BJP
British Journal of Pharmacology
British Journal of Pharmacology (2016) 173 1302–1313
1302
RESEARCH PAPER Anti-oxidative effect of AST-120 on kidney injury after myocardial infarction Correspondence Hideki Fujii, Division of Nephrology and Kidney Center, Kobe University Graduate School of Medicine, 7-5-2, Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017, Japan. E-mail: [email protected]
Received 8 June 2015; Revised 10 December 2015; Accepted 17 December 2015
Hideki Fujii1, Yuriko Yonekura1, Yusuke Yamashita2, Keiji Kono1, Kentaro Nakai1, Shunsuke Goto1, Mikio Sugano2, Sumie Goto2, Ayako Fujieda2, Yoshiharu Ito2 and Shinichi Nishi1 1
Division of Nephrology and Kidney Center, Kobe University Graduate School of Medicine, Kobe, Japan, and 2Biomedical Research Laboratories, Kureha Corporation, Tokyo, Japan
BACKGROUND AND PURPOSE Chronic kidney disease (CKD) is a crucial risk factor for cardiovascular disease (CVD), and combined CKD and CVD further increases morbidity and mortality. Here, we investigated effects of AST-120 on oxidative stress and kidney injury using a model of myocardial infarction (MI) in rats.
EXPERIMENTAL APPROACH At 10 weeks, male spontaneously hypertensive rats (SHR) were divided into three groups: SHR (n = 6), MI (n = 8) and MI + AST-120 (n = 8). AST-120 administration was started at 11 weeks after MI. At 18 weeks, the rats were killed, and blood and urine, mRNA expression and renal histological analyses were performed. Echocardiography was performed before and after MI.
KEY RESULTS At 18 weeks, the BP was significantly lower in the MI and MI+AST-120 groups than in the SHR group. Elevated levels of indoxyl sulfate (IS), one of the uremic toxins, in serum and urine were reduced by AST-120 treatment, compared with the MI group. Markers of oxidative stress in urine and serum biomarkers of kidney injury were decreased in the MI+AST-120 group compared with the other two groups. Renal expression of mRNAs for kidney injury related-markers were decreased in the MI+AST-120 group, compared with the MI group. In vitro data also supported the influence of IS on kidney injury. Immunohistological analysis showed that intrarenal oxidative stress was reduced by AST-120 administration.
CONCLUSIONS AND IMPLICATIONS Serum IS was increased after MI and treatment with AST-120 may have protective effects on kidney injury after MI by suppressing oxidative stress.
Abbreviations 8-OHdG, 8-hydroxydeoxyguanosine; BNP, brain natriuretic peptide; CKD, chronic kidney disease; CTGF, connective tissue growth factor; CVD, cardiovascular disease; EF, ejection fraction; FS, fractional shortening; IS, indoxyl sulfate; KIM-1, kidney injury molecule-1; LAD, left anterior descending; L-FABP, L-type fatty acid binding protein; LV, left ventricle; LVM, left ventricular mass; MI, myocardial infarction; NGAL, neutrophil gelatinase-associated lipocalin
DOI:10.1111/bph.13417
© 2016 The British Pharmacological Society
Effect of AST-120 on kidney injury
BJP
Tables of Links TARGETS
LIGANDS
Other proteins
BNP, brain natriuretic peptide
L-FABP, L-type fatty acid binding protein
CTGF,connective tissue growth factor TGF-β1
These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are permanently archived in the Concise Guide to PHARMACOLOGY 2015/16 (Alexander et al., 2015).
Introduction
Methods
Chronic kidney disease (CKD) is well known as a very important risk factor associated with the occurrence of cardiovascular disease (CVD) and consequent mortality (Go et al., 2004; Ninomiya et al., 2005). However, detailed mechanisms underlying the cardio-renal association remain unclear. Among the many possible factors, increased oxidative stress and inflammation play a role in the pathogenesis of both CKD and CVD (Prabhakar et al., 2007; Sirker et al., 2007; Cottone et al., 2008; Fujii et al., 2010). Although there are many reports of investigations of the mechanisms of CVD in CKD, to our knowledge, there is little information about the mechanisms of the progression of kidney injury in patients with cardiac dysfunction. Indoxyl sulfate (IS), which is one of the uremic toxins, is known to accumulate in correlation with declining renal function. Particularly, serum IS levels are elevated in patients with advanced-stage CKD and associated with the prognosis and occurrence of CVD events (Barreto et al., 2009). Furthermore, a recent study reported that serum IS levels are significantly and independently associated with CVD events even in patients without CKD after adjustment of renal function (Shimazu et al., 2013). An experimental study using animal models has shown that serum IS levels were raised after myocardial infarction (MI) (Lekawanvijit et al., 2013). Several previous studies have reported that IS accelerates the progression of CKD and CVD and that increased oxidative stress plays a key role in this mechanism (Nakagawa et al., 2006; Shimoishi et al., 2007; Fujii et al., 2009). In preliminary experiments, we confirmed the elevation of serum IS levels and increased urinary excretion of IS after MI in rats. Here, we have treated rats with MI with an orally administered charcoal adsorbent, AST-120, that is known to reduce the levels of circulating uremic toxins such as IS and indole acetic acid. Treatment with AST-120 attenuated oxidative stress produced by uremic toxins in CKD (Nakagawa et al., 2006; Shimoishi et al., 2007; Fujii et al., 2009). Such treatment potentially prevented histological and functional damage to kidney and the cardiovascular system in patients and in an animal model of CKD (Nakagawa et al., 2006; Fujii et al., 2009; Fujii et al., 2011). In this study, we report the effects of dietary AST-120 on oxidative stress and kidney injury, using a model of MI in rats with hypertension.
