Dietary fibers produce energy when metabolized, but not all dietary fibers are metabolized by digestive enzymes[16]. The gut microbiota of the large intestine contains enzymes that are able to metabolize these fibers and recover additional energy[17,18].

Undigested proteins are degraded into peptides, amino acids and other metabolites in the large intestine. Some of these metabolites are dangerous to the body and could cause diseases as colorectal cancers and kidney dysfunction[19]. The MEROPS database documented that the composition of the large intestine microbiota may contains different proteases responsible for inducing the production of different meta-bolites[20,21]. The gut microbiota also exerts important actions on lipids, bile salts and polyphenols.

The structural integrity of the intestinal epithelium is essential to avoid a dangerous increase in permeability. The maintenance of structural integrity is essential for the microbiota. In normal conditions, cytokines produced in the gut may back diffuse in small quantities passing through the gut barrier. The barrier function of the tight junction in dysbiosis condition, may be weakened by several endotoxins of some pathogens as Escherichia coli (E. coli), Clostridium difficile and Clostridium perfrigens. In this condition of dysbiosis, the diffusion of citokines such as interleukin 4, interleukin 1 beta, tubular necrosis factor alpha and interferon gamma is increased[22-26].

The gastrointestinal tract represents a bidirectional barrier between the gut microbiota and the gut immune system[27]. The barrier is composed of three layers: the mucus layer, the antimicrobial peptides (AMPs) and the IgA system.

Mucin glycoproteins secreted by goblet cells form a layer over the epithelia to restrict bacterial adhesion. This layer prevents the adherence of commensal microbiota to gut epithelial cells, limiting the bacterial adhesion[28]. A second layer is represented by AMPs secreted by epithelial cells. AMPs include α and β defensins secreted by the epithelium and mediated by cytosolic nucleotide-binding oligomerization domain-containing protein 2[29,30]. C-type lectins activate Toll-like receptors to limit bacterial penetration through the gut barrier[31].

The third layer is composed of the IgA system. Dendritic cells (DCs) located beneath the epithelial dome of Peyer’s patches take up bacteria, migrate to mesenteric lymph nodes and induce B cells to differentiate into IgA plasma cells that secrete IgA[32,33].

Renal transplant patients, in addition to receiving relevant immunosuppressive therapy in the first period after transplantation, receive several antibiotic treatments as a prophylactic measure to avoid infections.

All these drugs extensively modify the human microbiota, principally at the gut and urinary tract levels. Historically, since the initiation of renal transplantation, when very high doses of cyclosporine A were used, gingival overgrowth was observed as an important side effect. This change was related to modifications of the oral microbiota and generation of pathobionts[78].

In a pilot study, Lee et al[79], performed polymerase chain reaction in samples from 26 kidney transplant recipients and documented a change in the microbiota between the pre- and posttransplant periods. The results are shown in Table 3.

Firmicutes were the most abundant bacteria detected pre- and posttransplantation, but their quantity posttransplantation was lower than in healthy subjects[80]. The same study reported posttransplantation an increase in the abundance of Bacteroides that included infective pathogens such as E. coli and Klebsiella pneumoniae[81].

Overall, the study by Lee and colleagues documented a dysbiosis that was later confirmed by other studies. A recent review from Xiao et al[82] on microbiota modifications in response to solid organ transplantation highlighted an increase in the abundance of pathogenic Proteobacteria, which might represent the cause of infectious diseases occurring after transplantation.

These data were confirmed by a recent study by Swarte et al[83] that confirmed a reduction in the abundance of Firmicutes with variability among the species. The most significant reduction was observed for Streptococcus thermophilus and Blautiawexlerae.

Overall these authors observed an increase in the abundance of Proteobacteria (E. coli) and a decrease in the abundance of Actinobacteria posttransplantation. The increase in Proteobacteria has already been proposed as a marker of dysbiosis[84]. Additionally, the same study observed a reduction in SFCAs producing bacteria after transplantation. In particular, reductions in the abundance of Eubacterium rectale, Coprococcuscatus and Roseburia were observed. All these bacteria produce SCFAs[85] that exert beneficial effects on the kidney and increase the number of Tregs, reducing systemic inflammation[86,87]. The use of proton pump inhibitors, of MMF and aging were the prevalent determinants of this form of dysbiosis[88,89].

Another study[90] analyzed the gut microbiota in 142 kidney transplant recipients. The authors detected potential pathogens, such as Clostridium difficile and E. coli in 30% of patients. These pathogens were not associated with diarrhea, as expected.

A different study[91] observed that major changes in the microbioma occur in the first month after transplantation, with substantial differences among patients. The authors concluded that longitudinal analyses should be performed to provide more information.

