The Effects of Sacran, a Sulfated Polysaccharide, on Gut Microbiota Using Chronic Kidney Disease Model Rats

Abstract

The aim of this study was to investigate the beneficial effects of sacran, a sulfated polysaccharide, on renal damage and intestinal microflora, in 5/6 nephrectomy rats as a model for chronic kidney disease (CKD). 5/6 Nephrectomy rats were divided into sacran treated and non-treated groups and examined for lethality after 4 weeks. The 5/6 nephrectomy rats were also divided into three groups: sacran treated, non-treated and AST-120 treated groups, and treated orally in a concentration-dependent manner for 4 weeks. Renal function was estimated by biochemical and histopathological analyses. Metagenomic analysis of feces from each group after 4 weeks was also performed and changes in intestinal microflora were compared. The administration of sacran to CKD rats at ≥19 mg/d increased their survival. In addition, the sacran-treated group improved CKD-related parameters in a concentration-dependent manner, and the inhibitory effect of 40 mg/d of sacran was comparable to that of AST-120. The changes in the intestinal microflora of the sacran treated group were positively correlated with an increase in the number of Lactobacillus species, which are known to be rich in beneficial bacteria, and the increment of this beneficial bacteria was negatively correlated with the concentration of indoxyl sulfate, a uremic toxin, in plasma. These results strongly suggest that the oral administration of sacran could contribute to the stabilization of intestinal microflora in CKD rats and to the reduction of oxidative stress as well as the inhibition of progression of CKD.

INTRODUCTION

Uremic toxins are defined as compounds that accumulate in the body due to the progression of renal dysfunction and, in chronic kidney disease (CKD) patients, they can have adverse effects on various uremic symptoms.^1,2) Indoxyl sulfate (IS) is a representative uremic substance, that binds to serum proteins at a binding rate of over 90%.³⁾ It is also produced by the gastrointestinal absorption of indole produced from dietary tryptophan by the action of Escherichia coli and other intestinal microflora, followed by hydroxylation and conjugation with sulfate in the liver.^4,5) IS is largely present in the blood bound to albumin and is excreted via the kidney. In hemodialysis patients, the concentration of IS in the blood can increase up to 40 to 60 times the normal level.⁶⁾ The accumulation of IS induces an increased production of free radicals in tubular epithelial cells and is also known to be involved in increased stress in blood vessels. Therefore, the adsorption of indole in the gastrointestinal tract and its elimination in the feces is considered to be one of the more important and relevant therapeutic strategies for reducing the progression of kidney injury and oxidative stress.^7,8)

Kremezin^® (AST-120), an oral spherical-shaped adsorbent, is a drug that was approved in 1991 to reduce serum IS concentrations by adsorbing indole, a precursor of IS, in the gastrointestinal tract followed by excretion via the feces, thereby improving uremic symptoms and delaying the need for dialysis in CKD patients. However, AST-120 is not recommended for use in CKD patients due to difficulties associated with its administration and side effects that include gastrointestinal symptoms such as constipation. Therefore, the use of AST-120 in patients after the introduction of maintenance hemodialysis therapy is not recommended, despite the fact that IS, which is strongly protein-bound, cannot be effectively removed by ordinary hemodialysis and remains highly concentrated in the blood, even after a hemodialysis session.

As a new beneficial effect of saran, it was recently proposed to be used as an adsorbent of lipids substances in non-alcoholic steatohepatitis (NASH).⁹⁾ Sacran, a type of new sulfated polysaccharide, is a biomaterial with excellent water solubility and safety, and is expected to be used in pharmaceuticals of the future. Thus, if sacran could be used as a health food rather than as a medicine, it might be possible to start its administration at an earlier stage than in the traditional pre-dialysis uremic state, in which AST-120 is prescribed as a uremic adsorbent.¹⁰⁾ Further, fucoidan, a sulfated polysaccharide that is similar to sacran, also has a reno-protective effect by lowering serum creatinine and urea nitrogen levels.¹¹⁾ Fucoidan, a sulfated polysaccharide has also been used as a prebiotic and changes in the intestinal environment, including intestinal microflora, were reported in the pathogenesis of CKD, suggesting that controlling the balance of intestinal microflora is important in terms of preventing the progression of CKD.^12,13)

Given the above information, we initiated a study of the nephroprotective and prebiotic effect of sacran as a function of its concentration as an adsorbent of uremic toxins on CKD rats and compared the findings with those of AST-120 or a non-treatment group.

