Original Paper Pharmacology 2013;92:245–256 DOI: 10.1159/000355060

Received: June 14, 2013 Accepted after revision: August 14, 2013 Published online: November 12, 2013

Nicorandil Protects against Ischaemia-Reperfusion Injury in Newborn Rat Kidney Yu-Jing Zhang Ai-Qi Zhang Xia-Xia Zhao Zhi-Liang Tian Li Yao Department of Pediatrics, Second Affiliate Hospital of Harbin Medical University, Harbin, PR China

Abstract Ischaemia-reperfusion injury (IRI) is the predominant cause of acute kidney injury. Nevertheless, the underlying molecular mechanisms are still unclear. The current study investigated the effects of nicorandil on ATP-sensitive potassium (KATP) channels and the potential signal transduction pathway(s) in a rat kidney IRI model and in cultured tubular HK-2 cells subjected to oxygen and glucose deprivation/reoxygenation (OGD/R) injury. The standard procedure for IRI was performed in newborn rat kidneys. Pretreatment with nicorandil (10 mg/kg) 2 h prior to induction of IRI improved renal function, attenuated tubule damage, and prevented apoptosis of tubule cells, infiltration of neutrophils and macrophages, and production of inflammatory cytokines interleukin (IL)-6, IL-17 and tumour necrosis factor-α. Ischaemiareperfusion-induced reduction of KIR6.2 was restored to normal levels by nicorandil. The activation of the phosphoinositide-3-kinase (PI3K)-Akt-nuclear factor (NF)-κB axis was detected in this rat kidney IRI model, which was blocked by nicorandil. The renoprotection of nicorandil against IRI was

© 2013 S. Karger AG, Basel 0031–7012/13/0926–0245$38.00/0 E-Mail [email protected] www.karger.com/pha

abolished by its inhibitor glibenclamide (1 mg/kg). Similar results were obtained in OGD/R-damaged HK-2 cells. Taken together, our findings demonstrated the specific renoprotective role of nicorandil in the newborn rat IRI kidney by decreasing the production of inflammatory cytokines, and restoring the expression of KIR6.2 potentially through the PI3K-Akt-NF-κB axis. © 2013 S. Karger AG, Basel

Introduction

Renal ischaemia is the predominant cause of acute kidney injury. Reperfusion is essential for the survival of the ischaemic kidney [1]. Nevertheless, reperfusion itself can cause further renal injury termed as ischaemia-reperfusion injury (IRI) due to many mediators such as depletion of ATP, infiltration of neutrophils and macrophages, production of cytokines and oxidants, as well as generation of reactive oxygen species [2, 3]. Many studies about IRI in other organs, especially in acute cardiac failure, demonstrated that the activation of ATP-sensitive potassium (KATP) channels is involved in the IR-induced cellular injury [4, 5]. An earlier study found that KATP channels were activated in renal IRI by using an isolated perfused rat kidney model [6]. Zhi-Liang Tian, MD Department of Pediatrics Second Affiliate Hospital of Harbin Medical University 246 Xuefu Road, Nangang District, Harbin, Heilongjiang Province, 150086 (PR China) E-Mail tianzhiliang999 @ yahoo.com.cn

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Key Words Kidney ischaemia-reperfusion injury · Newborn rat · Nicorandil · Glibenclamide · ATP-sensitive potassium channel

in the left kidney by clamping the renal vascular pedicle for 30 min. Sham operations were performed in the same manner except that the renal vascular pedicle was not clamped. After 24 h of reperfusion, the rats were anaesthetized, then blood samples were taken from the angular artery and spot urine was collected from the bladder. The left kidney was removed and the cortex tissues were immediately frozen at –80 ° C for extraction of RNA and protein as well as the detection of myeloperoxidase (MPO) activity, or fixed in 4% phosphate-buffered paraformaldehyde solution for histological analysis and immunohistochemistry staining.  

 

Measurement of Renal Function A blood urea nitrogen (BUN) detection kit was used to measure the level of BUN (Arbor Assays, USA). The serum creatinine (sCr) levels were measured with the Jaffe method (creatinine test Wako, Japan). The urinary albumin (U-Alb), creatinine and β2-microglobulin (β2-MG) were determined by using the enzyme-linked immunosorbent assay and a Beckman Coulter DTX 880 plate reader (Exocell, USA). The concentration of urinary Na+ (U-Na+) was detected by an automatic biochemical analyser (Hitachi, Japan). Histological Examination of Kidney Paraffin-embedded kidney tissue sections (4 μm) were stained by using haematoxylin and eosin. The Jablonski grading scale (score 0–4) was applied to evaluate the overall proximal tubular damage: score 0 = normal; score 1 = necrosis of individual cells; score 2 = necrosis of all cells in adjacent proximal convoluted tubules, with survival of surrounding tubules; score 3 = necrosis confined to the distal third of proximal convoluted tubules, with band(s) of necrosis extending across the inner cortex, and score 4 = necrosis of all three segments of proximal convoluted tubules [10, 11]. Histological evaluations were performed and analysed blindly by an experienced renal pathologist. Immunohistochemistry Staining Paraffin-embedded kidney tissue sections (4 μm) were routinely deparaffinized, and then antigen retrieval was performed in TE buffer (10 mmol/l Tris-HCl, 1 mmol/l EDTA, pH 9.0) at 95 ° C in a water bath for 45 min. After cooling down and washing with water and phosphate-buffered saline (PBS), permeabilization was performed with 0.5% Triton/PBS for 10 min. Non-specific background was removed by incubating in block solution (10% normal goat serum/PBS) for 1 h. The primary antibodies rabbit anti-F4/80 and rabbit anti-caspase-3 (Abcam, USA) were diluted (1: 500) in block solution, and incubated at 4 ° C overnight. After washing with PBS 3 times, the slides were incubated with Alexa 594- or 488-conjugated goat anti-rabbit IgG (Invitrogen, USA) for 1 h. As blank control, primary antibody was replaced by block solution. The slides were mounted with Prolong gold antifage reagent (Invitrogen). The immunostained slides were viewed under a BX-51 light microscope (Zeiss, Germany). Twenty photographs of renal cortex per sample were randomly obtained with a digital camera at a magnification of ×400, and F4/80- or caspase-3-positive cells were analysed and counted blindly by using Image J.  

