The complex role of the human intestinal

The complex role of the human intestinal microbiota is emerging and its functions has been elucidated when considering energy metabolism, nutrient digestion, vitamin synthesis, epithelial defenses and immune responses [34]. The colon is responsible for salvage of energy and possibly nitrogen from carbohydrate and protein not digested in the upper gastrointestinal tract. Fermentation of the MK-8745 tyrosine and tryptophan by intestinal microbiota generates p-cresol and indole, respectively. After absorption, these are further metabolized to generate PCS and IS. IS and PCS circulate in equilibrium between a free solute fraction and a fraction bound to serum proteins. The best-characterized binding site is albumin Sudlow site II, for which IS and PCS are competitive binding inhibitors [35]. So far, most studies focused on the bioactivity of indoxyl sulfate. Indoxyl sulfate induces free radicals in renal tubular cells and glomerular mesangial cells [36], activating the nuclear factor-kappa B pathway [37]. The present study demonstrated that severe renal tubulointerstitial fibrosis was found in 5/6Nx rats, accompanied by increased serum levels of IS, PCS, and 24h urinary protein. As shown in Fig. 3, when concentration of IS, PCS changes, it means that concentration of indole, p-cresol, the precursor of IS, PCS has been already changed in the body. And the reason leading to the change of indole and p-cresol, is that the change of tryptophan, tyrosine metabolism and related gut microbiota. Almost all of p-cresol, indole only can be produced by gut microbiota in intestinal tract as well [39]. So the concentration of indole and p-cresol in feces may reflect the progression of CKD. At present, BUN and SCr is used as markers for the clinical diagnosis of CKD [38]. As shown in Fig. 3, there was a significant change in the concentration of p-cresol and indole between sham and 5/6Nx rats feces with CKD progression, which indicate that fecal indole and p-cresol might be an alternative biomarker to judge the progression of CKD. As mentioned before, PCS and IS both originate from bacterial protein fermentation in the large intestine. Colonic microbiota degrades tryptophan to indole. Further hydroxylation results in 3-hydroxy-indole, the majority of which is sulfonated to IS. In parallel, fermentation of tyrosine leads to the production of p-cresol and PCS [39]. The colon is the main source of many uremic solutes [40]. Studies have shown that CKD patients had increased number of bacteria that possess urease, uricase, p-cresol- and indole-forming enzymes, and reduced number of bacteria that possess short chain fatty acid forming enzymes [41]. In this study, we have found that the metabolism profile of amino acids in intestine was becoming more and more disordered over time, therefore, it is necessary to study the bacteria corresponding to the formation of amino acids in feces of 5/6Nx rats. In order to further prove the relationship between amino acid disorders and gut microbiota, we made 16S rDNA sequencing of two groups of rats. 16S rDNA sequencing is one of the high-throughput-sequencing-based methods used in gut microbiota analysis. Almost all the bacterial species in gut microbiota can be quantified through 16S rDNA sequencing, which has made this method into the mainstream [42]. Bifidobacterium can produce vitamin B1, B2, B6, B12 and alanine, valine, aspartic acid and threonine nutrients, which are necessary for human body [43]. The main function of Lactobacillus is to break down lactose and protein, help digestion and absorption [39]. Microorganisms such as Anaeroplasma in the gut can also metabolize peptides and proteins under anaerobic conditions [45]. Consistent with expectations, according to Fig. 5A, there was significant difference between the two groups in some microbial strains, such as Acinetobacter, Bilophila, Holdemania, Butyricimonas, Sutterella, Staphylococcus, Akkermansia, Coprobacillus, Lactobacillus, Anaeroplasma, Bifidobacterium. The abundance of microbial strains which related to the degradation of proteins and the production of amino acids have been changed compared with sham, which explains why there is a difference in the concentration of amino acids in the feces of the two groups of rats.