## 4 Discussion  
To develop an innovative symbiotic with the prospective purpose of being used in nephropathy, we evaluated different probiotics, antioxidants, and prebiotics. Starting from the evidence that CKD patients show an increased incidence of oxidative stress [35] and that, in nephropathy, oxidative unbalance is due to increased production of reactive oxygen species [36], we characterized different antioxidants for the development of an innovative symbiotic. The applied selection methods are primarily based on the evaluation of probiotics' ability to grow in the presence of antioxidants and prebiotics. Some antioxidants have antimicrobial activities [37; 38; 39] and, for this reason, the first analyses were carried out on probiotics in the presence of antioxidants. Twenty-five probiotics, previously isolated from animal and human feces or commercially available formulations, were used. The tested bacteria belonged to the two main groups of probiotics, _Bifidobacteria_ and lactobacilli. In the two last decades, evidence widely highlighted the beneficial roles of these two taxa on host  
Figure 11: Fecal metabolites (nmol/g wet fecal batch) detection of **(A)** uremic toxins by nuclear magnetic resonance (NMR) and **(B)** uremic toxins precursors by amino acid analysis in chronic kidney disease (CKD) fecal batches before (t0) and after simulated (in vitro) colonic fermentation (t24; 24 h at 37 \({}^{\circ}\)C) with and without the innovative symbiotic NatuREN G (NG). Fecal batches with healthy subjects (HC) feces were used as the control. The concentration of NMR compounds is summarized as the average of two independent analyses \(\pm\) SD. The concentration of FAAs is singularly reported (average expressed as a middle bar). * Significantly different compounds’ concentrations (multiple t-test using the Sidak-Bonferroni correction, \(p\) < 0.05) compared with the relative baseline (t0). health [40; 41; 42]. Both of these probiotics are recognized as the main saccharolytic bacteria of a healthy human gut [43]. We specifically focused on them also because of the critical lack of _Bifidobacterium_ and lactobacilli frequently found in CKD [8]. We selected those probiotics that, in the presence of antioxidants, showed the highest degree of acidification. Then, the same criterion was adopted for prebiotics evaluation, with the aim of selecting high SCFA-producing bacteria. In healthy conditions, SCFAs are the main energy source for intestinal epithelial cells; additionally, they are primarily involved in maintaining intestinal pH within optimal ranges [18]. Subsequently, we evaluated the in vitro effect(s) of the resulting combination of antioxidants and prebiotics. Experimental treatments were initially tested in fecal media prepared from feces collected from healthy subjects. Subsequently, the same was also performed with fecal media prepared from feces collected from CKD patients at stage IIID-IV, i.e., the worst condition before the ESRD stage when dialysis becomes essential. In the evaluation of the obtained results, we underline the widely recognized limitations that are innate in our experimental conditions due to the impact of sampling fecal microbiota communities [44]. However, many (12 out of 25) of the evaluated probiotics showed promising results, at least in our in vitro analyses. Therefore, the total free amino acids (FAAs) analysis was an intermediate step, playing a pivotal role in decreasing the mainly proteolytic metabolism typical of nephropathy [45]. In CKD, the dysbiotic, highly proteolytic, microbial patterns determine the increase in uremic toxins, e.g., IS and pCS, produced from urea and other nitrogenous compounds [8]. Moreover, due to low kidney functionality, CKD patients are unable to excrete waste metabolites (including urea, ammonia, and uremic toxins), which, in turn, contribute to pH increase in the intestinal environment [7]. Additionally, urea and ammonia are primarily involved in the tight junctions' disruption in CKD [46].