Probiotics and Antimicrobial Proteins

https://doi.org/10.1007/s12602-018-9509-5

Effects of Synbiotics and Probiotics Supplementation on Serum Levels

of Endotoxin, Heat Shock Protein 70 Antibodies and Inflammatory
Markers in Hemodialysis Patients: a Randomized Double-Blinded
Controlled Trial
Neda Haghighat 1 & Majid Mohammadshahi 1,2 & Shokouh Shayanpour 3 & Mohammad Hossein Haghighizadeh 4

# Springer Science+Business Media, LLC, part of Springer Nature 2019

Abstract
The aim of this study was to investigate the effects of probiotic and synbiotic supplementation on serum inflammatory markers,
endotoxin, and anti-HSP70 in hemodialysis (HD) patients. This study was a randomized, double-blind, placebo-controlled trial.
Seventy-five hemodialysis patients who met the inclusion and exclusion criteria were randomly assigned for 12 weeks to one of
the three arms: synbiotics [n = 23; 15 g of prebiotics, 5 g of probiotic powder containing Lactobacillus acidophilus T16,
Bifidobacterium bifidum BIA-6, Bifidobacterium lactis BIA-6, and Bifidobacterium longum LAF-5 (2.7 × 107 CFU/g each)],
probiotics [n = 23; 5 g probiotics as in synbiotic group with 15 g of maltodextrin in the sachet as placebo], and placebo [n = 19;
20 g of maltodextrin in the sachet]. Blood and feces were collected at baseline and after intervention. Serum high sensitive C-
reactive protein (hs-CRP), interleukin-6, endotoxin, and anti-heat shock protein 70 antibodies (anti-HSP70) were measured. The
number of fecal colonies was determined using the plate-counting method. The mean serum level of hs-CRP, anti-HSP70, and
endotoxin decreased significantly between groups (p = 0.007, p = 0.037, and p = 0.036, respectively). For the synbiotic group, the
mean changes in hs-CRP and IL-6 were significantly lower than for the placebo (p < 0.001 and p < 0.001, respectively) and
probiotic group (p = 0.011 and p = 0.008, respectively). Anti-HSP70 mean changes in the synbiotic and probiotic groups differed
from the placebo group (p = 0.002 and p = 0.013, respectively). Administration of synbiotics was more effective than probiotics
for improvement of inflammatory markers, endotoxin and anti-HSP70 serum levels. Trial registration number:
IRCT2017041233393N1

Keywords Synbiotics . Probiotics . Hemodialysis . Inflammation . Anti-HSP70

Introduction failure (CKD). In recent years, an imbalance in gut microbiota

has emerged as a factor contributing to systemic inflamma-
Chronic inflammation is a risk factor for cardiovascular dis- tion. Gut microbiota consists of trillions of microorganisms
ease, which is the main cause of mortality in chronic kidney with dominant gram-negative and gram-positive phyla.
Increased levels of ammonia in CKD patients increase the
pH of the stool, induce the growth of aerobic bacteria (gram-
* Majid Mohammadshahi
shahi334@gmail.com negative), and decrease levels of beneficial bacteria (gram-
positive) such as Bifidobacteria and Lactobacillus [1]. In ad-
1
dition to altered composition of the gut microbiome, the dis-
Nutrition and Metabolic Diseases Research Center, Ahvaz
ruption of the intestinal epithelial barrier structure and func-
Jundishapur University of Medical Sciences, Ahvaz, Iran
2
tion occurs in CKD patients. These abnormalities lead to
Hyperlipidemia Research Center, Ahvaz Jundishapur University of
translocation of bacteria and endotoxins, which contributes
Medical Sciences, Ahvaz, Iran
3
to systemic inflammation, uremic toxins, and various other
Department of Nephrology, Golestan Hospital, Ahvaz Jundishapur
morbidities in this population [2].
University of Medical Sciences, Ahvaz, Iran
4
Oxidative stress and inflammation induce the expression
Department of Biostatistics and Epidemiology, Faculty of Public
and release of HSPs, which are intra-cellular proteins. Their
Health, Ahvaz Jundishapur University of Medical Sciences,
Ahvaz, Iran upregulation protects cells under stressful conditions. In vitro