Animals All animal care and experimental procedures were in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee guidelines (Permit Number: 21120020). All efforts were made to minimize suffering and studies are reported in accordance with the ARRIVE guidelines (Kilkenny et al., 2010; McGrath and Lilley, 2015). Male spontaneously hypertensive rats (SHR) were obtained from SLC Japan Inc., Shizuoka, Japan. The rats were housed with food and water available ad libitum in lightcontrolled and temperature-controlled environments. At 10 weeks, the rats were randomly divided into three groups: sham-operated SHR (SHR, n = 6), inducedMI SHR (MI, n = 8) and induced-MI and AST-120-treated SHR (MI + AST-120, n = 8). To induce MI in rats, a previously described procedure (van Dokkum et al., 2004; Wakeno et al., 2006) was used. Briefly, the rats were anaesthetized with isoflurane, intubated and artificially ventilated. The thorax was opened, the heart was exteriorized and a ligature was placed around the left anterior descending (LAD) coronary artery. The heart was returned to its normal position, and the thorax was closed. A sham operation involved the same procedure, except for the LAD ligation. After briefly inflating the lungs, the thorax was closed, and the skin was sutured. Full supportive care was provided post-operatively. One week after the surgical procedures, rats in the SHR and MI groups continued on standard rodent diet and the rats in the MI+AST-120 group were fed the same diet containing 8% AST-120. There were no significant differences in the amount of food consumed (about 20g per day) between the experimental groups. Twenty-four-hour urine samples were collected from each rat using a metabolic cage at baseline, at 11weeks, i.e., one week after the surgical procedure and immediately before the rats were killed (ether anaesthesia) at 18weeks. Blood samples for serum measurements were collected from the left ventricle (LV) and the kidneys were removed for RNA extraction and histomorphological analysis. British Journal of Pharmacology (2016) 173 1302–1313
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Cell culture In the present study, we examined the effect of IS on the mRNA expressions of kidney injury related markers using proximal tubular cells (LLC-PK1). LLC-PK1 cells were maintained in 24-well plate with medium 199 supplemented with 10% FBS, 100 U·mL 1 penicillin and 100 μg·mL 1 streptomycin. LLC-PK1 cells were incubated at 37°C in a 5% CO2-humidified atmosphere with high concentration of IS (5 and 10 mM) for 6 or 24 h and low concentration of IS (0.5 and 1.0 mM) for 72 h.
Serum and urine measurements After centrifugation for 5 min at 800x g, the serum samples were stored at 80°C until analysis. Urine samples were also stored at 80°C for later analysis. Serum creatinine, serum urea nitrogen, urinary creatinine and urinary albumin (U-Alb) levels were measured using a UniCel DxC 600 (Beckman Coulter, Brea, CA, USA). Serum and urinary IS were measured by HPLC (Niwa et al., 1991), using a Shimadzu 10A system (Shimadzu, Kyoto,Japan). The urinary excretion of 8-hydroxydeoxyguanosine (U-8-OHdG) was determined using an ELISA kit (Japan Institute for Control of Aging, Shizuoka, Japan). Serum N-terminal pro-brain natriuretic peptide (NTpro-BNP) levels, TGF-β1, neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1) and L-type fatty acid binding protein (L-FABP) were also determined (NT-proBNP, NGAL and KIM-1: MSD®, Gaithersburg, MD, USA; TGF-β1 and L-FABP: R&D Systems, Minneapolis, MN, USA).
BP measurements Systolic BP measurements were performed in conscious, restrained rats by tail-cuff plethysmography (BP-98 A; Softron Co. Ltd., Tokyo, Japan). To reduce the possibility of stress artefacts, the rats were allowed to acclimatize for at least 15 min and the mean of multiple readings (at least 10) was recorded. BP was measured at baseline and at the end of the study period.
Echocardiographic measurements The rats were lightly anaesthetized with sodium pentobarbital (25 mg·kg 1 i.p.). Echocardiography was performed using a commercially available echocardiographic system (Aplio 50; Toshiba Medical Systems, Tochigi, Japan). A two-dimensional short-axis view of the LV was obtained at the papillary muscle level. These studies were performed at 9 and 18 weeks.
