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Exercise in Maintenance Hemodialysis Patients Induces Transcriptional Changes in Genes Favoring Anabolic Muscle

Joel D. Kopple^*,,, Huiyuan Wang^*, Richard Casaburi^*,, Mario Fournier, Michael I. Lewis^,, Wayne Taylor^|| and Thomas W. Storer^*,¶

^* Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center and ^¶ El Camino College, Torrance, and David Geffen School of Medicine at UCLA, UCLA School of Public Health, Burns and Allen Research Institute, Cedars-Sinai Medical Center, and ^|| Martin Luther King/Drew School of Medicine, Los Angeles, California

Correspondence: Dr. Joel D. Kopple, Division of Nephrology and Hypertension, Harbor-UCLA Medical Center, 1000 W. Carson Street, Torrance, CA 90509. Phone: 310-222-3891; Fax: 310-782-1837; E-mail: jkopple{at}labiomed.org

Received for publication July 26, 2006. Accepted for publication July 6, 2007.

	Introduction

Top Introduction Results Discussion Concise Methods Disclosures References

 
Exercise training increases exercise capacity of maintenance hemodialysis patients, but the cellular mechanisms responsible for this effect are unclear. We studied the effects of different forms of exercise training (endurance, strength, or a combination where patients underwent about one-half each of the endurance and strength training of the first two groups) on mRNA levels of genes in muscle that may contribute to increased exercise capacity. Before exercise training, muscle mRNA of insulin-like growth factor (IGF)-IEa, IGF-I receptor, IGF-II, IGF-binding protein (IGFBP)-2, and IGFBP-3 were lower in hemodialysis patients than normal controls. After approximately 21 weeks of exercise training, muscle mRNA increased significantly for all of these genes, as well as for IGF-IEc. Muscle mRNA for myostatin, a protein that inhibits skeletal muscle protein synthesis and muscle hypertrophy, decreased. The responses of mRNA to the different types of exercise training generally showed similar significant changes or trends. Muscle IGF-I protein also increased with exercise. Anthropometry, but not dual energy x-ray absorptiometry or bioelectrical impedance, showed a decrease in body fat and an increase in fat-free mass. In conclusion, exercise training in hemodialysis patients induces changes in skeletal muscle mRNA and increases muscle IGF-I protein, which may promote protein anabolism.

Maintenance hemodialysis (MHD) patients frequently describe malaise and display muscle weakness, sarcopenia, and intolerance to all but the mildest forms of exercise.^1–6 These factors can contribute to the impaired rehabilitation and constricted lifestyle and quality of life of these individuals.^1,3–5 Many studies indicate that exercise training by MHD patients improves their exercise capacity.^1–6 In healthy humans and mammals, improvement in exercise capacity is associated with the expression in skeletal muscle of many proteins, including those involved with increased enzyme activity and myosin heavy chains.^7–10 There are increased quantities of growth factor proteins that promote synthesis or suppress degradation of proteins. There are almost no studies concerning the cellular mechanisms that are responsible for improved exercise capacity in exercising MHD patients.

This study was undertaken in MHD patients to examine, in right vastus lateralis muscle, the effects of various types of exercise training on mRNA levels for various growth factors that stimulate or suppress protein synthesis or inhibit protein degradation. Normal sedentary adults served as control subjects.

	Results

Top Introduction Results Discussion Concise Methods Disclosures References

 
Eighty MHD patients completed baseline testing, including a muscle biopsy, and then underwent randomization to one of the four groups: Endurance training (ET), strength training (ST), endurance plus strength training (EST) or no training (NT). Seventeen patients (six ET, two ST, five EST, and four NT) dropped out because of illness, noncompliance, moving away, or possibly reluctance to undergo another muscle biopsy. Of these, two ET, one ST, and three EST patients dropped out before (five patients) or only after 1 wk of exercise training. As a result of a laboratory accident, one or more of the muscle biopsy specimens from 12 patients were destroyed. This left data from 51 patients who completed the study.

Characteristics of the 51 MHD patients and 20 normal control subjects are shown in Tables 1 and 2. MHD patients exercised for 21.5 ± 0.7 wk. No patient had insulin-dependent diabetes. The normal individuals had normal serum creatinine levels, and none had a history of kidney disease or hypertension. Mean age and serum albumin were similar in the two groups; hemoglobin and hematocrit were only slightly lower in the MHD patients, which probably reflects the effects of erythropoietin treatment.

Serum C-reactive protein (CRP), TNF-, and IL-6 concentrations in the MHD patients did not change with exercise training or in the NT group (Table 2). Serum CRP and IL-6, obtained from all four pre-exercise MHD groups combined, did not differ from the initially obtained normal values. Pre-exercise serum TNF- was significantly greater than normal (P < 0.001). Dietary energy and protein intakes, before exercise, expressed in kcal/d or g/d or kcal/kg per d or g/kg per d, were significantly lower for the four groups combined (P < 0.05 to 0.01; Table 2) except for protein intake, expressed in g/kg per d. Dietary energy or protein intakes did not change with exercise training in any group or in the three exercising groups combined.

Skeletal muscle IGF-IEa mRNA in the four groups of MHD patients, before the onset of exercise training, was significantly lower than in the normal control subjects (Table 3). With exercise training, muscle IGF-IEa mRNA tended to increase in all groups of MHD patients, and this increase was significant in the ST and EST groups and in all three exercising MHD groups combined (Table 3). Skeletal muscle IGF-IEc mRNA also tended to be lower than normal in the MHD groups, although these values were not significantly different (Table 3). With exercise training, IGF- IEc tended to increase in all three groups, and this increase was significant in the EST group and in all three exercising groups combined (Table 3). The IGF-I receptor (IGF-IR) mRNA before exercise training in the four MHD groups combined was significantly lower than in the normal group. By the end of exercising, IGF-IR mRNA increased significantly in the three exercising groups combined.

IGF binding protein-2 (IGFBP-2) mRNA in the MHD patients, before commencing exercising, was lower than normal (Table 3). With exercise training, IGFBP-2 mRNA increased significantly only in the ET group and in all exercising groups combined. IGFBP-3 mRNA in the MHD patients, before exercising, was not significantly lower than normal values. With exercise training, IGFBP-3 mRNA rose significantly in the three groups of exercising patients combined.

Before commencement of exercise training, there were no significant differences in the myostatin mRNA levels between the four groups of MHD patients combined and the normal control subjects (Table 3). After exercise training, there was some decrease in myostatin mRNA in each of the three exercising groups (ET 22%; ST 23%; EST 39%) and some increase in the NT group (29%). This decrease was statistically significant (P < 0.05) for all three groups combined. The fall in myostatin mRNA in the ET, ST, and EST groups combined was also significantly different from the change in the NT group (P < 0.05).

In the 29 MHD patients who had mid-training muscle biopsies, mRNA was also assayed for IGF-IEa, IGF-IEc, IGF-IR, IGF-II, IGF-IIR, IGFBP-2, IGFBP-3, and myostatin. In general, the same changes in mRNA levels present in the final muscle specimens were already present as trends in the mid-training biopsies (data shown only in Figure 1). By coincidence, the mean IGF-IEc values in the patients who underwent three muscle biopsies tended to be greater than in the patients who underwent two biopsies. We are unable to identify a cause for this discrepancy (e.g., there were no differences between the two groups with regard to age, gender, presence or absence of diabetes, or proportions of patients undergoing each type of exercise intervention).

View larger version (13K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Time course of IGF-IEc mRNA levels (A) and IGF-II mRNA levels (B) in right vastus lateralis muscle of MHD patients undergoing various types of exercise training or no exercise training. Lines indicate mean values for each group of MHD patients.

No MHD patient or normal individual manifested edema. There were no significant differences in the pre-exercise anthropometric measurements in the four MHD patients combined versus the normal control subjects when the two genders were considered together. In the male patients considered separately, the same anthropometric similarities and differences were found except for a lower proximal thigh muscle area in the four MHD groups combined at baseline (187.7 ± 7.5 cm²) as compared with the normal men (215.3 ± 11.2 cm²; P = 0.044). The number of female patients studied was considered to be too small for separate statistical comparisons. After exercise training, there was a significant decrease in triceps and subscapular skinfold thicknesses in all three exercise groups combined (Table 5). Most striking, after exercise, anthropometry-derived percentage of body fat and total body fat decreased and fat-free mass (FFM) increased significantly in all exercising patients combined.

	Discussion

Top Introduction Results Discussion Concise Methods Disclosures References

 
This study indicates that in right vastus lateralis muscle of sedentary MHD patients, there were decreased mRNA levels for a number of factors that may stimulate protein anabolism, suppress protein degradation, and promote accrual of protein. In particular, the mRNA for IGF-IEa, IGF-IR, IGF-II, and IGFBP-2 were decreased. Exercise training was associated with an increase in muscle mRNA for many of these growth factors, namely, IGF-IEa, IGF-IEc, IGF-IR, IGF-II, IGFBP-2, and IGFBP-3. Moreover, myostatin mRNA fell significantly. As might be expected, there was no change in the mRNA for any growth factor in the NT patients. As a result of these gene transcription changes, most of the mRNA levels were closer to normal at the end of exercise training. The only exceptions were IGFBP-2 mRNA, which after exercise training tended to be greater than normal, and myostatin mRNA, which tended to be less than normal. These findings indicate that in clinically stable, sedentary MHD patients, long-term exercise training induces a pattern of changes in gene transcription that would be likely to promote protein synthesis and reduce protein degradation.

Muscle IGF-I peptide was significantly increased whereas muscle IGF-II protein was normal in the MHD patients before they exercised. The significant further increase in muscle IGF-I protein with exercise in the ET and ST groups and in the three exercising groups combined (Table 4) is consistent with the rise in the skeletal muscle mRNA levels for IGF-IEa and IGF-IEc. These results provide further evidence that exercise training engenders an anabolic response in sedentary MHD patients. The absence of a rise in muscle IGF-II despite the rise in IGF-II mRNA with exercising may reflect posttranscriptional or posttranslational factors. It is also possible that the IGF-II protein levels rose transiently during exercise training and then fell back to normal by the time of the biopsy.

Skeletal muscle IGF-I promotes skeletal muscle protein synthesis and hypertrophy, preserves architecture, and suppresses protein degradation.¹¹ It seems that circulating IGF-I has little effect on muscle hypertrophy,¹² and most skeletal muscle IGF-I is synthesized in situ.¹³ In this regard, Nindl et al.¹⁴ described a decrease in serum total IGF-I, ternary IGF-I (IGF-I, IGFBP-3, and the acid labile substance), and the IGF-I/IGFBP-3 ratio during resistance training in 10 maintenance dialysis patients. In this study, serum IGF-I was normal and serum IGF-II was elevated before exercising, and neither peptide changed with exercise in our MHD patients (Table 4). Similar to our findings, MacDonald et al.¹⁵ described normal serum IGF-I and IGFBP-3 in 17 MHD patients; however, muscle IGF-I was low in their patients, whereas we observed increased muscle IGF-I that rose further with exercise (Table 4). The low muscle IGF-I may have been due to the wasted state of their patients, as indicated by anthropometry and body composition measurements, whereas our patients did not seem wasted. This discrepancy might be explained by the fact that our MHD patients were a more selected group of healthier individuals because they were required to undergo exercise training in our study.

Three isoforms of IGF-I are recognized in humans: IGF-IEa, IGF-IEb, and IGF-IEc. These isoforms are generated by splice variants of the IGF-I gene.¹⁶ IGF-IEa and IGF-IEc are present in skeletal muscle, whereas IGF-IEb is found primarily in liver and is also detected in muscle. These reductions in mRNA for IGF-IEa, IGF-IR, and IGF-II may predispose to the myofiber atrophy and sarcopenia that are described in MHD patients^17–19 and reduce their ability to remodel muscle protein. Although IGF-IEc mRNA was not significantly reduced in the MHD patients, the same trend toward lower mRNA values was observed for this growth factor as for IGF-IEa mRNA (Table 3). IGF-IEc, also called mechanogrowth factor, is of special interest for exercise training, because it may play a particularly important role in promoting skeletal muscle physical capacity and hypertrophy.²⁰

Myostatin is inhibitory to skeletal muscle protein synthesis and muscle hypertrophy.²¹ The finding that myostatin fell with exercise training is again consistent with a protein synthetic response to exercise training. These results are consistent with the findings of Sun et al.,²² who described an increase in IGF-I mRNA and a decrease in myostatin mRNA in plantaris muscle of rats with chronic renal failure after the rats were subjected to a chronically increased workload on that muscle.

The dietitian-performed anthropometry indicated that the MHD patients who underwent exercise training experienced a decrease in triceps and subscapular skinfold thicknesses, percentage of body fat, and total body fat mass and an increase in FFM (Table 5). The DEXA and bioelectrical impedance studies showed no changes in body composition, except for reduced bone mineral density with exercise training observed by DEXA. The reason for this discrepancy is not known. Because DEXA is often considered a more reliable measure of body composition and because the DEXA and bioelectrical impedance measurements were in agreement, we conclude that there probably were no changes in FFM or fat mass in these patients. Although no patient or normal individual had evidence of edema, MHD patients cannot regulate their water balance well; therefore, it is possible that undetected increases or reductions in total body water may have obscured our ability to detect the effects of exercise on muscle mass by DEXA or bioelectrical impedance measurements.

The increase in skeletal muscle mRNA levels for growth factors in our exercising MHD patients is similar to those reported with exercising normal humans and various animals^7–10; however, in normal humans and animals, the type of training that they undergo (e.g., endurance versus strength) influences the specific muscle mRNA levels that increase with exercise,^7–10 and skeletal muscle hypertrophy tends to occur only with resistance training.^7–10

These distinctions were not observed in our MHD patients. After exercise training, there was a significant increase in mRNA levels for one growth factor each with ET and ST and with three growth factors for EST (Table 3). The mRNA that increased with ET was for IGFBP-2, which might not be considered a growth factor. Moreover, in groups for which the mRNA levels did not change significantly, the mean values for mRNA usually moved in the same direction, as was found for the significant changes in mRNA.

There are several possible explanations for the similar responses in all three groups of exercising MHD patients. First, because of the severe degree of deconditioning and comorbidity of the MHD patients, most patients were not able to exercise vigorously; this may have obscured the ability of the various exercise groups to develop different responses to exercising. Second, the patients undergoing ST or EST might have shown more muscle hypertrophy if their training had been based on a high percentage of the maximum weight exertion performed one time (1-RM) rather than the 5-RM, which reduced their resistance load. We chose this lower resistance technique as a safety measure to reduce the risk for tendon rupture and bone fractures that have been reported in MHD patients who were subjected to high-resistance loads.^6,23,24 Also, for this latter reason and because they were deconditioned and our focus was on developing exercise capacity rather than muscle hypertrophy, the ST and EST patients generally spent their first 8 wk performing less intensive exercise training and increased the intensity of their resistance training slowly. Our objective was more focused on improving strength than muscle hypertrophy per se; therefore, we did not use such techniques as multiple sets of multiple exercises with a relatively short rest interval and eight to 12 repetitions to muscle failure, which are more effective at recruiting type IIx fibers (previously identified as type IIb or fast-twitch glycolytic fibers) in human skeletal muscle (see Sale²⁵ for motor unit recruitment patterns). Similarly, the ET patients, in general, did not exercise very vigorously (see also the Concise Methods section).

Third, many MHD patients seem to have a myopathy that might impair the response to exercise training.^2,26–28 Fourth, there may be metabolic factors in advanced renal failure that inhibit or facilitate certain mRNA responses so that the effects of different types of exercise training are obscured.²⁹ In this regard, all exercising groups manifested evidence of chronic inflammation as indicated by the significantly elevated serum TNF- and the NS trend toward raised serum CRP and IL-6.

Finally, because the sample sizes in the various exercising groups were small, there is the possibility of a type B statistical error (see the Concise Methods section). Against this possibility is the finding that the trends toward changes in mRNA levels with exercising were virtually the same in all groups. Indeed, when the responses of all three exercising groups were pooled, a significantly increased mRNA was observed for every growth factor measured, except for myostatin, a growth inhibitor that decreased significantly, and for the IGF-II receptor, which did not change.

Our results indicated a greater increase in muscle gene transcripts for IGF-IEc and IGFBP-3 in men versus women in all three exercise training groups combined. Bamman et al.³⁰ described a greater increase in strength and in myofiber hypertrophy in older men versus older women who where undergoing similar resistance (strength) training. These differences could not be attributed to different expressions of IGF-I or myogenin mRNA, to serum IGF-I or dehydroepiandrosterone sulfate concentrations, or to changes in serum testosterone levels.

A limitation of this study is the relatively small numbers of patients in each group, which prevented a rigorous comparison of possible differences in responses to exercise training in the three exercising groups. Also, the uneven number of dropouts in the various groups could reflect a treatment bias; that most of the people who dropped out did so before commencing exercise training or were in the NT group makes this a less likely possibility. Finally, all muscle mRNA and peptide measurements were from the right vastus lateralis muscle, which may not reflect the growth factor mRNA or peptide responses in other skeletal muscles.

The exercise training regimens in this study may have also reduced muscle protein degradation. Muscle mRNA increased for some growth factors that suppress protein degradation as well as stimulate protein synthesis. Such growth factors include IGF-I and IGF-II.³¹ In addition, the increased mRNA for the IGF-I receptor or IGFBP might have inhibited protein degradation. In this regard, we have observed a significant reduction in the 14-kD fragment of actomyosin in our patients undergoing ET and EST but not ST or NT.³² Du et al.³³ described this 14-kD fragment as an indicator of the degradation rate of actomyosin protein in skeletal muscle via the caspase-3 system.

It is somewhat puzzling that our body composition measurements did not show clear evidence for increased muscle mass with exercise training; however, a rise in muscle mRNA levels and growth factor proteins with exercise training may increase protein synthesis and reduce degradation of selected proteins without engendering muscle hypertrophy. In normal mammals, ET or ST increases the synthesis of many skeletal muscle proteins that are associated with enhanced physical capacity.⁹ These changes include increased mitochondria number, particularly of the subsarcolemmal type; vascular endothelial growth factor; capillary density; fibronectin; carnitine palmitoyltransferase; and GLUT4, among others.^7,8,10,34,35 There is a redistribution of myosin heavy-chain isoforms and of the activities of many other enzymes involved with energy generation.^7,8,10 Many of these changes are associated with an increase in skeletal muscle mRNA levels for the respective proteins and also for a number of growth factors, including IGF-I and IGF-II.^7,8,10,34,35 These structural and biochemical changes in skeletal muscle are thought to contribute to the increased physical exercise capacity that occurs with ET or ST. To our knowledge, this study is the first to show that similar changes in skeletal muscle mRNA in MHD patients for certain growth factors can occur in response to ET and/or ST. It is possible that these changes in skeletal muscle mRNA observed with exercise training in this study may contribute to structural and biochemical changes in muscle that might improve the exercise capacity of MHD patients.

	Concise Methods

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Patients
Inclusion criteria were clinically stable MHD patients who were aged 25 to 65 yr and had been undergoing MHD thrice weekly for at least 6 mo. Exclusion criteria were no history of hospitalization or systemic infection for at least 3 mo; active cancer other than basal cell carcinoma; severe heart, lung, or liver disease; poorly controlled hypertension; acute or chronic inflammatory disease including tuberculosis or acquired immunodeficiency disease; insulin-dependent diabetes; severe osteoporosis, neuropathy, or musculoskeletal disease; amputations involving the lower extremities; or a joint infirmity that would prevent participants from exercising. Healthy adults who were not disqualified by these exclusionary criteria and who were of similar age range, gender distribution, and racial/ethnic mix as the MHD patients served as control subjects.

Only MHD patients and normal control subjects who gave a history of a sedentary lifestyle and no recent regular manual labor, sports activity, or exercise training were studied. The dorsal pedal artery:brachial artery BP ratio was 0.90 in all MHD patients and normal subjects. This study was approved by the Human Subjects Committee at Harbor-UCLA Medical Center; informed written consent was obtained from each participant.

Exercise Training Regimens
Patients first underwent exercise testing, including an exercise electrocardiogram and a muscle biopsy. They were then randomly assigned to one of four treatment arms: ET, ST, EST, or NT. Exercise training was performed thrice weekly immediately before the onset of a hemodialysis treatment for ST and during the first 60 min of each hemodialysis session for ET. Exercise training was always performed under the direct supervision of an experienced exercise trainer, who operated under the direction of one of the authors (T.W.S.). The planned term of exercise training was 18 wk; exercise was sometimes extended as a result of scheduling problems or because occasionally patients who became ill had their exercise training period extended to allow several weeks of continuous exercise before their postexercise tests were conducted. Normal control subjects underwent baseline testing but did not undergo exercise training. All study participants were strongly encouraged not to perform sports activity or exercise training, other than what was assigned by their treatment arm, and not to increase their physical activity at work.

Exercise was preceded by 5 to 10 min of warmup and stretching. ET was performed with a SCIFIT PRO II Recumbent Cycle Ergometer (Sci-Fit Pro, Tulsa, OK), which maintains a constant work rate even when pedaling rate changes. During weeks 1 through 4, patients exercised at approximately 50% of their peak oxygen consumption. Some patients exercised only in 4:1 work/rest intervals because of debility. Initial exercise duration, including interval training, was 20 min, which was extended to 30 min as tolerated. Attempts were made to convert interval training to continuous training. During weeks 5 through 8, duration of exercise was advanced to 40 min with the same intensity. During the remainder of training, exercise duration was kept at or progressively increased to 40 min, and work intensity was increased as tolerated.

ST was performed with a Life Fitness leg extension/leg curl combination and a Life Fitness leg press/calf extension combination apparatus (Life Fitness, Schiller Park, IL). During weeks 1 through 4, patients performed one set of each exercise at 12 to 15 repetitions at 70% of the 5-RM,⁶ resting 1 min between sets. During weeks 5 through 8, patients performed two sets of this exercise, with resistance increasing as tolerated. During the remaining weeks, patients increased their exercise, as tolerated, to three sets of six to eight repetitions and resistance to 80% of a newly determined 5-RM score. With EST, ET and ST were each conducted for approximately one half of the time but with the same intensity expended by patients undergoing ET or ST and with the same progression of training (i.e., EST patients performed a combination of approximately one half of the ET work effort and also one half of the ST work effort).

Randomization Procedure
Patients who completed baseline testing were assigned in random order to one of the four exercise groups: ET, ST, EST, or NT. Randomization was performed using a computer-generated set of random numbers stratified by exercise training center and gender and blocked in sets of four.

Skeletal Muscle Biopsy
Biopsies of the right vastus lateralis muscle were performed as described previously³⁶ on MHD patients at baseline, before commencing exercise training, and at the end of the study before cessation of exercise training and on nonexercising MHD patients at the same time intervals. After the study began, the protocol was revised so that the last 29 MHD patients underwent an additional muscle biopsy 13.0 ± 1.1 wk after the first biopsy. Muscle biopsies were always performed between 9:00 a.m. and 11:00 a.m. on the day after a hemodialysis treatment in patients who were fasted from 10:00 p.m. the previous night.³⁶ For the mid- and end-exercise biopsies, patients continued exercise training up through the day before the muscle biopsy was performed. Normal control subjects underwent biopsy twice under identical conditions to the patients, except that they were not receiving hemodialysis or exercising. Individuals were fasted from 10:00 p.m. the night before the biopsy until the end of the procedure.³⁶

Real-Time PCR Amplification of mRNA for IGF-I (the Splice Variants IGF-IEa and IGF-IEc), IGF-II, IGF-IR, IGF-IIR, IGFBP-2, IGFBP-3, and Myostatin
Total RNA was extracted from skeletal muscle and quantified, and first-strand cDNA was synthesized as described previously with Random Primer p[dN]6 and AMV Reverse Transcriptase.³⁶ Real-time PCR was performed with the ABI Prism Sequence Detection System 7000, as described previously,³⁶ to facilitate SYBR-green fluorescence quantification (Applied Biosystems, Foster City, CA). Glyceraldehyde-3-phosphate dehydrogenase mRNA levels were assayed as internal controls. Primers with the following oligonucleotides were developed for IGF-I, IGF-IR, IGF-II, IGF-IIR, IGFBP-2, IGFBP-3, and myostatin as described previously³⁶ or as follows: IGFBP-2, forward 5'-AGCAGGTTGCAGACAATG-3' and reverse 5'-AGCTCCTTCATACCCGACT-3'; IGFBP-3, forward 5'-GAACTTCTCCTCCGAGTCC-3' and reverse 5'-CTTGTCACAGTTGGGAATGT-3'.

Protein Assay for Muscle IGF-I and IGF-II
Acid/ethanol extraction was used to separate IGF-I and IGF-II from muscle samples as described previously.³⁷ The DSL-5600 IRMA and DSL-2600 kits (Diagnostic System Laboratories, Webster, TX) and accompanying protocols were used, respectively, for the IGF-I and IGF-II assays.³⁶

Protein Assay for Serum IGF-I and IGF-II
Acid/ethanol solution (12.5%/87.5% vol/vol) was used to extract serum IGF-I and IGF-II.³⁷ A Nichols Institute Diagnostics IGF-I 100T kit (San Clemente, CA) was used for the serum IGF-I assay. The serum IGF-II assay followed the same procedure as specified in the DSL-2600 kit.³⁶

CRP, IL-6, and TNF- Assays
CRP, IL-6, and TNF- were measured as described previously.³⁶ The Wako Diagnostics (Richmond, VA) CRP-UL kit was used for the CRP assay. The R&D Systems (Minneapolis, MN) IL-6 and TNF- kits were used for the IL-6 and TNF- assays, respectively.

Nutrient Intake, Clinical Laboratory Measurements, and Anthropometry
Nutrient intake was determined and serum for IGF-I, IGF-II, CRP, IL-6, and TNF- measurements were obtained at the time of the first and last muscle biopsies. Blood obtained immediately before a midweek hemodialysis was measured for serum creatinine, bicarbonate, and albumin and for blood hemoglobin and hematocrit in the Davita Central Laboratory. Harbor-UCLA General Clinical Research Center research nutritionists assessed nutrient intake by 3-d dietary diaries and dietary interviews and performed anthropometry as described previously, except that mid-arm muscle circumference was calculated without adjustment for bone mass.^38–40 Proximal-thigh and mid-thigh muscle areas were measured as follows^38,39:

Mid-calf muscle areas were assessed as described previously.⁴⁰ Skinfold thicknesses were measured in the biceps, triceps, and subscapular area, and total body fat was calculated as described previously.^38–41 Lean body mass or FFM was calculated from anthropometry as the difference between body weight and total body fat. DEXA measurements for assessment of body composition were performed with Dual Energy X-Ray Absorptiometry (Model QDR; Hologic, Bedford, MA).

Bioelectrical impedance was measured with a RJL BIA Quantum instrument (model 101Q; RJL Systems, Mt. Clemens, MI), which applies an 800-µA current at a frequency of 50 KHz. Patients reclined on an examination table, with limbs not touching each other. Measurements were made on the side of the body that did not contain a hemodialysis access. The measurements were performed according to the National Institutes of Health.⁴² Electrodes were placed between the distal prominences of the radius and ulna, the distal end of the third metacarpal, between the median and lateral malleoli at the ankle, and at the distal end of the third metatarsal. FFM was calculated from the measurements of resistance made at 50 kHz by using previously validated predictive equations as supplied by the manufacturer.⁴³

Power Analysis
The a priori power analysis for this clinical trial was based on the heterogenicity of changes in muscle myostatin mRNA in our previous study in MHD patients after they had undergone a mean of 8.9 wk of ET.²⁸ On the basis of these data, we estimated that in this study there would be at least an 80% power to detect a significant decrease in myostatin mRNA between the NT group and an exercise group if there were at least 17 individuals in each group and if the NT patients have no change in myostatin mRNA and the exercise group has at least a 50% change,

Statistical Analyses
Statistical analyses to assess differences between pre- and postexercise testing values were performed with repeated measures two-way ANOVA (using groups and time) with the Sigma STAT software (version 3.0; SPSS, Chicago, IL). When statistically significant differences (P < 0.05) were detected among all four groups of patients, significant differences for pre- versus postexercise values within individual groups were tested with Tukey test. Significant differences for pre- versus postexercise values for all three groups of exercising MHD patients combined were tested using the paired t test. The pre-exercise values in all four groups of patients combined were compared with the measurements in the normal control subjects with a nonpaired t test. Data are expressed as the means ± SE or, for nonparametric sets, as the median and interquartile (25th and 75th percentiles) range of values. Statistical significance was taken as P < 0.05.

	Disclosures

Top Introduction Results Discussion Concise Methods Disclosures References

 
None.

	Acknowledgments

 
This study was supported in part by National Institutes of Health grant 5 RO1 DK054457-04 and General Clinical Research Center grant MO1-RR00425.

These data were presented, in part, at the 36th and 37th annual meetings of the American Society of Nephrology; November 12 through 17, 2003; San Diego, CA; and October 29 through November 1, 2004; St. Louis, MO.

We thank Linda Miladinovich, RN, Jooree Choi, MD, Patricia Oleskie, Jennifer Zitterkoph, RD, Merlyn Dubria, RN, Karina Romero, and Stephanie Griffiths for excellent technical support and Peter Christensen, PhD, for statistical advice. R.C. holds the Gancell/Burns Chair in the Rehabilitative Sciences.

	Footnotes

 
Published online ahead of print. Publication date available at www.jasn.org.

	References

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