Study setting
The study is being conducted at an academic institution in association with a local nephrology practice. The study has been reviewed and approved by the Institutional Review Board of the academic institution. This Board assumes the responsibility for monitoring the study. Any adverse events are required to be reported to the IRB in compliance with policies of this Board. Any changes to the original protocol are reviewed and approved by the IRB before implementation in this study. If additional analyses are to be performed that are not listed in the original consent form, consent will be required prior to performing any new analyses.
Eligibility criteria
Inclusion criteria
Sixty stage 3–4 CKD (GFR 15–59 ml/min/m2) patients between the ages 30–75 years are being recruited for this study, which is being completed over a 3-year period. Other inclusion criteria include the ability of the individual to comply with all aspects of the study protocol and to be able to give consent independently.
Exclusionary criteria
Patients are excluded if they have any of the absolute contraindications to exercise as defined by the American College of Sports Medicine (ACSM) [18]; if they have received a kidney transplant; if they have received antibiotic therapy within the previous month, or if they have taken prebiotic or probiotic supplements within the month prior to the start of the study [19]; if they have any gastrointestinal condition that prohibits their use of resistant starch [19], or if they have been involved in a structured exercise program (i.e., exercising 3 days per week at a moderate intensity for at least the previous 3 months). Individuals who are pregnant are also excluded from this study.
Recruitment
A member of the research team (EE), has been given access to the patient database at the local nephrology practice participating in this study. She screens the list of patients and approaches those who meet the eligibility criteria for the study at their next scheduled appointment. While patients are waiting to see their nephrologist, she explains the details of the study to them. If they indicate a willingness to participate, she gives them the medical history form to take into the appointment with them. If the nephrologist approves of the patient’s participation in the study, EE obtains informed consent from the patient and enrolls them into the study at that time. Individuals are free to withdraw from the study at any time as stated in the IRB approved consent form. Individuals who experience adverse reactions from taking the supplement will be removed from the study without penalty.
Confidentiality
All forms with participant information for this study are stored in a locked filing cabinet in the PI’s office with access limited to specified members of the research team. Upon entry to the study, participants are given a number and this number is then used on all documents, apart from the consent form and within the database. The study data are stored in a password protected database with access limited to the PI, the statistician and members of the research team tasked with data entry and verification.
Testing sessions and timeline
Participants are asked to attend a total of four testing sessions (see Fig. 1), all conducted at the academic institution’s Human Performance Laboratory. During the first visit, anthropometric variables of, height (Detecto, Webb City, Missouri), weight and body composition via bioelectrical impedance (BC-418,Tanita,Tokyo, Japan) are assessed along with the determination of peak oxygen uptake (VO2peak) using a computerized metabolic cart (MAX-II, AEI Technologies, Pittsburg, PA), that is calibrated according to manufacturer’s specifications. The cardiopulmonary exercise test is performed on a motor driven treadmill with a Q-stress system (Welch Allyn, Skaneateles Falls, NY) using the Modified Bruce protocol. Resting cardiovascular (CV) variables (i.e., central and peripheral blood pressure, pulse wave velocity and pulse wave analysis) are assessed using applanation tonometry (SphygmoCor Excel, AtCor Medical Inc., Itasca, IL) after a 10-min rest period in the supine posture. One week later, patients return to the Human Performance Laboratory for their second baseline visit following at least an 8-h fast. On arrival, a blood sample is taken, and processed by spinning in a refrigerated centrifuge (4 °C, and spun at 3000 rpms for 15 min). The plasma is then removed and placed in 10 storage tubes and stored in a − 80° freezer for later analysis. Prior to visit 2, participants collect a stool sample at home and complete a 3-day food record documenting the brand, portion size, and preparation methods of all foods and beverages consumed. Both the stool sample and the food records are brought to the lab for visit 2. Following baseline testing, subjects are assigned randomly to one of 4 groups ensuring equal number of subjects in each group: Group A receives the prebiotic supplement with usual care but no prescribed exercise, Group B receives the prebiotic supplement and performs supervised aerobic exercise training at a moderate intensity for 16 weeks, Group C receives the placebo (cornstarch) supplement along with 16 weeks of moderate intensity aerobic exercise training, while group D receives the usual care and the placebo but no supervised exercise training. The randomization was performed by the study statistician prior to the start of the study.
Follow-up testing
All of the aforementioned variables (except the stool sample, and the VO2peak test) are reassessed after 8 and 16 weeks of participation in the study. The final stool sample and the post-training VO2peak test are done after week 16. All post-tests are performed at least 48- h following the completion of the final training session to avoid the residual effect of the final training session.
Analysis of biomarkers
Analysis of inflammatory and oxidative stress markers
Venous blood samples are being collected at BL, Wk8 and Wk16. A 21- or 23-gauge needle (BD Vacutainer® Safety Lok and Vacutainer® one-use holder, Becton, Dickinson and Company, Franklin Lakes, NJ) is used to collect two, 10 mL of blood at each of the time point and placed into an appropriate collection tubes (EDTA Monoject Vacutainer, Coviden, Mansfield, MA) by trained personnel. Plasma is obtained from the EDTA tubes through centrifugation immediately after collection at 1500 g for 15 min at 4 °C. Plasma samples are being stored at − 80 °C until ELISA assays are conducted for the analyte panel, including: CRP, malondialdyhide (MDA), and nitrate/nitrite (Cayman Chemical, Ann Arbor, MI), Cytokine panel (TNFα, IL-6, IL-10), Enothelin-1, (Millipore, Burlington, MA), MCP-1 (Eagle Biosciences, Amherst, NH), and 8-isoprostane (Cell Biolabs, San Diego, CA). All samples will be measured in duplicate, in accordance with manufacturers’ recommended protocol and be within acceptable variance and above sensitivity provided from the manufacturer.
Analysis of uremic toxins
The concentration of IS and PCS in the plasma is measured by reverse-phase HPLC. In brief, 0.24 M sodium octanoate is added in plasma as a competitor to replace non-covalent binding of p-cresyl sulfate and indoxyl sulfate to albumin. After 5 min incubating at room temperature, the plasma is deproteinized by adding 4 parts of cold acetone to 1 part of plasma. The mixture is then centrifuged at 3000×g for 20 min at 4 °C and the supernatant is measured by HPLC (Waters 1525 binary HPLC system coupled with 2475 fluorescence detector) [20].
Analysis of stool samples
Stool will be used for microbial assessment by 16 s RNA sequencing (MiSeq platform, 2 × 300 bp). The pipelines of QIIME2 with DADA2 will be used to perform demultiplexing sequences, quality control, feature table construction and taxonomy assignment [21]. Taxonomic assignment (from kingdom to genus level) of the representative sequences of each sample will be performed using the Silva 132 pre-trained Naive Bayes classifier and the q2-feature-classifier plugin in the QIIME2 pipeline. Alpha-diversity will be calculated by observed species, Chao1, and Shannon indices using QIIME2. To compare microbial profiles between samples, weighted (quantitative) and unweighted (qualitative) variants of UniFrac and Bray-Curtis will be measured. Principal coordinate analysis (PCoA) will be applied on the resulting distance matrices to generate two-dimensional plots. Permutational multivariate analysis of variance (PERMANOVA) will be used to statistically test for significant differences of the microbial profile between groups. Hierarchical clustering analysis will be performed to show a visual interpretation heatmap of the similarity of microbial taxa and function gene among groups. In terms of downstream microbiome analysis, centered Log Ratio Transformation (CLR) will be applied to transform the microbiome composition. The longitudinal model (MetaLonDA) [22] and the generalized estimating equation (GEE) model [23] will be performed to compare the dynamics of microbial composition over time between groups. Within each arm, the repeated ANOVA with permutation test will be used to analyze microbial composition.
Interventions
Nutrition protocol
Participants are randomized to receive a supplement of either resistant starch (intervention) or waxy cornstarch (control). The study dietitian (DC) is the only “unblinded” member of the research team, and is responsible for providing the correct supplement to participants, without revealing the randomization scheme to other team members or participants. At the completion of the study, DC reveals the contents of the supplement to the participant. The resistant starch is composed of 40% digestible starch and 60% resistant starch and is provided as Hi-Maize® 260 resistant starch (Hi-Maize 260, Ingredion, Bridgewater, NJ). The control is Amioca® corn starch (AMIOCA, Ingredion, Bridgewater, NJ), is nearly completely digestible. For week 1, the dose of supplements is 15 g/day. The dose is then increased to 33 g/day for weeks 2–16. Participants are instructed to blend their supplement into commonly consumed foods/beverages such as yogurt, soups, oatmeal, coffee, or juice. This daily dose was selected to provide a minimum of 30 g resistant starch/day as previously reported by Sirich et al. [9], and allow for the 10% variability in the sachet weight as determined by the commercial packing company. Sirich et al. [9] utilized a similar protocol with good gastrointestinal tolerance. Participants are provided with dosing suggestions and recipes to promote good compliance. Supplements are provided to participants as daily doses in sachets labeled as either “Supplement 1” or “Supplement 2”. Participants are issued half of their daily doses of supplement at visit 2, and the remaining daily doses of supplement are provided at visit 3. Participants are instructed to return any unused supplement at the completion of the study. A research assistant contacts the subjects biweekly during the study to determine if the subject had experienced any complications with consuming the supplement and to encourage compliance. Three-day food records are administered at baseline, and week 16 and entered into Food Processor software (ESHA) for nutrient analysis -to assess between-group differences in dietary intake of nutrients which may impact upon renal function (i.e. protein, potassium, phosphorus).
Exercise protocol
Individuals assigned to the exercise groups participate in supervised training (3 x per week at 55–65% VO2 peak for 45–50 min) for 16 weeks in the Wellness Center of the institution. Each exercise session begins with a 3–5 min warm-up at a low intensity and static flexibility exercises. Participants build up their exercise tolerance to complete 45–50 min of moderate intensity continuous aerobic exercise using a combination of exercise apparati, depending upon their preference. The heart rates that corresponded to 55–65% of measured VO2 peak from the exercise test are used to monitor exercise intensity along with perceived exertion to make sure that subjects are working within a safe, but effective range. At the completion of each workout, subjects are taken through a cool-down procedure involving static stretching. Blood pressure is assessed before and after all exercise sessions and if these values are abnormal for the individual, they are not allowed to exercise on that day. Blood glucose is assessed in persons with diabetes prior to and after each exercise training session. If the pre-exercise value is less than 100 mg/dl the individual is given a drink containing approximately 15 g of carbohydrate. However, if the baseline plasma glucose level exceeds 250–300 mg/dl with ketones present, the individual is not permitted to exercise on that day in compliance with ACSM guidelines [18].
Outcomes
The primary outcomes of this study are systemic markers of inflammation: CRP,TNFα, IL-6, IL-10, MCP1, markers of oxidative stress, MDA, 8-isoprostanes F2a, uremic toxins, PCS and IS and the faecal microbiota composition. All biochemical markers will be assayed by well-established labs that are blinded to the group assignments of the participants.
Secondary outcomes include indices of vascular function; carotid-femoral arterial stiffness as pulse wave velocity (PWV), resting peripheral and central SBP/DBP (measured by applanation tonometry), endothelin-1, and nitrate/nitrite.
Statistical analyses
An Intention-To-Treat design will be used in this study. Effects of treatment on all dependent variables will be assessed by a 2 (prebiotic supplement or control) by 2 (aerobic exercise or control) by 3 (time) mixed factor ANOVA. The effect of the prebiotic supplement will be determined by examining the prebiotic supplement x time interaction, the effect of exercise will be determined by examining the aerobic exercise x time interaction, and the combined effect of the supplement and exercise will be determined by examining the 3-way supplement x exercise x time interaction. Significant interactions will be explored through simple effects analysis. The main variables of interest are inflammatory markers, particularly IL-6, and IL-10.
Data auditing
Prior to performing the final study analyses, data will be checked by the statistician who will be assisted by research assistants in this process. These individuals will be blinded to the group assignment of the participants.
Sample size estimates
A study investigating the effects of aerobic exercise on inflammation reduction [24] demonstrated effect sizes (Cohen’s d) varying between .40 and .77 for IL-6 and IL-10 and their ratio, which would be considered a medium to large effect. Few studies have investigated the effect of prebiotic supplements on inflammation, though similar magnitude effect sizes were reported for a prebiotic supplement intervention on changes in free circulating uremic solutes indoxyl sulfate and p-cresol sulfate, key indicators of adverse outcomes of CKD [9], though those changes were not found to be significant in that small sample study. Based on these studies, we anticipate medium effect sizes, and with power set at .80, and alpha set at .05, a sample size of 60 (15 per group) is predicted to be sufficient for detecting significant two-way interactions specified in the statistical analysis section. To account for any dropouts from the protocol, missing data will be addressed through multiple imputation, and results of all analyses averaged over multiple imputed datasets. All variables in the analysis will be examined for assumptions for all statistical tests. Data will also be examined in a per-protocol analysis, using only data from participants in all groups who completed 70% or more of their assigned exercise sessions and/or supplement use and who provided outcome data on the relevant variables.
Dissemination policy
Upon the completion of data collection, the PI and the research team will endeavor to publish the findings of this trial in peer-reviewed journals whatever the outcome of the trial. The PI will also submit the findings to be presented at relevant professional meetings.