MATERIALS AND METHODS

Materials

Sacran (Mw: 1.6 × 10⁷) was provided by Green Science Materials Co. (Kumamoto, Japan). All other reagents and solvents were of commercial grade.

Study of Optimal Dosage Based on the Free Intake of Sacran Using CKD Rats

Male Wister rats (7-week-old male, 200–220 g) with 5/6 nephrectomies as CKD rats were purchased from SLC Japan Ltd. (Shizuoka, Japan). After 2 weeks of pre-housing, the rats were divided into two groups: one receiving sacran (0.25%, N = 8) and the other receiving water only (N = 8). Diets were given ad libitum. The lethality of each treatment group at 4 weeks was compared. Animal experiments were carried out with the approval of the Ethics Committee of Sojo University (Permission No. 2020-P-14).

Reno-Protective Study of Sacran by Daily Oral Administration for 4 Weeks Using CKD Rats

Male Wister rats (7-week-old male, 200–220 g) with 5/6 nephrectomies as CKD rats were purchased from SLC Japan Ltd. After 2 weeks of pre-housing, the non-treated group received water, the sacran treated group received 0.5 mL (10 mg/d) and 2 mL (40 mg/d) of a 2% (w/v) sacran solution, and the AST-120-treated group received 1 mL (40 mg/d) of a 4% (w/v) water suspension followed by oral flushing with 1 mL of water at 4 weeks. The diet was ad libitum and each sample was administered daily at 19:00. Animal experiments were carried out with the approval of the Ethics Committee of Sojo University (Permission No. 2020-P-14).

Blood Analyses

At 4 weeks after the administration of each sample, blood was collected and kidneys were removed and stored at −80 °C. Renal function parameters were measured according to previously reported methods.^7,14,15)

Evaluation of the Plasma Antioxidant Potential (PAO) of Sacran

The antioxidant capacity of the obtained plasma was determined using an antioxidant capacity assay kit “PAO” (Nikken-Seil Co., Ltd., Shizuoka, Japan).^16,17)

Observation of Renal Tissue in CKD Rats after Treatment with Sacran

The kidneys of CKD rats at the end of the study were fixed in 10% neutral buffered formalin solution and paraffin-embedded sections (2.5 µg) were prepared and stained with Masson’s trichrome. Tissue images of each sample were obtained using a BZ-X700 (KEYENCE, Osaka, Japan).^9,18,19)

The Evaluation of Indole Adsorption Capacity

A 5 mg/mL aqueous sacran solution and an AST-120 suspension were incubated in 0–300 µg/mL aqueous indole solution at 120 rpm for 24 h at room temperature. The solutions were filtered by centrifugation for 1 min at 6000 rpm in a VIVASPIN 500 (Vivascience AG, Germany), and the filtrates were quantified by determined using a UV spectrophotometer at wavelengths of 270 nm. Equilibrium constants and maximum adsorption volumes were calculated using the following Langmuir’s adsorption isotherm equation.

  

• q: Indole adsorption amount, q_m: Maximum adsorption amount, K: Equilibrium constant, C: Sample concentration

DNA Extraction from Fecal Samples and Bacterial Community Analysis

After 4 weeks of treatment, fecal samples were suspended in the buffer solution of the fecal collection kit (Anicom Holdings, Inc., Japan). Subsequently, the extraction of DNA from the faces and the analysis of the intestinal microflora by the QIIME2 software package were sent to and analyzed by Anicom Holdings. Specifically, the V3–V4 region of bacterial 16S ribosomal RNA genes was subjected to PCR amplification. This region showed the best results for accuracy of taxonomic classification.^19–21)

Histologic Examination of Renal Tissues

Renal abnormalities were evaluated after 4 weeks of treatment. The kidneys were removed, weighed, and fixed with 10% phosphate buffered formalin. The tissue was then dehydrated at room temperature by passage through a graded series of ethanol and then embedded in paraffin. The prepared tissue was cut into 2.5 µm sections and treated with Masson’s trichrome for routine histology and morphometric studies. The ratio of renal tubular degeneration was measured using 20 randomly selected microscopic fields by two independent pathologists who were blind with respect to the experimental data. Tissue sections were examined by light microscopy using BZ-X810 (KEYENCE).