studies indicate that HSPs, especially HSP70, play an impor-
tant role in the protection against cell damage caused by an
increase in urea [3]. An emerging body of data supports the
role of circulating HSPs and their antibodies on inflammatory
response as predictors of atherogenesis [4]. Studies have
shown that increased concentrations of several anti-HSP anti-
bodies in patients with end-stage of renal disease on or off
dialysis. Musial et al. found that antibodies against HSPs in-
creased in dialyzed patients versus the controls [5]. Anti-
HSP70 was significantly higher in HD patients than in auto-
mated peritoneal dialysis (APD) patients. Thus, HD patients
may more susceptible to cardiovascular disease than APD
patients [6].
Strategies aimed at lowering systemic inflammation and
endotoxins as well as the use of probiotics and prebiotics to
normalize the gut microbiome and epithelial barrier disruption
could be beneficial [2]. Probiotics are live microorganisms
that improve the gut microbiota profile by decreasing the aer-
obic microorganism count and production of uremic toxins.
Prebiotics are non-digestible carbohydrates that improve the
growth and activity of probiotics. A combination of probiotics
and prebiotics (synbiotics) may result in synergistic effects on
gastrointestinal health which may indirectly benefit the in-
flammation in dialysis patients [7]. Phosphates, potassium
binders, and dietary restriction on potassium-rich foods, in-
cluding fruits and vegetables, may be prescribed to avert
hyperkalemia will have an increased affect to the gut
microbiome because fruits and vegetables are major sources
of indigestible complex carbohydrates (fiber) that are the main
nutrients for normal colonic bacteria [8].
This study investigated the effects of synbiotic and probi-
otic supplementation in hemodialysis patients. Although there
is evidence of the beneficial effects of oral supplementation
with prebiotics, probiotics, and synbiotics in CKD patients on
or off of dialysis [8], few studies have demonstrated the ben-
efits of synbiotics on inflammation and endotoxins, particu-
larly for hemodialysis patients. Furthermore, previous studies
on the effects of inflammation on anti-HSP70 have focused on
increasing intra-cellular HSPs in response to stress and are
mainly in vitro experiments [9]. We hypothesize that the
anti-inflammatory effect of synbiotics and probiotics will de-
crease anti-HSP70 levels in serum.
Materials and Methods
Trial Design
The study was a double-blind, randomized, placebo-
controlled clinical trial. The study was conducted according
to the guidelines of the Helsinki Declaration and all proce-
dures involving human patients were approved by the Ethics
Committee of Ahvaz University of Medical Sciences
Probiotics & Antimicro. Prot.
(university ethics code: IR.AJUMF.REC.1395.812). Written
informed consent was obtained from all patients before initi-
ating the study. The study was registered as a clinical trial with
the Iranian Registry of Clinical Trials (registration number:
IRCT2017041233393N1, http://www.irct.ir).
Participants
The 75 clinically stable hemodialysis (HD) patients with arte-
riovenous fistula aged 30 to 65 years receiving HD thrice
weekly at dialysis centers of Ahvaz University of Medical
Sciences for at least 3 months prior to the onset of the study
were enrolled. Dialysis duration was 3 to 4.5 h per session,
three times per week, with a blood flow of 250 mL/min and a
dialysate flow of 500 mL/min. Patients who had undergone
kidney transplants or were likely to receive a transplant, those
medically diagnosed with severe infections, diabetes, malig-
nancy, chronic liver disease, Crohn’s disease, or ulcerative
colitis, those using a central catheter for hemodialysis access,
had amputated limbs, were pregnant and patients using cata-
bolic drugs, antioxidant vitamin supplements, prebiotics,
probiotics, synbiotics, or antibiotics within 1 month of study
commencement were excluded.
Protocol
This was a double-blind randomized placebo study. Patients
were randomly assigned to one of the three groups. The first
group received synbiotics (n = 25), the second received
probiotics (n = 25), and the third received a placebo (n = 25).
Patients were allocated to a study group by simple randomi-
zation using a computer-generated random binary list. The
nephrologist performing the original visit was responsible
for enrollment and randomized group assignment. Neither
the patients, the nephrologists, nor the nutritionist performing
further patient evaluation were aware of the group assignment.
For all patients, the baseline clinical evaluation included
anthropometric examination, body composition, and blood
chemistry assessment with serum anti-HSP70, endotoxin,
hs-CRP, and IL6 measurement. The frequency of gastrointes-
tinal symptoms was assessed using the gastrointestinal symp-
tom rating scale (GSRS). Dietary intake of energy, macronu-
trients, and fiber were monitored by 24-h food recall for two
weekdays and one weekend day and physical activity levels
were assessed by the international physical activity question-
naire (IPAQ). The IPAQ and GSRS were obtained at the be-
ginning and end of the study. A nutritionist completed the
questionnaires by direct interview. These daily nutrient intakes
were determined using Nutritionist 4 (N-squared Computing;
USA). All participants provided the 3-day food recalls on
