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

3. FMT

In recent years, FMT has been acknowledged as an impactful strategy to manipulate the gut microbiome [10]. The first report of an FMT application derives from ancient Chinese medicine for treating severe diarrhea, while its first clinical employment was documented in the 20th century for the management of pseudomembranous colitis [123]. Currently, FMT represents the first-line strategy for resolving recurrent Clostridium difficult infection (CDI). Similarly to solid organ transplantation, the conceptualization behind the FMT procedure is based on the replacement of a dysbiotic flflora with a healthy microbiome. In clinical practice, FMT concerns the inoculation of fecal material from a healthy subject in the diseased gut of a recipient patient. Hence, the administration of healthy microbiomes purposes is to decolonize and repopulate the intestinal environment with a stable microbial community [124]. Based on this evidence, fecal transplantation could represent a novel and promising treatment for different dysbiosis-associated disorders. 

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FMT Procedure 

Similarly to solid organ transplantation, the fecal transfer procedure demands stool collection from healthy donors [125]. Importantly, the workflow concerning donor eligibility is based on rigorous hematological and stool analysis to avoid the transfer of transmittable diseases such as HIV, Hepatitis, syphilis, C. dificile, protozoa, and helminths [126]. Additionally, potential donors undergo a medical interview to check for any history of infectious diseases or gastrointestinal and metabolic disorders. Of note, following the donation, the collected stool can be either directly transplanted into the recipient or frozen for later use. As stated in the European guidelines for FMT application, different routes can be employed for fecal transfer [126]. Depending on the GI tract site, the healthy microbiota can be conducted by the lower (colonoscopy or rectal enema) or upper (nasogastric or nasojejunal) route [127]. Widely employed, the colonoscopy demonstrated a success rate of 90% for resolving rCDI. On the other hand, fecal administration by enema is employed only when colonoscopy is inaccessible. Finally, due to the high risks of tissue perforation, the upper GI tract route is hardly ever used [127]. Interestingly, in view of improving invasiveness and patient compliance, a novel capsule-mediated FMT approach was developed in recent years for treating recurrent C. diffificile infections [128,129]. Notably, the so-called oral FMT is based on the encapsulation of lyophilized microbiota into a 0/00 gastro-resistant capsule, which excellently vehiculates the healthy-associated flflora into the recipient’s intestine [130]. Clinical trials assessed the effectiveness of oral-fecal transplants for resolving rCDI, with a success rate of 90% associated with lower unfavorable events [131–133]. Moreover as reported by a few meta-analyses, the capsulo-mediated FMT strongly improved the microbiota tolerability compared with the traditional routes of delivery [131]. 

4. FMT and Kidney Disease 

Although FMT exerts a primary function in the modulation of the gastrointestinal flflora, its beneficial effects extend to a systemic level in regulating inflflammation, oxidative stress, and metabolic disorders. Based on this evidence, in recent years, the application of FMT was implemented in many dysbiosis-associated diseases but also in extra-intestinal disorders, such as metabolic syndrome and neurodegenerative diseases [10]. Recently, mounting data have highlighted the detrimental effects of dysbiosis-associated uremic toxicity in kidney disease. In these settings, a broad number of strategies focused on gut microbiota modulation, such as biotic supplementation, have demonstrated positive results in lowering the circulating levels of PBUTs. On the other hand, a substantial amount of data produced contradictory findings [134]. Furthermore, despite their time-limited effect, the strategies based on pre-, pro-, and syn-biotics provide early evidence that the whole microbiota repopulation may exert a long-term effect. Thus, the FMT could represent an auspicious strategy in preserving renal injury from the deleterious effects of gut-derived PBUTs (Figure 2). Research is in its infancy and prior to the application of FMT on patients, researchers are focusing on in vivo studies.

Figure 2. The potential effect of FMT on kidney disease. Abbreviations: CKD, chronic kidney disease; AKI, acute kidney injury; PBUTs: protein-bound uremic toxins; an CVD, cardiovascular disease.

