As renal function decreases, waste products called uremic toxins accumulate in the blood, and also increase in the intestinal epithelium [8,9,13]. This change in the gut promotes the colonization of bacteria that can use urea as an energy source by producing the enzymes urease or uricase. Urease is a cytosolic enzyme that catalyzes the hydrolysis of urea to ammonia and carbon dioxide. Consequently, the markedly increased formation of ammonia increases the gut pH, mediates enterocolitis by the breakdown of gut epithelial tight junctions, and facilitates endotoxemia and systemic inflammation [7-9,13-16]. The uric acid originating from purine metabolism is mainly excreted via the kidney; however as renal function decreases, the colon replaces the kidney as the primary site of uric acid excretion [9,15,16]. The high concentration of uric acid increases the bacteria containing urease and uricase and influences changes in the gut microenvironment via the same pathway. Several studies have demonstrated that CKD patients have significant changes in the gut microbiota with loss of α-diversity, β-diversity, and richness [8-10]. In a study of 24 stable ESRD patients, Wong et al. [17] also showed reduction in the Lactobacillaceae and Prevotellaceae families, which can express the butyrate kinase gene involved in protective processes, and confirmed that most of the microbiota has urease, uricase or tryptophanase activity; 12 of 19 bacteria families have urease activity (including Alteromonadaceae, Cellulomonadaceae, Clostridiaceae), five have uricase activity (Cellulomonadaceae, Dermabacteraceae, Micrococcaceae, Polyangiaceae, and Xanthomonadaceae), and three have tryptophanase activity (Clostridiaceae, Enterobacteriaceae, and Verrucomicrobiaceae). This alteration of gut microbiota can promote the acceleration of dysbiosis and its metabolic activities, and lead to the change of gut structure called leaky gut syndrome. Vaziri et al. [16] reported a marked reduction in the amount of tight junction-related proteins such as claudin-1, occludin, and zonula occludens 1 in the colonic mucosa of animals with CKD. In addition, Farhadi et al. [18] showed several histological changes such as reduction of villous height, elongation of the crypts, and infiltration of the lamina propria with inflammatory cells in the intestines of CKD patients. These pathological changes in the gut suggest that translocation of bacteria and the influx of endotoxins across the intestinal wall can contribute to inducing systemic inflammation in CKD.

The classic CKD diet is generally composed of light meals containing low concentrations of sodium, potassium and phosphate, to reduce their adverse metabolic effects [19]. This type of diet limits the consumption of potassium-rich foods such as fruits, vegetables, nuts, and high-fiber products. Indigestible carbohydrates are not only essential nutrients for the metabolism of gut microbiota but also precursors of short-chain fatty acids (SCFAs) related to immune regulation, glucose and lipid metabolism, gut integrity, and appetite [4,19,20]. Therefore, the CKD diet contributes to a decrease in the production of SCFAs and increases the amino nitrogen load, which can transform into uremic toxins [8,20]. CKD patients are commonly prescribed multiple drugs including iron-containing compounds, phosphate binders, and antibiotics [20,21]. This long-term and high pill burden for CKD patients can extensively influence the composition and richness of the gut microbiota.