Materials and Methods Establishment of a Newborn Rat Kidney IRI Model All animal experiments were approved by the Experimental Animal Ethics Committee of Harbin Medical University (Harbin, China). Seven-day-old male Wistar rats weighing 12–18 g were purchased from the Animal Centre of the Second Affiliate Hospital of Harbin Medical University (Harbin) and randomly divided into 4 groups: (1) sham-operated control group (n = 9); (2) IR group, including 30 min of left renal ischaemia followed by 24 h of reperfusion (n = 12); (3) IR group additionally treated with 10 mg/ kg of nicorandil injected intraperitoneally 2 h prior to IR (n = 12), and (4) IR group additionally treated with 10 mg/kg of nicorandil and 1 mg/kg of glibenclamide injected intraperitoneally 2 h prior to IR (n = 12). The stock solutions of nicorandil (Sigma, USA) and glibenclamide (Sigma) were made in 0.9% NaCl (pH 7.4) and ethanol, respectively, and diluted in saline before intraperitoneal injection (total volume: 0.2 ml). After anaesthesia with diethyl ether, each rat was put on a heating pad to keep temperature at 37 ° C, and the dorsum was opened at the midline, then the right kidney was removed. IR was induced  

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Real-Time Polymerase Chain Reaction Total renal RNA was extracted with an RNA Purification Kit (Life Technologies, USA), and 2 μg of RNA was used to synthesize complementary DNA by using the SuperScript First-Strand Syn-

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Nicorandil, 2-nicotinamidoethyl-nitrate ester, an opener of the KATP channel, has a protective effect on rat myocardial IRI mainly based on the opening of the mitochondrial KATP channels and the lowering of the Ca2+ overload [4, 5]. A decade ago, it was found that in a cultured rat mesangial cell, nicorandil exerts inhibitory effects on mesangial cell proliferation potentially via the protein kinase G pathway [7]. In anti-Thy1 experimental mesangioproliferative glomerulonephritis, a renoprotective role of nicorandil was reported by preventing the overexpression of fibronectin, transforming growth factor-β, platelet-derived growth factor, and type I collagen [8]. Moreover, in a rat remnant kidney model of chronic kidney disease, nicorandil prevented albuminuria, glomerular and tubulo-interstitial injury by reducing oxidative stress through increasing the anti-oxidant system in glomerular podocytes and blocking the pro-oxidative systems of xanthine oxidase in macrophages [9]. These findings suggested that nicorandil has renoprotective effects in either acute or chronic kidney diseases. Just recently, Shimizu et al. [10] reported that pretreatment with nicorandil ameliorated IR-induced kidney injury in adult rats by preventing the reduction of KIR6.2. However, the underlying molecular mechanisms by which nicorandil plays a renoprotective role in renal IRI is elusive. The present study investigated both the renoprotection of nicorandil and, behind signal transduction, pathway(s) of nicorandil against IRI in a newborn rat kidney model and in cultured tubular HK-2 cells subjected to oxygen and glucose deprivation/reoxygenation (OGD/R) injury.

Table 1. Primer sequence, annealing temperature, and product size of real-time PCR

