J Huazhong Univ Sci Technol [Med Sci] 34(4):516-520,2014 DOI 10.1007/s11596-014-1308-y J Huazhong Univ Sci Technol［Med Sci］ 34(4):2014
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Glucocorticoid Receptor Agonist Dexamethasone Attenuates Renal Ischemia/Reperfusion Injury by Up-regulating eNOS/iNOS* Jiong ZHANG (张 炯)1, 2, Jun-hua LI (李俊华)1, Le WANG (王 艻)1, Min HAN (韩 敏), Fang XIAO (肖 芳)3, Xiao-qin LAN (兰小勤)3, Yue-qiang LI (李月强)1, Gang XU (徐 钢)1#, Ying YAO (姚 颖)1# 1 Department of Nephrology, 3Department of Gastroenterology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China 2 Department of Nephrology, Subsidiary of the Sichuan Academy of Sciences & Sichuan Provincial People's Hospital, University of Electronic Science and Technology, Chengdu 610072, China © Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2014
Summary: The aim of this study was to determine the effect of dexamethasone (DEX) on renal ischemia/reperfusion injury (IRI). C57BL/6 mice were randomly divided into Sham group, IRI group and DEX group. The mice in IRI and DEX groups subjected to renal ischemia for 60 min, were treated with saline or DEX (4 mg/kg, i.p.) 60 min prior to I/R. After 24 h of reperfusion, the renal function, renal pathological changes, activation of extracellular signal-regulated kinase (ERK) and glucocorticoid receptor (GR), and the levels of iNOS and eNOS were detected. The results showed DEX significantly decreased the damage to renal function and pathological changes after renal IRI. Pre-treatment with DEX reduced ERK activation and down-regulated the level of iNOS, whereas up-regulated the level of eNOS after renal IRI. DEX could further promote the activation of GR. These findings indicated GR activation confers preconditioning-like protection against acute IRI partially by up-regulating the ratio of eNOS/iNOS. Key words: ischemia/reperfusion injury; dexamethasone; nitric oxide synthase
Acute kidney injury (AKI) is a common clinical complication which is associated with high mortality and morbidity[1]. It is frequently occurring as a result of sepsis, hypovolemia, and hypotension and ischemia reperfusion injury[2–5]. As a major cause of AKI, renal ischemia/reperfusion injury (IRI) frequently occurs following transplantation, cardiothoracic and vascular surgery[6–8]. The pathogenesis of IRI is complex and the studies show that apoptosis[2], nitric oxide (NO)[9, 10], and inflammation[4], etc. are involved in the renal dysfunction. NO is a commonly seen compound which plays an important role in IRI[9, 10]. NO is metabolically produced by NO synthase (NOS) from L-arginine, oxygen, and nicotinamide adenine dinucleotide phosphate. The NOS family has three isoforms: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS)[11, 12]. In the kidney, eNOS and iNOS are the key molecules for catalyzing the synthesis of NO. eNOS is constitutively present in the plasma membranes of endothelial cells[13] and produces small quantities of NO to maintain kidney vascular homeostasis during stable conditions[13]. In contrast, under stress, such as bacterial infection, inflammtion and IRI, iNOS is up-regulated in various cells, including tubular epithelial cells, endothelial cells, etc., and iNOS produces large amounts of NO to cope with stressful conditions; the large amounts of NO in turn Jiong ZHANG, E-mail: [email protected] # Corresponding authors, Ying YAO, E-mail: [email protected]; Gang XU, E-mail: [email protected] * This project was supported by grants from the National Natural Science Foundation of China (Nos. 81170686, 81100498, 81100264, 81100485 and 81370798), and the Ministry of Education of China (No. 311028).