In conclusion, dysbiosis after renal transplantation is related to an imbalance between the indigenous microbiota and the pathobionts. This imbalance is related principally to the need for immunosuppressant and prophylactic and therapeutic antimicrobial agents[92].

The metabolic and clinical consequences of dysbiosis are represented by a higher incidence of acute rejections, acute infections, interstitial fibrosis, posttransplant diarrhea, reduced production of protective agents such as SCFAs by the gut microbiota, and modification of immunosuppressant levels in the blood.

Several experimental studies conducted in animals have documented en effect of the gut microbiota on immune responses that lead to transplant rejection[93].

Few studies have been conducted in the humans on this topic.

In the aforementioned study by Lee et al[79], the differences in the fecal bacteria composition of patients with and without rejection are shown in Table 4.

In one recent study[84], the microbiota was evaluated pre- and posttransplant in 60 patients who received a renal transplant.

Samples from urine, oral swabs, rectal swabs and blood were evaluated for up to 6 mo after transplantation.

In the study, the most relevant changes in the microbiota principally verified in the first month after transplant, when the immunosuppressive treatment was heavier because of the induction therapy. Further modifications in the microbiota were verified in the first six months after transplantation. In urine samples and in oral swab samples, changes were verified principally in the phylum Proteobacteria. In the rectal swab samples, Firmicutes were the bacteria whose composition changed more frequently.

Significant changes in Leptotrichia, Neisseria and Actinobacteria were observed in five patients who experienced acute rejection. Four patients experienced late acute rejection and displayed significant changes in Anaerotruncus, Coprobacillus and Coprococcus.

The same authors of the study on acute rejection[94] documented that similar changes in the microbiota were also associated with a higher incidence of urinary tract infections.

In particular, in four patients with posttransplant infections, the abundance of the genus Anaerotruncus of Firmicutes was markedly decreased compared to the other patients.

Several factors may cooperate with dysbiosis to generate infections, as shown in Table 5. This higher incidence of both urinary and gastrointestinal infections was also reported in the aforementioned studies by Lee et al[79] and Chan et al[95].

In a recent study[96], a transplant patient with recurrent urinary infections recovered after fecal microbiota transplantation (FMT), which induced a marked decrease in the abundance of E. coli in the urinary microbiota.

In conclusion, according to these studies, some microbial species may exert a protective effect on the mucosal surface under normal conditions, and when the microbiota changes, pathobionts and aggressive phenotypes appear to induce renal dysfunction.

The hypothesis that urinary dysbiosis is principally responsible for the development of interstitial fibrosis of the graft was based on the findings that patients affected by interstitial fibrosis/tubular atrophy (IF/TA) had abnormalities in the urinary microbiota with appearance of pathobionts and, consequently, in the immune response. Two studies, conducted in humans[97,98] detected antibodies directed against E. coli LPS, a powerful activator of the immune system via TLR4 receptor in the biopsies of patients affected by IF/TA.

In a recent study of transplant patients, Modena et al[99] collected urine samples from 25 patients at two time points after kidney transplantation (approximately 1 mo and 6 mo after transplantation). All these patients demonstrated developed IF/TA in surveillance biopsies collected 6 mo after transplantation.

These samples were compared with 23 patients with normal surveillance biopsies and stable renal function at 6 mo after transplantation.

At six months after transplantation, patients affected by IF/TA displayed decreased abundances in the Lactobacillus and Streptococcus genera along with an increase in the abundance of no dominant species.

The authors concluded that the urinary microbiota, modified posttransplantation, may contribute to IF/TA development by altering the host immune response.

IF/TA is associated with a loss of the indigenous dominant resident urinary microbiota and an increase in the abundance of pathobionts or nonresident, pathogenic bacteria.

The phenomenon of IF/TA may be mediated by myofibroblasts, as has already been documented in the gut, where gut dysbiosis potentially leads to intestinal fibrosis[100]. Myofibroblasts may be derived from transdifferentiation processes such as the epithelial to mesenchymal transition or endothelial to mesenchymal transition. These processes may be induced and aggravated by modifications in the indigenous microbiota.

In conclusion, myofibroblasts may play a relevant role in inducing IF/TA either at the gut or renal level, and the indigenous microbiota might have regulatory and protective functions under normal conditions.

Diarrhea represents a severe complication after kidney transplantation, affecting approximately 20% of patients[101], and it represents an important cause of graft loss and death[102]. However, its etiology is still being discussed, and a clear diagnosis not available for approximately 85% of transplanted patients affected by diarrhea. With the exception of the few cases that are ascribed to a specific infection and the presence of pathogens, the diarrhea etiology is often ascribed to the use of immuno-suppressants, in particular MMF. However, a reduction in the MMF dose is dangerous and may lead to an increased risk of allograft rejection[103].

