Fecal Microbiota Transplantation in Reducing Uremic Toxins Accumulation in Kidney Disease: Current Understanding And Future Perspectives

Abstract: During the past decades, the gut microbiome emerged as a key player in kidney disease. Dysbiosis-related uremic toxins together with pro-inflammatory mediators are the main factors in a deteriorating kidney function. The toxicity of uremic compounds has been well-documented in a plethora of pathophysiological mechanisms in kidney disease, such as cardiovascular injury (CVI), metabolic dysfunction, and inflflammation. Accumulating data on the detrimental effect of uremic solutes in kidney disease supported the development of many strategies to restore eubiosis. Fecal microbiota transplantation (FMT) spread as an encouraging treatment for different dysbiosis-associated disorders. In this scenario, flourishing studies indicate that fecal transplantation could represent a novel treatment to reduce the accumulation of uremic toxins. Here, we present the state-of-the-art concerning the application of FMT on kidney disease to restore eubiosis and reverse the retention of uremic toxins. 

Keywords: fecal microbiota transplantation; PBUTs; chronic kidney disease; acute kidney injury; kidney transplantation; uremic toxins; oral FMT 

Key Contribution: Uremic toxins figure as the chief contributors to the development of uremic complications during acute or chronic kidney impairment, and they harm several physiological functions. Based on this evidence, the employment of FMT for kidney disease could represent a promising strategy for PBUT reduction.

CLICK HERE TO KNOW CISTANCHE SUPPLEMENTS FOR KIDNEY DISEASE

1. The Gut Microbiome in Health and Kidney Disease 

The healthy gut microbial ecosystem consists of trillions of microorganisms that play a pivotal function in maintaining homeostasis by influencing metabolic, oxidative, and cognitive status and immune defense against pathogen infections. Each individual displays a unique microbial profile in early life depending on their gestational date of birth, type of delivery, milk feeding methods, sex, and gender. In adulthood, this healthy native microbiota remains relatively stable, despite several factors including body mass index (BMI), exercise, dietary habits, pharmacological therapies (e.g., antibiotics), and aging that can alter its composition [1]. According to large-scale studies, higher microbial diversity and richness in phyla, genera, and families is associated with healthier and advantageous intestinal status [2,3]. More specifically, the abundance of some enterotypes as Bifidobacterium Bififidum, Lactobacillus acidophilus, or Streptococcus thermophilus has been widely described to be beneficial for an effective immune response [4,5]. Furthermore, bacteria belonging to Clostridiaceae, Bififidobacteriaceae, and Bacteroidaceae families have been found in microbial communities of centenarians, suggesting that this phenomenon could reduce age-related immune system dysfunction [6]. 

In this scenario, dysbiosis represents both a structural and a functional alteration of the microbiome that is closely associated with a specifific disease. Importantly, the comparative metagenomic analysis shows the differences in gut microbiome profiles between pathological and non-pathological conditions. For example, chronic diseases (i.e., rheumatoid arthritis, inflflammatory bowel disease (IBD), diabetes, etc.) are characterized by lower microbial diversity and richness, higher levels of harmful bacteria, and an abnormal Firmicutes/Bacteroidetes ratio [7–9]. On the other hand, these studies also suggest that differences in a microbiome profile occur between diseases. 

Over the last decade, the mutual crosstalk between the gut microbiome and human disease enticed growing consideration in numerous intestinal and extra-intestinal diseases, such as chronic inflammatory diseases, metabolic dysfunction, neurological disorder, and cardiovascular disease [10]. In the context of renal disease, it still needs to be clarified whether intestinal dysbiosis represents a cause or a consequence since a noxious cycle was recognized between uremia and gut microbiome [11]. A large plethora of data established that the alteration in the microbiome arrangement represents a consequence of kidney injury and strongly drives its exacerbation due to the accumulation of manifold bacterial-derived toxins [11,12].