weeks 0, 3, 6, 8, and 12 of intervention (Table 2).
Throughout intervention, the patients were requested to main-
tain stable dietary intakes, physical activity, and medication
Probiotics & Antimicro. Prot.
and not to consume any supplements other than the one pro-
vided to them by this trial.
Intervention
After basal records, patients were provided symbiotic, probi-
otic, or placebo supplementation to be taken at home. The
supplements were prescribed for 12 weeks as 5 g of powder
to be dissolved in water four times a day with meals. As
reported by the manufacturer, each synbiotic sachet contained
Lactobacillus acidophilus T16 (IBRC-M10785),
Bifidobacterium bifidum BIA 6, Bifidobacterium lactis BIA-
6, and Bifidobacterium longum LAF-5 (2.7 × 107 CFU/g
each) in sachets and 15 g of prebiotics, including three fiber
types: 5 g of fructooligosaccharides (FOS), 5 g of galacto-
oligosaccharides (GOS), and 5 g of inulin.
The probiotic group ingested 5 g of the same probiotic
powder as the synbiotic group and 15 g of maltodextrin pow-
der instead of prebiotics as the placebo in the sachets. The
placebo powder was comparable in color, texture, and taste
to the synbiotics and probiotics, but contained only maltodex-
trin. Supplement packaging was done by Tak Gen Zist
Pharmaceutical Company.
The main criteria for selecting the bacterial strains in the
synbiotic and probiotic formulation was a decrease in hs-CRP,
IL-6, and endotoxin levels. On the basis of these criteria,
strains from the Lactobacillus and Bifidobacteria genera were
considered as suitable candidates for the purpose of this trial
[10–13]. The prebiotic component and dosage were based on
mechanistic benefits described in previous studies [14]. To
prevent side effects of high-dose synbiotics, such as flatulence
and bloating, which have previously been reported [10], we
advised patients to consume the synbiotic, probiotic, and pla-
cebo supplements in four separate doses (5 g per serving).
Treatment Adherence
Participants were given synbiotics, probiotics, or a placebo
each week for consumption and were instructed to return all
unused supplements at each visit. The remaining supplements
were counted to determine the number of supplement sachets
taken by the participants. To increase compliance, all subjects
received short messages on their cell phones reminding them
to take the supplements each day and it was suggested that
they be consumed in four separate doses. To monitor treat-
ment adherence, stool samples were collected from patients at
baseline and the end of the study to investigate changes in the
fecal microbiota colonies.
Biochemical Analyses
Blood samples were drawn from each subject in the morning
after 12 h of overnight fasting before the midweek dialysis
treatment using the slow flow/stop pump technique. The se-
rum was separated (15 min; 3000×g; 4 °C) and stored at −
80 °C until analysis. Circulating levels of serum anti-HSP70
antibodies were assessed using an enzyme-linked immunosor-
bent assay (ELISA) kit (ab133063; Abcam; USA) with intro-
and inter-assay CVs of < 10%. The serum level of IL-6 (CK-
E10140; Eastbiopharma; China), hs-CRP (CAN-CRP-4360;
Biochem; Canada), and endotoxins (CK-E10840;
Eastbiopharma; China) were measured by using commercially
available ELISA kits with intra-assay and inter-assay coeffi-
cients of variation of 10% and 12%, respectively. The number
of Lactobacillus and Bifidobacterium colony-forming units
(CFU) in the feces was determined in a fecal sample collected
by spontaneous evacuation.
Statistical Analysis
The Kolmogorov–Smirnov test was used to test the distribu-
tion of variables and the results were expressed as mean ± SD.
The Leven test was used to determine the equality of variance.
For comparison of quantitative variables at the beginning and
end of the study and to compare the mean change in these
variables during the study, one-way ANOVA or the
Kruskal–Wallis test were used between groups. The changes
between groups were compared using the Bonferoni post-hoc
pairwise comparison. To compare variations in the quantita-
tive variables in each group, the paired t test or Wilcoxon test
was used. Repeated-measures ANOVA between groups was
used to compare the mean dietary intake within groups for
time effect, interaction of time, and intervention. The p value
of 0.05 was considered statistically significant and analysis
was performed in SPSS 19.0.
Results
Characteristics of Patients
The baseline characteristics of the 75 participants who com-
pleted the study are shown in Table 1. From the initial 75
patients, 10 (2 synbiotic, 2 probiotic, and 6 control) withdrew
from the study. A total of 65 patients who met the inclusion
and exclusion criteria were included in the final analysis. The
mean age was 48.04 ± 2.10 for the synbiotic group, 46.21 ±
2.39 for the probiotic group, and 45.47 ± 2.46 for the control
group. Figure 1 is a consort flow chart of the study. There were
no significant differences in the baseline characteristics be-
tween groups. Repeated-measures ANOVA (Greenhouse–
Geisser correction) between groups showed no significant
changes (p > 0.5) in dietary intake within or between groups
(Table 2). No serious adverse effects, especially gastrointesti-
nal symptoms, were reported during the study. The global
profile of the fecal microbiota of patients was altered by the
 Probiotics & Antimicro. Prot.

