Effects of Exercise Using Electrical Muscle Stimulation in End Stage Kidney Disease
Phase: NA
Conditions: Dialysis
Sponsor: University of Iowa

Study Title: Effects of Exercise by Neuromuscular Stimulation in Dialysis Patients 
NCT03517553 
Document Date: 11/11/2013 

 
1
 
 
 
 
 
 
 
Effects of Exercise using Electrical Muscle Stimulation in End Stage Kidney Disease 
 
 
 
 
 
 
 
Protocol EMS-HD-100 
Version 3 
11/11/13 
 
 
 
 
 
Investigators 
 
Bradley S. Dixon, MD 
Manish Suneja, MD 
Richard Shields, PhD 
 
 

 
2
A. Significance 
 
We propose a pilot feasibility study to determine if exercise delivered by passive electrical muscle 
stimulation (EMS) in patients with end stage renal disease (ESRD) on hemodialysis improves physical 
fitness and insulin resistance, outcome markers associated with morbidity and mortality in this 
population.  
 
Every year more than 105,000 people in the U.S. initiate hemodialysis for end stage renal disease 
(ESRD) facing the grim prognosis of a mortality rate that is 20% per year.1 Mortality rates on 
hemodialysis have improved over the years. Nevertheless, compared to the general population, excess 
mortality for patients initiating dialysis is more than 50-fold higher for young women under 45 years of 
age and nearly 3-fold higher for patients over 75 years of age.2 For age-matched patients, having 
ESRD requiring dialysis is associated with a significantly higher mortality than having cancer, 
diabetes, heart failure, stroke or an acute MI.1 Nearly half of the deaths on hemodialysis are attributed 
to cardiovascular causes. Patients on hemodialysis average 1.9 hospital admissions per year, mostly for 
cardiovascular causes (volume overload) and infections. The need for re-hospitalization within 30 days 
of discharge is 36%, nearly double that of patients hospitalized without ESRD.  
 
Despite the high mortality rate there are no interventions proven to reduce mortality in this 
population.3 Large randomized trials have examined the effect of dialysis dose, normalization of 
anemia, use of statins, lowering phosphate with a non-calcium binder, reduction in homocysteine level, 
the antioxidant vitamin E, human growth hormone and cinacalcet with no improvement in overall 
mortality.3  Frequent hemodialysis 6 times per week reduces left ventricular hypertrophy and may 
reduce morbidity and mortality but this is not a feasible option for the majority of patients who are on 
in-center maintenance hemodialysis 3 times per week.4 Clearly new approaches to improve morbidity 
and mortality for in-center hemodialysis patients are needed.  
 
Low physical capacity is a common, modifiable risk factor strongly associated with mortality and 
morbidity in ESRD patients that is ripe for intervention.5-9 Cardiovascular fitness and muscle strength 
in ESRD patients is reduced to 50-80% that of untrained healthy controls.10,11 Exercise training in 
patients with CKD can improve aerobic capacity, muscle strength, some nutritional measures and 
health-related quality of life and lower blood pressure.12,13 In addition, exercise training has been 
reported to improve multiple intermediate outcomes associated with mortality in hemodialysis patients 
including: endothelial function14, heart rate variability15,16, depression15 and insulin resistance17.  
 
Despite these benefits, there are no trials of exercise training on survival in patients with ESRD on 
hemodialysis. The effect of exercise training on survival has been reported in patients with systolic 
heart failure with mixed results.18,19 A meta-analysis of 9 randomized controlled trials reported that 
exercise training significantly reduced mortality and death or admission to the hospital.18  However, a 
subsequent large multicenter randomized controlled trial of 2331 patients with systolic heart failure 
(HF-ACTION trial) found a non-significant reduction in all-cause death or hospitalization (HR=0.93, 
P=0.13).19 A major limitation of most exercise trials including the HF-ACTION trial is that compliance 
with exercise training wanes with time and does not achieve prescribed target levels, particularly when 
performed in a home-based, non-supervised setting.12 In addition, it is hard to motivate older, more 
debilitated, sedentary or depressed individuals who might derive the most benefit from exercise 
training to participate. 
 
To address these concerns, we propose a pilot feasibility study to test a novel approach using 
passive electrical muscle stimulation (EMS) given 3 times a week to both quadriceps muscle groups 
while on hemodialysis for 16 weeks to determine whether this improves physical fitness, insulin 
resistance and other outcome markers associated with well being, morbidity and mortality in this 
population. In patients without CKD, EMS has been reported to improve peak VO2 and endothelial 

 
3
function, increase muscle strength and 6 minute walk time, improve insulin sensitivity and decrease 
inflammatory cytokines (tumor necrosis factor-alpha, TNFα and interleukin-6, IL-6).20-28 In 
preliminary studies of patients with metabolic syndrome, we also have evidence that 8 weeks of EMS 
can reduce inflammatory cytokines (IL-6 and TNFα) and improve insulin sensitivity (as assessed by 
the homeostatic model assessment, HOMA-IR, unpublished data). However, patients with ESRD may 
be slow to respond to exercise and generally at least 12 weeks of exercise is recommended to observe 
an effect.12  
 
EMS appears safe and may be beneficial to perform during hemodialysis.29  In one acute study of 
patients on hemodialysis, passive EMS was found to increase phosphate and urea removal while 
helping to maintain blood pressure.29  The improved phosphate removal is attributed to increased 
muscle perfusion allowing better equilibration of phosphate from the intracellular compartment into 
the blood stream. Elevated serum phosphate is linked to poor outcomes for hemodialysis patients.30 
Control of serum phosphate is difficult for many patients on hemodialysis and administration of 
phosphate binders with meals is one of the major contributors to the excessively high pill burden for 
these patients.31  Lowering this burden would likely improve a patient’s quality of life. Overall, we 
expect EMS will be safe during hemodialysis and will lower the threshold for patients to initiate and 
sustain exercise training.  Administration as part of the routine dialysis prescription will help promote 
long-term exercise compliance in a supervised setting. Improvement in phosphate control and blood 
pressure stabilization may be additional benefits.  
 
Insulin resistance as measured by HOMA-IR and the leptin-adiponectin ratio (LAR) will be the 
primary outcome measure. Insulin resistance in muscles is a known consequence of chronic kidney 
disease32 and independently associates with cardiovascular mortality.33 HOMA-IR and LAR are known 
to correlate strongly with insulin resistance as measured by the gold standard, hyperinsulinemic 
euglycemic clamp (HEGC) technique in patients with ESRD.34 HOMA-IR and LAR can be measured 
by a blood test and are technically more feasible for these intervention studies. Insulin resistance in 
CKD may be mediated in part by adipokines released from increased visceral fat stores.35-37 Exercise 
training can reduce visceral fat, which may be one mechanism leading to improvement in insulin 
resistance.38,39  Hence, estimation of changes in visceral fat (by DXA and anthropometric 
measurements) will be important secondary outcomes for this clinical trial.  
 
Elevations in inflammatory cytokines such as IL-6 are strongly associated with mortality in ESRD 
40-42 and the effect of EMS on these cytokines will be measured. Other secondary outcomes of 
importance include measurement of change in aerobic capacity, quadriceps strength, mobility, nutrition 
and malnutrition-inflammation score (MIS), serum markers of oxidative stress and effect on depression 
and quality of life.   
 
B. Innovation 
 
Passive exercise provided by EMS 3 times a week is an innovation that could finally bring the 
established benefits of regular exercise to hemodialysis patients. The benefits of passive EMS are 
similar to traditional exercise programs (vida supra). However, it addresses many of the barriers that 
limit implementation of traditional exercise programs in this population. Barriers to exercise include 
physiological, behavioral, psychological, and social/financial factors, including medical co-
morbidities, patient motivation, inanition, depression, and the availability of suitable tools to overcome 
physical limitations (e.g. inability to walk due to a diabetic foot ulcer).  Passive EMS which can be 
provided in the hemodialysis unit addresses each of these barriers. While it requires consent (like 
hemodialysis), passive EMS recommended by the dialysis provider can overcome lack of motivation, 
inanition and depression. By reducing depression, improving mobility and enhancing quality of life, 

 
4
passive EMS may reinforce its own use. Passive EMS should be safe and effective to use in most all 
patients on hemodialysis, with the exception of patients with pacemakers. Administration during the 
dialysis session addresses patient concerns regarding lack of time for exercise and access to suitable 
tools to exercise. It will also improve intradialytic clearance of phosphate and may stabilize 
intradialytic blood pressure that could have additional benefits in this population. Moreover, exercise 
training achieves the best results when it is supervised. Provision within the dialysis unit as part of the 
dialysis prescription provides the external motivation and social support of dialysis unit staff and other 
patients to help sustain this treatment. Finally, if passive EMS were shown to improve outcomes and 
lower annual cost per patient for in-center hemodialysis it would be readily adopted by dialysis 
providers and third-party payers.  
 
C. Approach 
 
 1. Overview.  We propose a clinical study to test the feasibility and efficacy of 16 weeks of passive 
exercise induced by electrical muscle stimulation (EMS) to improve markers of insulin resistance 
associated with morbidity and mortality in hemodialysis patients (Figure 1). EMS is provided 
sequentially for 15 minutes to both the right and left quadriceps muscle groups three times a week. The 
primary outcome measure will be improvement in insulin resistance measured using the homeostatic 
model assessment (HOMA-IR) and leptin-adiponectin ratio (LAR).  
 