Measurement of Transforming Growth Factor β₁ (TGF-β₁) in Renal Tissue

After homogenization of CKD rat kidney tissue samples (10 mg), total RNA was prepared by the following method. Reverse transcription was demonstrated as described in a previous report.²²⁾ The following specific primers were used: glyceraldehyde 3-phoshate dehydrogenase (GAPDH), F: 5′-GGCACAGTCAAGGCTGAGAATG-3′, R: 5′-ATGGTGGTGAAGACGCCAGTA-3′; TGF-β₁, F: 5′-TGATACGCCTGAGTGGCTGTC-3′, R: 5′-CATTGATTTCCACGTGGAGTAC-3′.

Statistics

The experimental data are presented as the mean ± standard deviation. The statistical significance of differences between groups was analyzed with one-way ANOVA with Tukey–Kramer’s test (Table 1), two-way ANOVA, log-rank test, and least squares regression analysis. For all analyses, p < 0.05 was considered to indicate statistical significance.

Table 1. Effects of Sacran Administration on Renal Function of CKD Model Rats

	Non-treatment	Sacran 10 mg	Sacran 40 mg	AST-120
Body weight (g)	356 ± 13	336 ± 16	343 ± 10	324 ± 7*
SBP (mmHg)	197 ± 16	150 ± 8**	131 ± 5**	141 ± 13**
Plasma Cr (mg/dL)	1.4 ± 0.10	0.9 ± 0.10**	0.8 ± 0.10**	0.8 ± 0.03**
Plasma BUN (mg/dL)	90 ± 8	61 ± 5**	58 ± 5**	62 ± 2**
Plasma IP (mg/dL)	6.4 ± 0.5	6.2 ± 0.1	5.3 ± 0.4**	5.5 ± 0.1**
Plasma IS (µM)	31.6 ± 9.7	22.0 ± 3.2	12.2 ± 5.2**	6.7 ± 1.2**
Plasma PAO (µM)	329 ± 11	385 ± 17**	389 ± 24**	374 ± 12**
TGF-β1/GAPDH	1.21 ± 0.37	1.01 ± 0.25	0.58 ± 0.13*	0.54 ± 0.25*

Data are averages ± standard deviation (n = 4–6 rats per group). * p < 0.05, ** p < 0.01, vs. non-treatment CKD rats.

RESULTS

Evaluation of Indole Adsorption Capacity of Sacran in Vitro

The capacity for sacran to adsorb indole in vitro was compared with that for AST-120, and the results for indole adsorption after 24 h showed a lower adsorption capacity than that for AST-120 (Fig. 1). The maximum adsorption capacity (q_m) for sacran, as calculated from the Langmuir adsorption isotherm equation was about 22% that of AST-120 (q_m of sacran: 29.3 mg/g, qm of AST-120: 129.5 mg/g).

Fig. 1. Evaluation of the Capacity of Sacran and AST-120 to Absorb Sacran

White or black circles denote sacran or AST-120, respectively. Data are averages ± standard deviation (n = 3). ** p < 0.01 vs. AST-120 group. The statistical significance of differences between groups was analyzed with two-way ANOVA. For all analyses, p < 0.05 was considered to indicate statistical significance.

Study of Optimal Dosage by the Free Intake of Sacran Using CKD Rats

We initially investigated the effect of sacran on CKD in 5/6 nephrectomized rats. As shown in Fig. 2a, the survival rate was increased in the treated group compared to the non-treated group. Further, the survival rate was significantly increased in CKD rats in the case of a daily intake of more than 19 mg of sacran (Fig. 2b).

Fig. 2. Lethality in CKD Rats by the Ingestion of Sacran under the Following Conditions (a) with and without Sacran, and (b) with an Average Daily Intake Standard of 19 mg of Sacran

(a) After 2 weeks of pre-housing, the rats were divided into two groups: one receiving sacran (0.25%, N = 8) and the other receiving water only (N = 8). Diets were given ad libitum. The lethality of each treatment group at 4 weeks was compared. The difference in survival rate was determined by log-rank test, and the difference was estimated significant when the risk rate was less than 5%. (b) The daily intake of sacran was measured in the sacran-treated group, which was divided into two groups based on a daily intake of 19 mg, and the mortality rates were compared. The difference in survival rate was also determined by log-rank test, and the difference was estimated significant when the risk rate was less than 5%.

Effects of Sacran on Body Weight, Vital, and Plasma Parameters

Based on the results in the above section, the dose of sacran was set at 10 and 40 mg, and the effect on CKD after 4 weeks of sacran treatment was compared with that of the uremic adsorbent AST-120. As shown in Table 1, following the oral administration of sacran, the recovery of several important renal disease-related parameters was observed, and this recovery was found to be concentration-dependent. Particularly noteworthy was the significant reduction in plasma IS, creatinine (Cr), blood urea nitrogen (BUN) and inorganic phosphorus (IP) levels after 4 weeks of the oral administration of sacran and AST-120 compared to the non-treated group. Further, PAO after 4 weeks was also significantly increased in the sacran treated and AST-120 treated groups compared to the untreated group.

Effects of Sacran on Intestinal Microflora

Although the capacity to adsorb indole for sacran in vitro was smaller than that of AST-120, the reno-protective and antioxidant effects for the sacran treated group were similar to those of the AST-120 treated group. Therefore, we focused on the variation in gut microbiota in the intestinal tract. We extracted DNA from the feces of each sample after 4 weeks of treatment, performed a metagenomic analysis, and compared the changes in the intestinal microflora between the groups. As a result, among the five major bacterial phyla of the human commensal flora, Firmicutes, Bacteroidota, Actinobacteria, Proteobacteria, and Verrucomicrobia, the sacran treated group showed an increasing trend for Bacteroidota and a decreasing trend for Firmicutes compared to the non-treated and AST-120 treated groups (Fig. 3a). More than 80% of the bacteria found in the intestinal microflora are typically comprised of Alistipes, Bacteroides, Clostridium, Dorea, Eubacterium, Faecalibacterium, Lactobacillus, Prevotella, Porphyromonas, and Ruminococcus genera, which exist in only two phyla, Bacteroidota and Firmicutes. Therefore, we next focused on these genera and compared the variation in the number of these genera among the three groups.²³⁾ The results showed a significant increase in the number of Lactobacillus genus only in the sacran treated group compared to the non-treated group (Fig. 3b). Interestingly, there was a negative correlation between the plasma IS concentration and the number of Lactobacillus genus (Fig. 3c, p < 0.05, r = 0.58), and a positive correlation was observed between the number of Lactobacillus genes and plasma antioxidant potential (Fig. 3d, p < 0.05, r = 0.74).

Fig. 3. Effects of Sacran and AST-120 on Intestinal Microflora Using CKD Rats

Change in the bacterial community at the phylum level (a) and change of lactobacillus (b) at the genius level. Correlation between the number of lactobacillus and serum IS (c) or plasma antioxidant potential (d). Data are averages ± standard deviation (n = 4–6 rats per group). * p < 0.05 vs. CKD rats. The statistical significance of differences between groups was analyzed with one-way ANOVA with Tukey–Kramer’s test (b) and least squares regression analysis (c). For all analyses, * p < 0.05 was considered to indicate statistical significance.

Histological Observation of Kidneys Tissue Treated with Sacran

Figure 4 provides information on kidney samples from CKD rats, and sacran treated CKD rats after staining with Masson’s trichrome (MT) (Fig. 4). The Renal fibrosis ratio, as detected by the MT staining of kidney sections, was calculated based on microscopy observations. Approximately 11% of the area was stained in CKD rats, while the positive area had decreased to 7% by the administration of sacran and this decrease was concentration-dependent, indicating that sacran protects the kidney from the progression of CKD. Therefore, these results show that the sacran treated CKD rats had significantly less kidney injuries and this damage regarding renal fibrosis with the increment of TGF-β₁, was concentration dependent, compared with the CKD rats (Fig. 4, Table 1).

Fig. 4. Histological Observation of Kidneys Tissue Treated with Sacran and AST-120

(a) Whale of Masson’s trichrome (MT) staining. (b) MT staining of the enlarged image of kidney (scale bars indicate 200 µm). (c) MT staining of the enlarged image of kidney (scale bars indicate 50 µm).

DISCUSSION

In this study, we report on an investigation of the inhibitory effect of sacran as a function of its concentration, on the progression of CKD. Since fucoidan, has been reported to exert a reno-protective effect and is therefore an effective prebiotic, we started our study using sacran, which is also a sulfated polysaccharide.^11,24) Indole adsorption capacity was evaluated in vitro, and the maximum adsorption capacity (q_m) of sacran was found to be about 22% that of AST-120 (maximum adsorption capacity of sacran q_m: 29 mg/g, maximum adsorption capacity of AST-120 q_m: 130 mg/g) (Fig. 1). The survival rate was found to be increased in the sacran treated group compared to the non-treated group, and the survival rate increased significantly when the daily intake of sacran was 19 mg or higher (Fig. 2). Based on these results, the daily dose of sacran was set at 10 and 40 mg, and the inhibitory effect at these concentrations on CKD was compared with that of AST-120. The results show that both biochemical and antioxidant parameters were improved in the sacran treated group compared with non-treated group and that this improvement was concentration-dependent. Indeed, reflecting on the fact that the in vitro indole adsorption capacity of sacran was lower than that of AST-120, the IS lowering effect by sacran appears to be smaller than that by AST-120 (Table 1). On the other hand, when considering the inhibitory effect of sacran on NASH, it is quite possible that there are other reasons for the lowering effect of blood indole concentrations that was observed in the in vivo studies. Therefore, we next investigated the relationship between the administration of sacran and changes in the intestinal microflora as a potentially new inhibitory mechanism of CKD.