Originally, the fecal transplantation procedure was applied to elucidate the role of intestinal dysbiosis in kidney diseases (Table 2). Uchiyama et al. elegantly demonstrated that the transplantation of CKD-derived microbiomes reproduced the uremic phenotype in healthy, germ-free mice. After the fecal transplantation, the recipient mice showed a profound dysbiosis associated with high levels of circulating uremic toxins, including PHS, IS, and HA. Moreover, the overproduction of such toxins induced sarcopenia, insulin resistance, and intestinal permeability in healthy recipient mice [85]. Conforming with these data, Li and co-workers demonstrated that the fecal contents of streptozotocin-treated DN mice induced a microbiome alteration associated with high levels of TMAO and LPS in antibiotic-treated recipients. The recipient mice displayed a high grade of renal damage correlated with elevated levels of bacterial toxins when transplanted with the contents of DN mice with severe proteinuria (≥300 mg/24 h). On the other hand, low-grade injury associated with a lower concentration of TMAO and LPS was induced when recipients were transplanted with fecal microbiota from mice with moderate proteinuria (<300 mg/24 h) [24]. Notably, a disease-associated microbial pattern was also detected between the two groups of DN mice, indicating that the dissimilarity in microbiome patterns can influence the different outcomes of DN.

Comparably, Wang and collaborators evaluated that the fecal microbiota of individuals with advanced CKD worsened renal injury when transferred in CKD rodents. The authors show that the dominance of several bacteria, including Eggarthella lenta and Fusobacterium nucleatum, was closely related to the increase in many PBUTs, such as HA, PHS, PCS, and IS. Furthermore, the transplantation of healthy donor stool in diseased rodents improved the circulating level of uremic solutes, such as creatinine, urea, and PBUTs [14]. Yang and colleagues further explored the relationship between microbiota and AKI by using the microbiota transplantation procedure. Specifically, they observed that the post-AKI microbiome strongly influenced the severity of the ischemia/reperfusion injury when administered in germ-free mice. Altogether, these findings corroborated the causal link between the detrimental microbiome and kidney disease [27].

In the last two years, few in vivo studies have explored the implementation of FMT in renal disease, supporting the therapeutic contributions of microbiota replacement in uremia (Table 2). These attempts aimed to correct PBUT levels, inflflammation, and metabolic dysfunction. Liu et al. explored the effects of FMT on 1/2 nephrectomy-induced CKD rats using fecal contents from sham-operated donors. Of note, before the transplantation, the CKD group was sterilized by an antibiotic cocktail in order to deplete the resident flflora. Fecal administration restored the gut eubiosis of CKD rodents by affecting the enrichment of Lactobacillaceae (L. johnsonii and L. intestinal). Interestingly, the FMT strongly lowered the blood levels of a large number of PBUTs, including IS, PCS, PHS, TMAO, and Phenylacetyl glycine. In addition, the sham-related microbiome improved renal injuries and the inflflammatory status when transplanted in CKD animals [135]. A similar study was performed by Barba et colleagues using adenine-induced CKD mice. In this study, it was mainly observed that the transfer of healthy mice microbiota in CKD recipients was able to correct the uremic dysbiosis, resulting in the reduction of plasma tyrosine-derived PBUTs. Moreover, the improvement of glucose tolerance and IR was further observed after three incidences of FMT administrations. These findings suggest that the reduction of PCS and PCG was effective in ameliorating CKD-related metabolic complications [136].

