NFκ-B signaling pathway

Journal Pre-proof Nicorandil combats doxorubicin–induced nephrotoxicity via amendment of TLR4/P38 MAPK/NFκ-B signaling pathway Ali Khames Abd El-twab, Marwa M. Khalaf, Amany M. Gad, Ola M. Abd El-raouf, Mohamed Ahmed Kandeil PII:

S0009-2797(19)30720-3

DOI:

https://doi.org/10.1016/j.cbi.2019.108777

Reference:

CBI 108777

To appear in:

Chemico-Biological Interactions

Received Date: 1 May 2019 Revised Date:

16 July 2019

Accepted Date: 31 July 2019

Please cite this article as: A. Khames Abd El-twab, M.M. Khalaf, A.M. Gad, O.M. Abd El-raouf, M.A. Kandeil, Nicorandil combats doxorubicin–induced nephrotoxicity via amendment of TLR4/P38 MAPK/NFκ-B signaling pathway, Chemico-Biological Interactions (2019), doi: https://doi.org/10.1016/ j.cbi.2019.108777. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

Editors-in-Chief

1

Chemico-biological interactions

2

Dear Editor:

3

Kindly find our attached revised manuscript, entitled: Nicorandil combats

4

doxorubicin-induced

TLR4/P38

5

MAPK/NFκ-B signaling pathway which we submit for consideration for

6

publication following revision in Chemico-biological interactions

7

We believe that this study would be of interest to your readers. The current

8

study aims to highlight on the ameliorative effect of nicorandil (anti-

9

anginal and cardioprotective drug) on doxorubicin-induced nephrotoxicity

10

experimentally in rats. This manuscript focuses on describing the

11

underlying mechanisms that mediate its activities targeting inflammatory &

12

oxidative stress and apoptotic markers mentioning previous trials and what

13

is added by our study, and focusing on the pathway that is included greatly

14

by doxorubicin; TLR4/MAPK P38/NFκ-B expression pathway that is

15

significantly induced by doxorubicin and is expected to be suppressed by

16

nicorandil. We emphasized our results and conclusions by western blot

17

technique.

this manuscript is complied with the scope of "Chemico-

18

biological interactions" journal because it discusses how nicorandil

19

prevented doxorubicin toxicity and this is the scopes of "Chemico-

20

biological interactions" journal.

21

nephrotoxicity

1

via

amendment

of

Thank you for your consideration.

22

Best regards

23

Correspondence: All correspondence should be addressed to:

24

Dr. Marwa Mahmoud Khalaf,

25

Department of Pharmacology and Toxicology, Faculty of Pharmacy, Beni-

26

Suef University.

27

Postal code: 62514, Salah Salem Street, Beni-Suef, Egypt

28

E-mail: [email protected]

29

[email protected]

30

Tel.: 00201002784548

31

• Nicorandil ameliorated doxorubicin-induced nephrotoxicity.

32

• Nicorandil restored the oxidant/antioxidant balance.

33

• Nicorandil suppressed inflammatory signaling pathway TLR4/MAPK

34

P38/NF-κb/TNF-α.

35

Nicorandil regulated BAX/Bcl-2 apoptotic pathway

36

Title: Nicorandil

37

combats

doxorubicin–induced

nephrotoxicity

via

38

amendment of TLR4/P38 MAPK/NFκ-B signaling pathway

39

Ali Khames Abd El-twab1, Marwa M. Khalaf2, Amany M. Gad3, Ola M.

40

Abd El-raouf4, Mohamed Ahmed Kandeil5.

41

2

1

Department of Pharmacology and Toxicology, Faculty of Pharmacy,

42

Deraya university, Minia, Egypt.

43

1,3,4

44

Department of Pharmacology, National Organization for Drug Control

and Research (NODCAR), Cairo, Egypt.

45

2,5

46

Department of Pharmacology and Toxicology, Faculty of Pharmacy,

Beni-Suef University, Beni-Suef, Egypt.

47

Running Title: Nicorandil combats doxorubicin-induced nephrotoxicity

48

via amendment of TLR4/P38 MAPK/NFκ-B signaling pathway

49

* Correspondence: All correspondence should be addressed to:

50

Dr. Marwa Mahmoud Khalaf,

51

Department of Pharmacology and Toxicology, Faculty of Pharmacy, Beni-

52

Suef University.

53

Postal code: 62514, Salah Salem Street, Beni-Suef, Egypt

54

E-mail: [email protected]

55

[email protected]

56

Tel.: 00201002784548

57

3

Abstract

58

Nicorandil ameliorated doxorubicin-induced nephrotoxicity; this study

59

aimed to show and explain the mechanism of this protection. A precise

60

method was elucidated to study the effect of nicorandil on doxorubicin-

61

induced nephrotoxicity in rats depending on the critical inflammation

62

pathway TLR4/ MAPK P38 / NFκ-B. Adult male rats were subdivided

63

into four groups. The 1st group was normal control, the 2nd group received

64

nicorandil (3 mg/kg; p.o., for 4 weeks), the 3rd group received doxorubicin

65

(2.6 mg/kg, i.p., twice per week for 4 weeks), and the fourth group was

66

combination of doxorubicin and nicorandil for 4 weeks.