Histological and immunohistochemical analyses To evaluate cardiac and kidney injury, the heart and kidneys were removed, weighed and fixed with 10% formaldehyde, dehydrated at room temperature through an ethanol series, embedded in paraffin and cut into in 3 μm sections. These sections were stained with haematoxylin–eosin for routine histology and azan for morphometric studies. Myocardial fibrosis due to MI was graded semi-quantitatively on a scale of 0 to 4 (fibrosis score; normal to severe) in 20 random microscopic fields by two independent investigators, blinded to the experimental treatments. The formation of 8-OHdG, which is a sensitive oxidative stress indicator in the kidney tissue, was assessed with anti8-OHdG monoclonal antibodies raised in rats (Japan Institute 1304
British Journal of Pharmacology (2016) 173 1302–1313
for Control of Aging, Shizuoka, Japan). In brief, kidney slices were pre-incubated with blocking agents (Simple Stain Rat; Nichirei, Tokyo, Japan) and then incubated with the primary antibodies mentioned previously for 60 min at room temperature. A universal immunoperoxidase polymer (Histofine Simple Stain MAX PO, anti-rat and anti-rabbit; Nichirei, Tokyo, Japan) was used for immunostaining. The 8-OHdGpositive intraglomerular and tubular cells in the kidney tissue were counted in 20 random microscopic fields to give 8OHdG-positive cell scores. All evaluations were performed in a blinded manner.
RNA extraction and RT-PCR Total RNA was extracted from the rat kidneys and LLC-PK1 cells using an ISOGEN kit (Wako Pure Chemicals Industries, Ltd., Osaka, Japan) according to the manufacturer’s instructions. Total RNA from the rat kidneys and LLC-PK1 cells was used as the template for cDNA synthesis in a 20 μL volume with the SuperScript First-Strand Synthesis System (Invitrogen, CA, USA) using the oligo-dT hexamer as per the manufacturer’s instructions. The reaction mixture was incubated at 65°C for 5 min and placed on ice for 1 min. Another reaction mixture was then added to it, and the mixture was incubated at 50°C for 50 min, followed by 85°C for 5 min and chilled on ice for 1 min. Next, 1 μL of RNase H was added, and the mixture was incubated at 37°C for 20 min. The synthesized cDNA was stored at 20°C until PCR analysis. Real-time PCR was performed using a LightCycler 350 s Real-Time PCR System (Roche, Mannheim, Germany) with the LightCycler FastStart DNA Master SYBR Green I Kit (Roche). The analysis was performed with the second derivative maximum method of the LightCycler software (version. 4.0; Roche). The relative amount of the sample mRNA was normalized to the GAPDH mRNA. For PCR analysis, we used the following primers: rat TGF-β1 (5′CCTGGAAAGGGCTCAACAC-3′, 5′-CAGTTCTTCTCTGTGGA GCTGA-3′), rat NF-κB (5′-ACTGCTCAGGCCCACTTG-3′, 5′TGTCATTATCTCGGAGCTCATCT-3′), rat NADPH oxidase 4 (NOX4; 5′-GAACCCAAGTTCCAAGCTCA-3′, 5′-GCACAAAGGTCCAGAAATCC-3′), rat connective tissue growth factor (CTGF; 5′-GCACAAAGGTCCAGAAATCC-3′, 5′-CCGGTAGGT CTTCACACTGG-3′) and rat GAPDH (5′-TGGGAAGCTGGTC ATCAAC-3, 5′-GCATCACCCCATTTGATGTT-3′), pig TGF-β1 (5′-TGCCGGAACCTGTATTGCTC-3′, 5′-CTGGTATAGCTCC ACGTGCT-3′), pig NF-κB (5′-CTGAGTGCTGCTCCTTCCAA-3′, 5′-CCTGGATCACGTCAATGGCT-3′), pig NOX4 (5′-ATCCCAAGGATGACTGGAAACC-3′, 5′-ACCGGAAGGACTGGATGTCT3′), pig CTGF (5′-CAACGCTTCTTGCAGACTGG-3′, 5′-GC TCAAACTTGACGGGCTTG-3′) and pig GAPDH (5′-CCAT CACCATCTTCCGAG-3, 5′-GAGATGATGACCCTCTTGGC-3′).
Data and statistical analysis The study design and analysis conform to the recent guidance on experimental design and analysis in pharmacology (Curtis et al., 2015). Results are presented as mean ± SEM. We used the computer software application STATVIEW 5.0 (SAS Institute, Cary, NC, USA) for all statistical analyses. The significance of the differences between two groups was analysed by Student’s t-test. The differences among three groups were assessed by one-way ANOVA followed by the Tukey–Kramer test. P < 0.05 was considered statistically significant.