Uremic toxins can be classified based on their site of origin: endogenous (mammalian metabolism), exogenous (diet), or microbial [22,23]. Among these original site-specific toxins, three molecules including indoxyl sulfate (IS), p-cresyl sulfate (pCS), and trimethylamine N-oxide (TMAO), are microbiota-derived uremic toxins involved in the development of complications including cardiovascular disease and mortality as well as the pathogenesis and progression of CKD. IS, a protein-binding uremic toxin and one of the microbiota-derived metabolites, is synthesized from dietary tryptophan by bacterial fermentation, and is excreted in the urine. IS converted from indole in the liver contributes to peripheral vascular disease and thrombosis of vascular access [22-24]. Its serum concentration increases in proportion to a decrease in renal function. In a prospective study of 268 CKD patients [24], Wu et al. [24] reported that the baseline concentration of IS can be a predictor of loss of renal function. Some experimental studies have demonstrated that IS mediates the renal expression of genes related to tubulointerstitial fibrosis, such as transforming growth factor β1 and a tissue inhibitor of metalloproteinases [23,25]. Another experimental study showed that mouse podocytes exposed to IS for 8 weeks exhibited a pro-inflammatory phenotype, perturbed actin cytoskeleton, decreased expression of podocyte-specific genes, and decreased cell viability [26]. pCS is a colonic fermentation product of tyrosine and phenylalanine catabolism by anaerobic gut bacteria. Once p-cresol is absorbed, it is conjugated with other substances in the liver, where a sulfate group is added [6-8]. In a CKD rat model, pCS increased the production of reactive oxygen species (ROS), activating nicotinamide adenine dinucleotide phosphate oxidase and increasing caspase-3 activity, leading to an increased apoptosis ratio [27]. In another experimental study of half-nephrectomized mice, IS or pCS activated the intrarenal renin-angiotensin-aldosterone system (RAAS) and induced interstitial fibrosis and glomerulosclerosis [28]. TMAO is synthesized from dietary choline, phosphatidylcholine, and L-carnitine. Pelletier et al. [29] reported that the serum TMAO concentration negatively correlates with the glomerular filtration rate (GFR) in patients with CKD. Tang et al. [30] showed that an elevated level of TMAO was associated with the degree of tubulointerstitial fibrosis in an animal model and poorer overall survival in CKD patients. Stubbs et al. [31] also reported that TMAO increases the phosphorylation of SMAD3, an important regulator of fibrosis, and consequently enhances atherosclerosis and thrombosis, leading to an increased incidence of coronary artery disease. Therefore, TMAO has been proposed as a potential surrogate marker to detect early cardiovascular risk in patients with CKD. These accumulating data suggest that increased microbiota-derived metabolites are definite risk factors that contribute to CKD development and progression, and attempting to reduce microbiota-derived uremic toxins seems to be a reasonable therapeutic strategy for CKD patients.

The gut microbiota can provide a large number of potential immunostimulatory bacterial products. Recently, this has been considered a critical exogenous trigger of host immune dysfunction [4,5]. The leaky gut in CKD patients can facilitate the influx of microbiota-derived products into the hepatic portal system and systemic circulation across the intestine wall. A lipopolysaccharide (LPS) is derived from the cell wall component of gram-negative bacteria [32-34]. LPS induces systemic inflammation via a cascade of inflammatory responses. Lipid A is a component of LPS and serves as the microbe-specific molecular signal that binds to the surface receptor complexes of immune cells (monocytes/macrophages), which comprise toll-like receptor-4 (TLR4) and myeloid differentiation (MD) factor-2. The formation of the TLR4-MD2-LPS complex activates the signaling pathway controlling the expression of inflammatory genes, leading to excessive production of pro-inflammatory cytokines such as interleukin-1β (IL-1β), IL-6, and tumor necrosis factor α [33-35]. TLR4 is also highly expressed in the endothelial cell, vascular smooth muscle cell and adventitial fibroblast. Activated TLR4 promotes the recruitment of monocytes, transformation of macrophages to foam cells, and the expression of adhesion molecules, ROS, and pro-coagulant activity by intracellular inflammatory pathways mediated through nuclear factor-κB and mitogen-activated protein (MAP) kinases [32-35]. Ultimately, these processes can initiate and promote atherosclerosis. McIntyre et al. [36] demonstrated that circulating bacterial endotoxin/LPS levels are increased in all stages of CKD and reach the maximum in dialysis patients, and suggested that systemic inflammation is also a strong and independent predictor of mortality in CKD. Szeto et al. [32] also showed that circulating endotoxin in patients undergoing peritoneal dialysis is related to systemic inflammation and features of atherosclerosis.

The gut is the second-most innervated organ in the body, and the gut microbiota communicates with the nervous system by producing several hormones and neurotransmitters [4,7]. The central and peripheral nervous systems regulate the circulatory system by modulating the sympathetic nervous system, RAAS, and pituitary hormone release. Jazani et al. [37] demonstrated that the gut microbiota activates the hypothalamic-pituitary-adrenal (HP2020-10-25A) axis and increases the secretion of serotonin and other neurotransmitters. Onal et al. [38] reported that Bifidobacteriaceae, Lactobacillaceae, and Prevotellaceae species can synthesize neurotransmitters such as γ-aminobutyric acid (GABA) and acetylcholine (ACh) and can promote production of intestinal incretin, glucagon-like peptide-1 and -2 (GLP-1, -2), and the gut hormone peptide YY. GABA is known to stimulate natriuresis and to suppress renal sympathetic nerve activity [9,38]. ACh and GLP-1 have been reported to increase the GFR by renal vasodilation and reducing angiotensin II [7,9,37]. Therefore, gut dysbiosis with reduction of bacteria species such as Bifidobacteriaceae and Lactobacillaceae in CKD causes activation of the RAAS and augmentation of sympathetic outflow and, ultimately, leads to HTN and CKD progression. In addition, changes in GLP and peptide YY can influence energy homeostasis by reduced energy expenditure, lipolysis, and insulin sensitivity and secretion, leading to obesity, hypercholesterolemia, insulin resistance, and/or DM. These are important risk factors for CKD development and can also contribute to CKD progression [4,33,38].