67

Nephrotoxicity was assessed by biochemical tests through measuring

68

Kidney function biomarkers such as [serum levels of urea, creatinine,

69

albumin and total protein] besides renal kidney injury molecule-1 (KIM-1)

70

and cystatin C], oxidative stress parameters

such as [renal tissue

71

malondialdehyde (MDA), reduced glutathione (GSH), SOD, catalase and

72

nrf-2], mediators of inflammation such as [Toll like receptor 4 (TLR-4),

73

Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB),

74

p38 MAPK, Interleukin 1 beta (IL-1 β), and Tumor necrosis factor alpha

75

(TNF-α)] and markers of apoptosis [BAX and Bcl-2 in renal tissue].

76

Finally, our data were supported by histopathology examination.

77

4

Nicorandil pretreatment resulted in a significant decrease in nephrotoxicity

78

biomarkers, oxidative stress markers, inflammatory mediators and

79

prevented apoptosis through decreasing BAX and increasing Bcl-2 in renal

80

tissues. Nicorandil prevented all the histological alterations caused by

81

doxorubicin. Nicorandil is a promising antidote against doxorubicin-

82

induced nephrotoxicity by neutralizing all toxicity mechanisms caused by

83

doxorubicin through normalizing inflammatory cascade of TLR4/ MAPK

84

P38 / NFκ-B.

85

Key words: Nicorandil, Doxorubicin, Nephrotoxicity, Inflammation,

86

Apoptosis.

87

5

1. Introduction

88

Doxorubicin (DOX), an anthracycline derivative, is commonly

89

used to treat a variety of malignant neoplasms including, solid tumors such

90

as breast, pulmonary, uterine, ovary and cervix cancer as well as leukemia

91

and haematopoietic tumors (Akindele et al., 2018). Its continuous usage

92

alone in clinical settings is hindered owing to its life-threatening outcomes

93

on organs such as heart, kidney, liver and testis (Ren et al., 2016, Nagai et

94

al., 2018, Zidan et al., 2018).

95

Doxorubicin

nephrotoxicity

is

a

critical

factor

during

96

chemotherapy (Lee and Harris, 2011). Previous studies focused on

97

doxorubicin nephrotoxicity but there is no antidote that can prevent this

98

toxicity completely. Doxorubicin nephrotoxicity can be attributed to the

99

mitochondrial electron transfer reactions besides accumulated calcium ions

100

(Danmaigoro et al., 2018). In addition, inflammatory reaction and

101

apoptosis are causative factors (Shaker et al., 2018). Previous studies

102

evaluated the doxorubicin nephroathy mainly through measuring oxidative

103

stress parameters (Nagai et al., 2018), while others studied the effect of

104

hyperuricemia (Khames et al., 2017).

105

Several efforts have recently been made to ameliorate the

106

adverse effects of DOX. An approach to ameliorate doxorubicin related

107

toxicity is to use drug which engender a pharmacological intervention and

108

6

promising therapeutic strategy for the treatment or prevention of renal

109

disorders related to ROS overproduction.

110

Nicorandil, a mitochondrial ATP-dependent potassium (KATP)

111

channel opener, was used for the treatment of angina through increasing

112

coronary circulation (Simons and Laham, 2016, Kallistratos et al., 2017)

113

and was considered to be cardioprotective agent on ischaemic myocardium

114

(Abdel‐‐Raheem et al., 2013, Zhang et al., 2018). beside inhibiting

115

apoptosis in cardiac tissues (Nagata et al., 2003).

116

Nicorandil is a powerful anti-oxidant drug that has been previously

117

reported to exert great protection against oxidative stress in several

118

conditions (Qi et al., 2015, Ravindran et al., 2017) such as prevention of

119

oxidative stress in streptozotocin-induced diabetes (Mano et al., 2000).

120

Nicorandil was also used for the prevention of contrast‐induced

121

nephropathy (Ma et al., 2018). In addition, nicorandil has anti-

122

inflammatoy effects as shown by its anti-neuroinflammation effects in

123

astrocytes (Dong et al., 2016). Through anti-apoptotic effects, nicorandil

124

was pereviously reported to improve cardiac function and integrity after

125

coronary micro embolisation (He et al., 2018).

126

Our study assessed the involvement of toll like receptor 4

127

(TLR4) as a novel and significant inflammatory pathway. Toll like receptor

128

4 is a part of the innate immunity (Nair, 2015) and mediate inflammatory

129

7

response (Molteni et al., 2016) by inducing nuclear factor kappa B (NFκ-

130

B).

131

Currently, there is no information available about the protective effects of

132

the KATP channel openers on doxorubicin injury in the kidney. In the

133

present study, we hypothesized that nicorandil might prevent the renal

134

injury caused by doxorubicin and we investigated the effects and the

135

possible mechanisms of action of the KATP channel opener, nicorandil, on

136

the DOX injury in the rat kidney through TLR4 and NFκ-B signalling

137

pathway.

138

2. Material and Methods:

139

2.1. Ethics statement:

140

Animals were adapted for two weeks in the animal house before the start of

141

the study. Experimental methods were conducted according to the ethical

142

guidelines for investigations in experimental rats and were advisable by the

143

Research Ethical Committee of Faculty of Pharmacy, Beni-Suef University

144

(Beni-Suef, Egypt) to agree with the instructions of the Care and Use of

145

experimental rats (ILAR, 1996). Unnecessary aggressive deal with animals

146

was avoided. Animals were quietly handled; vigorous handling, pressure,

147

and tough maneuver was avoided.

