PART Ⅰ: The Administration Of The Synbiotic Lactobacillus Bulgaricus 6c3 Strain, Inulin And Fructooligosaccharide Decreases The Concentrations Of Indoxyl Sulfate And Kidney Damage
Mar 23, 2022
ali.ma@wecistanche.com
AlonsoJerez-Morales,JoseS.Merino,Sindy T.Diaz-Castillo & et al.
1. Introduction
Chronic kidney disease (CKD) is considered a permanent alteration of the kidney structure, its function, or, in the most severe cases, both. CKD directly affects the health and life quality of the individual [1,2]. At the physiological level, CKD caused changes frequently observed are increased serum levels of creatinine, cystatin Cor ureic nitrogen [2]. The intensified surveillance and the awareness in the population have increased CKD diagnoses reaching, for example, to be determined that a 15% of the USA population is affected by this pathology [3]. The progression of the disease, as well as the effects of the above mentioned metabolites, have a serious impact in multiple systems of the body, including the cardiovascular and the gastrointestinal systems, being the presence of these metabolites associated to an increased mortality in the former and affecting assimilation, motility, and permeability in the latter [4,5]. The gastrointestinal alterations provoke an increased absorption of metabolites in the intestine contributing to sustaining a systemic inflammation which, in turn, affects the composition of the normal microbiota and its metabolic functions and may cause intestinal dysbiosis [6].
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The association between CKD and intestinal dysbiosis might not be explained only by changes in the diet but also, besides, by the increase of the luminal pH, secondary to an increased concentration of ammonium [7]. End-stage CKD patients have marked differences in the abundance of several bacterial taxa in their feces when compared to healthy individuals [8]. Particularly, at the bacterial genus level, CKD patients show an increase of Enterorhabdus, Blautia, Ruminococcus, Parasutterella, Bacteroides, Clostridium (sensu stricto),Escherichia, Shigella, and Allobaculum and a decrease of Akkermansia, Bifidobacterium, Clostridium IV,Lactobacillus, Lactococcus, Leuconostoc, Alkaliphilus, and Pseudomonas [9-11]. Either by the poor renal depuration or by the intestinal dysbiosis, there is a decrease of bacterial genera capable to metabolize and inhibit pathogens that produce toxic metabolites, resulting in their increase [12]. Wong et al. [8] demonstrated that patients suffering advanced CKD showed an increase of bacterial groups possessing enzymes such as urease, uricase, and those producing p-cresol and indole..
The accumulation of uremic retention metabolites (URMs) in the blood, including indoxyl sulfate (IS), p-cresyl sulfate (pCS), triethylamine, and trimethylamine N-oxide (TMAO)[5] are postulated as accelerators of CKD [13]. IS(Indoxyl sulfate), the best-known uremic indole, is secreted by the proximal convoluted tubules of the kidney and it originates from the metabolism of tryptophan by the bacterial tryptophanase. The evidence from animal models shows that IS(Indoxyl sulfate) can affect the renal function and it can damage cells of the tubules generating oxidative stress in endothelial cells, inhibiting repair mechanisms, and favoring interstitial tubular fibrosis because it increases the genic expression of TGF-1, of tissue inhibitor of metalloproteinases (TIMP-1)and of collagen [14]. Nevertheless, the unbalance of the intestinal microbiota, and the concomitant production of toxic metabolites can be modified. It has been described that supplementation with certain probiotics and prebiotics may reduce the production of certain uremic toxins and restore the normal microbiota [5]. For example, meta-analysis studies have concluded that CKD patients receiving supplementation with probiotic strains increased their anti-inflammatory cy-tokines and decreased their levels of uremic toxins [15,16]. Some studies reported that the administration of synbiotics, a combination of probiotics and prebiotics, is capable to decrease the progression of CKD with favorable results [12].Within the context of the present work, inulin-type fructans are relevant prebiotics because they have shown their capacity to decrease the progression of CKD [17] and to reduce URMs, such as pCS and IS(Indoxyl sulfate) [18]. Panza et al. (2017) administered the probiotics L.acidophlilus and Bifidobacterium longum plus fructooligosaccharides (FOS) in patients with stage 2-5 CKD and reported that untreated patients had significantly more pCS and are (Indoxyl sulfate) in the blood than patients who received the synbiotic treatment [19]. In this work, we first evaluated the capability of probiotic strains to reduce IS(Indoxyl sulfate) in vitro to select the most promising one. Then, this strain was combined with the prebiotics inulin and fructooligosaccharides (FOS)to obtain a putative synbiotic. These prebiotics was selected because they have shown that they can decrease pCS and IS(Indoxyl sulfate) concentration in blood [17,18]. Finally, the efficiency of the putative synbiotic was tested in vivo, evaluating blood IS(Indoxyl sulfate) and creatinine levels and kidney and heart damage using a rat model (Sprague-Dawley rats) including kidney deficient (nephrectomized) and untreated animals.