Table 1 Baseline characteristics

of participants Variable1 Placebo (n = 19) Probiotic (n = 23) Synbiotic (n = 23) p value2

Age (years) 45.47 (10.76) 46.21 (11.49) 48.04 (10.11) 0.72

BMI 23.38 (4.31) 22.12 (3.81) 23.85 (5.44) 0.42
Gender Male 10 (52.6) 12 (52.2) 12 (52.2) 0.99
Female 9 (47.4) 11 (47.8) 11 (47.8)
Kt/V
Baseline 1.57 (0.28) 1.46 (0.25) 1.37 (0.29) 0.070
Week 12 1.54 (0.22) 1.41 (0.23) 1.39 (0.19) 0.058
GIS score
Baseline 11.26 (3.31) 11.13 (2.76) 10.95 (3.67) 0.95
Week 12 11.68 (2.61) 10.34 (2.7) 10.3 (2.45) 0.92
Physical activity 0.63

METs-h/day
Baseline 24.2 (2.6) 24.3 (2.6) 24.1 (2.3)
Week 12 24.3 (2.4) 24.4 (2.4) 24.2 (2.1)
BUN (mg/dl) 60.74 (20.35) 59.04 (15.11) 58.82 (14.70) 0.92
Cr (mg/dl) 6.33 (3.36) 6.02 (2.40) 7.11 (2.84) 0.41
Duration of hemodialysis, no (%) 0.803
6 m–23 m 4 (21.1%) 5 (21.7%) 5 (21.7%)
24 m–59 m 4 (21.1%) 5 (21.7%) 8 (34.8%)
60 m–120 m 11 (57.9%) 13 (56.5%) 10 (43.5%)
Diabetes (%) 12 (63.2%) 15 (65.2%) 16 (69.6%) 0.907
Hypertension (%) 12 (63.2%) 19 (82.6%) 18 (78.3%) 0.331