148

2 materials:

149

8

- Doxorubicin: 2.62 mg/kg i.p twice weekly for 4 weeks (Elsherbiny

150

and El-Sherbiny, 2014a).

151

- Nicorandil: 3 mg/kg/day orally for 4 weeks (Ahmed and El-maraghy,

152

2013a).

153

All drugs and chemicals will be obtained from Sigma Chemical

154

Company (St Louis, USA).

155

2.3. Animals

156

Male adult Sprague-Dawley rats (200g ± 20) were purchased from the

157

animal house of national organization for drug control and research, Dokki,

158

Egypt. Animals were adapted at the experimental area of the National

159

Organization for Drug Control and Research (NODCAR, Dokki, Egypt).

160

Animals had no limitations to food and drink. They were kept at 20–25˚C

161

with 12-h light–dark cycle

162

2.4. Experimental design and sampling

163

Rats were randomly divided into four groups (n=10-12 per each),

164

The 1st group served as normal control group received saline (vehicle for

165

nicroandil) orally for 4 weeks and administered saline (0.5 ml/Rat, i.p.)

166

twice a week as vehicle for Dox.

167

9

The 2nd group received nicorandil only (3 mg/kg/day, orally for 4 weeks)

168

(Ahmed and El-Maraghy, 2013b).

169

The 3rd group were injected with a cumulative dose of doxorubicin 21

170

mg/kg, i.p. twice per week, for 4 weeks) (Elsherbiny and El-Sherbiny,

171

2014b).

172

The 4th group received doxorubicin and nicorandil for 4 weeks.

173

Nicorandil was given one hour before doxorubicin injection. At the end

174

of the experiment blood samples were collected from retro-orbital sinus

175

plexus and sera were separated by centrifugation at 1000g for 10 min and

176

stored at −80°C for serological measurements of the kidney function tests.

177

Afterwards, animals were sacrificed by decapitation under anesthesia, both

178

kidneys of each animal were dissected out, and portions of them were fixed

179

in 10% formalin solution for histopathological examination while the

180

remainder portions were homogenized in 50 mM phosphate buffer (pH

181

7.4), and stored at -80°C till estimations of biochemical and western blot

182

examinations.

183

2.5. Biochemical analysis

184

2.5.1. Assessment of renal functions

185

Colorimetric assay kits for the measurement of the level of blood

186

urea nitrogen (BUN), serum creatinine, serum albumin and total protein

187

10

using (biomed diagnostics, Cairo, Egypt). Rat Cystatin-C (Cys-C) as well

188

as Rat Kidney injury molecule (KIM-1) ELISA Kits were obtained from

189

Mybiosource (San Diego, CA, USA) and were used for measurement of

190

serum cystatin and KIM-1, respectively. All procedures were performed

191

according to the manufacturers’ instructions.

192

2.5.2. Measurement of renal oxidative stress estimation

193

The renal content of thiobarbituric acid reactive substances (TBARS)

and

glutathione

(GSH),

activity of

195

myeloperoxidase, catalase (CAT), and superoxide dismutase (SOD) were

196

measured using the Mybiosource ELISA kits (San Diego, CA, USA)

197

according to the manufacturer’s instructions.

198

2.5.3. Measurement of renal Toll like receptor 4 (TLR-4), Nuclear

199

factor kappa-light-chain-enhancer of activated B cells (NF-κB),

200

Nuclear factor erythroid 2–related factor 2 (Nrf-2) and P38 mitogen-

201

activated protein kinases (p38-MAPK)

202

Protein levels of TLR-4, NF-κB, Nrf-2, and p38-MAPK were assessed

203

using Western blot technique using TGX Stain-Free™ Fast Cast™

204

Acrylamide Kit (SDS-PAGE) which was provided by Bio-Rad

205

Laboratories, TNC, USA Catalog. NO. 161-0181.

206

11

as

well

as

the

194

The Western Blot analysis technique was applied (using V3 Western

207

WorkflowTM Complete System, Bio-Rad R _ Hercules, CA). Briefly,

208

proteins were extracted from tissue homogenates using ice-cold radio-

209

immuno precipitation assay buffer supplemented with phosphatase and

210

protease

mM

211

phenylmethylsulphonyl fluoride, 2 mg/mL aprotinin, and 0.5 mg/mL

212

leupeptin) then centrifugated at 12,000 rpm for 20 min. Bradford assay

213

method was used to estimate the protein concentration of each sample. The

214

same amounts of protein (20–30 ??g of total protein) were isolated by gel

215

electrophoresis (10% acrylamide gel) depending on a Bio-Rad Mini-

216

Protein II system. The protein was moved to polyvinylidene difluoride

217

membranes (Pierce, Rockford, IL) with a Bio- Rad Trans-Blot system.

218

Then, PBS was used for washing membranes then blocked for 1 h at

219

ambient temperature with 5% (w/v) skimmed milk powder in PBS.