paradoxically damage kidney tissue by forming nitrogen peroxide[14–18]. Therefore, the ratio of eNOS/iNOS plays an important role in regulating renal function. It is reported that IRI leads to the decreased eNOS expression and increased iNOS expression, which destroys sources of NO balance in renal tissues[9, 10]. Otherwise, the sources of NO imbalance finally impair renal function after IRI[11–13]. Dexamethasone (DEX) is one of the glucocorticoids (GCs) which are widely used in clinical practice for their anti-inflammatory and immunosuppressive effect[14]. Recently, GCs have been shown to inhibit extracellular signal-regulated kinase (ERK) signal pathway. ERK is a classical signal pathway which is involved in regulating the ratio of eNOS/iNOS[15]. We hypothesize that DEX can decrease ERK activation through regulating eNOS/iNOS to protect kidney against acute IRI[16]. In this study, the protective effect of DEX on renal IRI, and the possible mechanisms were investigated. 1 MATERIALS AND METHODS 1.1 Animals and Grouping Male C57BL/6 mice (Hua Fukang Experimental Animal Center, Beijing, China) were given a standard laboratory diet and water ad libitum. The protocol was approved by the Institutional Animal Care and Use Committee of Tongji Medical College, Huazhong University of Science and Technology (China). At the start of the experiments, the mice were 8–10 weeks of age, weighing 25–30 g. After a minimum 7-day acclimation period, the mice were randomly allocated into three groups: (1) IRI group, in which mice were subjected to
517
J Huazhong Univ Sci Technol［Med Sci］34(4):2014
renal ischemia for 1 h (n=6); (2) DEX group, in which mice were administered DEX (4 mg/kg, i.p.) 1 h prior to I/R (n=6); (3) sham-operated (Sham) group, in which mice were subjected to identical surgical procedure without occlusion of both renal pedicles (n=6). The dose of DEX used in this study was referred to that reported in the previous literature[17]. 1.2 Renal IRI Model Briefly, mice were anaesthetized with intraperitoneal injection of 1% sodium pentobarbital solution (6 mL/kg). Following abdominal incisions the renal pedicles were bluntly dissected and a microvascular clamp was placed on the left renal pedicle for 60 min. During the procedure, mice were kept well-hydrated with warm saline and at a constant temperature of 32°С in an infant incubator. After removal of the clamp, the contralateral kidney was removed. Thereafter, incisions were sutured, and the animals were allowed to recover with free access to food and water. Mice were sacrificed 24 h after reperfusion, and the blood samples and the left kidneys were harvested for further analysis. 1.3 Assessment of Renal Function Blood samples were obtained from the inferior vena cava 24 h after reperfusion. Blood urea nitrogen (BUN) and serum creatinine (SCr) levels were measured to assess the renal function. 1.4 Renal Pathological Changes At 24 h post-reperfusion, renal tissue samples harvested from mice were fixed in formalin and then embedded in paraffin. Four-μm thick sections were stained with periodic acid-Schiff (PAS) staining[18]. The pathological changes in the cortex and medulla were observed by a pathologist in a blinded fashion. The percentage of pathological changes in the cortex and medulla was scored using a semi-quantitative scale designed to evaluate the degree of infarction, tubular vacuolization and cast formation on a 5-point scale based on injury area of involvement as follows: 0,
516
Glucocorticoid Receptor Agonist Dexamethasone Attenuates Renal Ischemia/Reperfusion Injury by Up-regulating eNOS/iNOS* Jiong ZHANG (张 炯)1, 2, Jun-hua LI (李俊华)1, Le WANG (王 艻)1, Min HAN (韩 敏), Fang XIAO (肖 芳)3, Xiao-qin LAN (兰小勤)3, Yue-qiang LI (李月强)1, Gang XU (徐 钢)1#, Ying YAO (姚 颖)1# 1 Department of Nephrology, 3Department of Gastroenterology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China 2 Department of Nephrology, Subsidiary of the Sichuan Academy of Sciences & Sichuan Provincial People's Hospital, University of Electronic Science and Technology, Chengdu 610072, China © Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2014
Summary: The aim of this study was to determine the effect of dexamethasone (DEX) on renal ischemia/reperfusion injury (IRI). C57BL/6 mice were randomly divided into Sham group, IRI group and DEX group. The mice in IRI and DEX groups subjected to renal ischemia for 60 min, were treated with saline or DEX (4 mg/kg, i.p.) 60 min prior to I/R. After 24 h of reperfusion, the renal function, renal pathological changes, activation of extracellular signal-regulated kinase (ERK) and glucocorticoid receptor (GR), and the levels of iNOS and eNOS were detected. The results showed DEX significantly decreased the damage to renal function and pathological changes after renal IRI. Pre-treatment with DEX reduced ERK activation and down-regulated the level of iNOS, whereas up-regulated the level of eNOS after renal IRI. DEX could further promote the activation of GR. These findings indicated GR activation confers preconditioning-like protection against acute IRI partially by up-regulating the ratio of eNOS/iNOS. Key words: ischemia/reperfusion injury; dexamethasone; nitric oxide synthase
Acute kidney injury (AKI) is a common clinical complication which is associated with high mortality and morbidity[1]. It is frequently occurring as a result of sepsis, hypovolemia, and hypotension and ischemia reperfusion injury[2–5]. As a major cause of AKI, renal ischemia/reperfusion injury (IRI) frequently occurs following transplantation, cardiothoracic and vascular surgery[6–8]. The pathogenesis of IRI is complex and the studies show that apoptosis[2], nitric oxide (NO)[9, 10], and inflammation[4], etc. are involved in the renal dysfunction. NO is a commonly seen compound which plays an important role in IRI[9, 10]. NO is metabolically produced by NO synthase (NOS) from L-arginine, oxygen, and nicotinamide adenine dinucleotide phosphate. The NOS family has three isoforms: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS)[11, 12]. In the kidney, eNOS and iNOS are the key molecules for catalyzing the synthesis of NO. eNOS is constitutively present in the plasma membranes of endothelial cells[13] and produces small quantities of NO to maintain kidney vascular homeostasis during stable conditions[13]. In contrast, under stress, such as bacterial infection, inflammtion and IRI, iNOS is up-regulated in various cells, including tubular epithelial cells, endothelial cells, etc., and iNOS produces large amounts of NO to cope with stressful conditions; the large amounts of NO in turn Jiong ZHANG, E-mail: [email protected] # Corresponding authors, Ying YAO, E-mail: [email protected]; Gang XU, E-mail: [email protected] * This project was supported by grants from the National Natural Science Foundation of China (Nos. 81170686, 81100498, 81100264, 81100485 and 81370798), and the Ministry of Education of China (No. 311028).