Promising results have also been found in models of DN rodents treated with healthy microbiota. In this context, the investigation by Hu et al. evidenced that the repopulation of dysbiotic microbiota from healthy donor GI-flora improved the tubular lesion by ameliorating the apoptosis of TECs in DN rats. Furthermore, FMT enriched the amount of acetate-producing Prevotellaceae, Ruminococcaceae, and Lactobacillaceae families, leading to the correction of cholesterol homeostasis. Finally, the decrease in serum IL-6 suggested an improvement in systemic inflflammation [137]. In a similar fashion, Lu et al. demonstrated that DN rats that were transplanted with healthy flflora normalized the altered insulin pathway in podocytes by reducing the circulating levels of microbiota-derived acetate. Acetate reduction was also associated with GPR43 downregulation in podocytes. Curiously, FMT was also able to recover both the morphology and number of podocytes [138]. More recently, an in vivo study found that the replacement of dysbiotic microbiota with healthy communities signifificantly ameliorated the metabolic complications associated with diabetic nephropathy in leptin-deficient BTBR mice. The fecal bacteriotherapy downregulated the expression of TNF-α in enterocytes and reestablished gut permeability [139]. Despite the lack of PUBTs analysis in these studies, it can be speculated that the recovery of metabolic complications may be associated with a decrease in several PBUTs, including PCS, PCG, and IS, due to their involvement in metabolic dysfunction. The beneficial effects of fecal transplantation were further explored in IgA nephropathy, where dysbiosis is involved in driving its pathogenesis by influencing the host immune response [140]. In this scenario, Lauriero and collaborators demonstrated that the graft of healthy human microbiota mitigated inflflammation and improved kidney injury and glucose tolerance in a mice model of IgAN. Additionally, when transplanted, the healthy microflora reversed the accumulation of dysbiosis-related cresols and indoles and intensified the gut production of several SCFAs [140].

On the other hand, despite the evidence concerning the beneficial effects of fecal bacteriotherapy on CKD, there is a limited number of data that support the implementation of FMT in AKI. Emal et al. detected that the depletion of the intestinal flflora via antibiotic treatment was able to attenuate kidney injury in the I/R model of AKI. Furthermore, when compared with the antibiotic-untreated group, the sterilized mice exhibited an improvement in renal dysfunction related to the reduction of several pro-inflammatory factors (i.e., TNF, IL-6, MCP-1, and MIP). Additionally, the gavage of fecal contents from untreated donors restored the granulocyte influx and the expression of chemokine receptors in resident macrophages. However, they further reported that FMT did not influence the renal function of AKI recipients [141]. In contrast to this observation, the study by Nakade et al. indicated that the treatment of healthy microbiota for twelve weeks signifificantly alleviated acute renal injury and intestinal dysfunction [142].

Finally, within the field of kidney disease, the clinical use of FMT was documented only in two different case reports regarding CKD patients with intestinal discomfort (Table 2). In the investigation by Zhao et al., the 20-week fecal transplantation from healthy donors to IgAN recipients signifificantly lowered the proteinuria and alleviated intestinal distress by improving α and β diversity [143]. Consistent with this finding, Zhou et colleagues reported that FMT decreased the levels of small water-soluble toxins and reversed intestinal edema and diarrhea in a patient suffering from membranous nephropathy [144]. The oral FMT treatment, using encapsulated fecal microbiota derived from a healthy gut, was used to treat a patient with Focal Segmental Glomerulosclerosis. The administration of twenty capsules once a week for a total of three weeks was able to refurbish the lipidic profile and the relative proportion of various microbial families, including Prevotellaceae and Bacteroidaceae. Moreover, the authors showed a reduction in several proinflammatory mediators [145]. 

Traditionally, fecal transplantation is performed using feces collected by untreated healthy donors (Table 2). However, a small group of studies showed that the stimulation of donor microbiota with biotic supplements before transplantation could represent an encouraging strategy in augmenting the effect of FMT. For example, the fecal contents of resveratrol-treated rodents demolished the elevated ratio of PBUTs-producing bacteria, including Firmicutes, Tenericutes, Deferribacteres, and Enterococci, when implanted in CKD recipients. The manipulated microbiota was able to reverse the gut injury and inflflammation in diseased rodents [146]. In another study, healthy mice treated with the probiotic Astragalus membranaceus were used as donors of fecal microbiota. When transplanted, the A. membranaceous-supplemented microbiota strongly improved intestinal permeability and increased the proportion of Akkermansia and Lactobacillus in CKD mice. Moreover, the reinforced microbiome alleviated glomerular dysfunction and tubular fibrosis [147]. Despite the fact that the levels of PBUTs were not reported, it can be hypothesized that the demolition of gut dysbiosis together with the recovery of the intestinal barrier may have limited their blood accumulation. Finally, Zheng et al. developed an engineered microbiota by micro-encapsulating a synthetic cocktail of different bacteria. Interestingly, they reported that its administration markedly lowered several small solutes, including urea and creatinine, in both mice and swine models of AKI and CKD [148]. Altogether, these data lay the foundation for promising “reinforced FMT” approaches which could represent a new frontier in personalized therapies to modulate the gut microbiome

Table 2. Summary of in vivo studies based on the application of fecal transplantation in kidney disease. 