In the pilot study by Lee et al[79], the authors observed a reduction in the commensal indigenous microbiota, such as Ruminococcus, Dorea and Coprococcus, in 26 renal transplant patients affected by diarrhea. In addition, they did not detect pathogens such as Clostridium difficile or norovirus in fecal specimens. These findings prompted the hypothesis that in the majority of patients, gut dysbiosis rather than the presence of pathogens may represent an important cause of posttransplant diarrhea. In a recent study by Lee et al[104], fecal specimens from 25 patients presenting diarrhea in the first three months after transplantation were compared with 46 patients who did not develop diarrhea. In the diarrhea group, the abundance of the genera Eubacterium, Anaerostipes, Coprococcus, Romboutsia, Ruminococcus, Dorea, and Faecalibacterium were significantly decreased, while the abundance of the genera Lachnoclostridium, Escherichia and Enterococcus were significantly increased. Table 6 provides a detailed description of the data. Many of the bacteria that were present at lower abundance in the diarrhea group belong to the Lachnospiraceae and Ruminococcaceae families[105] and contribute to metabolic functions essential for gut health[106]. Utilizing the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States Analysis[107], 9 metabolism-related pathways were decreased in the diarrhea group. The decrease in the abundance of these indigenous microbiota bacteria in the subjects affected by diarrhea contributes to the development of an abnormal metabolic status, which might lead to diarrhea.

Interestingly, a similar decrease in the abundance of protective bacteria was also observed in nontransplant patients affected by diarrhea[108].

Notably, the specimens from transplanted patients with diarrhea were negative for known bacterial and protozoan pathogens that cause diarrhea.

Finally, two transplanted patients affected by persistent diarrhea underwent FMT from allogeneic donors. Diarrhea resolved in the first month after FMT, and the abundances of 13 protective bacteria taxa increased with a simultaneous decrease in the abundances of the 3 identified pathobionts or pathogenic bacterial taxa[96,108].

SCFAs are produced in the gut by the indigenous microbiota and have a trophyc action on the gut epithelium. In addition, these substances exert an anti-inflammatory effect on the whole body and regulate immune cells.

Ninety-five percent of SFCAs are represented by acetic acid, propionic acid, butyric acid and valeric acid, all of which are derived from saccharolytic fermentation. Under normal conditions with a microbiota producing normal quantities of SCFAs, several beneficial effects have been documented after transplantation both in animals and in humans.

In humans, SCFAs increase the expression of antimicrobial peptides secreted to the external surface by epithelial cells[109]. Studies in vitro or in animals documented that SCFAs modulate the production of immune mediators, including IL-18 and other cytokines and chemokines[110], regulate the differentiation, recruitment and activation of immune cells, including neutrophils[111], DCs, macrophages[112] and T lympho-cytes[113].

Finally, Wu et al[114] documented, in a murine kidney transplantation model, that SCFAs are able to induce donor-specific tolerance by inducing the production of T regulatory cells[114].

Andrade-Oliveira et al[115] evaluated the effect of SFCAs on a mouse model of ischemia-reperfusion[115].

In the animals, the treatment with SCFAs improved renal function after ischemia-reperfusion injury, reduced the apoptosis, inhibited NFkB activation and nitric oxide production and reactive oxygen species production. All these actions of SCFAs are summarized in Table 7.

In mice, SCFAs decrease the activation of bone marrow-derived DCs and inhibit their function as antigen presenting cells[115].

In conclusion, the authors showed that SFCA supplementation reduces inflammation in their model and improves ischemia-reperfusion injury.

To our knowledge, few studies have been conducted in humans. A recent study by Lee et al[116] studied 168 kidney transplant recipients and divided the patients according to whether they had higher levels of butyrate-producing bacteria (BPG) or low levels of BPG. The posttransplant administration of antibiotics was associated with a decrease in BPG levels. These patients have a higher incidence of respiratory tract infections.

For the first time, the clinically beneficial effects of higher butyrate levels and posttransplant-induced dysbiosis were documented in transplanted men and may induce higher infection rates.

Similarly, in another study on transplanted humans, 51 renal transplanted recipients have been followed up to 12 mo after transplantation to study the serum levels of uremic toxins as p cresyl sulfate, p cresyl glucoronide, indoxyl sulfate, TMAO and phenylacetylglutamine. The results were compared with CKD control patients with similar renal function. The study documented that after transplantation the colonic microbiota derived uremic retention solutes decreases. As the urinary excretion is lower in transplanted patients, this fact suggests an independent effect after transplantation on intestinal uptake and a different colonic microbial metabolism and absorption[117].

