Gut Microbiota In Chronic Kidney Disease

Microorganisms inhabiting the human organism are currently referred to as a microbiota (formerly – micro-flora) and the set of their genomes is called a micro-biome. It includes not only bacteria but also fungi, viruses and archaea. A microbiota colonizes, among others, the skin, the upper respiratory tract, the auditory meatus, the genital tract and the entire human gastrointestinal testinal tract. In the human gastrointestinal tract, there are 10¹⁴ microorganisms, which is equal to 10-fold number of eukaryotic cells in which microbial cells constitute 1.5–2 kg of human body weight. The greatest number and diversity of microorganisms occur in the large intestine [55]. Scientific research conducted in the last few years has shown that microorganisms have the ability to communicate (cross-talk) between each other and the host cells. The diverse network of connections and transmission of signals creates a complicated ecosystem which is also the basis for maintaining the state of the host’s metabolic and immune homeostasis.

The first contact with microorganisms takes place already in foetal life. The presence of the DNA of bacteria whose contact with the foetus stimulates its immune system [64] has been confirmed in the placenta. In the postnatal period, the composition of the microbiota is affected by: the mode of delivery (caesarean section vs. natural birth), diet, hygiene or the necessity of taking antibiotics. The full development of human intestinal microbiota is observed in adulthood [34].

The composition of intestinal microbiota varies between individuals, and homogeneous microbiota exists within small populations. The possibility of carrying out thorough genetic tests allowed for isolating and accurate characterisation of the so-called enterotypes, or permanent systems of intestinal microbiomes. Enterotypes are characteristic for a given host and differ in the type of dominant bacteria. Three enterotypes have been distinguished in humans: Bacteroides, Prevotella and Ruminococcus [3]. These include the following bacteria: Bacteroides, Prevotella, Ruminococcus, Faecalibacterium, Eubacterium, Lactobacillus, Clostridium, Dorea [44]. A given enterotype affects the composition of the entire intestinal flora by inhibiting or promoting the growth of other bacteria; it also specializes in the synthesis of specific compounds [54]. Bacteroides produces biotin, Prevotella – thiamine, and Ruminococcus specializes in the synthesis of heme [48]. Genomic diversity amongst enterotypes leads to variability in the functioning of metabolic pathways, including differences in obtaining energy from food [54]. It resulted in the creation of a hypothesis whereby knowledge of the enterotype of a given host will allow the selection of a personalized diet, as well as medications [10].

In healthy condition, the bacteria of intestinal flora remain in symbiosis with the host, which means the phenomenon of two different species coexisting, beneficial to both sides. In the intestine, there are conditions favouring growth of anaerobic bacteria which convert the non-absorbed carbohydrates supplied with food through the process of fermentation. The energy-yielding nutrients produced therefrom are later used by the host’s cells. Short-chain fatty acids (SCFA) are the most important of these products. The butyrate formed in the course of their biochemical transformation is the main energy substrate for enterocytes. Its appropriate concentration determines the correct metabolism of these cells, and thus the maintenance of the integrity of the intestinal barrier [11]. The intestinal barrier through the presence of tight junctions and the transmembrane transport system enables complex communication between the host and the intestinal microbiota, mainly in the immune system. Proteins that build tight junctions are primarily: the peripheral membrane protein ZO-1(zonula occludens – 1 ZO1) and the proteins connecting the membranes of neighbouring cells (transmembrane proteins) – occludin and claudin [58].

A change in the environment in which a microbiota exists: the presence of toxins, diet, antibiotic therapy, stress or medications taken cause the dominant bacteria and the compounds synthesized by them to change, which leads to damaging the intestinal barrier. This is the case, among others, in chronic kidney disease [2].

Chronic kidney disease (CKD) is a condition with a complex aetiology. The most common causes of this disease are lifestyle diseases: diabetes and hypertension. Its development is also promoted by immunological and genetic diseases as well as congenital malformations. The severity of this disease is assessed, among others, by the decrease in glomerular filtration rate (GFR) and is divided into five stages. In stage I of CKD, glomerular filtration is not yet impaired but morphological changes in the kidneys may appear, e.g. urolithiasis, cysts. In stage II, GFR falls below 89 ml/min/1.73 m², however it is higher than 60 ml/min/1.73 m². Stage III is characterized by a further reduction in glomerular filtration and is between 59 and 30 ml/min/1.73 m². Stage IV is GFR between 29–15 ml/min/1.73 m², and the last stage is V, in which GFR falls below 15 ml/min/1.73 m² [7].

The essence of CKD is not only the retention of uremic toxins, impaired production of erythropoietin, calcium-phosphate management disorders and lack of the active form of vitamin D, but also persistent inflammation. Its source is, among others, impaired integrity of the intestinal barrier, which occurs in uraemia [48]. In this situation, the pro-inflammatory molecules and bacterial toxins leak from the gastrointestinal tract into the bloodstream of the host. What is most often listed in this case is bacterial lipopolysaccharides (LPS), integral components of the outer membrane of Gram-negative bacteria and cyanobacteria, which play a key role in bacteria life processes, but also constitute their main pathogenic factor [5]. The presence of the fragments and metabolites of bacteria outside the intestinal lumen induces and sustains an inflammatory response. This pathomechanism is also superimposed by: a decreased clearance of cytokine, intravenous iron administration in patients undergoing dialysis and finally the haemodialysis procedure itself (use of filtration membranes, dialysis fluids) [7]. As previously mentioned, in the case of CKD, there is an accumulation of a number of uremic toxins, including urea and uric acid. It has been observed that their presence in the gastrointestinal tract lumen results in both quantitative and qualitative changes in the intestinal microbiota. Their excessive concentration makes them become alternative metabolic substrates for bacteria. The bacteria possessing urease, uricase and p-cresol-forming enzymes (Alteromondacea, Cellulomondacea, Clostridiacea, Dermabacteracea, Enterobacteriacea, Halomonadacea, Methylocaccaceae, Micrococcaceae, Moraxellaceae, Polyangiaceae, Pseudomonadaceace and Xanthomonadaceae) are beginning to dominate. On the other hand, those that metabolize non-absorbable carbohydrates (Bifidobacterium and Lactobacillus) [11, 62] are dying. Urease breaks down urea into ammonia. The increase in the synthesis of ammonia contributes to the increase of pH, and thus leads to damage to tight junctions in the intestinal epithelial cells and the destruction of the intestinal barrier [59]. In a study carried out by Vaziri et al., it was proved that the metabolic changes result in the impairment of function and the destruction of proteins by – and the intramembranes forming part of tight junctions – ZO1 protein, claudin and occluding [59].

Other, coexisting factors that adversely affect the integrity of the intestinal barrier include: swelling of the intestinal wall which occurs in CKD, excessive use of diuretics, aggressive ultrafiltration during haemodialysis, gastrointestinal bleeding, infiltration of the intestinal epithelial lamina propria by lymphocytes [17, 19, 33, 37]. Uremic toxins inhibit activity and reduce the number of transmembrane transporters, which is very important from the clinical point of view, because it may lead to changes in the pharmacokinetics of certain drugs and impair their metabolism in CKD patients [31].

The type of diet and eating habits in this group of patients should be mentioned as well. They have a huge impact on the content of the intestinal flora. Often this disease is accompanied by a lack of appetite, nausea and reluctance to accept certain foods [6]. Patients with CKD should control the amount of fluid intake and also limit the consumption of proteins and products rich in phosphorus and potassium to prevent hyperphosphatemia and hyperkalaemia. Unfortunately, this also involves limiting the consumption of vegetables and fruit. This is not beneficial because it has been proved that a diet rich in fibre and low in red meat, sodium and simple sugars increases the survival rate of patients with CKD [23, 24].