Effect of AST-120 on kidney injury
BJP
Table 1 Animal characteristics at baseline (11 weeks)
SHR (n = 6)
MI (n = 8)
MI + AST-120 (n = 8)
Body weight (g)
280.7 ± 4.2
259.4 ± 4.7
261.3 ± 4.5
SBP (mmHg)
178.1 ± 3.9
158.1 ± 3.5a
158.5 ± 4.1b
Cr (mg·L
1
) 1
BUN (mg·L
Ccr (mL·min
) 1
·100 g
1
)
-1
S-IS (mg·L )
1.9 ± 0.1
2.0 ± 0.2
1.8 ± 0.9
211 ± 7
212 ± 3
210 ± 3
1.12 ± 0.07
1.14 ± 0.06
1.18 ± 0.57
2.6 ± 1 1
U-IS (mg·day
U-Alb (mg·day
) 1
)
2.8 ±1.9
2.4 ±0.2
2.55 ± 0.24
2.63 ± 0.12
2.36 ± 0.11
101.1 ± 10.9
95.9 ± 9.8
97.1 ± 8.8
373 ± 39
994 ± 182a
1017 ± 199b
EF (%)
84.9 ± 0.9
62.2 ± 5.0a
60.6 ± 6.1b
FS (%)
49.4 ± 1.1
31.5 ± 3.6a
30.8 ± 3.6b
NT-proBNP (pg·L
1
)
SHR versus MI, P < 0.05. SHR versus AST-120, P < 0.05. SHR, spontaneously hypertensive rats; MI, myocardial infarction; SBP, systolic BP; Cr, creatinine; BUN, blood urea nitrogen; Ccr, creatinine clearance; S-IS, serum indoxyl sulfate; U-IS, urinary indoxyl sulfate; U-Alb, urinary albumin; NT-proBNP, N-terminal pro brain natriuretic peptide; EF, ejection fraction; FS, fractional shortening. a
b
Table 2 Animal characteristics at 18 weeks
SHR (n = 6)
MI (n = 8)
MI + AST-120 (n = 8)
Body weight (g)
319.8 ± 4.7
312.7 ± 3.7
315.8 ± 3.2
SBP (mmHg)
190.2 ± 3.2
164.9 ± 6.5a
169.3 ± 6.7b
Cr (mg·L
1
BUN (mg·L
) 1
Ccr (mL·min
) 1
U-Alb (mg·day
·100 g 1
1
)
)
Kidney weight ratio (g·100 g
1
)
2.0 ± 0.1
1.7 ± 0.1
1.6 ± 0.1
225 ± 7
219 ± 4
225 ± 4
1.21 ± 0.07
1.28 ± 0.05
1.35 ± 0.09
216.7 ± 18.6
263.5 ± 30.1
221.7 ± 20.7
0.41 ± 0.01
0.39 ± 0.01
0.38 ± 0.01
SHR versus MI, P < 0.05. SHR versus AST-120, P < 0.05. SHR, spontaneously hypertensive rats; MI, myocardial infarction; SBP, systolic BP; Cr, creatinine; BUN, blood urea nitrogen; Ccr, creatinine clearance; U-Alb, urinary albumin. a
b
Table 3 Cardiac parameters at 18 weeks
FS (%) EF (%)
MI (n = 8)
MI + AST-120 (n = 8)
44.9 ± 1.5
21.6 ± 3.1a
26.0 ± 3.9b
80.9 ± 1.42
LVM (g) Heart weight (g) 1
)
47.2 ± 5.7
a
54.3 ± 6.3b
1.11 ± 0.05
a
0.97 ± 0.02b
0
3.18 ± 0.40
a
3.30 ± 0.42b
1.26 ± 0.04
1.52 ± 0.09a
1.44 ± 0.05
0.40 ± 0.01
a
0.46 ± 0.02
0.89 ± 0.01
Cardiac fibrosis score
Relative heart weight. (g·100 g
SHR (n = 6)
0.49 ± 0.03
SHR versus MI, P < 0.05. SHR versus AST-120, P < 0.05. SHR, spontaneously hypertensive rats; MI, myocardial infarction; EF, ejection fraction; FS, fraction shortening; LVM, left ventricular mass. a
b
British Journal of Pharmacology (2016) 173 1302–1313
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Figure 1 Serum and urinary IS levels in the SHR, MI and MI + AST-120 groups at 18 weeks. At 11 weeks, rats were divided into three groups: sham-operated SHR (SHR, n = 6), induced-MI SHR (MI, n = 8), and induced-MI and AST-120-treated SHR (MI + AST-120, n = 8) and then followed until 18 weeks. At 18 weeks, blood and urine samples were collected. Serum (A) and urinary IS (B) levels were measured using HPLC. As reference, we measured the serum and urinary IS levels in age-matched Wistar Kyoto rats (WKY, n = 10). These levels were significantly lower in the WKY group than in the SHR group (P < 0.05; Student’s t-test). Serum and urinary IS levels were significantly reduced in the MI + AST-120 group compared to those in the other groups. † P
British Journal of Pharmacology
British Journal of Pharmacology (2016) 173 1302–1313
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RESEARCH PAPER Anti-oxidative effect of AST-120 on kidney injury after myocardial infarction Correspondence Hideki Fujii, Division of Nephrology and Kidney Center, Kobe University Graduate School of Medicine, 7-5-2, Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017, Japan. E-mail: [email protected]
Received 8 June 2015; Revised 10 December 2015; Accepted 17 December 2015
Hideki Fujii1, Yuriko Yonekura1, Yusuke Yamashita2, Keiji Kono1, Kentaro Nakai1, Shunsuke Goto1, Mikio Sugano2, Sumie Goto2, Ayako Fujieda2, Yoshiharu Ito2 and Shinichi Nishi1 1
Division of Nephrology and Kidney Center, Kobe University Graduate School of Medicine, Kobe, Japan, and 2Biomedical Research Laboratories, Kureha Corporation, Tokyo, Japan
BACKGROUND AND PURPOSE Chronic kidney disease (CKD) is a crucial risk factor for cardiovascular disease (CVD), and combined CKD and CVD further increases morbidity and mortality. Here, we investigated effects of AST-120 on oxidative stress and kidney injury using a model of myocardial infarction (MI) in rats.
