## 4 Materials and Methods
### Clinical Trial: Patients and Study Design  
A randomized, placebo-controlled, pilot study in HD patients was set up to verify the hypothesis that an innovative combined approach with the use of a symbiotic and an innovative dialysis cartridge would be effective in reducing circulating levels of microbial uremic toxins and inflammation. The study was carried out in accordance with the Helsinki Declaration (IV Adaptation) and the European Guidelines for Good Clinical Practice. The protocol of the study was approved by the Ethical Committee of the Azienda Ospedaliero-Universitaria Consorziale Policlinico of Bari, Italy (Authorization nr. 0642; 17 May 2017). Written informed consent was obtained from all patients. The authors confirm that all ongoing and related trials for this intervention are registered in the ClinicalTrials.gov registry database; because of administrative issues that led to a delay in the ClinicalTrials.gov registration, we registered the trial after the enrolment of the participants started (Identifier nr. NCT03946176). Sixteen volunteers were enrolled according to the following inclusion/exclusion criteria: (i) inclusion criteria were hemodialysis patients, aged between 30 and 65 years, with CKD stage V and having undergone Bicarbonate HD for at least 6 months, body mass index (BMI) between 18.5 and 29.9, omnivorous diet, signature of informed consent; (ii) exclusion criteria were use of antibiotics or probiotics 30 days before the recruitment, chronic gastrointestinal diseases, systemic inflammatory disease, suspected or clinical diagnosis of malignancy, chronic liver disease, corticosteroid therapy or immunosuppressive drugs, psychiatric conditions that reduce compliance with therapeutic protocols. At the enrolment (T0), patients were randomized for the use of the symbiotic NATUREN G (\(n=7\)) or placebo (\(n=6\)) for 8 weeks. NATUREN G(Farmalabor SRL) consisted of a mixture of probiotics (_Lactobacillus Casei_ LC4P1 \(3.2\times 10^{9}\), _Lactobacillus Bulgicus_ SP5 \(3.2\times 10^{9}\), and _Bifidobacterium Animals_ BLC1 \(3.2\times 10^{9}\)), prebiotics (tructoligosaccharides 5 g and inulin 5 g), and natural antioxidants (a mix of quercetin 0.13 g, resveratrol 46 ug, and proanthocyanidins 25 ug). During their last week of either symbiotic or placebo supplementation, all the patients underwent a cycle of three hemodialysis sessions using DVB-PVP cartridge (DVB HD). At T0, after taking the symbiotic/placebo (T7w) and after dialysis with DVB-PVP (T8w), routine blood parameters were evaluated, while blood and fecal samples were collected. At each time point, information about food habits, by means of a food frequency questionnaire, were collected, while Gastrointestinal Symptom Rating Scale and Bristol stool scale questionnaires were administered by the patients to evaluate gastrointestinal symptoms and stool type, respectively. IS and PCS plasma levels were measured pre- and post-hemodialysis at each time point, as reported in Figure 3. Patients were blinded for the use of the symbiotic/placebo, while they were aware of the use of innovative sorbent cartridges. At the end of the innovative hemodialysis cycle, patients were returned to traditional dialysis. The efficacy of innovative hemodialysis was compared with standard bicarbonate hemodialysis (BHD) for each patient. Primary outcome: change in serum concentration of PCS. Secondary outcomes: change in serum concentration of IS, inflammatory markers. Exploratory outcomes: modulation of gut microbiota composition, change in gastrointestinal (GI) symptoms and stool type, change in serum concentration of trimethylamineoxide. The study was carried out at the ambulatories of the Nephrology and Dialysis Unit of our Department. No incentive was provided to the volunteers. RRs for IS and PCS were calculated according to the following equation:  
RR = \((1-\text{Cpost-corr}/\text{Cpre})\times 100\) (%)using corrected Cpost (Cpost-corr) as reported by [PERSON] and coworkers (2016).