BMI, body mass index; GIS, gastrointestinal symptoms; BUN, blood urea nitrogen; Cr, creatinine; pH, potential of
hydrogen; METs, metabolic equivalents. 1 Mean (SD) for quantitative and frequency (%) for qualitative variables.
2
Using one-way ANOVA or chi-square

consumption of the synbiotic or probiotic sachets compared to
the placebo.
Primary Outcome
As shown in the Table 3, the results revealed that, at the end of
12 weeks of intervention, there was a significant decrease in
anti-HSP70 in the synbiotic group compared to baseline (p =
0.008); however, no significant changes were observed in the
probiotic (p = 0.19) and placebo (p = 0.08) groups. Furthermore,
the present study found that there was a significant mean change
in anti-HSP70 in the synbiotic and probiotic groups compared to
the placebo (p = 0.002 and p = 0.01 respectively).
Secondary Outcomes
The inflammatory indices in all groups are shown in Table 3.
At the beginning of the study, there was no significant differ-
ence between groups for inflammatory biomarkers and endo-
toxin. We found significant differences in the mean hs-CRP
and endotoxin values between groups at the end of study (p =
0.007 and p = 0.036, respectively). The administration of
synbiotics significantly decreased the hs-CRP compared to
the placebo (p = 0.005). The change in mean hs-CRP in the
synbiotic group was significantly different from the probiotic
and placebo groups (p = 0.01 and p < 0.001, respectively).
The mean IL6 at the end of the study was not significantly
different between groups (p > 0.05). The mean IL6 decreased
significantly in the synbiotic and probiotic groups compared
to the placebo group (p < 0.001 and p = 0.01, respectively).
The mean endotoxin in the synbiotic group was significantly
lower than in the placebo at the end of the study (p = 0.032).
The mean changes in the synbiotic group were significantly
different from the placebo (p = 0.038).
Discussion
An increasing number of studies show that HSP and anti-HSP
are present in serum and are elevated in some chronic diseases
[4]. This study assessed whether or not serum anti-HSP70
would decrease with the use of synbiotics or probiotics as
anti-inflammatory agents. The synbiotic supplementation in
our previous study showed a significant decrease in ICAM-1
as a vascular inflammatory marker [11]. In this study, there
were significant decreases in serum hs-CRP, IL6, endotoxin,
Probiotics & Antimicro. Prot.

Fig. 1 The consort flow diagram

Assessed for eligibility
indicating patient enrollment into
the synbiotic, probiotic, and
placebo group (n = 90)

Enrollment
Excluded (n = 15)

Not meeng inclusion criteria

(n = 7)

Refused to parcipate
(n = 8)

Randomized (n = 75)
Allocation

Allocated to probioc Allocated to synbioc Allocated to placebo

(n = 25) (n = 25) (n = 25)

Lost to follow up Lost to follow up Lost to follow up

Follow up

(n = 0) (n = 1) (Candidate to (n = 4) (Infecon)
transplant)
Disconnued (n = 2) Disconnued
intervenon (n = 2) (n = 1) Disconnued intervenon
(errac supplement intervenon (medicaon
consumpon) (medicaon changing)
changing)

Analyzed (n = 23) Analyzed (n = 23) Analyzed (n = 19)

Analysis

Excluded from analysis Excluded from analysis Excluded from analysis

(n = 0) (n = 0)
(n = 0)

and anti-HSP70 levels. Probiotic supplementation showed a
decrease in hs-CRP, IL6, and endotoxin and a significant de-
crease in anti-HSP70. These changes suggest that there was a
decrease in inflammation, which may explain the decrease
observed in serum anti-HSP70 in the synbiotic and probiotic
groups.
Probiotic bacteria, particularly the gram-positive
Lactobacillus and Bifidobacterium species, are known to
modify GI microbiota and have been shown to reduce
systemic inflammation and serum endotoxins [12, 13, 15,
16]. However, this data is scarce among CKD and HD pa-
tients. Wang et al., in an RCT on 39 peritoneal dialysis pa-
tients, demonstrated that probiotic supplementation
(B. bifidum, B. catenulatum, B. longum, L. plantarum) for
6 months decreased TNF-α, IL-5, IL-6, and endotoxin and
increased the anti-inflammatory cytokine IL-10 [12].
Probiotics containing E. faecalis, Clostrydium butyricum,
Bacillus mesentericus, or Lactobacillus johnsonii reduced
 Probiotics & Antimicro. Prot.

