furosemide or indomethacin injection have been used in rats to predispose them to injury [4,6–8]. Water deprivation is regarded as an appropriate pretreatment method because it is easy to prepare with no pharmacological or surgical procedures. Previous studies have shown that a prolonged dehydration period (3 days) before high-osmolar contrast medium (HOCM) administration promotes renal function deterioration [4]. However, clinical data have demonstrated a significantly lower frequency of CI-AKI in patients with impaired renal function who received low-osmolar contrast medium (LOCM), leading to the adoption of LOCM in clinical practice instead of HOCM [1]. We speculate that water deprivation alone might not be a sufficient pretreatment for the induction of AKI in rats exposed to LOCM. In the present study, we compared multiple pretreatment plans and found that dehydration for 3 days plus furosemide injection 20 min before contrast medium administration is optimal to induced CI-AKI. Under this condition, we obtained adequate and reproducible
elevation of SCr and histopathologic changes of kidney tissue consistent with the changes observed in human CI-AKI. Therefore, we believe that the model described here is a suitable model of experimental nephropathy and is comparable to the human CI-AKI. Acknowledgments This work was supported by the Grant Numbers 30800453, 30971185, 81370399, 81070239, 81100200, 81100199, 81270374 and 81170192 from the National Natural Science Foundation and the Grant Numbers 10JC1409400, 11JC1407100, 11ZR1421600 and 12140902600 from the Shanghai Municipal Natural Science Foundation. References [1] McCullough PA. Contrast-induced acute kidney injury. J Am Coll Cardiol 2008;51: 1419–28. [2] Zhang T, Shen LH, Hu LH, et al. Statins for the prevention of contrast-induced nephropathy: a systematic review and meta-analysis. Am J Nephrol 2011;33:344–51. [3] Idee JM, Bonnemain B. Reliability of experimental models of iodinated contrast media-induced acute renal failure. From methodological considerations to pathophysiology. Invest Radiol 1996;31:230–41. [4] Toprak O, Cirit M, Tanrisev M, et al. Preventive effect of nebivolol on contrast-induced nephropathy in rats. Nephrol Dial Transplant 2008;23:853–9. [5] Schrier RW, Wang W, Poole B, et al. Acute renal failure: definitions, diagnosis, pathogenesis, and therapy. J Clin Invest 2004;114:5–14. [6] Heyman SN, Brezis M, Reubinoff CA, et al. Acute renal failure with selective medullary injury in the rat. J Clin Invest 1988;82:401–12. [7] Agmon Y, Peleg H, Greenfield Z, et al. Nitric oxide and prostanoids protect the renal outer medulla from radiocontrast toxicity in the rat. J Clin Invest 1994;94:1069–75. [8] Gazi S, Altun A, Erdogan O. Contrast induced nephropathy: preventive and protective effects of melatonin. J Pineal Res 2006;41:53–7.
