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ARTICLE Beneficial effects of an intradialytic cycling training program in patients with end-stage kidney disease Carole Groussard, Myriam Rouchon-Isnard, Céline Coutard, Fanny Romain, Ludivine Malardé, Sophie Lemoine-Morel, Brice Martin, Bruno Pereira, and Nathalie Boisseau

Abstract: In chronic kidney disease (CKD), oxidative stress (OS) plays a central role in the development of cardiovascular diseases. This pilot program aimed to determine whether an intradialytic aerobic cycling training protocol, by increasing physical fitness, could reduce OS and improve other CKD-related disorders such as altered body composition and lipid profile. Eighteen hemodialysis patients were randomly assigned to either an intradialytic training (cycling: 30 min, 55%–60% peak power, 3 days/week) group (EX; n = 8) or a control group (CON; n = 10) for 3 months. Body composition (from dual-energy X-ray absorptiometry), physical fitness (peak oxygen uptake and the 6-minute walk test (6MWT)), lipid profile (triglycerides (TG), total cholesterol, high-density lipoprotein, and low-density lipoprotein (LDL)), and pro/antioxidant status (15-F2␣-isoprostanes (F2-IsoP) and oxidized LDL in plasma; superoxide dismutase, glutathione peroxidase, and reduced/oxidized glutathione in erythrocytes) were determined at baseline and 3 months later. The intradialytic training protocol did not modify body composition but had significant effects on physical fitness, lipid profile, and pro/antioxidant status. Indeed, at 3 months: (i) performance on the 6MWT was increased in EX (+23.4%, p < 0.001) but did not change in CON, (ii) plasma TG were reduced in EX (–23%, p < 0.03) but were not modified in CON, and (iii) plasma F2-IsoP concentrations were lower in EX than in CON (–35.7%, p = 0.02). In conclusion, our results show that 30 min of intradialytic training, 3 times per week for 3 months, are enough to exert beneficial effects on the most sensitive and reliable marker of lipid peroxidation (IsoP) while improving CKD-associated disorders (lipid profile and physical fitness). Intradialytic aerobic cycling training represents a useful and easy strategy to reduce CKD-associated disorders. These results need to be confirmed with a larger randomized study. Key words: chronic kidney disease, exercise training, oxidative stress, physical fitness, lipid profile, isoprostanes. Résumé : Chez les patients souffrant d’insuffisance rénale chronique terminale (« CKD »), le stress oxydatif (« OS ») joue un rôle central dans le développement des maladies cardiovasculaires. Cette étude pilote se propose de tester l’hypothèse selon laquelle un protocole d’entraînement aérobie intradialytique sur bicyclette ergométrique, en améliorant la condition physique permet de diminuer le OS et les désordres associés a` la pathologie (composition corporelle et profil lipidique). 18 patients hémodialysés ont été répartis aléatoirement dans deux groupes : un groupe entraîné (« EX »; n = 8) pendant la dialyse (bicyclette ergométrique : 30 min, 55–60 % de la puissance pic, 3 jours/semaine) ou dans un groupe de contrôle (« CON »; n = 10), et ce, pendant 3 mois. Nous avons mesuré au début de l’intervention et 3 mois plus tard, la composition corporelle (absorptiométrie a` rayons X en double énergie), la condition physique (consommation maximale d’oxygène et test de marche de 6 min (« 6MWT »)), le profil lipidique (triglycéride (« TG »), cholestérol total, lipoprotéine de haute densité, lipoprotéine de faible densité (« LDL »)) et le statut pro/antioxydant (dans le plasma :15-F2-isoprostanes (« F2-IsoP ») et LDL oxydées; dans les érythrocytes : l’activité de la superoxyde dismutase et de la glutathion peroxydase, le rapport glutathion réduit/glutathion oxydé). Le protocole d’entraînement intradialytique ne modifie pas la composition corporelle, mais améliore la condition physique, le profil lipidique et le statut pro/antioxydant. En effet, après 3 mois, nous observons : (i) une amélioration de la performance au 6MWT dans le groupe EX (+23,4 %, p < 0,001), sans amélioration dans le groupe CON, (ii) une diminution des TG plasmatiques dans le groupe EX (–23 %, p < 0,03), sans amélioration dans le groupe CON et (iii) une plus faible concentration des F2-IsoP plasmatiques dans le groupe EX comparé au groupe CON (–35,7 %, p = 0,02) après 3 mois. Pour conclure, nos résultats démontrent que 30 min d’entraînement intradialytique, 3 fois par semaine pendant 3 mois suffisent a` exercer des effets bénéfiques notamment sur le marqueur le plus sensible et fiable de la peroxydation lipidique (IsoP) tout en luttant contre certains désordres associés a` la pathologie (amélioration du profil lipidique et de la condition physique). De plus, notre étude illustre la faisabilité d’un programme de pédalage pendant les séances de dialyse et souligne que l’activité physique devrait faire partie intégrante de la prise en charge du patient hémodialysé. Ces résultats doivent être confirmés par d’autres études randomisées a` grande échelle. [Traduit par la Rédaction] Mots-clés : néphropathie chronique, entraînement physique, stress oxydatif, condition physique, profil lipidique, isoprostanes.

Received 4 November 2014. Accepted 17 December 2014. C. Groussard, L. Malardé, S. Lemoine-Morel, and B. Martin. Laboratory “Movement, Sport and Health Sciences” (M2S), Rennes 2 University–ENS Cachan, Avenue Robert Schuman, Campus de Ker Lann, F-35170 Bruz, France. M. Rouchon-Isnard, C. Coutard, and F. Romain. AURA Auvergne, 8 rue du Colombier, F-63400 Chamalières, France. B. Pereira. University Hospital Center (CHU) of Clermont-Ferrand, Biostatistics Unit, DRCI, F-63000 Clermont-Ferrand, France. N. Boisseau. Laboratoire des Adaptations Métaboliques a` l'Exercice en conditions Physiologiques et Pathologiques (AME2P), Université Blaise Pascal, Campus Universitaire des Cézeaux, Bât. Biologie B, 5 impasse Amélie Murat, TSA 60026, CS 60026, 63178 Aubière Cedex, France. Corresponding author: Nathalie Boisseau (e-mail: [email protected]). Appl. Physiol. Nutr. Metab. 40: 550–556 (2015) dx.doi.org/10.1139/apnm-2014-0357

Published at www.nrcresearchpress.com/apnm on 8 May 2015.

Groussard et al.

Introduction Chronic kidney disease (CKD) is a serious public health problem worldwide that substantially reduces quality of life and significantly affects patients’ short-term and long-term survival (Tonelli et al. 2006). This pathology is characterized by a progressive loss of renal function; in end-stage kidney disease (ESKD), the patient requires dialysis or kidney transplantation to survive. Patients with CKD (especially those with ESKD) are at high risk of cardiovascular diseases (CVD), which are the leading cause of mortality (Baigent et al. 2000). Although the prevalence of traditional cardiovascular risk factors (such as hypertension, diabetes, and low high-density lipoproteins (HDL)) is elevated in these patients, the extent and severity of associated cardiovascular morbidity and mortality remain disproportionate to traditional risk factor profiles. Consequently, nontraditional risk factors such as increased oxidative stress (OS) and increased inflammation are postulated to be important contributors to cardiovascular complications (Himmelfarb 2004). Patients with CKD suffer from a variety of comorbid diseases in addition to cardiovascular disorders; this creates a vicious cycle that gradually leads to inactivity (Painter 2005), which in turn reduces physical function and increases mortality (O’Hare et al. 2003). Few strategies are available to reduce the progression of CKD and its associated disorders. In a range of disease conditions (obesity, diabetes, etc.), exercise training is well known to have beneficial effects on health (Hawley and Holloszy 2009; Bird and Hawley 2012). Moreover, physical training may also prevent physical deconditioning by increasing aerobic fitness (Storer et al. 2005) and may reduce OS (Gomez-Cabrera et al. 2008). While the benefits of physical training in patients with CKD have been extensively studied by evaluating physical fitness using objective laboratory tests (i.e., cardiorespiratory fitness through peak oxygen uptake (V˙O2peak)) and (or) physical performance tests (i.e., the 6-minute walk test (6MWT)) (Painter 2005; Segura-Orti and Johansen 2010; Heiwe and Jacobson 2014), less is known about the effects of aerobic exercise training on OS in this population. To our knowledge, only 2 studies have evaluated the potential benefits of aerobic physical training on OS in patients with CKD (Pechter et al. 2003; Wilund et al. 2010), and both assessed plasma lipid peroxidation by the most widely used method, thiobarbituric acid reactive substances, which is known to lack sensitivity and specificity (Janero 1990). Therefore, the aim of the present study was to evaluate the impact of an intradialytic aerobic exercise training program (cycling) on OS markers, including the most reliable lipid peroxidation marker, 15-F2␣-isoprostanes (F2-IsoP) (Roberts and Morrow 2000). Physical fitness and other variables that are commonly altered in patients with CKD (lipid profile and body composition) were also evaluated. We hypothesized that intradialytic aerobic exercise training would improve pro/antioxidant status and lipid profile.

Materials and methods Subjects Twenty patients with ESKD (stage 5; 5 females, 15 males) in maintenance hemodialysis were recruited from 2 autodialysis centers through the dialysis association AURA Auvergne. All patients were treated by conventional hemodialysis 3 times a week. ‘Health and medical history questionnaires were used to determine patients’ eligibility for the study. The inclusion criteria were as follows: (i) age = 20–85 years, (ii) dialysis for at least 2 years, (iii) consent of the patient’s cardiologist, (iv) no orthopedic problems that prevented cycling during dialysis, and (v) no participation in another study. None of the patients had been exercising regularly before starting the study. Eligible subjects were randomly divided into 2 groups. Ten patients participated in the intradialytic exercise training study (EX group). A comparison group, designated as non-exercising patients (CON group), consisted of 10 individuals

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who received no intradialytic exercise training but participated in assessments for all measures before and after the 3-month study period. It was not possible to blind participants or researchers to group assignment. Written informed consent was obtained from all participants prior to the beginning of the experiment. This pilot program conformed to the principles of the Declaration of Helsinki and was approved by the local Ethics Committee: CPP Sud est VI, VI-AU818, Clermont-Ferrand, France. Experimental protocol Clinical testing and measurements At baseline (T0) and 3 months later (T3, at the end of the exercise training program), all patients underwent clinical examination (anthropometric and body composition measurements), a physical fitness evaluation (V˙O2peak, 6MWT), and laboratory data collection as described below. During the clinical examination, height, weight, and body mass index (BMI = weight (kg)/[height (m)]2) were determined for each subject. Dual-energy X-ray absorptiometry was used to assess body composition, including total and lower-limb fat-free mass (FFM) and fat mass (FM) (QDR-4500A, Hologic Inc., Waltham, Mass., USA). Physical fitness was assessed using cardiopulmonary exercise testing (V˙O2peak and the 6MWT). For measurements of V˙O2peak, each participant completed a maximal incremental cardiopulmonary exercise test on an electrically braked cycle ergometer (Cardiosoft, Marquette Hellige, Munzinger, Germany) with 12-lead electrocardiography (Asept Inmed, Quint Fonsegrives, France). Oxygen consumption was measured by a breath-by-breath gas analyzer (CareFusion, Masterscreen CPX, Houten, The Netherlands), and the power output (watts) was also recorded. Subjects began with a warm-up at 10 to 50 W, depending on age, sex, and physical capacity. The power output was then increased at a rate of 10 W/min until exhaustion. The test was performed until volitional exhaustion or until the subject exhibited any of the criteria for the termination of an exercise test as recommended by the American College of Sports Medicine. The duration of the test was between 12 and 15 min. The 6MWT was used as an index of aerobic capacity and was performed without any assistance in a quiet hospital corridor (25 m long) as described in detail by Fitts et al. (Fitts and Guthrie 1995). Exercise training intervention After group assignment and baseline testing, subjects in the EX group underwent a 3-month intradialytic aerobic exercise training program consisting of cycling 3 days/week on specialized cycle ergometers (OxyCycle II, Kinou Medical) adapted on the subject’s dialysis chair. Cycling was performed in a seated position during the first 2 h of dialysis. During the first exercise session, subjects cycled for 15 min at a tolerable pace. The duration was then gradually increased by 5 min during the first week and then by 10 min during the second week to reach 30 min after 2 weeks of training. Before and after each session, respectively, subjects performed a 5-min warm-up on the specialized cycle ergometer and a 5-min cool-down. The workload was set at 55%–60% of the peak power ouput achieved during the pretraining incremental exercise test. Heart rate was monitored during each exercise training intervention to control the intensity over time. All sessions were supervised by a professional team with expertise in physical activity. Subjects were instructed to continue cycling for 30 min at a constant pedal frequency of 50 r/min. Nutritional assessment Participants were asked to eat and drink normally and not to consume any antioxidant supplements. They completed a 7-day diet record 1 week before the beginning of the protocol (T0) and 1 week after the end of the 3-month exercise training period (T3). Published by NRC Research Press

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Table 1. Patient characteristics at baseline (T0) and at 3 months (T3) in control (CON) and intradialytic training (EX) groups. CON (n = 10) T0 Age (y) Sex Anthropometric measurements Weight (kg) Height (cm) BMI (kg/m2) % Fat mass Fat-free mass (kg) Fat-free mass, lower limbs (kg) Hemodialysis variables S-creatinine (␮mol/L) Albumin (g/L) Transthyretin (g/L) Hemoglobin (g/dL) eKt/V Time on dialysis (mo) Dialysis prescription (h/wk) Comorbidity (Charlson index)

EX (n = 8) T3

T0

T3

68.4±3.7 7 m, 2 f 70.4±6.8 161.8±3.0 26.5±1.8 27.2±2.7 49.4±3.5 14.9±1.3

69.5±6.6 161.8±3.0 26.2±1.9 27.3±2.8 48.7±3.4 14.8±1.4

633.2±53.7 623.7±48.4 31.50±0.79 31.91±0.74 0.31±0.02 0.31±0.02 12.6±0.6 12.5±0.4 1.47±0.07 1.32±0.11 41.2±8.1 11.9±0.1 5.7±1.5

66.5±4.6 5 m, 3 f 78.1±6.4 162.9±4.3 29.4±2.1 32.2±3.1 51.8±4.7 16.3±1.8

78.3±6.0 162.9±4.4 29.5±1.9 32.4±3.2 52.2±4.9 16.3±1.9

713.4±99.0 712.2±99.8 33.50±0.66 31.81±0.60 0.29±0.02 0.28±0.02 12.2±1.7 11.3±1.7 1.31±0.10 1.42±0.10 36.6±8.2 12.2±0.7 6±1.7

Note: Values are means ± SE. BMI, body mass index; f, female; eKt/V, equilibrated Kt/V; h/wk, hours of dialysis per week; m, male.

Kilocalorie, macronutrient, and antioxidant intakes were analyzed by the same technician using Nutrilog 2.5 software. Blood sample preparation and analysis For each participant, blood samples (15 mL) were collected before a dialysis session both at the beginning of the experiment (before training) to establish the baseline (T0) and 3 months later to determine the effect of the exercise training protocol (T3). Biochemical parameters Blood samples were collected in dry vacutainer tubes the week before and 1 week after the end of the training program. The following biochemical parameters were measured in the fasting condition before dialysis: hemoglobin, albumin, transthyretin, serum potassium, phosphate, calcium, alkaline phosphatase, calcium–phosphorus product, and blood urea nitrogen. The following parameters were measured at the end of the dialysis session: urea to calculate the eKt/V, creatinine, calcium, phosphate, sodium, and potassium. All variables were measured using an autoanalyzer (Olympus Inc.) at the Gen-Bio laboratory (ClermontFerrand, France). Lipid profile Blood samples were collected in lithium heparin vacutainer tubes from subjects in a fasted state. Plasma total cholesterol, HDL-cholesterol, and triglyceride (TG) concentrations were measured using a UniCel DxC system (Beckman Coulter) by a cholesterol oxidase method (synchron CHOL) for total cholesterol, a direct homogeneous method (synchron HDLd) for HDL-cholesterol, and a lipase/glycerol kinase method (synchron TG GPO) for TG. The lowdensity lipoprotein (LDL) subfraction was indirectly quantified using the Friedewald equation (Friedewald et al. 1972). Pro/antioxidant status Blood samples were collected from non-fasted subjects. Blood was drawn into EDTA vacutainer tubes and prepared for analysis immediately after collection. For oxidized glutathione (GSSG) measurement, 100 ␮L of whole blood was added to 10 ␮L of scavenger, provided in the GSH/GSSG’ kit, and immediately stored at –80 °C. For reduced glutathione (GSH) and glutathione peroxidase (GPx) measurements, 50 ␮L and 150 ␮L of whole blood, respectively, were collected and immediately stored at –80 °C. For measurement of superoxide dismutase (SOD) activity, 500 ␮L of whole

Table 2. Physical fitness and lipid profile at baseline (T0) and at 3 months (T3) in control (CON) and intradialytic training (EX) groups.

Physical fitness V˙O2peak (L/min) V˙O2peak (mL/(min·kg)) Peak power (W) Peak power (W/kg) Lipid profile Total cholesterol (g/L) HDL (g/L) LDL (g/L) TG (g/L)

CON (n = 10)

EX (n = 8)

T0

T0

T3

T3

0.99±0.14 1.01±0.11 1.12±0.16 1.15±0.20 13.4±1.1 15.3±0.6 14.7±2.1 14.3±2.3 66.0±6.0 72.0±4.9 80.5±12.9 88.6±14.0 0.97±0.05 1.11±0.06 1.06±0.17 0.99±0.19 1.53±0.09 0.47±0.03 0.83±0.08 1.20±0.13

1.63±0.09 1.71±0.15 1.61±0.13 0.47±0.04 0.39±0.04 0.42±0.05 0.91±0.09 0.94±0.16 0.89±0.12 1.09±0.13 1.90±0.43 1.46±0.33*

Note: Values are means ± SE. HDL, high-density lipoprotein-cholesterol; LDL, low-density lipoprotein-cholesterol; TG, triglycerides; V˙O2peak, oxygen uptake at maximal load. *Significant difference between T0 and T3, p < 0.05.

blood was centrifuged (1500g, 10 min, 4 °C) and plasma was removed. Then, the erythrocytes were washed according to the assay manufacturer’s recommendations. For the rest of the assays, blood was immediately centrifuged at 1500g for 10 min at 4 °C to separate the plasma (Universal 320R, Hettich Zentrifugen, Germany). For F2-IsoP, butylated hydroxytoluene (0.05%) was added to the plasma to prevent oxidation. The rest of the plasma was aliquoted (including 300 ␮L for oxidized LDL (oxLDL)) and stored at –80 °C. SOD and GPx activities were determined using the Ransod and Ransel kits, respectively (Randox, Montpellier, France). The GSH/ GSSG ratio was determined using the GSH/GSSG-412 kit (Bioxytech, Oxis International Inc., Portland, Ore., USA). oxLDL was determined spectrophotometrically with a competitive enzyme-linked immunosorbent assay (Immunodiagnostik). F2-IsoP was measured by liquid chromatography mass spectrometry as described previously (Youssef et al. 2009). Statistical analysis Data are presented as the mean ± standard error of the mean (SE). All statistical analyses were performed using Statistica 7.1 software. The nonparametric Mann–Whitney test was used to compare anthropometric data between the 2 groups. To study the influence of the intradialytic exercise program on the measured Published by NRC Research Press

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Fig. 1. The 6-minute walk test (6MWT) responses at T0 and T3 in control (CON) and intradialytic training (EX) groups.

parameters, 2-way analysis of variance (group, time, and group × time interaction) was conducted after checking the binomial probability distribution and variance. Tukey’s post hoc test was used when the interaction was significant. Differences were considered statistically significant at p < 0.05.

Results Patient characteristics Two patients of the EX group withdrew. The first one moved out of the area and the second one underwent kidney transplantation. Thus, 8 subjects were included in the EX group and 10 in the CON group. Time on dialysis (months), dialysis prescription (hours per week), and comorbidity (Charlson index) were not different between the 2 groups (Table 1). Similar eKt/V values were observed in the CON and EX groups, indicating similar dialysis adequacy. The nutritional status of the patients was not different between the 2 groups (creatinine, albumin, and transthyretin levels) (Table 1). No significant differences were reported between the 2 groups for age, body weight, height, BMI, % FM, total FFM, and FFM of the lower limbs. The 3-month period did not affect any anthropometric parameters in the CON or EX group (Table 1). Nutritional assessment Analysis of food records showed no significant difference in nutritional intake between the 2 groups at T0. Moreover, statistical analysis of the food surveys showed no difference in food habits or antioxidant consumption in either group between the beginning and the end of the study (data not shown). Thus, inspection of the subjects’ diaries did not indicate any deviations from the protocol that may have affected the results. Physical fitness assessment V˙O2peak and the 6MWT were not different at baseline between the 2 groups (Table 2). Whereas no changes in relative or absolute V˙O2peak or peak power were observed between T0 and T3 in either group, the distance walked during the 6MWT test increased by 23.4% in the EX group (p < 0.001) but did not change in the CON group (376 ± 20 and 406 ± 18 at T0 vs. 406 ± 29 and 500 ± 30 at T3 for CON and EX, respectively) (Fig. 1).

Lipid profile The lipid profiles were not different between the CON and EX groups at T0 (Table 2). The 3-month training program showed beneficial effects on plasma TG levels in the EX group (–23% at T3 compared with T0; 1.90 ± 0.43 vs. 1.46 ± 0.33 g/L; p < 0.03). No change in plasma TG concentrations was observed in the CON group. Other lipid profile markers (HDL, LDL, total cholesterol) were unchanged throughout the intervention period. Pro/antioxidant status Markers of pro/antioxidant status are presented in Table 3 and Fig. 2. No difference appeared at T0 between the 2 groups. After the 3-month period, no change was observed in these markers except for F2-IsoP (Fig. 2). Indeed, the EX group exhibited a significantly lower F2-IsoP value at T3 compared with the CON group (p = 0.02).

Discussion Our pilot program showed that an intradialytic aerobic cycling program had beneficial effects on physical fitness and lipid profile and prevented aggravation of OS in patients with ESKD. Furthermore, this study is the first to report a significant difference in plasma F2-IsoP (the most sensitive and reliable marker of lipid peroxidation) between trained and untrained patients at the end of an intervention protocol. Effects of exercise training on body composition and lipid profile The 3-month intradialytic aerobic training program did not induce significant changes in the body composition of the ESKD patients. Thus, the exercise training protocol failed to modify FM in the EX group. The frequency (3 times/week) and duration of the sessions (30 min) and the length of the protocol (3 months) were probably insufficient to achieve the energy expenditure needed to alter total fat mass. In parallel, food intakes did not change between T0 and T3 in either group (from a qualitative point of view and in terms of total kilojoules consumed). Muscle atrophy is regularly observed in patients with ESKD. This loss of FFM contributes to the vicious cycle that induces Published by NRC Research Press

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Table 3. Pro/antioxidant status at baseline (T0) and at 3 months (T3) in control (CON) and intradialytic training (EX) groups. CON (n = 10) T0 Oxidative stress markers Ox-LDL (U/L) GSH/GSSG Antioxidant enzyme activity GPx/g Hb SOD/g Hb

EX (n = 8) T3

T0

T3

38.6±4.6 249±93

39.7±4.6 335±120

41.5±3.2 436±165

40.6±3.4 367±101

81.2±0.05 1361.2±160.3

74.9±9.9 1490.31±184.4

99.6±11.3 1372.1±182.6

84.9±8.9 1425.6±115.8

Note: Values are means ± SE. GPx, glutathione peroxidase activity; GSH/GSSG, ratio between reduced and oxidized glutathione; Hb, hemoglobin; Ox-LDL, oxidized low-density lipoprotein; SOD, superoxide dismutase activity.

Fig. 2. Plasma F2-IsoP at T0 and T3 in control (CON) and intradialytic training (EX) groups.

physical inactivity (Painter 2005) and favors morbidity and mortality (O’Hare et al. 2003). In our study, the exercise training protocol did not modify FFM in the EX group. This result confirms previous findings that resistance training (muscular strengthening) must be combined with aerobic training to induce muscle hypertrophy (Kouidi et al. 1998). Another ESKD-associated disorder is dyslipidemia. Hypertriglyceridemia is the most common blood lipid abnormality in patients with CKD and is considered a risk factor for CVD (Green et al. 1983; Jeppesen et al. 1998). Fortunately, the training program significantly reduced plasma TG (–23%), indicating an improvement of the lipid profile. This result indicates that our training program was a sufficient and successful lipid-lowering therapy. Our results are consistent with the study of Goldberg et al. (1983), in which a similar training protocol decreased plasma TG by 33% and increased HDL by 16%. In our study, HDL increased by only 9%. The relatively high HDL level in the EX group before the beginning of the study may explain this result. Changes in dietary intake cannot explain this result, since no modifications of kilocalorie, macronutrient, and antioxidant intakes were observed during the intervention (data not shown). Effects of training on physical fitness Physical fitness was evaluated by an objective laboratory test, i.e., V˙O2peak, and by a field test that measures physical performance of a standardized task, i.e., 6MWT. Although aerobic exercise training had no effect on V˙O2peak, it improved the distance

walked (+23.4%). We can explain this apparent discrepancy as follows. As evidenced in a recent meta-analysis, training-induced changes in V˙O2peak are positively correlated with exercise training duration. The most important changes have been observed in patients who perform combined aerobic and strength training for 6 months or more on non-dialysis days (Smart and Steele 2011). Moreover, even if the gold standard test used to evaluate functional capacity is the determination of V˙O2max (Painter 2005), it is unlikely that patients with ESKD will reach V˙O2max, because of functional limitations including bone, joint, and/or muscle pain and muscle fatigue. Consequently, the V˙O2peak test (which measures the highest oxygen uptake in a symptom-limited test) or other indirect tests such as walk tests are preferred. Furthermore, walk tests are more appropriate in the clinical setting to assess physical fitness in subjects with low functional capacity, such as patients with ESKD (American Thoracic Society 2002). The significant increase in distance walked is of fundamental interest because walking capacity is considered a better indicator than physiological exercise capacity, as it assesses capability as well as fitness and reflects the ability to perform activities that are similar to those of everyday life, i.e., walking (Koufaki and Kouidi 2010). The magnitude of improvement in the distance walked after the training program is in the upper range of previously reported increases of between 5% and 18% after aerobic training programs (Parsons et al. 2006) and is considered to be clinically relevant (Wise and Brown 2005). This result indicates that a 3-month intPublished by NRC Research Press

Groussard et al.

radialytic aerobic cycling program improves physical fitness and walking ability, leading to a better quality of life (Painter et al. 2000) and increased survival in these patients (Sietsema et al. 2004). Effects of training on pro/antioxidant status OS appears to play a central role in the development and progression of CVD and its complications. Many clinical studies have demonstrated that patients with CKD are prone to chronic OS (Martin and Goeddeke-Merickel 2005), owing to impaired pro/ antioxidant balance, but the exact causes are still debated. In healthy or pathological subjects, training was demonstrated to be a useful approach to decrease OS (Miyazaki et al. 2001; Rodriguez et al. 2012). Indeed, the transient and low level of reactive oxygen species (ROS) induced by acute exercise stimulates adaptive mechanisms such as increased antioxidant defense (Gomez-Cabrera et al. 2008) and reduced ROS production (Venditti et al. 1999; Coelho et al. 2010), which leads to decreased resting OS (Miyazaki et al. 2001; Rodriguez et al. 2012). We found no significant differences in oxLDL, which is believed to be one of the major factors involved in the pathogenesis of atherosclerosis. Moreover, even though we failed to detect a significant decrease in F2-IsoP in the EX group after the 3-month training program, there was a significant difference in plasma F2-IsoP concentration at T3 between EX and CON subjects, indicating lower OS in the trained group. Because F2-IsoP is considered the most reliable biomarker of OS, and since OS is thought to elevate CVD risk by increasing endothelial dysfunction (Vogiatzi et al. 2009), we propose that intradialytic training could reduce CVD risk. In humans, very few studies have investigated the effects of exercise training on OS in patients with CKD (Pechter et al. 2003; Wilund et al. 2010). Our study is the first to measure plasma F2-IsoP in response to aerobic training. Since training did not increase antioxidant enzyme activities, the significant difference in F2-IsoP at the end of the training period between the EX and CON groups may be due to decreased ROS production. Indeed, Coelho et al. (2010) observed a training-induced decrease in OS markers that was explained not by upregulation of antioxidant enzyme activity but rather by decreased superoxide anion production. This decrease could be due to upregulation of uncoupling mitochondrial proteins and/or to acceleration of electron transport by an increase in the adenosine diphosphate supply (Coelho et al. 2010). In our study, antioxidant enzyme activities were not upregulated by training. In healthy and pathological patients, upregulation of antioxidant enzyme activity and (or) content is observed when training improves V˙O2max (Ohno et al. 1988; Ennezat et al. 2001; Miyazaki et al. 2001), which was not the case in our study, probably because of the short duration of the protocol (3 months) and of each session (30 min). The difference between the results we obtained for SOD/GPx activities (no change with training) and F2-IsoP (lower value at T3 for EX vs. CON) could be explained by the tissue location (red blood cells for SOD/GPx activities and plasma for F2-IsoP). Indeed, plasma is a crossroad for F2-isoPs, which are produced in situ and subsequently removed from the cell membrane (by a phospholipase A2) before ending up in the plasma and the urine (Morrow et al. 1992). Choice of training program While the majority of studies have focused on interdialytic prescription, the training program proposed in our study of ESKD patients was an intradialytic one. We preferred an intradialytic training program for several reasons. First, we expected better compliance with an intradialytic protocol, which does not require extra visits. Indeed, previous work has shown very low adherence when exercise sessions are performed on non-dialysis days (Konstantinidou et al. 2002). In our study, we lost only 2 trained subjects: one moved out of the area and the second underwent kidney transplantation. Moreover, intradialytic training allows better patient’ monitoring and provides motivation in a structured environment. Second, the time spent at a hemodialysis ses-