On the whole, the uremic intestinal community is typically marked by a high proportion of Actinobacteria, Bacteroides, and Firmicutes, which is rarely described in healthy conditions [13]. According to many metagenomic studies, CKD patients show alterations in the expression of 16S rRNA associated with the Enterobacteriaceae family (e.g., Enterobacter, Klebsiella, and Escherichia genera), indicating that the Gram-negative Proteobacteria represent a fundamental constituent of uremic flflora [13,14]. The abundance of urease, which is uricase accompanied by tryptophanase-tyrosine phenol-lyase positive bacteria (e.g., Actinomycetia, Methylococcaceae, Micrococcineae, Pseudomonadales, Alteromonadales, Micrococcales, Halomonadaceae, and Pseudomonadaceae), was recognized as the hallmark of uremic dysbiosis, due to its proteolytic activity in producing uremic toxins [15,16]. Additionally, elevated growth of Bacteroidaceae and Clostridiaceae has been associated with systemic inflflammation [17]. On the other hand, the strong reduction in the relative proportion of both Lactobacilli and Actinobacteria phylum together with the lower proliferation of Prevotellaceae and Bacteroidacee families reflect a decline in short-chain fatty acids (SCFAs) production [17,18]. Of note, it has also been shown that differences in gut microbiome profiles occur not only between CKD stages but also between kidney diseases characterized by different phenotypes patterns. For instance, it was shown that hemodialysis (HD) patients exhibit a disproportion in Gammaproteobacteria and Firmicutes when compared with pre-dialysis patients [13,19]. Furthermore, IgA nephropathy (IgAN) seems to be characterized by the high amount of many microbial groups, including Streptococcus and Paraprevotella [20,21]. Interestingly, several studies indicated that the enrichment of Escherichia-Shigella is increased in diabetes-associated kidney damage [22]. On the other hand, the overgrowth of Anaerosporobacter and Blautia was associated with metabolic dysfunctions in diabetic nephropathy (DN) [23,24]. Interestingly, recent evidence has indicated that intestinal dysbiosis also occurs in acute kidney injury (AKI) [25]. For instance, Andrianova et al. demonstrated by an in vivo study that alterations in the microbiome composition occurred following renal ischemia/reperfusion injury (IRI), and several bacteria including Rothia and Staphylococcus were linked to the high degree of injury [26]. In line with this research, Yang and their co-workers showed that the AKI-related microflora contributed to the exacerbation of renal damage, inflflammation, and intestinal permeability when transplanted in germ-free animals [27]. Finally, more recent data highlighted the involvement of dysbiosis and uremic toxins after solid organ transplantation, including kidney transplantation. In detail, the 16S analysis of renal transplanted patients detected a profound disruption of microbial diversity associated with the enrichment of uremic toxins producing Proteobacteria and Enterobacteriaceae [28,29]. Based on this evidence, strategies aimed to restore eubiotics and the levels of microbiome-related metabolites could represent a promising therapy for kidney disease. However, an alteration in microbiome composition is not necessarily negative; microbiome composition can be modified and accommodated by interventions such as the Mediterranean diet, supplements (probiotics, prebiotics, and Ω-3 Fatty Acids) or exercise that influence the inflflammatory state, which can decelerate CKD progression [30–32].

The growing knowledge of the detrimental effect of dysbiosis in kidney disease has supported the development of several targeted strategies to restore the level of uremic toxins. In the first instance, the therapeutic interventions based on biotic supplements aimed to prevent the generation of PBUTs by rebalancing the gastrointestinal flora equilibrium. However, a great number of conflating elements, such as the period of administration, bacterial amount, and strain selection impede the result interpretation and the method standardization. In this scenario, the manipulation of microbiota by FMT could represent a novel treatment to reduce uremic toxicity in patients with CKD. Therefore, this extensive review highlights the current knowledge of the role of fecal transplantation in the context of kidney disease, providing novel insight into the FMT-based strategy to correct the levels of uremic toxins. 

