Dietary Fermented Soy Extract and Oligo-Lactic Acid Alleviate Chronic Kidney Disease in Mice via Inhibition of Inflammation and Modulation of Gut Microbiota

Chronic kidney disease (CKD) is a global epidemic with an increasing prevalence worldwide. Effective preventive strategies are urgently needed. This study aimed to investigate the effect of nutraceutical components, a fermented soybean product (ImmuBalance, IMB) and an oligo-lactic acid product (LAP), on the prevention of adenine-induced CKD in mice. Female C57BL/6 mice were randomly assigned into following experimental groups: negative control; model control; and models treated with IMB at 250 or 1000 mg/kg body weight (BW), LAP at 1000 or 2000 mg/kg BW, and IMB/LAP combinations. The CKD model was established by intraperitoneal injection of adenine daily for 4 weeks, and treatments started 2 weeks before adenine injection and ended after 10 weeks. Compared with the model control, the treatments did not significantly alter the body weight or food intake. Both IMB and LAP, especially their combination, significantly inhibited tubular dilation, tubulointerstitial degeneration or atrophy, interstitial chronic inflammation and acute inflammation in the kidneys of CKD mice, and significantly decreased serum cystatin C levels. IMB or LAP significantly reversed CKD-associated increases of circulating and kidney levels of inflammatory cytokines, circulating levels of kidney injury biomarkers, and kidney levels of stem cell biomarkers, and significantly reversed CKD-associated reduction of cecum Clostridium leptum group. Our results suggest that dietary supplementation of IMB or LAP may significantly delay the development and/or progression of CKD.


Introduction
Chronic kidney disease (CKD) is a slow and progressive loss of kidney function, and poses serious health problems. It is estimated that about 15% of the U.S. population, or 37 million U.S. adults, would have had CKD in 2019 [1]. CKD is an important contributor to morbidity and mortality from noncommunicable diseases, and this disease should be actively addressed to meet the United Nation's (UN) Sustainable Development Goal target to reduce premature mortality from non-communicable

Quantitative Polymerase Chain Reaction (qPCR) for Determination of Gut Microbiota
The mouse cecum was collected and microbial genomic DNA was extracted from 200 mg of cecal sample using the E.Z.N.A. ® Stool DNA Kit (D4015, Omega Bio-Tek, Inc., Norcross, GA, USA)/QIAamp DNA Stool mini kit (Qiagen) according to the manufacturer's instructions. Total DNA was quantified and its purity was assessed using NanoDrop™ 2000 spectrophotometers (ThermoFisher, Carlsbad, CA, USA). Nuclease-free water was used as a blank.
The amount of total microbiota was estimated using the universal primers, Uni331F and Uni797R, which amplified a conserved region of the 16S rRNA for most of the common microbiota [14]. The following representative dominant/subdominant groups from four major phyla of gut microbiota were chosen: Atopobium cluster, Bifidobacterium genus, Bacteroides fragilis group, Clostridium coccoides group, Clostridium perfringens group, Desulfovibrio genus, Enterobacteriaceae family, Lactobacillus genus, Clostridium leptum group, and Prevotella genus. qPCR assays were performed using SYBR Green qPCR Master Mix on a CFX384 Touch™ Real-Time PCR Detection System (BioRad Laboratories, Hercules, CA, USA). The amplification protocol consisted of 1 cycle of 95 • C for 20 s, followed by 40 cycles of 95 • C for 15 s, appropriate annealing temperature for 30 s and 72 • C for 35 s, and finally 1 cycle of 60-94 • C with 0.5 • C increments, 15 s dwell time. The results were normalized to 16S ribosomal (universal) DNA sequences and expressed as the relative difference using the 2 ∆∆Ct method.

Statistical Analysis
Data were expressed as the group mean ± standard deviation and analyzed by one-way analysis of variance (ANOVA) test, followed by multiple comparison of least-significant difference (equal variances assumed) or Dunnett's T3-test (equal variances not assumed) to evaluate the difference of parametric samples among groups. When raw data or log-transformed data did not meet the statistical criteria for the assumption of normality showing equal variance, the nonparametric Kruskal-Wallis or Mann-Whitney test was used to determine statistical differences, and Bonferroni correction was used to correct the p-value. Differences were considered to be statistically significant at p < 0.05. A Spearman correlation analysis was used to investigate the relationship between histopathology and biomarkers. All analyses were carried out using IBM SPSS Statistics version 20.0 and GraphPad Prism 5 Software.