EXPERIMENTAL APPROACH At 10 weeks, male spontaneously hypertensive rats (SHR) were divided into three groups: SHR (n = 6), MI (n = 8) and MI + AST-120 (n = 8). AST-120 administration was started at 11 weeks after MI. At 18 weeks, the rats were killed, and blood and urine, mRNA expression and renal histological analyses were performed. Echocardiography was performed before and after MI.
KEY RESULTS At 18 weeks, the BP was significantly lower in the MI and MI+AST-120 groups than in the SHR group. Elevated levels of indoxyl sulfate (IS), one of the uremic toxins, in serum and urine were reduced by AST-120 treatment, compared with the MI group. Markers of oxidative stress in urine and serum biomarkers of kidney injury were decreased in the MI+AST-120 group compared with the other two groups. Renal expression of mRNAs for kidney injury related-markers were decreased in the MI+AST-120 group, compared with the MI group. In vitro data also supported the influence of IS on kidney injury. Immunohistological analysis showed that intrarenal oxidative stress was reduced by AST-120 administration.
CONCLUSIONS AND IMPLICATIONS Serum IS was increased after MI and treatment with AST-120 may have protective effects on kidney injury after MI by suppressing oxidative stress.
Abbreviations 8-OHdG, 8-hydroxydeoxyguanosine; BNP, brain natriuretic peptide; CKD, chronic kidney disease; CTGF, connective tissue growth factor; CVD, cardiovascular disease; EF, ejection fraction; FS, fractional shortening; IS, indoxyl sulfate; KIM-1, kidney injury molecule-1; LAD, left anterior descending; L-FABP, L-type fatty acid binding protein; LV, left ventricle; LVM, left ventricular mass; MI, myocardial infarction; NGAL, neutrophil gelatinase-associated lipocalin
DOI:10.1111/bph.13417
© 2016 The British Pharmacological Society
Effect of AST-120 on kidney injury
BJP
Tables of Links TARGETS
LIGANDS
Other proteins
BNP, brain natriuretic peptide
L-FABP, L-type fatty acid binding protein
CTGF,connective tissue growth factor TGF-β1
These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are permanently archived in the Concise Guide to PHARMACOLOGY 2015/16 (Alexander et al., 2015).
Introduction
Methods
Chronic kidney disease (CKD) is well known as a very important risk factor associated with the occurrence of cardiovascular disease (CVD) and consequent mortality (Go et al., 2004; Ninomiya et al., 2005). However, detailed mechanisms underlying the cardio-renal association remain unclear. Among the many possible factors, increased oxidative stress and inflammation play a role in the pathogenesis of both CKD and CVD (Prabhakar et al., 2007; Sirker et al., 2007; Cottone et al., 2008; Fujii et al., 2010). Although there are many reports of investigations of the mechanisms of CVD in CKD, to our knowledge, there is little information about the mechanisms of the progression of kidney injury in patients with cardiac dysfunction. Indoxyl sulfate (IS), which is one of the uremic toxins, is known to accumulate in correlation with declining renal function. Particularly, serum IS levels are elevated in patients with advanced-stage CKD and associated with the prognosis and occurrence of CVD events (Barreto et al., 2009). Furthermore, a recent study reported that serum IS levels are significantly and independently associated with CVD events even in patients without CKD after adjustment of renal function (Shimazu et al., 2013). An experimental study using animal models has shown that serum IS levels were raised after myocardial infarction (MI) (Lekawanvijit et al., 2013). Several previous studies have reported that IS accelerates the progression of CKD and CVD and that increased oxidative stress plays a key role in this mechanism (Nakagawa et al., 2006; Shimoishi et al., 2007; Fujii et al., 2009). In preliminary experiments, we confirmed the elevation of serum IS levels and increased urinary excretion of IS after MI in rats. Here, we have treated rats with MI with an orally administered charcoal adsorbent, AST-120, that is known to reduce the levels of circulating uremic toxins such as IS and indole acetic acid. Treatment with AST-120 attenuated oxidative stress produced by uremic toxins in CKD (Nakagawa et al., 2006; Shimoishi et al., 2007; Fujii et al., 2009). Such treatment potentially prevented histological and functional damage to kidney and the cardiovascular system in patients and in an animal model of CKD (Nakagawa et al., 2006; Fujii et al., 2009; Fujii et al., 2011). In this study, we report the effects of dietary AST-120 on oxidative stress and kidney injury, using a model of MI in rats with hypertension.