29.3 (5.4)
55.6 (5.3)
12.6 (2.4)

13.1 (6.9)
30.2 (5.2)
12.3 (2.4)

Data are presented as mean (SD) for dietary intakes. Obtained from the repeated measure ANOVA test. EI, energy intake; carb, carbohydrate; PRO, protein; SFA, saturated fatty acid; MUFA, mono-
1 (.2)

4.6 (.8)
serum endotoxin levels in other populations [13, 15]. Sabico
et al. (2018) found that diabetic patients with daily multi-strain

5
probiotic supplementation of B. bifidum W23, B. lactis W52,

29.2 (4.4)

12.3 (2.4)

12.6 (2.7)
12.2 (3.3)
L. acidophilus W37, L. brevis W63, L. casei W56,

.9 (.1)

4.6 (.7)
53.6 (6)

30.9 (9)
4 L. salivarius W24, L. lactis W19, and L. lactis W58 for
6 months showed significantly reduced endotoxin, TNFα,
IL-6, and CRP levels [16]. Another study showed that similar
55.1 (6.1)
12.4 (2.2)

12.2 (2.1)
12.7 (1.4)
31 (6.3)
1.1 (.2)

4.7 (.9)
29.1 (4)
probiotic supplementation for 3 months failed to reduce endo-
toxins in diabetic patients [17].
3

Probiotic studies showed an array of strains and dosages.

55.4 (5.9)
12.7 (2.4)

13.2 (1.9)
1 (.2)

4.7 (.9)
29.7 (4)

13.5 (3)
30.3 (5) Adequate dosing of probiotics, due to the diversity of GI sur-
Placebo (n = 19)

vival rates of probiotic strains, is not known yet. In other

2

chronic diseases, therapeutic benefits of probiotics are highly

29.1 (5.8)

13.3 (1.2)
57 (6.8)
12.4 (2.4)

12.7 (5.1)
30.4 (5.3)
1 (.1)

4.7 (.9) strain specific, indicating that a specific mix of bacterial gen-
era and dosages may be required for the CKD population [18].
1

Thus, in the present study, insignificant decreases in hs-CRP,

28.4 (5.4)

12.4 (2.3)

11.1 (2.4)
12.5 (8.1)
55.7 (6.8)

29.3 (6.8)

IL-6, and endotoxin may be due to the relatively short treat-

.9 (.1)

4.6 (.8)

ment period or the different dosage and strain of probiotic

Dietary intakes of study participants throughout the study in baseline (1), week 3 (2), week 6 (3), week 8 (4), week 12 (5)

used. In agreement with our results, Borges et al. (2018) tested

5

3 months of probiotic supplementation (Streptococcus

13 (2.8)
29.1 (4.4)
54.8 (6.6)
12.5 (2.2)

29.6 (8.7)
.9 (.1)

4.6 (.7)
12 (4)

thermophilus, L. acidophilus, B. longum, 90 billion CFU/

day) and observed no effect for hs-CRP and IL-6 in HD pa-
4

tients [19]. Natarajan et al. (2014) found no effect on CRP

13.1 (1.6)
29.2 (3.9)
52.6 (5.8)
12.4 (2.2)

33.4 (5.3)
12.8 (2.5)
.9 (.2)

4.7 (.8)

level for 22 HD patients after 2 months of probiotic supple-

mentation (S. thermophilus KB 19, L. acidophilus KB 27, and
3

B. longum KB 31, 180 billion CFU/day) [20].

13.2 (2.7)
28.2 (4.1)
54.8 (5.8)

30.8 (5.2)
13 (2.1)

The synbiotic treatment was more effective in compari-

1 (.2)

4.7 (.8)
12.9 (2)

son with the probiotic treatment for decreasing hs-CRP,

Probiotic (n = 23)

IL6, and endotoxin. The serum levels of hs-CRP were sig-

2

nificantly more effective. The possible explanation is that

13.1 (4.8)
12.8 (2.1)
57.1 (6.5)