Table 2. Cont

Abbreviations: IR, insulin resistance; CKD, chronic kidney disease; AKI, acute kidney injury; IS, Indoxyl sulfate; PHS, phenyl sulfate; HA, hippuric acid; IL; DN, diabetic nephropathy; DKD, diabetic kidney disease; STZ, streptozotocin; TMAO, Trimethylamine N-Oxide, lipopolysaccharide; SCFAs, short chain fatty acids; ESRD, end-stage renal disease; PAG, phenylacetylglutamine; PCS, P-cresyl sulfate; TGF-β1, transforming grow factor β1; IRI, ischemia-reperfusion injury; TNF-α; IFN-γ, interferon-γ; PCG, p-cresyl glucuronide; BTBRob/ob mice, black and tan brachyurous obese mutant mice; IgAN, IgA nephropathy; BAFF, B cells’ activating factor; GI, gastrointestinal; MN, membranous nephropathy; and NA, not assessed.

Effects of FMT on Kidney Transplantation 

The relevant data support the interconnection between gut microbial profiles and the outcomes in renal transplantation [149]. Great amounts of research have highlighted that microflora alteration is induced by the elevated administration of immunosuppressants and antimicrobial-prophylactic drugs to prevent graft failure and infections. The detrimental microflora is usually marked by the alteration of an alpha/beta diversity correlated with high amounts of Proteobacteria [28,29,150,151]. The microbiota profile alters the host’s immune response through the activation of numerous pivotal factors (i.e., Myd-88 or TLR-9) and affects the over-proliferation of many lymphocyte populations, worsening the functional outcome of the graft [112,152]. Another important aspect is related to the link existing between dysbiosis and infectious complications [111]. Magruder et al. demonstrated that dysbiosis could be connected with the occurrence of a urinary tract infection (UTI) in transplanted patients [153]. As mentioned previously, the evaluation of metabolic profiles has been considered a hallmark of bacterial richness and diversity [149]. Poesen and colleagues observed that PBUTs derived from kidney-transplanted patients were reduced in patients with CKD [117]. This finding was further sustained by the evidence that the changes in the severity of gut bacteria were achieved immediately after transplantation and ameliorated after twelve months after transplantation [154]. The accumulated knowledge supports the notion that diet and biotic supplements could represent therapeutic approaches in modulating the GI microflora and ameliorating the outcome of the graft. Until now, there have been limited data concerning the efficiency of fecal transplantation in preventing the accumulation of PBUTs. Stripling et al. reported the effectiveness of fecal bacteriotherapy in a kidney recipient with rCDI [155]. In another study, the authors demonstrated that FMT led to the resolution of recurrent CDI in eight patients who were non-transplant [156]. 

5. FMT: A Mixed Blessing for Kidney Disease

Intestinal dysbiosis is closely linked to kidney disease. Moreover, the high accumulation of a large amount of microbiota-derived PBUTs is strongly associated with metabolic dysfunction, inflflammation, and CVI [85,87,157]. In recent years, the use of oral supplements together with different dialysis settings has been designed to counteract the accumulation of uremic solutes. However, the effectiveness of these strategies remains controversial [134]. Beyond the lowering of uremic toxins, strong evidence supports the pivotal impact of FMT in restoring uremia-related complications by ameliorating the gut uremic milieu. Moreover, in many pathologic conditions, fecal transplantation was related to the improvement of bacterial richness [158–160]. In line with these data, the application of FMT in kidney disease indicates that gut recolonization with a healthy microbiome is able to re-establish a stable microbial community in the gut of the recipient [135,136,140]. Moreover, as a result of FMT, a strong reduction of many PBUTs was observed, especially those derived from the proteolytic metabolism of tyrosine, tryptophan, and choline [135,136,140]. Based on this observation, it can be assumed that gut repopulation could alleviate the detrimental effect of uremic toxins.

