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Title Page Protective Effects of Curcumin on Acute Gentamicin-Induced Nephrotoxicity in Rats Running title: Curcumin on gentamicin-induced nephrotoxicity Liyu He1, Xiaofei Peng1, Jiefu Zhu1, Guoyong Liu2, Xian Chen1, Chengyuan Tang1, Hong Liu1, Fuyou Liu1, Youming Peng1 The author affiliations: 1.Nephrology Department, 2nd Xiangya Hospital, Central South University, Key Lab of Kidney Disease and Blood Purification in Hunan, 139 Renmin Road, Changsha, Hunan 410011, People's Republic of China.2. Department of Nephrology, The First Affiliated Hospital of Changde Vocational Technical College, Changde, Hunan 415000, People's Republic of China. Corresponding author: Youming Peng (1)Full mailing address: Nephrology Department, 2nd Xiangya Hospital, Central South University, Key Lab of Kidney Disease and Blood Purification in Hunan, 139 Renmin Road, Changsha, Hunan 410011, People's Republic of China. (2)Tel: +8673185292064
Fax number：+8673185295843
(3)Email: [email protected] Conflict of interest The authors have no conflicts of interest to disclose. Key Words: oxidative stress; apoptosis; Nrf2; HO-1; Sirt1 Word account：4100 All other Authors have read the manuscript and have agreed to submit it in its current form for consideration for publication in the Journal
Page 2 of 27
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Abstract Background Gentamicin-induced nephrotoxicity is one of the most common causes of acute kidney injury (AKI). The phenotypic alterations that contribute to acute kidney injury include inflammatory response and oxidative stress. Curcumin (CUR) has broad biological functions particularly antioxidant. This study was designed to evaluate the renoprotective effect of CUR treatment on gentamicin-induced AKI. Methods Gentamicin-induced AKI was established in female Sprague-Dawley rats. Rats were treated with curcumin 100 mg/kg/BW by intragastric once daily followed by gentamicin sulfate solution intraperitoneal at a dose of 80 mg/kg/BW for 8 consecutive days. At 3 and 8 days, the rats was sacrificed, kidneys and blood samples were collected for further analysis. Results Gentamicin administered animals showed marked deterioration of renal function together with higher levels of neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule 1 (KIM-1) in the plasma compared to the control. Animals that underwent intermittent exposure to CUR treatment exhibited significant improvements in renal functional parameters. We also observed that treatment with CUR significantly attenuated renal tubular damage, apoptosis, and oxidative stress. CUR treatment exerted anti-apoptosis and anti-oxidative effects by up-regulating Nrf2/HO-1 and Sirt1 expression. Conclusions Our data clearly demonstrated that CUR protected the kidney from GM-induced AKI via an amelioration of oxidative stress and apoptosis of renal tubular cells, thus brought a light of hope for ameliorating GM-induced
Page 3 of 27
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nephrotoxicity. Keywords: oxidative stress; apoptosis; Nrf2; HO-1; Sirt1
Introduction Over the past 2 decades, rapid increases in the incidence of acute kidney injury (AKI) have been reported, highlighting a growing contribution to the public health burden of advanced kidney disease (Siew and Davenport 2014). Drug or toxicant induced AKI approximately 20 percent of community-
and hospital-acquired episodes (Yano
2013). Gentamicin, as an aminoglycoside class of bactericidal antibiotic, is employed in the treatment of severe gram-negative bacterial infections. However, its clinical use has been limited because of gentamicin-induced AKI. The precise mechanism of gentamicin nephrotoxicity is not well elucidated. The phenotypic alterations that contribute to acute kidney injury include inflammatory response, oxidative stress and hemodynamic changes (Zorov 2010). Nuclear factor erythroid 2-related factor 2 (Nrf2) regulates an expansive set of antioxidant/detoxification genes including acting in synergy to remove ROS/RNS through sequential enzymatic reactions. In the nucleus, Nrf2 binds to the antioxidant response elements (ARE) in the promoter regions of Nrf2 target genes including heme oxygenase-1 (HO-1). HO-1 exhibits low basal expression levels in most cells and tissues, is highly up-regulated by a wide variety of oxidative stress stimuli (Gozzelino et al. 2010). Due to its regulatory pattern, induction of HO-1 has generally been considered to be an adaptive cellular response against the toxicity of oxidative stress
Page 4 of 27
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4
(Paine et al. 2010). Nrf2-mediated HO-1 induction contributes to antioxidant capacity has been demonstrated by multiple disease models, including cardiomyopathy in type 2 diabetic mice (Zhang et al. 2014b), rat model of transient global cerebral ischaemia (Lee et al. 2014), and endotoxic shock-induced acute lung injury in rabbits (Yu et al. 2014). Recently, Nrf2/HO-1 activation also shows protective effect in aristolochic acid-induced acute kidney injury (Wu et al. 2014a) and intestinal ischemia-reperfusion induced acute renal injury (Sun et al. 2013). Silent information regulator 1 (Sirt1), a key member of the mammalian sirtuin family, is a type of histone deacetylase whose activity is dependent on nicotinamide adenine dinucleotide (NAD+). Using many histones and non-histone proteins as substrates, Sirt1 possesses remarkable anti-oxidative capacity (Radak et al. 2013). The current data also point that Sirt1 is upstream regulators for fasting-induced activation of the NRF2-ARE pathway in the antioxidant response (Kulkarni et al. 2014). Curcumin (CUM) is an active ingredient of polyphenolic curcuminoids extracted from Curcuma longa Linn. CUR has been well recognized as a dietary spice for centuries and its pharmacological activity have been studied in various animal models and clinical investigations which exerts anti-inflammatory and antioxidant effects (Boonla et al. 2014b; Wu et al. 2014b). In cisplatin-induced nephrotoxicity mice model, renal dysfunction and renal tubular necrosis scores were attenuated by CUR treatment through its anti-inflammatory effects by suppressing the pro-inflammatory factors NF-κB and COX-2 as well as promoting downstream markers Nrf2 and HO-1 in kidney (Sahin et al. 2014; Ueki et al. 2013). Recently, a study also shows that CUR
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pretreatment could attenuate ischemia reperfusion injury by reducing ischemia reperfusion-induced mitochondrial oxidative damage through the activation of SIRT1 signaling (Yang et al. 2013). Although its role as a protective agent against renal oxidative damage mediated by gentamicin has been investigated in previous studies (Ali et al. 2005; Farombi and Ekor 2006), the underlying mechanism of the anti-apoptosis and anti-oxidant effect of CUR has not been elucidated until recently. Therefore, this study was designed to evaluate the anti-apoptosis and antioxidant effect of CUR pretreatment on gentamicin-induced AKI. The involvement of Nrf2/HO-1 and SIRT1 signaling in mediating the protective mechanisms of CUR was also evaluated.
Materials and methods Animals and reagents The protocol of this study was approved by Institutional Animal Ethical Committee of The Second Xiangya Hospital. Young (6- to 7-week-old) female Sprague-Dawley rats weighing 180-210g were purchased from Slaccas Animal Laboratory (Shanghai, China), and housed under controlled environmental conditions (temperature 22°C, 12 h darkness period). Animals were given free access to water and fed a standard laboratory diet. Curcumin was purchased from Sigma Aldrich (HPLC grade, ≥99.5%) (St. Louis,MO, USA). Pentobarbital sodium was purchased from Dainippon Sumitomo Pharma Co., Ltd. (Osaka, Japan). Gentamicin was purchased from Tianjin Pharmaceutical
Page 6 of 27
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Company, China
Experimental Protocol Rats were randomly assigned into four groups (n=12 each): (i) control group: rats received a daily intraperitoneal (i.p.) injection of 1 ml saline for 8 days; (ii) gentamicin group (GM): received a daily i.p. injection of gentamicin sulfate solution at a dose of 80 mg/kg/BW for 8 days, which is well known to cause significant nephrotoxicity in rats; (iii) curcumin group (CUM): curcumin was suspended in a volume of
2ml olive oil. Rats were treated with curcumin 100mg/kg/day by
intragastric once daily for 8 days. This dose of curcumin was selected on the basis of existed data (Boonla et al. 2014a). (iv) GM+CUR group: rats treated with curcumin (100 mg/kg by gavage) followed by gentamicin for 8 consecutive days. Six rats each group was sacrificed under anesthesia on days 3 and 8 after the last gentamicin injection, respectively. Blood sample was taken from the abdominal aorta as this method can minimize blood transfusions. Kidneys were harvested. One kidney was for the analysis of histology, immunohistochemistry. The other was frozen in liquid nitrogen and stored at – 80℃ for further assay.