When glomerular filtration rate (GFR) decreases, adverse metabolic processes are accelerated. Indoxyl sulphate (IS) is the final product of the degradation of tryptophan in indol pathway. The first stage of this pathway is initiated by tryptophanase – an enzyme synthesized by Escherichia coli bacterium which is part of the intestinal flora. As a result of this process indole is formed, which is absorbed into the bloodstream through the intestinal barrier and undergoes sulfonation in the liver. Under the right conditions, IS is excreted from the organism with urine [37]. IS concentrations observed in patients with CKD with impaired renal filtration function are higher than in healthy subjects – basing on research conducted the relationship between IS concentration and the progression of kidney disease and the development of vascular complications has been demonstrated [28]. With the use of the established cell lines and animal models, the negative influence of IS on the proximal cells of the renal tubules causing their progressive damage has been proved. Interstitial fibrosis and glomerulosclerosis have also been observed [37]. A study conducted by Leong et al. confirmed that in patients with CKD in stages I–IV the risk of the progression of kidney disease associated with high levels of IS is increased [28].

Trimethylamine N-oxide (TMAO) is also included in the group of uremic toxins. It originates from choline, phosphatidylcholine and L-carnitine, supplied with food, with the participation of intestinal bacteria [8]. In the study conducted by Hoyles et al., it was observed that Enterobacteriacea metabolizes TMAO trimethylamine oxide most effectively [20]. High concentrations of TMAO are associated with an increased risk of the development and progression of kidney disease, which was confirmed, among others, in the Framingham Heart Study with the participation of 1,500 participants [46]. In turn, in studies conducted on mice, the influence of TMAO on the development of tubulointerstitial fibrosis and increased collagen deposition in the kidneys has been demonstrated [8].

In recent years, the significant impact of the microbiome on human health has been recognized. This led to the flourishing of research into the therapeutic possibilities of diseases through exerting an impact on bacterial flora. This also applies to chronic kidney disease. The use of prebiotics, probiotics, and synbiotics is one of the proven methods of therapeutic interventions in the case of symbiosis disorders. Prebiotics are specific nutrients, containing demainly fibre, oligosaccharides, such as inulin. They promote the growth of beneficial intestinal bacteria and by changing the pH, they affect the intestinal barrier status and also increase the activity of intestinal hormones such as GLP-1 (glucagon-like peptide-1). Probiotics are selected strains of microorganisms, able to survive in the human body and exert a beneficial effect on health. They must be characterised by the ability to adhere to the intestinal epithelium so that they are not removed from them in a short time (e.g. during diarrhoea). In addition, they must be resistant to hydrochloric acid contained in the stomach, digestive enzymes, bile acids. The preparation combining probiotics and prebiotics is called a synbiotic [48, 51]. Probiotics may be of natural origin, e.g. fermented products – mainly dairy products, such as yogurt or kefir, pickled products, or commercially produced preparations taken orally [16]. Although they have been used for many years and are generally considered safe, they may also have pathogenic significance. Cases of generalized infections caused by various probiotic strains have been reported. Most cases were associated with Saccharomyces boulardi and cerevisae fungemia. However, bacterial infections caused by Lactobacillus, Bacillus subtilis, and Bifidobacterium breve strains have also been found. In the literature one can also find descriptions of endocarditis caused by probiotic Streptococcus strains. Some of these cases may have been the result of the transfer of bacteria to the area of intravenous puncture through the hands of the staff, who previously administered probiotics. Another negative aspect may be altered metabolism in the intestinal lumen. Administration of a probiotic increases the intestinal epithelial oxygen requirement or increases the formation of lactate, which can lead to dangerous complications. Moreover, in theory, probiotics may overstimulate the intestinal immune system or spread antibiotic-resistant genes among other intestinal bacteria. As of now, these fears have not been justified [13]. To date, numerous studies have been carried out using a variety of bacterial strains, mainly of the genera Lactobacillus, Streptococcus and Bifidobacterium and prebiotics [48]. The action of probiotics is complex and specific to individual bacterial strains. It consists, among others, in sealing barrier joints, increasing mucin production, and protecting enterocytes from apoptosis. In addition to the intestinal barrier protective effect, they have an antagonistic effect on the pathogenic intestinal flora by secreting bacteriocins and competition in occupying intestinal niches. The stimulating effect on immunity and the formation of regulatory lymphocytes is also important[25]. A significant reduction in the levels of harmful metabolites, such as cresol compounds, indoxyl sulphate, or urea nitrogen (BUN, blood urea nitrogen), has been obtained in studies. In one of the studies, an increase in glomerular filtration was observed after prebiotics were applied. The examined patients with chronic kidney disease first consumed 1.6 g of fibre per day for 2 weeks. Then the dietary fibre supply was increased to 23 g per day for 4 weeks. The disadvantage of this study was the small number of participants – 13 people, although a statistically significant decrease in creatinine was obtained, persisting for at least up to 4 weeks after the end of the nutritional intervention [49]. These effects are achieved mainly by creating a suitable environment for the growth of sucrose bacteria at the expense of proteolytic substances, increased absorption of nitrogen compounds by the intestinal flora, lower production of harmful metabolites, and their excretion with stool. An appropriate diet increases the production of short-chain fatty acids, possessing the capacity for “sealing” the intestine [48]. Fatty acids also have a direct metabolic and immunomodulatory effect by directly impacting the FFAR1, FFAR2, and FFAR3 receptors. In this way, they increase leptin levels, stimulate the activity of regulatory lymphocytes, and reduce the amount of reactive forms of oxygen [14]. Other potentially beneficial effects of prebiotics depend on the activation of glucagon-like peptide-1 (GLP-1), the YY peptide (PYY-anorexigenic neuropeptide), and other intestinal hormones. They consist in lowering body weight, improving lipid metabolism, and reducing insulin resistance [51]. However, a study conducted in the population of peritoneal dialysis patients proved a lower level of proinflammatory cytokines and endotoxin after a 6-month period of consuming probiotics from the strains of Bifidobacterium and Lactobacillus [39]. The analysis of the NHANES cohort indicates a lower severity of albuminuria in people consuming probiotics and yogurt, at least 3 times a week. Unfortunately, in the study based on completing nutrition questionnaires, no specific type of probiotics was indicated [39, 63]. In studies on rats, it has been proved to slow down the progression of kidney disease, and even to prolong the survival after the use of probiotics of the strains of Bacilluspasteurii or Lactobacillus sporogenes [39, 51].

There are also numerous observational and interventional studies on humans and animals, proving the benefits of taking an increased amount of fibre. It may have both the form of oligosaccharides or resistant starch. A large observational study on a population of approximately 14,000 confirmed the reduction of the signs of inflammation and overall mortality along with an increase in fibre intake [36, 45]. On the other hand, the results of some studies do not confirm the benefits of using fibre in CKD. In a randomized, double-blind study involving 40 patients with CKD in stage III–IV, the therapeutic effect of arabinoxylan administered over 4 weeks was not proved [42]. Arabinoxylan is a compound derived from the walls of plant cells, mainly cereal products, being a polymer of arabinose and xylose.

Currently, the most promising results are obtained through the use of synbiotics. Studies using different bacterial strains combined with inulin and other probiotics have led to a decrease in the concentration of toxic metabolites, mainly p-cresol sulphate, inflammatory parameters, and even a slowdown in CKD progression. The research was conducted on various groups of patients, both dialyzed and remaining in the third and fourth stage of CKD. After the application of the strains of Lactobacillus acidophilus, a decrease in the serum concentration of dimethylamine was obtained, whereas after the administration of Bifidobacterium longum – a decrease in the serum concentration of indoxyl sulphate, homocysteine, and triacylglycerols. The disadvantage of these studies is the relatively small number of examined groups (up to 40 people) and they require confirmation in a larger population [39]. A recently published meta-analysis of randomized trials using probiotics in patients with stage III–V CKD indicates a reduction in p-cresol sulphate and an increase in the concentration of interleukin-6. The use of probiotic did not have a significant statistical effect on the concentration of creatinine, nitrogen in urea, and other toxins, or on the concentration of haemoglobin and C-reactive protein (CRP). The meta-analysis was based on eight studies, covering a total of 261 patients. Patients received eight different probiotic preparations at different doses. Among the administered bacterial strains, there were: Lactobacillus: acidophilus, gasseri, bulgaricus, plantarum, casei, salivarius, sporogenes, Bifidobacterium: bifidum, longum, infantis, breve, and Streptococcus thermophilus [22].

An interesting possibility for treating microbiota disorders is the modification of the bacterial genome used in the form of probiotics in order to adjust their metabolism to give the greatest benefits to CKD patients. In theory, it is possible to deliver bacteria with a modified genome to the gastrointestinal tract of patients, equipped with enzymes breaking down uremic toxins. Currently, there are no practical tests assessing such possibilities [39, 41, 51].

Other potential possibilities of exerting influence include the use of antibiotics which kill abnormal intestinal flora or inhibitors of fermentation of undigested carbohydrates, such as acarbose, which leads to the inhibition of the digestion of polysaccharides by intestinal cells, providing an energy substrate for intestinal bacteria [48]. The use of rifaximin, a broad-spectrum bactericidal antibiotic, has a proven effect of reducing the production of trimethylamine oxide by removing intestinal bacteria. More specific effects can be obtained by blocking bacterial enzymes such as TMA – lyase and tryptophanase, to prevent the production of toxins without a bactericidal effect. So far, however, there has been a lack of studies proving the safety and efficacy of such procedure in humans [43]. Lubiprostone, a CIC-2 chloride channel activator used in the treatment of constipation, may also contribute to restoring the intestinal microbial balance by multiplying bacteria of the genus Lactobacillus and Prevotella [39].

Yet another approach is proposed which does not directly affect the intestinal microbiota, but rather counteracts the negative effects of uremic toxin formation. It involves the use of substances which bind harmful metabolites in the digestive tract. Studies have been carried out using a carbon compound called AST-120 with respect to the activity of sevelamer hydrochloride. A reduction in the concentration of indoxyl sulphate in the blood was observed; moreover, administration of AST-120 to patients with ESRD during the pre-dialysis period led to a reduction in overall mortality in this group of patients [48].

Among the new potential therapies impacting the intestinal microbiome there is also canagliflozin. This antidiabetic medication, in addition to blocking SGLT-2 glucose transporters in the renal tubules, also has a weak inhibitory effect on SGD-1, sodium-dependent glucose transporters and SGLT-1 galactose found in the intestines. A study on mice with renal failure indicates that canagliflozin inhibits the absorption of carbohydrates in the initial section of the small intestine, leading to their greater concentration in the final section of the small intestine and large intestine. This affects the development of potentially beneficial bacteria, e.g. of the genus Bifidobacterium. The production of short-chain fatty acids is increased and the formation of p-cresol sulphate and, to a lesser extent, indoxyl sulphate is clearly reduced. This opens up new possibilities and the need for research on SGLT-1 inhibitors as a drug restoring intestinal eubiotism [35].

In recent years, there has been a growing interest in intestinal microbiota in the context of its impact on human health. Thanks to the development of bioinformatics and progress in the field of DNA sequencing techniques, the relationship between altered intestinal microbiota and the occurrence of many diseases, including chronic kidney disease, has been proved. Altered intestinal flora stimulates the production of metabolic toxins and increases their absorption in the intestine, which in turn leads to the progression of kidney disease and the development of its complications. It is believed that restoring the normal intestinal flora in these patients can slow down the disease and reduce the risk of complications. The results of preclinical studies on potential therapeutic options are promising, however, there is still a need for clinical trials in large populations to establish unambiguous treatment strategies in patients with chronic kidney disease. Perhaps in the future, modifying the composition of the intestinal flora will allow better treatment and control of kidney diseases.