Animals All animal care and experimental procedures were in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee guidelines (Permit Number: 21120020). All efforts were made to minimize suffering and studies are reported in accordance with the ARRIVE guidelines (Kilkenny et al., 2010; McGrath and Lilley, 2015). Male spontaneously hypertensive rats (SHR) were obtained from SLC Japan Inc., Shizuoka, Japan. The rats were housed with food and water available ad libitum in lightcontrolled and temperature-controlled environments. At 10 weeks, the rats were randomly divided into three groups: sham-operated SHR (SHR, n = 6), inducedMI SHR (MI, n = 8) and induced-MI and AST-120-treated SHR (MI + AST-120, n = 8). To induce MI in rats, a previously described procedure (van Dokkum et al., 2004; Wakeno et al., 2006) was used. Briefly, the rats were anaesthetized with isoflurane, intubated and artificially ventilated. The thorax was opened, the heart was exteriorized and a ligature was placed around the left anterior descending (LAD) coronary artery. The heart was returned to its normal position, and the thorax was closed. A sham operation involved the same procedure, except for the LAD ligation. After briefly inflating the lungs, the thorax was closed, and the skin was sutured. Full supportive care was provided post-operatively. One week after the surgical procedures, rats in the SHR and MI groups continued on standard rodent diet and the rats in the MI+AST-120 group were fed the same diet containing 8% AST-120. There were no significant differences in the amount of food consumed (about 20g per day) between the experimental groups. Twenty-four-hour urine samples were collected from each rat using a metabolic cage at baseline, at 11weeks, i.e., one week after the surgical procedure and immediately before the rats were killed (ether anaesthesia) at 18weeks. Blood samples for serum measurements were collected from the left ventricle (LV) and the kidneys were removed for RNA extraction and histomorphological analysis. British Journal of Pharmacology (2016) 173 1302–1313
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H Fujii et al.
Cell culture In the present study, we examined the effect of IS on the mRNA expressions of kidney injury related markers using proximal tubular cells (LLC-PK1). LLC-PK1 cells were maintained in 24-well plate with medium 199 supplemented with 10% FBS, 100 U·mL 1 penicillin and 100 μg·mL 1 streptomycin. LLC-PK1 cells were incubated at 37°C in a 5% CO2-humidified atmosphere with high concentration of IS (5 and 10 mM) for 6 or 24 h and low concentration of IS (0.5 and 1.0 mM) for 72 h.
Serum and urine measurements After centrifugation for 5 min at 800x g, the serum samples were stored at 80°C until analysis. Urine samples were also stored at 80°C for later analysis. Serum creatinine, serum urea nitrogen, urinary creatinine and urinary albumin (U-Alb) levels were measured using a UniCel DxC 600 (Beckman Coulter, Brea, CA, USA). Serum and urinary IS were measured by HPLC (Niwa et al., 1991), using a Shimadzu 10A system (Shimadzu, Kyoto,Japan). The urinary excretion of 8-hydroxydeoxyguanosine (U-8-OHdG) was determined using an ELISA kit (Japan Institute for Control of Aging, Shizuoka, Japan). Serum N-terminal pro-brain natriuretic peptide (NTpro-BNP) levels, TGF-β1, neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1) and L-type fatty acid binding protein (L-FABP) were also determined (NT-proBNP, NGAL and KIM-1: MSD®, Gaithersburg, MD, USA; TGF-β1 and L-FABP: R&D Systems, Minneapolis, MN, USA).
BP measurements Systolic BP measurements were performed in conscious, restrained rats by tail-cuff plethysmography (BP-98 A; Softron Co. Ltd., Tokyo, Japan). To reduce the possibility of stress artefacts, the rats were allowed to acclimatize for at least 15 min and the mean of multiple readings (at least 10) was recorded. BP was measured at baseline and at the end of the study period.
Echocardiographic measurements The rats were lightly anaesthetized with sodium pentobarbital (25 mg·kg 1 i.p.). Echocardiography was performed using a commercially available echocardiographic system (Aplio 50; Toshiba Medical Systems, Tochigi, Japan). A two-dimensional short-axis view of the LV was obtained at the papillary muscle level. These studies were performed at 9 and 18 weeks.