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sion is a period of forced inactivity, which contributes to further degradation of physical function. An intradialytic training program during this period reduces the side effects of inactivity and provides the benefits of exercise. Third, increased blood flow induced by exercise would increase the removal of solutes such as toxins or urea (Parsons et al. 2006). The advantages of intradialytic training have been discussed by Cheema et al. (2005), who recommended its incorporation into routine dialysis care. In our study, only one subject dropped out of the protocol for nonmedical reasons. This high degree of participant approval was probably due to the stimulation provided by aerobic exercise during dialysis, interest in the activity, the social aspect (2 patients shared the same exercise session), and the positive effects on health.

Conclusion The main objectives of the therapeutic management of CKD (including ESKD) are to slow disease progression (Ruggenenti et al. 2001) and prevent cardiovascular complications (Oberley et al. 2000; Zoccali et al. 2002). Our pilot program demonstrates that an intradialytic aerobic cycling training program has beneficial effects on physical fitness (by increasing the distance walked during the 6MWT) and lipid profile (by lowering plasma TG) and prevents increased basal OS (without aggravating F2-IsoP, which is the most reliable and specific marker of lipid peroxidation). Intradialytic aerobic cycling training represents a useful and easy strategy against hypertriglyceridemia, loss of physical function, and increased OS. These results are very encouraging, considering the relatively low duration of our protocol (only 3 months). Thus, a regular intradialytic aerobic cycling program that can be easily transferred to any dialysis center needs to be developed. Of course, these results must be confirmed in a larger randomized study. Conflict of interest statement The authors declare no conflict of interest.

Acknowledgements This study was supported by the following partners: Amgen, Baxter, Hemotech, Meditor, Roche, and ANCA (Association des Néphrologues Centre Auvergne). We would like to thank all the participants, the technicians and engineers (especially Luz Lefeuvre of the M2S laboratory and Marine Perrot and Franck Enjolras from the AME2P laboratory), and the medical staff of Durtol, especially the cardiologist team of the cardiothoracic clinic, who contributed to the study (Dr Cuenin, Dr Moisa, and Dr D’Agrosa-Boiteux). This study also received technical assistance from Dr Pincemail of the Laboratory of the University Hospital of Liège (Belgium) for oxLDL measurement and from Dr Forte of the Gen-Bio laboratory (Clermont-Ferrand, France) for biochemical analysis of renal function. We also thank Professor Denis Fouque for his advice during the publication period.

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