Figure 1. 
 
 
 
 
The efficacy of the exercise training will be assessed by change in aerobic capacity measured by 
peak VO2 and quadriceps muscle strength. Additional measures will include measurement of fat and 
EMS 
Visit 9 
8 week EMS 
Outcomes 
-Muscle strength 
-DXA 
-VO2 peak 
 
Visit 10 
Blood 
Muscle Biopsy 
Visit 12 
16 week EMS 
Outcomes 
-Muscle strength 
-DXA 
-VO2 peak 
 
Visit 13 
Blood 
Muscle Biopsy 
 
4 week Care 
Optimization 
 
EMS Training:  
3x/week, 30 min/session 
Visit 1 
Enroll & 
Baseline 
data 
-Blood 
-Muscle 
strength 
 
No EMS 
Training 
Visit 14 
4 wk Post-
EMS 
Outcomes 
-Blood 
-Muscle 
strength 
Visit 2 
Pre-EMS data 
-Muscle strength 
-DXA 
-VO2 peak 
 
Visit 3 
Blood 
Muscle Biopsy 
Start EMS training 
Care Optimization  
EMS Study Visits  
Visits 4-8, 11 
Muscle Biopsies-3 (optional) 

 
5
muscle content by dual energy X-ray absorptiometry (DXA) and anthropometry, measures of physical 
activity, nutritional status, depression, quality of life, anemia, mineral metabolism, dialysis blood 
pressure and serum levels of inflammatory cytokines and markers of oxidative stress.  
 
Following enrollment, baseline data and selected outcome markers will be assessed. Then all 
patients will undergo a 4-week care optimization phase during which time we will review the patient's 
dialysis care and make adjustments (if necessary) to meet standard of care guidelines as defined by 
CMS, KDOQI and KDIGO (Table 1). Outcome markers will be reassessed again after completion of 
the 4-week care optimization phase. At this time all patients will also undergo a baseline exercise test 
to measure peak VO2 and a DXA scan for body composition analysis before starting EMS training. 
 
Table 1 
Parameter 
Optimized care goals 
Vascular access  
Discontinuation of central venous catheter and use of arteriovenous access 
Fluid removal  
Optimize dry weight. Limit UF rate to ≤13 ml/kg/hr 
Solute removal  
Minimum dialysis time ≥ 3.5 hours. Goal 4 hours. kT/V ≥ 1.4 
Acid-base  
Pre-dialysis HCO3 between 22-27 mEq/L 
Anemia 
Hemoglobin 9.5-11 g/L, if on erythropoietin; Iron saturation ≥20% 
Ca, PO4, PTH 
Calcium, PO4 within normal levels; PTH ≤600 pg/ml  
 
 
EMS training will be done while the patient is on hemodialysis. At the first EMS training session, 
optimal EMS settings will be established and patients will be taught how to apply the electrodes and 
use the muscle stimulator. For patients who agree we will also do a muscle biopsy of the quadriceps 
(vastus lateralis) muscle at this first training visit before starting EMS training. Patients will be seen at 
the next dialysis session and again after 7 days to confirm that they are doing the EMS training 
properly. Patients are then seen at Day 14, 28, 42, 56, 84 and 112 after starting EMS training to assess 
for any adverse events, monitor progress with EMS training and adjust the EMS settings to make sure 
they are at the optimal tolerated intensity of muscle stimulation. The EMS training will continue for 16 
weeks. At Day 56 and Day 112 (8 and 16 weeks) of EMS-training all the primary and secondary 
outcome variables including exercise stress test for peak VO2 and DXA scan will be performed. For 
patients who agree we will also perform a muscle biopsy at a dialysis visit around Day 56 and Day 112 
(not the same day as the exercise stress test). One month after stopping EMS training the patient will 
be seen and the outcome variables will be repeated except for the DXA scan and the exercise stress 
test.    
 
2. Intervention.  EMS is delivered to the quadriceps muscle groups using a neuromuscular electrical 
stimulator (using either the EMPI 300PV or its replacement, the Continuum device, EMPI, Inc). The 
device generates electrical impulses that are delivered to nerves stimulating the muscles through 
electrodes applied to the skin surface. Patients are taught how to place the adherent electrode pads over 
the quadriceps muscles on the upper thigh a few inches below the anterior iliac crest and just above the 
patella bilaterally. The electrodes will be applied at home or after coming to the dialysis unit just prior 
to each dialysis session. The patient’s legs are extended straight on the reclining dialysis chair. The 
device is set to deliver biphasic electrical stimulation at 1-20 Hz and a pulse duration of 400 µS applied 
for 1-12 seconds on and 1-12 seconds off.  The stimulus intensity (typically 10-100 mA) is gradually 
increased to the maximum tolerable level for muscle contraction. The patient will feel contraction of 
the muscles and may feel mild shortness of breath and tingling of the skin but otherwise is expected to 
tolerate the stimulation well. The stimulation is delivered to one side for 15 minutes and then to the 

 
6
opposite side for 15 minutes. This will be repeated at each dialysis session 3x/week. The first 
intervention will be observed through the entire treatment. If the patient feels excessively fatigued or 
other untoward signs the stimulator will be turned off until they feel better and then restarted to 
achieve the full treatment time. 
 
At each study visit the study coordinator will confirm that the settings are correct and readjust the 
maximal tolerated stimulus intensity as needed to account for training. Patients will also be encouraged 
to be active and exercise as much as possible outside of the dialysis unit.  
 
The total duration of the study for any patient is expected to be 6 months. We plan to complete the 
study of all patients within 2 years.  
 
 
3. Study population. 
 
Patients on hemodialysis three times a week at participating study sites within the UIHC dialysis 
program will be eligible to participate.   
 
3.1 Inclusion & exclusion criteria. 
 
Inclusion criteria include: age over 18 year old, able to provide informed consent, on maintenance 
hemodialysis for end stage renal disease and expected to stay in a participating dialysis unit for at least 
6 months.   
 
Exclusion criteria include: presence of a cardiac pacemaker or presence of any other implanted 
electrical stimulation device, uncontrolled hypertension (SBP>170 mmHg), unstable angina, heart 
attack within the last month, expected survival <6 months, unsuitable for participation based on 
physician assessment, unwilling or unable to give informed consent.  
 
4. Outcome measures. 
4.1 Primary Outcome.  
 
The primary outcome measure will be the change in insulin resistance as determined by either 
HOMA-IR or LAR after 8 and 16 weeks of EMS training.  The analysis will also be stratified by 
whether patients achieved optimum dialysis care goals at randomization to see if optimization of care 
influenced the response to EMS. The correlation between change in HOMA-IR or LAR with change in 
aerobic capacity (peak VO2) and quadriceps strength after 16 weeks training and with maximal EMS 
training intensity (in mV) achieved will be assessed to determine if change in insulin resistance 
depends on intensity of training.    
 
4.2. Secondary outcomes measures. 
 4.2.1. Aerobic capacity and quadriceps strength. The effect of EMS on quadriceps muscle strength 
will be assessed 5 times (Visits 1, 2, 9, 12 and 14  in Figure 1) : at baseline, pre-EMS training, at 8 and 
16 weeks of EMS training and again 4 weeks after stopping EMS training. Quadriceps muscle strength 
will be determined using a belt-stabilized portable dynamometer.43 Change in peak VO2 will be 
measured 3 times (Visits 2, 9 & 12): pre-EMS training and after 8 and 16 weeks of EMS training. Peak 
VO2 will be determined using graded exercise treadmill test using a Balke protocol. Cycle ergometry 
will be used to determine peak VO2 in patients who can’t walk on a treadmill but can do cycling. This 
will determine if EMS produced an increase in quadriceps strength or improvement in aerobic capacity 
after 8 and 16 weeks of training. It will also determine how rapidly the effect on muscle strength is lost 
after stopping EMS training.   
 

 
7
4.2.2. Body and leg composition changes. The effect of EMS on total body fat and lean mass, visceral 
fat mass and regional thigh fat and lean mass will be determined by DXA scanning. DXA scanning 
will be done 3 times: pre-EMS training and after 8 and 16 weeks of EMS training. The DXA results 
will be supplemented by measuring the effect of EMS on anthropometric measures including: body 
mass index (BMI), waist (minimal circumference) to hip (maximal gluteal protuberance) ratio, 
anterior-posterior diameter at the umbilicus and thigh circumference (1/2 way between the anterior 
iliac crest and upper border of the patella, bilaterally). The anthropomorphic studies will be performed 
5 times: at baseline, pre-EMS, after 8 and 16 weeks of EMS training and 4-weeks after stopping 
training. Collectively these studies will determine whether EMS had an effect on body fat or muscle 
composition. Of particular interest is change in visceral/abdominal fat and change in quadriceps 
muscle mass after 8 and 16 weeks of EMS training.  
 
4.2.3. Measures of physical activity. Changes in physical activity will be assessed at 5 time points: 
baseline, pre-EMS, after 8 and 16 weeks of EMS training and 4 weeks after stopping EMS training 
using a standardized 6-minute walk test and by monitoring 48 hour activity level between dialysis 
sessions using a wristwatch activity monitor. The 6-minute walk test is determine by measuring the 
distance a patient can walk on a standardized track within 6 minutes at whatever pace they choose to 
walk.  
 