Laboratory Analyses Plasma creatinine and blood urea nitrogen concentration (BUN) were measured using commercial kits from BioAssay Systems to indicate renal function. Plasma neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule 1 (KIM-1) levels
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7
were quantified using a commercially available ELISA kit (Cusabio Biotech, Wuhan, China). Malondialdehyde (MDA) formation was utilized to quantify a naturally occurring product of lipid peroxidation and measured as thiobarbituric acid reactive material. It was determined using supernatant of the renal cortical homogenate, according to the manufacturer’s protocol (TBARS Assay Kit, Cayman Chemical Company, USA). Prior to biochemical analysis, kidneys of each group were also homogenized and used for the analysis of enzymatic as well as non-enzymatic antioxidants. Glutathione (GSH) concentration, superoxide dismutase (SOD) activity and Catalase (CAT) activity were measured spectrophotometricallty according to the method previously reported (Farombi and Ekor 2006) using commercial kits (NeoBioscience Technology Co., Ltd. Beijing, China).
DHE and Tunnel Assay Dihydroethidium (DHE) was used to assess the production of intracellular superoxide anion (O2-). The TUNEL procedure was used to gauge apoptosis following the manufacturer’s instructions (Roche, Basel, Switzerland).
RTRT-PCR Total RNA was isolated from kidney of individual mice using TRIzol (Takara, Dalian, China). cDNA was synthesized using M-MLV RTase cDNA Synthesis Kit (Takara, Dalian, China) according to the manufacturer's instructions. Real-time PCR was performed with ABI Prism 7300 Sequence Detection system (Applied
Page 8 of 27
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Biosystems) using the SYBR® Premix189 Ex Taq™ II (Takara, Dalian, China). The applied primers for Real-time qPCR were shown in Table.1. The gene expression from each sample was analyzed in duplicates and normalized against the internal control gene GAPDH.
Western blot analysis Western blot was performed according to our previous procedures (He et al. 2013). Membranes were incubated with the following primary antibodies, respectively: rabbit antibody to Nrf2 (1:200 Biorbyt, Cambridge, UK), HO-1 (1:500 MBL International Corporation, Nagoya, Japan)) and Sirt1 (1:1000 MyBioSource, LLC, San Diego, CA, USA). β-Actin-specific antibody (1: 1,000; Abcam, Cambridge, UK) was used for loading controls on stripped membranes. Horseradish peroxidase-conjugated secondary antibodies were applied, and enhanced chemiluminescence (Thermo, Rockford, Ill., USA) was used to visualize the bands.