Interestingly, we found that sacran caused an increase in the Bacteroidota/Firmicutes (B/F) ratio in the intestines of CKD model rats by approximately 1.2-fold (untreated group B/F ratio = 0.56, sacran treated group B/F ratio = 0.67) (Fig. 3a). The B/F ratio is frequently used as an indicator of chronic disease, and, as an indicator, the higher the ratio, the better. In fact, a fermentable dietary fiber composed of high amylose maize-resistant starch type 2 (HAMRS2) has been shown to cause an increase in the B/F ratio and, in CKD rat models, ultimately resulting in a markedly improved kidney function.^25,26) In addition, the number of Lactobacillus genera was significantly increased in the sacran group, compared to the non-treated group. Lactobacillus genera have been reported to generally act directly on intestinal cells, where they increase the expression of tight junction proteins and regulate the membrane permeability of substances.²⁷⁾ It has also been suggested that a decrease in the number of Lactobacillus genera in the intestines of patients with renal failure may increase the membrane permeability of uremic precursors such as indole.²⁸⁾ In fact, we also observed a significant negative correlation between the number of Lactobacillus genera and plasma IS concentration (Fig. 3c, p < 0.05, r = 0.58). These results suggest that a decrease in the number of Lactobacillus genera not only leads to a decrease in tight junction function but also to an increase in the uptake of uremic substances such as indole, which may contribute to the progression of CKD. It has been reported that the administration of probiotics including Lactobacillus to 5/6 nephrectomized CKD rats prolonged their life span, caused a decrease in plasma BUN levels, and that the administration of probiotics including Lactobacillus to peritoneal dialysis patients resulted in a decrease in the levels of blood inflammatory cytokines and endotoxins. In addition, the administration of probiotics containing Lactobacillus to peritoneal dialysis solutions has been reported to reduce the plasma levels of inflammatory cytokines and endotoxins, indicating that Lactobacillus based gut microbiota may be a novel therapeutic agent for the treatment of uremia in CKD patients.^28,29) Although an increase in Lactobacillus genera has been reported in CKD model rats treated with AST-120,³⁰⁾ this is the first comparison of a new prebiotic, i.e., sacran, and the rate of increase in Lactobacillus genera. In this study, the increase in Lactobacillus genera caused by AST-120 was less than that of sacran. Although sacran has a lower capacity to adsorb indole than AST-120, it may contribute to the stabilization of the intestinal microflora, thereby inhibiting the progression of CKD.^31,32) In addition, a significant positive correlation was observed between the Lactobacillus genera and PAO (Fig. 3d, p < 0.05, r = 0.74), suggesting that a sacran treatment may contribute not only to the progression of renal failure but also to a reduction in the level of oxidative stress. Furthermore, histological observations of kidney sections after 4 weeks of the sacran treatment showed less renal tissue damage and less renal fibrosis compared to the non-treated group (Fig. 4). In fact, the production of TGF-β₁, which plays a central role in the pathogenesis of renal fibrosis, was also suppressed by sacran treatment⁸⁾ (Table 1). These results suggest that the oral administration of sacran may contribute to the inhibition of CKD progression and a reduction in oxidative stress by stabilizing the intestinal microflora, especially an increase in Lactobacillus genera, in addition to its indole adsorption capacity. On the other hand, as previously reported, a trend towards increased Lactobacillus species was observed in the case of AST-120 administration,³⁰⁾ but this increase was not significant. This may be due to the variability of the data of individual rats, and further studies with a larger number of cases will be needed to confirm this conclusion.

The quantification of indole in the feces of CKD rats showed that the fecal excretion of indole tended to be increased by sacran and this increase was concentration dependent (data not shown). Interestingly, although pre-tested, the amount of indole excreted in feces tended to be higher than that in the AST-120 group. In addition to the adsorption effect of sacran in the gastrointestinal tract, the high viscosity and water absorption of sacran may inhibit the absorption of uremic substances in the gastrointestinal tract. In fact, due to its high water-holding capacity, which is 5 times larger than that of hyaluronic acid,³³⁾ sacran has been applied as a transdermal drug carrier for use in the treatment of atopic dermatitis and wound healing.^34,35) In the future, it will be necessary to investigate the adsorption capacity and water absorption of uremic substances using sacran solutions with different viscosities to clarify the details of the mechanisms responsible for these observations.

In the present study, we found that the sulfated polysaccharide, sacran reduced the concentration of indole, a uremic substance, in feces by its adsorption in the gastrointestinal tract and its excretion in the feces by inhibiting its absorption due to its viscosity, thereby efficiently reducing oxidative stress and inhibiting the progression of CKD by acting as a prebiotic. While AST-120, has been approved for use as a uremic toxin adsorbent, it is only used for short periods of time, and typically starts when uremic symptoms appear before dialysis induction and stopping when dialysis is initiated for insurance purposes.³⁶⁾ However, because sacran is listed as a functional food, it would be expected to have an effective nephroprotective effect when used starting at the early stage of renal failure when oxidative stress initially appears. These findings provide useful basic data for the application of sacran in the pharmaceutical field. Previous studies in our laboratory have shown that surface-deacetylated chitin nanofibers (SDACNFs) adsorbed indole and exerted inhibitory effects on the progression of CKD and oxidative stress.^7,14) In the present study, we propose that sacran, like SDACNFs, suppresses oxidative stress and inhibits the progression of CKD. These results strongly suggest that sacran has a multifaceted effect on the progression of CKD by altering the mix of intestinal microflora as a prebiotic in addition to the above effects.

Acknowledgments

This work was partly supported by Grants-in-Aid from the Japan Society for the Promotion of Science (JSPS), a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology (Grant Nos. 23790211 and 26460248), Japan.

Conflict of Interest

The authors declare no conflict of interest.

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