Exercise to Prevent Muscle Mass and Functional Loss in Elderly Dialysis Patients
Phase: NA
Conditions: Chronic Kidney Disease
Sponsor: VA Office of Research and Development

 
 
 
 
 
 
 
 
 
Exercise to Prevent Muscle Mass and Functional Loss in Elderly Dialysis Patients  
 
           NCT #01990495 
 
           Date: 5/31/2020

02a. Research Plan 
 
Background and Significance.   
 
               Prevalence and Consequences of Chronic Kidney Disease. In the United States, the proportion 
of the population over 65 years is predicted to grow from 12.4% (35 million) in 2000 to 19.2% (71 million) 
in 2030 (1). Thus, the proportion of elderly dialysis patients that currently comprise approximately half of 
the incident adult US dialysis population can be expected to increase (2). Aging alone leads to a loss of 
muscle mass and strength with decreased function, increased atrophy, and eventually frailty following bed 
rest (1; 3; 4). Many of the causes of age-related wasting are the same as those in maintenance 
hemodialysis (MHD) patients, including nutritional and hormonal derangements, oxidative stress, inactivity 
and co-morbid conditions (5;6). In elderly dialysis patients the situation is compounded, since uremia per-
se causes a loss of muscle mass and function and together with the aging process predisposes these 
patients to the development of frailty with an increase in fall-related injuries, a decrease in quality of life 
(QOL) and an increase in morbidity and mortality (7; 8). In chronic kidney disease (CKD) physical function 
declines as renal failure progresses (9) and in those new to dialysis the vast majority (up to 95%) report 
significantly impaired physical function and reduced activity which is worse in the elderly (10). Older 
dialysis patients have even more co-morbidities and functional limitations; the death rate of prevalent 
dialysis patients increases by 2-fold in those aged 66-74 and approximately 3-fold in those aged 75-79 vs. 
those <65 years (2;11). Moreover, Kurella-Tamura (12), studying elderly nursing home residents with 
multiple co-morbidities, noted that during the first dialysis year, 58% died, 29% had a decrease in 
functional status, and physical function was maintained in only 13%. Current recommendations to prevent 
and manage aging events in patients with MHD include provision of an adequate nutrient intake and 
regular exercise (3-6). Exercise-based rehabilitation is cost effective and has been demonstrated to 
improve muscle mass, muscle function, and QOL in MHD patients (13;14). This highlights the need for 
rehabilitation during treatment in patients with CKD, whether they undergo dialysis or conservative 
management. However, implementation of rehabilitation as a standard treatment modality in patients with 
CKD is lacking (13). 
 
 
                                                Mechanisms of Loss of Muscle Mass in CKD. Several processes operative in uremia contribute to 
loss of muscle mass and function by affecting protein turnover. These processes include reduced nutrient 
intake, inflammation, acidosis, anemia, oxidative stress, resistance to and/or deficiencies in anabolic 
hormones (15), and reduced physical activity and disuse due to concomitant illnesses. Together these 
alterations lead to an increase in muscle proteolysis, largely because of activation of the ubiquitin-
proteasome system (16), while protein synthesis may also be diminished (17; 18). Other proteolytic 
systems including lysosomal and caspase systems may also be involved (16-18). Maneuvers to correct or 
prevent muscle wasting in uremia have included correction of acidosis, anabolic hormone therapy, and 
exercise (14; 15; 19; 20).  Provision of adequate dietary protein and calories can help, but in some 
patients supplements may be required (21; 22). Abnormalities in amino acid (AA) levels are present in 
CKD (23; 24). Plasma levels of several essential AA (EAA) especially branched-chain AA (BCAA) 
including leucine, are reduced (25), while non-EAA levels may be abnormally elevated (26). Numerous 
metabolic abnormalities at the cellular level have also been described in CKD (25). Regarding the 
increased catabolism and loss of AA that occurs during MHD, intradialytic parenteral or enteral AA and 
calories acutely stimulate whole body and muscle protein synthesis and inhibit whole body (22; 26), but 
not muscle proteolysis (27). Of note, net muscle protein accretion can be augmented by concurrent 
exercise (20; 28). The nutrient effect appears to arise from correction of AA deficiencies and stimulation of 
insulin release. In addition, EAA, especially the BCAA leucine, directly activates the mTOR signaling 
pathway, which promotes translation initiation of protein synthesis. Whether the AA stimulated mTOR 
signaling response is intact in patients with end-stage renal disease (ESRD) is at present unknown. In rats 
with CKD, it has been reported that leucine-stimulated insulin secretion is impaired (29). Nevertheless, in 

a recent study with rats with CKD we observed that leucine does stimulate the mTOR anabolic signaling 
pathway, albeit less effectively than in normal controls (30). In normal humans, EAA are highly effective in 
stimulating protein synthesis if ingested at the time of resistance exercise, a maneuver that also activates 
mTOR signaling, but earlier in the signaling pathway (31) (Fig 1).  
 
 
      Effects of Exercise Training in CKD. While not a uniform finding (32), several studies in 
advanced CKD and ESRD have reported that exercise increases the mass of the exercised muscle, 
though not necessarily lean body mass (33-35). Failure to do so may reflect the local response to 
exercising just one set of muscles (e.g. lower limbs). In contrast, regular exercise involving trunk, leg and 
arm muscles increases total body K content, a measure of body cell mass (33). In general, resistance 
exercise in particular is effective in increasing muscle mass, muscle fiber area, improving strength and 
function and even reducing inflammation in CKD (33-35). Interestingly, it was reported that prolonged 
endurance exercise can increase muscle fiber size in ESRD (32-36), which is not the usual response in 
normal subjects.  Nevertheless, endurance exercise appears to be therapeutic, since it improves muscle 
strength as well as cardiopulmonary function (13; 14; 35-37). Regular endurance exercise also has a 
marked effect on cardiovascular risk factors in ESRD subjects (14). Both intensive and moderate aerobic 
training have been shown to improve cardiovascular performance in MHD subjects, and studies have 
consistently shown an increase in peak VO2 following rehabilitation programs, with an average increase of 
17% (14; 36; 37).  Peak VO2 is the gold standard for cardiopulmonary fitness since it incorporates 
elements of cardiac output, metabolic changes in skeletal muscle, vascular function, and muscle strength 
(38; 39). Moreover, peak VO2 is an independent predictor of survival in MHD patients (40). In studies 
employing combined endurance and resistance exercise in MHD, significant increases in muscle mass 
and function have been demonstrated (41; 42). Indeed, clinical trials and guidelines on physical activity 
and health in non-renal subjects suggest that peak VO2, muscular strength, and endurance are optimally 
improved by programs using combined aerobic and resistance exercise vs. either one alone (38; 39). 
Because of its potential benefits, there have been numerous short-term studies of resistance and/or 
endurance exercise training in CKD patients in different venues including facility-based, MHD unit-based 
and even home-based exercise training (14).  Overall, studies in patients with CKD have suggested that in 
addition to improving muscle mass, strength and functionality, regular exercise has a salutary effect on 
cardiac and respiratory function, endothelial function, insulin sensitivity, lipid levels, inflammation, 
psychosocial status and QOL, and has been shown to be safe (14; 43). The potential cardiovascular 
benefits of exercise may be particularly important for ESRD patients, who have a high prevalence of 
cardiovascular disease that accounts for nearly half their deaths (2; 44). 
 
   
     However, most of the exercise studies in patients with MHD have involved small numbers, lack 
randomized controls, were of short duration, and enrolled relatively young subjects in relatively good 
condition. Notably, there is a dearth of studies specifically targeting the elderly (5). In addition, there is 
limited information on the cellular mechanisms underlying the salutary effect of exercise in humans with 
ESRD (45-47). Moreover, the precise relationship between muscle wasting with loss of function and poor 
long-term outcomes in elderly ESRD patients remains to be established. The extent to which muscle 
atrophy is a consequence of underlying disease processes and the extent to which it actually contributes 
to outcomes is also unclear (47). Thus, there is a need for large-scale prolonged studies to establish the 
true long-term benefits of exercise on morbidity and mortality in this population. It may be that given the 
natural limit of lifespan and the co-morbid conditions common in elderly MHD patients (8), the main 
benefits of exercise are improvements in physical function, activities of daily living, QOL, and a decrease 
in adverse events (e.g. falls and hospitalizations, which are more frequent in the elderly dialysis patient 
than the general population) (7). Sorely needed as a prerequisite to such large, long-term studies is 
development of a low-cost exercise program that will be relatively easy to administer and adhere to, and 
that will form part of routine ESRD care (13; 47). In this regard a recent short-term study reported the 
feasibility and potential effectiveness of home-based exercise in dialysis patients, half of whom were over 

age 60 (48). Lastly, a small number of studies of exercise training among elderly CKD subjects, not yet on 
dialysis, have also shown promising results on multiple domains of physical function (5).  
  
    
  Protein Signaling in CKD. The mechanism whereby resistance exercise induces muscle 
enlargement in uremia is incompletely understood, though from our work and others (45; 49), a change in 
the balance between local IGF-1 (up), and myostatin (down) and also activation of anabolic signaling 
pathways, especially the PI3K/ mTOR pathway, which is depressed in CKD, appear to be important (50). 
Exercise also increases muscle androgen receptor expression in normals (51). Among the factors 
regulating muscle mass, IGF-1 plays a key anabolic role (52; 53). IGF-1 is synthesized under the influence 
of growth hormone (GH) and nutrition (54). In skeletal muscle, expression of IGF-1, including the IGF-1Ea 
mRNA transcript and the splice variant mechano growth factor (MGF), is also regulated by mechanical 
stimuli induced by exercise independent of GH, and plays a key role in exercise-induced hypertrophy (52; 
53). Indeed, in uremic rats we showed that muscle work overload effectively stimulates local IGF-1 
expression, despite resistance to GH-induced IGF-1 expression (49). In addition, expression of myostatin, 
an inhibitor of muscle growth which is elevated in CKD, decreased. These responses provide explanations 
for the beneficial effects of exercise in MHD patients. Indeed, the same response to exercise was 
subsequently observed in MHD subjects (45). In ESRD skeletal muscle IGF-1 and IGF-1 receptor mRNA 
levels are reported to be reduced (55) and in patients on MHD with muscle wasting, IGF-1 peptide levels 
are low, even when serum levels are unchanged (56). Generally, serum and muscle IGF-I levels are 
normal or even elevated in adults with ESRD (55; 56), but are reduced in the severely malnourished 
wasted subject (54). IGF-1 bioavailability may be reduced because of increased serum IGF binding 
proteins (57). Also contributing to uremic muscle wasting is impaired IGF-1, and insulin signaling, 
mediated through the IRS-PI3K/Akt signal transduction pathway (50; 58). Normally, activation of Akt/ PKB, 
leads to mTOR phosphorylation which then activates the 70-kDa ribosomal S6 kinase (p70S6K), 
eventually leading to increased protein synthesis (59; 60). Akt activation also suppresses proteolysis (26). 
Notably, the human studies described above were performed in relatively young ESRD populations and 
data specifically for the elderly are not available. 
 
           The signaling pathways through which IGF-1 stimulates muscle growth are similar to those 
activated by mechanical stimuli alone [Fig 1], and it is difficult to distinguish between these stimuli, 
especially as mechanical stimulation leads to a local increase in IGF-1 (53; 61). Both mechanical and IGF-
1 induced activation of the Akt/mTOR pathway are essential for promoting muscle growth and repair (53; 
59). How mechanical stimuli activate the PI3-kinase/Akt/mTOR pathway and cause muscle hypertrophy 
are poorly understood (61).  One way is through growth hormone-independent stimulation of local IGF-1 
expression (52; 53). mTOR signaling is also directly activated by EAA, particularly leucine, and then 
mTOR via its downstream signaling proteins, up-regulates mRNA translation initiation and thereby 
stimulates protein synthesis (Figure 1) (30; 62). Thus mTOR appears to serve as a nutrient sensor for 
EAA, but how this occurs is unknown (104). AA also elicit an anabolic response by increasing insulin 
secretion, but the signaling responses to AA and insulin are independent though complimentary (62). 
From these studies it is clear that AA function more than simply serving as a key protein synthesis 
substrate. 
 
Figure 1.  IGF-1/insulin, exercise and amino acid activated signaling 
pathways in skeletal muscle. IGF-1 and insulin receptor binding activates 
the IRS-1 PI3K-mTOR and the calcineurin/NFAT and RAS/MAPK pathways 
leading to increased protein synthesis and decreased proteolysis. IGF-1 
also induces muscle repair and growth. Exercise acts upstream of mTOR 
via Akt/PKB to promote muscle fiber adaptation and growth. Mechanical 
stress also acts by stimulating local IGF-1 production. Branched chain 
amino acids, especially leucine, directly activate mTOR and in turn its 
downstream mediators, eukaryocytic initiation factors (eIF)4E binding 

protein 1 and possibly eiIF4G to enhance assembly of eIF4G with eIF4E and 70-kDa ribosomal S6 kiinase 1, 
thereby stimulating  mRNA translation initiation and ultimately protein synthesis (62). Figure from (30). 
 
            The activated PI3K/Akt pathway also inhibits protein degradation. It does so by inactivating the 
FOXO transcription factors that increase expression of the atrophy-related ubiquitin ligases, atrogin-
1/MAFbx and probably MuRF-1, and thus suppresses proteolysis mediated through the ubiquitin-
proteasome pathway (16). Caspases, an important component of the degradation process, cleave 
complex proteins into smaller products rapidly degraded via the ubiquitin-proteasome system (16). In 
CKD and other catabolic states, PI3-kinase/Akt signaling is depressed (50; 58) and consequently protein 
synthesis and FOXO inactivation are depressed. This permits FoxO to stimulate the E3 enzymes 
atrogin-1 and muscle ring finger-1 (MuRF-1), favoring proteolysis through the ubiquitin pathway. 
Inflammatory cytokines such as TNF- , may also increase degradation by activating NFK- B, which 
leads to increased expression of the E3 enzyme MuRF-1 (16). Protein synthesis is attenuated and 
degradation through the ubiquitin-proteasome pathway is accelerated, especially if acidosis is present. 
Caspase-3 activity is increased, accelerating actinomysin cleavage with increased formation of a 14-kD 
actin fragment, a muscle proteolysis marker (16; 46). Interestingly we and others noted in uremic 
rodents that by simply increasing muscle workload, the IRS-1/PI3-kinase/Akt signaling pathway can be 
fully activated and this leads to a decrease in proteolysis, with a reduction in actin fragment level, 
increased protein synthesis and muscle hypertrophy (50; 63). In MHD patients, endurance exercise 
decreases the elevated fragment levels (45). 
 
 
  There has been intense interest in establishing whether the anabolic response to exercise can 
be enhanced by providing supplemental EAA (64-66). In normals, leucine-rich supplements are 
especially effective in promoting muscle protein synthesis and when combined with resistance exercise 
cause an even greater increase in muscle protein synthesis than exercise alone (64; 67). A similar 
response is seen with endurance exercise (68). Among commonly used protein supplements, whey is of 
special interest. It is readily digestible, is rich in EAA (~50g/100g/protein), particularly leucine 
(14g/100g), has a low Na, K, and phosphorus content, is inexpensive, and is effective in enhancing 
muscle protein synthesis (66; 69; 70). Hulmi (71) reported that whey acutely increased anabolic 
signaling in resistance exercise-trained normal subjects over 10.5 weeks. Regular exercise with whey 
supplementation caused a greater increase in muscle mass than exercise alone. Commonly a 
carbohydrate is added to the whey or AA supplement to stimulate insulin release and inhibit proteolysis 
induced by exercise (72). This combination also stimulates anabolic signaling induced by endurance 
exercise (68). Since in ESRD alterations in serum and intracellular AA profile with reduced serum BCAA 
levels are common (17; 23), provision of EAA at the time of exercise is likely to be especially effective. 
However, it is conceivable that there may be resistance to the signaling response to AA as there is for 
insulin/IGF-1 in CKD (50; 58). In healthy elderly subjects there is some impairment to AA-induced 
protein synthesis (73), and this can be overcome by increasing the leucine load (66), particularly in the 
form of whey protein (74). In a study with CKD rats, described below, we found that leucine alone largely 
corrects and stimulates the anabolic signaling defect present in these rats (30). Since leucine also 
stimulates protein synthesis in non-exercising humans including the elderly, it is recommended that the 
diet of the elderly include leucine-rich foods to prevent and treat sarcopenia (6; 75).  
 
Specific Aims: 
 
    Specific Aim 1. To devise a safe, low-cost aerobic and resistance exercise regimen  to counteract 
the loss of muscle mass and function common in elderly MHD patients that is easily implemented, suitable 
for the home and to which most subjects will adhere. 
 
 
    Specific Aim 2. To elucidate the mechanisms whereby a program of exercise-based rehabilitation 
counteracts muscle wasting in elderly ESRD patients.   
 

 
    Specific Aim 2A. To determine whether the mTOR anabolic signaling response to exercise or a 
high-calorie leucine-rich protein load is intact in elderly MHD patients and whether the response to exercise 
is enhanced by providing this supplement at the time of exercise.  
 
Hypotheses:  
 
  
    Hypothesis # 1. Regular exercise, by counteracting muscle wasting, enhancing cardiopulmonary 
and muscle function, and reducing cardiac risk factors, will improve muscle mass, cardiopulmonary 
function, and psychosocial health in elderly MHD patients. We anticipate that our approach to implementing 
a home-based rehabilitation program, modeled on a program effective in cardiac patients, in a rigorous 
randomized clinical trial will provide an innovative and relatively low-cost approach to the management of 
ESRD patients. It is our hope that this proposal will lead to future studies in which long-term outcomes of 
elderly MHD subjects will be improved. 
 
    
 
    Hypothesis # 2. The mechanisms whereby regular exercise counteracts muscle wasting in 
elderly ESRD patients are explained by changes in local skeletal muscle expression of IGF-1, androgen 
receptors and myostatin, and by stimulating Akt signaling pathways that promote protein synthesis and 
inhibiting proteolysis.   
 
    
  
    Hypothesis # 2A. Provision of leucine-rich protein at the time of acute exercise will stimulate 
skeletal muscle anabolic signaling in uremia. By augmenting exercise-induced signal transduction, this will 
increase the anabolic response to exercise.  
 
 
Significance: 
 
              Significance of Specific Aim 1. As described above, muscle wasting with frailty and loss of muscle 
function and reduced exercise capacity are common in advanced CKD. This is particularly true in elderly 
CKD patients, and these factors have been shown to be associated with increases in morbidity and 
mortality (5; 11). These issues also compromise the elderly patient who might otherwise be suitable for 
transplantation (75). One simple approach to these problems is an exercise program, an approach that has 
been effective in the long-term management of patients with cardiovascular disease (76), including the 
elderly (77; 78). Such programs in patients with cardiovascular disease have been consistently associated 
with improved survival rates (79). Exercise programs, at least in the short term, also appear to be effective 
in the management of MHD patients (14; 80), but there have been logistic, economic and compliance 
barriers to implementing exercise regimens in these patients, and the long-term impact on morbidity and 
mortality has not been established. Thus, despite evidence of short-term benefits, at least in younger 
patients, exercise is not part of routine MHD patient care. The primary thrust of this application is therefore 
to devise an effective, safe and low-cost home-based exercise regimen that is easily implemented and in 
which a substantial number of elderly MHD patients could potentially participate and adhere to, and that 
improves surrogate markers of long-term benefit. We do realize that given natural lifespan limits and the 
co-morbid conditions common in the elderly, the main benefits of exercise in elderly MHD patients are likely 
to be improvements in physical function, activities of daily living and QOL, and a reduction in adverse 
events such as falls and need for medical attention. If the present study yields a positive outcome, then it 
will form the basis of a broader study to examine the long-term outcomes of this regimen in both elderly 
and younger patients on MHD. The results could also have important implications for non-renal patients 
with other catabolic diseases. Thus, this study holds the potential for serving to increase QOL and to 
reduce morbidity and possibly mortality rates in many dialysis patients and at considerably lower cost than 
other approaches under consideration, such as daily dialysis. Moreover, if effective, the study may serve as 
a model for implementing home-based exercise for advanced CKD patients not needing MHD, and for 
establishing clinic-based exercise programs for MHD patients unable to undergo home exercise. 
 

 
  Significance of Specific Aim 2. We propose that regular exercise acts largely by: (a) inducing 
changes in local skeletal muscle expression of IGF-1, androgen receptors and myostatin; and (b) by 
stimulating Akt signaling pathways that promote protein synthesis and inhibiting proteolysis. This aim will 
provide novel information regarding the impact of uremia on molecular events that regulate muscle mass in 
response to exercise training in CKD. In addition, this may lead to novel strategies for preventing muscle 
wasting in patients with CKD, and provide an impetus for the use of rehabilitation as a treatment strategy 
for patients suffering from this condition. 
 
 
     Significance of Specific Aim 2A. We postulate that leucine supplementation will stimulate muscle 
anabolic signaling in uremia by augmenting exercise-induced signal transduction, and will increase the 
anabolic response to exercise. This information will help elucidate the mechanisms whereby exercise 
counteracts muscle wasting in elderly ESRD patients. This analysis will also provide new information 
regarding nutrient supplementation and exercise-induced molecular events in uremia and may well serve 
as the basis for a more extensive study that combines regular exercise and dietary intervention as 
treatment options for managing protein energy wasting in elderly patients.  
 
       Relevance to the VA Patient Care Mission  
 
 
    CKD is a common condition in the VA; there are approximately 18,000 such individuals with end-
stage CKD requiring MHD currently being treated in the VA system, and this condition is responsible for 
approximately 18% of all deaths in the VA (81,82). The number of persons affected by CKD is projected to 
increase by 35% between 2005 and 2015 (83; 84). Moreover, CKD is typically associated with other co-
morbid conditions, including cardiovascular disease, resulting in frequent hospitalizations and a mortality 
rate ranging between 15 and 30 times that of age-matched healthy individuals (85). It has been estimated 
that a patient with end-stage chronic renal failure requires healthcare resources costing the VA Health Care 
System more than $50,000 annually, making CKD the second most costly condition in the VAHCS, second 
only to spinal cord injury (82). Notably, the majority of these costs are not attributable to dialysis, but rather 
to renal and other medical/surgical hospitalizations. This underscores the frequent and costly co-morbidities 
that are associated with advanced CKD.  Moreover, total Medicare expenditures in 2005 for an elderly (≥67 
years) dialysis patient (similar in age to our population) have been estimated to be >$100,000 per year.  
Costs for MHD treatment in the VA system are expected to double over the next 10 years (81). Exercise-
based rehabilitation programs have been demonstrated not only to improve functional capabilities of 
patients with CKD (including those requiring MHD) through improvements in muscle mass, strength and 
cardiovascular function, but rehabilitation has also been shown to reduce disability and hospitalizations (14; 
43; 86). However, these results have come largely from relatively small research-based studies and a major 
gap exists between these research findings and the clinical implementation of rehabilitation as a treatment 
modality in MHD patients.  A home-based rehabilitation program that can be easily applied at minimal cost 
has the potential to substantially reduce healthcare costs associated with this condition.  
 
            We have recently implemented a National Institutes of Health-sponsored home-based rehabilitation 
program in patients with abdominal aortic aneurysm (AAA) disease, in which we observed marked 
improvements in exercise capacity, and in which no adverse events related to exercise testing or training 
occurred (87). Exercise training as a treatment modality in this population was novel, and the home-based, 
case management exercise model provided a training stimulus that would not have been possible with a 
conventional center-based program. We similarly anticipate that that a home-based exercise program in 
patients with MHD will be “user-friendly” and will effectively counteract muscle wasting, improve 
cardiopulmonary and muscle function, quality of life, and surrogate markers of long-term outcomes. New 
insight into mechanisms whereby exercise and nutrient supplementation induce an anabolic response in 
CKD will be provided that may assist in developing novel strategies to improve outcomes. Our broader goal 
is to assess the long-term effects of such a program at multiple centers. If the program described in the 
current proposal were to be implemented on a wider scale, the overall health of patients with advanced 
CKD requiring MHD could be markedly improved, and healthcare cost savings in the VAHCS could be 

substantial. Moreover, if effective, the study may serve as a model for implementing home-based exercise 
for advanced CKD patients not needing MHD, and for establishing clinic-based exercise programs for MHD 
patients unable to undergo home exercise. Finally, this program could serve as a model for the 
management of Veterans with other chronic disease states such as advanced COPD.     
 
Research Design and Methods 
           
          General study design:  MHD subjects aged 65-80 years with impaired exercise capacity and 
function will be randomized into two groups (30/group). One group will undergo a home-based exercise 
program and the other usual care over a 12 week period. This subject number allows for a drop-out rate of 
20%, while maintaining 80% power to detect a significant difference in our primary end-point, peak VO2. 
After enrollment, all subjects will be encouraged to consume at least 1.2 g/kg/protein/day throughout the 
study. The 12-week program will include a 2-week clinic-based exercise instruction period with testing at 
baseline before intervention and after 6 and 12 weeks. Measures at baseline and 12 weeks will include 
body composition by anthropometrics and dual energy X-ray absorptiometry (DEXA), thigh muscle volume 
and composition by CT scan, muscle strength and function, cardiopulmonary exercise testing, QOL, 
cognitive function and nutritional, inflammatory, lipid and biochemical status. Muscle biopsies will be 
obtained at baseline and at 12 weeks and will be assayed for signaling proteins, and gene and protein 
expression of key muscle mass regulators. More limited interim testing will be performed at 6 weeks. In 
addition, two to three days after the 12-week study, a pilot sub-study will be performed to test whether a 
high-calorie leucine-rich protein supplement enhances exercise-stimulated anabolic signaling. At this time 
muscle biopsies will be taken. Overviews of the study measurements and timeline are presented in 
Appendices I and II.  
 
            For recruitment we will draw on a large patient population within 30-40 miles of the VA Palo Alto 
Health Care System (VAPAHCS) with travel reimbursement provided. For example, the nearby Satellite 
Healthcare Centers have agreed to actively support the identification and recruitment of subjects, with their 
Chief Medical Officer, Dr. Schiller, serving as a significant contributor to this study. They operate 30 stand-
alone dialysis centers within a 40 mile radius the VA Hospital in Palo Alto. These centers treat a total of 
5,000 dialysis patients, with approximately 25% of these patients on home dialysis. At any given time, 
approximately 2% of their total patient population is enrolled in a clinical trial. There are 6 Satellite 
Healthcare clinics within 10 miles of the VA Hospital in Palo Alto, which provide MHD services to roughly 
1,000 patients; these clinics will be a primary source of patients for the study. At Santa Clara Valley 
Medical Center, a Stanford affiliate, ≈300 patients are treated with MHD and this facility has agreed to refer 
study subjects. Smaller dialysis clinics such as VAPAHCS (Dr. Rabkin), Stanford Medical Center (Dr. 
Chertow) and community nephrologists (letters attached) will also serve as resources. Combined, these 
resources provide a pool of ≈2,000 subjects in close proximity to our center. From prior experience, ≈ 5% of 
subjects will meet inclusion criteria and agree to participate. This will provide an adequate enrollment pool. 
While we expect the recruitment of subjects to reflect the distribution of women and minorities in our area, 
a concerted effort will made to recruit and include them in the study.   
 
 
Study population and inclusion/exclusion criteria:  Males and females aged 65-80 years 
undergoing MHD for at least three months and without other active/uncontrolled disease will be studied. 
Exercise and usual care groups will be matched by age, body mass index (BMI), MHD duration, and 
protein intake using a stratified randomization approach.  
      
           Inclusion criteria.  MHD subjects with impaired exercise capacity and function will be considered for 
the study. Subjects will be required to be in the peak VO2 range of 10 to 20 ml/kg/min, equivalent to 
moderate functional impairment in patients with heart failure (88; 89). Subjects will be required to have 
dialysis treatment for >3 months with an average Kt/V ≥ 1.2, and be able to perform exercise safely. Racial 

and ethnic composition will reflect the diverse composition of the ESRD population in counties from which 
patients are referred.   
          
           Exclusion criteria.  Current activity > 2 hrs/wk of moderate intensity exercise, temporary vascular 
access, uncontrolled diabetes mellitus, active vasculitis, active autoimmune disease, malignancy, severe 
obesity (BMI > 35), alcoholism or other recreational drug use, unstable cardiac disease (abnormal exercise 
test, angina, uncontrolled arrhythmias or myocardial infarction within three months), peripheral vascular 
disease (claudication with exercise), lung, liver or intestinal disease, those who are medically unstable and 
subjects who have received anabolic, catabolic or cytotoxic medications in the past 3 months. We will also 
exclude subjects with excessive previous exposure to radiation. Although “excessive" in regards to 
previous radiation exposure has not been defined by consensus panels and there are no national 
standards or regulatory guidelines that define this, we will not consider subjects for the study who have 
undergone a CT scan or any procedure with similar radiation exposure within the last year. 
 
 
           Screening.  MHD subjects will be screened for eligibility and to ensure that they can safely complete 
the exercise program. Screening tests will include: Part 1: (a) cardiovascular risk factors (including smoking 
history, blood lipids, CRP,  blood pressure), and VA cardiovascular disease score; (b) resting 12 lead ECG; 
(c) medical history and physical function questionnaires; (d) Kidney Disease QOL-36 instrument (90); (e) 
the VAPAHCS  Physical Activity Questionnaire (91); (f) chair raise test; and (g) six-minute walk test. Part 1 
screening results will be collected and presented for evaluation by the investigators in the Cardiology 
Service.  If the subject is approved, they will proceed with Part 2 testing, which will include a treadmill 
exercise test with 12 lead ECG and measurement of peak oxygen consumption. If the subject is considered 
sufficiently low-moderate risk following the exercise test and shows no evidence of severe functional 
impairment on the 6-min walk test (<200 meters) and cardiopulmonary exercise test (<10 ml/kg/min), 
he/she will be scheduled for additional baseline testing. 
 
 
Group Assignment. After screening and baseline testing, an independent third party investigator (Dr. 
Heidenreich, letter attached) will match MHD subjects on age, body mass index (BMI), MHD duration, and 
protein intake, and assign them randomly to the exercise or usual care study groups. At the end of 12 
weeks of study this investigator will assign half of the subjects in each group to receive a blinded nutritional 
supplement (high calorie whey or placebo). The blind will be broken on completion of the study.   
 
 
 
 Dietary Control. Prior to entry into the study the subjects will be encouraged to consume 1.2 g 
protein/kg/day, per Kidney Disease Outcomes Quality Initiative (KDOQI) recommendations (92) and 
advised to maintain a steady intake. However, we realize that the recommended intake may not be 
achieved in some subjects. In order to adjust for variation in protein intake we will measure the subject’s 
protein intake on 3 occasions during the month prior to entry into the study. Then when assigning subjects 
to the study groups, measured protein intake will be taken into account by the third part study assigner, so 
that the protein intake in the 2 study groups will be similar. Protein intake will be monitored by three-day 
dietary records and the normalized protein equivalent of nitrogen appearance (nPNA,), a reflection of 
protein intake in stable conditions. nPNA is calculated from the urea generation rate by means of  kinetic 
modeling using measurements of pre- and post-dialysis urea levels  plus any urea lost in the urine in 
patients with residual renal function. After entry into the study nPNA is calculated weekly for the first two 
study weeks, then monthly. During the study period, there will be regular bi-weekly follow up counseling by 
a dietician in an effort to ensure that both groups meet target dietary protein intakes 
 
 
 
  Safety Measures. Before screening, subjects will be informed of the nature of the study and the 
procedures to be performed, and will sign an approved consent form. Measures to monitor and ensure 
safety include a screening ECG, maximal exercise test, and medical approval (see screening). The 
cardiovascular exercise lab has extensive experience in conducting clinical studies and has a staff of 
experienced personnel. We do not expect any long-term side effects from the proposed interventions. The 
exercise program is well balanced and varied with aspects of strength and aerobic training. Subjects will 

have an adaptation period of several weeks in which they will progress at their own rate toward the desired 
intensity and duration goals. During the lead-in period, exercise variety and relatively modest energy output 
of the program will minimize risk of musculoskeletal and cardiovascular injury. Subjects will not take part in 
any other research project without investigator approval. All procedures will be performed by trained 
personnel in a hospital setting with emergency medical staff available. All samples and data will be stored 
and analyzed using subject codes to assure participant confidentiality. Interventions employed, including 
exercise testing and training, are of low to moderate risk.  
 
 
  Retention of Subjects and Compliance to the Intervention. We will select subjects highly motivated 
to improve and maintain their health. Thus, we expect high compliance and low attrition rates (up to 20% 
drop out). During recruitment, emphasis will be placed on the importance of completing the 12-week 
program. According to our power calculations (detailed below), we will need to retain at least 23 
subjects/group to maintain a power of 80%. On completion of the study, subjects in the exercise group will 
receive a small payment ($350) for the time and inconvenience of 14 exercise clinic and lab visits. Subjects 
in the usual care group will receive $75 for their participation (4 clinic and lab visits). Participants in the 
muscle biopsy substudy will receive an additional $250. To encourage compliance and continued 
participation in the exercise program, the investigators will provide regular feedback including weekly 
telephone calls and encouragement regarding exercise and assistance with diets, scheduling etc., and will 
encourage subjects to return to the exercise clinic for evaluation and reinforcement every two weeks during 
the study. 
 
 
 
A.  Specific Aim 1 is designed to develop an effective, safe, low-cost aerobic and resistance 
exercise regimen.  Because of the focus on improving cardiopulmonary function as well as muscle 
function and mass, both resistance and aerobic exercise will be components of the rehabilitation program. 
The program is modeled on a successful combined in-home/in-center study program administered by Dr. 
Myers at the VAPAHCS rehabilitation center for patients with cardiovascular disease with a mean age of 72 
years (87). In general, exercise rehabilitation is highly effective in patients with cardiovascular disease (76), 
including the elderly (77; 78), and is associated with improved survival rates (76; 77; 79). Subjects 
randomized to the exercise group will undergo 12 weeks of exercise, including a 2-week ramp-up period 
conducted at the VAPAHCS rehabilitation facility. Subjects will be required to attend the facility twice a 
week on non-dialysis days during the initial 2 weeks of the study. These sessions will consist of 45-60 min 
of supervised exercise that combines aerobic and resistance training, including warm-up and cool-down. 
Throughout the study, subjects will be asked to perform 45 minutes of unsupervised aerobic exercise of 
their choice (such as walking) on non-dialysis days.  Following the initial 2-week supervised period, 
subjects will be instructed to exercise on their own, and surveillance methods will be employed as 
previously described (87; 93) and outlined below. Following the 12-week study period, all subjects will 
undergo muscle biopsies. The following end-points will be measured at baseline and after the 12 week 
study period: 
 
             Primary Endpoint. Peak VO2 is the primary outcome measure and is the gold standard for 
cardiopulmonary fitness. It incorporates elements of cardiac, pulmonary and muscle function known to 
improve with exercise. 
 
            Secondary Endpoints. These include lower and upper body strength, body composition, thigh 
muscle mass, muscle biopsy for protein signaling, quantitative muscle morphology and gene expression, 
daily activities, QOL, laboratory measures of cardiovascular risk factors, nutritional and inflammatory 
parameters as detailed below. 
 
 
            Together, these measures should allow us to determine whether the home-based exercise regimen 
is effective in counteracting loss of muscle function and mass common in elderly MHD patients, along with 
reducing cardiovascular risk. 
 

Exercise training, detailed description. Initially, all subjects will undergo four supervised exercise 
sessions over two weeks. The purpose of these sessions is to familiarize the patients to their individualized 
training program, assess stability during exercise, ascertain that they understand their exercise prescription 
and how to use activity and heart rate logs, and provide guidelines and education in terms of what is 
expected of them during the study. Subjects will then exercise at home, with an in-clinic exercise session 
repeated every two weeks for the duration of the study. The biweekly exercise sessions will be used to 
ensure stability and compliance, to review activity logs, and to modify the exercise prescription as 
appropriate. Guidelines for patient monitoring, safety, and prescription outlined by the AHA, American 
College Sports Medicine, and AACPR will be followed (76; 94; 95). 
 
             Supervised in-clinic exercise sessions. These will include 5 min warm-up and cool-down sessions 
before and after a combination of continuous aerobic (treadmill walking, cycle ergometry, arm ergometry, 
rowing, stair climbing) and resistance exercise. Exercise intensities will initially be targeted to achieve 60% 
of HR reserve for 30 min; exercise intensity will be documented for each patient by frequent (5 min) 
recordings of heart rate and perceived effort. Progression of exercise intensity will be individualized in 
accordance with established guidelines (76; 94-96), but in general the goal will be to increase intensity and 
duration to 70 to 80% heart rate reserve and 45 min, respectively. Perception of effort will be targeted to 
12-14 on the Borg scale. All in-house sessions and activity surveillance will be supervised by certified 
exercise physiologists. A nephrologist (R. Rabkin), cardiologist (V. Froelicher) and a clinical fellow will 
address patients’ clinical concerns as necessary. As many MHD patients have a blunted HR response to 
exercise or are taking β-blockers, measured maximal heart rate will be used to develop exercise 
prescriptions, and patients will remain on their β-blockers for testing. 
 
            During the initial supervised sessions, resistance exercise will involve an introduction to a low-
resistance, high-repetition regimen including upper and lower body major muscle groups under 
individualized supervision in accordance with established guidelines (76; 96). Resistance exercises will 
include leg press, leg extension, leg flexion, chest press, shoulder press, row, and lat pull-down. During the 
initial sessions, subjects will perform 12 to 15 repetitions at 70% of the 5-repitition maximum with a 
minimum 2-minute rest period between sets. Subjects will gradually increase to two sets of these 
exercises, with increasing resistance as tolerated. Subjects will be given hand-held weights and Thera-
Bands in accordance with their capabilities, and instructed on their use at home based on their 
individualized prescription (see attached VAPAHCS brochures, Appendices II and III). Thera-Bands are 
special latex bands used for resistance exercise and have been effectively employed in MHD subjects (48). 
 
 
Home training sessions. Subjects will receive detailed instructions on individualized exercise 
prescriptions, including how to monitor exercise intensity using heart rate and perceived exertion, and how 
to use heart rate and activity tracking devices (see below). Cycle ergometers will be provided for home use, 
but the subjects will be encouraged to achieve an individualized, targeted exercise stimulus using walking, 
stair stepping, and other available modes of exercise. Activity logs will be given to each patient to record 
activities, their intensity, duration, HR, and pedometer steps. 
 
Detailed description of measurements: 
 
(1)  Vitals: Body weight, height, resting blood pressure and resting heart rate will be measured at the start 
of each testing appointment. 
 
(2)  Fitness, functional and cardiopulmonary testing:    
       (a) Peak aerobic capacity (peak VO2): Peak VO2 is considered the gold standard for cardiovascular 
fitness (39). Aerobic capacity will be determined on a treadmill using an individualized ramp protocol (97) 
with collection of continuous ventilatory gas exchange responses (38; 39).            
       (b) Exercise testing: Symptom/sign limited ramp treadmill tests will be performed on all patients at 
baseline and after 12 weeks of participation (97). A thorough review of clinical history, medications and risk 
factors will performed at the time of baseline exercise testing. Exercise capacity will be measured directly 

using ventilatory gas exchange techniques. The Duke Treadmill and VA Prognostic Scores (38; 98) will be 
used to help estimate risk and assess suitability for exercise. Patients with evidence of significant coronary 
artery disease as determined by profound ischemic markers, ominous arrhythmias, or resting ECGs that 
confound the recognition of ischemia (bundle branch block, more than 1.0 mm resting ST depression, 
paced rhythm) will be excluded. Patients with hemodynamic instability or inability to exercise, those with 
complicating illnesses or questionable motivation to sustain prolonged training will also be excluded. The 
exercise tests will be performed, analyzed, and reported according to a standardized protocol and utilizing 
a computerized database (99). 
         (c) Measurement of strength: Strength will be measured at baseline and after the 12-week study 
period using leg extension 1 repetition maximum for lower body strength and chest press 1 repetition 
maximum for upper body strength. Maximal isometric strength will be measured using a hand-grip 
dynamometer. All assessments will be performed using standardized guidelines, including appropriate 
warm-up and familiarization trials, instruction, joint positioning, and rest periods between repeat tests (100).  
        (d) Six-minute walk test: This test incorporates elements of balance as well as strength and aerobic 
capacity. A standardized 6-min walk test (39) will be performed at baseline and after the 12 week study 
period. A 100 foot distance will be marked in a corridor, and patients will be instructed to walk from end to 
end at their own pace, attempting to cover as great a distance as possible within 6 minutes. The test will be 
supervised by a research assistant, who will provide standardized verbal encouragement at 30-second 
intervals. Distance covered is expressed in meters.   
       (e) Chair raises: Balance and strength will be measured using chair raises, a surrogate for muscle 
power. Subjects will be asked to rise with arms folded on the chest from a standard seat (46 cm high) and 
then return to a sitting position. Time to complete five repetitions and number of repetitions that can be 
completed in 60 seconds will be recorded. Chair raise capacity, validated against more extensive 
assessments (101), correlates strongly with muscle cross-sectional area and is reduced in ESRD (102).   
       (f) Additional monitoring of daily activities: In addition to formal training sessions, subjects will be 
encouraged to increase their daily activities and to exercise at a moderate individualized intensity for a 
minimum of 45 min each day they do not exercise at the hospital facility. Energy expenditure will be 
documented with a pedometer, a heart rate monitor and by intermittent accelerometer usage. We have 
recently validated these methods of estimating energy expenditure during home-based exercise (93). 
Brochures that we have used for instructing patients on the use of TheraBands at home are presented in 
Appendices III and IV, respectively. Patients will complete daily activity logs (Appendix V), and phone calls 
will be made weekly to monitor compliance, to complete the Veterans Exercise Testing Study (VETS) 7-
day Activity Recall Questionnaire (Appendix VI), and to address any clinical concerns. Energy expenditure 
will be expressed in terms of kcals/week and MET/hours/week. If patients in the exercise group fail to meet 
their exercise goals, they will be strongly encouraged to increase their activity and monitoring will be 
increased. This approach is used at our facility (87; 93) and the PI is experienced in monitoring and 
surveillance of physical activity with these devices (93). 
 
(3)  Body composition and muscle area:   
        (a) Anthropometrics:  Abdominal girth and thigh circumference.   
        (b) Total body lean mass and percent body fat will be assessed using whole body DEXA. Scans 
will be performed in duplicate at baseline and 12 weeks. DEXA has been effectively used for measuring 
body composition in ESRD patients, is recommended by the National Kidney Disease Outcomes Quality 
Initiative (K-DOQI) as a reference method to assess body composition in ESRD (92), and has been shown 
to be responsive to exercise training in numerous studies, including conditions associated with muscle 
wasting such as HIV and osteoporosis (103). As water is measured by DEXA in the fat-free compartment 
and thus may confound compositional assessment, MHD subjects will be carefully examined for fluid 
retention before scanning. Scans will be performed the day after dialysis at estimated “dry weight”. 
 

       (c) Thigh muscle mass and fat composition by computerized tomography (CT). Mid-thigh CT of the 
non-dominant limb will be performed a day after MHD, with subjects in the supine position with the thigh 
muscle relaxed. Axial CTs will be obtained at the midpoint between the trochanter of the femur and the 
superior border of the patella, using slice thickness of 2.5 mm. The radiographic film will be digitally 
scanned for analysis with a personal computer. The analyses will be performed by Dr. McIntire (Co-
Investigator), who has extensive experience analyzing CT images in patients with CKD. He will be blinded 
to study group when processing the images. Thigh muscle cross sectional area (a measure of muscle 
mass) and interstitial adipose tissue embedded in muscle (reflects muscle quality) will be measured and 
computed. Measurements of mid-thigh subcutaneous and total fat will also be obtained.  
 
(4) Muscle biopsy measurements:   
        (a) Biopsy procedure. Vastus lateralis muscle will be biopsied under local anesthesia and sterile 
conditions with a Bergstrom needle. This procedure will be performed by Dr. Rabkin (Co-PI), who has 
extensive experience in this area. A ~150 mg sample will be obtained by applying suction to the needle at 
the time of biopsy as described previously (104), trimmed of fat and connective tissue and bisected. One 
part will be frozen at -70°C, and the remainder will be mounted in embedding medium and frozen. Biopsies 
will be taken at the beginning and end of the 12 week study period as detailed below. 
 
 
(5) Biochemical tests:   
 
 (a) Nutritional parameters: Serum albumin, pre-albumin and glucose will be measured in the 
PAVAHCS clinical lab. Serum IGF-I and IGF binding proteins (IGFBP1, 2, 3 and 5) will be measured by 
ELISA and Western ligand blots as previously described in our laboratory at 0 and 12 weeks (105).  
 
 (b) Insulin sensitivity from fasting serum glucose and insulin levels at 0, 6 and 12 weeks: 
Insulin resistance appears in early CKD and is associated with an increase in cardiovascular disease (106). 
Resistance is characterized by an increase in serum insulin levels relative to serum glucose levels and will 
be evaluated using the Homeostasis Model Assessment of Insulin Resistance (HOMA-IR). Fasting glucose 
and insulin samples will be obtained and the HOMA-IR calculated using the formula of fasting insulin 
(µU/ml) times fasting glucose (mmol/l) divided by 22.5. HOMA-IR is used frequently in epidemiologic 
studies and has been validated against a euglycemic-hyperinsulemic clamp technique (r=0.82-0.88) as an 
estimate of insulin sensitivity (107). We anticipate that insulin sensitivity, measured by HOMA-IR, will 
improve with exercise, though a recent study in MHD subjects failed to show any benefit of aerobic 
exercise (107). However, it is possible that this may have reflected a suboptimal exercise program. Insulin 
samples will be batched and analyzed with commercial kits in the PIs lab.  
 
(c) Lipid panel: (Fasting total cholesterol, LDL, HDL, ApoB, very low density lipoprotein, 
lipoprotein(a) and triglycerides). Individuals with ESRD are reported to have a high prevalence of 
dyslipidemia, particularly hypertriglyceridemia, and are at greater risk for CVD (44). The benefits of aerobic 
exercise on lipid profiles in non-CKD patients are well established, but the results in MHD patients are 
mixed (14). Lipid panels will be performed by the VA clinical laboratory at baseline and 12 weeks.  
 
(d) C-reactive protein (CRP),  inflammatory cytokines and complete blood count (CBC): 
Serum CRP and inflammatory cytokines such as IL-6 and TNF-alpha are elevated in many individuals with 
advanced  renal failure, but can be reduced with resistance exercise (34). Batched serum samples will be 
assayed in the Co-PIs lab (Dr. Rabkin) for high sensitivity CRP levels by nephelometry, and IL-6 and TNF-
alpha with ELISA kits at 0, 6 and 12 weeks.  
 
(e) Total Testosterone, steroid hormone binding globulin and bioavailable TT will be 
measured in the VAPAHCS clinical laboratory. Measurements will be made at baseline and after 12 weeks.  
 
(f) Renal biochemistry: BUN, Creatinine, CA, phosphate electrolytes, and bicarbonate will be 
monitored monthly.  
 
(6) Questionnaires:  
 
    (a) Activity questionnaires & logs (Appendices IV and V).  All subjects will keep daily activity 
and treatment logs. Daily logs will record compliance to the exercise regimen and quantify any changes in 

physical activity during the study. Daily activity logs are helpful in completing the VETS 7-Day Activity 
Recall Questionnaire, which will be completed during a weekly phone call with a staff member (93).  
 
     (b)  Diet: Subjects will be instructed on completing accurate three-day food records at the start of 
the study. Dietary records will be collected at 0, 4, 8, and 12 weeks. Dietary analysis will be performed 
using Nutritionist 5 (First Data Bank, San Bruno, CA) and subjects meet with a clinical dietician after each 
analysis to discuss results and make proper adjustments. nPNA will be used to assess protein intake. 
           (c)  Health Related QOL (HRQOL): HRQOL is a sensitive and validated means to assess a 
patient’s perceived well-being in physical, psychological, and social areas of health (90). ESRD 
patients have depressed HRQOL scores, but the literature indicates that physical activity can reduce 
depression, improve physical function, psychological well-being and social and family interactions, thus 
also improving HRQOL in ESRD (109). The Kidney Disease QOL-comprehensive instrument  to evaluate 
QOL will be used (90). Dr. Kurella-Tamura (Co-Investigator) has published extensively in the area of QOL 
in CKD, and she will oversee these evaluations.    
 
B.  Approach to Specific Aim 2. This pilot sub-study is targeted to elucidate the mechanisms 
whereby exercise training ameliorates or protects against muscle wasting in elderly ESRD patients 
and to test whether a nutrient supplement can enhance the anabolic response to exercise.   
Since this is a pilot sub-study carried out with a limited number of subjects after completion of the main 12 
week study, we have adopted a unilateral exercise protocol in which the resting contralateral limb serves 
as a control. This protocol will allow us to gain considerable insight into the salutary molecular processes 
activated by exercise in ESRD subjects and to test whether these processes can be enhanced by provision 
of a leucine-rich supplement. In brief, two to three days after completing the primary 12-week study, 
subjects from both the exercise and usual care groups will be studied in which one limb is exercised for 15 
minutes, after which the vastus lateralis muscles of both the exercised and the contralateral resting limb will 
be biopsied. Half the subjects in each of these 2 main groups (12 subjects from each group) will be 
matched by BMI and age and randomized to either a placebo mixture or a high calorie nutrient supplement. 
The nutrient supplement will consist of a high-calorie leucine-rich protein (whey protein) given at the start 
and end of the exercise session. The study consists of the following 4 groups, each undergoing acute, 
unilateral exercise: 
 
Group 1.  Regular exercise group plus placebo 
Group 2.  Regular exercise group plus nutrient supplement  
Group 3.  Usual care group plus placebo  
Group 4.  Usual care group plus nutrient supplement  
 
An outline of the experimental protocol is illustrated in Figure 2, and a detailed protocol follows.  
 

Biopsies will be obtained from the dominant limb after acute exercise and from the non-dominant limb 
following rest. A single biopsy of each limb will taken from each subject. Tissue taken from the biopsies will 
undergo molecular analysis to answer the following questions: 
 
1. 
What sustained molecular processes, activated by regular exercise, might be contributing to 
the maintenance or improvement of muscle mass in MHD subjects who participate in a 12 week 
exercise program? To this end, molecular events in the resting dominant limb of participants given 
placebo in regularly exercised (Group 1) and usual care (Group 3) subjects will be compared.  We will test 
the postulate that regular exercise acts largely by inducing local changes in the expression of key 
regulators of muscle mass and also by stimulating the Akt/mTOR signaling pathways that serve to promote 
protein synthesis and inhibit proteolysis. We anticipate that a 12-week program of rehabilitation will 
increase muscle mass as measured by CT and DEXA scans, and improve skeletal muscle function via 
several interrelated mechanisms. These include changes in the balance between IGF-1 and myostatin, 
increased androgen receptor expression, changes in expression of components of the ubiquitin-
proteasome and calpain proteolytic systems, suppression of inflammatory cytokines, and increased 
anabolic signaling. This is illustrated in Figure 3.  
 
 
2.  What impact does acute exercise have on anabolic signaling in regular exercise versus usual 
care subjects?  To answer this question we will measure activity (phosphorylation) of the IRS-1/AKT and 
mTOR signaling transduction pathways in the acutely exercised (and the contralateral resting) limbs of 
regular exercised subjects ingesting placebo (Group 1). The same measurements will be made in the 
muscle of the corresponding usual care group (Group 3) (Figure 4).  Activity of the anabolic IRS-1/AKT and 
mTOR signaling pathways in the exercised limb will be compared with these responses in the contralateral 
resting limb of the same subject. We anticipate that acute exercise will activate anabolic signaling in both 
groups and that the response will be more vigorous in the regular exercise group.  
 
 
 

3. Does ingestion of a high-calorie leucine-rich protein (whey) supplement enhance acute exercise-
stimulated anabolic signaling?  (Specific Aim 2A). The effect of a nutritional supplement on acute 
exercise-induced signal transduction will be assessed by comparing IRS-1/AKT and mTOR signaling in a 
unilateral acutely exercised limb of regularly exercised subjects ingesting the supplement (Group 2) versus 
the acutely exercised limb of comparable subjects ingesting placebo (Group 1). A similar comparison will 
be made between the exercised limbs of usual care subjects ingesting the supplement (Group 4) versus 
placebo (Group 3) (Fig 5). 
 
 
We anticipate that the leucine-rich supplement will enhance the acute exercise-stimulated mTOR 
signaling pathway and that the response will be more marked in the group undergoing the 12 week 
exercise program. This thesis is based on reports that in healthy young subjects, a leucine-rich supplement 
added to a resistance exercise program stimulates muscle protein synthesis, mass and strength (64; 72) 
and although delayed in the elderly, the anabolic response is effectively stimulated by this regimen (73).  In 
this regard, Hulmi reported that whey protein enhanced exercise-induced signal transduction 
(phosphorylation of p70S6K) by nearly five-fold over exercise alone in young healthy subjects (71).  
 
 
In summary, we anticipate that a 12-week program of exercise rehabilitation will increase muscle 
mass and improve skeletal muscle function and that this is mediated by several different processes that we 
aim to identify. In this regard we expect that basal anabolic signaling will be higher in the resting muscle of 
the regular exercise group versus the resting muscle of the usual care group, a potential carry over effect 
of regular exercise. In addition, we anticipate that while the high calorie whey supplement will cause an 
increase in anabolic signaling in the resting muscle of both groups, the response will be greater in the 
regular exercise group, again possibly reflecting a carryover effect of regular exercise.  If a carryover effect 
is indeed present, then this would support the hypothesis that regular exercise is beneficial even in ESRD.  
We also anticipate that acute exercise alone will increase anabolic signal transduction, and that this 
response will be enhanced by the high calorie whey supplement. If true, this holds the prospect of 
developing a simple therapeutic strategy to boost the anabolic response to regular exercise and 
thereby enhance the functional and cardiopulmonary benefits of exercise in patients with ESRD.  
  
Detailed Protocol for Nutrient Supplementation, Acute Exercise, and Biopsy for Specific Aim 2. An outline 
illustrating the acute exercise, biopsy, and nutrient supplementation protocol for Specific Aim 2 is presented 
in Figure 2. On a morning the day before regular MHD and after an overnight rest, both regular exercise 
and usual care subjects will consume a light protein-free breakfast between 7 and 8 am and will be studied 
three hours later after a 60-min rest. Subjects in both groups will then will begin a unilateral dominant lower 
limb exercise session lasting ≈15 min, with the contra-lateral limb at rest. At the start of the exercise 
session, subjects will ingest half the high calorie whey or placebo so as to compensate for AA deficiencies 
common in MHD subjects. The other half of the supplement will be ingested at the end of the exercise 
session so as to enhance anabolic signal transduction. This will be followed by 60 minutes of rest, then 
muscle biopsies will be performed on both limbs to assess anabolic signal transduction.  

 
The unilateral exercise will be performed using seated single knee extension exercise against 
resistance. Subjects will be seated with the hips flexed at 90ο with their back against a backrest at a ≈110ο 
angle. The leg pad will be positioned ≈5 cm above the ankle and the rotation arm will be positioned so that 
the knee is bent to 90ο. After a warm-up consisting of 8-10 repetitions with minimal resistance, the weight 
will be adjusted to 70% of the one repetition maximum. The unilateral 1-RM will be determined by a post-
training period strength test, performed on a different day. Five sets of 10 repetitions will be performed, with 
2 minutes of rest periods between sets. The load will be adjusted during the course of the session due to 
fatigue such that each subject is able to perform 10 repetitions during each set. 
 
 
 The supplement, high-calorie whey, (300 ml) comprises 24 g whey protein (contains ≈3 g leucine) with 
30g carbohydrates (sucrose) in liquid, and the placebo contains a liquid, dextrose 24 g plus sucrose 30g. 
The amount of whey (Nestle Healthcare) prescribed is guided by studies in normal subjects (66; 69; 70; 
110) including the elderly (70; 74). We do not anticipate any adverse effects from nutrient or placebo if 
taken as prescribed. Whey has a low NA, K and phosphorus content (60,140 and 60 mg/serving 
respectively). The vehicle for dissolving the powdered supplement will be of the subject’s choice, but is 
limited to fluids permissible for ESRD such as apple juice and will be pre-tested for taste by the subject. 
Whey is also available in a variety of flavors and in long term studies has been included in baked goods or 
sprinkled over foods. 
                 
Detailed molecular analysis of skeletal muscle.  The following items will be measured using methods 
established in Dr. Rabkin’s laboratory. Note that receptor and signaling proteins will be measured by 
western immunoblots; mRNA levels by real-time quantitative PCR, and selected degradative proteins by 
immunobloting and serum and local IGF-1 by ELISA. 
 
 
  mRNA levels of local regulators of muscle mass:  Total IGF-1 (all transcripts), the Ea transcript (a 
generalized tissue form and main form in liver, and the IGF-1Eb transcript, MGF, a loading sensitive and 
more potent form than the Ea form). Myostatin, and receptors for IGF-1, insulin, growth hormone and 
androgens. 
 
  mRNA levels of inflammatory cytokines IL-6 and TNFα.   
 
  Signaling protein levels and their phosphorylated forms activated by IGF-1, insulin, EX or AA, 
including IRS-1, Akt, mTOR, p70S6K, rpS6, and 4EBP-1.  
 
  Protein degradative pathway; mRNA levels of FOXO1 and 3, the atrogenes Murf-1 and 
MAFbx/atrogin-1, E3α, and the 20S proteasome α7 subunit and their proteins when antibodies are 
available. Muscle specific calpain 3 (p84) mRNA and if sufficient tissue, the mRNA for the ubiquitous 
calpains calpain 1 and 2 and calpastatin) will be determined. The 14 kDa actin protein fragment, a 
caspase-3 activity product that serves as an indirect measure of its activity (170), and LC3-II protein, a 
marker of autophagy, will be determined (111).                                                                              
 
  DNA content as an indirect measure of myonuclear number (112).                                                                      
 
  RNA content, an indirect measure of total tissue ribosomal content and protein translational capacity 
(49).     
  
     Subject Burden. We anticipate that baseline evaluations for each subject will require two-2 hour 
visits, with the exercise test and questionnaires performed on a separate day from the CT and biopsy 
evaluations. The initial visit will include questionnaires, health history, DEXA, exercise test and blood panel. 
The second visit will include the CT scan. For the exercise group, the total number of visits to the hospital 
will be approximately 14 (including exercise training sessions), and for the usual care group the total 
number of visits will be approximately 4. 
 
 
Limitations of the study and anticipated problems.   
 

(1) Though subject number is relatively small, the study is rigorous and sufficiently powered to achieve our 
aims and takes into account subject compliance. To our knowledge this is the first randomized controlled 
study to test whether home-based exercise is feasible and effective in elderly MHD subjects. Ideally we 
would have liked to include healthy sedentary subjects to serve as controls for comparison and also more 
extensive testing of nutrient supplementation, but this would have increased the required group sample 
sizes dramatically and encumbered the study. However, there is a large body of rigorous studies 
documenting the effects of exercise in healthy individuals, including signal transduction.                                             
(2) The study is relatively short in duration (3 months/subject), and surrogate markers of long-term 
outcomes such as peak VO2, serum albumin, and cardiac risk factors are used. Nevertheless, if the study 
outcome shows that our exercise training program is efficacious, then this will form the basis for a 
multicenter, long-term outcomes study. Thus, this study could be of great value in determining whether a 
long-term study of home-based exercise with or without nutrient supplementation is merited, and will be 
invaluable in the design of such a study.                                                                     
(3)  Recruitment of MHD subjects that meet our eligibility criteria and are prepared to undergo a large 
battery of tests and an exercise program involving considerable time requires highly motivated subjects 
and dedicated personnel. In terms of recruitment, the PIs and Co-investigators are accomplished in this 
area, with Dr. Schiller and staff at Satellite Healthcare being particularly experienced in recruiting and 
conducting ESRD clinical trials. Furthermore, as discussed earlier under the general study design, we will 
be able to draw on a large patient population, ~2,000, within 30-40 miles of the VAPAHCS.  
(4) The study sample will include subjects who, in addition to MHD, will have other co-morbidities. We 
recognize that a great deal of motivation will be required for compliance with the exercise program. Some 
subjects may not be able to sustain 30 continuous minutes of aerobic exercise initially, and will need to be 
gradually progressed as tolerated. The cardiac rehabilitation group at the VAPAHCS under Dr. Myers’ 
direction has significant experience with monitoring, surveillance, and motivating patients to sustain 
physical activity, and will apply similar techniques in the current proposal. We have also built a 20% 
dropout rate into the sample size estimates, as we anticipate that some patients will not complete the 
program.  
(5)  Study subjects, though functionally impaired, are likely to be highly motivated and in better condition 
than many other elderly MHD patients. However, given the consequences of losing muscle function, a low-
cost regimen beneficial to even a modest subset of patients, would seem worth implementing. Also, this 
program could serve as a model to test in younger patients, among whom many more may be able to 
participate.        
(6) Since the exercise training program consists of endurance and resistance exercise, and we anticipate 
that the daily activities of the subjects will increase in response to the exercise, we will be unable to define 
the role played by each type of training in any of the responses that occur. To define these respective 
roles, separate studies of resistance and endurance exercise would be required, but this is beyond the 
scope of this study. 
(7) The study of nutrient supplementation is limited to the acute response at the time of exercise on one 
occasion only. It would be preferable to also study the long-term effects of this combination, but to ensure 
recruitment to achieve our primary goal of testing home-based exercise training, we have limited this study 
to a pilot. A wider study would require increasing the subject number several fold.  If positive, this pilot will 
form the basis of a more extensive study. 
(8) As the nutrient supplement to be administered contains several soluble proteins and AA, we will not be 
able to attribute any responses to a specific AA. This is well beyond the scope of the present proposal. 
(9) While each of the questionnaires has been widely used and shown to be valid, each is subjective. The 
accuracy of the responses depends upon subject’s recollection and the judiciousness with which 
researcher and subject conduct the interview. For example, though the KDQOL has been demonstrated to 
be reliable and valid (90), some domains have a higher internal reliability than others. In addition, there is 
some evidence to suggest that subjects participating in a study tend to overestimate their compliance to 

exercise and diet (113). Our research group has extensive experience in using these tools; particular 
emphasis will be placed on taking the necessary time to obtain responses as accurately as possible.  
 
                                                    
 
 
Statistics.  Sample Size Computation and Power Analysis.   
  Primary endpoint, Specific Aim 1.  For this analysis we have used data from a recent 12-week 
study by Storer et al (36) on the effects of endurance exercise versus no exercise (usual care) on 
cardiorespiratory and skeletal muscle function in MHD patients. Change from baseline for peak VO2 was 
22% (an increase) in the exercise group and 6% (a decrease) in the usual care group. These findings 
are consistent with an average increase from baseline of 17% in MHD patients participating in exercise-
based rehabilitation programs reported in the literature (14). Similarly, our primary outcome will be 
comparison of the change in peak VO2 for patients randomized to exercise vs. usual care. To calculate 
the desired sample size we used a two group t-test of equal means and calculated the per group sample 
size (East 5, Cytel Inc, copyright 2007). Given the desired values for the standardized effect size (the 
absolute value of the difference in group mean changes divided by the common standard deviation) and 
overall level of significance and power of the test, the required sample was calculated. To detect a 
difference in group mean changes of 17% in peak VO2 for exercise vs. usual care, given a common 
standard deviation of 20% (based on Storer et al (36), using a standardized effect size of 0.85), 23 
subjects/group would be required to complete the study to achieve statistical power of 80% with a two-
sided test of significance at the 0.05 level. Appendix VII shows how the required sample size varies by 
the standardized effect size for power of 0.80 (solid curve) and 0.85 (dashed curve). Appendix VIII 
provides curves showing how the power varies by the standardized effect size for a per group sample 
size of 23 (solid curve) and 26 (dashed curve).  For example, a per group sample of 23 provides power 
of about 0.17 and 0.38 to detect a small (0.3) and medium (0.5) standardized effect size, respectively. 
To allow for a 20% dropout rate we plan to enroll 30 subjects per group, or a total of 60. The 20% 
dropout rate is based on the PI’s experience (87), and other similar research studies. 
 
 
   Secondary analyses, Specific Aim 2. In this specific aim we will analyze molecular changes in 
muscle biopsied at baseline and at the end of the study. IGF-1 is a key muscle growth factor; Kopple et 
al (45) reported that IGF-1Ea mRNA levels rose ~100% in 12 MHD subjects undergoing 21.5 weeks of 
combined resistance and endurance exercise. We plan to compare changes in IGF-1 mRNA levels from 
baseline to those levels at the end of 12 weeks of rehabilitation or usual care. Muscle will be obtained 
from limbs at rest in the exercise and usual care subjects as described above (n=12/group) and we will 
consider a 50% change in IGF-1Ea mRNA to be a clinically meaningful difference. While there are no 
data available in MHD subjects to estimate the standard deviation of the change in these molecular 
indices from baseline, Appendix VIII may be used to estimate the power associated with an assumed 
standardized effect size that is small (0.3), medium (0.5) or large (0.8).  
 
 
     Secondary analyses, Specific Aim 2A. In this specific aim we will assess the nutrient-enhanced 
exercise-induced protein signal transduction pathway in subjects undergoing exercise versus usual care. 
Specifically, we will compare the change in activity (phosphorylation) of the mTOR signal transduction 
pathway induced by unilateral exercise alone versus unilateral exercise plus nutrient supplementation. This 
will be a pilot study of 12 subjects in each group. Since this is a novel measurement and there are no data 
in MHD subjects describing the variance of this response, a power analysis has not been carried out. 
Change in activity will be taken to equal the difference in activity between the unilateral exercise limb and 
the contra-lateral resting limb of the same subject. We will also examine the effect of the supplement alone 
vs. placebo on signal transduction in the resting limb of unilateral exercise and usual care subjects. Of 
note, Hulmi (71) reported that whey protein enhanced exercise-induced signaling (phosphorylated p70S6K) 

by nearly 5-fold over exercise alone in young healthy subjects, while Drummond (73) noted a similar 
increase in the elderly following EAA ingestion.                                                                                               
 
            Statistical analysis plan. Comparisons of clinical and demographic data between groups at 
baseline will be performed by the appropriate statistical test (for continuous variables, t-test for normally 
distributed data and Wilcoxon test for non-normal data; for categorical variables, chi-square test or Fisher’s 
exact test). These comparisons will be performed at baseline only to assess adequacy of randomization. 
Comparisons between groups for cardiopulmonary exercise test, muscle biopsy, energy expenditure, 
cognitive function, and other functional measures before and after the study period will be performed by 
multivariate ANOVA; post-hoc comparisons will be made using the Bonferroni method. To analyze 
continuous variables measured at three or more time points we will use linear mixed effects regression 
(114). To explore the relationships between a given outcome and other variables, we will use multiple 
regression techniques. Since the reliability of a fitted regression model may be reduced if too many 
predictors are used (114), we will limit the number of predictors to four variables in each model (≤1/10 of the 
total). For the primary outcome, change in peak VO2, the specific independent variables in the model will 
include exercise compliance (average energy expended in kcals/week), change in muscle strength, and 
change in muscle mass. Missing data will be managed using the multiple imputation method which is 
preferable to older methods and described in detail by Schafer & Olsen (1999).  
 
 
For Specific Aim 2 (effect of acute and chronic exercise on muscle wasting), we will employ a linear 
model, using period (baseline vs. 4 months) and treatment (exercise training vs. usual care) as independent 
variables along with their interaction (Bonferroni method). The intention to treat principal will be applied for 
all analyses.   
                                                                                 
            Timeline.  We anticipate that we will recruit ≈15 MHD subjects/year during the 4 years of the study. 
We expect that it will take four years to complete the study, with much of the final year dedicated to 
completing sample processing and data analysis. We do not expect much delay in getting started as there is 
an established exercise research program at our facility. In addition, the techniques for biopsying muscle as 
well as measuring muscle structure and cellular biology are established in the PI’s (Rabkin) research 
laboratory. Finally, both the Satellite Healthcare Dialysis research group (Dr. Schiller) and Dr. Chertow’s 
group (Stanford University) have vast experience in recruiting and performing clinical trials in MHD patients. 
A study Timeline of Project Tasks is presented in Appendix I. 
 
 
           Plan for Dissemination. Dissemination will be a major focus of year 4. We believe the results of this 
study will lend themselves well to presentations at the VA RR&D meeting, American Society of Nephrology, 
and a number of rehabilitation meetings (AACVPR, ACSM). The Co-PIs will devote efforts during years 3 
and 4 to abstract presentations and manuscript preparation. We anticipate that there will be results on 
functional and psychosocial outcomes that will be appropriate for publication in the Journal of Rehabilitation 
Research and Development, Journal of Cardiopulmonary Rehabilitation and Prevention, and Archives of 
Physical Medicine and Rehabilitation. Physiological results will be appropriate for the Journal of Applied 
Physiology and Nephrology journals, including Journal of the American Society of Nephrology, American 
Journal of Kidney Diseases, and others.  
 
PRELIMINARY STUDIES 
 
             Exercise training studies. The Co-PI (Dr. Myers) and one of the Co-Investigators on the current 
proposal (Dr. Froelicher) have previously conducted a 3-year NIH-funded Specialized Center of Research 
(SCOR) project of exercise training in patients with cardiovascular disease (115; 116), and are currently 
conducting an NIH Specialized Center of Clinically-Oriented Research (SCCOR) project on the effects of 
exercise therapy in patients with abdominal aortic aneurism disease (87; 93). The proposed project will use 
existing exercise testing and training facilities at the VA Palo Alto Medical Center, and methods similar to 
the current NIH SCCOR project will be used in the current proposal. 

 
              Dr. Myers’ research focus has been in the areas of exercise testing, training, and epidemiology in 
patients with coronary artery disease, chronic heart failure, and spinal cord injury. Since January 1992, he 
has directed the exercise research laboratory at the Palo Alto VAHCS.  Dr. Myers has a more than 25 year 
body of work related exercise testing and training in patients with cardiovascular disease. He has served 
on writing groups for guidelines developed by the American Heart Association (AHA), American College of 
Sports Medicine (ACSM), American Thoracic Society (ATS), American Association of Cardiovascular and 
Pulmonary Rehabilitation (AACVPR), and the European Society of Cardiology Working Group on 
Prevention and Rehabilitation. He is a recent recipient of the AACVPR Established Investigator Award, and 
is a Senior Research Career Scientist Award recipient from the VA. The fifth edition of his textbook co-
authored with Dr. Froelicher, “Exercise and the Heart: Clinical Concepts” was published in April 2006.    
 
             Dr. Myers has also published extensively in the area of exercise training among patients with 
chronic heart failure (CHF). The focus of these studies has been on the application of MRI to quantify the 
effects of training on the myocardial remodeling process. He and his colleagues were the first to employ 
this technology to address a controversy that arose in the 1990s, in which it was suggested that exercise 
training could lead to myocardial damage. Using a high intensity training protocol and a series of 
randomized trials, it was demonstrated that training did not cause further myocardial damage among 
patients with CHF (117-120). These results have since been confirmed by studies performed in Italy, 
Canada, the US and elsewhere, and have been influential in the development of rehabilitation guidelines in 
these patients. Dr. Myers’ followed these initial studies by applying a novel MRI tagging technique, in which 
the myocardium could be visualized more closely in 3 dimensions, including rotational displacement during 
systole and diastole (120). These studies demonstrated no adverse effects of training on myocardial size, 
function, or rotation velocity during systole up to one year after undergoing training, and an improvement in 
relaxation velocity in a subgroup of patients with non-ischemic cardiomyopathy.     
 
              An ongoing NIH SCCOR project entitled, “Effects of exercise therapy in abdominal aortic 
aneurysm (AAA) disease” (Myers and Dalman, Co-PIs, Froelicher, Co-Investigator) is in its 6th year, and 
employs exercise testing and training methods that are similar to those in the current proposal (87; 93).  
The purpose of the SCCOR project is to test the effectiveness of exercise training to reduce abdominal 
aortic aneurysm risk, limit small aneurysm progression, and modify biologic markers of AAA disease. The 
study includes 3 arms; one to identify signature protein profiles of AAA disease, another the application of 
MRI to develop and validate hemodynamic computational models in AAA progression, and the 3rd to 
investigate the effects of 3 years of exercise training on AAA size, aneurysm risk, and physiologic 
responses to exercise. Some early results of this trial are presented below. 
 
             Safety, efficacy, and adherence to a home-based exercise training program in patients with 
cardiovascular disease.  Myers J, et al. Effects of exercise training in patients with abdominal aortic 
aneurysm (AAA): Preliminary results from a randomized trial. J Cardiopulm Rehabil Prev 30: 374-383, 
2010, and Myers J et al. Cardiopulmonary exercise testing in abdominal aortic aneurysm: Profile, safety, 
and mortality estimates. Eur J Cardiovasc Prev Rehabil 18:459-466, 2011. Figure 6 below shows changes 
in exercise capacity in 57 subjects with AAA disease after 1-year of home-based exercise training at the 
VA Palo Alto rehabilitation facility. At the 3-year evaluation point, significant differences between exercise 
and usual care groups were also observed for peak VO2, VO2 at the ventilatory threshold, and other indices 
of the training response, such as a reduction in submaximal heart rate. Using a case-management 
approach and the application of weekly phone calls, activity logs, heart rate monitors and pedometers, 
subjects in the exercise group expended an average of >2,000 kcals/week of recreational energy 
expenditure. In a substudy, these methods of activity surveillance have been validated (Myers J, Dupain M, 
Vu A, Powell A, Smith K, Dalman R, on behalf of the Stanford AAA SCCOR Investigators. Agreement 
between activity monitoring devices during home rehabilitation: A substudy of the AAA STOP Trial. J Phys 
Act Aging, in press, 2012).   

 
 
 
 
 
 
  Figure 6. Bar graphs showing 42% increase in exercise time in the exercise group and no change in usual 
care subjects following 1-year of exercise training in patients with abdominal aortic aneurysm disease (86). 
Changes in exercise capacity were similar at the completion of the full trial, after up to 3 years of home-
based exercise training. 
  
     Data on the approach to activity monitoring and surveillance. In the current proposal, methods for 
monitoring and surveillance of physical activity will be employed that are similar to the above-mentioned 
ongoing NIH trial. In our NIH trial, we observed that subjects achieved 98±7% of their target heart rates 
during monitoring (87). Using 7-day activity recall instruments to quantify weekly activity, the mean overall 
energy expenditure for subjects in the exercise group after 1 year of participation was 2269±1207 
kcals/week (shown in the figure below). This is roughly double the CDC/AHA recommendations for 
physical activity, and is significantly higher than the recently-published HF-ACTION trial (≈500 kcals/week) 
and other rehabilitation studies. This degree of exercise stimulus resulted in a 42% increase exercise 
capacity on the 1-year treadmill test, while no changes were observed in controls. Thus, the approach 
proposed is likely to be successful in achieving an adequate training stimulus.    
 
Figure 7. Average energy expenditure over 1 year in the exercise group (from reference #86) 
 
             Studies on muscle wasting in kidney failure.. Dr. Rabkin (Co-PI) is a Professor of Medicine, 
Emeritus Active at Stanford University, and a Staff Nephrologist at the Palo Alto VAHCS.  The focus of his 
research is on the mechanisms of muscle wasting in CKD with the goal of identifying potential strategies 
for the prevention and treatment of this disorder. The current proposal reflects an effort to translate his 
many animal findings to a clinical population with CKD. The following briefly highlights some of this work 
relevant to the current proposal.  
  
            Chronic uremia attenuates growth hormone induced signal transduction in skeletal muscle. Sun D, 
Zheng Z, Tummala P, Oh J, Schaefer F, Rabkin R:  J Am Soc Nephrol 15:2630-6, 2004. Resistance to 
growth hormone contributes to muscle wasting in uremia.  To test whether impaired growth hormone 
signaling is a cause of the resistance to growth hormone and to elucidate its mechanisms, we studied 

**
PF
CRF
PF
CRF
2 days
7 days
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0
100
200
300
400
500
Relative IGF-1 mRNA level
CON
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muscle growth hormone signaling and action in rats with surgically induced chronic renal failure.  As we 
observed previously, growth hormone stimulated IGF-1 expression was depressed in CRF and evidence 
is provided that the resistance to growth hormone is caused at least in part by impaired JAK2-GHR-
STAT5 phosphorylation and nuclear STAT5 translocation. Furthermore, this study demonstrated that the 
attenuated JAK2 –STAT5 signaling may be caused by at least two different processes.  One involves 
depressed phosphorylation of the signaling proteins because of increased suppression of cytokine 
signaling-2 expression that may be linked to low-grade inflammation. The other may involve increased 
signaling protein dephosphorylation because of heightened protein-tyrosine phosphatase activity.  
 
            Work-induced changes in skeletal muscle IGF-1 and myostatin gene expression in uremia.  Sun 
DF, Chen Y, Rabkin R. Kidney Int 70:453-9, 2006. Resistance to GH induced IGF-1 gene expression 
contributes to uremic muscle wasting. Since exercise stimulates muscle IGF-1 expression independent of 
growth hormone, we tested whether work overload could increase skeletal muscle IGF-1 expression in 
uremia and thus bypass the defective growth hormone action. In addition, to provide insight into the 
mechanism of uremic wasting and the response to exercise we examined myostatin expression.  
Unilateral plantaris muscle work overload was initiated in uremic and pairfed normal rats by gastrocnemius 
tendon ablation with the contralateral plantaris as a control.  Some rats were growth hormone treated for 7 
days. Work overload stimulated a gain in weight of the plantaris muscle in both groups and corrected the 
uremic muscle atrophy.  As shown in Figure 8 below, growth hormone increased plantaris IGF-1 mRNA 
levels >2 fold in pairfed rats but the response in chronic renal failure was severely attenuated.  
 
         
 
 
 
 
 
 
 
 
 
Fig 8. Effect of GH on muscle IGF-1 mRNA levels       Fig 9. Effect of work overload on muscle IGF-
1 mRNA levels 
 
Work overlaod increased the IGF-1 mRNA levels significantly in both the chronic renal failure and pairfed 
groups, albeit less brisk in renal failure (Figure 9). However after 7 days IGF-1 mRNA levels were elevated 
similarly, >2 fold, in both groups. Of note, the atrophied uremic plantaris muscle basal myostatin mRNA 
levels were increased significantly and normalized after an increase in work-overloaded rats suggesting a 
myostatin role in the wasting process (Figure 10).  
 
 
 
 
 
 
Fig 10.  Myostatin levels in CRF and response to 
work overload 
 
 
 
In summary, these findings provide insight into the 
mechanisms of skeletal muscle wasting in uremia and the hypertrophic response to exercise, and suggest 
that alterations in the balance between IGF-1 and myostatin play an important role in these processes. 
 

             Increased workload fully activates the blunted IRS-1/PI3-kinase/Akt signaling pathway in 
atrophied uremic muscle. Chen Y, Sood S, Biada J, Roth R, Rabkin R.  Kidney Int. 2008. In this study of 
muscle wasting and recovery in chronically uremic rats, we set out to characterize the mechanisms 
whereby resistance exercise increases muscle mass, despite the presence of impaired signaling through 
the IRS-1/PI3-kinase/Akt pathway and insensitivity to IGF-1.  Uremic and sham nephrectomized (SN) rats 
with surgically induced unilateral plantaris muscle work overload were studied, with the contralateral 
plantaris muscle serving as a nonloaded control. In these control muscles signaling protein levels were 
similar in the chronic renal failure and SN groups despite atrophy of the chronic renal failure muscle. 
However, basal state activation (phosphorylation) of the IRS-1/PI3-kinase/Akt signaling pathway was 
depressed in the uremic rat muscle.  Of note, even though the basal activity of the IRS-1/PI3-kinase/Akt 
signaling pathway was depressed in the uremic atrophied plantaris muscle, simply increasing the muscle 
workload effectively activated this signaling pathway and phosphorylation of the signaling proteins in the 
work-overloaded plantaris muscle was similar in both groups of rats (Figure 11 below).  Work overload 
induced a significant increase in local IGF-1 and mechano-growth factor expression and also a significant 
increase in signaling protein levels. Together these changes provide an explanation for the work-induced 
increase in signal transduction.  Importantly, the work-induced signaling response in the uremic rats was 
similar to that observed in non-uremic rats and promoted changes that we observed consistent with a 
decrease in muscle protein degradation, an increase in protein synthetic capacity and an increase in 
myonuclear number. 
 
 
 
 
 
 
 
 
 
 
 
 
 
Fig 11. Levels of phosphorylated signaling protein levels in control and work overloaded plantaris muscle 
of SN (sham nephrectomy) and chronic renal failure rats. Phosphorylation of IRS-1 and Akt is depressed 
in the control (con) non-loaded muscle of the renal failure group. Work overload stimulated signaling 
protein phosphorylation similarly in both groups of rats.   
 
We concluded that increasing the work of atrophied muscle in uremia can fully activate the depressed 
basal IRS-1/PI3-kinase/Akt signaling pathway and it appears to do so by increasing IGF-1 and 
mechano growth factor expression and increasing the levels and phosphorylation the IRS-1/PI3 
kinase/Akt/ mTOR signaling proteins. This in turn appears to account for the increase in the reduced 
uremic muscle mass, which reached a level comparable to that seen in normal rat. 
 
            Leucine stimulated mTOR signaling is partly attenuated in skeletal muscle of chronically uremic 
rats.  Chen Y, Sood S, McIntire K, Roth R, Rabkin R. Am J Physiol Endocrinol Metab. 2011. The 
branched chain amino acid leucine stimulates muscle protein synthesis in part by directly activating the 
mTOR signaling pathway. Furthermore, leucine, if given in conjunction with resistance exercise, enhances 
the exercise-induced mTOR signaling and protein synthesis. In this study we tested whether leucine can 
activate the mTOR anabolic signaling pathway in uremia and whether it can enhance work overload-
induced signaling through this pathway. CKD and control rats were studied after 7 days of surgically 
induced unilateral plantaris muscle work overload and a single leucine or saline load. In the basal state 
4E-BP1 phosphorylation was modestly depressed in non-work overload muscle of CKD rats while rpS6 

phosphorylation was nearly completely suppressed. After oral leucine mTOR, S6K1and rpS6 
phosphorylation increased similarly in both groups while the phospho-4E-BP1 response was modestly 
attenuated in CKD. In work overload CKD muscle leucine augmented mTOR and 4EBP1 phosphorylation, 
but its effect on S6K1 phosphorylation was attenuated and it failed to stimulate rpS6 phosphorylation.  
In summary, this study has established that the chronic uremic state impairs basal signaling 
through the mTOR anabolic pathway, an abnormality that may contribute to muscle wasting. However 
despite this abnormality, leucine can stimulate this signaling pathway in CKD, though its 
effectiveness is partially attenuated, especially in skeletal muscle undergoing sustained work overload. 
Thus, even though there is some resistance to leucine in CKD, these results suggest a potential role for 
leucine rich supplements in the management of uremic muscle wasting. 
 
Description of key relevant studies published by co-investigators. 
Dr Chertow (Co-Investigator) is Professor Medicine /Nephrology and Chief of Nephrology at Stanford 
University and a leader in the field of outcomes research in CKD.  Dr. Kurella-Tamura (Co-Investigator) is 
a Staff Nephrologist/Gerontologist VAPAHCS and Assistant Professor of Medicine/Nephrology at Stanford 
University. She is a widely recognized authority on cognitive disorders in the CKD population. Some 
representative studies relevant to the current proposal are presented below.  
 
           Cognitive impairment in chronic kidney disease.  Kurella-Tamura M, Chertow GM, Luan J, and 
Yaffe K.  J Am Ger Soc; 52:1863-1869, 2004. The prevalence of cognitive impairment and its association 
with the severity of kidney disease has not been defined.  A cross-sectional study of cognitive function was 
performed in 160 ambulatory persons with ESRD and CKD not yet on dialysis. The performance on three 
cognitive function tests assessing global cognitive function (3MS), executive function (Trails B), and verbal 
memory (California Verbal Learning Trial, or CVLT) was compared to published norms. Overall, 17% of 
subjects met established criteria for global cognitive impairment.  Global impairment was present in 27% 
with ESRD and 15% with advanced CKD, but in no subjects with mild to moderate CKD (p=0.02).  
Unadjusted and adjusted mean scores on the 3MS, Trails B, and CVLT immediate and delayed recall were 
significantly worse for subjects with ESRD than for subjects with CKD or published norms (p<0.001 for all 
comparisons). Scores on the Trails B (p<0.001) and CVLT immediate (p=0.01) and delayed recall (p<0.001) 
were significantly worse for subjects with CKD not requiring dialysis than for published norms. In addition, 
the fraction of subjects with impairment on the 3MS and Trails B increased with decreasing kidney function. 
These results demonstrate that significant impairment in cognitive function is present in both CKD and 
ESRD, and suggest kidney disease directly contributes to cognitive impairment.   
 
            Significance of frailty among dialysis patients. Johansen KL, Chertow GM, Jin C, Kutner NG. J Am 
Soc Nephrol. 2007 Nov;18(11):2960-7. The aim of this study was to determine the prevalence and 
predictors of frailty among a cohort of incident dialysis patients and to determine the degree to which frailty 
was associated with death and hospitalization. A cohort of 2,275 adults was studied who participated in 
the Dialysis Morbidity and Mortality Wave 2 study, of whom two-thirds met our definition of frailty: a 
composite construct that incorporated poor self-reported physical functioning, exhaustion/fatigue, low 
physical activity, and undernutrition. Multivariable logistic regression analysis suggested that older age, 
female sex, and hemodialysis (rather than peritoneal dialysis) were independently associated with frailty. 
Cox proportional hazards modeling indicated that frailty was independently associated with higher risk of 
death (adjusted hazard ratio [HR] 2.24, 95% confidence interval [CI] 1.60–3.15) and with the combined 
outcome of death or hospitalization (adjusted HR 1.63, 95% CI 1.41–1.87). Therefore, frailty is extremely 
common and is associated with adverse outcomes among incident dialysis patients. Given its prevalence 
and consequences, increased research efforts should focus on interventions aimed to prevent or 
attenuate frailty in the dialysis population.