Histological and immunohistochemical analyses To evaluate cardiac and kidney injury, the heart and kidneys were removed, weighed and fixed with 10% formaldehyde, dehydrated at room temperature through an ethanol series, embedded in paraffin and cut into in 3 μm sections. These sections were stained with haematoxylin–eosin for routine histology and azan for morphometric studies. Myocardial fibrosis due to MI was graded semi-quantitatively on a scale of 0 to 4 (fibrosis score; normal to severe) in 20 random microscopic fields by two independent investigators, blinded to the experimental treatments. The formation of 8-OHdG, which is a sensitive oxidative stress indicator in the kidney tissue, was assessed with anti8-OHdG monoclonal antibodies raised in rats (Japan Institute 1304
British Journal of Pharmacology (2016) 173 1302–1313
for Control of Aging, Shizuoka, Japan). In brief, kidney slices were pre-incubated with blocking agents (Simple Stain Rat; Nichirei, Tokyo, Japan) and then incubated with the primary antibodies mentioned previously for 60 min at room temperature. A universal immunoperoxidase polymer (Histofine Simple Stain MAX PO, anti-rat and anti-rabbit; Nichirei, Tokyo, Japan) was used for immunostaining. The 8-OHdGpositive intraglomerular and tubular cells in the kidney tissue were counted in 20 random microscopic fields to give 8OHdG-positive cell scores. All evaluations were performed in a blinded manner.
RNA extraction and RT-PCR Total RNA was extracted from the rat kidneys and LLC-PK1 cells using an ISOGEN kit (Wako Pure Chemicals Industries, Ltd., Osaka, Japan) according to the manufacturer’s instructions. Total RNA from the rat kidneys and LLC-PK1 cells was used as the template for cDNA synthesis in a 20 μL volume with the SuperScript First-Strand Synthesis System (Invitrogen, CA, USA) using the oligo-dT hexamer as per the manufacturer’s instructions. The reaction mixture was incubated at 65°C for 5 min and placed on ice for 1 min. Another reaction mixture was then added to it, and the mixture was incubated at 50°C for 50 min, followed by 85°C for 5 min and chilled on ice for 1 min. Next, 1 μL of RNase H was added, and the mixture was incubated at 37°C for 20 min. The synthesized cDNA was stored at 20°C until PCR analysis. Real-time PCR was performed using a LightCycler 350 s Real-Time PCR System (Roche, Mannheim, Germany) with the LightCycler FastStart DNA Master SYBR Green I Kit (Roche). The analysis was performed with the second derivative maximum method of the LightCycler software (version. 4.0; Roche). The relative amount of the sample mRNA was normalized to the GAPDH mRNA. For PCR analysis, we used the following primers: rat TGF-β1 (5′CCTGGAAAGGGCTCAACAC-3′, 5′-CAGTTCTTCTCTGTGGA GCTGA-3′), rat NF-κB (5′-ACTGCTCAGGCCCACTTG-3′, 5′TGTCATTATCTCGGAGCTCATCT-3′), rat NADPH oxidase 4 (NOX4; 5′-GAACCCAAGTTCCAAGCTCA-3′, 5′-GCACAAAGGTCCAGAAATCC-3′), rat connective tissue growth factor (CTGF; 5′-GCACAAAGGTCCAGAAATCC-3′, 5′-CCGGTAGGT CTTCACACTGG-3′) and rat GAPDH (5′-TGGGAAGCTGGTC ATCAAC-3, 5′-GCATCACCCCATTTGATGTT-3′), pig TGF-β1 (5′-TGCCGGAACCTGTATTGCTC-3′, 5′-CTGGTATAGCTCC ACGTGCT-3′), pig NF-κB (5′-CTGAGTGCTGCTCCTTCCAA-3′, 5′-CCTGGATCACGTCAATGGCT-3′), pig NOX4 (5′-ATCCCAAGGATGACTGGAAACC-3′, 5′-ACCGGAAGGACTGGATGTCT3′), pig CTGF (5′-CAACGCTTCTTGCAGACTGG-3′, 5′-GC TCAAACTTGACGGGCTTG-3′) and pig GAPDH (5′-CCAT CACCATCTTCCGAG-3, 5′-GAGATGATGACCCTCTTGGC-3′).
Data and statistical analysis The study design and analysis conform to the recent guidance on experimental design and analysis in pharmacology (Curtis et al., 2015). Results are presented as mean ± SEM. We used the computer software application STATVIEW 5.0 (SAS Institute, Cary, NC, USA) for all statistical analyses. The significance of the differences between two groups was analysed by Student’s t-test. The differences among three groups were assessed by one-way ANOVA followed by the Tukey–Kramer test. P < 0.05 was considered statistically significant.
Effect of AST-120 on kidney injury
BJP
Table 1 Animal characteristics at baseline (11 weeks)
SHR (n = 6)
MI (n = 8)
MI + AST-120 (n = 8)
Body weight (g)
280.7 ± 4.2
259.4 ± 4.7
261.3 ± 4.5
SBP (mmHg)
178.1 ± 3.9
158.1 ± 3.5a
158.5 ± 4.1b
Cr (mg·L
1
) 1
BUN (mg·L
Ccr (mL·min
) 1
·100 g
1
)
-1
S-IS (mg·L )
1.9 ± 0.1
2.0 ± 0.2
1.8 ± 0.9
211 ± 7
212 ± 3
210 ± 3
1.12 ± 0.07
1.14 ± 0.06
1.18 ± 0.57
2.6 ± 1 1
U-IS (mg·day
U-Alb (mg·day
) 1
)
2.8 ±1.9
2.4 ±0.2
2.55 ± 0.24
2.63 ± 0.12
2.36 ± 0.11
101.1 ± 10.9
95.9 ± 9.8
97.1 ± 8.8
373 ± 39
994 ± 182a
1017 ± 199b
EF (%)
84.9 ± 0.9
62.2 ± 5.0a
60.6 ± 6.1b
FS (%)
49.4 ± 1.1
31.5 ± 3.6a
30.8 ± 3.6b
NT-proBNP (pg·L
1
)
SHR versus MI, P < 0.05. SHR versus AST-120, P < 0.05. SHR, spontaneously hypertensive rats; MI, myocardial infarction; SBP, systolic BP; Cr, creatinine; BUN, blood urea nitrogen; Ccr, creatinine clearance; S-IS, serum indoxyl sulfate; U-IS, urinary indoxyl sulfate; U-Alb, urinary albumin; NT-proBNP, N-terminal pro brain natriuretic peptide; EF, ejection fraction; FS, fractional shortening. a
b
Table 2 Animal characteristics at 18 weeks
SHR (n = 6)
MI (n = 8)
MI + AST-120 (n = 8)
Body weight (g)
319.8 ± 4.7
312.7 ± 3.7
315.8 ± 3.2
SBP (mmHg)
190.2 ± 3.2
164.9 ± 6.5a
169.3 ± 6.7b
Cr (mg·L
1
BUN (mg·L
) 1
Ccr (mL·min
) 1
U-Alb (mg·day
·100 g 1
1
)
)
Kidney weight ratio (g·100 g
1
)
2.0 ± 0.1
1.7 ± 0.1
1.6 ± 0.1
225 ± 7
219 ± 4
225 ± 4
1.21 ± 0.07
1.28 ± 0.05
1.35 ± 0.09
216.7 ± 18.6
263.5 ± 30.1
221.7 ± 20.7
0.41 ± 0.01
0.39 ± 0.01
0.38 ± 0.01
SHR versus MI, P < 0.05. SHR versus AST-120, P < 0.05. SHR, spontaneously hypertensive rats; MI, myocardial infarction; SBP, systolic BP; Cr, creatinine; BUN, blood urea nitrogen; Ccr, creatinine clearance; U-Alb, urinary albumin. a
b
Table 3 Cardiac parameters at 18 weeks
FS (%) EF (%)
MI (n = 8)
MI + AST-120 (n = 8)
44.9 ± 1.5
21.6 ± 3.1a
26.0 ± 3.9b
80.9 ± 1.42
LVM (g) Heart weight (g) 1
)
47.2 ± 5.7
a
54.3 ± 6.3b
1.11 ± 0.05
a
0.97 ± 0.02b
0
3.18 ± 0.40
a
3.30 ± 0.42b
1.26 ± 0.04
1.52 ± 0.09a
1.44 ± 0.05
0.40 ± 0.01
a
0.46 ± 0.02
0.89 ± 0.01
Cardiac fibrosis score
Relative heart weight. (g·100 g
SHR (n = 6)
0.49 ± 0.03
SHR versus MI, P < 0.05. SHR versus AST-120, P < 0.05. SHR, spontaneously hypertensive rats; MI, myocardial infarction; EF, ejection fraction; FS, fraction shortening; LVM, left ventricular mass. a
b
British Journal of Pharmacology (2016) 173 1302–1313
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H Fujii et al.
Figure 1 Serum and urinary IS levels in the SHR, MI and MI + AST-120 groups at 18 weeks. At 11 weeks, rats were divided into three groups: sham-operated SHR (SHR, n = 6), induced-MI SHR (MI, n = 8), and induced-MI and AST-120-treated SHR (MI + AST-120, n = 8) and then followed until 18 weeks. At 18 weeks, blood and urine samples were collected. Serum (A) and urinary IS (B) levels were measured using HPLC. As reference, we measured the serum and urinary IS levels in age-matched Wistar Kyoto rats (WKY, n = 10). These levels were significantly lower in the WKY group than in the SHR group (P < 0.05; Student’s t-test). Serum and urinary IS levels were significantly reduced in the MI + AST-120 group compared to those in the other groups. † P