220

Following blocking, the blots were developed using antibodies for TLR4,

221

NF-κB, Nrf-2, p38-MAPK and beta actin supplied by Thermoscientific

222

(Rockford, IL) and incubated overnight at pH 7.6 at 4◦C with gentle

223

shaking. After washing, peroxidase-labeled secondary antibodies were

224

added and the membranes were incubated at 37◦C for 1 h. Band intensity

225

examined and analyzed by the imaging system ChemiDocTM with Image

226

LabTM software version 5.1 (Bio-Rad Laboratories, Hercules, CA). The

227

inhibitors

(50

mmol/L

12

sodium

vanadate,

0.5

results are expressed as arbitrary units after comparison to the normal -

228

actin protein expression

229

2.5.4. Measurement of (TNF-α) and Interleukin 1 beta (IL-1 β)

230

Rat TNF-α and IL-1 β were estimated according to the ELISA kits

231

manufacturer guide obtained from RayBiotech Inc. (Parkway, LaneSuite

232

Norcross, GA).

233

2.5.5. Measurement of protein expression of Bax/Bcl2

234

B-cell lymphoma 2 (Bcl-2) protein, Bcl-2-Associated-X-protein (Bax) and

235

Bax/Bcl-2 ratio were assessed using western blot technique, Protein levels

236

of Bax/Bcl2 in renal tissues of different treatment groups (1–4) were

237

assessed using Western blot technique. Where proteins were extracted

238

using TRIzol reagent, and protein concentrations were estimated using

239

Bradford method. The primary antibodies used were raised in rabbit anti-

240

Bcl-2 antibody (13-8800 Thermo Fisher Scientific), anti-Bax antibody

241

(MA5-14003 Thermo Fisher Scientific).

242

2.5.6. Renal histopathological examination

243

Kidney specimens were taken from many rats in each group and were

244

stabilized in 10% formal saline for one day. Washing was carried out by

245

water and dehydrated by diluted methyl and ethyl alcohol that is serially

246

diluted and were used for dehydration. Samples were kept in paraffin wax

247

13

at 56 degree in oven for one day. Renal tissues blocks 4 microns thickness

248

were made by slidge microtome. Then the sections were added to glass

249

slides, paraffin is removed , hematoxylin & eosin stain are used for the

250

examination by light electric microscope (Suvarna et al., 2018).

251

2.6. Data and Statistical analysis

252

Results were expressed as mean ± SE. Statistical analysis was performed

253

using the SPSS version 16 (Chicago, IL, USA), while the graphs were

254

drawn using a prism computer program (GraphPad software Inc. V5, San

255

Diego, CA, USA). Statistical analysis was carried out using one-way

256

analysis of variance (ANOVA) followed by Tukey-Kramer Multiple

257

Comparison Test as a post hoc test. Probability values of less than 0.05

258

were considered statistically significant.

259

3. Results

260

3.1.

Effect of nicorandil on serum levels of urea nitrogen,

261

creatinine, total protein and albumin and on renal tissue

262

contents of KIM-1 and Cystatin C in doxorubicin-induced

263

nephrotoxicity in rats:

264

Normal control values for the serum levels of urea, creatinine, total protein

265

and albumin were 33.17 ± 1.9 mg/dl, 0.18 ± 0.013 mg/dl, 5.59 ± 0.15

266

mg/dl and 3.5 ± 0.25 mg/dl, respectively. Normal control values for the

267

14

renal contents of KIM-1 and cystatin C were 2.202 ± 0.09 pg/mg.tissue and

268

0.6617 ± 0.05 pg/mg.tissue. Doxorubicin significantly increased serum

269

levels of urea nitrogen and creatinine associated with a significant decline

270

in total protein and albumin levels as compared to the normal control

271

group. Additionally, doxorubicin significantly increased renal contents of

272

KIM-1 and cystatin C.

273

However, co-treatment with nicorandil significantly abolished the increase

274

of urea nitrogen, creatinine, KIM-1 and cystatin C levels and counteracted

275

the reduction of total protein and albumin, respectively, as compared to

276

doxorubicin group (Table 1).

277

3.2.

Effect of nicorandil on the renal oxidative stress biomarkers in

278

doxorubicin-induced nephrotoxicity in rats:

279

Normal control values for the renal contents of MDA and GSH and the

280

renal activities of SOD, CAT, Nrf-2 and MPO were 5.350 ± 0.41 nmol/g.

281

tissue, 71.15 ± 7.24 mg/g tissue, 5.89 ± 0.26 mg/g tissue, 120.0 ± 2.6 mg/g

282

tissue and 39.25 ± 5.86 mg/g tissue respectively.

283

Doxorubicin treatment resulted in a marked increase in the renal MDA

284

content and MPO activity associated with a marked decrease in renal GSH

285

content as well as a significant decrease in Nrf-2, SOD and CAT activities,

286

respectively, as compared to the normal control group

287

15

Treatment with nicorandil significantly decreased the renal MDA content

288

and MPO activity and nearly restored renal GSH content and Nrf-2, SOD

289

and CAT activities to normal control values as compared to doxorubicin

290

group (Fig. 1A-F).

291

3.3.

Effect of nicorandil on the renal inflammatory mediators in

292

doxorubicin-induced nephrotoxicity in rats:

293

Normal control values for TLR4, P38 MAPK, NF-κB, IL.1 β and TNF-α

294

were 1.00 ± 0.01, 1.005 ± 0.001, 1.007 ± 0.003, and 24.88 ± 2.121 pg/g

295

tissue, respectively.