Histopathological Examinations and Immunohistochemistry Kidney slices were fixed in 10% formalin, embedded in paraffin wax, cut into 5 µm sections and stained with hematoxylin and eosin. The tissues were evaluated by light microscopy. For immunohistochemical stains, in brief, sections were rehydrated and antigens retrieved using heated citrate. After the incubation with blocking buffers, tissue sections were exposed sequentially to the primary
Page 9 of 27
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9
antibody, the horseradish peroxidase-coupled secondary antibodies (Vectastain Elite; Vector Laboratories, Peterborough, UK). The signals were developed with DAB Peroxidase Substrate Kit (Vector Laboratories). All immunohistochemical analyses were repeated at least three times and representative images were presented
Statistical Analysis Data are expressed as means ± standard error (SE). Comparisons among groups and two groups were evaluated by ANOVA and Mann-Whitney U test, respectively. Values of p
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1
Title Page Protective Effects of Curcumin on Acute Gentamicin-Induced Nephrotoxicity in Rats Running title: Curcumin on gentamicin-induced nephrotoxicity Liyu He1, Xiaofei Peng1, Jiefu Zhu1, Guoyong Liu2, Xian Chen1, Chengyuan Tang1, Hong Liu1, Fuyou Liu1, Youming Peng1 The author affiliations: 1.Nephrology Department, 2nd Xiangya Hospital, Central South University, Key Lab of Kidney Disease and Blood Purification in Hunan, 139 Renmin Road, Changsha, Hunan 410011, People's Republic of China.2. Department of Nephrology, The First Affiliated Hospital of Changde Vocational Technical College, Changde, Hunan 415000, People's Republic of China. Corresponding author: Youming Peng (1)Full mailing address: Nephrology Department, 2nd Xiangya Hospital, Central South University, Key Lab of Kidney Disease and Blood Purification in Hunan, 139 Renmin Road, Changsha, Hunan 410011, People's Republic of China. (2)Tel: +8673185292064
Fax number：+8673185295843
(3)Email: [email protected] Conflict of interest The authors have no conflicts of interest to disclose. Key Words: oxidative stress; apoptosis; Nrf2; HO-1; Sirt1 Word account：4100 All other Authors have read the manuscript and have agreed to submit it in its current form for consideration for publication in the Journal
Page 2 of 27
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Abstract Background Gentamicin-induced nephrotoxicity is one of the most common causes of acute kidney injury (AKI). The phenotypic alterations that contribute to acute kidney injury include inflammatory response and oxidative stress. Curcumin (CUR) has broad biological functions particularly antioxidant. This study was designed to evaluate the renoprotective effect of CUR treatment on gentamicin-induced AKI. Methods Gentamicin-induced AKI was established in female Sprague-Dawley rats. Rats were treated with curcumin 100 mg/kg/BW by intragastric once daily followed by gentamicin sulfate solution intraperitoneal at a dose of 80 mg/kg/BW for 8 consecutive days. At 3 and 8 days, the rats was sacrificed, kidneys and blood samples were collected for further analysis. Results Gentamicin administered animals showed marked deterioration of renal function together with higher levels of neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule 1 (KIM-1) in the plasma compared to the control. Animals that underwent intermittent exposure to CUR treatment exhibited significant improvements in renal functional parameters. We also observed that treatment with CUR significantly attenuated renal tubular damage, apoptosis, and oxidative stress. CUR treatment exerted anti-apoptosis and anti-oxidative effects by up-regulating Nrf2/HO-1 and Sirt1 expression. Conclusions Our data clearly demonstrated that CUR protected the kidney from GM-induced AKI via an amelioration of oxidative stress and apoptosis of renal tubular cells, thus brought a light of hope for ameliorating GM-induced
Page 3 of 27
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3
nephrotoxicity. Keywords: oxidative stress; apoptosis; Nrf2; HO-1; Sirt1
Introduction Over the past 2 decades, rapid increases in the incidence of acute kidney injury (AKI) have been reported, highlighting a growing contribution to the public health burden of advanced kidney disease (Siew and Davenport 2014). Drug or toxicant induced AKI approximately 20 percent of community-
and hospital-acquired episodes (Yano
2013). Gentamicin, as an aminoglycoside class of bactericidal antibiotic, is employed in the treatment of severe gram-negative bacterial infections. However, its clinical use has been limited because of gentamicin-induced AKI. The precise mechanism of gentamicin nephrotoxicity is not well elucidated. The phenotypic alterations that contribute to acute kidney injury include inflammatory response, oxidative stress and hemodynamic changes (Zorov 2010). Nuclear factor erythroid 2-related factor 2 (Nrf2) regulates an expansive set of antioxidant/detoxification genes including acting in synergy to remove ROS/RNS through sequential enzymatic reactions. In the nucleus, Nrf2 binds to the antioxidant response elements (ARE) in the promoter regions of Nrf2 target genes including heme oxygenase-1 (HO-1). HO-1 exhibits low basal expression levels in most cells and tissues, is highly up-regulated by a wide variety of oxidative stress stimuli (Gozzelino et al. 2010). Due to its regulatory pattern, induction of HO-1 has generally been considered to be an adaptive cellular response against the toxicity of oxidative stress