2. The Gut–Kidney Axis 

When the glomerular filtration rate decreases, a considerable amount of nitrogenous toxic catabolites (i.e., urea and urates) accumulate into the blood of CKD patients [33]. Against this background, the removal of these toxins is supported by the intestine, resulting in their retention in the gut lumen. As a result, the uremic milieu promotes a sustained dysbiosis characterized by the disequilibrium of proteolytic bacteria to the detriment of the saccharolytic communities. Consequently, the strong urease activity along with intensive proteolytic fermentation enhances the conversion of urea and amino acids (e.g., tyrosine, tryptophan) in toxic compounds, named uremic toxins [11]. Several studies demonstrated that the retention of such compounds strongly affects the integrity of gut mucosa by triggering a leaky gut and local inflflammation. In line with this evidence, a marked impairment of epithelial junctions including CLDN, OCLN, and Zonula occludens- 1, was observed within the intestinal barrier of several animal models of CKD [34–36]. Additionally, the translocation of a huge number of toxins in the circulation together with the triggering of the immune response branch causes systemic hyperinflammation and exerts multi-organ damage [37] (Figure 1).

Figure1.The detrimental effect of microbiota-derived uremic toxin accumulation related to kidney disease.

2.1. Uremic Toxins in CKD 

Uremic toxins figure as the chief contributors to the development of uremic complications during acute or chronic kidney impairment and they harm several physiological functions. According to their molecular weight and characteristics, they are usually categorized into small solutes (<0.5 kDa), middle-weight molecules (0.5–60 kDa), and protein-bound uremic toxins (PBUTs) [38]. Small solutes (creatinine, urea) are usually successfully removed by conventional hemodialysis techniques. The second category includes peptides and proteins with middle-molecular weight molecules, such as b2 microglobulin and alfa1-macroglobulin. In patients with normal kidney function, renal elimination accounts for 30–80% of total removal, while during renal injury, the removal of such compounds may be signifificantly altered. The final category of uremic toxins (PBUTs) includes relatively low molecular weight molecules that present specifific ionic or hydrophobic characteristics through which they strongly bind to albumin in the blood [39,40]. In patients with normal kidney function, they are usually eliminated by organic anion transporters (OATs) in the proximal tubules [41]. Although conventional hemodialysis is the main technique used for the reduction of uremic toxins, it has been demonstrated that it is most effective in eliminating small water-soluble compounds, while the removal of middle-weight compounds and PBUTs is very limited (reduction rate <30–35%), due to the strong protein-bond of such compounds and the usual pores’ cutoff of low-efflux (LF) membranes that avoid albumin loss and the consequent hypoalbuminemia [39,42]. Moreover, the increase in the number of HD sessions and/or the treatment time may improve small and middle molecule removal, but not for PBU molecules. Only the unbound portion of PBUTs could be efficiently removed by HD, due to their low molecular weight [39,43,44]. The reduction rate of PBUTs by conventional HD is listed in Table 1. Interestingly, most of the gut-derived uremic toxins including indoxyl sulfate (IS), p-cresyl sulfate (PCS), p-cresyl glucuronide (PCG), indol-3-acetic acid (IAA), and hippuric acid (HA), belong to the PBUTs group. On the other hand, the bacterial metabolite Trimethylamine-N-Oxide (TMAO) is grouped as small water-soluble molecules. The metabolic pathways of the most relevant uremic toxins together with their characteristics are summarized in Table 1. Briefly, phenol-derived PCS and PCG originate from tyrosine metabolism, while IS and IAA derive from tryptophanase-positive bacteria. Notably, tryptophan metabolism was also implicated in the kynurenine pathway [45]. 

Table 1. Summary of microbially produced uremic toxins, class, precursor, property, and related toxic effect. 

The toxicity of PBUTs has been well-documented in a plethora of pathophysiological mechanisms in kidney disease, such as cardiovascular injury (CVI), metabolic dysfunction, and inflflammation [64,65]. 

In the past years, mounting studies have corroborated the role of PBUTs in cardiovascular dysfunction and capillary rarefaction. For instance, in vivo, and in vitro experiments established that indoles and phenols exert a role in vascular leakage by promoting apoptosis and affecting the integrity of the adherent junctions in endothelial cells [66,67]. Additionally, more recent evidence indicated that increased oxidative stress represents the most relevant consequences of endothelial damage in patients displaying the highest level of uremic toxins [68]. At the cardiac level, gut-related toxins were determined to trigger reactive oxygen species (ROS) origination by upregulating the NADPH oxidases (NOX) activity [69]. This finding was closely associated with gap junction damage in cardiac muscle cells resulting in cardiomyocyte dysfunction [70]. According to several in vitro studies, PBUTs modulate the endothelial cells' senescence by downregulating the expression of klotho leading to vascular hypertrophy via the endothelial/mesenchymal transition [71,72]. 