29.7 (5.1)
29 (3.7)

4.7 (.8)
1 (.1)

12.5 (1)

administration of probiotics and prebiotics as a synbiotic

with a synergic and incorporated influence on gut flora
1

stability and gut milieu may increase efficiency, as was

29.5 (5.4)
52.8 (6.1)
12.2 (2.3)

30.4 (6.3)

4.6 (.8)
.9 (.2)

seen in the present study. Although consistent with our

13.7 (8)
13.1 (2)

findings, it has been shown that probiotics alone have prov-

5

en ineffective in attenuating systemic inflammation, uremic

29.4 (8.8)

12.7 (2.7)
28.4 (4.4)
53.6 (6.3)

11.8 (3.9)
12.3 (2.4)

toxicity, and adverse clinical outcomes in CKD patients

4.5 (.7)
.9 (.1)

[21, 22]. In a recently published review, Vaziri et al. ex-

unsaturated fatty acid; PUFA, poly unsaturated fatty acid

plained that uremia, dietary, and medicinal regimens asso-

4

ciated with CKD create an unfavorable biochemical milieu

31.6 (5.5)
28.6 (4.1)
54.5 (5.5)

12.34 (1.1)
12.7 (1.1)
.9 (.1)

4.6 (.8)
12.4 (2)

for the introduction of probiotic bacteria.

HD patients who have significant uremia levels recorded
3

higher urea influx into the intestinal lumen. Disruption of the

29 (4.7)
55 (5.9)

30.6 (5.3)
13 (1.9)
12.6 (2.1)

12.1 (3.5)

epithelial barrier caused by the administration of probiotics

1 (.2)

4.7 (.8)
Synbiotic (n = 23)

made it impossible to improve gut dysbiosis [2]. Moreover,

the greater improvement in the synbiotic group may have been
2

caused by the effect of the prebiotics. Prebiotics play an im-

31.8 (5.8)
29 (5.5)
57.1 (6.4)
11.8 (1.8)

12.9 (5.3)
0.9 (0.2)

4.6 (.9)
13.1 (1)

portant role by improving the production of SCFAs, the gut

barrier, alterations in intestinal bacteria, and control of the
1

systemic and mucosal immune responses [7]. In the present

PRO, gr/kg
EI, kcal/kg

MUFA, %
PUFA, %
Variables

study, the synbiotic group consumed exclusively 15 g of dif-

Carb, %

SFA, %
PRO,%

FAT, %
Table 2

ferent prebiotics, GOS, FOS, and inulin, which maintained

fermentation throughout the colon. In agreement with our
Probiotics & Antimicro. Prot.

Table 3 Changes in biochemical parameters in treatment groups

Variable1 Placebo (n = 19) Probiotic (n = 23) Synbiotic (n = 23) p2

hs-CRP (ng/ml) Baseline 5413.1 (2496.7) 5741.9 (1782.2) 6154.7 (1984.9) 0.51
Week 12 6418.2 (1469.2)c 5366.4 (1901.6) 4580.1 (1654.9)a 0.007
Changes 1005.0 (1488.5) b, c − 375.5 (1198.8) a, c − 1574.6 (1414.1) a, b < 0.001
p3 0.009 0.14 < 0.001
IL-6 (ng/L) Baseline 135.84 (87.16) 151.06 (71.29) 143.54 (89.19) 0.83
Week 12 144.48 (81.60) 150.63 (70.15) 122.31 (81.82) 0.439
Changes 8.70 (26.50)c − 0.42 (15.80)c − 21.23 (25.35)a, b < 0.001
p3 0.046 0.07 0.001
Anti-HSP70 (ng/ml) Baseline 334.00 (124.96) 304.95 (136.61) 348.30 (149.08) 0.56
Week 12 375.63 (136.37) 287.69 (112.32) 293.95 (107.75) 0.037
Changes 41.63 (100.40)a, b − 17.26 (61.40)b − 54.34 (90.04)a 0.002
p3 0.08 0.19 0.008
Endotoxin (IU/L) Baseline 706.71 (106.31) 638.60 (176.90) 699.94 (215.60) 0.37
Week 12 707.60 (145.26) c 619.46 (169.20) 587.88 (130.99) a 0.036
Changes 0.89 (104.71)c − 19.13 (104.68) − 112.06 (198.09)a 0.028
p3 0.97 0.39 0.028

hs-CRP, high sensitive C-reactive protein; IL-6, interleukin-6; anti-HSP70, heat shock protein 70. 1 Mean (SD). 2 Using one-way ANOVA for hs-CRP,
IL-6, HSP70, and endotoxin. 3 Using paired sample t test. Using Wilcoxon signed-rank test. a, b, c Similar letters show significant differences
between groups

results, prebiotic supplementation in a clinical randomized
controlled trial of 124 HD patients followed for 6 weeks
showed a significant decrease in plasma IL-6, IL-8, hs-CRP,
and lipid profile levels [23]. A recent meta-analysis of RCTs
done on probiotics, prebiotics, and synbiotics in CKD with or
without dialysis revealed that a prebiotic dose of > 5 g/day
was shown to effect microbiota diversity. A threshold dose
of 15–20 g/day may be required to benefit HD patients [18].
Only the probiotic group exhibited a decline in anti-HSP70
levels with no significant change in inflammatory markers,
suggesting the possibility of a direct effect of probiotics on
anti-HSP70. Further studies aimed at elucidating the specific
mechanisms of reducing the serum anti-HSPs level are re-
quired. In vitro studies have shown that probiotics inhibit the
nuclear factor-kB of the inflammatory pathway and at the
same time induce the expression of HSP25 and HSP70 [9].
Intra-cellular HSPs guarantee cell tolerance against a variety
of stressors as a part of the defense system. The release of
HSPs from cells may be caused by damage or through a se-
cretory pathway. The known interaction of HSPs and their
antibodies with the cells of the immune system make them
atherogenesis indicators [24]. There has been some discussion
on the relationship between inflammation marker levels and
serum anti-HSP70 levels [25]. It appears that synbiotic and
probiotic supplementation modulates the immune system and
reduces inflammation. Endotoxins may influence the level of
anti-HSP70 antibodies. In agreement with our findings, recent
randomized clinical trials showed a decrease in the level of
several HSP antibodies by anti-inflammatory agents [26–28].
The most important strength of this study among the few
studies which assess probiotic and synbiotic effects alone in
HD patients is that this is the first clinical trial which compares
synbiotic and probiotic intervention. Moreover, in synbiotic
therapy, we used three prebiotics in order to facilitate fermen-
tation throughout the colon. FOS and GOS completely
fermented in the proximal part of the colon and inulin may
assist the extension of fermentation to the distal part of the
colon [29].
This study has some limitations. Although previous studies
using the real-time PCR sequencing method to measure gut
microbiota have demonstrated that synbiotic or probiotic sup-
plementation improved the gut microbiome profile, the cur-
rent study used additional quantitative testing to assess the
variation of gut microbiota and counted fecal bacteria colo-
nies, which may not reflect a reliable gut microbiome profile.
The short duration of the study limited the statistical power for
detection of changes in parameters in the probiotic and
synbiotic groups.
The current study found some evidence that the
synbiotic supplementation compared to the placebo and
probiotic supplementation was more effective in the im-
provement of cardiovascular risk factors, at least partial-
ly through the attenuation of inflammatory markers.
Probiotic supplementation reduced anti-HSP70 levels. It
is clear that further work is needed to determine the
mechanism of probiotic effects on anti-HSP70 in serum
and whether probiotics directly or indirectly cause
changes in anti-HSP70 levels.

Compliance with Ethical Standards
The study was conducted according to the guidelines of the Helsinki
Declaration and all procedures involving human patients were approved
by the Ethics Committee of Ahvaz University of Medical Sciences (uni-
versity ethics code: IR.AJUMF.REC.1395.812). Written informed con-
sent was obtained from all patients before initiating the study.
Conflict of Interest The authors declare that they have no conflicts of
interest.
Publisher’s Note Springer Nature remains neutral with regard to jurisdic-
tional claims in published maps and institutional affiliations.
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