Page 4 of 27
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4
(Paine et al. 2010). Nrf2-mediated HO-1 induction contributes to antioxidant capacity has been demonstrated by multiple disease models, including cardiomyopathy in type 2 diabetic mice (Zhang et al. 2014b), rat model of transient global cerebral ischaemia (Lee et al. 2014), and endotoxic shock-induced acute lung injury in rabbits (Yu et al. 2014). Recently, Nrf2/HO-1 activation also shows protective effect in aristolochic acid-induced acute kidney injury (Wu et al. 2014a) and intestinal ischemia-reperfusion induced acute renal injury (Sun et al. 2013). Silent information regulator 1 (Sirt1), a key member of the mammalian sirtuin family, is a type of histone deacetylase whose activity is dependent on nicotinamide adenine dinucleotide (NAD+). Using many histones and non-histone proteins as substrates, Sirt1 possesses remarkable anti-oxidative capacity (Radak et al. 2013). The current data also point that Sirt1 is upstream regulators for fasting-induced activation of the NRF2-ARE pathway in the antioxidant response (Kulkarni et al. 2014). Curcumin (CUM) is an active ingredient of polyphenolic curcuminoids extracted from Curcuma longa Linn. CUR has been well recognized as a dietary spice for centuries and its pharmacological activity have been studied in various animal models and clinical investigations which exerts anti-inflammatory and antioxidant effects (Boonla et al. 2014b; Wu et al. 2014b). In cisplatin-induced nephrotoxicity mice model, renal dysfunction and renal tubular necrosis scores were attenuated by CUR treatment through its anti-inflammatory effects by suppressing the pro-inflammatory factors NF-κB and COX-2 as well as promoting downstream markers Nrf2 and HO-1 in kidney (Sahin et al. 2014; Ueki et al. 2013). Recently, a study also shows that CUR
Page 5 of 27
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5
pretreatment could attenuate ischemia reperfusion injury by reducing ischemia reperfusion-induced mitochondrial oxidative damage through the activation of SIRT1 signaling (Yang et al. 2013). Although its role as a protective agent against renal oxidative damage mediated by gentamicin has been investigated in previous studies (Ali et al. 2005; Farombi and Ekor 2006), the underlying mechanism of the anti-apoptosis and anti-oxidant effect of CUR has not been elucidated until recently. Therefore, this study was designed to evaluate the anti-apoptosis and antioxidant effect of CUR pretreatment on gentamicin-induced AKI. The involvement of Nrf2/HO-1 and SIRT1 signaling in mediating the protective mechanisms of CUR was also evaluated.
Materials and methods Animals and reagents The protocol of this study was approved by Institutional Animal Ethical Committee of The Second Xiangya Hospital. Young (6- to 7-week-old) female Sprague-Dawley rats weighing 180-210g were purchased from Slaccas Animal Laboratory (Shanghai, China), and housed under controlled environmental conditions (temperature 22°C, 12 h darkness period). Animals were given free access to water and fed a standard laboratory diet. Curcumin was purchased from Sigma Aldrich (HPLC grade, ≥99.5%) (St. Louis,MO, USA). Pentobarbital sodium was purchased from Dainippon Sumitomo Pharma Co., Ltd. (Osaka, Japan). Gentamicin was purchased from Tianjin Pharmaceutical
Page 6 of 27
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Company, China
Experimental Protocol Rats were randomly assigned into four groups (n=12 each): (i) control group: rats received a daily intraperitoneal (i.p.) injection of 1 ml saline for 8 days; (ii) gentamicin group (GM): received a daily i.p. injection of gentamicin sulfate solution at a dose of 80 mg/kg/BW for 8 days, which is well known to cause significant nephrotoxicity in rats; (iii) curcumin group (CUM): curcumin was suspended in a volume of
2ml olive oil. Rats were treated with curcumin 100mg/kg/day by
intragastric once daily for 8 days. This dose of curcumin was selected on the basis of existed data (Boonla et al. 2014a). (iv) GM+CUR group: rats treated with curcumin (100 mg/kg by gavage) followed by gentamicin for 8 consecutive days. Six rats each group was sacrificed under anesthesia on days 3 and 8 after the last gentamicin injection, respectively. Blood sample was taken from the abdominal aorta as this method can minimize blood transfusions. Kidneys were harvested. One kidney was for the analysis of histology, immunohistochemistry. The other was frozen in liquid nitrogen and stored at – 80℃ for further assay.
Laboratory Analyses Plasma creatinine and blood urea nitrogen concentration (BUN) were measured using commercial kits from BioAssay Systems to indicate renal function. Plasma neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule 1 (KIM-1) levels
Page 7 of 27
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7
were quantified using a commercially available ELISA kit (Cusabio Biotech, Wuhan, China). Malondialdehyde (MDA) formation was utilized to quantify a naturally occurring product of lipid peroxidation and measured as thiobarbituric acid reactive material. It was determined using supernatant of the renal cortical homogenate, according to the manufacturer’s protocol (TBARS Assay Kit, Cayman Chemical Company, USA). Prior to biochemical analysis, kidneys of each group were also homogenized and used for the analysis of enzymatic as well as non-enzymatic antioxidants. Glutathione (GSH) concentration, superoxide dismutase (SOD) activity and Catalase (CAT) activity were measured spectrophotometricallty according to the method previously reported (Farombi and Ekor 2006) using commercial kits (NeoBioscience Technology Co., Ltd. Beijing, China).
DHE and Tunnel Assay Dihydroethidium (DHE) was used to assess the production of intracellular superoxide anion (O2-). The TUNEL procedure was used to gauge apoptosis following the manufacturer’s instructions (Roche, Basel, Switzerland).
RTRT-PCR Total RNA was isolated from kidney of individual mice using TRIzol (Takara, Dalian, China). cDNA was synthesized using M-MLV RTase cDNA Synthesis Kit (Takara, Dalian, China) according to the manufacturer's instructions. Real-time PCR was performed with ABI Prism 7300 Sequence Detection system (Applied
Page 8 of 27
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8
Biosystems) using the SYBR® Premix189 Ex Taq™ II (Takara, Dalian, China). The applied primers for Real-time qPCR were shown in Table.1. The gene expression from each sample was analyzed in duplicates and normalized against the internal control gene GAPDH.
Western blot analysis Western blot was performed according to our previous procedures (He et al. 2013). Membranes were incubated with the following primary antibodies, respectively: rabbit antibody to Nrf2 (1:200 Biorbyt, Cambridge, UK), HO-1 (1:500 MBL International Corporation, Nagoya, Japan)) and Sirt1 (1:1000 MyBioSource, LLC, San Diego, CA, USA). β-Actin-specific antibody (1: 1,000; Abcam, Cambridge, UK) was used for loading controls on stripped membranes. Horseradish peroxidase-conjugated secondary antibodies were applied, and enhanced chemiluminescence (Thermo, Rockford, Ill., USA) was used to visualize the bands.
Histopathological Examinations and Immunohistochemistry Kidney slices were fixed in 10% formalin, embedded in paraffin wax, cut into 5 µm sections and stained with hematoxylin and eosin. The tissues were evaluated by light microscopy. For immunohistochemical stains, in brief, sections were rehydrated and antigens retrieved using heated citrate. After the incubation with blocking buffers, tissue sections were exposed sequentially to the primary
Page 9 of 27
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9
antibody, the horseradish peroxidase-coupled secondary antibodies (Vectastain Elite; Vector Laboratories, Peterborough, UK). The signals were developed with DAB Peroxidase Substrate Kit (Vector Laboratories). All immunohistochemical analyses were repeated at least three times and representative images were presented
Statistical Analysis Data are expressed as means ± standard error (SE). Comparisons among groups and two groups were evaluated by ANOVA and Mann-Whitney U test, respectively. Values of p