The effect of several uremic toxins on vascular dysfunction may also be due to their ability to modulate the downstream expression of many intracellular miRNAs in endothelial cells [73,74]. Altogether, this evidence highlights that uremic toxins may represent the missing player between endothelial damage and uremia-related CVI. On the other hand, PBUTs induced detrimental consequences on systemic inflflammation. For instance, Ito et colleagues evaluated that the over-expression of E-selectin was mediated by PCS via JNK/NF-kB pathways, which resulted in the enhancement of leukocyte rolling on endothelial cells and organ extravasation [75]. More recently, several groups have found a cause–effect link between uremic solutes and the expression of certain pro-inflammatory cytokines [76]. At the renal level, the accumulation of PBUTs was demonstrated to increase the ROS levels in renal tubular epithelial cells (TEC) and mesangial cells by upregulating the NOX activity [77–80]. Furthermore, it was shown that the alteration of several cellular pathways including AhRs, NF-kB, and p53, in response to the elevated level of PBUTs, was associated with interstitial fibrosis and renin-angiotensin system (RAS) activation [49,81,82]. Moreover, klotho downregulation represents the hallmark of AKI and CKD and is strongly correlated with the senescence of renal cells [83]. The growing data suggest that PBUTs are involved in klotho hypermethylation by affecting the expression of several methyltransferases, probably via an NF-kB-mediated mechanism [84]. 

Finally, many experimental data found a strong relationship between uremic dysbiosis and CKD-related insulin resistance (IR) [85]. IR is indeed recognized as an important clinical condition that affects metabolic complications, sarcopenia, and cardiovascular injury in the CKD population. Moreover, the alterations in insulin signaling in CKD turn into kidney lipodystrophy, which is characterized by adipocyte dysfunction [85]. It is worth noting that adipocytes stimulated with blood from CKD subjects fixed an IR phenotype characterized by impaired glucose uptake [86]. Additionally, Stocker-Pinto et al. demonstrated that a ROS imbalance occurred when the adipocytes were stimulated with uremic solutes [87]. To elucidate this mechanism, Koppe et al. demonstrated that IR together with the redistribution of body fat was detected when healthy mice were treated with PCS [59]. Altogether, these data indicate that PBUTs seem to be the missing key in exacerbating the metabolic complications in uremia. Interestingly, some of the uremic mediators related to the decline of kidney function seem directly associated with cognitive decline. For instance, the elevated accumulation of several small water-soluble solutes including uric acid, guanidino compounds, and asymmetric dimethylarginine (ADMA), were found to be implicated in neurotoxicity [88–90]. Additionally, many studies indicated that higher levels of IS and PCS increased neuroinflammation, resulting in the cognitive impairment of patients with CKD [91,92]. Moreover, indole and cresol can cause BBB impairment via AhR activation, leading to inflflammation and oxidative stress [92]. Homocysteine (Hcy), which is produced through the intestinal metabolic transmethylation of methionine to cysteine, has been linked to neuronal damage in many neurological diseases. Patients with CKD who show elevated concentrations of plasma Hcy are known to suffer from cognitive and motor impairment [93]. The proposed mechanism involves the overstimulation of the N-methyl-D-aspartate receptors (NMDAR) [94]. Neuroinflammation represents another detrimental factor in CKD-associated cognitive impairment since pro-inflammatory mediators (IL-1β, IL-6, TNF, and TGF-β) and immune cells exacerbate the cognitive decline in CKD patients [95]. The disruption of BBB during inflflammation allows the interaction of cytokines with the neurotrophic factor (BDNF) in the central nervous system (CNS). Recently, the kynurenine pathway was found to be strongly implicated in the context of brain disorders. Several neuroactive metabolites can be catabolized from tryptophan, including kynurenine, 3-hydroxykynurenine (3HKYN), picolinic acid, and quinolinic acid (QUIN) [96]. The condition of inflflammation can drive the upregulation of the kynurenine pathway, which lowers the synthesis capacity of serotonin from tryptophan. Elevated levels of pro-oxidative 3-HKYN in the CNS lead to increased neuronal apoptosis, due to its involvement in reactive oxygen species (ROS) formation, via superoxide and H2O2 generation [97,98]. QUIN is produced by microglia and penetrated by macrophages [99]. Excessive synthesis of QUIN leads to ROS formation via the excitation of the NMDA receptor [100,101]. This increases lipid peroxidation, nitric oxide levels, protein decomposition, and cytoskeletal destabilization [101]. 