The aforementioned hypothesis that gut microbioma metabolites such as SCFAs could induce donor-specific tolerance through the induction of regulatory T cell differentiations[114], introduces the chapter on the relationship between microbiota and tolerance.

This relationship is well known in the development of immune tolerance in children. Indeed, in the first 1000 d of life, the early exposure of food allergens to indigenous intestinal microbiota induces tolerance through activation of Tregs and subsequent production of TGFβ and IL-10[118].

In a recent study, Colas et al[119] examined the urinary microbiota of 86 renal transplant patients. Patients were divided into 3 groups: Normally immuno-suppressed with stable renal function, minimally immunosuppressed, and spontaneously tolerant patients. Differences in microbiota profiles were observed, and a unique and specific urinary microbiota was detected in patients with spontaneous tolerance characterized by a clear Proteobacteria profile. The profile was different in patients stratified according to gender (higher in males) and inversely correlated with the quantity of immunosuppressive drugs.

The Proteobacteria detected in tolerant subjects included Janthinobacterium, Clostridia and Firmicutes. Janthinobacterium is known to produce an indole-derived peptide with antiproliferative and anti-inflammatory activities[120,121]. Clostridia exert an anti-inflammatory effect by producing SCFAs[122]. Firmicutes produce indole derivatives[123] and polyphosphate[124] with anti-inflammatory activities.

In conclusion, the indigenous microbiota may favor the induction of tolerance, but the use of immunosuppressants modifying the microbiota may represent an obstacle to the development of the tolerance state.

Bilateral actions between the microbiota and immunosuppressive drugs have been identified. On one hand, the microbiota may modify the absorption and the meta-bolism of immunosuppressants; on the other hand, immunosuppressants may modify the indigenous microbiota.

The vast majority of studies on this issue have been conducted on calcineurine inhibitors.

Several studies have extensively documented that factors such as age, gender, race and CYP3A5 polymorphisms influence the absorption and metabolism of immuno-suppressants and account for interindividual variability such that the individual dosing is not the same for all patients.

Recently, the gut indigenous microbiota or the pathobionts have been suspected to exert a powerful effect, justifying the different metabolism from one patient to another and in the same subject.

The assumption of other drugs, such as antibiotics, modifying the indigenous microbiota may account for this variability[125-128].

Lee et al[129] examined the microbiota in the fecal specimens of 19 patients who received a kidney transplant and were on tacrolimus (TAC) as the principal immuno-suppressive therapy. All patients received the same prophylactic antibiotic therapy to avoid biases. Patients were divided into two groups according to the need to receive increasing TAC doses (Dose Escalation Group) or not (Dose Stable Group). By examining the microbiota, the authors found a significantly higher level of Faecalibacterium prausnitzii in patients from the Dose Escalation Group than in patients from the Dose Stable Group. In addition, Faecalibacterium prausnitzii was the most significant factor justifying the need to increase the TAC dose. Even if a large quantity of TAC is absorbed by the small intestine, it may also be absorbed in the colon[130]. Although the Lee’s study is a pilot one, the results raise the question of the relevance of microbiota and of Faecalibacterium prausnitzii, particularly on TAC trough levels, which are also important due to the narrow therapeutic index of TAC.

In a different study, Guo et al[131] incubated Faecalibacterium prausnitzii cells in vitro with TAC. The authors detected a compound named M1 that is a cheto-produced metabolite of TAC with a less powerful immunosuppressant. The authors measured a large quantity of M1 in the stool samples of patients with a larger quantity of Faecalibacterium prausnitzii in the stool.

In addition, the same study documented that other bacteria, such as Clostridia and Bacteroidales, are able to convert TAC into M1 metabolites. The authors conclude that several commensal microbiota may metabolize TAC in the gut to less powerful compounds, explaining the differences in TAC exposure in transplant recipients.

On one hand, the microbiota may alter the metabolism of immunosuppressants; on the other hand, immunosuppressants may alter the gut indigenous microbiota. The study by Gibson et al[132] reviewed this topic extensively. Unfortunately the vast majority of studies have been conducted on calcineurine inhibitors and very few have examined renal transplantation.

The studies by Zhang et al[133] and by Lee et al[129] documented the effect of TAC on the gut microbiota in renal transplant recipients. Other studies[134] analyzed the same phenomenon in liver transplant recipients. Zaza et al[135] examined the microbiota in patients receiving TAC + MMF or everolimus + MMF, but they did not observe any difference.

In the pilot study by Lee et al [79], patients with early corticosteroid withdrawal had fewer Clostridiales and Erysipelotrichaeles in the microbiota, but the difference was not statistically significant.

Finally, a recent study[136] documenting that encapsulated cyclosporine A does not change the composition of the human indigenous microbiota is worth mentioning.