4.2.4. Malnutrition-inflammation (MIS) assessment. The MIS is a 10 item test which includes 
assessment of change in patient dry weight over 3-6 months, dietary intake, gastrointestinal symptoms, 
functional capacity, major co-morbidities, decreased fat mass, signs of muscle wasting, BMI, serum 
albumin and serum TIBC.44  It has been validated as a predictor of mortality in hemodialysis patients. 
44,45  MIS will be assessed at 5 time points: baseline, pre-EMS, after 8 and 16 weeks of EMS training 
and 4-weeks after completion of EMS training.  
 
4.2.5. Depression assessment. Assessment of depression will be performed using the Patient Health 
Questionaire-9 (PHQ-9) at 5 time points: baseline, pre-EMS, after 8 and 16 weeks of EMS training and 
4-weeks after completion of EMS training. The PHQ-9 has been validated as a screening tool for 
detection of depression in ESRD patients with a score ≥ 10 having both a sensitivity and specificity of 
92% for detecting depression compared to the Structured Clinical Interview for Diagnostic and 
Statistical Manual of Mental Disorders, Fourth Edition as the gold standard.46 PHQ-9 has been used as 
a screening tool for depression in the ACCORD trial and shown to associate with overall mortality. 47 
 
4.2.6. Quality of life will be assessed using the Kidney Disease Quality of Life-36 (KDQOL-36) form 
at 5 time points: baseline, pre-EMS, after 8 and 16 weeks of EMS training and 4-weeks after 
completion of EMS training. The KDQOL-36 is a validated standardized form used to assess quality of 
life in hemodialysis patients that is associated with morbidity and mortality.48,49 
 
4.2.7. Inflammatory cytokines. The level of inflammatory cytokines, particularly IL-6 and the 
inflammatory marker, C-reactive protein (CRP) are strongly associated with mortality in dialysis 
patients 50,51 and can be modulated by exercise52. To determine if EMS can decrease the levels of these 
inflammatory cytokines, we will measure IL-6, TNFα and CRP at 5 time points: baseline, pre-EMS, 
after 8 and 16 weeks of EMS training and 4-weeks after completion of EMS training. 
 

 
8
4.2.8. Oxidative stress markers. Markers of oxidative stress are increased in patients with ESRD 53-55 
and associated with increased mortality.56,57 Treatment with the antioxidant alpha tocopherol has been 
reported in a small study to decrease mortality.58  Chronic exercise training can reduce oxidant stress 
and may be one mechanism whereby exercise improves cardiovascular outcomes.59,60 To determine 
whether EMS alters markers of oxidative stress we will measure serum F2-isoprostane (a measure of 
lipid oxidation), protein thiols (a measure of protein oxidation) and the ratio of reduced to oxidized 
glutathione (GSH/GSSG) in erythrocytes.61-63  To assess the effect of EMS on antioxidant levels we 
will measure erythrocyte glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase 
(CAT) activity.64 These markers will be measured at 5 time points: baseline, pre-EMS, after 8 and 16 
weeks of EMS training and 4-weeks after completion of EMS training. 
 
4.2.9. Anemia is a marker of inflammation65 and associated with morbidity and mortality in dialysis 
patients.66,67 The blood levels of hemoglobin, iron saturation and ferritin, as well as the dose of 
erythropoietin and iron administered are tracked monthly as routine standard of care for hemodialysis 
patients. We will collect each of these variables from the patient chart starting with the labs drawn just 
before enrollment and continuing through labs drawn 4 weeks after completing the EMS training. We 
will plot this data to see whether any change occurred during treatment with EMS.   
 
4.2.10. Serum phosphorus is associated with mortality for hemodialysis patients.68  Control of 
phosphorus levels using phosphate binders taken 3 times a day with meals is one of the major causes of 
excessive pill burden for these patients.31 EMS has been shown to increase intra-dialytic phosphate 
removal and may help lower serum phosphate or reduce pill burden for these patients.29 Serum 
phosphorus, calcium, and PTH as well as dose of phosphate binders prescribed are recorded monthly 
for hemodialysis patients as part of standard of care. We will collect each of these variables from the 
patient chart starting with the labs drawn just before enrollment and continuing through labs drawn 1 
month after completing the EMS training. We will plot this data to see whether any change occurred 
during treatment with EMS. 
 
4.2.11. Blood pressure and dialysis hypotension. Maintenance of blood pressure on dialysis can be a 
challenge for some patients, particularly those with large intra-dialytic fluid gains, and is associated 
with poor outcomes.69 A prior study of EMS during hemodialysis found that EMS produced a 
statistically significant increase in BP and fewer hypotensive episodes compared to no EMS.29 For the 
first EMS treatment in each patient we will directly record BP and pulse at 15-minute intervals before, 
during and after treatment with EMS. Thereafter, we will collect the BP and pulse data that is recorded 
(approximately every 15-30 minutes) as standard of care for every hemodialysis session. We will 
compare the average hemodialysis BP and pulse during the 4-week “care optimization” phase to that 
obtained during the EMS treatment and the 4-week post-EMS treatment phases to determine if EMS 
was associated with any change in BP or pulse. We will also analyze the frequency of dialysis sessions 
with a hypotensive episode (defined as SBP<90 mmHg or need for IV saline for hypotensive 
symptoms) during EMS compared to non-EMS treatment.   
 
4.2.12. Muscle gene expression. Gene expression profiling of mRNA extracted from a skeletal muscle 
biopsy will be performed to give insight into the biological pathways activated by EMS both acutely 
and with chronic training in hemodialysis patients. Patients will be allowed to opt out of any of the 
muscle biopsy samples and still participate in the rest of the study. For these studies a muscle biopsy 
will be obtained as previously described70 from the vastus lateralis muscle prior to the first EMS 

 
9
treatment and after 8 and 16 weeks of EMS training to determine the chronic effect of EMS training on 
muscle gene expression. Gene expression analysis will be done using Affymetrix Human Exon 1.0 ST 
arrays as previously described.70  We will first examine specific genes that are highly overexpressed or 
under-expressed after acute and chronic exercise compared to the baseline (pre-EMS). We will also 
look for biological pathways activated or suppressed by acute and chronic exercise specifically looking 
at pathways relating to muscle glucose metabolism and insulin response, mitochondrial biogenesis and 
function, muscle phenotype, growth and energy metabolism, oxidant stress and inflammation. A future 
goal of this research is to compare the gene expression response to EMS in this study with that seen in 
patients with normal renal function and varying levels of CKD to see if the gene response profile is 
qualitatively different in patients with varying levels of kidney function. We are also interested how 
the gene expression profiles seen with EMS compare to that seen with traditional volitional aerobic 
exercise to determine if EMS activates different genes or biological pathways than volitional exercise. 
 
5.  Visits & Assessments.  
 
Patients will be identified from the population of patients on regular maintenance hemodialysis at one 
of the University of Iowa dialysis units. Potential subjects will be approached at a dialysis visit and 
informed of the study.  
5.1. Visit 1. If they indicate an interest in participating we will proceed with obtaining written 
informed consent and confirm that they meet all the inclusion criteria and none of the exclusion 
criteria.  
- Obtain informed consent to participate and consent to access additional appropriate medical records 
- Review inclusion and exclusion criteria 
 
 Baseline data collection (Day -35 to -28). Performed at dialysis unit or CRU as appropriate. Up to 4 
hours time.  
- Record demographics and baseline disease characteristics 
- Record past medical history and current review of symptoms 
- Record current dialysis orders 
- Record episodes of dialysis hypotension (SBP<90 or symptoms needing saline, last 6 HD sessions) 
- Record baseline exercise status (minutes of exercise per week) 
- Record concomitant medications 
- Malnutrition-inflammation (MIS) assessment 
- Kidney Disease Quality of Life-36 (KDQOL-36) assessment 
- Patient Health Questionaire-9 (PHQ-9) 
- Record sitting blood pressure and pulse 
- Physical examination by a physician 
- 6-minute walk test 
- Record height, weight, waist circumference, hip circumference  
- Thigh circumference bilaterally 
- Record most recent standard dialysis monthly lab tests. 
ο Monthly labs (predialysis): albumin, alkaline phosphatase, bicarbonate, blood urea nitrogen, 
calcium, chloride, complete blood count, creatinine, iron studies, ferritin, hemoglobin A1c (if 
available), phosphorus, potassium, sodium, total protein, 25 hydroxy vitamin D. eGFR 
calculation by modified MDRD equation from enzymatic creatinine determination) 
ο Monthly labs (postdialysis): blood urea nitrogen. Calculated urea reduction ratio from pre and 

 
10
post dialysis BUN. 
 
- Fasting blood sample (30 ml) for: 
ο Insulin and glucose 
ο Interleukin-6 (IL-6), tumor necrosis factor alpha (TNFα), C-reactive protein 
ο Glutathione (reduced and oxidized forms), glutathione peroxidase (GPx), superoxide dismutase 
(SOD), catalase (CAT), F2 isoprostane and protein thiols. 
 
- Measure quadriceps muscle strength (bilaterally) 
- 48 hour activity monitoring (using wrist watch monitor between dialysis sessions). 
 