2.2. Uremic Toxins in AKI 

Compared with CKD, interest in uremic toxins in AKI was in its infancy in 2009, and the existence of gut–kidney crosstalk in AKI was frequently debated. Today, it is wellestablished through a variety of experimental animal studies and clinical trials that acute renal damage negatively shapes the microbiota composition [25]. Importantly, the AKI– microbiota relationship is bidirectional: on the one hand, AKI can cause dysbiosis; on the other hand, the microbial shift influences the severity of the renal injury. To corroborate this theory, Jang et al. first demonstrated that after IRI induction, germ-free rodents displayed the worst creatinine and histological damage when compared to the controls [102]. Next, in the context of renal IRI, hypoxia itself can alter the ratio between aerobic and anaerobic populations [103]. More recently, Andrianova et al. demonstrated by an in vivo study that alterations in the microbiome composition occurred following IRI [26]. The connection between uremic toxins and an AKI occurrence has been strengthened by several studies [94]. Indoles and cresols are well-known toxins, and their circulating level has been connected with CVI and poor survival rates in end-stage renal disease [104,105]. Interestingly, the rising concentration of PBUTs in AKI is correlated with the severity, according to the RIFLE criteria [106]. In AKI rodents, the depletion of IS and PCS production alleviated renal damage [107]. As already discussed, the gut microbiota composition is of pivotal importance, not only for uremic toxins, as it can also influence immune responses. In addition, germ-free AKI rodents showed higher activation of NK cells and CD-8+ cells compared with non-sterile controls. More specifically, the “sterile immunity” showed the over-proliferation of the Th17 population. Th17 lymphocyte affects kidney dysfunction by releasing interleukin-17, which triggers a strong inflflammatory response in the renal environment [108]. Moreover, the gut barrier could also be injured during AKI as the concentrations of uremic toxins increase. Additionally, circulating endotoxin levels stimulate a systemic and inflflammatory effect. Against this scenario, one of the most recent interventional studies aiming to modulate AKI by uremic toxins/microbiome modulation was performed by Dong et al. [109]. The authors showed that antibiotic treatment was able to ameliorate the severity of acute renal damage.

In conclusion, while uremic toxins could represent the essential factors of AKI, there is only a limited amount of data available, as the majority of findings are derived from experimental animal models, which have severe limitations. Moreover, despite the advancements of dialysis technologies, the poor prognosis of AKI patients could be connected with microflora-related compounds, which exert multi-organ dysfunction, especially in kidneys, where its accumulation augments the pre-existent tubular and vascular injury resulting in the delay of renal recovery. Uremic toxins are the central actors in the multi-organ breakdown and they negatively affect renal recovery after acute injury. Thereby, research on the optimal renal replacement therapy cartridge that is able to clear specifific toxins or pharmacological therapies with the effect of bacteria metabolites strongly requires further exploration. 

