Pediatr Nephrol DOI 10.1007/s00467-014-2971-8

ORIGINAL ARTICLE

Effects of acute exercise on markers of inflammation in pediatric chronic kidney disease: a pilot study Keith K. Lau & Joyce Obeid & Peter Breithaupt & Vladimir Belostotsky & Steven Arora & Thanh Nguyen & Brian W. Timmons

Received: 26 March 2014 / Revised: 16 September 2014 / Accepted: 18 September 2014 # IPNA 2014

Abstract Background Children and adolescents with chronic kidney disease (CKD) are chronically exposed to high levels of inflammation, placing them at an increased risk of secondary health complications. Regular exercise may represent an effective therapy to reduce inflammation. The aims of this pilot study were to determine the effects of acute exercise on inflammation and immune cell counts in CKD. Methods Nine children and adolescents (4 males) with CKD stages III–V performed a graded exercise test to determine peak oxygen uptake (VO2peak). Following a 10-min break, participants cycled for 20 min at 50 % of VO2peak. Blood samples were collected before and after the exercise period for the determination of complete blood counts, natural killer cells (NKbright, NKdim) and circulating progenitor cell (CPC) counts, as well as interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) concentrations. Results Complete blood counts and NKdim cell and CPC counts were unchanged with exercise. Following exercise, NKbright cell counts increased (7.4±4.3 vs. 12.2±8.3×106 cells/L; p=0.02), while trends were observed for an increase in IL-6 (2.1±2.2 vs. 2.7±2.6 pg/mL; p=0.08), decrease in TNF-α (4.5±1.2 vs. 4.2±1.0 pg/mL; p=0.08) and an increase in the IL-6:TNF-α ratio (0.6±0.7 vs. 0.8±0.8; p=0.07). Conclusions Our findings suggest that acute exercise may create an anti-inflammatory environment in children and adolescents with CKD stages III–V. K. K. Lau (*) : V. Belostotsky : S. Arora Division of Nephrology, Department of Pediatrics, McMaster University, 1280 Main Street West, HSC 3A50, Hamilton, Ontario, Canada L8S 4K1 e-mail: [email protected] J. Obeid : P. Breithaupt : T. Nguyen : B. W. Timmons Child Health & Exercise Medicine Program, McMaster University, Hamilton, Ontario, Canada

Keywords Exercise . Youth . Anti-inflammatory . Progenitor cells . Cytokines . Flow cytometry

Introduction The management of children and adolescents with chronic kidney diseases (CKD) has been hampered by the lack of effective treatment strategies other than minimizing the effects of risk factors, such as hypertension and proteinuria [1]. The current literature suggests that regular exercise may play a beneficial and potentially therapeutic role in adults with CKD, leading to improvements in health-related markers such as blood pressure control and inflammatory cytokines [2, 3]; however, the scarce attention has been paid to such benefits in children and adolescents. Moreover, despite the purported benefits of exercise, children, adolescents and adults with CKD often present with reduced levels of physical activity and aerobic fitness compared with their healthy counterparts [4–11]. Although the potential benefits of regular exercise have been described across all stages of CKD [2, 3], it would be inappropriate to assume that these lines of evidence in adults are also applicable to children and adolescents, particularly in the case of inflammation, given that their systems are still maturing [12]. However, emerging evidence suggests that regular exercise can have an anti-inflammatory effect, brought about by the cumulative effect of specific episodes of exercise [13] which alter the systemic balance between anti- and proinflammatory cytokines [3]. Indeed, it has been suggested that exercise-induced changes in the interleukin-6 (IL-6) to tumor necrosis factor-alpha (TNF-α) ratio may be an important variable to monitor in children and adolescents with a chronic inflammatory disease [14]. Moreover, exercise can also mobilize immune cells, including natural killer (NK) cells that express either high or low levels of the cell surface marker

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CD56 [15] and blood stem cells (i.e. CD34+ cells) that have been implicated in improving cardiovascular health via vascular repair and regeneration [16]. Thus, the potentially broad anti-inflammatory effects of exercise render it an appealing adjunctive therapy to individuals with chronic inflammatory conditions like CKD. However, to date, studies that have examined the effects of a specific episode of exercise on markers of inflammation and immune cell counts in children and adolescents with CKD are lacking. Therefore, the aims of this pilot study were to determine the effects of acute exercise on the systemic levels of IL-6, TNF-α, NKbright (immunoregulatory), NKdim (cytotoxic) cells and CD34+ (hematopoietic and circulating progenitor) cell populations in children and adolescents with CKD stages III–V.

Methods Participants All children and adolescents with CKD stages III–V who were being followed at the McMaster Children’s Hospital Chronic Kidney Disease and Transplant Clinic were asked to participate in this study. After exclusion of patients who were unwilling or unable to complete all study procedures, five girls and four boys were enrolled in this study, which was approved by the Hamilton Health Sciences/Faculty of Health Sciences Research Ethics Board. Each participant and their parent or guardian provided written informed assent and consent, respectively, prior to participation. Three participants were CKD stage III, five were CKD stage IV and one participant was CKD stage V, but not yet receiving dialysis. Kidney dysplasia was the primary cause of renal disease in seven patients, and two had previously suffered an acute kidney injury. Borderline anemia was seen in one patient, and another presented with mild metabolic acidosis. Parathyroid hormone level adjusted for CKD stage was elevated in three participants. Detailed participant characteristics are provided in Table 1. Study overview Participants were invited to attend one testing session that coincided with a regularly scheduled clinic visit. A resting blood sample was taken prior to exercise as part of standard clinical care. This was followed by assessments of height (Harpenden Stadiometer; C.M.S. Weighing Equipment Ltd., London, UK), body weight (platform scale; Rice Lake Weighing Systems, Rice Lake, WI), percentage body fat (InBody520; Biospace Co. Ltd., Seoul, Korea) and selfreported Tanner stage based on pubic hair development in boys and breast development in girls. A graded exercise test was completed on a cycle ergometer (Corival exercise bike; Lode, Groningen, The Netherlands) to determine peak O2

Table 1 Participant characteristics Total n=9 (4 males)

Mean ± SD

Range

Age (years) Height (cm) Body mass (kg) Body mass index (BMI) (kg·m−2) BMI percentilea % Body fat Tanner stage Hemoglobin (g/dL) eGFR (mL·min−1 per 1.73 m2)

13.6±2.6 151.0±12.7 50.8±12.8 21.3±2.8 71.3±26.6 24.6±9.3 2.7±1.0 12.7±0.9 29.5±13.1

9.4–17.6 129.8–182.0 24.7–99.5 14.7–30.0 13.8–96.7 7.2–36.5 1–5 11.5–14.0 13.0–58.1

eGFR Estimated glomerular filtration rate according to Jie et al. [48] a

BMI percentiles are based on the Centers for Disease Control growth charts, National Center for Health Statistics, http://www.cdc.gov/ growthcharts/clinical_charts.htm

uptake (VO2peak) and peak workload (Wpeak). Breath-bybreath measurements of O2 uptake and CO2 production were made using a calibrated metabolic cart (Vmax Encore; Viays Healthcare Inc., Conshohocken, Pa). VO2peak was defined as the highest 30-s oxygen uptake value, and Wpeak as the highest achieved power output, prorated to the time completed in the final stage. Because we did not test a control group, VO2peak and Wpeak values were expressed as the percentage predicted based on our laboratory’s reference data using height and sex. Heart rate (HR) was monitored continuously throughout the test using a Polar HR monitor (Polar FT1; Polar Electro Inc., Lake Success, NY). Upon completion of the test, each participant was given 10 min of rest, following which he/she performed 20 min of submaximal cycling at a workload set to 50 % of their measured VO2peak. HR was monitored continuously throughout the exercise period to ensure appropriate workload, and breath-by-breath gas was collected over the final 5 min to confirm that participants were exercising at 50 % of their measured VO2peak. A second blood sample was collected following exercise. Our aim was for this sample to be collected after 5 min of recovery, but due to difficulties in obtaining the sample from some patients the actual times ranged from 5 to 35 min. Approximately 15 mL blood was collected at rest and post-exercise for processing to determine complete blood counts (performed by the McMaster Core Facility), NK cell and CD34+ cell counts, as well as cytokine concentrations. NK and CD34+ cells Both NK cells and CD34+ cells were assessed by flow cytometry as described by Timmons et al. [15] and Duda et al. [17], respectively. Briefly, 100 μL of whole blood was incubated with CD3 (fluorescein isothiocyanate, FITC) and CD56 (phycoerythrin, PE). CD34+ cells were labeled in peripheral blood mononuclear cells (PBMCs), which were isolated from the

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remaining whole blood sample by density gradient centrifugation a ccording to the manufacturer protocols (Lymphoprep™; Axis-Shield, Oslo, Norway). Upon isolation, PBMCs were incubated with an Fc-receptor blocking reagent and then incubated with CD31 (FITC), CD34 (allophycocyanin), CD45 (PerCP) and CD133 (PE). Following incubation with their respective antibodies, both samples were lysed, fixed and stored at 2–8 °C until further analysis. NK and CD34+ cells were enumerated within 24-h of sample collection on a 15-color BD LSRII flow cytometer (McMaster Flow Cytometry Facility, McMaster University) and subsequently analyzed with FlowJo (ver. 8.7 for MacIntosh; Tree Star Inc., Ashland OR). Specific cell populations of interest were: (1) CD3 −CD56 dim (NK dim ); (2) CD3−CD56 bright (NKbright); (3) total CD34+ cells; (4) CD31+CD34brightCD45dimCD133+ cells (circulating progenitor cells, CPCs). Cell counts are expressed in both concentrations as well as percentage of lymphocytes (for NK cells) or PBMCs (for CPCs) and are corrected for changes in blood volume with exercise [18]. Cytokines The concentrations of IL-6 and TNF-α were determined as per the manufacturer’s instructions using commercially available enzyme-linked immunosorbent assays (ELISA; R&D Systems, Minneapolis, MN). All analyses were performed in duplicate. Intra-assay coefficients of variation were 5.7 % for IL-6, and 2.8 % for TNF-α. Cytokine data were adjusted for changes in plasma volume. Statistical analyses All variables were assessed for normality using the Shapiro– Wilk test. Differences in resting and post-exercise cell counts and cytokine concentrations were examined using either a dependent sample t test for normally distributed data or the Wilcoxon signed rank test for non-parametric data. Significance was set at p≤0.05. All statistical analyses were performed in SPSS (ver. 20.0; IBM, Chicago, IL), and data are reported as the mean± standard deviation (SD), with ranges where appropriate. In light of the pilot nature of this study, we also calculated effect sizes and report these for each variable as: (post-exercise value – pre-exercise value) ÷ pooled SD.

Results Exercise data Each participant completed both exercise tests with no complications or adverse events. The mean (±SD) VO2peak and

Wpeak were 33.7±11.5 ml×kg−1 ×min−1 and 2.1±0.6 Watts (W)/kg, which represent approximately 85.0±22.5 and 67.2± 18.3 %, respectively, of the predicted values. During the 20min cycling test, participants exercised at 50.2±2.3 % of their VO2peak. Data on the exercise-related variables are presented in detail in Table 2. Immune cells Total counts of leukocytes, neutrophils, lymphocytes and monocytes were unchanged with exercise (Table 3; p>0.05). Similarly, no significant differences were observed in the concentration or percentage of NKdim cells and CPCs (Table 3). NKbright cell concentration increased following exercise (7.4±4.3 vs. 12.2±8.3×106 cells/L; p=0.02), as did the percentage of NKbright cells (0.4±0.2 vs. 0.6±0.3 %; p<0.01). Cytokines Trends towards an increase in IL-6 (p=0.08) concentration, a decrease in TNF-α (p=0.08) concentration and an increase in the IL-6:TNF-α ratio from 0.58±0.67 to 0.8±0.8 (p=0.07, ES =0.25; Fig. 1) were observed.

Discussion The purpose of this pilot study was to determine the effect of a specific episode of exercise on markers of inflammation and immune cell counts in children and adolescents with CKD stages III–V. We found that NKbright cells were elevated in the Table 2 Exercise parameters Exercise parameters

Mean ± SD

Range

Maximal exercise test HRpeak (beats·min−1) VO2peak (L·min−1) VO2peak (% predicted) VO2peak/kg (mL×min−1 ×kg−1) Wpeak (Watts) Wpeak (Watts/kg) Wpeak (% predicted) Submaximal exercise test

175±23 1.6±0.4 85.0±22.5 33.7±11.5 106.2±26.3 2.1±0.6 67.2±18.3

129–192 0.7–2.6 54.4–117.7 18.1–50.2 47.5–200.0 1.2–3.2 44.3–94.6

35.4±11.0 16.8±5.8 17.0±6.0 100.4±4.5

10–100 9.1–25.1 9.2–28.0 92.2–111.6

Workload (Watts) Target VO2 (mL·min−1 ·kg−1) Measured VO2 (mL·min−1 ·kg−1) % of target VO2

SD, Standard deviation; HR, heart rate; VO2peak, peak oxygen uptake; Wpeak, peak workload

Pediatr Nephrol Table 3 Blood parameters Blood parameters

Resting

Total leukocytes (×109 cells/L) 7.5±1.5 (6.4, 8.7) Neutrophils (×109 cells/L) 4.8±1.2 (3.8, 5.7) 9 Lymphocytes (×10 cells/L) 2.0±0.6 (1.5, 2.5) Monocytes (×109 cells/L) 0.5±0.1 (0.4, 0.6) Regulatory natural killer cells (NKbright) Concentration (×106 cells/L) 7.4±4.3 (3.8, 11.0) % of lymphocytes 0.4±0.2 (0.2, 0.5) dim Cytoxic NK cells (NK ) Concentration (×106 cells/L) 227.4±207.8 (53.7, 401.2) % of lymphocytes 10.9±6.8 (5.2, 16.6) Progenitor cells (CD34+) Concentration (×106 cells/L) 1012.0±590.3 (558.2, 1465.7) % of PBMCs 41.4±21.6 (24.7, 57.9) Circulating progenitor cells (CPCs) Concentration (×106 cells/L) 0.3±0.3 (0.1, 0.5) % of PBMCs 0.01±0.01 (0.002, 0.02) Interleukin-6 (pg/mL) 2.1±2.2 (0.4, 3.8) Tumor necrosis factor-α (pg/mL)

4.5±1.2 (3.6, 5.4)

Post-exercise

Change

p value Effect sizea

7.6±1.7 (6.2, 9.0) 4.9±1.5 (3.7, 6.1) 1.9±0.5 (1.5, 2.3) 0.6±0.1 (0.5, 0.7)

0.1±0.6 (−0.4, 0.5) 0.1±0.7 (−0.4, 0.7) −0.1±0.1 (−0.3, 0.2) 0.03±0.1 (−0.02, 0.1)

0.75 0.63 0.43 0.20

0.04* 0.09 0.14 0.20

12.2±8.3 (5.2, 19.2) 0.6±0.3 (0.3, 0.9)

4.8±4.5 (1.1, 8.6) 0.3±0.2 (0.1, 0.4)

0.02 0.01

0.76 1.01

162.8±75.8 (99.5, 226.2) 9.1±4.2 (5.6, 12,6)

−64.6±160.0 (−198.3, 69.2) −1.8±1.7 (−5.9, 2.2)

0.38a 0.32

0.46 0.33

982.2±467.4 (622.9, 1341.4) −29.8±296.5 (−257.7, 198.1) 0.77 41.2±18.4 (27.0, 55.3) −0.1±12.6 (−9.8, 9.6) 0.98

0.06 0.01*

0.3±0.2 (0.2, 0.4) 0.01–0.01 (0.007, 0.02) 2.7±2.6 (0.7, 4.7)

−0.02±0.2 (−0.2, 0.1) 0.72 0.002±0.007 (−0.003, 0.007) 0.63b 0.6±1.0 (−0.2, 1.4) 0.08b

0.11 0.03* 0.27

4.2±1.0 (3.4, 5.1)

−0.3±0.4 (−0.6, 0.04)

0.26

0.08

*Significant at p≤0.05 Data are presented as the mean ± SD, with the 95 % confidence interval (95 % CI: lower bound, upper bound) given in parenthesis PBMC, Peripheral blood mononuclear cell a

Effect sizes were calculated as: (post-exercise value – pre-exercise value) ÷ pooled SD

b

Wilcoxon signed rank test for non-parametric data

early recovery period following an acute bout of exercise, with a trend towards a higher IL-6:TNF-α ratio. Notwithstanding the small sample size, these novel findings suggest that exercise may create an anti-inflammatory environment and be a potentially appropriate anti-inflammatory therapy for these patients. Studies in adults with pre-dialysis CKD have shown these patients have elevated levels of pro-inflammatory cytokines and suffer from more infectious complications secondary to disturbances in their immune systems [19, 20]. Children and adolescents with pre-dialysis CKD are also known to have

Fig. 1 The interleukin-6: tumor necrosis factor-alpha (IL-6:TNF-α) ratio at rest and following 20 min of cycling for each participant. Dashed line Mean values

similar perturbations [21, 22]. Inflammation may lead to the progression of renal disease, highlighting the need for the identification of novel strategies, such as exercise, to ameliorate inflammation in patients with CKD [23]. Despite a recent study involving adults with CKD [24], to our knowledge no studies have as yet examined the anti-inflammatory potential of exercise in the pediatric CKD population. We demonstrated a trend towards an increase in IL-6 and a decrease in TNF-α during the early recovery period following a well-tolerated bout of exercise in pediatric patients with CKD stages III–V. Although our results should be interpreted with caution in light of the small sample size and variability in responses, Viana and colleagues [24] reported that among their adult patients with CKD an acute bout of 30 min of walking also increased IL-6 levels by approximately 22 % and mobilized neutrophils, lymphocytes and monocytes into the peripheral circulation. Unfortunately, these authors did not measure TNF-α levels, although they did observed that soluble TNF receptors did not change immediately after the walking exercise. Thus, the available data suggest that specific episodes of exercise may alter inflammatory mediators among adults and children with CKD. The anti-inflammatory properties of exercise-induced IL-6 are well established [13] and likely reflect activation of the

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“classic” signaling pathway of IL-6, rather than “trans-signaling”, which is associated with the pro-inflammatory effects of IL-6 pathology [25]. IL-6 has also been found to induce the expression of vascular endothelial growth factor, a potent angiogenic factor that may be important for muscle adaptation during physical training [26]. In contrast to IL-6, TNF-α is a more prototypical proinflammatory cytokine with various catabolic effects on the human body. It promotes the initiation of inflammatory responses to infection [27] and inhibits protein synthesis in skeletal muscle [28, 29]. The balance between IL-6 and TNF-α has been recognized as an important antiinflammatory marker of specific episodes of exercise [14, 30]. The exercise-induced shift in the IL-6:TNF-α ratio observed in our patients is consistent with the results of other studies involving healthy children and adolescents [31, 32]. That this effect of exercise appears to be intact in children and adolescents with CKD suggests they may benefit from exercise as an anti-inflammatory therapy. The recent finding by Viana and colleagues [24] that IL-6 levels were lower in individuals who participated for 6 months in a walking program (150 min per week) than in the control group supports this notion. Future investigations should focus on the effects of an exercise intervention on systemic markers of inflammation in pediatric patients. We also measured immune cells known to be responsive to exercise, namely NK cells and CD34+ cells, with a focus on the early recovery period. NK cells are directly involved in defense mechanisms against various infections [33] and display cytotoxicity against foreign and tumor cells [34]. NK cells have two major subsets: CD56bright and CD56dim, which are characterized by immunoregulatory and cytotoxic activity, respectively [35]. NK cells are also differentially mobilized in response to exercise [36], with an increase in the ratio of NKbright to NKdim cells during the early recovery period following exercise. We examined the responses of NK cell subsets to exercise in children and adolescents with CKD and observed higher counts of NKbright cells during the early recovery period, without any changes in NKdim cells, thereby confirming a higher ratio, as previously reported in healthy children and adolescents [15, 37]. An exercise-induced redistribution of NK cell subsets observed following the end of exercise may reflect a process of homeostatic recovery and adaptation in response to physiological stress. NKbright cells express an abundance of angiogenic growth factors [38], possess an enhanced capacity for cytokine production and express elevated levels of adhesion molecules integral for tissue homing [39], characteristics that make them wellsuited to help facilitate the early adaptive response to exercise stress. Patients with CKD are prone to cardiovascular complications, and this increased risk is not reversed even after renal transplantation [40]. CD34 is the surface marker on

hematopoietic stem cells [41], and CD34+ stem cells have been shown to induce angiogenesis in animal models [42]. Recent studies also support the therapeutic use of CD34+ stem cells in the treatment of cardiovascular diseases [43–45]. Given the responsiveness of these cells to exercise in healthy populations [46], it is plausible that exercise mobilization of CD34+ cells represents one mechanism by which being active provides protection against cardiovascular disease. In this study, we did not observe significant changes in CD34+ cells, possibly because the timing of our post-exercise blood sample missed a transient increase in these cells. Alternatively, it is possible that the ability to mobilize CD34+ cells was blunted in the presence of renal dysfunction. There is evidence that the concentration of these cells under resting conditions is reduced in children and adolescents with CKD [47, 48], but our study is the first to examine their responsiveness to a specific episode of exercise. We have previously observed a blunted CD34+ response to a specific episode of exercise in children with juvenile idiopathic arthritis, another chronic inflammatory disease of childhood, compared to healthy controls (Obeid et al., unpublished observations 2012). Future work should determine the optimal sampling strategy and duration and intensity of exercise needed to effectively mobilize CD34+ cells in children and adolescents with CKD to confirm the extent to which exercise can meaningfully impact these cells. Notwithstanding the novel findings of our study, a number of limitations must be considered when interpreting our results. Since this was a pilot study, our sample size is small and the patients have different underlying etiologies for CKD. Using the results provided herein, a future study can be designed to better represent the different stages of CKD. While we were interested in the early recovery period following exercise, additional blood samples would have provided a clearer representation of the time-course of changes in markers of inflammation and immune cell counts. We did not assess functional activity of NK cells or CD34+ cells, so our interpretation is limited to the exercise-induced changes in cell counts.

Conclusion The results of this pilot study in a small group of children and adolescents with CKD stages III–V demonstrate preliminary evidence of an anti-inflammatory effect of exercise that needs to be confirmed in a larger study. The exercise was welltolerated, and it was feasible to assess various markers of inflammation and immune cell counts. Our results can be used to design a larger study to examine the potential benefits of exercise training in reducing inflammation in children and adolescents with CKD.

Pediatr Nephrol Acknowledgements We wish to thank the participants and their families for their time and effort, as well as Andrew Kuo for his technical assistance. BW Timmons is supported by a CIHR New Investigator Award. This research was made possible with funding from the Department of Pediatrics, McMaster University and Natural Sciences and Engineering Research Council of Canada.

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