In recent years, a large number of data demonstrated that fecal transplantation is able to induce recipient homeostasis by modulating inflflammation and metabolic dysfunction [126,161]. In CKD, the retention of PBUTs is associated with a wide range of metabolic disorders including insulin resistance and dyslipidemia [85]. Interestingly, the data presented in this study show that fecal transplantation may represent a novel approach to restoring glucose intolerance, insulin signaling, and triglyceride and cholesterol levels. Moreover, FMT ameliorated the systemic inflflammation in CKD and AKI recipients, since a reduction of many pro-inflammatory mediators (e.g., IFN-γ, IL-6, TNF-α, and IL-1β) was observed after the treatment [27,135,145,146]. Comparable results were observed in many other diseases in which fecal transplantation alleviated inflflammation and the immune response [161–163]. Hence, the loss of pathogenic flflora followed by the reduction of several microbial toxins such as LPS and PBUTs may clarify, at least in part, the modulation of the local immune system leading to the downregulation of inflflammation.

Finally, several data show that gut dysbiosis represents a pivotal mechanism in modulating the renin-angiotensin-aldosterone system (RAAS) in kidney disease [164]. In detail, few pieces of evidence suggest that the RAAS activation could be, at least in part, linked to the microbiota-derived PBUTs. Consistently with this statement, experimental studies demonstrated that dysbiosis-related uremic toxins are involved in promoting kidney injury by activating the intrarenal angiotensin II [165,166]. Based on this evidence, the improvement of RAAS after FMT may represent the missing link involved in the improvement of renal injury [77,135,139,142]. 

Despite its growing experimentation in animal models of kidney disease, there is no scientific guideline for the best approach. On the one hand, various procedures in fecal contents processing, storage, and delivery, are not well standardized. Additionally, the frequency and duration of FMT administration can profoundly affect FMT results. Finally, the use of antibiotics before transplantation to deplete the dysbiotic flflora remains controversial. Of note, particular focus should be placed on the experimental model of kidney disease including adenine-treated mice, 5/6 nephrectomy

6. Conclusions

In recent years, FMT has been accepted as an impactful strategy to manipulate the gut microbiome. Currently, FMT represents a first-line therapy for resolving rCDI. C. difficult fecal transplantation is demonstrated to be an auspicious strategy in treating a great number of disorders, including kidney disease. In this scenario, several in vivo studies shed light on FMT applications in the context of CKD as a novel strategy for regulating PUBUT levels. Although it produced promising results, the method’s standardization including the delivery route, fecal amount, and the time of administration, should be wellestablished before human application. From a practical standpoint, for the treatment of several dysbiosis-associated chronic diseases, the FMT probably needs to be administered over a long period. For instance, in CKD, where dysbiosis represents a result of chronic renal injury, a single FMT administration could be ineffective over the long term. Based on this observation, it can be hypothesized that capsule-mediated FMT, due to its safety and low invasiveness, could represent the future direction to achieve “chronic fecal microbiota repopulation” therapy. Finally, the next generation of approaches could be represented by “reinforced FMT” therapies to personalize the microbiome interventions. 

Author Contributions: Conceptualization, G.C., and L.G.; writing—original draft preparation, G.C., A.S., R.F., M.F., M.T.C., P.P., A.D., R.P. and L.G. writing—review and editing, G.C.; supervision P.P. and L.G. All authors have read and agreed to the published version of the manuscript. 

Funding: This research received no external funding. 

Institutional Review Board Statement: Not applicable. 

Informed Consent Statement: Not applicable.

Data Availability Statement: Not applicable. 

Acknowledgments: L.G. acknowledges the support from the program PON “Ricerca e Innovazione” 2014–2020 and FSC, Asse 2, Azione II, 2 Cluster-Ricerca Industriale e Sviluppo Sperimentale-Area di Specializzazione “Salute”-Project name: BIOMIS-Costituzione della Biobanca del Microbiota intestinale e Salivare Umano: Dalla Disbiosi alla Simbiosi-Grant number ARS01_01220 and from SNIPS “Sottoprodotti Naturali da matrici vegetal valorizzati per Preparazioni dalle Elevate proprietà Salutistiche” project, Mis. 16, PSR Puglia2014/2020. 

Conflicts of Interest: The authors declare no conflict of interest

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