296

Doxorubicin treatment resulted in a significant increase in renal content of

297

TLR4, P38 MAPK, NF-κB, IL.1 β and TNF-α as compared to the normal

298

control group.

299

Co-treatment with nicorandil significantly normalized renal content of

300

TLR4, P38 MAPK, NF-κB, IL.1 β and TNF-α as compared to doxorubicin

301

group (Fig. 2A-D).

302

3.4.

Effect of nicorandil on renal antiapoptotic markers in doxorubicin-induced nephrotoxicity in rats:

303 304

Normal control values for the pro-apoptotic protein BCL2 Associated X

305

protein (Bax), and anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) were

306

1.012 ± 0.01 and 1.018 ± 0.016.

307 16

Doxorubicin increased apoptotic markers significantly as compared to the

308

normal control group.

309

As shown in Fig. (3A-C) after nicorandil administration the level of Bax

310

was significantly decreased while Bcl-2 level was significantly increased in

311

the renal tissue resulting in inhibition of apoptosis as compared with

312

doxorubicin group.

313

3.5. Effect of nicorandil on histopathological examination of renal tissues in doxorubicin-induced nephrotoxicity in rats.

314 315

As shown in Figure 4, kidney sections obtained from the normal control

316

and the control nicorandil groups showed normal histological appearance

317

of kidney architecture.

318

Meanwhile, doxorubicin-treated group showed focal inflammatory cells

319

infiltrations with few fibroblastic cells proliferation were detected in

320

between the tubules and glomeruli at the cortex. The endothelial cells

321

lining the tufts of the glomeruli showed vacuolization. The cortical stromal

322

blood vessels showed congestion as well as perivascular oedema. The

323

corticomedullary portion showed focal fibrosis and focal haemorrhage

324

inbetween the tubules. There was swelling in the lining tubular epithelium

325

with obliteration in the tubular lumen

326

17

while other tubules at the

corticomedullary portion had vacuolar degeneration in the lining

327

epithelium (Fig.4),(table 2)

328

However, nicorandil co-treated group showed mild focal inflammatory

329

cells infiltration, the tubules at the corticomedullary portion showed

330

vacuolar degeneration

331

4. Discussion

332

Cancer chemotherapy usually demolishes the normal physiological

333

homoeostasis and affects multiple organs during the course of treatment.

334

Despite its extensive clinical utilization in the fight against a variety of

335

human malignancies (Akindele et al., 2018), treatment with the

336

conventional doxorubicin (DOX) is limited because of its multiorgan

337

toxicities including renal damage and nephrotoxicity (Wang et al., 2000),

338

cardiac, pulmonary, testicular and hematological toxicities (Singal et al.,

339

2000a).

several

340

mechanisms including free radical production as well as inflammatory,

341

apoptotic and hyperuricemic effects (Khames et al., 2017b).

342

In the present study, i.p. doxorubicin produced severe increase in serum

343

levels of BUN, creatinine, KIM and cystatin C, while it significantly

344

decreased serum levels of albumin and total protein. These results are in

345

agreement with (Öz and İlhan, 2006, Kramer et al., 2009;, Jaćević et al.,

346

Doxorubicin

deteriorates

18

renal

function

through

2018, Qiao et al., 2018). These deteriorations in renal function are

347

suggested to be attributed mainly to the increased production of free

348

radicals and ROS in renal tissues that resulted in reaction of these free

349

radicals with proteins of the nephron causing structural and functional

350

changes of renal tubules and glomeruli.

351

In the present study, Kim-1 was significantly elevated in the Dox-treated

352

group. This result was consistent with the earlier reports of (Lateef et al.,

353

2014, Wang et al., 2015).

354

Kidney injury molecule-1 is a type 1 membrane protein that is expressed at

355

negligible levels in normal rat kidneys. However, it is reported to be

356

massively induced in tubules after ischemic or toxic injury in rats

357

(Ichimura et al., 1998). Tubular kidney injury molecule-1 (Kim-1) is

358

induced in acute renal injury and is known to be reversible. In addition,

359

Kim-1 is also induced in chronic renal damage (Huo et al., 2010). The

360

mechanism of Kim-1 induction by doxorubicin nephropathy was not

361

specifically studied. However, its expression was found to occur in a wide

362

array of renal conditions in association with early tubular damage (Kramer

363

et al., 2009). It is plausible in this study that proteinuria was the marker for

364

renal injury.

365

The present results also showed that the level of cystatin C was

366

significantly elevated in the Dox-treated group. In agreement, Wang et al.

367

19

(2015) reported that, animals treated with doxorubicin (6 mg-kg) for 15

368

days showed a significant increase in cystatin C level.

369

Cystatin C in the blood is usually filtered through the glomeruli, being

370

absorbed in the proximal tubule of the kidney. Dysfunction of the renal

371

tubule leads to raised level of cystatin C in the body. many studies proved

372

that serum cystatin C is preferred than creatinine as an indicator of renal

373

failure involving defective glomerular functions (Dharnidharka et al.,

374

2002).

375

Oxidative stress was incriminated to be the main mechanism responsible

376

for DOX-induced nephrtoxicity (Khames et al., 2017a). Mansouri et al.