2.3. Uremic Toxins in Kidney Transplantation 

Kidney transplantation represents one of the most effective treatments for patients with end-stage renal disease since it signifificantly improves the survival rate and ameliorates the quality of life. Moreover, the implementation of immunosuppressive therapies was shown to reduce acute rejection episodes and increase organ survival. On the other hand, it leads to an increase in complications related to the reduced immunocompetence of transplanted patients, such as infections, with the subsequent requirement of antimicrobial therapy. Kidney transplantations are often associated with infectious complications, which increase the mortality rate of recipients. Over the previous years, it has been demonstrated that kidney transplantation induces perturbations in the gastrointestinal flflora that could act as key players in transplant-associated infections [110]. Moreover, gut dysbiosis can influence the immune system of the recipient and recent evidence has highlighted a relationship between alterations in gastrointestinal communities and poor outcomes in renal transplant recipients [111]. Although it remains to be clarified whether this condition of gut dysbiosis is strictly related to kidney transplantation or is a common element for all patients with end-stage renal disease, it has been reported that substantial differences characterize the microbial pattern of recipients in comparison with healthy subjects [29,111]. In particular, it has been shown that the gut microbial populations of kidney transplant recipients are characterized by the prevalence of Firmicutes, whereas an increase in Proteobacteria was detected within fifteen days after transplantation [29]. Gut dysbiosis represents a risk factor for the optimal functioning of renal graft and can adversely affect the outcome of kidney transplantation through alterations of both the host’s immune system and inflflammatory cytokines production [111]. Indeed, gut dysbiosis can be associated with an impairment of the gastrointestinal barrier integrity, which can cause bacterial displacement into the systemic circulation that triggers the pro-inflammatory response. These conditions can cause graft inflflammation and, in the end, graft rejection via the autoreactive and alloreactive lymphocyte [111,112]. Microbial metabolism is essential in producing uraemic retention solutes, such as PBUTs. The toxicity of PBUTs has been well-documented in a plethora of pathophysiological mechanisms in kidney disease [113–115]. However, the impact of renal transplantation on these toxins has not been completely explored so far. Liabeuf et al. reported that the IS amount is markedly lower in transplanted recipients after 12 months than in non-transplanted CKD subjects with similarly estimated glomerular filtration rates [116]. Additionally, kidney transplantation strongly lowered the blood levels in a large amount of PBUTs, including phenols, and to a smaller degree, indoles [117]. Several other uremic compounds such as HA, PHS, IAA, and kynurenines, have also been described as signifificantly reduced in the first week after transplantation [118], although their reduction seems to not be correlated with the amelioration of neurocognitive functions. Taken together, these results demonstrated that kidney transplantation can affect uremic toxins levels since their accumulation largely declined after renal engraftment. Thus, it could be speculated that the transplant itself is able to influence the microbiome diversity, the leaky gut, and consequently, the adsorption of such toxins [117]. This condition could be dependent on immunosuppression and prophylactic antimicrobial therapy with the consequent reduction and/or alteration of PBUT production.

Microbiota-derived metabolites may have a negative impact on the outcomes of graft survival and function, since high levels of PCS and IS can cause the production of pro-fibrotic molecules and inflflammatory cytokines from renal tubular cells, resulting in increased tubulointerstitial fibrosis, cellular injury, and nephrotoxicity [58,119]. Korytowska et al. in 2021 demonstrated that salivary IS can be employed as a non-invasive diagnostic marker in order to recognize the loss/deterioration of the graft function (DoGF) more than a year following kidney transplantation [120]. The study was carried out on 92 kidney transplant recipients and, although it presents some limitations to the proposed model, this study assessed the role of IS as a potential predictor of DoGF and as a useful marker to prevent graft failure, and therefore, extending the survival and the functioning of the transplanted kidney [120]. Nevertheless, in the case of some uremic toxins, different and contrasting results, with respect to those previously described, have been reported. For example, levels of middle-molecular uremic toxin fibroblast growth factor 23 (FGF23) remain high even after kidney transplantation [121]; alternatively, plasma levels of the small molecule asymmetric dimethylarginine (ADMA) increase immediately after kidney transplantation, and its levels reduce over the weeks without this being reflected in an improvement of renal graft function [122]. The understanding of the biological mechanisms underlying these findings is still partial, partly because of the limited number of studies carried out so far on this topic. Thus, it is clear that these results highlight the need for further investigation of how uremic toxins may affect renal transplantation and vice versa, with the aim of implementing specifific therapeutic interventions to improve the outcome of kidney transplantations. 

Supportive Service:

Email:wallence.suen@wecistanche.com 

Whatsapp/Tel:+86 15292862950

Shop:

https://www.xjcistanche.com/cistanche-shop