Effects of IMB and LAP Treatments on Body Weight
There was no significant difference in initial body weight among all groups (p > 0.05) ( Figure 1A). Induction of CKD significantly reduced final body weights and food intake in all model groups (p < 0.05) ( Figure 1B,C). Compared with the model control, dietary treatments did not significantly alter final body weights ( Figure 1B) or food intake ( Figure 1C) (p > 0.05).

Statistical Analysis
Data were expressed as the group mean ± standard deviation and analyzed by one-way analysis of variance (ANOVA) test, followed by multiple comparison of least-significant difference (equal variances assumed) or Dunnett′s T3-test (equal variances not assumed) to evaluate the difference of parametric samples among groups. When raw data or log-transformed data did not meet the statistical criteria for the assumption of normality showing equal variance, the nonparametric Kruskal-Wallis or Mann-Whitney test was used to determine statistical differences, and Bonferroni correction was used to correct the p-value. Differences were considered to be statistically significant at p < 0.05. A Spearman correlation analysis was used to investigate the relationship between histopathology and biomarkers. All analyses were carried out using IBM SPSS Statistics version 20.0 and GraphPad Prism 5 Software.

Effects of IMB and LAP Treatments on Body Weight
There was no significant difference in initial body weight among all groups (p > 0.05) ( Figure  1A). Induction of CKD significantly reduced final body weights and food intake in all model groups (p < 0.05) ( Figure 1B, 1C). Compared with the model control, dietary treatments did not significantly alter final body weights ( Figure 1B

Effects of IMB and LAP Treatments on Kidney Inflammation and Damage
The effects of treatments on kidney inflammation and damage were evaluated histopathologically by H & E, PAS, and trichrome staining. The statistical analysis results of histopathological scores of kidney tissues are presented in Table 2, and the representative results are shown in Figure 2. Tubular dilation without basement membrane thickening was considered as an

Effects of IMB and LAP Treatments on Kidney Inflammation and Damage
The effects of treatments on kidney inflammation and damage were evaluated histopathologically by H & E, PAS, and trichrome staining. The statistical analysis results of histopathological scores of kidney tissues are presented in Table 2, and the representative results are shown in Figure 2. Tubular dilation without basement membrane thickening was considered as an acute process and was used to evaluate the acute change. Tubular dilation along with other features were evaluated under the tubular atrophy. Tubular atrophy, interstitial fibrosis, and chronic inflammation were evaluated as chronic changes. The kidney tissues in the adenine-treated mice showed tubular dilation (Figure 2A), acute inflammation (Figure 2A), tubulointerstitial degradation ( Figure 2B), interstitial chronic inflammation ( Figure 2B), capillary widening ( Figure 2C), thickened glomeruli basement membrane ( Figure 2C), and tubulointerstitial fibrosis ( Figure 2D), whereas the kidney tissues from normal control mice did not show histopathological lesions ( Figure 2E-G). These histopathological parameters were improved by IMB and/or LAP treatments ( Figure 2H-J, images were from one mouse treated with the IMB-H/LAP-H combination group).  Compared with the NC, the MC showed significantly higher levels of tubular dilation, tubulointerstitial degeneration/atrophy, chronic inflammation, and acute inflammation (p < 0.05 in all histopathological lesions). The administration of LAP-L significantly alleviated the kidney inflammation and damage, and inhibited tubular dilation (p < 0.05), tubulointerstitial degeneration/atrophy (p < 0.05), and chronic inflammation (p < 0.05), and IMB-H significantly inhibited tubulointerstitial degeneration/atrophy (p < 0.05). In particular, the IMB-H/LAP-H combination significantly inhibited all measured histopathological parameters (p < 0.05), and the IMB-L/LAP-L combination significantly alleviated tubular dilation and interstitial chronic inflammation (p < 0.05). These results suggest that the combination of IMB and LAP, especially at the high doses, may further enhance the protective effect on kidney damage/inflammation, although the apparent combination effect (additive or synergistic) was not obvious. While IMB and LAP treatment alone showed some beneficial effects, no clear dose-dependent effect was determined. Compared with the NC, the MC showed significantly higher levels of tubular dilation, tubulointerstitial degeneration/atrophy, chronic inflammation, and acute inflammation (p < 0.05 in all histopathological lesions). The administration of LAP-L significantly alleviated the kidney inflammation and damage, and inhibited tubular dilation (p < 0.05), tubulointerstitial degeneration/atrophy (p < 0.05), and chronic inflammation (p < 0.05), and IMB-H significantly inhibited tubulointerstitial degeneration/atrophy (p < 0.05). In particular, the IMB-H/LAP-H combination significantly inhibited all measured histopathological parameters (p < 0.05), and the IMB-L/LAP-L combination significantly alleviated tubular dilation and interstitial chronic inflammation (p < 0.05). These results suggest that Nutrients 2020, 12, 2376 7 of 15 the combination of IMB and LAP, especially at the high doses, may further enhance the protective effect on kidney damage/inflammation, although the apparent combination effect (additive or synergistic) was not obvious. While IMB and LAP treatment alone showed some beneficial effects, no clear dose-dependent effect was determined.

Histopathology
Grade Tubulointerstitium degeneration/atrophy Interstitial chronic inflammation Acute inflammation Data were presented as the number of mice that had the damage/inflammation in each group, and were transferred to frequency, followed by the analysis of Kruskal-Wallis test among all groups and then Mann-Whitney test between two groups.

Effects of IMB and LAP Treatments on Circulating Levels of Cytokines and Kidney Injury Biomarkers
Figure 3 ( Figure 3A-F) shows that the serum levels of cytokines IP-10, VEGF, MCP-1, IL-6, and IFN-γ in the MC were significantly increased compared with the NC (p < 0.05), whereas the serum level of IL-12p70 in the MC was non-significantly increased (p > 0.05) ( Figure 3E). Compared with that of the MC, MCP-1 levels were significantly reduced in all treatment groups except the IMB-L group (p < 0.05, Figure 3C), IL-6 levels were significantly reduced in the LAP-L and the IMB-H/LAP-H groups (p < 0.05, Figure 3D), IL-12p70 levels were significantly reduced in the LAP-H and the IMB-L/LAP-L groups (p < 0.05, Figure 3E), and IFN-γ level was significantly reduced in the IMB-H/LAP-H group (p < 0.05, Figure 3F).
Compared with that of the NC, the serum levels of kidney injury biomarkers TIMP-1, cystatin C, lipocalin-2, and clusterin in the MC were significantly increased (p < 0.05, Figure 3G-J, respectively). Compared with that of the MC, cystatin C levels were significantly decreased in all treatment groups except the LAP-H group (p < 0.05, Figure 3H), TIMP-1 levels were significantly reduced in the IMB-H, LAP-L, IMB-L/LAP-L, and IMB-H/LAP-H groups (p < 0.05, Figure 3G), and lipocalin-2 levels were significantly reduced in the LAP-L group (p < 0.05, Figure 3I). On the other hand, clusterin levels were not significantly altered by the treatments ( Figure 3J).

Effects of IMB and LAP Treatments on the Expression Levels of Inflammatory Cytokines in Kidney
As shown in Figure 4, when compared with the NC group, the MC group showed significantly increased expression levels of MCP-1, IL-1, IL-6, TLR-4, TNF-α, F4/80, TGF-β1, and IL-1β genes in kidney (p < 0.05). When compared with the MC, the experimental treatments of IMB or LAP significantly reduced the expression levels of IL-1 ( Figure 4B Figure 4H). The IMB-H/LAP-H treatment also reduced the expression level of MCP-1 ( Figure 4A). It is also important to note that the IMB or LAP treatments reduced adenine-induced expression of MCP-1, IL-1, TLR-4, F4/80, TGF-β1, and IL-1β genes in kidney to the levels that were comparable to the NC (p > 0.05). These results fur ther supported that IBM or LAP could significantly reduce kidney inflammation levels in kidney.

Effects of IMB and LAP Treatments on the Expression Levels of Inflammatory Cytokines in Kidney
As shown in Figure 4, when compared with the NC group, the MC group showed significantly increased expression levels of MCP-1, IL-1, IL-6, TLR-4, TNF-α, F4/80, TGF-β1, and IL-1β genes in kidney (p < 0.05). When compared with the MC, the experimental treatments of IMB or LAP significantly reduced the expression levels of IL-1 ( Figure 4B Figure 4A). It is also important to note that the IMB or LAP treatments reduced adenine-induced expression of MCP-1, IL-1, TLR-4, F4/80, TGF-β1, and IL-1β genes in kidney to the levels that were comparable to the NC (p > 0.05). These results further supported that IBM or LAP could significantly reduce kidney inflammation levels in kidney.

Effects of IMB and LAP Treatments on the Expression Levels of Stem Cell-Related Genes in Kidney
Compared with the NC, the MC showed significantly elevated CD44, CD133, CD11β, Pax-2, vimentin, Wnt-4, Wt-1, and nestin mRNA levels in kidney ( Figure 5, p < 0.05). The treatments with LAP-H and the two combinations ameliorated the imbalance changes of CD44, CD133, CD11β, and Pax-2 mRNA levels compared with the positive control (p < 0.05), and the treatments with IMB (low or high dose) and the combination of IMB-H/LAP-H reduced Wt-1 and nestin mRNA levels (p < 0.05).

Effects of IMB and LAP Treatments on the Expression Levels of Stem Cell-Related Genes in Kidney
Compared with the NC, the MC showed significantly elevated CD44, CD133, CD11β, Pax-2, vimentin, Wnt-4, Wt-1, and nestin mRNA levels in kidney ( Figure 5, p < 0.05). The treatments with LAP-H and the two combinations ameliorated the imbalance changes of CD44, CD133, CD11β, and Pax-2 mRNA levels compared with the positive control (p < 0.05), and the treatments with IMB (low or high dose) and the combination of IMB-H/LAP-H reduced Wt-1 and nestin mRNA levels (p < 0.05).

Effects of IMB and LAP Treatments on the Expression Levels of Stem Cell-Related Genes in Kidney
Compared with the NC, the MC showed significantly elevated CD44, CD133, CD11β, Pax-2, vimentin, Wnt-4, Wt-1, and nestin mRNA levels in kidney ( Figure 5, p < 0.05). The treatments with LAP-H and the two combinations ameliorated the imbalance changes of CD44, CD133, CD11β, and Pax-2 mRNA levels compared with the positive control (p < 0.05), and the treatments with IMB (low or high dose) and the combination of IMB-H/LAP-H reduced Wt-1 and nestin mRNA levels (p < 0.05).

Effects of IMB and LAP Treatments on Gut Microbiota
We also determined the effect of treatments on alteration of gut microbiota community in the mouse model. Compared with that in the NC, gut Clostridium leptum group level was significantly decreased in the MC, whereas this decrease was significantly reversed by the treatments (Figure 6D, p at least <0.05, except the IMB-L/LAP-L group owing to high variation). Compared with the NC, the MC group had a non-significant decrease of Clostridium coccoides group level, whereas the treatments of IMB-L and LAP-H significantly increased Clostridium coccoides group levels ( Figure 6C, p at least <0.05). Compared with the MC, the treatment of 250 mg/kg of IMB significantly increased levels of Bifidobacterium genus, Bacteroides fragilis group, and Clostridium leptum group (p < 0.05). The levels of other gut microbiota groups were not significantly altered by the treatments (Supplement Figure S1). Values are expressed as Mean±SD, n=8. Within each panel, values with the superscript symbols "*" and "#" are significantly different from the NC and MC, respectively, P at least <0.05.

Effects of IMB and LAP Treatments on Gut Microbiota
We also determined the effect of treatments on alteration of gut microbiota community in the mouse model. Compared with that in the NC, gut Clostridium leptum group level was significantly decreased in the MC, whereas this decrease was significantly reversed by the treatments (Figure 6D, p at least <0.05, except the IMB-L/LAP-L group owing to high variation). Compared with the NC, the MC group had a non-significant decrease of Clostridium coccoides group level, whereas the treatments of IMB-L and LAP-H significantly increased Clostridium coccoides group levels ( Figure 6C, p at least < 0.05). Compared with the MC, the treatment of 250 mg/kg of IMB significantly increased levels of Bifidobacterium genus, Bacteroides fragilis group, and Clostridium leptum group (p < 0.05). The levels of other gut microbiota groups were not significantly altered by the treatments (Supplement Figure S1).

Correlation Analysis between Histopathological and Metabolic Parameters
Spearman correlation analysis was applied to determine the association between histopathological parameters and molecular biomarkers in blood or kidney tissues. As shown in Table 3 serum levels of kidney injury markers KIM-1, TIMP-1, cystatin C, lipocalin 2, clusterin, and OPN were significantly positively correlated with all histopathological parameters of kidney lesions (except no significant correlation between interstitial chronic inflammation and KIM-1, or clusterin). Serum levels of cytokines IP-10, VEGF, IL-6, and IFN-γ were significantly positively correlated with

Correlation Analysis between Histopathological and Metabolic Parameters
Spearman correlation analysis was applied to determine the association between histopathological parameters and molecular biomarkers in blood or kidney tissues. As shown in Table 3 serum levels of kidney injury markers KIM-1, TIMP-1, cystatin C, lipocalin 2, clusterin, and OPN were significantly positively correlated with all histopathological parameters of kidney lesions (except no significant correlation between interstitial chronic inflammation and KIM-1, or clusterin). Serum levels of cytokines IP-10, VEGF, IL-6, and IFN-γ were significantly positively correlated with all histopathological parameters of kidney lesions. Kidney gene expression levels of inflammatory cytokines IL-6, TNF-α, TGF-β1, and TLR-4 were significant positively correlated with all histopathological parameters of kidney lesions (except no significant correlation between TLR-4 and interstitial chronic inflammation). Kidney gene expression levels of stem cell markers CD133, Pax-2, CD11β, CD44, vimentin, and nestin were also significantly positively correlated with all histopathological parameters of kidney lesions.
The correlation analysis also showed that Desulfovibrio genus was negatively correlated with all histopathology inflammation and damage parameters (p < 0.05), and that Prevotella genus was negatively correlated with tubular dilation and interstitial acute inflammation (p < 0.05).

Discussion
In this study, we evaluated the effects of two novel nutritional components, IMB and LAP, on inhibition of inflammation and kidney injury, and alteration of gut microbiota in the adenine-induced CKD mouse model. With a few exceptions, both IMB and LAP, especially their combinations, significantly inhibited tubular dilation, tubulointerstitial degeneration or atrophy, interstitial chronic inflammation, and acute inflammation in kidneys of the CKD mice. Molecular biomarkers determination showed that IMB or LAP significantly reversed CKD-associated increases of circulating and kidney levels of inflammatory cytokines, circulating levels of kidney injury biomarkers, and kidney levels of stem cell biomarkers. The correlation analysis further indicated significant positive correlations of histopathological markers of kidney lesions to measured molecular markers, with a few exceptions. Gut microbiota analysis also showed that IMB or LAP reversed CKD-associated reduction of Clostridium leptum group and Clostridium coccoides group; the correlation analysis showed significantly negative correlation between histopathology parameters of kidney lesions and gut Desulfovibrio genus or Prevotella genus species.
CKD is associated with an increased acute and chronic pro-inflammatory state [3,15]. While inflammation is one of the core pathological features of CKD, it may play a causal role in the development of kidney injury, and contribute to the progression of CKD by inducing release of pro-inflammatory cytokines [15]. The adenine-induced CKD mouse model has been reported to recapitulate several key characteristics of kidney injury and CKD, especially those related to inflammation [12,13], and it has been applied for evaluating the effects of natural products on prevention and treatment of CKD [16,17].
In this study, we found significant reductions in most of the pro-inflammatory markers. The circulating levels of TNF-α, IFN-γ, IL-1, IL-6, and TLR-4 were increased by adenine, but were attenuated by LAP and/or IMB treatment (Figures 3 and 4). Similarly, adenine significantly induced expression levels of MCP-1, IL-1, IL-6, TLR-4, TNF-α, F4/80, TGF-β1, and IL-1β genes in kidney, and most of these increases were attenuated by IMB and/or LAP treatments (Figure 4), which is consistent with the histopathological confirmation of inflammation. Altogether, these findings support that IMB and/or LAP alleviate the kidney injury in CKD at least in part via inhibition of inflammation.
Our results showed that serum levels of TIMP-1, cystatin C, and lipocalin2, markers of kidney injury [18], were high in the adenine-induced CKD mice, but TIMP-1 and cystatin C was significantly reduced by the treatments. The serum level of TIMP-1, which promotes cell proliferation and has anti-apoptotic functions, was increased at the highest level in the adenine-induced CKD mice, and was significantly reduced by these nutritional compounds. To further understand the kidney injury repair mechanism, we determined the gene expression levels of kidney stem cell-related markers. We observed increased expression of key stem cell markers CD44, CD133, Pax-2, CD11β, vimentin, and nestin in the kidney tissues of affected mice, which were all attenuated in the treatment groups ( Figure 5). In the human kidney, the expression of CD133 characterizes a population of resident scattered cells with resistance to damage and the ability to proliferate [19,20]. CD133 itself appears to play a functional role in renal tubular repair through maintenance of proliferative responses and the control of senescence [21]. Pax2 [22] is a marker of mature podocytes and is essential for the phenotypic conversion from mesenchymal stem cells to tubular epithelial cells during kidney development. Nestin and CD44 are also involved in cellular proliferation and migration [23][24][25]. Vimentin [26], as a kidney stem cell marker, maintains cells shape and integrity of the cytoplasm, and stabilizes cytoskeletal interactions. WT-1 is now known to have an important role in kidney progenitor cells during development [27]. As kidney injury and kidney stem cell-related markers were increased owing to the CKD, and were significantly or non-significantly reduced by the IMB and/or LAP treatments, the results suggest that, while kidney stem cells related repair is needed in kidney injury, and the effective treatments with IMB and/or LAP reduced the requirement of kidney stem cells for repair.
Microbiota metabolism is emerging as a modifiable risk factor in nephrology, and nutritional manipulation of gut microbiota may play an important role in the prevention and management of CKD [28]. The gut microbiota alterations in CKD was reported by others [29][30][31][32][33], and appear to be the result of metabolic changes. Specifically, CKD patients were reported to have reduced Bifidobacteriaceae and Lactobactria levels and increased Bacteroides levels [34,35]. Interestingly, kidney functions and systemic inflammation were improved by Lactobactria treatment in the rat and dog models of CKD [35,36]. Our gut microbiome analysis exhibited alterations in a number of microbial species in the adenine-induced CKD mice model group that were reversed in the treatment groups ( Figure 6 and Supplementary Figure S1). However, our data indicate that LAP and IMB do not exert their effects in CKD through altering those bacteria mentioned in other studies. We observed a significant decrease in Clostridium leptum levels in the model control mice, which was significantly increased in most of the treatment groups. Some nutritional components such as partially hydrolyzed guar gum could increase Clostridium leptum levels and decrease inflammation in a mouse model of colitis [37]. A decrease in Clostridium leptum group was also reported in inflammatory bowel diseases and inflamed mucosa of patients with ulcerative colitis [38]. Therefore, more studies in this arena may unravel the link between the decrease in Clostridium leptum levels and increased inflammation in CKD. Further correlation analysis also found significantly negative correlation of histopathology inflammation and damage parameters to Desulfovibrio genus (Table 3). Inflammatory bowel diseases and inflamed mucosa of patients with ulcerative colitis had a decreased Desulfovibrio group level [39]. However, Desulfovibrio is also considered as a proinflammatory bacteria [40]. Therefore, the functional role of Desulfovibrio in CKD needs further investigation.

Conclusions
In conclusion, our animal study demonstrated that novel bioactive components, IMB and LAP, significantly inhibited the development and progression of CKD associated with the inhibition of inflammation in kidney tissues and in circulation, improvement of stem cell-based kidney repair, and modulation of gut microbiota. Our results provide essential preclinical evidence to support further investigation on applying IMB and/or LAP for the prevention and alleviation of CKD and associated kidney injury. Funding: This research was funded by Nichimo Biotics Co., Ltd., Japan and LifeTrade Co., Ltd., Japan.