377

(2017) reported that, the mechanisms of doxorubicin nephrotoxicity

378

included oxidative stress status characterized by high amount of the

379

produced free oxidative radicals and a defect in the endogenous antioxidant

380

defense mechanisms causing an imbalance in the normal oxygen metabolic

381

pathway. First, a semiquinone metabolite is formed after adding an electron

382

to the quinone form of doxorubicin and finally, the quinone structure is

383

rapidly reformed by reduction of the molecular oxygen to ROS (Liu et al.,

384

2009). ROS produces hydrogen peroxide (H2O2) and hydroxyl radicals

385

(OH· ) and these products can attack DNA, oxidize it and finally induce

386

apoptosis in both normal and tumoral tissue cells (Al-Dalaen and Al-

387

Qtaitat, 2014).

388

20

Results of the current investigation revealed that, DOX administration led

389

to a significant reduction in GSH content, Nrf-2 level as well as the

390

activities of SOD and catalase enzymes. However, there was a significant

391

increase in MDA content and MPO activity. These findings are in the

392

agreement with (Sun et al. 2016, Oyagbemi et al., 2017, Qiao et al.,

393

2018).

394

A possible explanation for these results may be the consumption of GSH as

395

result of DOX-induced lipid peroxidation directly through semiquinone

396

structure or indirectly through production of ROS (Ashour et al., 2011).

397

The oxidant metabolites of doxorubicin, the semiquinone form, in presence

398

of oxygen can produce hydrogen peroxide and other oxidative free

399

radicals. In addition, another mechanism for doxorubicin-induced oxidative

400

stress is dependent on the presence of iron, where doxorubicin-iron

401

complex oxidizes oxygen to give hydrogen peroxide and other ROS

402

resulting in accumulation of MDA and MPO with the consumption of

403

antioxidant enzymes (Deavall et al., 2012). Nuclear factor erythroid-2

404

related factor 2 (Nrf2) is a master transcription factor in controlling the

405

basal and inducible expression of a battery of antioxidant genes and other

406

cytoprotective phase II detoxifying enzymes (Li et al., 2009). Kabel et al.

407

(2018) and Zhao et al. (2018) reported that, administration of DOX caused

408

21

a significant decrease in Nrf-2 level resulting in hepatotoxicity and

409

cardiotoxicity.

410

Another important finding in the present study was that, DOX increased

411

the inflammatory mediators namely; TLR4, P38 MAPK, NF-κB and TNF-

412

α in renal tissues. These results are in harmony with (Zhu et al., 2015, Min

413

et al., 2016, Yao et al., 2017). This inflammatory response can be

414

explained on the basis of oxidative stress, because free radicals produced

415

by the semiquinone form of doxorubicin after consumption of natural anti-

416

oxidant enzymes induces renal tissue injury leading to inflammatory

417

response.

418

This finding provides a new answer to what causes systemic inflammation

419

in the cancer patients receiving doxorubicin treatment. Actually , evidences

420

obtained through clinical studies or in animal experiments have proved that

421

doxorubicin caused serious systemic inflammation in vivo, including

422

hepatitis, nephritis, phlebitis, and mucositis throughout the digestive tract,

423

with an increase in the inflammatory cytokines levels in the circulation

424

(Wang et al., 2016). LPS, The heat shock proteins, ROS and chemicals

425

can bind TLR4 starting an inflammatory cascade by activating mitogen-

426

protein kinases (MAPK) that activate NFκB transcriptin factor. Finally

427

stimulation of NFκB induces protein synthesis and increases the levels of

428

the inflammatory mediators IL-1β and TNF-α (Baeza-Raja and Munoz-

429

22

Cánoves, 2004, Vyas et al., 2014).

NF-κB activation through TLR4

430

stimulation by the anticancer drug induces the expression of many

431

cytokines such as interleukin and TNF-α inducing cellular apoptosis and

432

death (Kumar et al., 2004).

433

Results of the present investigation showed that, one of the important

434

causes of doxorubicin-induced nephrotoxicity is apoptosis and activating

435

proapoptic markers as evidenced by the significant increase observed in

436

BAX expression and the significant decrease observed in Bcl-2 expression.

437

These results are in agreement with (Hoshi et al., 2017, Sun et al., 2018).

438

Apoptosis induced by DOX is considered to be a logic response for

439

inflammatory and oxidative mediators which triggers and activate pro-

440

apoptic agents. MAPK can motivate NF-κB and activate downstream

441

genes to further modulate the inflammatory responses which would

442

regulate the pathological state. Therefore, ROS production, increased

443

inflammatory cytokines and MAPK signaling, facilitate the activation of

444

NF-κB transcription factors and apoptotic genes as well (Imam et al.,

445

2018). (Park et al., 2012) reported that, the activation of p38 MAPK

446

induces apoptosis. In addition, the pro-apoptotic proteins such as BAX and

447

the anti-apoptotic proteins such as Bcl-2, and the tumor suppressor protein

448

p53 are also involved in doxorubicin-induced apoptosis. These findings

449

were in full agreement with the results of the present study that, NF-κB and

450

23

the activation of MAPK pathways are involved in the development of

451

doxorubicin-induced nephropathy. It is also clear in this study that

452

apoptotic changes in renal tissue were due to ROS mediated NF-κB

453

activation. Moreover, the generation of ROS promotes lipid peroxidation

454

which is reported to induce apoptotic changes in the cells as reported

455

previously by (Park et al., 2014) that the apoptotic changes in the cells are

456

due to loss of mitochondrial membrane integrity and p53 activation.

457

The present study proved that, nicorandil pretreatment protected against

458

doxorubicin-induced nephrotoxicity as evidenced by the decreased plasma

459

levels of urea, creatinine, KIM and cystatin. This nephroprotective

460

potential of nicorandil could be explained by its vasdilatory effect

461

enhancing the renal perfusion and excretion. Similar results suggested that

462

nicorandil ameliorates nephrotoxicity in rat (Shimizu et al., 2011, Ozturk

463

et al., 2017).

464

Results of the present investigation showed that, nicorandil significantly

465

decreased the elevated MDA content and myeloperoxidase activity in the

466

kidney, while restored the depleted GSH content and the activities of the

467

anti-oxidant enzymes Nrf-2, SOD and catalase. These results are thinkable

468

to be due to the free radical scavenger property of nicoranil and its ability

469

to neutralize ROS produced by doxorubicin. This agreed with the results of

470

(Gupta and Sharma, 2014, Ozturk et al., 2017, Zhu et al., 2018).

471

24

In this study, the anti-inflammatory effect of nicorandil was proved

472

through its ability to decrease TLR 4, p38 MAPK, NF-κB , IL-1 beta and

473

TNF–α as compared to the doxorubicin group. This is in the harmoney

474

with the results of (Heywood and Thomas, 2002, Kawamura et al., 2005,

475

Zhao et al., 2014) and could be explained by the nitric oxide donating

476

effect of nicorandil (Naito et al., 1994, Wei et al., 2003) and its effect on

477

potassium channels (Hongo et al., 2005).

478

Additionally, it has been observed in the present study that nicorandil

479

significantly ameliorated the apoptotic effect of doxorubicin through

480

decreasing Bax content and increasing Bcl-2 content in renal tissues. The

481

strong antiapoptotic effect of nicorandil is thought to be through the anti-

482

oxidative and free radical capturing ability of nicorandil besides its ability

483

to inhibit p38 MAPK pathway resulting in inhibiting all signals of cell

484

death (Yu et al., 2013).Additionally, the released NO causes vasodilatory

485

effect and inhibits inflammation due to bradykinin that is decreased in

486

various models of renal tissue apoptosis (El-Kashef, 2018). This is similar

487

to the results of (Yu et al., 2015, He et al., 2018). (Nagata et al., 2003)

488

reported that, nicorandil prevents the inflammatory cascade caused by

489

doxorubicin and thereby prevents the activation of pro-apoptotic proteins

490

and oxidative stress induced apoptosis.

491

25

Histopathological results confirmed our findings through improvement of

492

structural changes of kidney induced by doxorubicin. Where nicorandil

493

prevented the congestion of the renal blood vessels,

the perivascular

494

edema, focal fibrosis and haemorrhage in between the tubules in addition

495

to maintaining the renal tubules integrity keeping the filtration,

496

reabsorptive power and renal function.

497

From the previous results and conclusions, it could be deduced that the

498

mechanisms behind doxorubicin nephrotoxicity include oxidative stress,

499

inflammation and apoptosis. Nicorandil is a promising drug that could be

500

used concomitantly with cancer chemotherapy in order to prevent the

501

expected toxicity through its anti-oxidant, anti-inflammatory and anti-

502

apoptotic mechanisms. Furthermore, the ability of nicorandil to prevent

503

doxorubicin-induced cardiotoxicity

504

gives it a great importance and

priority in treating doxorubicin adverse effects than any other agent.

505

In conclusion

506

The present study concluded that DOX induced severe nephrotoxicity by

507

promoting oxidative, inflammatory and apoptotic mechanisms. Nicroandil

508

attenuated DOX-induced apoptosis and inflammation through suppression

509

of oxidative stress mediated activation of TLR4/MAPKp38/NF-κB

510

signaling pathways. Therefore, it is advisable to be used concomitantly

511

with DOX to reduce its nephrotoxicity.

512

26

Acknowledgments:

513

The authors would like to thank Prof. Dr. A. Bakear (Pathology

514

Department, Faculty of Veterinary Medicine, Cairo University, Cairo,

515

Egypt) for assistance in the histopathological examinations.

516

Disclosure of Conflict of interest

517

The authors have read the journal's policy on disclosure of potential

518

conflicts of interest and they all declare no personal or financial conflict of

519

interest.

520

Authorship Statement

521

All authors have read the journal’s authorship statement and agree to it.

522

5. References

523

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696 697 698 699 700 701 702

31

Table1: Table1: Effect of Nicorandil on serum levels of urea, creatinine, total protein, albumin, albumin, Kidney injury moleculemolecule-1 and cystatin in doxorubicindoxorubicin-induced nephrotoxicity in rats.

Group

Control saline Nicorandil Doxorubicin Nicorandil + Doxorubicin

Serum urea

Serum

Serum total

Serum albumin

Kidney injury

Serum cystatin

(mg/dl)

creatinine

protein

g/dl

moleculemolecule-1

Unit

(mg/dl)

g/dl

pg/ml

pg/ml

33.17 ± 1.9

0.18 ± 0.013

5.59 ± 0.15

3.48 ± 0.25

2.2 ± 0.09

0.66 ± 0.05

30.33 ± 1.35b

0.45 ± 0.01b

5.51 ± 0.14b

3.5 ± 0.34b

2.13 ± 0.1b

0.63 ± 0.06 b

83.83 ± 2.79a

1.64 ± 0.12 a

3.77 ± 0.06a

2.4 ± 0.22a

12.17 ± 0.35a

2.862 ± 0.11a

58.83 ± 2.13ab

0.70 ± 0.07ab

4.98 ± 0.03ab

3.3 ± 0.23ab

5.65 ± 0.37ab

1.23 ± 0.17ab

1

Table 2: Effect of Nicorandil on histopathological alterations in doxorubicin-induced nephrotoxicity in rats.

