The Synbiotic Reducing Indoxyl Sulfate And Chronic Kidney Disease

ali.ma@wecistanche.com

PART Ⅱ: The Administration of the Synbiotic Lactobacillus bulgaricus 6c3 Strain, Inulin and Fructooligosaccharide Decreases the Concentrations of Indoxyl Sulfate and Kidney Damage in a Rat Model

AlonsoJerez-Morales,JoseS.Merino,Sindy T.Diaz-Castillo & et al.

Abstract

Indoxyl sulfate (IS) is involved in the progression of chronic kidney disease (CKD) and its cardiovascular complications. One of the approaches proposed to decrease IS(Indoxyl sulfate) is the administration of synbiotics. This work aimed to search for a probiotic strain capable to decrease serum IS(Indoxyl sulfate) levels and mixit with two prebiotics(inulin and fructooligosaccharide (FOS)) to produce a putative synbiotic and test it in a rat CKD(chronic kidney disease) model. Two groups of Sprague-Dawley rats were nephrectomized. One group (Lac)received the mixture for 16 weeks in drinking water and the other no (Nef).A control group (C) included sham-nephrectomized rats. Serum creatinine and IS(Indoxyl sulfate) concentrations were measured using high-performance liquid chromatography with diode array detector(HPLC-DAD). Optical microscopy and two-photon excitation microscopy was used to study kidney and heart samples. The Lac group, which received the synbiotic, reduced IS(Indoxyl sulfate) by 0.8% while the Nef group increased it by 38.8%. Histological analysis of kidneys showed that the Lac group increased fibrotic areas by 12% and the Nef group did it by 25%.The synbiotic did not reduce cardiac fibrosis. Therefore, the putative synbiotic showed that function reducing IS(Indoxyl sulfate) and the progression of CKD(chronic kidney disease) in a rat model, but no heart protection was observed.

Keywords: CKD; indoxyl sulfate; CVD; renal fibrosis; synbiotic

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3. Discussion

Considering its significant increase in worldwide prevalence, the difficulties and cost of treatment, and because it is one of the main risk factors for cardiovascular disease [13,20], CKD(chronic kidney disease) is becoming one of the main public health challenges. Presently, scientific evidence supporting a close relationship between intestinal health and renal health commonly referred to as the intestinal-renal axis is growing [21]. The communication/interaction between the intestinal microbiota and its host is patho-physiologically relevant, particularly in CKD(chronic kidney disease) patients [22]. In these patients, the increase of uremic compounds in the blood may affect the composition and metabolism of the microbiota and also favor the growth of bacterial groups which produce these metabolites [21,22]. It has been reported that, in individuals suffering dysbiosis, the microbiota can produce over 200 serum metabolites [17]. All the above implies an increase of intestinal permeability which may result in translocation of metabolites, endotoxins, and viable bacteria into the bloodstream [23]thus favoring chronic inflammation, increasing cardiovascular risk, and worsening uremic toxicity. Nevertheless, the intestinal microbiota unbalance may be reverted, for example, using prebiotics, probiotics, or their combination as synbiotics. In this context, it has been described that supplementation with certain probiotics and prebiotics might reduce the production of certain uremic toxins and restore normal microbiota [5]. Our results provide evidence that, from the 84 bacterial strains tested in vitro in this study, three strains showed the ability to significantly reduce [Sin the culture medium.These results are similar to those reported by other authors [24,25]. The mechanism by which IS(Indoxyl sulfate) is reduced remains uncertain yet. It has been postulated that IS(Indoxyl sulfate) may bind to the cell wall of probiotics and then be eliminated with feces [25] or that some microorganisms possess enzvmes capable to degrade IS(Indoxyl sulfate) [26]. Whether these postulated mechanisms are correct or not, it has been widely reported that probiotics restore the normal microbiota in individuals having CKD(chronic kidney disease) caused by dysbiosis [27].

The use of prebiotics and probiotics as an approach to decrease the progression of CKD(chronic kidney disease) has been extensively investigated. Various prebiotics have demonstrated to be use-ful to reduce different dysbiosis metabolites, as peritoneal dialysis supplement [17,18]. Lai et al. [28] compared the effect of a low protein diet and the supplementation of this diet with inulin (19g/day)in stage3 or 4 CKD(chronic kidney disease) patients. They reported that the dietary intervention had little effect on the microbiota, only reducing the frequency of genus Lacto-bacillus, but when inulin was added it was possible to increase the levels of Bifidobacterium.

Another beneficial effect of inulin was a reduction of intestinal pH, ureic nitrogen, of TNF-a and NOX2 in blood, limiting the increase of URMs and reducing inflammation [28]. Nevertheless, it has been reported that the addition of probiotics to the diet generates much more significant effects, leading to the use of prebiotics and probiotics mixtures (synbiotics)to potentiate their microbiota modulating activity. Alla and Sadeek [29]evaluated the administration of Arabic gum and L. casei Shirota to Wistar rats suffering induced CKD(chronic kidney disease). Their results showed that the treatment was able to reduce blood concentrations of urea, creatinine, and uric acid. Similarly, it has been extensively reported that the use of prebiotics, probiotics, and synbiotics possess a modulating effect on the intestinal microbiota. This is an important issue because, under dysbiosis, uremic toxins(particularly indole derivatives)have shown to activate the pregnane X receptor(PXR) which regulates the expression of TLR4. The resulting increased inflammation affects the intestinal barrier augmenting its permeability to various metabolites and allowing the translocation of bacteria [11]. In the present work, the treatment consisted in the administration of a synbiotic including inulin, FOS(Fructooligosaccharide) and L. bullgaricus 6c3 strain. Rocchettiet al. [30] demonstrated that a different L. bulgaricus strain, combined in a synbiotic, decreased IS and PCS serum levels when used associated with an innovative dialysis treatment in HD patients.

Differences in the serum creatinine levels were observed between the control and nephrectomized(Lac and Nef)rats indicating that the uremic model was successfully established. Nevertheless, no significant difference was observed when comparing groups Lac and Nef. That is to say, there was no difference in creatinine levels if the treatment was administered or not to CKD(chronic kidney disease) rats, an observation consistent with other reports [24,31,32]. Thus, the contribution of creatinine measurements was to confirm the kidney deficiency achieved by nephrectomy. On the other hand, serum IS(Indoxyl sulfate) levels increased significantly in the Nef group (38.8%)but not in the synbiotic administered Lac group whose IS(Indoxyl sulfate) level was similar to that of the control group. Thus, the synbiotic was able to avoid the damaging increase of IS(Indoxyl sulfate) concentrations despite the presence of induced CKD(chronic kidney disease).Several works have reported similar results when administering prebiotics [28], probiotics [24] or synbiotics [29]because they have the capacity to decrease IS(Indoxyl sulfate) in different models of study. Wuet al.[31]administered L.acidophilus LB to5/6th nephrectomized Sprague-Dawley rats and evaluated 35 metabolites in feces. Interestingly, some metabolites (3-(3-hydroxyphenyl) propionic acid, amebamide, benzopyrene, aspartyl-glutamine, phenethylamine glucuronide, and T2 toxin tetrol) increased five times in nephrectomized rats; nevertheless, their levels were restored to normal levels atter treatment.These results are in agreement with our observations because the administration of the synbiotic maintained IS(Indoxyl sulfate) levels similar to that of the control group.

It has been reported that IS(Indoxyl sulfate) induces a local increase of reactive oxygen species (ROS)in the renal tissue which, in turn, activate the nuclear factor kappa B(NF-kB) and activate the oxidative stress and the production of pro-inflammatory cytokines [11]. IS is capable to enter into cells of convoluted tubules using the transporter for organic anions and, once there, it increases ROS and NF-kB, leading to a decrease of E-cadherin and an increase of the transforming growth factor β(TGF-β) and smooth muscle actin alfa(α-SMA).The result is an epithelial-mesenchymal transition producing renal fibrosis [33]. Similarly, one of the main kidney protecting mechanisms of L.bulgaricus 6c3 is its anti-inflammatory activity achieved decreasing IS levels. Our observations showed that the kidney fibrotic area in animals of the nephrectomized Nef group (25% of the fibrotic area) was twice as large as that of synbiotic administered nephrectomized Lac rats(12%of fibrotic area)and sham-nephrectomized control rats; thus, the treatment with the synbiotic was effective to reduce the fibrotic area of the kidney in CKD(chronic kidney disease) rats. Theoretically, this protecting effect can be explained by an alteration of the intestinal lumen which, in turn, could reduce IS(Indoxyl sulfate) serum concentrations. Feng et al.[34] demonstrated significant changes between nephrectomized and non-nephrectomized rats involving 13 bacterial species belonging to genera Blautia, Escherichia_Shigella, Bacteroides, Allobaculum Clostridium_IV and 291 metabolites; thus, emphasizing the impact of CKD(chronic kidney disease) on the microbiota and URMs. Moreover, they also provided evidence that the administration of two prebiotics(Poria cocos and poricoic acid A) modified the intestinal lumen and restored the intestinal normal microbiota. Both prebiotics mitigated inflammation and oxidative stress through the inhibition of the IKBo /NF-kBIpathway and normalized serum urea and creatinine as well as o-SMA expression. So far, to the best of our knowledge, this is the first work reporting the use of two-photon excitation microscopy in this type of studies and,interestingly, although there is evidence of damage in the glomerular corpuscle (Masson trichrome stain), the fibrotic lesions were only observed in the convoluted tubules by two-photon excitation microscopy. This observation supports those reports mentioning that the main deleterious effect of the most studied URMs is, precisely, on the cells of the convoluted tubules of the kidney [33].

It has been reported that IS(Indoxyl sulfate) induces cardiac fibrosis with the expression of fibrotic proteins(TGF-β1, SMA, and type 1 collagen), cardiac hypertrophy, and oxidative stress [35], and an increasing number of publications is confirming the negative effects of IS(Indoxyl sulfate) on the cardiovascular system. Kaminski et al. [36] studied 51 patients in CKD(chronic kidney disease) stages 1 to 5 and reported that the accumulation of IS(Indoxyl sulfate) was correlated with disturbances of some factors affecting parameters of the hemostatic system of CKD(chronic kidney disease) patients, such as H2O2 and plasma Cu/Zn superoxide dismutase. It has been also reported that the administration of a probiotic strain can reduce the biomarkers of inflammation in cardiovascular disease and reduce its associated risks [37,38]. Our results indicated that, although serum IS(Indoxyl sulfate) was significantly reduced, the administration of the synbiotic did not avoid the development of cardiac fibrosis. In fact, it has been postulated that, despite the damage produced by IS(Indoxyl sulfate) in the cardiovascular system, the main URMs affecting the heart are p-cresol [39]and TMAO [40], metabolites not evaluated in this study. Nevertheless, some authors state that differences among the results of different studies are the consequence of the variability of the study models. Karbowska and coworkers [41], working with Wistar rats and C57BL6/cmdb mice, reported the effect of acute exposure to IS on the thrombotic process, clotting, coagulation and erythrocyte osmotic resistance among other parameters. They reported that 0.1 mM IS in blood was correlated with fibrin generation and collagen induced aggregation in PRP and that up to 1 mM plasmatic IS(Indoxyl sulfate) had no effect on erythrocyte hemolysis. In our work, using Sprague-Dawley rats, the highest IS(Indoxyl sulfate) levels reached were nearly 0.0025 mM in the nephrectomized group not consuming the synbiotic (Nef). IS levels above 0.0025 mM might have revealed a correlation between creati-nine and are (Indoxyl sulfate) levels. Regarding our results on the hematocrit of all three animalgroups, they are consistent with the report of Karbowska and coworkers. They also concluded that IS(Indoxyl sulfate) may be one crucial factor of heart damage. Since we also observed heart damage with lower IS(Indoxyl sulfate) levels, perhaps our model using nephrectomized Sprague-Dawley rats might contribute to detecting other key factors causing heart damage. Finally, it must be emphasized that the higher levels of IS reported by Karbowswka and coworkers when compared to those reported in the present work can probably be the consequence that they injected up to 100 mg/kg of body weight of IS into the animals while our IS levels were the consequence of 5/6th nephrectomy of them. Although the increase of publications pertaining the administration of probiotics in cardiac and other diseases there is still a long path to walk before comprehending how prebiotics and probiotics contribute to our health.

4. Conclusions

The results here reported demonstrate that the synbiotic formulated using the strain L. bulgaricus 6c3, inulin, and FOS(Fructooligosaccharide) is capable to reduce IS(Indoxyl sulfate) both in vitro and in the blood of Sprague-Dawley rats used model. Moreover, it was able to reduce the progression of renal fibrosis when administered daily but it was unable to avoid the development of cardiac fibrosis.