paradoxically damage kidney tissue by forming nitrogen peroxide[14–18]. Therefore, the ratio of eNOS/iNOS plays an important role in regulating renal function. It is reported that IRI leads to the decreased eNOS expression and increased iNOS expression, which destroys sources of NO balance in renal tissues[9, 10]. Otherwise, the sources of NO imbalance finally impair renal function after IRI[11–13]. Dexamethasone (DEX) is one of the glucocorticoids (GCs) which are widely used in clinical practice for their anti-inflammatory and immunosuppressive effect[14]. Recently, GCs have been shown to inhibit extracellular signal-regulated kinase (ERK) signal pathway. ERK is a classical signal pathway which is involved in regulating the ratio of eNOS/iNOS[15]. We hypothesize that DEX can decrease ERK activation through regulating eNOS/iNOS to protect kidney against acute IRI[16]. In this study, the protective effect of DEX on renal IRI, and the possible mechanisms were investigated. 1 MATERIALS AND METHODS 1.1 Animals and Grouping Male C57BL/6 mice (Hua Fukang Experimental Animal Center, Beijing, China) were given a standard laboratory diet and water ad libitum. The protocol was approved by the Institutional Animal Care and Use Committee of Tongji Medical College, Huazhong University of Science and Technology (China). At the start of the experiments, the mice were 8–10 weeks of age, weighing 25–30 g. After a minimum 7-day acclimation period, the mice were randomly allocated into three groups: (1) IRI group, in which mice were subjected to
517
J Huazhong Univ Sci Technol［Med Sci］34(4):2014
renal ischemia for 1 h (n=6); (2) DEX group, in which mice were administered DEX (4 mg/kg, i.p.) 1 h prior to I/R (n=6); (3) sham-operated (Sham) group, in which mice were subjected to identical surgical procedure without occlusion of both renal pedicles (n=6). The dose of DEX used in this study was referred to that reported in the previous literature[17]. 1.2 Renal IRI Model Briefly, mice were anaesthetized with intraperitoneal injection of 1% sodium pentobarbital solution (6 mL/kg). Following abdominal incisions the renal pedicles were bluntly dissected and a microvascular clamp was placed on the left renal pedicle for 60 min. During the procedure, mice were kept well-hydrated with warm saline and at a constant temperature of 32°С in an infant incubator. After removal of the clamp, the contralateral kidney was removed. Thereafter, incisions were sutured, and the animals were allowed to recover with free access to food and water. Mice were sacrificed 24 h after reperfusion, and the blood samples and the left kidneys were harvested for further analysis. 1.3 Assessment of Renal Function Blood samples were obtained from the inferior vena cava 24 h after reperfusion. Blood urea nitrogen (BUN) and serum creatinine (SCr) levels were measured to assess the renal function. 1.4 Renal Pathological Changes At 24 h post-reperfusion, renal tissue samples harvested from mice were fixed in formalin and then embedded in paraffin. Four-μm thick sections were stained with periodic acid-Schiff (PAS) staining[18]. The pathological changes in the cortex and medulla were observed by a pathologist in a blinded fashion. The percentage of pathological changes in the cortex and medulla was scored using a semi-quantitative scale designed to evaluate the degree of infarction, tubular vacuolization and cast formation on a 5-point scale based on injury area of involvement as follows: 0,
