Short-Term Effects of Pre/Probiotics on P-Cresol and Indoxyl-Sulphate Serum Concentrations During the Various Stages of Chronic Kidney Disease

Background: The uremic syndrome is provoked by a progressive number of compounds that are normally excreted by kidneys in healthy individuals. Indoxylsulphate (IXS) and p-cresylsulphate (PCS), have been found increased in subjects with end stage renal disease (ESRD) creating great harm to biological systems; these uremic toxins come from the intestinal bacterial fermentation of the proteins. The aim of our study is to evaluate the short-term effects after an administration of pre / probiotics in CKD patients, regarding the production and then the serum concentrations of free IXS and PCS (i.e. non-protein bound fraction) and total IXS and PCS ( i.e. sum of unbound and protein bound fraction). Methods: In our study , 26 patients with CKD stage 2-5 associated with hypertension and / or diabetes mellitus type 2 were enrolled, and administered with 2 g/day dose of pre-probiotics for four months: mixed oligofructose (prebiotic component) + Lactobacillus acidophilus and Bifi dobacterium longum (probiotic component). In all patients, at the beginning of the study, kidney function tests, glucose metabolism, PTH and blood uric acid were evaluated. Free and total PCS and IXS were also measured. 20 control subjects with normal renal function were considered in relation to the same parameters. After 4 months 19 patients were re-evaluated in relation to the same parameters. Statistical differences were studied using the Student-t paired and unpaired tests. Results: The baseline values of IXS and PCS of the 26 patients were signifi cantly higher compared with the normal subjects and importantly increased with the transition to the higher stage of CKD. The values in stage 2 3 CKD were signifi cantly lower in respect to stage 4 – 5 CKD. Higher mean values of IXS and PCS in 12 diabetic subjects were highlighted, although not statistically signifi cant compared to 14 hypertensive non-diabetic patients. The data after the use of pre probiotics in 19 patients that completed the treatment protocol (5 patients were out of the study for non-compliance of the processing and 2 patients for dialysis entrance), showed increased concentrations of free and total IXS and PCS. Considering renal function, the use of pre probiotics increased the concentration of free and total PCS and IXS in all conditions, while remaining signifi cantly higher in patients at stage 4-5 rather than in the ones at the stage 2-3. The use of pre probiotics increased the IXS and PCS serum concentrations, remaining signifi cantly higher in diabetics rather than in hypertensive patients. In all periods, both baseline and after the uptake of pre-probiotics, the other measured parameters didn’t change except serum PTH that decreased signifi cantly and Calcium increased even if not signifi cantly Conclusions: In conclusion IXS and PCS can be considered as kidney function markers as well as have systemic toxic effects. Diabetes seems to increase the concentration of the two metabolites. The use of pre-probiotics should be started in the early stages of kidney failure and certainly for periods longer than four months. Pre-probiotics could aid in preventing renal osteodystrophy. Research Article Short-Term Effects of Pre/Probiotics on P-Cresol and Indoxyl-Sulphate Serum Concentrations During the Various Stages of Chronic Kidney Disease Filomena Panza2, Diletta Duranti1, Ralli Chiara2, Matteo Basile1, Marco Bagnati1, Giorgio Bellomo1 and Ennio Duranti2* 1Department of Pathology and Clinical Laboratories AUO Novara 2UOC Nephrology and Dialysis Unit, Hospital of Arezzo Dates: Received: 14 March, 2017; Accepted: 29 April, 2017; Published: 03 May, 2017 *Corresponding author: Ennio Duranti, UOC Nephrology and Dialysis Unit, Hospital of Arezzo, Italy. E-mail: https://www.peertechz.com


Introduction
The uremic syndrome is provoked by a progressive number of compounds that are normally excreted by kidneys in healthy individuals. At least 90 compounds, often called uremic toxins, like indoxylsulphate (IXS) and p-cresylsulphate (PCS), have been found increased in subjects with ESRD creating great harm to biological systems [1]; these uremic toxins come from the intestinal bacterial fermentation [2] of the proteins. These solutes are considered not only biomarkers of renal function, but according to some authors they contribute to the development of kidney disease. The accumulation of these compounds has a negative impact on many body functions, especially on the cardiovascular system, as recently proved by several authors who underline the association among serum PCS, cases of general mortality and cardiovascular mortality either in chronic kidney disease (CKD) or in its fi nal stage [3][4][5]. In addition, in a study of Schepers et al., it has been shown that PCS stimulates the basic leukocyte activity with pro-infl ammatory effects, inhibiting the activated leukocyte function and inducing endothelial disorder [6], a condition that could induce cardiovascular events. PCS, a phenol 108 Da MW, is a terminal product of protein catabolism, produced by intestinal bacteria that metabolize tyrosine and phenylalanine [7,8].
As regards IXS it is metabolized by the liver from the indol, which is produced by the intestinal fl ora as a metabolite of tryptophan. IXS causes an endothelial disorder of the uremia, promoting the proliferation of smooth muscle cells through the activation of growth factors derived from platelets and inducing a signifi cant production of free radicals by endotelail cells. IXS appears to have a clinically important role in aortic stiffness and vascular calcifi cation [9][10][11].
PCS and IXS both come from bacterial fermentation of the proteins in the large intestine: the colonic microbiota degrades tryptophan to indole. Therefore in renal failure conditions, the altered intestinal bacterial metabolism changes serum concentrations of IXS and PCS, so we wonder wether it is possible to induce a decrease of their serum concentrations.
At this regard it was found that the intake of prebiotic inulin, enriched with oligofructose, could signifi cantly reduce serum concentrations of PCS and IXS [12]; therefore clinical studies investigating the role of prebiotics and / or probiotics in CKD patients, would be useful in order to evaluate the possible positive impact on the evolution of chronic renal failure prevention. The aim of our study is to evaluate the shortterm effects after an administration of pre / probiotics in CKD patients, regarding the production and then the serum concentrations of free IXS and PCS (i.e. non-protein bound fraction) and total IXS and PCS (i.e. sum of unbound and protein bound fraction).

Materials and Methods
In our study , 26 patients with CKD stage 2-5 associated with hypertension and / or diabetes mellitus type 2 (Table 1) were enrolled, and administered with 2 g/day dose of preprobiotics for four months: mixed oligofructose (prebiotic component) + Lactobacillus acidophilus and Bifi dobacterium longum (probiotic component). In all patients, at the beginning of the study, kidney function tests, glucose metabolism, PTH and blood uric acid were evaluated. Free and total PCS and IXS were also measured. The two metabolites were analyzed in fresh or frozen serum sample (in fact the stability of the compounds allows the two conditions). The method involves the denaturation and precipitation of serum total proteins for the separation of the supernatant, on which the total PCS and IXS will be measured (sum of the protein bound fraction and the unbound fraction). The free fraction of these metabolites is achieved by centrifugal fi ltration in order to remove the binder's proteins. The dosage is performed by HPLC / MSMS using PCS-D4 as internal standard. At the same time 20 control subjects with normal renal function were considered in relation to the same parameters (Table 1).
After 4 months of daily intake of pre-probiotics, 19 patients were re-evaluated in relation to the same parameters.
Statistical differences were studied using the Student-t paired and unpaired tests.

Results
The baseline values of free and total IXS and PCS of the 26 patients were signifi cantly higher compared with the 20 normal subjects (Table 1)  the free PCS that was signifi cantly higher, P <0.02), compared to 14 hypertensive non-diabetic patients ( Table 2).
The 4-months re-evaluated data after the use of preprobiotics in 19 patients (5 patients were out of the study for non-compliance of the processing and 2 patients for dialysis entrance) that completed the treatment protocol, showed increased concentrations of free and total IXS and PCS (Table   3). Considering renal function (7 patients in stage 2-3 CKD vs 12 patients in stage 4-5 CKD) the use of pre -probiotics increased the concentration of free and total PCS and IXS in all conditions, while remaining signifi cantly higher in patients at stage 4-5 rather than in the ones at the stage 2-3 (Tables 4,5 ). Considering the 9 diabetics patients and the 10 hypertensive non-diabetics patients, the use of pre -probiotics increased the free and total IXS and PCS serum concentrations, remaining signifi cantly higher in diabetics rather than in hypertensive patients (Tables 6,7). In all periods, both baseline and after the uptake of pre-probiotics, the other measured parameters didn't change, except serum PTH that decreased signifi cantly and Calcium increased even if none signifi cantly (Table 8).

Discussion
Decades ago, nephrologists already thought that intestinal       putrefaction products, in particular " the indican", were detectable in the blood of patients with "renal failure" and they played an important role in renal failure symptoms [13,14]. These studies showed that the concentration of indican a tryptophan derived from the oxidation product of indole, was higher in the blood of uremic patients rather than in the control subjects , and that several other aromatic compounds as phenols, cresols or aromatic oxyacids would produce strong oxidizing reactions up to trigger the formation of IXS and PCS [13,14]. Therefore our data seem to confi rm what has already been reported by Wu et al., that IXS and PCS signifi cantly increase with decreasing renal function and they can be regarded as valid markers for the progression of CKD [5]. This and between concentrations of PCS and IXS [5,9]. The data of our study confi rmed this theory, so we agree that from a in an increase of their production and urinary excretion [3].
Therefore the concentration of serum IXS and PCS in uremic patients can be decreased by switching to a low-protein diet [6]. IXS and PCS are metabolites of tyrosine and phenylalanine, and they are converted to acid 4-hydroxyphenylacetic by intestinal bacteria, before being decarboxylated [7]. The main bacteria that contribute to the IXS and PCS rot are both aerobic (predominantly enterobacteria) and anaerobes (especially Clostridium perfringens) [8]. During the CKD, changes in the intestinal fl ora stimulate the overgrowth of specifi c bacteria that are producers of IXS and PCS [8]. The administration of antibiotics reduces its urinary excretion, as a result of bacterial clearance that produces them [15,16]. However, they can be reduced by orally administration of AST-120 absorbent (not sold in Italy), with a consequent serum decrease of IXS and PCS [18]. The gastric acids suppression, using omeprazole, promotes malabsorption of proteins and fermentation, resulting in an increase of their production [11]. Other environmental factors, such as the use of pre-probiotics may help to change the bacterial fl ora and thereby reduce the putrefaction which is the basis of the IXS and PCS production and absorption. Therefore, a diet with a small amount of animal protein [6], as well as an extra administration of Lactobacillus [16][17][18], decreases the IXS and PCS production. In our experience, an observation period of 4 months of daily intake of pre-probiotics associated with a low-protein diet (0.6-0.8 g of proteins per kg of body weight, based on the CKD stage), was not suffi cient to prove the effi cacy in all CKD stages; it is our opinion that their use should start and carried on for indefi nite time, in the early stages of CKD (stages 2-3) and that it would be useless in later stages of CKD (stages 4-5). 19 patients after 4 months of pre-probiotic intake showed a signifi cant reduction in PTH and a slight increase in serum total calcium despite therapy had remained unchanged since the beginning of the study. In this context the work of Campbell JM noted that bacteria of probiotic component, produce short chain fatty acids which decrease PTH followed by an increase in Calcium absorption via their solubilisation [19]. So in our opinion pre-probiotics could help the therapeutical strategy against osteodistrophy, during the various stages of CKD, in combination with vitamin D or other calcimimetics .

Conclusions
In conclusion IXS and PCS can be considered as kidney function markers as well as have systemic toxic effects. Diabetes seems to increase the concentration of the two metabolites. The use of pre-probiotics should be started in the early stages of kidney failure and certainly for periods longer than four months. Pre-probiotics could aid in preventing renal osteodystrophy.