Increasing evidence supports the concept that dysbiosis in CKD patients is associated with CKD-related complications such as HTN, cardiovascular events, mineral and bone disorder (MBD), and cognitive dysfunction [33,38]. The shift towards a predominantly proteolytic fermentation pattern in CKD promotes the increase of microbiota-derived toxins and increases systemic inflammation.

Cardiovascular disease is a main cause of morbidity and mortality in CKD patients. Beyond the gut-kidney axis, several reports suggest that diverse mechanisms including enhanced ROS production, leukocyte activation, pro-inflammatory cytokine production, myocyte hypertrophy, and RAAS activation contribute to the development and progression of cardiovascular disease, calling these relationships between the gut and heart the gutheart axis [7,38-41]. Lin et al. [42] showed that elevated levels of pCS and IS are associated with increased mortality in CKD patients while pCS, but not IS, is associated with an increased risk of cardiovascular events. A recent meta-analysis studying TMAO in 19,256 patients also demonstrated that elevated levels of TMAO and its precursors were associated with a 1.7-fold increased risk of major acute cardiovascular events and all-cause mortality in comparison with low TMAO [43].

Psychiatric disease is common in CKD patients and is associated with increased morbidity and mortality [9,39,44,45]. Although estimates vary, studies have shown that combined depression and cognitive dysfunction is present in approximately 20% to 25% of CKD patients [7-9,44,45]. Growing evidence suggests that gut dysbiosis promotes dysregulation of the HPA axis with overproduction of glucocorticoids, alterations in levels of neurotransmitters, and activation of pro-inflammatory cytokines, called the gut-brain axis [39,45-48]. Gut microbiota-derived toxins play an important role in the pathogenesis of cognitive dysfunction by direct toxicity or other putative factors such as oxidative stress, inflammation, endothelial dysfunction and vascular calcification [7,45,46]. IS crosses the blood-brain barrier through the organic anion transporter-3 (OAT-3), and may accumulate in the brain in CKD due to dysfunctional OAT-3 [46]. Elevated IS induces inflammation and apoptosis of human astrocytes and glial cells through oxidative stress induction and MAP kinase pathway inhibition [45-48]. Aryl hydrocarbon receptor (AhR), known as a ligand-activated transcriptional factor, is mainly expressed in the hippocampus, cerebral cortex, and cerebellum, and has been implicated in sensorimotor and cognitive functions [7,9,47,48]. Elevated IS activates AhR in astrocytes, which is likely to promote further oxidative stress, leading to sensorimotor and cognitive dysfunction in CKD [47,48]. In a clinical study with 260 hemodialysis (HD) patients, Lin et al. [47] showed that the circulating free form of IS is significantly associated with lower cognitive function test scores, particularly in cognitive abilities screening instrument domains of memory, mental manipulation, and language ability. Further clinical studies based on molecular regulation in the expression of uremic toxins are needed in CKD patients.

CKD-MBD is a syndrome recently re-named to embody the biochemical, skeletal, and cardiovascular pathophysiology, not limited to bone disease [49]. CKD-MBD is associated with poor outcomes in terms of myocardial infarction, stroke, bone fracture, and all-cause mortality [50]. Growing evidence indicates elevated uremic toxins are involved in the development of bone abnormalities in CKD [51]. Mozar et al. [50] demonstrated that IS inhibited both osteoclast differentiation and bone-resorbing activity in monocyte/macrophage cellular models. Nii-kono et al. [52] also found that IS suppressed parathyroid hormone (PTH)-stimulated intracellular cyclic adenosine monophosphate production and decreased PTH receptor expression in a primary osteoblast culture from mouse calvaria. In a clinical study with 47 HD patients, Goto et al. [53] showed that IS correlated negatively with alkaline phosphatase, particularly bone-specific alkaline phosphatase, regardless of intact PTH activity. These findings strongly suggested that IS not only inhibits osteoblast function but also has an inhibitory effect on osteoclasts and PTH and, thus, could affect bone remodeling in CKD patients.