2. Results
2.1.Isolation of Strains Capable to Reduce IS(Indoxyl sulfate) In Vitro
From 36 samples of dairy products,84 strains were isolated in MRS agar supplemented with bromophenol blue (MRS-BFB), used as a differential medium. This medium allows differentiating lactic acid bacteria. At pH 3 colonies will be colored yellow and at pH close to 5 they will be colored blue [19]. All the 84 strains isolated were tested in vitro to determine their ability to mediate the reduction of IS(Indoxyl sulfate). For this, strains were cultured in MRS broth supplemented with 2.8 mM IS(Indoxyl sulfate) for 48 h, and then IS(Indoxyl sulfate) was measured by high-performance liquid chromatography (HPLC).Strains lc2, 6c3, and VIc2 were able to significantly reduce (p<0.05) the concentration of the compound (Table 1). Furthermore, the three strains were cultured together for 48 h in the presence of IS(Indoxyl sulfate) to evaluate if they acted synergistically to reduce IS(Indoxyl sulfate). When all three strains were cultured together in the presence of IS(Indoxyl sulfate), the concentration of IS(Indoxyl sulfate) was also significantly reduced but less than that achieved by the strains alone, ruling out the possibility of synergistic activity(Table1).
Table 1. In vitro decrease, if indoxyl sulfate(IS), is evaluated by high-performance liquid chromatography (HPLC), mediated by bacterial strains.
2.2. Identification of the Selected Strains
The three strains selected due to their ability to reduce IS(Indoxyl sulfate) were identified by 16S rRNA sequencing after PCR amplification of DNA.Strain 1c2 was a Gram-positive chain forming coccus which, in the molecular analysis, amplified with the LacZ primers for Streptococcus thermophilus. Strains 6c3 and VIc2 were both Gram-positive bacilli and amplified with LbG primers to identify genus Lactobacillus. The analysis of the 16SrRNA sequencing allowed to identify strain lc2 as Streptococcus thermophilus(97.3% similarity;Access Number: AY188354.1),strain 6c3 as Lactobacillus bulgaricus(99.7%similarity; Access Number: CR954253),and strain VIc2 as Lactobacillus acidophilus (99.5%similarity; Access Number: AY773947.1).Only one of the three strains was selected to prepare the synbiotic and to carry on the following assays. Streptococcus thermophilus 1c2 strain was discarded because it showed a low growth rate. From the remaining two strains, the Lactobacillus bul-garicus 6c3 strain was selected because it showed better probiotic functionality and safety characteristics than the Lactobacillus acidophilus VIIc2 strain.
2.3. Effect of the Synbiotic in Blood Creatinine and IS(Indoxyl sulfate) Levels
The probiotic L. bulgaricus 6c3 was lyophilized in association with the prebiotics Inuline and FOS, being the concentration of the probiotic 10° CFU/g of lyophilized product. Ten grams of synbiotic were administered daily, in the drinking water, to the group of nephrectomized synbiotic treated Sprague-Dawley rats(Lac). The nephrectomized untreated group (Nef) and the non-nephrectomized control group did not receive the syn-biotic. Serum creatinine and IS(Indoxyl sulfate) levels of the rats were measured by HPLC at the beginning (one week after nephrectomy) and the end of the assay. Moreover, the hematocrits of the animals of all groups were recorded. The variation of serum creatinine levels at the end of the assay were significantly different (p<0.05) in the Nef and Lac groups,increasing 127%and 121%, respectively, when compared to their initial values(Figure 1). The control group showed a non-significant creatinine increase of only 2%. The increase of creatinine serum levels in the Nef and Lac groups was significantly higher than that of the control group but not significantly different between the Nef and Lac groups. Nevertheless, all three groups maintained their creatinine levels within de normal limits for these rats. On the other hand, serum IS(Indoxyl sulfate) levels significantly increased only in the Nef group corresponding to the nephrectomized (renal deficient) animals not administered the synbiotic (38.8%)(p <0.05). IS(Indoxyl sulfate) levels showed a non-significant 0.5% increase in the control group and a non-significant 0.8% decrease in the Lac group(Figure 2). When the groups of nephrectomized animals were compared, the group not consuming the synbiotic (Nef) showed a significant higher level of IS(Indoxyl sulfate) in comparison with the group who consumed the synbiotic(Lac). There was no significant difference between the Lac group and the control group. The analysis of the data searching for a possible correlation between creatinine and IS(Indoxyl sulfate) levels showed that this correlation was not present (Supplementary Materials: Figures S1-S3). Finally, the results of the hematocrits showed no significant differences among the three groups (Figure 3).
Figure 1. (a) Serum creatinine levels of Sprague-Dawley rats at the beginning and the end of the 16 weeks administration of the synbiotic. (b)Differences of the means for final creatinine concentrations with 95% Tukey simultaneous confidence intervals (If an interval does not contain zero, the corresponding means are significantly different). Nef: nephrectomized rats not receiving the synbiotic, Lac: nephrectomized rats receiving the synbiotic, Control: sham-nephrectomized rats.
Figure 2. (a) Serum indoxyl sulfate (IS) levels of Sprague-Dawley rats at the beginning and at the end of the 16 weeks administration of the synbiotic.(b)Differences of the means for final IS(Indoxyl sulfate) concentrations with 95% Tukey simultaneous confidence intervals(If an interval does not contain zero, the corresponding means are significantly different). Nef: nephrectomized rats not receiving the synbiotic, Lac: nephrectomized rats receiving the synbiotic, Control:sham-nephrectomized rats.
2.4.The Histological Analysis Showed that the Administration of the Sybiotic Decreased the Progression of CKD
At the end of the experiment, rats were euthanized to obtain samples from the left kidneys and hearts to be processed for histological analysis. The fibrotic area was determined by the Masson's trichrome (MT) stain using the informatic tool Image-Pro Plus 3.0 (Media Cybernetics, Rockville, MD, USA)to calculate the percentage of damaged (i.e., fibrotic)area(Table 2) and by two-photon excitation microscopy, a method that detects collagen by the emission of two-photons, causing the collagen to generate a second harmonic, which can be visualized by confocal microscopy getting a specific image close to the organization and distribution of collagen inside of the tissue. When comparing the fibrotic areas of the kidneys, evaluated by the MT stain, it was possible to observe that fibrotic areas, were 25% larger in the Nef group but only 12% larger in the Lac group, administered the snbiotic when compared to sham-nephrectomized control animals. Lesions observed with this staining procedure were located at the parietal layer of the Bowman's capsule and the basement membrane of the proximal and distal convoluted tubules of the animals of groups Nef and Lac. In the case of the Nef group, including the animals not administered the synbiotic, a loss of cytoplasm of the cells of the convoluted tubules was observed with concomitant development of fibrotic tissue. When the tissues of the kidneys were analyzed by two-photon excitation microscopy, the collagen was observed in all samples having a mesh-type conformation. Groups Nef and Lac showed more lesions than the control group, in which only one fibrotic area was found (Figure 4). No lesions were found in the renal corpuscles in any sample when observed under two-photon excitation microscopy.
Table 2. Percentage of fibrotic area in Masson's trichrome stained kidney histological sections. Results are expressed as mean + standard error.
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| Figure 3. Hematocrit values of Sprague-Dawley rats at the end of the 16 weeks administration of the synbiotic. Nef: nephrectomized rats not receiving the synbiotic, Lac: nephrectomized rats receiving the synbiotic, Control:sham-nephrectomized rats. | Figure 4. Histological sections of kidneys of Sprague-Dawley rats.(A-C):Masson's trichrome stain of nephrons. Fibrotic areas are observed in blue (arrows).(D-F): same tissue observed by two-photon excitation microscopy. Fibrotic tissue is observed in green. Nef: nephrectomized rats not receiving the synbiotic, Lac: nephrectomized rats receiving the synbiotic, Control:sham-nephrectomized rats. Scale bar∶ 100 μum for（A-C） micrographs and 50 μm for （D-F） micrographs. |
2.5.The Administration of the Synbiotic Did Not Attenuate Cardic Fibrosis
In the heart, the MT stain showed the presence of fibrotic areas in the tissue of nephrectomized rats, while these areas were absent in the control animals. The induction of CKD in rats generates cardiac fibrosis which was not prevented by the administration of the synbiotic treatment in nephrectomized rats (Figure 5).
Figure 5. Masson's trichrome(MT)staining of the heart of Sprague-Dawley rats.Fibrotic areas are stained in blue (arrows). Nef: nephrectomized rats not receiving the synbiotic, Lac: nephrectomized rats receiving the synbiotic. Control:sham-nephrectomized rats. Scale bar 100 um.