Gene

Primer

Temperature, °C

Product size, bp

Reference

TNF-α

F: 5′-ccacgtcgtagcaaaccaccaag-3′ R: 5′-caggtacatgggctcatacc-3′

60

316

12

IL-6

F: 5′-ggagttccgtlttctacctgg-3′ R: 5′-gccgagtagacctcatagtg-3′

60

275

12

IL-17

F: 5′-atcaggacgcgcaaacatg-3′ R: 5′-tgatcgctgctgccttcac-3′

62

143

13

GAPDH

F: 5′-aaacccatcaccatcttcca-3′ R: 5′-gtggttcacacccatcacaa-3′

60

198

14

Detection of Myeloperoxidase Activity MPO activity, indicative of neutrophil infiltration into tissue, was measured by using a Colorimetric Myeloperoxidase Assay Kit (Abcam). Briefly, the equally sized kidney samples were homogenized in 4 vol of MPO Assay Buffer, and then centrifuged (13,000 g, 10 min) to remove insoluble material. We added 5 μl triple parallel samples in a 96-well plate, and adjusted the final volume to 50 μl in each well with MPO Assay Buffer. Then, we added 50 μl of the reaction mix to each well, and incubated it for 30 min, then added 2 μl of stop mix and incubated it another 10 min to stop the reaction. Then, we added 50 μl TNB reagent/standard to the standard wells at this time, and mixed well. After 5 min, the optical density was read at 412 nm, and the optical density of colour upon decrease in TNB was: ΔA412 nm = Abackground – Asample. MPO activity in samples can then be calculated as: MPO activity = B/(ΔT × V) × sample dilution factor = nmol/min/mm = mU/ml (where B is the TNB amount in nanomoles from the standard curve, T is the time in minutes of the first incubation before stop mix, V is the pre-adjusted sample volume in millilitres added to the reaction well). The n-fold change of MPO activity was shown in this study.

OGD/R Assay The OGD assay was performed as previously described [15, 16]. OGD solution is a balanced glucose-free salt solution (pH 7.4) consisting of 116 mmol/l NaCl, 5.4 mmol/l KCl, 0.8 mmol/l MgSO4, 1 mmol/l NaH2PO4, 26 mmol/l NaHCO3 and 1.8 mmol/l CaCl2. Before use, pure nitrogen gas was bubbled through for 15 min to remove oxygen from the solution. When 80% confluence was achieved, HK-2 cells were washed sequentially with warmed HEPES buffer solution (120 mmol/l NaCl, 5.4 mmol/l KCl, 0.8 mmol/l MgCl2, 1.8 mmol/l CaCl2, 15 mmol/l anhydrous D-glucose and 20 mmol/l HEPES) and the warmed OGD solution. Then, the cells were cultured with 1 ml of the warmed OGD solution and incubated in airtight gas chambers for the indicated period exposed to 95% nitrogen and 5% CO2 at 37 ° C. After OGD treatment, plates were removed from the gas chamber, the OGD solution was replaced with the warmed culture medium and placed in a 5% CO2 incubator at 37 ° C for 24 h. To evaluate the effects of nicorandil on tubular cell injury, nicorandil (1–100 μmol/l) was incubated for 1 h prior to OGD treatment. To counteract the role of nicorandil, glibenclamide (0.1–10 μmol/l) was included during OGD treatment.  

 

 

 

Cell Viability Analysis Cell viability was evaluated by using a Vybrant® MTT Cell Proliferation Assay Kit (Molecular Probes, USA). Briefly, 4 × 105 cells were resuspended in 100 μl of phenol red-free fresh medium. Then, 10 μl of 12 mmol/l MTT solution was added and incubated at 37 ° C for 2 h followed by incubation with 100 μl of sodium dodecyl sulphate-HCl solution for 4 h at 37 ° C in a humidified chamber. We mixed each sample again and read absorbance at 570 nm. Cell viability was calculated as a percentage of the optical density of formazan in the treated group against that in the control.  

 

 

 

HK-2 Cell Culture HK-2, a stabilized human kidney proximal tubular epithelial cell line (ATCC, USA), was cultured in high glucose-GlutaMaxDMEM media supplemented with 10% fetal bovine serum (Gibco, USA) and 1% penicillin/streptomycin. Cells were grown at 37 ° C in a humidified atmosphere with 5% CO2, adding fresh growth medium every 2 days.

Western Blot Kidney tissue and HK-2 cells were lysed in the modified radioimmunoprecipitation assay buffer (1% Nonidet P-40, 0.05 mol/l HEPES, pH 7.5, 150 mmol/l NaCl, 1.5 mmol/l MgCl2, 1 mmol/l ethylene glycol tetraacetic acid, 10% glycerol, 0.1% sodium dodecyl sulphate, 0.5% deoxycholic acid sodium salt) with the complete protease and phosphatase inhibitors (Roche, USA). Nuclear proteins were extracted for analysing nuclear factor (NF)-κB (p65) expression by using a Nuclear Extraction kit (Thermo Scientific,

Nicorandil Ameliorates Newborn Rat Kidney IRI

Pharmacology 2013;92:245–256 DOI: 10.1159/000355060

 

 

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thesis System (Invitrogen). The reverse transcription reaction without reverse transcriptase was used as negative control. Subsequently, 1 μl of complementary DNA was mixed (total volume: 20 μl) with 2 μl of 10× SYBR green PCR buffer, 1.2 μl of 25 mmol/l MgCl2, 1.6 μl of 2.5 mmol/l dNTPs, 1 μl of 5 mmol/l specific PCR primers (table 1), and 0.2 μl of 5 U/μl Taq Gold DNA polymerase for real-time PCR by using the GeneAmp 5700 system (PE Biosystems, USA). The quantity of inflammatory cytokine mRNA was corrected by using housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). To verify the specific amplification, PCR products from normal kidney were separated on a 1.5% agarose gel containing 0.4 mg/l ethidium bromide.

0.015 0.010 0.005

10.0 7.5 #

5.0

*

2.5

Ct l

+

N ic + Gl i

IR

+

#

125

*

100 75 50 25 N ic + Gl i

IR

+

e

N ic

+

IR

l Ct

N ic + Gl i N ic

IR

+

IR

+

Ct

IR

0 l

0

d



*

150 U-Na+ (mmol/l)

*

N ic

+ IR



12.5

c

IR

175

*

IR

Ct l

N ic + Gl i

IR

15.0 DŽ2-MG:Cr (ng/μg)

b

+

a

N ic

IR

+

IR

0 Ct l

0

#

N ic + Gl i

20

*

N ic

40

#

0.020

*

+

60

0.025



*

IR

80

0.030

20 18 16 14 12 10 8 6 4 2 0

IR

#

* BUN (mmol/l)

100



*

0.035

*

IR

sCr (μmol/l)

0.040



*

120

U-Alb:Cr (mg/mg)

140

Fig. 1. Effects of nicorandil (Nic) on renal function in the newborn rat IRI model. Ctl = Control; Gli = glibenclamide. IR-mediated renal injury was evaluated by measuring sCr (a), U-Alb (b), BUN (c), β2-MG (d) and U-Na+ concentrations (e). U-Alb and β2-MG were normalized against urinary creatinine. Data are shown as means ±

SD, n = 12. * p < 0.05 versus Ctl; # p < 0.05 versus IR; † p < 0.05 versus IR + Nic.

 

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Pharmacology 2013;92:245–256 DOI: 10.1159/000355060

ed. After washing with TTBS solution, the membrane was then incubated for 15 min in Stripping Buffer (Thermo Scientific), reblocked in 5% milk/TTBS solution, and reprobed with anti-total PI3K and anti-total Akt antibody, respectively. Statistical Analysis All data were described as means ± SD and analysed by using GraphPad Prism 4.03. Statistical analysis was performed by using one-way ANOVA. A p value ≤0.05 was considered as statistically significant.

Results

Effects of Nicorandil on Renal Function in the Newborn Rat IRI Model Renal function was evaluated by measuring the sCr and U-Alb as the biomarker of glomerular damage, urine β2-MG, U-Na+ and BUN as the biomarker of proximal tubular damage. U-Alb and β2-MG were normalized Zhang/Zhang/Zhao/Tian/Yao

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USA). Fifty micrograms of total protein were separated on 7.5% sodium dodecyl sulphate polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose membranes (Thermo Scientific). Non-specific binding was blocked with 5% not-fat milk powder in Tris-buffered saline containing 0.05% Tween-20 (TTBS) for 1 h. The following primary antibodies were incubated overnight at 4 ° C: rabbit anti-KIR6.1 and rabbit anti-KIR6.2 antibody as well as rabbit anti-sulphonylurea receptor 1 (SUR1) antibody (Abcam), rabbit anti-ERK1/2, mouse anti-phospho-ERK1/2, rabbit anti-Akt, mouse anti-phospho-Akt, mouse anti-phosphoinositide-3-kinase (PI3K), mouse anti-phospho-PI3K, mouse antiGAPDH and mouse anti-β-actin antibody (Cellular Signaling, USA), mouse anti-NF-κB (p65) and mouse anti-histone H1 antibody (Santa Cruz, USA). After washing 3 times with TTBS, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (Thermo Scientific) for 1 h at room temperature. The enhanced chemiluminescence reagent (Thermo Scientific) was used to detect the bound antibodies. The specific protein bands were detected, scanned and quantified with Typhoon 9400 (GE Healthcare Life Sciences, USA). Stripping and reprobing was performed for PI3K and Akt detection. Briefly, phospho-PI3K and phospho-Akt were first detect-

Color version available online

a

4.0

Jablonski score



*

3.5

*

3.0 2.5 2.0 #

1.5

*

1.0 0.5

Bl o

+

N ic

IR

N ic

IR

+

IR

+

Ct

l

0

c

b Fig. 2. Effects of nicorandil (Nic) on kidney histology in the newborn rat IRI model. Ctl = Control; Gli = glibenclamide. a Haematoxylin and eosin staining was performed in kidney tissue sections from IR rats. Bar = 25 μm. b Boxed regions of a were proportionally magnified to display the inflammatory cell infiltrations (arrows), the detached necrosis cell debris (arrowheads), and tubule casts (asterisks). Bar = 25 μm. c The Jablonski score of the overall proximal tubular damage was evaluated blindly and analysed statistically. Data are shown as means ± SD, n = 6. * p < 0.05 versus Ctl; # p < 0.05 versus IR; † p < 0.05 versus IR + Nic.

Effects of Nicorandil on Kidney Histology in the Newborn Rat IRI Model Haematoxylin and eosin staining displayed that in comparison with control, severe acute tubular injury was observed in IR rat kidneys, presenting with brush border loss, inflammatory cell infiltrations, detached necrosis cell debris, and tubule casts (fig.  2a, b). No significant Nicorandil Ameliorates Newborn Rat Kidney IRI

damage was found in glomeruli and distal tubules in IR rat kidneys. The histological changes in the proximal tubules were obviously ameliorated in IR rats pretreated with nicorandil. Severe damage of tubules was also observed in rats subjected to IR after pretreatment with nicorandil and glibenclamide (fig. 2a, b). The Jablonski score in the IR rat kidney was significantly higher than that in the control rat kidney. Pretreatment with nicorandil alone, not combined with glibenclamide, remarkably improved Jablonski scores though it did not return them to control levels (fig. 2c). Effects of Nicorandil on Tubular Cell Apoptosis in the Newborn Rat IRI Model As compared with control, caspase-3 staining-positive cells increased dramatically in the IR group, which was obviously attenuated by nicorandil alone. The inhibition Pharmacology 2013;92:245–256 DOI: 10.1159/000355060

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against urinary creatinine. IR caused a significant increase in all the 5 indexes as compared with control. Notably, β2-MG and U-Na+ increased much more dramatically than sCr, U-Alb and BUN in IR rats. In IR rats, nicorandil alone remarkably decreased the level of U-Alb, β2-MG and U-Na+, and returned sCr and BUN to control levels. The renoprotection of nicorandil against IRI in the rat kidney was completely abolished by glibenclamide (fig. 1).

Color version available online

Caspase-positive cells

125

*

100



#

75

*

50

#

25

Gl i

N ic

+

IR

+

N ic

IR

IR

+

Ct l

0

b

a

*

0.05



#

*

IR

+

Ct

IR IR + N Nic ic + Gl i

0

+

– 36

#

0.10

IR

GAPDH

0.15

l

– 35



0.20

l

Caspase-3

*

Ct

kDa – 26

Bcl-2 Caspase-3

0.25

IR IR + N Nic ic + Gl i

Bcl-2

Protein level (relative to GAPDH)

N ic + IR

IR

IR

Ct

l

+

N ic

+

Gl i

0.30

Fig. 3. Effects of nicorandil (Nic) on tubule cell apoptosis in the newborn rat IRI model. Ctl = Control; Gli = Glibenclamide. a Immunofluorescence staining of caspase-3 was performed. Bar = 25 μm. b As compared with control, caspase-3-positive cells increased remarkably in the IR group, which was attenuated by the pretreatment with nicorandil alone. The protective role of nicorandil was

abolished by glibenclamide. c The specific band of caspase-3 and Bcl-2 was detected. d Normalized against GAPDH, the densitometry of caspase-3 and Bcl-2 was quantified and analysed statistically. Data are shown as means ± SD, n = 6. * p < 0.05 versus Ctl; #  p < 0.05 versus IR; † p < 0.05 versus IR + Nic.

effects of nicorandil on caspase-3 were abolished by glibenclamide (fig. 3a, b). The specific band of caspase-3 and Bcl-2 was detected (fig. 3c). In comparison with control, caspase-3 increased significantly while Bcl-2 decreased significantly in the IR group. The IR-induced upregulation of caspase-3 and downregulation of Bcl-2 was inhibited by nicorandil. The effects of nicorandil on caspase-3 and Bcl-2 were blocked by glibenclamide (fig. 3d).

control, cells positive for F4/80 staining increased drastically in the IR group, which was significantly attenuated by nicorandil alone. The inhibition role of nicorandil on macrophage and neutrophil infiltration was partially abolished by glibenclamide (fig. 4b). MPO is a lysosomal protein of the neutrophil. Compared to control, MPO activity increased significantly in the IR group. The IR-induced activation of MPO was totally prevented by the pretreatment with nicorandil alone, not double pretreatment with nicorandil and glibenclamide (fig. 4c).

Effects of Nicorandil on Inflammatory Cell Infiltrations in the Newborn Rat IRI Model Immunofluorescence staining by the macrophage marker F4/80 was performed (fig. 4a). As compared with 250

Pharmacology 2013;92:245–256 DOI: 10.1159/000355060

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d

c

150

#

*

100 50

3 2

Color version available online

4

#

1

+

N ic + Gl i

Ct l

0

+ IR

IR

+

IR

N ic

IR

IR

+

Ct l

0

*

Gl i

*

200



5

N ic



#

250

IR

300

*

6

+

*

N ic

n-fold change of MPO activity

F480-positive cell number

350

c

b

a Fig. 4. Effects of nicorandil (Nic) on inflammatory cell infiltration

control, MPO activity increased significantly in the IR group, which was decreased evidently by the pretreatment with nicorandil alone. The combined pretreatment with nicorandil and glibenclamide attenuated the inhibitory function of nicorandil on F4/80 and MPO activity in the rat IRI model. Data are shown as means ± SD, n = 6. * p < 0.05 versus Ctl; # p < 0.05 versus IR; † p < 0.05 versus IR + Nic.

in the newborn rat IRI model. Ctl = Control; Gli = glibenclamide. a Immunofluorescence staining of macrophage marker F4/80 was performed. Bar = 25 μm. b In comparison with control, F4/80positive cells increased drastically in the IR group, which was significantly attenuated by the pretreatment with nicorandil alone. The protective role of nicorandil was abolished by glibenclamide. c The activity of kidney tissue MPO was evaluated. Compared to

12

bp 400 300 200

IL-6 mRNA

RT(–)

TNF-į IL-6 GAPDH IL-17 TNF-į IL-6 GAPDH IL-17

RT(+)