Resistant starch (RS) is a type of carbohydrate that is incompletely digested by human pancreatic amylases. High amylose maize-resistant starch type 2 (HAMRS2), a kind of RS found in starchy foods such as potato, corn, and banana, reaches the large intestine and serves as an energy source for beneficial bacteria such as Bifidobacterium and Lactobacillus [54,55]. Several studies have demonstrated the beneficial effects of HAMRS2 on CKD progression, decreased microbial diversity, and an increased Bacteroidetes-to-Firmicutes ratio in HAMRS2-fed rats [54-56]. Vaziri et al. [56] also reported a favorable effect of this type of RS on reducing oxidative stress and inflammation, and restoring intestinal epithelial tight junctions in a CKD rat model. Krishnamurthy et al. [57] revealed that high fiber diets showed low inflammation and decreased all-cause mortality in a study analyzing data from 14,543 participants in the National Health and Nutrition Examination Survey III. Despite these potential renoprotective effects, high fiber diets have a limitation in alimentotherapy for patients with advanced stages of CKD due to their potassium and phosphorus content. Therefore, practical cooking techniques and counselling should be provided for safe intake in CKD patients.

Prebiotics are nondigestible food ingredients that can help stimulate the growth of the selected or a limited number of bacteria in the colon [58,59]. Inulin, fructo-oligosaccharides, galacto-oligosaccharides, soya-oligosaccharides, xylo-oligosaccharides, and pyrodextrins are commonly used as prebiotics. They promote the growth of Bifidobacteria and Lactobacilli species, while suppressing other groups of bacteria such as Bacteroides, Clostridia, and Enterobacteria species [59]. Meijers et al. [60] also demonstrated that serum concentrations of IS and pCS were significantly reduced by the oral intake of oligofructose-enriched inulin in 22 HD patients. The prebiotic lactulose also improved kidney function by modifying gut microbiota and inhibiting the production of uremic toxins in 10-week-old adenine-induced CKD Wistar/ST male rats. In addition, lactulose decreased serum levels of creatinine and blood urea nitrogen (BUN) and ameliorated CKD progression by suppressing tubulointerstitial fibrosis [61]. Probiotics are defined as live microorganisms that confer a health benefit on the host when administered in adequate amounts [62]. Probiotics commonly consist of living bacteria such as Bifidobacteria species, Lactobacilli and Streptococci [63]. A pilot clinical trial in patients with CKD stages 3 and 4 showed significantly decreased BUN and improved quality of life after treatment with the Renadyl (Kibow Biotech Inc., Newtown Square, PA, USA) formulation of Lactobacillus acidophilus, Streptococcus thermophilus, and Bifidobacterium longum over 6 months [63]. However, the follow-up randomized controlled trial in 22 patients failed to show decreased uremic toxins or the sensation of enhanced well-being [64]. Some of the benefits of probiotics could be explained by persistent uremia-induced alterations in the gut biochemical milieu as well as by dietary or medicinal regimens that produce an unfavorable microenvironment for the symbiotic microbiota. Synbiotics are the combination regimen of prebiotics and probiotics. A randomized clinical trial by Guida et al. [65] showed a suppressive effect of short-term synbiotic treatment on plasma p-cresol levels without improvement of gastrointestinal symptoms in 30 CKD patients after 4 weeks. A recent multicenter study in 42 HD patients showed an improvement of gastrointestinal symptoms and decreased C-reactive protein after 2 months of treatment [66]. The recent randomized controlled trial of the effects of pre/pro/synbiotics is summarized in Table 2 [63,67-76].

Fecal microbiota transplantation (FMT), also known as a stool transplant, is another option to modify the gut microbiota [77,78]. However, there are few data about FMT for the treatment of CKD, except the use of FMT for the treatment of Clostridium difficile infection, which is a common complication in patients with CKD undergoing HD [77,78]. Further research is needed to understand how FMT affects the progression of CKD.

AST-120, an oral charcoal adsorbent, is widely used as a therapeutic agent in CKD patients to absorb circulating uremic toxins and precursors such as IS. Several animal studies have shown that, in addition to the change in composition of the microbiota, AST-120 decreased serum IS levels, reduced the production of ROS by endothelial cells, and impeded subsequent oxidative stress and inflammation [79,80]. Several clinical studies have also shown that AST-120 reduced uremic symptoms, proteinuria and time-to-dialysis in CKD patients [79,81]. However, a recent meta-analysis reported that AST-120 reduced serum IS levels but showed no significant improvement of renal function and all-cause mortality [82]. Sevelamer (hydrochloride or carbonate), a polyphosphate binder, is noted for its ability to bind uremic toxins. Although some experiments and/or clinical studies report beneficial effects on reducing uremic toxins, such as serum IS and pCS levels, the results of the clinical studies published to date are conflicting [83].