5.2. Visit 2. Pre-EMS training data collection (Day -7 to 0).  Performed at dialysis unit or CRU where 
specified. Time required up to 4 hours.  
 
 
- Record any new medical conditions, symptoms or complaints 
- Record current dialysis orders 
- Record episodes of dialysis hypotension (SBP<90 or symptoms needing saline, last 6 HD sessions) 
- Record current exercise status (minutes of exercise per week) 
- Record concomitant medications 
- Malnutrition-inflammation (MIS) assessment 
- Kidney Disease Quality of Life-36 (KDQOL-36) assessment 
- Patient Health Questionaire-9 (PHQ-9) 
- Record vital sign measurements (predialysis or non-dialysis day, same timing as baseline measures) 
- 6-minute walk test 
- Record weight, waist circumference, hip circumference  
- Thigh circumference bilaterally 
- Record most recent dialysis monthly lab tests. (Drawn no more than 7 days prior to optimization 
visit) 
ο Monthly labs (predialysis): albumin, alkaline phosphatase, bicarbonate, blood urea nitrogen, 
calcium, chloride, complete blood count, creatinine, iron studies, ferritin, hemoglobin A1c (if 
available), phosphorus, potassium, sodium, total protein, 25 hydroxy vitamin D. eGFR 
calculation by modified MDRD equation from enzymatic creatinine determination) 
ο Monthly labs (postdialysis): blood urea nitrogen. Calculated urea reduction ratio from pre and 
post dialysis BUN. 
 
- Fasting blood sample (30 ml) for: 
ο Insulin and glucose 
ο Interleukin-6 (IL-6), tumor necrosis factor alpha (TNFα), C-reactive protein 
ο Glutathione (reduced and oxidized forms), glutathione peroxidase (GPx), superoxide dismutase 
(SOD), catalase (CAT), F2 isoprostane and protein thiols. 
 
-  Measure quadriceps muscle strength (bilaterally) 
- 48 hour activity monitoring (using wrist watch monitor between dialysis sessions). 
 
-  DXA scan (performed in CRU) 

 
11
-  Perform exercise stress test to determine peak VO2 (performed in CRU) 
 
5.3 Visit 3. Initiate EMS training (Day 0).  Performed in dialysis unit and CRU. 60-75 minutes. 30 
minutes extra for optional muscle biopsy, if performed.  
 
- Perform pre-EMS muscle biopsy before starting dialysis (optional) 
- EMS training is done while on hemodialysis starting approximately 30 min after dialysis initiation.  
ο Record pre-EMS blood pressure and pulse 
ο Initiate EMS training on quadriceps muscle of leg not used for muscle biopsy for 15 minutes 
while on dialysis 
ο Measure and record BP and pulse before and just prior to completion of EMS training. 
ο Record any symptoms related to EMS 
ο Train patient how to use EMS device.  
 
 
5.4 . Visits 4-8, 11. EMS training visits (Day 2/3 and 7). Performed in dialysis unit. Time required up 
to 45 minutes.  
 
- Record any new medical conditions, symptoms or complaints 
- Observe patient initiate and perform EMS training. 
 ο Adjust stimulus intensity to maximum tolerated by patient for both legs 
- Record EMS setting used. 
- Record pre-dialysis sitting BP and pulse 
- Record post-dialysis sitting BP and pulse 
 
 
5.5  Visits 9 &12. Outcome assessment visits (Day 56 & 112 ± 7 days). Performed in dialysis unit or 
CRU where specified. Time required up to 4 hours.  
 
- Record any new medical conditions, symptoms or complaints 
- Record current dialysis orders 
- Record episodes of dialysis hypotension (SBP<90 or symptoms needing saline, last 6 HD sessions) 
- Record current exercise status (minutes of exercise per week) 
- Record concomitant medications 
- Malnutrition-inflammation (MIS) assessment 
- Kidney Disease Quality of Life-36 (KDQOL-36) assessment 
- Patient Health Questionaire-9 (PHQ-9) 
- Record vital sign measurements (predialysis or non-dialysis day, same timing as baseline measures) 
- 6-minute walk test 
- Record weight, waist circumference, hip circumference  
- Record thigh circumference bilaterally 
- Record most recent dialysis monthly lab tests.  
ο Monthly labs (predialysis): albumin, alkaline phosphatase, bicarbonate, blood urea nitrogen, 
calcium, chloride, complete blood count, creatinine, iron studies, ferritin, hemoglobin A1c (if 
available), phosphorus, potassium, sodium, total protein, 25 hydroxy vitamin D. eGFR 
calculation by modified MDRD equation from enzymatic creatinine determination) 

 
12
ο Monthly labs (postdialysis): blood urea nitrogen. Calculated urea reduction ratio from pre and 
post dialysis BUN. 
 
- DXA scan (performed in CRU) 
- Measure quadriceps muscle strength (bilaterally) 
- Perform exercise stress test to determine peak VO2 (performed in CRU) 
- 48 hour activity monitoring (using wrist watch monitor between dialysis sessions). 
 
5. 6   Visits 10 & 13 Outcome assessment visits (Day 56 & 112 ± 7 days).  
- Fasting blood sample (30 ml) for: 
ο Insulin and glucose 
ο Interleukin-6 (IL-6), tumor necrosis factor alpha (TNFα), C-reactive protein 
ο Glutathione (reduced and oxidized forms), glutathione peroxidase (GPx), superoxide dismutase 
(SOD), catalase (CAT), F2 isoprostane, protein thiols. 
- Muscle biopsy (optional) 
 
5.7. Visit 14. Post-EMS visit (Day 142 - 149). Performed in dialysis unit. Time required 2-3 hours.  
- Record any new medical conditions, symptoms or complaints 
- Record current dialysis orders 
- Record episodes of dialysis hypotension (SBP<90 or symptoms needing saline, last 6 HD sessions) 
- Record current exercise status (minutes of exercise per week) 
- Record concomitant medications 
- Malnutrition-inflammation (MIS) assessment 
- Kidney Disease Quality of Life-36 (KDQOL-36) assessment 
- Patient Health Questionaire-9 (PHQ-9) 
- Record vital sign measurements (predialysis or non-dialysis day, same timing as baseline measures) 
- 6-minute walk test 
- Record weight, waist circumference, hip circumference  
- Record thigh circumference bilaterally 
- Record most recent dialysis monthly lab tests. (Drawn no more than 7 days prior to optimization 
visit) 
ο Monthly labs (predialysis): albumin, alkaline phosphatase, bicarbonate, blood urea nitrogen, 
calcium, chloride, complete blood count, creatinine, iron studies, ferritin, hemoglobin A1c (if 
available), phosphorus, potassium, sodium, total protein, 25-hydroxy vitamin D. eGFR 
calculation by modified MDRD equation from enzymatic creatinine determination) 
ο Monthly labs (postdialysis): blood urea nitrogen. Calculated urea reduction ratio from pre and 
post dialysis BUN. 
 
- Fasting blood sample (30 ml) for: 
ο Insulin and glucose 
ο Interleukin-6 (IL-6), tumor necrosis factor alpha (TNFα), C-reactive protein 
ο Glutathione (reduced and oxidized forms), glutathione peroxidase (GPx), superoxide dismutase 
(SOD), catalase (CAT), F2 isoprostane, protein thiols. 
 
- Measure quadriceps muscle strength (bilaterally) 
- 48 hour activity monitoring (using wrist watch monitor between dialysis sessions). 

 
13
 
6. Patient risks. 
 
6.1. Risk attributed to use of EMS device: 
 
6.1.1.  Muscle stimulation. Most patients will feel muscle contractions and perhaps some tingling of 
the skin but this should not be painful or harmful. There may be some muscle soreness initially as 
expected with new exercise but this should abate in a few days. The risks are minimal.  
 
6.1.2  Muscular tetany. If EMS is misused it could cause muscular tetany leading to muscle pain and 
muscle breakdown (rhabdomyolysis). The stimulation intensity will be monitored so the risk will be 
minimal. Patients will have control of the EMS device to turn it off or decrease the stimulation 
intensity if it is causing pain. They will be monitored clinically for signs of excessive muscle pain and 
told to decrease the stimulus intensity or cut down the length of training as needed to prevent this 
complication. 
 
6.1.3. Pacemaker or electrical stimulator complications. The electrical impulses generated by the EMS 
device might interfere with a cardiac pacemaker or other CNS/nerve stimulators. Patients with any 
implanted electrical device will be excluded from the study. 
 
6.1.4. Electric shock. If the device is not properly maintained or defective then it could deliver an 
electric shock. This risk will be minimized by maintaining and using the device per manufacturer’s 
instructions and have biomedical engineering check the device before use. 
 
6.1.5. Skin burns. This is theoretically possible if the electrodes are kept in place and stimulated for 
prolonged periods of time. The risk is minimal in the current study since skin contact time is limited. 
 
6.1.6. Skin bruising. Skin blood vessels may be damaged by the electrical stimulation and bruising 
could develop. The risk is minimal and will be monitored. EMS treatment parameters will be adjusted 
if this is clinically significant. 
 
 
6.2. Risks attributable to increased exercise from EMS or exercise stress tests for VO2 peak: 
 
Potential risks seen with the exercise stress testing for VO2 peak or EMS training are listed below 
along with the proposed management. All patients will have a formal exercise stress test to determine 
VO2 peak in the CRU before starting EMS. A physician will be immediately available for these tests. 
Problems seen with exercise will be detected during that initial testing and addressed before 
proceeding with EMS training. The cardiopulmonary demand of EMS will be less than the maximal 
exercise stress test.  
  
6.2.1. Angina or ischemic EKG changes. Exercise will be terminated and the patient monitored until 
the angina or EKG changes resolve. The study physician will be contacted. If the chest pain or EKG 
changes persist the patient will be transported to the ER for evaluation. Additional physician 
evaluation will be needed before the patient is allowed to continue in the study.  
 
6.2.2. Arrhythmia. Complex arrhythmias or second or third degree heart blocks will lead to immediate 
termination of the exercise stress test. The study physician will be contacted. The patient will be 

 
14
monitored until the arrhythmia resolves and the primary physician will be notified to follow-up on the 
results. Additional cardiology evaluation will be required before the patient can proceed in the study. 
  
6.2.3. Hypertension. Systolic BP >250 mmHg or diastolic > 120 mmHg will be grounds to terminate 
the exercise stress test and contact the study physician. The patient will be monitored until the blood 
pressure returns to baseline. The patient’s blood pressure management will be reviewed before 
proceeding into the EMS training phase of the study. An EMS session will be terminated for a systolic 
BP>200 or diastolic BP>110.  
  
6.2.4. Hypotension. Systolic BP dropping more than 20 mmHg during the exercise stress test will be 
grounds for terminating the test.  The study physician will consult with the primary care physician 
about further cardiology evaluation before proceeding with the EMS training. An EMS session will be 
terminated for a systolic BP<90.  
 
A prior study of EMS during hemodialysis found that EMS produced a statistically significant 
increase in BP (SBP increased by 8 mmHg and DBP increased by 3 mmHg) while the device was 
turned on compared to when it was turned off.29  Overall the average BP on dialysis was higher when 
people used EMS for 15 minutes out of every hour while on dialysis compared to no EMS (average BP 
= 125/66 with intermittent EMS compared to 121/64 when no EMS was performed). Fewer 
hypotensive episodes during a dialysis session were seen when patients used intermittent EMS 
compared to no EMS. 
 
6.2.5.  Fracture. Stress testing or EMS could lead to a bone fracture particularly in a patient with severe 
osteoporosis but this is considered unlikely to occur.  EMS is also being studied as a mechanism to 
prevent osteoporosis.  
  
6.2.5. Dizziness or confusion. Dizziness or confusion occurring during the exercise stress test or during 
an EMS training session is grounds for terminating the exercise and monitoring the patient until the 
symptoms resolve. The study physician will evaluate the circumstances of the event to decide whether 
further evaluation is needed and whether the patient can continue in the study.  
 
6.2.6. Hypoglycemia. Low blood sugar with exercise might occur particularly in patients who are 
diabetic. If symptoms of hypoglycemia occur then the exercise will be terminated and sugar 
administered to the patient. A blood sugar will be measured and the study physician notified. This is 
unlikely to occur during EMS as the patient will be on hemodialysis, which provides glucose in the 
dialysate.   
 
6.3. Risk of phlebotomy.  
 
Phlebotomy may cause discomfort due to needle puncture, hematoma formation, blood loss, 
infection or syncope. It will be performed at the time of dialysis from your dialysis needle by trained 
personnel to limit risk.  
 
6.4. Risk of radiation from a DXA scan.  
 
A DXA scan delivers a dose of radiation that is approximately one tenth that of a standard chest X-
ray and equivalent to the dose of radiation received during one-day exposure to natural background 
radiation.  
 

 
15
6.5. Risk of taking health and depression assessment questionnaires.  
 
Questions about personal health or depression might raise anxiety or concerns about personal 
health. If this occurs these will be addressed with the patient by a member of the study team or referred 
to their primary provider or social worker as appropriate.   
 
6.6. Risk of a muscle biopsy under ultrasound guidance.  
 
The muscle biopsy may cause transient pain from the injection of lidocaine, infection or hematoma 
at the biopsy site. There may be soreness or a small bump at the biopsy site for a few days that will 
resolve. Risks are limited by having the procedure done by trained investigators under aseptic 
technique using an automated biopsy gun (Temno) under ultrasound guidance.  
 
6.7. Risk of gene expression analysis.  
 
No risks are expected to occur from gene expression analysis. Gene expression analysis looks at 
mRNA expression and not DNA sequence. We will not be doing DNA analysis and will not 
investigate a patient’s genetic mutations or susceptibility to disease.  
 
 
6.7. Risk of loss of confidentiality.  
 
Data collected for this study will be labeled with a unique patient study number that does not 
identify the patient. The data will be stored in study binders in a secure locked room and electronically 
in folders on a network server that is password protected. The link between patient study number and 
patient identification information such as name or hospital number will be stored separately. Access to 
the study data will be limited to members of the study team who require access and regulatory bodies 
such as the IRB. Blood or tissue samples will be labeled and stored only with the de-identified patient 
study number.   
 
7. Safety monitoring plan 
 
An independent physician from the University of Iowa Department of Medicine who is not an 
investigator in this research study and has no conflicts of interest with the proposed research will be 
asked to periodically review patient safety data and provide written feedback on whether the study can 
continue as planned.  The reviews will occur every 3 months starting after the first patient is enrolled.  
The Independent Safety Monitor (ISM) will be provided a copy of all the adverse events that we have 
recorded for patients in the study. If an unexpected SAE occurs that is possibly or probably related to 
the study procedures or intervention then the ISM (and IRB) will be notified of this as soon as the 
investigators become aware of the event.  The ISM will be asked to provide written documentation of 
whether the study can continue as planned, should be modified or should be terminated. The IRB will 
be notified expeditiously if the ISM feels the study should be modified or terminated. If the ISM feels 
the study can continue as planned then this information will be provided to the IRB at the time of 
annual continuing reviews.  
 
8. Statistical analysis/Power calculations. 
 
This is a pilot feasibility and preliminary efficacy study to determine whether EMS can be 
delivered to this population and to assess the magnitude of the change and variability in the primary 
and secondary outcome measures. Data derived from this study will allow planning of a larger 
randomized controlled study. We plan to recruit up to 15 patients into the trial and complete the trial 
within 1 year. There will be 5 measurements of the primary and most of the secondary outcome 
variables in each patient. These will be performed at enrollment, after 4 weeks of “dialysis care 

 
16
optimization” before starting the EMS intervention, then at 8 and 16 weeks after initiation of the EMS 
intervention and finally 4 weeks after stopping EMS. The DXA scan, measurement of peak VO2 will 
only be performed three times (before starting EMS and then again at 8 and 16 weeks of EMS 
training). The optional muscle biopsy for gene expression analysis will be performed up to 4 times in 
patients who agree to this analysis (before and after the first EMS treatment, at 8 and 16 weeks of EMS 
training). Summary statistics will be used to describe and analyze the data.  Changes over time in the 
outcome variables will be analyzed using the Friedman analysis of variance by ranks to determine if 
there was a statistically significant effect of EMS. A P<0.05 will be considered statistically significant. 
Regression analysis will be used to determine if the change in HOMA-IR or LAR is correlated with the 
magnitude of the observed exercise training effect (i.e. the increase in peak VO2 or quadriceps muscle 
strength at 4 months) or maximum intensity of EMS stimulation used during training.  
 
Based on an estimated baseline mean (± SD) HOMA-IR score of 4.8 (± 1.7) mg/dl.µU/ml, a sample 
size of 15 patients will provide 80% power to detect a 28% decrease in the HOMA-IR score (by a 
paired t-test analysis) at the P=0.05 level.  This decrease in HOMA-IR score would be predicted to be 
associated with a clinically significant reduction in mortality.33    
  
 
9. Selected methods 
 
9.1. HOMA-IR and leptin-adiponectin ratio (LAR). HOMA-IR and LAR are both separate measures of 
insulin resistance. Patients will fast for 12 hours and withhold taking their morning insulin or oral 
antidiabetic agents prior to the test. Venous blood (10 ml) will be drawn into a red top tube (serum 
separator) placed on ice and centrifuged at 3,000 rpm at 4oC to separate serum. The serum is aliquoted 
and stored at -80oC until assay. Fasting serum insulin (µU/ml) is measured using an 
electrochemiluminescent assay and fasting serum glucose (mg/dl) by a hexokinase assay in the UIHC 
clinical chemistry lab. HOMA-IR score is calculated using the homeostatic model assessment equation 
(HOMA-IR = fasting glucose (mg/dl) x fasting Insulin (µU/mL) / 405). Serum leptin (ng/ml) and 
adiponectin (ng/ml) are measured by RIA using Linco kits (Linco, St. Charles, MO) in Dr Murry’s lab 
at Iowa.  
 
9.2. Exercise stress test for peak VO2. A supervised graded symptom-limited treadmill exercise test to 
determine peak VO2 will be performed before starting EMS training and again after 8 and 16 weeks of 
EMS training. This will establish baseline aerobic exercise capacity (peak VO2) and determine if there 
are any cardiopulmonary problems with performing exercise. The testing will be performed in the 
CRU using a Balke protocol. Briefly for the Balke protocol patients walk on a treadmill at a set speed 
(3.3 mph for men and 3.0 mph for women) with a stating grade of 0%. For men the grade is increased 
to 2% at 1 minute and 1% per minute thereafter. For women the grade is increased by 2.5% every 3 
minutes.  The study ends when the patient is exhausted and asks to stop. The total duration of exercise 
time is recorded. The study is performed with continuous EKG monitoring, continuous measurement 
of respiratory O2 uptake and CO2 excretion using a metabolic cart and blood pressure monitoring each 
minute. The monitoring starts at rest and is continued until the symptoms and vital signs return to near 
baseline levels.  The total study time is expected to be 30-45 minutes. The study is terminated early for 
angina, excessive shortness of breath or fatigue, dizziness or confusion, complex arrhythmias, second 
or third degree heart block, ECG changes suggestive of ischemia, excessive hypertension (systolic BP 
> 250 mmHg or diastolic BP > 120 mmHg) or hypotension (a sudden drop in systolic BP > 20 mmHg). 
The report will include the peak work rate achieved in METs and measured peak VO2, peak heart rate 
and blood pressure, and any abnormal signs or symptoms that occurred during or after the test. The 

 
17
ECG report will note any abnormalities in the EKG at rest, with exercise, and after return to baseline 
including the occurrence of any arrhythmias. If the EKG demonstrates ischemia changes, the time and 
double product at which the changes initially occurred will be specified. The time required for the 
exercise test is approximately 45 minutes. 
 
For patients who cannot walk on a treadmill we also can offer bicycle ergometry as an option.  
 
9.3. Quadriceps muscle strength. Quadriceps muscle strength will be measured in both legs five times: 
at baseline, after care optimization, at 8 and 16 weeks of EMS training and finally 4 weeks after 
stopping EMS training to determine the effect of EMS training on muscle strength. Muscle strength 
will be measured using a belt-stabilized hand held dynamometer (BSHHD) using a validated 
technique.43 Briefly, isometric knee extension strength (torque) will be measured with patients seated 
on an elevated chair and their knees bent at about 90o of flexion (i.e., the legs hanging vertical). 
Patients are stabilized to the chair with straps around proximal thighs and waist. A dynamometer-
stabilizing belt is passed around a bar secured behind the back legs of the chair and over a calibrated 
hand-held dynamometer that was placed against the anterior leg of the patient just proximal to the 
malleoli.  The distance from the estimated knee axis of rotation (lateral epicondyle of femur) to the 
midpoint of dynamometer is measured and recorded for calculation of torque. The patient is asked to 
perform three leg extensions separated by 30 seconds of rest. The first effort is submaximal to get the 
patient accustom to the procedure. Then two full strength efforts are performed and used for 
calculation of average peak force (N) and peak torque (N.m) generated. The test is performed 
sequentially on both legs at each measurement session. The total test time is approximately 15 minutes.  
 
9.4. Dual energy X-ray absorptiometry (DXA).  Advanced body composition analysis will be 
performed by DXA scan to measure total body and regional fat and lean mass before starting EMS 
training and again after 8 and 16 weeks of EMS training. Specific areas of interest will be in 
determining whether EMS training reduces visceral fat mass and produces the expected changes in 
thigh fat and muscle mass. The test is performed in the CRU at the University of Iowa using a 
Discovery A DXA scanner with the latest software version package (Hologic Inc., Bedford, MA). The 
advanced body composition analysis software package measures total and regional body composition 
and visceral fat mass (cm2) using a validated technique that produces comparable results to a CT scan 
with less cost and radiation exposure.71 The total radiation dose is less than one tenth that of a standard 
CXR and equivalent to one-day exposure to natural background radiation. Patients will be asked to 
report to the CRU dressed metal-free (no zippers, jewelry, piercings, underwire bra, etc.). Each study 
will take approximately 30 minutes.  
  
9.5. Circulating markers of inflammation (serum cytokines & CRP).  Serum levels of the pro-
inflammatory cytokines, interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFα) and the acute 
phase inflammation marker C-reactive protein (CRP) will be measured five times: at baseline, after 
care optimization, at 8 and 16 weeks of EMS training and finally 4 weeks after stopping EMS training 
to determine the effect of EMS training on inflammation.  Venous blood (10 ml) will be drawn into a 
red top tube (serum separator) placed on ice and centrifuged at 3,000 rpm at 4oC to separate serum. 
The serum is aliquoted and stored at -80oC until assay.  The assays are performed in Dr Daryl Murry’s 
lab at Iowa. Circulating IL-6 and TNFα will be analyzed using ELISA (Biosource International). C-
reactive protein will be quantified via immunoturbidimetric assay (Raichem). 
 
9.6. Oxidative stress markers. Markers of oxidative stress and erythrocyte antioxidant levels will be 

 
18
measured five times: at baseline, after care optimization, at 8 and 16 weeks of EMS training and finally 
4 weeks after stopping EMS training to determine the effect of EMS training on oxidative stress. 
Markers of oxidative stress include plasma F2 isoprostane (isomers of the cyclooxygenase-derived 
prostaglandin F2α (PGF2α ), plasma protein thiols and the ratio of reduced to oxidized glutathione 
(GSH/GSSG) in red blood cells. Venous blood is drawn into 10 ml tubes containing anticoagulant 
(EDTA), placed on ice until being centrifuged at 3,000 rpm at 4oC to separate plasma and RBCs. The 
plasma is aliquoted and stored at -80oC until assay. F2 isoprostanes are measured using liquid 
chromatography/mass spectroscopy (LC/MS) as described.72 Protein thiols are measured using 
Ellman’s reagent (5’5’ dithio-bis (2-nitrobenzoic acid)) that reacts with free thiol groups to cleave the 
disulfide linkage ultimately releasing 2-nitro-5-thiobenzoate (NTB-2) which can be quantitated 
spectrophotometrically by its absorbance at 412 nm.61  The RBC’s separated from plasma are used to 
measure the content of reduced (GSH) and oxidized (GSSG) glutathione by HPLC in Dr Beuttner’s lab 
at Iowa. Determination of the RBC cell count and size (MCV) allows determination of GSH and 
GSSG content per RBC. RBC’s separated from plasma are also used to measure the activity GPx, CAT 
and SOD by standard techniques used in Dr Beuttner’s lab.73 
 
9.7. Muscle biopsy. Muscle biopsies are done for the extraction of mRNA to analyze gene expression 
at 3 time points: immediately before and after 8 and 16 weeks of EMS training to determine the 
chronic effect of EMS training on gene expression. The muscle biopsies will be optional and the 
patient can participate in the study without agreeing to have the muscle biopsies. Percutaneous muscle 
biopsies are taken from the vastus lateralis muscle using a Temno biopsy needle (T1420; Cardinal 
Health) under ultrasound guidance as previously performed here.70 Up to 4 biopsies are performed to 
obtain a total of ~100-200 mg of muscle tissue which is sufficient for ~100-400 ng of total RNA. 
Muscle tissue is immediately placed into RNAlater (Ambion) and stored at -80oC until use. 
 
9.8. Gene expression profiling.  Gene expression profiling is done using the Human Exon 1.0 ST 
arrays (Affymetrix, Inc) which measures >17,000 different mRNA transcripts with about 4 probes per 
exon and 40 probes per gene. This recognizes both the gene being expressed and differences in 
alternative splice variants of that gene. To perform gene expression analysis total RNA must be 
extracted, converted to cDNA, the cDNA amplified, sense target cDNA prepared and then fragmented 
and labeled for detection prior to applying to the gene expression arrays.  
 
As previously described70, total RNA is extracted using TRIzol solution (Invitrogen) according to 
manufacturer’s instruction. Microarray hybridization will be performed at the University of Iowa DNA 
facility.70   Briefly, 50 ng total RNA is converted by Single Primer Isothermal Amplification (SPIA) to 
cDNA using the whole transcriptome (WT)-Ovation Pico RNA Amplification System (NuGEN 
Technologies, San Carlos, California). The amplified cDNA product is purified through a Qiagen 
MinElute Reaction Cleanup Column (Qiagen). A 4-µg quantity of amplified cDNA was used to 
generate sense target (ST)-cDNA using the WT-Ovation Exon Module (NuGEN Technologies), and 
again cleaned up with the Qiagen column. A 5-µg quantity of this product is fragmented (50-100 
bases) and biotin labeled using the NuGEN FL-Ovation cDNA Biotin Module (NuGEN Technologies) 
prior to use for gene expression profiling.  
  
For gene expression analysis the resulting biotin-labeled cDNA is mixed with eukaryotic 
hybridization buffer (Affymetrix, Inc., Santa Clara, California), placed onto Human Exon 1.0 ST 
arrays and incubated at 45oC for 18 h with 60-rpm rotation in a GeneChip hybridization oven (Model 
640; Affymetrix). After hybridization, the arrays are washed, stained with streptavidin–phycoerythrin 
(Molecular Probes, Inc., Eugene, Oregon), and signal amplified with anti-streptavidin antibody (Vector 

 
19
Laboratories, Inc., Burlingame, California) using a fluidics station (Model 450; Affymetrix). Arrays 
are scanned (Model 3000 7G scanner; Affymetrix) and data collected using the using GeneChip 
operating software (GCOS), version 1.4. 
 
In Affymetrix Human Exon 1.0 ST arrays, the log2 hybridization signal reflects the mean signal 
intensity of all exon probes specific for an individual mRNA, and is proportional to the level of that 
mRNA. We will perform two main comparisons: 1) the effect of acute exercise comparing the gene 
expression levels 4 hours after the first EMS training to baseline before training and 2) the effect of 
chronic EMS training (8 and 16 weeks) compared to the Pre-EMS training sample. We will restrict the 
analysis to mRNA’s that have a mean log2 hybridization signal of >5 in either the pre-EMS sample or 
the 8 or 16 week EMS treatment conditions. We will use paired t-tests to determine which of the 
remaining mRNA’s are present at a significantly different level (adjusting for the false discovery rate 
inherent in multiple testing) in the EMS treatment compared to the baseline sample. The list of 
differentially expressed genes will be examined for important biological pathways relating to muscle 
glucose metabolism and insulin response, mitochondrial biogenesis and function, muscle phenotype, 
growth and energy metabolism, oxidant stress and inflammation. We will also use bioinformatics tools 
such as Onto-Express to help translate the list of differentially expressed genes into functional 
categories such as biological process or cellular role based on the annotation using Gene Ontology 
terms. All microarray data will be deposited in the GEO database.  
 
10.  References 
1. 
U.S. Renal Data System, USRDS 2012 Annual Data report: Atlas of End-Stage Renal Disease 
in the United States, National Institutes of Health, National Institute of Diabetes and Digestive 
and Kidney Diseases, Bethesda, MD, . 2012. 
2. 
Carrero JJ, de Jager DJ, Verduijn M, et al. Cardiovascular and noncardiovascular mortality 
among men and women starting dialysis. Clin J Am Soc Nephrol. Jul 2011;6(7):1722-1730. 
3. 
Kramann R, Floege J, Ketteler M, Marx N, Brandenburg VM. Medical options to fight 
mortality in end-stage renal disease: a review of the literature. Nephrol Dial Transplant. Oct 8 
2012. 
4. 
Chertow GM, Levin NW, Beck GJ, et al. In-center hemodialysis six times per week versus 
three times per week. N Engl J Med. Dec 9 2010;363(24):2287-2300. 
5. 
Johansen KL. Exercise in the end-stage renal disease population. J Am Soc Nephrol. Jun 
2007;18(6):1845-1854. 
6. 
Matsuzawa R, Matsunaga A, Wang G, et al. Habitual Physical Activity Measured by 
Accelerometer and Survival in Maintenance Hemodialysis Patients. Clin J Am Soc Nephrol. 
Sep 13 2012. 
7. 
Kohl Ld, Signori LU, Ribeiro RA, et al. Prognostic value of the six-minute walk test in end-
stage renal disease life expectancy: a prospective cohort study. Clinics (Sao Paulo). 
2012;67(6):581-586. 
8. 
Koyama H, Fukuda S, Shoji T, et al. Fatigue is a predictor for cardiovascular outcomes in 
patients undergoing hemodialysis. Clin J Am Soc Nephrol. Apr 2010;5(4):659-666. 
9. 
Wilhelm-Leen ER, Hall YN, M KT, Chertow GM. Frailty and chronic kidney disease: the 
Third National Health and Nutrition Evaluation Survey. Am J Med. Jul 2009;122(7):664-671 
e662. 
10. 
Johansen KL, Painter P. Exercise in individuals with CKD. Am J Kidney Dis. Jan 
2012;59(1):126-134. 

 
20
11. 
Kettner-Melsheimer A, Weiss M, Huber W. Physical work capacity in chronic renal disease. 
Int J Artif Organs. Jan 1987;10(1):23-30. 
12. 
Heiwe S, Jacobson SH. Exercise training for adults with chronic kidney disease. Cochrane 
Database Syst Rev. 2011(10):CD003236. 
13. 
Segura-Orti E, Johansen KL. Exercise in end-stage renal disease. Semin Dial. Jul-Aug 
2010;23(4):422-430. 
14. 
Rus RR, Ponikvar R, Kenda RB, Buturovic-Ponikvar J. Effect of local physical training on the 
forearm arteries and veins in patients with end-stage renal disease. Blood Purif. 
2003;21(6):389-394. 
15. 
Kouidi E, Karagiannis V, Grekas D, et al. Depression, heart rate variability, and exercise 
training in dialysis patients. Eur J Cardiovasc Prev Rehabil. Apr 2010;17(2):160-167. 
16. 
Deligiannis A, Kouidi E, Tourkantonis A. Effects of physical training on heart rate variability 
in patients on hemodialysis. Am J Cardiol. Jul 15 1999;84(2):197-202. 
17. 
Goldberg AP, Geltman EM, Gavin JR, 3rd, et al. Exercise training reduces coronary risk and 
effectively rehabilitates hemodialysis patients. Nephron. 1986;42(4):311-316. 
18. 
Piepoli MF, Davos C, Francis DP, Coats AJ. Exercise training meta-analysis of trials in patients 
with chronic heart failure (ExTraMATCH). Bmj. Jan 24 2004;328(7433):189. 
19. 
O'Connor CM, Whellan DJ, Lee KL, et al. Efficacy and safety of exercise training in patients 
with chronic heart failure: HF-ACTION randomized controlled trial. Jama. Apr 8 
2009;301(14):1439-1450. 
20. 
Sillen MJ, Speksnijder CM, Eterman RM, et al. Effects of neuromuscular electrical stimulation 
of muscles of ambulation in patients with chronic heart failure or COPD: a systematic review 
of the English-language literature. Chest. Jul 2009;136(1):44-61. 
21. 
Mohr T, Dela F, Handberg A, Biering-Sorensen F, Galbo H, Kjaer M. Insulin action and long-
term electrically induced training in individuals with spinal cord injuries. Med Sci Sports Exerc. 
Aug 2001;33(8):1247-1252. 
22. 
Kawaguchi T, Shiba N, Maeda T, et al. Hybrid training of voluntary and electrical muscle 
contractions reduces steatosis, insulin resistance, and IL-6 levels in patients with NAFLD: a 
pilot study. Journal of gastroenterology. Jun 2011;46(6):746-757. 
23. 
Kawaguchi T, Shiba N, Takano Y, Maeda T, Sata M. Hybrid training of voluntary and 
electrical muscle contractions decreased fasting blood glucose and serum interleukin-6 levels in 
elderly people: a pilot study. Appl Physiol Nutr Metab. Apr 2011;36(2):276-283. 
24. 
Karavidas AI, Raisakis KG, Parissis JT, et al. Functional electrical stimulation improves 
endothelial function and reduces peripheral immune responses in patients with chronic heart 
failure. Eur J Cardiovasc Prev Rehabil. Aug 2006;13(4):592-597. 
25. 
Karavidas A, Parissis J, Arapi S, et al. Effects of functional electrical stimulation on quality of 
life and emotional stress in patients with chronic heart failure secondary to ischaemic or 
idiopathic dilated cardiomyopathy: a randomised, placebo-controlled trial. Eur J Heart Fail. Jul 
2008;10(7):709-713. 
26. 
Deftereos S, Giannopoulos G, Raisakis K, et al. Comparison of muscle functional electrical 
stimulation to conventional bicycle exercise on endothelium and functional status indices in 
patients with heart failure. Am J Cardiol. Dec 1 2010;106(11):1621-1625. 
27. 
Banerjee P, Caulfield B, Crowe L, Clark A. Prolonged electrical muscle stimulation exercise 
improves strength and aerobic capacity in healthy sedentary adults. J Appl Physiol. Dec 
2005;99(6):2307-2311. 

 
21
28. 
Griffin L, Decker MJ, Hwang JY, et al. Functional electrical stimulation cycling improves body 
composition, metabolic and neural factors in persons with spinal cord injury. Journal of 
electromyography and kinesiology : official journal of the International Society of 
Electrophysiological Kinesiology. Aug 2009;19(4):614-622. 
29. 
Farese S, Budmiger R, Aregger F, Bergmann I, Frey FJ, Uehlinger DE. Effect of 
transcutaneous electrical muscle stimulation and passive cycling movements on blood pressure 
and removal of urea and phosphate during hemodialysis. Am J Kidney Dis. Oct 
2008;52(4):745-752. 
30. 
Block GA, Klassen PS, Lazarus JM, Ofsthun N, Lowrie EG, Chertow GM. Mineral 
metabolism, mortality, and morbidity in maintenance hemodialysis. J Am Soc Nephrol. Aug 
2004;15(8):2208-2218. 
31. 
Chiu YW, Teitelbaum I, Misra M, de Leon EM, Adzize T, Mehrotra R. Pill burden, adherence, 
hyperphosphatemia, and quality of life in maintenance dialysis patients. Clin J Am Soc 
Nephrol. Jun 2009;4(6):1089-1096. 
32. 
DeFronzo RA, Alvestrand A, Smith D, Hendler R, Hendler E, Wahren J. Insulin resistance in 
uremia. J Clin Invest. Feb 1981;67(2):563-568. 
33. 
Shinohara K, Shoji T, Emoto M, et al. Insulin resistance as an independent predictor of 
cardiovascular mortality in patients with end-stage renal disease. J Am Soc Nephrol. Jul 
2002;13(7):1894-1900. 
34. 
Hung AM, Sundell MB, Egbert P, et al. A comparison of novel and commonly-used indices of 
insulin sensitivity in African American chronic hemodialysis patients. Clin J Am Soc Nephrol. 
Apr 2011;6(4):767-774. 
35. 
Landau M, Kurella-Tamura M, Shlipak MG, et al. Correlates of insulin resistance in older 
individuals with and without kidney disease. Nephrol Dial Transplant. Sep 2011;26(9):2814-
2819. 
36. 
Trirogoff ML, Shintani A, Himmelfarb J, Ikizler TA. Body mass index and fat mass are the 
primary correlates of insulin resistance in nondiabetic stage 3-4 chronic kidney disease patients. 
Am J Clin Nutr. Dec 2007;86(6):1642-1648. 
37. 
Axelsson J. The emerging biology of adipose tissue in chronic kidney disease: from fat to facts. 
Nephrol Dial Transplant. Oct 2008;23(10):3041-3046. 
38. 
Thompson D, Karpe F, Lafontan M, Frayn K. Physical activity and exercise in the regulation of 
human adipose tissue physiology. Physiol Rev. Jan 2012;92(1):157-191. 
39. 
Zanchi NE, Almeida FN, Lira FS, et al. Renewed avenues through exercise muscle contractility 
and inflammatory status. ScientificWorldJournal. 2012;2012:584205. 
40. 
Bologa RM, Levine DM, Parker TS, et al. Interleukin-6 predicts hypoalbuminemia, 
hypocholesterolemia, and mortality in hemodialysis patients. Am J Kidney Dis. 
1998;32(1):107-114. 
41. 
Pachaly MA, do Nascimento MM, Suliman ME, et al. Interleukin-6 is a better predictor of 
mortality as compared to C-reactive protein, homocysteine, pentosidine and advanced oxidation 
protein products in hemodialysis patients. Blood Purif. 2008;26(2):204-210. 
42. 
Beberashvili I, Sinuani I, Azar A, et al. IL-6 levels, nutritional status, and mortality in prevalent 
hemodialysis patients. Clin J Am Soc Nephrol. Sep 2011;6(9):2253-2263. 
43. 
Bohannon RW, Bubela DJ, Wang YC, Magasi SR, Gershon RC. Adequacy of belt-stabilized 
testing of knee extension strength. J Strength Cond Res. Jul 2011;25(7):1963-1967. 

 
22
44. 
Kalantar-Zadeh K, Kopple JD, Block G, Humphreys MH. A malnutrition-inflammation score is 
correlated with morbidity and mortality in maintenance hemodialysis patients. Am J Kidney 
Dis. Dec 2001;38(6):1251-1263. 
45. 
Rambod M, Bross R, Zitterkoph J, et al. Association of Malnutrition-Inflammation Score with 
quality of life and mortality in hemodialysis patients: a 5-year prospective cohort study. Am J 
Kidney Dis. Feb 2009;53(2):298-309. 
46. 
Watnick S, Wang PL, Demadura T, Ganzini L. Validation of 2 depression screening tools in 
dialysis patients. Am J Kidney Dis. Nov 2005;46(5):919-924. 
47. 
Sullivan MD, O'Connor P, Feeney P, et al. Depression predicts all-cause mortality: 
epidemiological evaluation from the ACCORD HRQL substudy. Diabetes Care. Aug 
2012;35(8):1708-1715. 
48. 
Lowrie EG, Curtin RB, LePain N, Schatell D. Medical outcomes study short form-36: a 
consistent and powerful predictor of morbidity and mortality in dialysis patients. Am J Kidney 
Dis. Jun 2003;41(6):1286-1292. 
49. 
Kimmel PL, Patel SS. Quality of life in patients with chronic kidney disease: focus on end-
stage renal disease treated with hemodialysis. Semin Nephrol. Jan 2006;26(1):68-79. 
50. 
Meuwese CL, Carrero JJ, Stenvinkel P. Recent insights in inflammation-associated wasting in 
patients with chronic kidney disease. Contrib Nephrol. 2011;171:120-126. 
51. 
Panichi V, Maggiore U, Taccola D, et al. Interleukin-6 is a stronger predictor of total and 
cardiovascular mortality than C-reactive protein in haemodialysis patients. Nephrol Dial 
Transplant. May 2004;19(5):1154-1160. 
52. 
Beavers KM, Brinkley TE, Nicklas BJ. Effect of exercise training on chronic inflammation. 
Clin Chim Acta. Jun 3 2010;411(11-12):785-793. 
53. 
Handelman GJ, Walter MF, Adhikarla R, et al. Elevated plasma F2-isoprostanes in patients on 
long-term hemodialysis. Kidney Int. May 2001;59(5):1960-1966. 
54. 
Miyata T, Sugiyama S, Saito A, Kurokawa K. Reactive carbonyl compounds related uremic 
toxicity ("carbonyl stress"). Kidney Int Suppl. Feb 2001;78:S25-31. 
55. 
Himmelfarb J, McMonagle E, McMenamin E. Plasma protein thiol oxidation and carbonyl 
formation in chronic renal failure. Kidney Int. Dec 2000;58(6):2571-2578. 
56. 
Bayes B, Pastor MC, Bonal J, Foraster A, Romero R. Oxidative stress, inflammation and 
cardiovascular mortality in haemodialysis--role of seniority and intravenous ferrotherapy: 
analysis at 4 years of follow-up. Nephrol Dial Transplant. Apr 2006;21(4):984-990. 
57. 
Stenvinkel P, Diczfalusy U, Lindholm B, Heimburger O. Phospholipid plasmalogen, a 
surrogate marker of oxidative stress, is associated with increased cardiovascular mortality in 
patients on renal replacement therapy. Nephrol Dial Transplant. Apr 2004;19(4):972-976. 
58. 
Coombes JS, Fassett RG. Antioxidant therapy in hemodialysis patients: a systematic review. 
Kidney Int. Feb 2012;81(3):233-246. 
59. 
Gomez-Cabrera MC, Domenech E, Vina J. Moderate exercise is an antioxidant: upregulation of 
antioxidant genes by training. Free Radic Biol Med. Jan 15 2008;44(2):126-131. 
60. 
Wilund KR, Tomayko EJ, Wu PT, et al. Intradialytic exercise training reduces oxidative stress 
and epicardial fat: a pilot study. Nephrol Dial Transplant. Aug 2010;25(8):2695-2701. 
61. 
Ramos LF, Shintani A, Ikizler TA, Himmelfarb J. Oxidative stress and inflammation are 
associated with adiposity in moderate to severe CKD. J Am Soc Nephrol. Mar 2008;19(3):593-
599. 
62. 
Morrow JD. The isoprostanes: their quantification as an index of oxidant stress status in vivo. 
Drug metabolism reviews. Aug-Nov 2000;32(3-4):377-385. 

 
23
63. 
Elokda AS, Nielsen DH. Effects of exercise training on the glutathione antioxidant system. Eur 
J Cardiovasc Prev Rehabil. Oct 2007;14(5):630-637. 
64. 
Dursun B, Dursun E, Suleymanlar G, et al. Carotid artery intima-media thickness correlates 
with oxidative stress in chronic haemodialysis patients with accelerated atherosclerosis. 
Nephrol Dial Transplant. May 2008;23(5):1697-1703. 
65. 
Kaysen GA. Biochemistry and biomarkers of inflamed patients: why look, what to assess. Clin 
J Am Soc Nephrol. Dec 2009;4 Suppl 1:S56-63. 
66. 
Volkova N, Arab L. Evidence-based systematic literature review of hemoglobin/hematocrit and 
all-cause mortality in dialysis patients. Am J Kidney Dis. Jan 2006;47(1):24-36. 
67. 
Avram MM, Blaustein D, Fein PA, Goel N, Chattopadhyay J, Mittman N. Hemoglobin predicts 
long-term survival in dialysis patients: a 15-year single-center longitudinal study and a 
correlation trend between prealbumin and hemoglobin. Kidney Int Suppl. Nov 2003(87):S6-11. 
68. 
Block GA. Therapeutic interventions for chronic kidney disease-mineral and bone disorders: 
focus on mortality. Curr Opin Nephrol Hypertens. Jul 2011;20(4):376-381. 
69. 
McIntyre CW. Haemodialysis-induced myocardial stunning in chronic kidney disease - a new 
aspect of cardiovascular disease. Blood Purif. 2010;29(2):105-110. 
70. 
Adams CM, Suneja M, Dudley-Javoroski S, Shields RK. Altered mRNA expression after long-
term soleus electrical stimulation training in humans with paralysis. Muscle & nerve. Jan 
2011;43(1):65-75. 
71. 
Micklesfield LK, Goedecke JH, Punyanitya M, Wilson KE, Kelly TL. Dual-energy X-ray 
performs as well as clinical computed tomography for the measurement of visceral fat. Obesity 
(Silver Spring, Md.). May 2012;20(5):1109-1114. 
72. 
Sircar D, Subbaiah PV. Isoprostane measurement in plasma and urine by liquid 
chromatography-mass spectrometry with one-step sample preparation. Clin Chem. Feb 
2007;53(2):251-258. 
73. 
Wagner BA, Buettner GR, Oberley LW, Darby CJ, Burns CP. Myeloperoxidase is involved in 
H2O2-induced apoptosis of HL-60 human leukemia cells. J Biol Chem. Jul 21 
2000;275(29):22461-22469.