Group

Control saline

Nicorandil

Doxorubicin

Doxorubicin + nicorandil

Histopathlgical alteration Tubular degeneration

_

_

++

_

Focal inflammatory cell infiltration

_

_

++

+

Focal fibrosis

_

_

++

_

Vaculisation of glomerular endothelium

_

_

+++

_

Congestion in blood vessels

_

_

++

_

Perivascular edema

_

_

++

Focal fibrosis

_

_

++

_

Focal haemorrhage

_

_

++

_

Degeneration in the tubules

_

_

++

_

+++ severe ++ moderate + mild 0 none 2

Statistical analysis was carried out by one way ANOVA followed by Tukey- Multiple Comparison Test. Each value represents the standard deviation of 8 rats (S.E.). .a Significantly different from normal control group value at p < 0.05 .b Significantly different from doxorubicin group value at p < 0.05

3

8

a

40

4

a

a 60 40

ab 20

0

0

b

1.5

ab

b

ab

100

100

b

Catalase ng/ g tissue

a

b

0

150

150

MPO ng/ g tissue

ab

2

20

0

6

MDA nmol/g tissue

ab

60

80

a 50

0

Nrf2 pg/ g tissue

GSH mg/g tissue

80

50

b

b

SOD Pg/ g tissue

100

1.0

ab 0.5

a 0.0

Groups

Figure 1 A-F : Effect of Nicorandil on renal content of GSH, MDA, SOD, MPO, Nrf2 and catalase in doxorubicin-induced nephrotoxicity in rats. Each value represents the mean of 8-10 rats ± standard error of the mean (SE.). Statistical analysis was carried out by one way ANOVA followed by Tukey Multiple Comparison Test. a b

Significantly different from normal control group value at p < 0.05. Significantly different from doxorubicin group value at p < 0.05.

nrf2 b actin

b

2 0

ab 4

b

2

a

100

100

ab

0

b

2

150

a

50

ab

4

0

0

150

IL.1B pg/ g tissue

P38 ng/ g tissue

ab

4

a

6

6

TNF Pg/ g tissue

NFĸb Pg/ g tissue

a 6

8

a

8

TLR4 ng/ g tissue

8

b

ab b

50

NFĸb P38

0

TLR4 b actin Groups

Figure 2A-D : Effect of Nicorandil on renal content of NFKb, P38 MAPK, TLR4, IL-1β and TNF-α in doxorubicininduced nephrotoxicity in rats. Each value represents the mean of 8-10 rats ± standard error of the mean (SE.). Statistical analysis was carried out by one way ANOVA followed by Tukey Multiple Comparison Test. a b

Significantly different from normal control group value at p < 0.05. Significantly different from doxorubicin group value at p < 0.05.

Bax pg/ g tissue

6

ab 4 2

b

0

BcL-2 pg/ g tissue

a

a

b

30

1.0

ab 0.5

a

Bax /Bcl-2 ratio

1.5

8

20

10

ab b

0

0.0

Bax BcL-2

Groups

b actin

b actin

Figure (3A-C) : Effect of Nicorandil on renal content of Bax and Bcl in doxorubicin-induced nephrotoxicity in rats. Each value represents the mean of 8-10 rats ± standard error of the mean (SE.). Statistical analysis was carried out by one way ANOVA followed by Tukey Multiple Comparison Test. a b

Significantly different from normal control group value at p < 0.05. Significantly different from doxorubicin group value at p < 0.05.

g

Figure 4: Effect of Nicorandil on kidney sections in doxorubicin-induced nephrotoxicity in rats stained with hematoxylin/eosin (H/E) and examined under the light microscope. Doxorubicin treated rats showed degenerative changes; focal inflammatory cells infiltration (DOX 1), The endothelial cells lining the tufts of the glomeruli showed vacuolization (DOX 2), The cortical stromal blood vessels showed congestion (DOX 3) , as well as perivascular oedema (DOX 4), The corticomedullary portion showed focal fibrosis (DOX 5), and focal haemorrhages in between the tubules (DOX 6), There were swelling in the lining tubular epithelium with obliteration in the tubular lumen (DOX 7), while other

tubules at the corticomedullary portion had vacuolar degeneration in the lining epithelium (DOX 8), There was no histopathological alteration as recorded in (nicorandil only) , Focal inflammatory cells infiltration was detected in between the degenerated tubules at the cortex (nicorandil + DOX).

• Nicorandil ameliorated doxorubicin-induced nephrotoxicity.

1

• Nicorandil restored the oxidant/antioxidant balance.

2

• Nicorandil suppressed inflammatory signaling pathway TLR4/MAPK

3

P38/NF-κb/TNF-α.

4

• Nicorandil regulated BAX/Bcl-2 apoptotic pathway

1

5

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: