Effects of Neuromuscular Electrical Stimulation During Hemodialysis on Peripheral Muscle Strength and Exercise Capacity

Accepted Manuscript Effects of neuromuscular electrical stimulation during hemodialysis on peripheral muscle strength and exercise capacity: a randomized clinical trial Ana Karla Vieira Brüggemann, Carolina Luana Mello, Tarcila Dal Pont, Deborah de Camargo Hizume Kunzler, Daniel Fernandes Martins, Franciane Bobinski, Wellington Pereira Yamaguti, Elaine Paulin PII:

S0003-9993(17)30009-6

DOI:

10.1016/j.apmr.2016.12.009

Reference:

YAPMR 56766

To appear in:

ARCHIVES OF PHYSICAL MEDICINE AND REHABILITATION

Received Date: 19 September 2016 Revised Date:

13 December 2016

Accepted Date: 15 December 2016

Please cite this article as: Brüggemann AKV, Mello CL, Pont TD, Kunzler DdCH, Martins DF, Bobinski F, Yamaguti WP, Paulin E, Effects of neuromuscular electrical stimulation during hemodialysis on peripheral muscle strength and exercise capacity: a randomized clinical trial, ARCHIVES OF PHYSICAL MEDICINE AND REHABILITATION (2017), doi: 10.1016/j.apmr.2016.12.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

ACCEPTED MANUSCRIPT Journal: ARCHIVES OF PHYSICAL MEDICINE AND REHABILITATION

RI PT

Neuromuscular electrical stimulation during hemodialysis

Effects of neuromuscular electrical stimulation during hemodialysis on peripheral muscle strength and exercise capacity: a randomized clinical

M AN U

SC

trial

Authors:

1

Ana Karla Vieira Brüggemann, 1Carolina Luana Mello, 1Tarcila Dal Pont, Deborah de Camargo Hizume Kunzler, 3Daniel Fernandes Martins, 4Franciane

TE D

2

EP

Bobinski, 5Wellington Pereira Yamaguti, 6Elaine Paulin

1

AC C

Titles and Institutions:

Master´s Degree Student in Physiotherapy at Santa Catarina State University –

UDESC. Florianopolis, SC – Brazil.

2

Phd in Medical Sciences from the University of São Paulo, São Paulo, SP –

Brazil, and Professor at Santa Catarina State University- UDESC, Florianopolis, SC – Brazil.

ACCEPTED MANUSCRIPT 3

Phd in Neurosciences from the Federal University of

Santa Catarina,

Florianopolis, SC – Brazil, and Professor at the University of Southern Santa Catarina- UNISUL, Florianopolis, SC – Brazil.

4

RI PT

Phd in Neurosciences from the Federal University of Santa Catarina,

Florianopolis, SC – Brazil, and Professor of the Undergraduate Program at

SC

Santa Catarina State University - UDESC, Florianopolis, SC – Brazil.

5

Phd in Experimental Physiopathology from the Medical School of the University

M AN U

of São Paulo, São Paulo, SP – Brazil, and Professor of the Master´s Program in Health Sciences at Sírio-Libanês Hospital.

6

Phd in Sciences from the University of São Paulo, São Paulo, SP – Brazil, and

TE D

Professor of Undergraduate and Graduate Physiotherapy Courses at Santa Catarina State University– UDESC, Florianopolis, SC – Brazil.

EP

Financial Support: CAPES.

AC C

No conflict of interest to declare.

Corresponding Author:

Ana Karla Vieira Brüggemann Centro de Ciências da Saúde e do Esporte – CEFID/UDESC Rua Pascoal Simone, 358 - Coqueiros - Florianópolis – SC CEP: 88080-350

ACCEPTED MANUSCRIPT Laboratório de Fisioterapia Respiratória – LAFIR E-mail: [email protected] Telephone: +55 (48) 3664-8602

RI PT

This study has been approved by the Research Ethics Committee of UDESC, under the Protocol number 08857612.2.0000.0118 as well as by the Clinical

AC C

EP

TE D

M AN U

SC

Trials.gov Registry (protocol number NCT02786849).

ACCEPTED MANUSCRIPT

1

Effects of neuromuscular electrical stimulation during hemodialysis on peripheral

2

muscle strength and exercise capacity: a randomized clinical trial

3 Key words: Chronic Renal Failure; Renal Dialysis; Electrical Stimulation; Muscle Strength;

5

Muscle Weakness.

RI PT

4

6 7

Abstract

SC

8 9

Objective: To evaluate the effects of neuromuscular electrical stimulation of high and low

11

frequency and intensity, performed during hemodialysis (HD), on physical function and

12

inflammation markers in patients with chronic kidney disease (CKD).

13

16 17

TE D

15

Design: Randomized clinical trial

Setting: Hemodialysis clinic.

EP

14

M AN U

10

Participants: 51 CKD patients were randomized into blocks of four by means of opaque

19

envelopes. They were divided into a group of high frequency and intensity neuromuscular

20

electrical stimulation (HG) and a group of low frequency and intensity neuromuscular

21

electrical stimulation (LG).

22

AC C

18

23

Intervention: HG was submitted to neuromuscular electrical stimulation with 50Hz and

24

medium intensity of 72.90mA, and LG used 5Hz and medium intensity of 13.85mA, 3 times

25

per week for one hour, during 12 sessions.

26 1

ACCEPTED MANUSCRIPT

27

Main Outcome Measures: Peripheral muscle strength, exercise capacity, levels of muscle

28

trophism marker [growth factor similar to insulin type 1 (IGF-1)] and levels of pro-

29

inflammatory [tumor necrosis factor (TNF-alpha)] and anti-inflammatory [Interleukin 10 –

30

(IL-10)] cytokines.

RI PT

31 Results: HG showed significant increase in right peripheral muscle strength

33

(155.35±65.32Nm versus 161.60±68.73Nm; p=0.01) and left peripheral muscle strength

34

(156.60±66.51Nm versus 164.10±69.76Nm, p=0.02) after training, which did not occur with

35

LG for both right muscle strength (109.40±32.08Nm versus 112.65±38.44Nm, p=0.50) and

36

left muscle strength (113.65±37.79Nm versus 116.15±43.01Nm; p=0.61). The distance of

37

the 6-minute walk test (6MWTD) increased in both groups: HG (435.55±95.81m versus

38

457.25±90.64m; p=0.02) and LG (403.80±90.56m versus 428.90±87.42m, p=0.007). The

39

groups did not differ in peripheral muscle strength and in the 6MWTD after the training

40

protocol. In HG, a correlation was observed between initial and final values for 6MWTD

41

and muscle strength. In LG, correlations occurred only between the 6MWTD and the initial

42

muscle strength. Only LG increased levels of IGF-1 (252.38±156.35pg/ml versus

43

336.97±207.34 pg/ml; p=0.03) and only HG reduced levels of IL-10 (7.26±1.81 pg/ml

44

versus 6.32±1.54 pg/ml; p=0.03). The groups showed no differences in the TNF-alpha

45

concentrations.

M AN U

TE D

EP

AC C

46

SC

32

47

Conclusion: CKD patients on HD improve exercise capacity after peripheral

48

neuromuscular electrical stimulation of high and low frequency and intensity. However, the

49

benefits on muscle and inflammatory outcomes seem to be specific for the adopted

50

strategy.

51

2

ACCEPTED MANUSCRIPT

List of Abbreviations: ANCOVA (analysis of covariance), ANOVA (analysis of variance),

53

BMI (body mass index), CKD (Chronic kidney disease), COPD (chronic obstructive

54

pulmonary disease), ELISA (Enzyme Linked Assay Immunosorbent), FEV1 (forced

55

expiratory volume in one second), FEV1/FVC ( ratio forced expiratory volume in one

56

second and forced vital capacity), FVC (forced vital capacity), HD (hemodialysis), GFR

57

(glomerular filtration rate), HF (heart failure), HG (high frequency and intensity

58

neuromuscular electrical stimulation group), HR (heart rate),

59

Hz (Hertz), ICD (Informed Consent Document), IGF-1 (Insulin Growth Factor 1), Kt/V (urea

60

clearance), IL-10 (interleukin-10), LG (low frequency and intensity neuromuscular electrical

61

stimulation group), LMS (left muscle strength), m (meter), pg/mL (picograms per mL), Nm

62

(Newton-meter), RMS (right muscle strength), s (seconds), SpO2 (oxygen saturation by

63

pulse oximetry), TNF-alpha (tumor necrosis factor), VAS (visual analogue scale), 6MWT

64

(6-minute walk test), 6MWTD (6-minute walk test distance), µs (microseconds).

M AN U

SC

RI PT

52

AC C

EP

TE D

65

3

66

ACCEPTED MANUSCRIPT

Chronic kidney disease (CKD) is a worldwide public health problem, which is associated with chronic inflammation and progressive loss of weight, peripheral muscle

68

strength and the ability to exercise1-4, and these changes are more pronounced in patients

69

on hemodialysis (HD)5-6. Recently, the terms “uremic sarcopenia”7 or “uremic muscle

70

dysfunction”8 have been used to describe the progressive and cumulative loss of muscle

71

mass and strength in CKD patients, which can be detected in the first stage of CKD In this

72

process of progression of renal injury, the role of inflammatory mechanisms, as mediators

73

of TNF-alpha and IL-6, has been increasingly demonstrated, as well as their role on

74

muscle catabolism, which leads to muscle wasting, and justifies the importance of the

75

study of adequate and specific CKD therapies9,10.

SC

M AN U

76

RI PT

67

Rehabilitation therapies through physical exercise have been introduced to this population11, with the aim to reduce muscle weakness, improve exercise capacity, and

78

decrease the risk of developing cardiovascular disease resulting from CKD. However, the

79

general health condition of CKD patients is acknowledged as being limited and aerobic

80

exercises may offer hemodynamic risks and acute cardiovascular complications12. This

81

shows the importance of introducing alternative rehabilitation programs with lower clinical

82

implications, such as neuromuscular electrical stimulation13. This neuromuscular

83

electrostimulation technique has been commonly used in patients with chronic obstructive

84

pulmonary disease (COPD) and heart failure (HF) in outpatient and inpatient basis. This

85

demonstrates beneficial results to maintain and increase mass, peripheral muscle strength

86

and exercise capacity14-17.

88

EP

AC C

87

TE D

77

Few studies have investigated the effects of neuromuscular electrical stimulation on

89

functional variables in CKD patients. A randomized study conducted by Dobsak et al.

90

(2012)13 showed positive effects of electrical stimulation on quadriceps and calf, with 10Hz

91

on peripheral muscle strength and exercise capacity in chronic renal failure patients during 4

ACCEPTED MANUSCRIPT

hemodialysis. However, the authors did not measure biochemical variables, such as

93

pro- and anti-inflammatory cytokines and muscle trophism markers, in order to

94

better understand the mechanisms involved in the improvement of the patients after

95

the training. In the pre-clinical field, a study in rats with nephrectomy-induced CKD18

96

showed that neuromuscular electrical stimulation of 20Hz minimized skeletal muscle

97

atrophy by improving muscle regeneration capacity and muscle proteins

98

metabolism.

RI PT

92

100

SC

99

Considering the peripheral muscle dysfunction among CKD patients, which affects both muscle composition (inflammatory mediators and muscle trophism) and

102

exercise capacity2,18,19, the effects of a 4-week training program with neuromuscular

103

electrical stimulation in CKD patients during hemodialysis should be further

104

investigated. Studies on neuromuscular electrical stimulation in CKD patients

105

available in the scientific literature use low frequency therapeutic strategies only.

106

Therefore, this study aimed at evaluating the effects of neuromuscular electrical

107

stimulation of high and low frequency and intensity on peripheral muscle strength,

108

exercise capacity, as well as biochemical markers of muscle changes and

109

inflammation in CKD patients during hemodialysis. The hypothesis of this study is

110

that high frequency and intensity training will bring more benefits for CKD patients.

112 113

TE D

EP

AC C

111

M AN U

101

Methods

114 115 116 117

This is a randomized, double-blind clinical trial with CKD patients who underwent hemodialysis. They were divided into two groups: patients who were 5

ACCEPTED MANUSCRIPT

submitted to high frequency and intensity neuromuscular electrical stimulation (HG), and

119

patients who were submitted to low frequency and intensity neuromuscular electrical

120

stimulation (LG). The patients were blind to group allocation. Randomization was stratified

121

according to sex, using blocks size of 4, and performed through opaque sealed envelope,

122

after the assessments. The groups completed a four-week neuromuscular electrical

123

stimulation training program. Drug therapy established for both groups remained

124

unchanged throughout the study period.

RI PT

118

126

SC

125

This study was approved by the Ethics Committee for Research Involving Human Beings of Santa Catarina State University – UDESC – (CAEE: 45904615.7.0000.0118), as

128

well as registered into the ClinicalTrials.gov (NCT02786849 registration number). The

129

training was conducted at the APAR VIDA Kidney Clinic in São José/SC and evaluations

130

were conducted at the Faculty of Sport and Health Sciences of Santa Catarina State

131

University (CEFID/UDESC) from July 2015 to May 2016.

133

TE D

132

M AN U

127

The baseline and final assessments were conducted on the first working day after hemodialysis. The final assessment was performed after 12 sessions of the training

135

protocol. At first, anthropometric measurements and vital signs were taken from all

136

patients, followed by assessment of peripheral muscle strength, pulmonary function test

137

and submaximal exercise capacity through the 6-minute walk test (6MWT). Furthermore,

138

CKD patients on hemodialysis were requested to present their latest blood tests including

139

urea clearance (Kt/V), creatinine and glomerular filtration rate. The assessment of

140

enzymatic markers of muscle trophism and of pro- and anti-inflammatory plasma cytokines

141

was carried out before and after the patients´ training protocol (Figure 1).

AC C

EP

134

142 143

The consecutive sampling method was used to recruit, select and assess 51 6

ACCEPTED MANUSCRIPT

144

patients of both sexes, undergoing hemodialysis treatment. Twenty- six patients

145

were allocated to HG, and 25 patients to LG. The inclusion and exclusion criteria

146

are in the supplementary material.

147 Inclusion criteria were as follows: (1) CKD patients undergoing hemodialysis

RI PT

148

regularly for at least 6 months (2) CKD patients aged 20 to 85 years, (3) CKD

150

patients in stable clinical condition and under medical supervision; (4) absence of

151

uncontrolled hypertension, recent ischemic heart disease (3 months or less),

152

unstable angina or severe cardiac arrhythmias; (5) absence of diseases

153

(respiratory, orthopedic and/ or neurological), which might limit the assessment

154

protocol and training; (6) patients who did not perform any form of physical exercise

155

or who exercised over six months ago.

M AN U

SC

149

156 157

Exclusion criteria were as follows: (1) inability to perform any of the study assessments (lack of understanding or cooperation); (2) clinical deterioration during

159

the research period; (3) clinical complications due to cardiorespiratory and/or

160

musculoskeletal reasons during the research period.

163 164 165

EP

162

The participants were informed of the procedures and objectives of the study

AC C

161

TE D

158

and signed an Informed Consent Document (ICD).

Intervention protocol

166 167

Isometric strength training on quadriceps muscle was performed with electrical

168

stimulation during one hour, 3 times a week. The participants were not instructed to

169

perform isometric voluntary contractions associated to NMES. The interventions were 7

ACCEPTED MANUSCRIPT

170

carried out in the first two hours of hemodialysis, in order to avoid physical stress in the

171

second half of the session, when the hemodynamic conditions of patients are unfavorable.

172

The patients chose to remain sitting or in dorsal decubitus on a chair during the

173

hemodialysis session, with their lower limbs extended on a footrest.

175

RI PT

174 Stimulation was applied using portable dual-channel muscle stimulator (Carci Fesmed IV, São Paulo, BR), with self-adhesive surface electrodes placed on the vastus

177

lateralis muscle (same direction of muscle fibers, one positioned 3 cm above the superior

178

border of the patella, and another 5 cm below the inguinal crease towards the anterior

179

superior iliac crest), and on the vastus medialis muscle (same direction of the muscle

180

fibers, one positioned 3 cm above the upper border of the patella, and another 5 cm below

181

the inguinal fold, obliquely, towards the groin).

M AN U

SC

176

182 183

The parameters for HG included: frequency of 50 Hertz (Hz), pulse width of 400 microseconds (µs), rise time and fall time of 2 seconds (s), on:off stimulation, initially with a

185

1:2 relation in the first week (10s of stimulation and 20s of rest, with the objective to adapt

186

and minimize the effects of muscular fatigue), to be increased to a 1:1 relation in the

187

second week (10s of stimulation of and 10s of rest). For LG, frequency of 5 Hz was used,

188

on:off stimulation time of 1:3 (10s of stimulation and 30s of rest) with pulse duration of

189

100µs. The current intensity was adjusted individually in the first application. In HG, the

190

intensity was gradually increased until visible muscle contraction could be observed: this

191

was the maximum intensity tolerated by the patient. In LG, the intensity was the least

192

noticeable by the patient. These values were monitored for further calculation of intensity

193

evolution throughout the training program.

AC C

EP

TE D

184

194

8

195

ACCEPTED MANUSCRIPT

Each session included a warm-up period, training and cool down period for both groups. The first minute of the warm-up period started with 20% of the intensity value

197

adopted in the previous session, with a gradual increase of 20% per minute until 5

198

minutes. Next, a 50-minute training session was performed, followed by a 5-minute

199

recovery period with gradual reduction of 20% of intensity each minute. The training

200

program was implemented by one physiotherapist.

RI PT

196

201

Before and after the training program, the patients were asked about their sensation

203

of fatigue/tiredness in the lower limbs, as well as sensation of dyspnea, using the modified

204

Borg scale. In the event of muscle pain, the intensity was assessed by visual analogue

205

scale (VAS). Blood pressure was measured in the beginning, after 30 minutes of training,

206

and at the end of the session. An oximeter (Rossmax - SB100 Fingertip, Taipei, Taiwan)

207

was used to measure heart rate (HR) every 10 minutes.

M AN U

SC

202

210 211

Assessments

To characterize the sample, age, sex, height, body mass, body mass index (BMI),

EP

209

TE D

208

urea clearance, creatinine, glomerular filtration rate and lung function were considered.

213

Peripheral muscle strength and submaximal exercise capacity were included into the main

214

objective of this study. Biochemical markers of muscle trophism and inflammation-induced

215

changes were the secondary objectives of this study. All assessments were performed by

216

the same examiner blinded for the patients' group allocation.

AC C

212

217 218

The right (RMS) and left (LMS) muscle strength were assessed using an isokinetic

219

dynamometer (Biodex System 4 Pro, New York, USA) in isometric mode. To measure the

220

knee extensors muscle strength, the patient was placed on a chair with hip flexion of 85º, 9

ACCEPTED MANUSCRIPT

and the lateral femoral condyle was aligned with the rotation axis. The trunk, hip and

222

evaluated lower limb were stabilized using belts to avoid compensation. The weight of the

223

limb under examination was calculated to correct the gravity and the maximum and

224

minimum angles were recorded. Following the familiarization with the equipment, the

225

patient performed five maximal voluntary isometric contractions. From the position at 60°

226

of knee flexion20,21, considering 0º of total length of the knee, the contraction should be

227

maintained for 5 seconds with a 75-second rest interval between each contraction. The

228

highest peak extensor torque value was recorded for further analysis.

SC

RI PT

221

229

Pulmonary function testing was performed with the NDD EasyOne portable digital

231

spirometer (Zurich, Switzerland), previously calibrated, in accordance with the guidelines

232

and criteria recommended by the American Thoracic Society and European Respiratory

233

Society22. The following parameters were obtained: forced vital capacity (FVC), forced

234

expiratory volume in one second (FEV1) and the FEV1/FVC ratio expressed in absolute

235

values and percentage of predicted normal values (FVC and FEV1 ≥ 80% of predicted and

236

FEV1/FVC≥0.7), according to the values determined by Pereira et al. (2007)23. In patients

237

with values lower than normal, salbutamol (400µg) inhaled bronchodilator was

238

administered, and the test was repeated 15 minutes later.

240

TE D

EP

AC C

239

M AN U

230

To assess the submaximal exercise capacity, the six-minute walk test was

241

performed, according to the guidelines of the American Thoracic Society24. The patients

242

were instructed to walk as far as possible over a period of 6 minutes in a 30-meter track.

243

They were motivated with standard phrases of encouragement every minute. If the

244

patients had limiting dyspnea or any other disabling discomfort, they could interrupt the

245

test without interrupting the execution time. Blood pressure was monitored before and after

246

each test, as well as HR, oxygen saturation by pulse oximetry (SpO2) and the sensation of 10

ACCEPTED MANUSCRIPT

247

dyspnea (modified Borg scale) during the test. The greatest distance between the two

248

tests was used for the analysis, and the reference values were those described by Britto et

249

al. (2013)25.

250 Assessments of urea clearance, creatinine, glomerular filtration rate and

RI PT

251

biochemical markers were carried out at the outpatient clinic, in the monthly blood

253

collection during hemodialysis. At first, the biochemical markers were assessed before

254

starting the protocol, and then, after 12 training sessions. Both the muscle trophism

255

marker [growth factor similar to insulin type 1 (IGF-1)] and the pro-inflammatory [tumor

256

necrosis factor (TNF-alpha)] and anti-inflammatory cytokines [interleukin-10 (IL-10)] were

257

analyzed using plasma of CKD patients during HD, initially, before starting the protocol

258

and after 12 sessions of training. Samples of 4 mL of blood were taken from existing

259

fistulas in patients; therefore, there was no need for additional invasive procedure for blood

260

collection. The blood was then stored in a vacutainer previously treated with lithium

261

heparin, and then centrifuged for 7 minutes at 400×g, in order to separate the plasma.

262

Then, aliquots of 350 µL were stored in a freezer at -80ºC, in order to measure the

263

markers to be studied (from each patient, in each period), using the appropriate Sandwich

264

ELISA (Enzyme Linked Assay Immunosorbent) kits (DuoSet ® ELISA, R & D Systems,

265

Minneapolis, MN, USA). For IGF-1, the Human IGF-1 Catalogue # DY291 with sensitive

266

range of 31.3 – 2000 picograms per mL (pg/mL) was used. For IL-10, the IL-10 Human

267

Catalogue # DY217B was used, with sensitivity range of 31.3 – 2000 pg/mL. And for TNF-

268

alpha, the Human TNF-alpha Catalogue #DY210 with a sensitivity range of 15.6 –1000

269

pg/mL was used.

AC C

EP

TE D

M AN U

SC

252

270 271

Statistical analysis

272 11

273

ACCEPTED MANUSCRIPT

An a priori sample size calculation was performed using the GPower 3.1 software based on the results of the study conducted by Dobsak et al. (2012)13. The peripheral

275

muscle strength values, which were obtained from the group that underwent electrical

276

stimulation before (185.4±53Nm) and after (222.4±36.6Nm) the treatment were used for

277

the calculation. The exercise capacity assessment values before (401.3±55m) and after

278

(428.9±69.2m) electrical stimulation were also used, where a similar effect was expected

279

to be observed. The significance level was set at 5% (80% test power), and therefore

280

there was a need to include 12 patients to respond the variable peripheral muscle

281

strength, and 34 patients for exercise capacity. Considering a 10% loss rate, 14 patients

282

would be needed to verify the effect on peripheral muscle strength, and 38 patients for the

283

effects on exercise capacity.

284

M AN U

SC

RI PT

274

The data were entered into the IBM SPSS (Statistical Package for Social Sciences)

286

version 20.0. The data were presented as mean ± standard deviation and 95% confidence

287

interval (CI95%). The Shapiro-Wilk test was applied to verify the data normality. The

288

Independent Sample t-Test was used to compare the behavior of the parametric variables

289

of age, HR, FEV1, FVC, FEV1/FVC, LMS and RMS, 6MWT, IGF-1, TNF-alpha and IL-10

290

between the groups, whereas the Mann-Whitney U test was used for nonparametric

291

variables of BMI, Kt/V, creatinine, GFR, dyspnea, fatigue and intensity. The repeated

292

measures ANOVA test was used to compare means before and after the use of electrical

293

stimulation. The categorical variable was analyzed by Chi-square test. The Pearson

294

correlation coefficient was used to verify the presence of a relationship between

295

respiratory muscle strength and exercise capacity. A linear regression analysis was

296

conducted to obtain the determination coefficient. The ANCOVA analysis was used in

297

order to reduce the variance between groups for the muscle strength variable, and

298

consequently, to adjust their mean values: Group (HG vs. LG) was the independent

AC C

EP

TE D

285

12

ACCEPTED MANUSCRIPT

299

variable, the initial left and right muscle strengths were the covariates, and the final right

300

and left muscle strengths were the dependent variable. The significance level for statistical

301

purposes was set at 5% (p<0.05).

302 The magnitude of the correlations was described according to Dancey and Reidy

RI PT

303 304

(2006)26, who consider the correlation coefficients between 0.10 to 0.30 as “weak”; 0.40 to

305

0.60 as “moderate”; and 0.70 to 1 as “strong”.

SC

306 307 Results

M AN U

308 309 310 311

A total of 51 patients were eligible for the study: 26 patients were randomized for HG; 25 for LG. Six and five patients were excluded from HG and LG, respectively. Thus,

313

40 patients completed the study. The exclusion criteria are shown in Figure 1.

TE D

312

314

The power analysis of a sample of 20 participants per group was calculated by a

EP

315

post hoc sample calculation, using the t-test in GPower 3.1 software. The RMS and LMS

317

mean values and 6MWTD before and after the training in both groups (HG and LG), with

318

their respective standard deviations, were also determined. In all analyses, the

319

significance level was set at 5%. In HG, for the right muscle strength, the effect value

320

calculation was 2 and the power for the sample was 1.0. For the left muscle strength,

321

effect was 2.33 and power was 1.0; and for 6MWD, effect was 4.2, producing power of 1.0.

322

In LG, for the right muscle strength, effect was 0.5 and power was 0.7; for the left muscle

323

strength, effect was 0.5 and power was 0.7; and for the 6MWT, the calculated effect was

324

7.9 and power was 1.0.

AC C

316

13

ACCEPTED MANUSCRIPT

325 326 327

Insert Figure 1 here

328

330

RI PT

329 In the baseline assessment, anthropometric and pulmonary variables, 6MWTD, Kt/V concentrations, creatinine, IGF-1, IL-10 and TNF-alpha (Table 1) showed no significant

332

differences between the groups. As for lung function, the mean values for both groups

333

remained within the normal range. GFR was significantly lower in LG; however, despite

334

such difference, the mean in both groups was within the range of classification compatible

335

with stage 5 CKD (GFR<15 mL/min/1.73m2), according to the Brazilian CKD Guidelines27.

336

For the RMS and LMS variables, the patients showed baseline differences between both

337

groups. This was an error by chance, and the analysis of covariance was used to adjust

338

these values by for post-training comparisons.

342 343 344

M AN U

TE D

341

Insert Table 1 here 1

EP

340

AC C

339

SC

331

HG showed a significant increase in right muscle strength (RMS) and left muscle

345

strength (LMS) after the training, which did not occur with LG for both the RMS and LMS

346

(Figures 2A and 2B). The ANCOVA analysis showed that the groups did not differ in mean

347

values of RMS and LMS after the training, even after adjustments for pre-training scores,

348

which demonstrated that HG had mean levels of RMS and LMS similar to LG, with

349

adjusted pre-training values. In the 6MWT, the 6MWTD increased in both groups.

350

However, the comparison between both groups showed no differences (Figure 2C). 14

ACCEPTED MANUSCRIPT

351 352 353

Insert Figure 2 here

354

356

RI PT

355 In HG, a strong correlation was observed between the initial RMS and 6MWTD, as well as a moderate correlation between the initial LMS and 6MWTD. In LG, these

358

correlations also occurred moderately between the 6MWTD and the initial RMS and initial

359

LMS. Right after the training, only HG presented correlations between the 6MWTD and the

360

RMS and LMS. LG showed no significant correlations after the training.

M AN U

SC

357

361 362 363

A moderate to strong correlation between age and initial e final muscle strength was observed only in HG. LG showed no relationship between age and muscle strength.

364

367

TE D

366

Correlation results are listed in the Supplementary Material.

Not all patients participated in the biochemical analysis because of the training

EP

365

protocol period. For concentrations of IGF-1, 11 patients were analyzed in HG and 10 in

369

LG. For IL-10 and TNF-alpha, 10 patients were analyzed from each group. The intra-group

370

results showed increased IGF-1 levels in LG only (Figure 3A). In the IL-10 analysis, HG

371

showed significantly reduced levels and LG showed no alterations (Figure 3B). Ultimately,

372

TNF-alpha remained the same in both groups (Figure 3C). No differences were found

373

between both groups for evaluated concentrations of growth factors and cytokines.

AC C

368

374 375 376

Further data from the training protocol effect, variables evaluated and analyzed correlations are in the supplementary material. 15

ACCEPTED MANUSCRIPT

377 378 379

Insert Figure 3 here

380

382

RI PT

381 Discussion

383

SC

384

This study demonstrated that CKD patients on hemodialysis, undergoing a high-frequency

386

and high intensity neuromuscular electrical stimulation therapy, as well as those submitted

387

to low frequency and low intensity therapy showed increased exercise capacity. However,

388

only HG showed improved peripheral muscle strength.

M AN U

385

389

A relationship was found between improved peripheral muscle strength and increased

391

6MWTD only in HG. This suggests that increased exercise capacity in LG occurred due to

392

other mechanisms. Importantly, the peripheral muscle strength was proportional to the

393

frequency of stimulation and the number of motor units recruited. Thus, the higher the

394

frequency, the greater the muscle torque and the motor recruitment will be, producing an

395

increase in strength28,29. Furthermore, low frequency may have stimulated muscle

396

endurance fibers, which explains the increased exercise capacity among the patients of

397

this group30. Additionally, a study with HF patients31 observed that a frequency below 20Hz

398

directs its activity to muscle endurance (type I fibers), reducing muscular fatigue.

399

Stimulation within this frequency range increases oxidative aerobic capacity of type I fiber,

400

improves peripheral vasodilation, and consequently leads to increased vascularization,

401

which would explain the increase in exercise tolerance in LG17.

AC C

EP

TE D

390

402 16

ACCEPTED MANUSCRIPT

In CKD, training with neuromuscular electrical stimulation has been little explored, when

404

the objective of the assessment (biochemical, functional or related to exercise capacity)

405

was to observe the effects on peripheral muscle32. The study conducted by Dobsak et al.

406

(2012) was the only study found in the literature that observed the effects of functional

407

variables in this population, but without studying biochemical markers13. In that study, the

408

authors compared the effect of aerobic training on a cycle ergometer and electrical

409

stimulation with parameters of a low-frequency therapy (10Hz, mode on:off 20:20s, pulse

410

width of 200µs for 60 minutes at quadriceps and calf - increased intensity to the maximum

411

final value of 60mA) with a control group, who had received no intervention. Training

412

sessions were conducted between the 2nd-3rd hours of HD three times per week, for a

413

total of 20 weeks. The authors found a significant increase in maximum quadriceps muscle

414

strength in the intervention group (electro stimulation and aerobic training), as well as in

415

6MWTD. There was an increase of 27.6 meters in the electrical stimulation group, with no

416

differences, when compared to aerobic training. In that study, even without a sham group,

417

patients did not report pain or discomfort, which suggests that low frequency during a 20-

418

week training program may be applicable and improve peripheral muscle strength as well

419

as the functional capacity of patients on hemodialysis. The current parameters used by the

420

authors are somewhat higher than those applied to LG in this study, which improved only

421

the exercise capacity with an average increase of 25.10meters after 4 weeks of treatment,

422

without increased peripheral muscle strength. Importantly, the final average intensity of LG

423

was 48.45mA, because the low intensity value was set based on the minimum perception

424

of the current reported by the patient.

AC C

EP

TE D

M AN U

SC

RI PT

403

425 426

Results that corroborate the findings of our study were observed in other populations, such

427

as in COPD33 and HF31 patients. These studies pointed out that low frequencies improve

428

exercise capacity, although significant effects on peripheral muscle strength could not be 17

ACCEPTED MANUSCRIPT

determined. Sillen et al. (2014)33 assessed 120 COPD patients with quadriceps muscle

430

weakness and compared neuromuscular electrical stimulation parameters of high (75Hz)

431

and low frequencies (15Hz) for 21 minutes, with training endurance of 8 weeks, 5 days per

432

week, twice a day on quadriceps and calf. The authors observed that the quadriceps peak

433

torque increased by 10.8 Nm in the group submitted to electrical stimulation with high

434

frequency (n=41). This did not occur in the low-frequency group (n=39) (1.4 Nm; p=0.43).

435

However, for the evaluation of exercise performance, both groups showed improved

436

6MWT values. Conclusively, for the strength training program, the high-frequency group

437

was as effective as the group undergoing quadriceps muscle strengthening. But,

438

regardless of the frequency, patients with peripheral muscle weakness improved their

439

exercise performance. In another study29 with patients with HF and healthy individuals, the

440

acute effect of frequencies of 15 and 50 Hz on peripheral muscle strength in both groups

441

was analyzed. In that study, the authors investigated the isometric peak torque at

442

maximum voluntary quadriceps contraction and at each frequency. They observed that the

443

isometric peak torque produced by electrical stimulation frequency of 50 Hz was higher

444

than that produced by electrical stimulation at 15 Hz, and both were lower than the peak

445

produced by maximal voluntary contraction in HF patients and in healthy individuals, with

446

no differences between the groups. In HF patients, neuromuscular electrical stimulation

447

seemed to improve 6MWD and peripheral muscle strength, similar to conventional training

448

with aerobic exercise34.

SC

M AN U

TE D

EP

AC C

449

RI PT

429

450

A systematic review32 aimed at investigating changes in enzyme activity, in the

451

composition of muscle fiber type, and in the size of muscle fibers of the lower limbs in

452

humans after a neuromuscular electrical stimulation therapy. The authors divided the

453

subjects into a group of high frequency (>50 Hz) and a group of low frequency (<20Hz),

454

and observed that lower frequencies improved exercise capacity, whereas higher 18

ACCEPTED MANUSCRIPT

frequencies were mostly recommended to improve muscle performance. Most studies with

456

low frequencies reported significantly increased activity of oxidative enzymes, whereas the

457

results for changes in muscle fiber composition and muscle size were conflicting. High

458

frequencies showed a significant increase in muscle size by 50% of all studies, as well as

459

increased peripheral muscle strength. These results are also in accordance with the study

460

conducted by Dal Corso et al. (2007)35, who observed that neuromuscular electrical

461

stimulation in COPD patients with frequency of 50Hz and pulse width of 400µs produced a

462

decrease of 9.8% in the type I fibers and a 12.5% increase in type II fibers.

SC

RI PT

455

463

This study observed that patients submitted to low frequency and intensity therapy

465

improved exercise capacity and increased blood markers, such as increased IGF-1

466

plasma levels, as well as no changes in pro-inflammatory, TNF-alpha and anti-

467

inflammatory IL-10 markers.

468

M AN U

464

Muscle metabolism in CKD is strongly influenced by IGF-14,18. In animal models, there is a

470

strong relation between the altered insulin/IGF1 signaling and muscle loss36, which will

471

increase the risk of early death in CKD patients4, when associated with risk factors, such

472

as heart or hormonal diseases. This current study observed that LG showed lower

473

baseline IGF-1 values compared to HG, but with no significant differences, which is in

474

agreement with the lower skeletal muscle strength values found in LG. After training, IGF-1

475

levels increased in LG only. This suggested that biochemical alterations for the production

476

of protein might be occurring. These would evolve into tissue and functional changes,

477

considering that IGF-1 has been recognized as one of the major factors responsible for

478

muscle growth coordination, increasing mass and muscle strength4,37. However, higher

479

plasma IGF-1 does not confirm its deposition to muscle tissue, since its release does not

480

have a specific target tissue37. Moreover, the musculoskeletal tissue, like other tissues,

AC C

EP

TE D

469

19

ACCEPTED MANUSCRIPT

produces cytokine locally37,38, which might explain why HG had not shown significantly

482

improved plasma IGF-1 level18, despite some functional gains. The significant increase in

483

IGF-1, observed in LG, may have occurred because the baseline values of severity

484

markers (GFR) and peripheral muscle strength were significantly lower. This reinforced the

485

findings previously reported in the medical literature, in which improved outcomes after

486

therapeutic intervention seem to be greater in more severe patients with increasing

487

functional impairment39-41.

RI PT

481

488

A study18 in rats with nephrectomy-induced CKD, which were submitted to neuromuscular

490

electrical stimulation at a frequency of 20Hz and an intensity of 1 mA current for 15

491

minutes during 15 days, aimed at identifying whether the low frequency electrical

492

stimulation would affect the IGF-1 signaling pathway. IGF-1 levels were measured in

493

serum and muscle. The authors observed that the serum IGF-1 was lower in CKD rats,

494

compared to sham rats (no CKD), and that the electrical stimulation had no significant

495

effects on serum levels of IGF-1 in both groups (sham and CKD who performed the

496

training). However, they found that electrical stimulation increased levels of muscle IGF-1

497

in both groups, indicating that increased IGF-1 in rats treated with electrical stimulation

498

was local, whose serum analysis was not capable of identifying such improvement. In this

499

experiment18, the authors could not observe significant effects immediately and up to 5

500

days after electrical stimulation of 20 Hz on the inflammatory TNF-alpha marker. Instead,

501

they found effects on the Interleukin-6 (IL-6), suggesting that electrical stimulation may

502

cause temporary acute inflammation, as if this was a common physiological response to

503

the exercise that leads to increased muscle mass42. Similarly, this study found no

504

alterations in TNF-alpha in both groups after training. However, IL-10, which regulates the

505

pro-inflammatory cytokines, such as TNF-alpha and IL-642, had a significant decrease only

506

in HG, which was not observed in LG.

AC C

EP

TE D

M AN U

SC

489

20

ACCEPTED MANUSCRIPT

507

There have been discussions about increased cardiovascular diseases in patients with

509

altered balance of cytokines due to the combination of decreased immune responses,

510

together with a persistent immune stimulation, which plays an important role in systemic

511

inflamation10. Studies have shown that inflammation increases cardiovascular risk and

512

mortality in CKD patients43,44, and low IL-10 levels in hemodialysis patients have been

513

associated with a significantly increased risk of death caused by cardiovascular diseases,

514

compared to patients with higher levels of IL-1045. However, for the results of this study, it

515

is important to consider that cytokines do not show effects as a single substance that acts

516

in a specific type of cell, because the increased cytokine immediately leads to a down-

517

regulation of several others, like a network of cytokines. Furthermore, many of the effects

518

of cytokines are local and non-systemic. A significant number of the effects are difficult to

519

be detected; therefore, it is reasonable to be careful with the interpretation of plasma

520

cytokine measurements42.

SC

M AN U

TE D

521

RI PT

508

This is the first randomized clinical trial to study the effects of two training frequencies and

523

different strategies to set the intensity current with neuromuscular electrical stimulation on

524

functional and biochemical variables in CKD patients during HD. According to the medical

525

literature, the main responders in trainings with electrical stimulation are those who endure

526

larger increase of intensity46. However, this study showed improved exercise capacity in

527

LG, even with low current intensity. Furthermore, LG showed lower sensation of muscular

528

fatigue. Thus, it may be suggested that the use of lower frequencies and intensities is

529

likely to be more comfortable for these patients, allowing muscular electrical stimulation to

530

be applied in patients with low tolerance to increased current intensity.

AC C

EP

522

531

21

ACCEPTED MANUSCRIPT

Finally, it could be observed that the treatment with neuromuscular electrical stimulation

533

for CKD patients may be an alternative to achieve functional benefits. The frequency and

534

intensity of electrical stimulation seem to have an important impact on the degree of

535

inflammatory and muscular response18. Further studies should investigate the effects of

536

high and low frequency currents in this population, in order to observe their effects on

537

muscle fibers type I and II, as well as on inflammatory cytokines and trophic modulators

538

directly on the muscle.

RI PT

532

SC

539 540 Limitations of the study

M AN U

541 542 543

Despite the pair analysis performed in this study, LG patients showed lower initial

545

peripheral muscle strength compared to HG. This chance error could not be controlled, but

546

it has been corrected for the analysis between the groups. HG showed a relationship

547

between age and right and left muscle strengths before and after the training, as well as a

548

higher GFR, which might explain the higher values found in the peripheral muscle strength

549

of this group.

EP

AC C

550

TE D

544

551

Another possible limitation of this study was the failure to complete an a priori sample size

552

calculation based on the study on 6MWT conducted by Dobsak et al. (2012)12, as well as

553

the inexistence of a control group. Although the target sample was not obtained, our post

554

hoc power analysis, which analyzed the sample of 20 patients in each group, showed high

555

power, indicating that our population responded to the proposed objective. Furthermore, it

556

would be interesting to control the level of physical activity in these patients, even though

557

this was not the main objective of the study. Ultimately, muscle resistance should have 22

ACCEPTED MANUSCRIPT

558

been measured by isokinetic dynamometer, which would confirm if improved 6MWD

559

results in LG were related to increased muscle resistance.

560 561 Conclusions

RI PT

562 563 564

CKD patients on hemodialysis improve exercise capacity after peripheral neuromuscular

566

electrical stimulation of high and low frequency and intensity. However, the benefits on

567

muscle and inflammatory outcomes seem to be specific for the adopted electrical

568

stimulation strategy.

M AN U

SC

565

569 570

574 575 576 577 578 579 580

TE D

573

Stimulator Carci Fesmed IV, São Paulo, BR.

EP

572

Suppliers

Oximeter Rossmax - SB100 Fingertip, Taipei, Taiwan.

AC C

571

Dynamometer Biodex System 4 Pro, New York, USA.

NDD EasyOne portable digital spirometer, Zurich, Switzerland.

581 582

DuoSet ® ELISA, R & D Systems, Minneapolis, MN, USA.

583 23

584

ACCEPTED MANUSCRIPT

G*Power Version 3.1.9.2. Franz Faul, Kiel University, Germany.

585 586

Statistical Package for Social Sciences; SPSS Inc.

587

589

RI PT

588 References

590

SC

591 1.

Fried LF. Boudreau R. Lee JS. et al. Kidney function as a predictor of loss of lean

593

mass in older adults: Health. Aging and Body Composition Study. J Am Geriatr Soc.

594

2007;55(10): 1578-1584

595

M AN U

592

2.

597

disease. Am J Clin Nutr. 2010;91(4):128S-1132S.

598

Workeneh BT. Mitch WE. Review of muscle wasting associated with chronic kidney

TE D

596

3.

600

conditions. Int J Biochem Cell Biol. 2013;45(10):2230-2238.

601

Wang XH. Mitch WE. Muscle wasting from kidney failure— a model for catabolic

EP

599

4.

Stenvinkel P. Carrero JJ. Von Walden F. Ikizler TA. Nader GA. Muscle wasting in

603

end-stage renal disease promulgates premature death: established. emerging and

604

potential novel treatment strategies. Nephrol Dial Transplant. 2015;0:1–8.

AC C

602

605 606

5.

National Kidney Foundation. KDOQI Clinical Practice Guideline for Diabetes and

607

CKD: 2012 Update. Am J Kidney Dis. 2012;60(5):850-886.

608 609

6.

Ikizler TA. Pupim LB. Brouillette JR. Levenhagen DK. Farmer K. Hakim RM. et al. 24

ACCEPTED MANUSCRIPT

610

Hemodialysis stimulates muscle and whole body protein loss and alters substrate

611

oxidation. Am.J.Physiol Endocrinol Metab. 2002;282(1):107-16.

612 7.

Fahal IH. Uraemic sarcopenia: aetiology and implications. Nephrol Dial

614

Transplant. 2014;29(9):1655-65.

615 8.

Souza VA. Oliveira D. Mansur HN. Fernandes NMS. Bastos MG. Sarcopenia na

617

doença renal crônica. J Bras Nefrol. 2015;37(1):98-105.

SC

616

RI PT

613

618 9.

Ruiz-Ortega M. Lorenzo O. Suzuki Y. Rupérez M. Egido J. Proinflammatory actions

620

of angiotensins. Curr Opin Nephrol Hypertens. 2001; 10(3):321-9.

621 622

10.

623

IL-10. IL-6. and TNFalpha: central factors in the altered cytokine network of uremia – the

624

good. the bad. and the ugly. Kidney Int. 2005;67(4):1216-33.

M AN U

619

TE D

Stenvinkel P. Ketteler M. Johnson RJ. Lindholm B. Pecoits-Filho R. Riella M. et al.

625 11.

Greenwood SA. Naish P. Clark R. O'Connor E. Pursey VA. Macdougall IC. Et al.

627

Intra-dialytic exercise training: a pragmatic approach. Journal of Renal Care.

628

2014;40(3):219-26.

629

AC C

EP

626

630

12.

Koh KP. Fassett RG. Sharman JE. Coombes JS. Williams AD. Effect of intradialytic

631

versus home-based aerobic exercise training on physical function and vascular

632

parameters in hemodialysis patients: a randomized pilot study. Am J Kidney

633

Dis. 2010;55(1):88-99.

634 635

13.

Dobsak P. Homolka P. Svojanovsky J. Reichertova A. Soucek M. Novakova

636

M. Dusek L. Vasku J. Eicher JC. Siegelova J. Intra-dialytic electrostimulation of leg 25

ACCEPTED MANUSCRIPT

637

extensors may improve exercise tolerance and quality of life in hemodialyzed patients. Artif

638

Organs. 2012;36(1):71-8.

639 14.

Bourjeily-Habr G. Rochester CL. Palermo F. Snyder P. Mohsenin V. Randomised

641

controlled trial of transcutaneous electrical muscle stimulation of the lower extremities in

642

patients with chronic obstructive pulmonary disease. Thorax. 2002;57(12):1045-9.

RI PT

640

643 15.

Kho ME. Truong AD. Brower RG. Palmer JB. Fan E. Zanni JM. et al.

645

Neuromuscular Electrical Stimulation for Intensive Care Unit–Acquired Weakness:

646

Protocol and Methodological Implications for a Randomized. Sham-Controlled. Phase II

647

Trial. Phys Ther. 2012;92(12):1564–79.

M AN U

SC

644

648 16.

Van Buuren F. Mellwig KP. Fründ A. Bogunovic N. Oldenburg O. Kottmann

650

T. Wagner O. Dahm JB. Horstkotte D. Fritzsche D. Electrical myostimulation:

651

improvement of quality of life. oxygen uptake and left ventricular function in chronic heart

652

failure Rehabilitation (Stuttg). 2014;53(5):321-6.

653

EP

TE D

649

17.

655

Neuromuscular electrical stimulation improves clinical and physiological function in COPD

656

patients. Respir Med. 2014;108(4):609-20.

657

Vieira PJ. Chiappa AM. Cipriano G Jr. Umpierre D.. Arena R. Chiappa GR.

AC C

654

658

18.

Hu L. Klein JD. Hassounah F. Cai H. Zhang C. Xu P. et al. Low-frequency electrical

659

stimulation attenuates muscle atrophy in ckd—a potential treatment strategy. J Am Soc

660

Nephrol. 2015;26(3):626-35.

661

26

ACCEPTED MANUSCRIPT

662

19.

Johansen KL. Shubert T. Doyle J. Soher B. Sakkas GK. Kent-Braun JA. Muscle

663

atrophy in patients receiving hemodialysis: Effects on muscle strength. muscle quality. and

664

physical function. Kidney International. 2003;63(1):291–7.

665 20.

Murphy AJ. Wilson GJ. Pryor JF. Newton RU. Isometric assessment of muscular

667

function: the effect of joint angle. Journal Appl Biomech 1995;11:205-15.

RI PT

666

668 21.

Smidt GL. Rogers MW. Factors contributing to the regulation and clinical

670

assessment of muscular strength. Physical Therapy. 1982;62(9):1283-90.

SC

669

M AN U

671 672

22.

Miller MR. Hankinson J. Brusasco V. Burgos F. Casaburi R. Coates A. et al.

673

Standardisation of spirometry. Eur Respir J. 2005;26:319-38.

674 23.

676

white adults in Brazil. J Bras Pneumol. 2007;33:397-406.

677

Pereira CA. Sato T. Rodrigues SC. New reference values for forced spirometry in

TE D

675

24.

Holland AE. Spruit MA. Troosters T. Puhan MA. Pepin V. Saey D. An official

679

European Respiratory Society/American Thoracic Society Technical Standard: field

680

walking tests in chronic respiratory disease. Eur Respir J. 2014;44(6):1521-37.

681

AC C

EP

678

682

25.

Britto RR. Probst VS. Andrade AFDD. Samora GAR. Hernandes NA. Marinho PEM.

683

et al. Reference equations for the six-minute walk distance based on a Brazilian

684

multicenter study. Brazilian Journal of Physical Therapy. 2013;17(6):556-63.

685 686

26.

Dancey C. Reidy. J. Estatística Sem Matemática para Psicologia: Usando SPSS

687

para Windows. 2006. Porto Alegre. Artmed. 27

ACCEPTED MANUSCRIPT

688 689

27.

SBN. Sociedade Brasileira de Nefrologia. Diretrizes Brasileiras de Doença Renal

690

Cronica. 2004.

691 28.

Gregory CM. Dixon W. Bickel CS. Impact of varying pulse frequency and duration

693

on muscle torque production and fatigue. Muscle Nerve. 2007;35(4):504-9.

RI PT

692

694 29.

Sbruzzi G1. Schaan BD. Pimentel GL. Signori LU. Da Silva AN. Oshiro MS. et al.

696

Effects of low frequency functional electrical stimulation with 15 and 50 Hz on muscle

697

strength in heart failure patients. Disabil Rehabil. 2011;33(6):486-93.

M AN U

SC

695

698 699

30.

Hannerz J. Discharge properties of motor units in relation to recruitment order in

700

voluntary contraction. Acta Physiol Scand. 1974;91(3):374-85.

701 31.

703

effects of chronic low-frequency stimulation of thigh muscles in patients with advanced

704

chronic heart failure. Eur Heart J 2004;25(2):136-43.

EP

705

Nuhr MJ. Pette D. Berger R. Quittan M. Crevenna R. Huelsman M. et al. Beneficial

TE D

702

32.

Sillen MJ1. Franssen FM. Gosker HR. Wouters EF. Spruit MA. Metabolic and

707

structural changes in lower-limb skeletal muscle following neuromuscular electrical

708

stimulation: a systematic review. PLoS One. 2013 Sep 3;8(9):e69391.

AC C

706

709 710

33.

Sillen MJ. Franssen FM. Delbressine JM. Vaes AW. Wouters EF. Spruit MA.

711

Efficacy of lower-limb muscle training modalities in severely dyspnoeic individuals with

712

COPD and quadriceps muscle weakness: results from the DICES trial. Thorax.

713

2014;69(6):525-31. 28

ACCEPTED MANUSCRIPT

714 34.

Sbruzzi G. Ribeiro RA. Schaan BD. Signori LU. Silva AM. Irigoyen MC. et al.

716

Functional electrical stimulation in the treatment of patients with chronic heart failure: a

717

meta-analysis of randomized controlled trials. Eur J Cardiovasc Prev Rehabil.

718

2010;17(3):254-60.

RI PT

715

719 35.

Dal Corso S. Napolis L. Malaguti C. et al. Skeletal muscle structure and function in

721

response to electrical stimulation in moderately impaired COPD patients. Respir Med.

722

2007;101(6):1236-43.

SC

720

M AN U

723 724

36.

Wang X. Hu Z. Hu J. Du J. Mitch WE. Insulin resistance accelerates muscle protein

725

degradation: Activation of the ubiquitin-proteasome pathway by defects in muscle cell

726

signaling. Endocrinology. 2006;147(9):4160-8.

727 37.

729

Horm IGF Res. 2014;24(5):157-63.

EP

730

Philippou U. Barton ER. Optimizing IGF-I for skeletal muscle therapeutics. Growth

TE D

728

38.

732

growth factor gene expression during work-induced skeletal muscle growth. Am J Physiol.

733

1990;259(1 Pt 1):E89-95.

734

DeVol DL. Rotwein P. Sadow JL. Novakofski J. Bechtel PJ. Activation of insulin-like

AC C

731

735

39.

Troosters T. Gosselink R. Decramer M. Exercise training in COPD: how to

736

distinguish responders from nonresponders. J Cardiopulm Rehabil. 2001;21(1):10-17.

737

29

ACCEPTED MANUSCRIPT

738

40.

Altenburg WA. de Greef MH. ten Hacken NH. Wempe JB. A better response in

739

exercise capacity after pulmonary rehabilitation in more severe COPD patients. Respir

740

Med 2012;106(5):694-700.

741 41.

Spruit MA. Augustin IM. Vanfleteren LE. Janssen DJ. Gaffron S. Pennings HJ. et al.

743

CIRO+ Rehabilitation Network. Differential response to pulmonary rehabilitation in COPD:

744

multidimensional profiling. Eur Respir J 2015;46(6):1625-35.

RI PT

742

SC

745 42.

Muñoz-Cánoves P. Scheele C. Pedersen BK. Serrano AL. Interleukin-6 myokine

747

signaling in skeletal muscle: a double-edged sword? FEBS J. 2013 Sep;280(17):4131-48.

M AN U

746

748 749

43.

Zimmermann J. Herrlinger S. Pruy A. Metzger T. Wanner C. Inflammation enhances

750

cardiovascular risk and mortality in hemodialysis patients. Kidney Int. 1999;55(2):648-58.

751 44.

753

Inflammation. malnutrition and atherosclerosis in end-stage renal disease: a global

754

perspective. Blood Purif. 2002;20(5):454-8.

EP

755

Nascimento MM. Pecoits-Filho R. Lindholm B. Riella MC. Stenvinkel P.

TE D

752

45.

Girndt M. Kaul H. Sester U. Ulrich C. Sester M. Georg T. Köhler H. Anti-

757

inflammatory interleukin-10 genotype protects dialysis patients from cardiovascular events.

758

Kidney Int. 2002;62(3):949-55.

AC C

756

759 760

46.

Vivodtzev I. Debigaré R. Gagnon P. Mainguy V. Saey D. Dubé A. et al. Functional

761

and muscular effects of neuromuscular electrical stimulation in patients with severe COPD:

762

a randomized clinical trial. Chest. 2012;141(3):716-25.

763 30

ACCEPTED MANUSCRIPT

764 765

Legends of figures and tables:

766 767 Figure 1 – Flow diagram with timeline of the study.

769

HG: group of patients submitted to high frequency and intensity neuromuscular electrical

770

stimulation; LG: group of patients submitted to low frequency and intensity neuromuscular

771

electrical stimulation.

SC

RI PT

768

772

Figure 2 – Intra-group comparisons before and after the electrical stimulation protocol.

774

HG: group of patients submitted to high frequency and intensity neuromuscular electrical

775

stimulation; LG: group of patients submitted to low frequency and intensity neuromuscular

776

electrical stimulation; Nm: Newton-meter; 6MWT: six-meter walk test; m: meter. Repeated

777

measures ANOVA test. * p<0.05.

TE D

778

M AN U

773

Figure 3 - Intra-group comparisons before and after the electrical stimulation protocol.

780

HG: group of patients who were submitted to high frequency and intensity neuromuscular

781

electrical stimulation; LG: group of patients who were submitted to low frequency and

782

intensity neuromuscular electrical stimulation; IGF-1: growth factor similar to Type 1

783

insulin; IL-10: interleukin 10; TNF-alpha: tumor necrosis factor-alpha; pg/mL: picograms

784

per milliliter. Repeated measures ANOVA test. * p<0.05.

AC C

785

EP

779

786

Table 1 – Characterization of the study sample.

787

HG: group of patients submitted to high frequency and intensity neuromuscular electrical

788

stimulation; LG: group of patients submitted to low frequency and intensity neuromuscular

789

electrical stimulation; M: men; W: women; BMI: body mass index; Kg/m2: kilograms per 31

ACCEPTED MANUSCRIPT

790

square meter; Kt/V: urea clearance; mg/dL: milliliter per deciliter; GFR: glomerular filtration

791

rate; mL/min/1.73m2: Milliliter per minute per square meter; Nm Newton-meter; 6MWTD: 6-

792

minute walk test distance; m: meter; IGF-1: growth factor similar to type 1 insulin; IL-10:

793

interleukin-10; TNF-alpha: tumor necrosis factor alpha; pg/ml: picograms per ml. * p<0.05.

AC C

EP

TE D

M AN U

SC

RI PT

794

32

ACCEPTED MANUSCRIPT Table 1 – Characterization of the study sample LG

(n = 20)

(n = 20)

Sex (M/W)

15/5

11/9

0.18

Age

52.65±13.79

60.50±12.53

0.07

[46,19-59,11]

[54,64-66,36]

31.59±11.52

28.59±6.58

[26,19-36,98]

[25,51-31,67]

1.21±0.19

1.28±0.16

[1,12-1,30]

[1,20-1,36]

10.39±2.94

10.60±2.55

[9,02-11,77]

[9,40-11,79]

M AN U

Kt/V

Creatinine (mg/dL)

GFR (mL/min/1.73m2) 11.91±11.06

7.26±2.41

109.40±32.08

[124,78-185,92]

[94,38-124,42]

156.60±66.51

113.65±37.79

strength (Nm)

[125,47-187,73]

[95,96-131,34]

Initial 6MWTD (m)

435.55±95.81

403.80±90.56

[390,71-480,39]

[361,41-446,19]

389.64±201.66

252.38±156.35

[254,16-525,12]

[140,53-364,23]

7.26±1.81

6.27±1.92

[5,87-8,66]

[4,78-7,75]

AC C

Initial left muscle

IGF-1 (pg/mL)

IL-10 (pg/mL)

0.35

0.35

0.96

0.009*

[6,13-8,39]

155.35±65.32

EP

strength (Nm)

TE D

[6,73-17,09] Initial right muscle

SC

BMI (Kg/m2)

p-value

RI PT

HG

0.008*

0.01*

0.28

0.10

0.27

ACCEPTED MANUSCRIPT TNF-alfa (pg/mL)

6.74±1.75

7.27±1.60

[5,58-7,94]

[6,16-8,34]

0.15

HG: group of patients submitted to high frequency and intensity neuromuscular electrical stimulation; LG: group of patients submitted to low frequency and

RI PT

intensity neuromuscular electrical stimulation; M: men; W: women; BMI: body

mass index; Kg/m2: kilograms per square meter; Kt/V: urea clearance; mg/dL:

milliliter per deciliter; GFR: glomerular filtration rate; mL/min/1.73m2: Milliliter per

SC

minute per square meter; Nm Newton-meter; 6MWTD: 6-minute walk test distance; m: meter; IGF-1: growth factor similar to type 1 insulin; IL-10:

M AN U

interleukin-10; TNF-alpha: tumor necrosis factor alpha; pg/ml: picograms per ml.

AC C

EP

TE D

* p<0.05.

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT