Study Design and Objectives This investigation is a single-arm, open-label pilot trial designed to assess the impact of a multispecies synbiotic supplementation on gut microbial metabolites, markers of inflammation, and gut-derived uremic toxins (GDUTs) over 12 weeks in adults with in a group of hemodialysis patients.
Sample Size Justification This pilot study targets 30 participants, which provides adequate power (80%, alpha 0.05) to detect a medium effect size (Cohen's d = 0.6) on the primary outcome, serum indoxyl sulfate levels. Calculations based on expected changes from 8.0 mg/L to 6.5 mg/L with SD of 2.5 mg/L indicate a required sample size of \~24; the sample size accounts for potential dropouts to ensure study robustness.
Study Procedures
* Baseline assessments:
* Collection of serum biochemical markers,
* Measurement of serum and urine uremic toxins,
* Quantification of microbial metabolites.
* Intervention:
Participants will receive Renobiome multispecies synbiotic containing 30 billion CFUs per capsule, including strains of Lactobacillus rhamnosus (strain ID pending), Lactobacillus salivarius LS 159, Lactobacillus pentosus LPE 588, and Lactococcus lactis LL 358.
* Dose: One capsule twice daily (morning and evening), with or without food, taken with room-temperature water.
* Storage: Capsules to be kept below 25°C, in a dry, light-protected environment.
* Follow-up and Monitoring:
* At 4 weeks: Monitoring and documentation of gastrointestinal symptoms and adverse events.
* At 12 weeks: Repeat evaluations identical to baseline, including blood and urine tests.
Compliance and Safety
* Adherence will be tracked via regular follow-up contacts.
* An adherence rate of 80-100% is considered acceptable.
* All adverse drug reactions and any unexpected events will be recorded throughout the study duration.
Biological Sample Collection and Laboratory Methods
• Blood Sampling: Serum Blood samples are collected separately from each participant. Blood samples are collected after an 8 to 12-hour fasting period, using sodium fluoride tubes, and kept on ice until transfer to the research laboratory. Once there, the samples are centrifuged at 3,000×g for 15 minutes at 4°C. The serum Blood samples are collected separately from each participant. Blood samples are collected after an 8 to 12-hour fasting period, using sodium fluoride tubes, and kept on ice until transfer to the research laboratory. Once there, the samples are centrifuged at 3,000×g for 15 minutes at 4°C. The serum aliquots (200 μL) is then transferred to sterile tubes and stored at -80°C until batch analysis.
* Biomarker Measurement:
* Before analysis, samples will be thawed at room temperature for 20 minutes.
* Quantification of inflammatory cytokines such as Interleukin-1(IL-1), Interleukin-6(IL-6), Tumor Necrosis Factor-alpha(TNF-α) using validated ELISA kits (Ziker Biological Technology Co., Ltd., Shenzhen, China).
* Measurement of microbial metabolites such as short-chain fatty acids (SCFA, e.g., butyric acid), indolelactic acid (ILA), and indolepropionic acid (IPA) will be conducted.
We measure serum levels of acetate, propionate, butyrate, and valerate, along with branched-chain SCFAs such as isobutyrate and isovalerate. These metabolite profiles are analyzed using ultraperformance liquid chromatography-mass spectrometry (UPLC-MS). In brief, 100 μL of each serum sample is mixed in 1.5-mL microtubes with 20 mg of NaCl, 10 mg of citric acid, 20 μL of 1 M HCl, and 100 μL of butanol. The mixture is vortexed for 2 minutes and then centrifuged at 18,000×g for 15 minutes. The resulting supernatant is transferred to fresh microtubes for analysis.
* Uremic Toxin Analysis:
* Concentrations of indoxyl sulfate (IS), p-cresyl sulfate (PCS),Indole-3-acetic (IAA), and indolelactic acid (ILA) in serum and urine will be determined by high-performance liquid chromatography-mass spectrometry (HPLC-MS) according to established protocols:
Briefly, for binding competition, 200μl serum to which we added 20μl 0.50mM 1-naphthalenesulfonic acid (internal standard) was vortex-mixed with 250μl 0.24M sodium octanoate (binding competitor).After incubation at room temperature for 5min, we added 2ml cold acetone to precipitate proteins. Following vortex-mixing and centrifuging at 4 ◦C, 1860×g for 20 min, the supernatant was transferred to 12mm×100mm, GL 14 glass test tubes and 2ml dichloromethane was added. After vortex-mixing and centrifuging at 4 ◦C, 1860×g for 10min, 200μl of the upper layer was transferred to glass autosampler vials, followed by addition of 20μl 1M HCl and 15μl was injected onto the HPLC. The HPLC analysis was performed on an Agilent 1100 series LC (Santa Clara, CA),and Agilent ChemStations software were used for the chromatographic analysis. The separation was carried out on a ZORBAX SB-C18 Solv Saver Plus HPLC column (5 μm, 3.0 mm×150 mm).at a flow rate of 0.6 ml/min. Mobile phase A is 0.2% trifluoroacetic acid in Milli-Q water and mobile phase B is 0.2% trifluoroacetic acid in acetonitrile. The analytical method consists of an isocratic run with 92% mobile phase A for 23 min.. Each analytical run was followed by a 1.3 min washout gradient to 100% B. Column temperature was 25 ◦C, and autosampler tray temperature was 6 ◦C. We quantified the analytes by using the analyte to standard peak area ratio on a Agilent 1100 High Performance Fluorescence detector G1321A and Agilent 1100 Series UV-Visible detectors G1314A. Detector settings were λex 260 nm/λem288nm for p-cresyl sulfate and λex 280 nm/λem 390nm for indoxyl sulfate, indole-3-acetic acid(IAA), indolelactic acid (ILA) and internal standard.