*

*

10 8 6 4

#

2

100

3.0

*

2.5 2.0

#

1.5 1.0

N ic + Gl i

*



*

4 3

#

2 1

0.5

Pharmacology 2013;92:245–256 DOI: 10.1159/000355060

N ic + Gl i +

IR

IR

d

N ic

+

IR

l Ct

N ic + Gl i N ic

IR

+

IR

+

IR

0 l

0

c

IR

5



*

TNF-įP51$

3.5

+

b

251

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Nicorandil Ameliorates Newborn Rat Kidney IRI

a

Ct

expression of inflammatory cytokines in the newborn rat IRI model. Ctl = Control; Gli = Glibenclamide. a PCR products from normal kidney were separated on agarose gel. The reverse transcription reaction without reverse transcriptase (RT) was used as negative control. The specific PCR band of TNF-α, IL-6, IL-17, and GAPDH was detected with the expected sizes of 316, 275, 143 and 198 bp, respectively. The mRNA expression of inflammatory cytokines IL-6 (b), IL-17 (c), and TNF-α (d) was analysed by real-time PCR. Data are shown as means ± SD, n = 6. * p < 0.05 versus Ctl; # p < 0.05 versus IR; † p < 0.05 versus IR + Nic.

IL-17 mRNA

Fig. 5. Effects of nicorandil (Nic) on mRNA

N ic

IR

IR

+

Ct

l

0

KIR6.1

kDa – 51

KIR6.2

– 40

*$3'+

– 36

0.6

Akt

– 60

#

0.7

0.4

#

0.3 0.2

*

*

0.1

#

– 65

– 85

+LVWRQH+1

– 37

kDa

#

l

+

Ct

N ic + Gl i +

IR

IR

d

cells. f The effect of nicorandil and glibenclamide on SUR1 was explored in OGD/R-damaged HK-2 cells and IRI rat kidneys (WK). g The effect of nicorandil and glibenclamide on PI3K, Akt and NF-κB was explored in OGD/R-damaged HK-2 cells. The PI3K inhibitor LY294002 was used as control. h The effect of nicorandil and glibenclamide on caspase-3 and Bcl-2 was explored in OGD/R-damaged HK-2 cells. Data are shown as means ± SD, n = 3 independent experiments. * p < 0.05, ** p < 0.01 versus Ctl; # p < 0.05 versus IR; † p < 0.05 versus IR + Nic.

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nicorandil was studied in HK-2 cells subjected to 1-hour OGD exposure. c The effect of glibenclamide was studied in HK-2 cells treated with nicorandil (10 μmol/l) for 1 h prior to 1-hour OGD exposure. d Expression of KIR and SUR1 was investigated in whole kidney (WK) and HK-2 cells. e The effect of nicorandil and glibenclamide on KIR6.2 was explored in OGD/R-damaged HK-2

0

N ic

IR

IR

+

Ct

l

0

#

1

N ic + Gl i

0.1

2

N ic

*

0.2

*

3

+

#

0.3



#

4

IR



*

5

IR

0.4

NF-NJ%KLVWRQH+1

0.5

Fig. 7. Protective role of nicorandil (Nic) on OGD/R-induced HK-2 cell damage. Ctl = Control; Gli = glibenclamide. a Different OGD exposure times were determined in HK-2 cells. b The role of

N ic + Gl i +

NF-NJ%

6

0.6

b

N ic

+ IR

kDa – 85 – 55

IR

c

*

0.7

IR

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channel opener and the proximal signalling pathway in the newborn rat IRI model. Ctl = Control; Gli = glibenclamide. a A specific band of KIR6.1 and KIR6.2 was detected in the rat kidney cortex. As compared with control, the abundance of KIR6.1, especially KIR6.2, decreased drastically in the IR group. Pretreatment with nicorandil alone or combination with glibenclamide significantly prevented the downregulation of KIR6.1 and KIR6.2. Protein expression of phospho-PI3K (b), phospho-Akt (c), and NF-κB (d) was analysed in the rat kidney cortex. Data are shown as means ± SD, n = 6. * p < 0.05 versus Ctl; # p < 0.05 versus IR; †  p < 0.05 versus IR + Nic.

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Nicorandil Ameliorates Newborn Rat Kidney IRI

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Effects of Nicorandil on K ATP Channel Opener and Potential Signal Transduction in the Newborn Rat IRI Model The specific band of KIR6.1 and KIR6.2 was detected in the cortex of the rat kidney. As compared with control, the protein expression of KIR6.1 and KIR6.2 decreased drastically in the IR group. Pretreatment with nicorandil alone significantly prevented IR-induced downregulation of KIR6.1 and KIR6.2. Glibenclamide showed no effects on the expression of KIR6.1 and KIR6.2 (fig. 6a). Compared to control, IR caused a great upregulation of phospho-PI3K (fig.  6b) and phospho-Akt (fig.  6c), which was totally inhibited by nicorandil alone. The inhibition role of nicorandil on active PI3K and Akt was attenuated by glibenclamide. As compared with control, protein expression of NF-κB (p65) in nuclei increased dramatically in the IR group. The IR-induced upregulation of NF-κB was prevented by the pretreatment with nicorandil alone, not by the combined pretreatment with nicorandil and glibenclamide (fig. 6d). Protective Role of Nicorandil on OGD/R-Induced Tubular Cell Damage The OGD exposure time was first determined. Cell viability analysis showed the induction of cell death after 1 h of OGD/R (fig. 7a). The role of nicorandil was studied on 1-hour OGD/R-induced HK-2 cell injury. Both 10 and 100 μmol/l of nicorandil significantly increased cell viability as compared with the OGD/R group, and no large difference was observed between 10 and 100 μmol/l (fig. 7b). Glibenclamide prevented the protective role of nicorandil (10 μmol/l) in HK-2 cells subjected to 1-hour 254

Pharmacology 2013;92:245–256 DOI: 10.1159/000355060

OGD/R injury, especially 10 μmol/l of glibenclamide (fig. 7c). KIR6.2 and SUR1 were detected both in the whole kidney and in HK-2 cells, while KIR6.1 was only detected in the whole kidney (fig. 7d). In HK-2 cells, KIR6.2 was decreased significantly in the OGD/R group, which was obviously prevented by nicorandil pretreatment. The upregulation role of nicorandil on KIR6.2 was not affected by glibenclamide (fig. 7e). SUR1 expression was decreased significantly both in OGD/R HK-2 cells and in IRI rat kidneys. Nicorandil pretreatment obviously prevented OGD/R- or IRI-induced reduction of SUR1. The upregulation role of nicorandil on SUR1 protein in OGD/R cells or IRI rat kidneys was abolished by glibenclamide (fig. 7f). PI3K and Akt were dramatically activated in OGD/Rinduced HK-2 cell injury, which was significantly prevented by nicorandil. The inhibition role of nicorandil on PI3K and Akt activation was abolished by glibenclamide. As compared with control, the fraction of NF-κB in nuclei was increased in OGD/R-treated HK-2 cells, which was prohibited by nicorandil. The inhibition role of nicorandil on the NF-κB level was abolished by glibenclamide. Additionally, the application of the PI3K inhibitor LY294002 prohibited the activation of Akt and translocation of NF-κB from the cytoplasm to nuclei in OGD/R HK-2 cells (fig.  7g). In comparison with control, caspase-3 increased significantly while Bcl-2 decreased significantly in OGD/R cells. The OGD/R-induced upregulation of caspase-3 and downregulation of Bcl-2 were inhibited by nicorandil. The effects of nicorandil on caspase-3 and Bcl-2 were prohibited by glibenclamide (fig. 7h).

Discussion

Although ischaemia is one of the major causes of acute renal failure, especially in infants and children, only a few therapeutic approaches are available to protect from IRI. The renoprotective role of nicorandil, an opener of KATP channels, has been reported in several acute kidney diseases including experimental kidney IRI [10]. This study investigated the effects of nicorandil on KATP channels and the potential signal transduction pathway(s) in a newborn rat kidney IRI model and in cultured tubular HK-2 cells subjected to OGD/R injury. In comparison with control, IR caused a significant increase in the biomarkers indicating both glomerular (sCr and U-Alb) and tubular (urinary β2-MG, Na+ and BUN) damage. The increased U-Na+ concentration in the IR group suggested Zhang/Zhang/Zhao/Tian/Yao

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Effects of Nicorandil on Inflammatory Cytokines in the Newborn Rat IRI Model The mRNA expression of inflammatory cytokines interleukin 6 (IL-6), IL-17, and tumour necrosis factor-α (TNF-α) was investigated by real-time PCR. To verify the specific amplification, PCR products from normal kidney were separated on agarose gel. The reverse transcription reaction without reverse transcriptase was used as negative control. The specific band of TNF-α, IL-6, IL-17 and GAPDH was detected with the expected sizes of 316, 275, 143 and 198 bp, respectively (fig. 5a). As compared with control, the mRNA level of IL-6, IL-17, and TNF-α in the kidney cortex increased significantly in the IR group. Pretreatment with nicorandil effectively inhibited the IR-induced upregulation of IL-17, TNF-α, and especially IL-6. The downregulation of nicorandil on IL-6, IL-17, and TNF-α was abolished by glibenclamide (fig. 5).

phages and neutrophils in renal tubule cells was abolished by glibenclamide. IL-6 is secreted by T cells and macrophages to stimulate the immune response, whereas TNF-α, a cytokine produced chiefly by activated macrophages in response to inflammatory stimuli, is a wellknown important mediator during IRI [19]. As a potent mediator that responds to immune reactions, IL-17 can recruit monocytes and neutrophils to the site of inflammation by increasing chemokine production in various tissues [20]. We also found that pretreatment with nicorandil effectively inhibited the IR-induced upregulation of IL-17, TNF-α, and especially IL-6, which was abolished by glibenclamide. These results provided evidence that macrophage and neutrophil infiltration and thus production of inflammatory cytokines play a crucial function in IR-mediated kidney injury, and that nicorandil specifically affects these inflammatory cells and cytokines in a direct or indirect manner. The KATP channel is a type of potassium channel that is gated by ATP. Mitochondrial KATP consists of KIR6.1 and KIR6.2, which are directly linked to changes in the metabolic environment such as hypoxia and ischaemia, etc. [21, 22]. Both KIR6.1 and KIR6.2 were detected in whole kidneys. Nevertheless, only KIR6.2 was detected in cultured tubular cells, suggesting that the renal tubular cell mainly expresses KIR6.2. Pretreatment with nicorandil alone significantly prevented the IR-induced downregulation of KIR6.1 and KIR6.2. However, Shimizu et al. [10] showed that IR induced the downregulation of only KIR6.2, not KIR6.1, in adult rat kidneys. In HK-2 cells, OGD/R injury also induced reduction of KIR6.2, which was prevented by nicorandil. The in vitro data demonstrated that KIR6.2, not KIR6.1, is mainly involved in IRinduced kidney injury, and that the renoprotective role of nicorandil might be implemented by directly upregulating the expression of KIR6.2. Interestingly, glibenclamide showed no effects on KIR6.1 and KIR6.2. Our results showed that SUR1 is present both in the whole kidney and in HK-2 cells. Moreover, nicorandil obviously prevented OGD/R- or IRI-induced reduction of SUR1. The upregulation role of nicorandil on SUR1 in OGD/R HK-2 cells or IRI rat kidneys was abolished by glibenclamide. These findings implied that glibenclamide might function by directly inhibiting SUR1. Finally, the potential signal transduction pathway was explored both in the newborn rat kidney IRI model and in HK-2 cells subjected to OGD/R injury. NF-κB is a protein complex that controls the transcription of DNA, and is involved in cellular responses to many stimuli [23, 24]. It has been demonstrated that the PI3K and Akt pathway

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an abnormality of tubular function, either the increase in Na+ excretion or the reduction of Na+ re-absorption in tubule cells. In an isolated perfused rat kidney model, IRI significantly decreased the glomerular filtration rate and increased the fractional excretion of sodium [6]. Nevertheless, urinary β2-MG and Na+ concentration increased much more dramatically than sCr, U-Alb and BUN in IR rats, implying that IR induced damage mainly in the renal tubules, not in the glomerulus, which is supported by no obvious histological alteration observed in glomeruli, while severe tubular damage is observed in proximal tubular cells. Shimizu et al. [10] also reported that renal IR induced a significant increase in sCr, U-Alb, and β2-MG, and that histological analysis showed no remarkable damage in glomeruli. Pretreatment with nicorandil alone remarkably decreased the level of U-Alb, β2-MG and U-Na+, and returned sCr and BUN to control levels, implying that nicorandil exerts both glomerular and tubular protection against IRI in the newborn rat kidney. In the isolated perfused rat kidney, pretreatment with diazoxide, another KATP opener, also reduced the postischaemic increase in sodium excretion [6]. However, Shimizu et al. [10] reported that nicorandil appears to provide a greater beneficial action against tubular dysfunction, rather than glomerular dysfunction. The application of nicorandil also drastically improved the Jablonski score, and decreased tubular cell apoptosis as proved by the reduction of caspase-3-positive cells, the decrease in caspase-3 expression and the induction of Bcl-2. In HK-2 cells, the MTT assay showed the induction of cell death after 1 h of OGD, which was prevented by nicorandil. The OGD/R-induced upregulation of caspase-3 and downregulation of Bcl-2 was also inhibited by nicorandil. Glibenclamide works by inhibiting the SUR1, the regulatory subunit of KATP channels, and has been shown to bind more efficiently to the ischaemic tissue [17]. Our results showed that the protective effects of nicorandil were completely abolished by glibenclamide both in vitro and in vivo. It was reported that glibenclamide could inhibit the diazoxide effect on the postischaemic excretion of sodium [6]. These findings suggested that sodium reabsorption is improved by KATP activation and that blockade of KATP channels during IRI has an injury-enhancing effect on renal epithelial function and histology. Cellular mediators of immunity, such as dendritic cells, T and B cells, as well as neutrophils and macrophages, contribute to the pathogenesis of kidney injury after IR [18]. Our results showed that the inhibitory role of nicorandil on the IR-induced infiltration of macro-

is responsible for the translocation of NF-κB from cytoplasm to nuclei [25]. We found that pretreatment with nicorandil effectively inhibited the OGD/R or IRI-induced activation of PI3K and Akt, as well as the increment of nuclear NF-κB, which was attenuated mostly by glibenclamide. Additionally, the application of the PI3K inhibitor LY294002 prohibited the activation of Akt and translocation of NF-κB from cytoplasm to nuclei, suggesting that the increment of nuclear NF-κB depends on the activation of PI3K and Akt in damaged tubular cells. It has been reported that a PI3K-driven, NF-κB-dependent transcriptional profile may play a critical role in promoting a micro-environment amenable to tumour progression [25]. Our findings suggested that the renoprotective role of nicorandil in the newborn rat IRI kidney might consist of decreasing the production of inflammatory cy-

tokines, and restoring the expression of KIR6.2 through directly inhibiting the PI3K-Akt-NF-κB axis. It should be noted that the pathogenesis of IRI is so much complicated that the true clinical potential of nicorandil remains to be clarified in future experimental studies and clinical trials.

Acknowledgement This work was supported by the Research Foundation from the Education Department of Heilongjiang Province.

Disclosure Statement The authors declare no conflict of interest.

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References

Nicorandil protects against ischaemia-reperfusion injury in newborn rat kidney.

Ischaemia-reperfusion injury (IRI) is the predominant cause of acute kidney injury. Nevertheless, the underlying molecular mechanisms are still unclea...
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