3.1 Lanthanum hydroxide has an effect on the overall structural in microbiota composition.
The goods_coverage was used to evaluate the total number of community species represented by the sequencing results. The goods_coverage of all groups was greater than 0.99, which suggested that the sequencing depth of the microbiome analysis was very deep and met the requirements (Figure 1A). The Rank-abundance curve can be used to explain two aspects of diversity, namely species abundance and species evenness. In the horizontal direction, the abundance of species is reflected by the width of the curve. The higher the abundance of the species, the larger the range of the curve on the horizontal axis. The shape (smoothness) of the curve reflects the uniformity of the species in the sample. The smoother the curve, the more even the species distribution. The W and G group have increased species abundance and more uniform species distribution compared with the M group (Figure 2A). Then, we calculated alpha diversity indices to evaluate the overall fecal microbiota richness and structural difference among these groups. We analyzed alpha diversity (α-diversity) indexes such as obserced_species, Chao 1, ACE and Simpson index values to determine changes in the composition of various bacterial species in the feces samples of different groups. The α-diversity ACE, Chao 1 and observed specie indexes were higher in the W and G groups of mice compared to the M group (P<0.05). The Simpson index in the W group is smaller than the M and G group, but there is no significant difference (Figure 1C-F). Next, we analyzed β-diversity indexes to identify differences in the gut microbial species among K, M and G groups of mice using Principal component analysis(PCA), Principal Coordinates Analysis (PCoA) and Non-metric Multidimensional Scaling (NMDS). The differences in the fecal microbiota among K, M and G groups were identified based on PCA (Figure 1G ), PCoA (Figure 1H ) and NMDS (Figure 1I ) of the weighted UniFrac distances for the 16S rRNA genes. It can be seen from PCA, PCoA and NMDS analysis that the M group is different from WT group. Moreover, after the administration of lanthanum hydroxide, the composition of the intestinal flora tended to be the K group. The above results demonstrate that lanthanum hydroxide improves the composition of whole intestinal flora in rats with chronic kidney disease.
3.2 Composition of gut microbiota of mice in each group of phylum and major differential microbial species.
We analyzed the differences in the abundance and composition of the gut microbial phylum and genus in the fecal samples of these groups using 16S ribosomal RNA (rRNA) sequencing. From the phylum-level analysis, we found that the predominant intestinal flora in the group mice were Bacteroides and Fimicutes. The relative abundance of Fimicutes in K, M and G groups were 75.1%, 61.2% and 74.2%, respectively. The relative abundance of Fimicutes in the M group was lower than other two groups (Figure 2A). In order to verify and further determine, the LEfSe was used to identify the specific phylotypes responding to K, M and G groups. We performed linear discriminant analysis (LDA) to determine LDA effect size (LEfSe) scores followed by Kruskal-Wallis and Wilcoxon tests. The main differential gut microbioal species between the K and other groups were s_Lactobacillus_intestinalis, g_Enterococcus, f_Enterococcaceae, s_Enterococcus_durans, g_unidentified_Lachnospiraceae, f_Moraxellaceae, and g_Acinetobacter. The main differential gut microbial species between M and other groups were f_Bacteroidaceae, g_Bacteroides and g_Parasutterella. The main differential gut microbial species between G and other groups were g_Turicibacter, c_unidentifiled_Actinobacteria, f_Bifidobacteriaceae, g_Bifidobacterium, s_Bifidobacterium_animalis, o_Bifidobateriales, g_Faecalibaculum, p_unidentified_Bacteria, g_unidentified_Bacteria, o_unidentified_Bacteria, g_Candidatus_Saccharimonas, f_unidentified_Bacteria, c_unidentified_Bacteria and g_Lacihabitams. According to the dominant flora in the three groups, we conducted a PICURES functional analysis and found that the dominant flora is mainly related to metabolism (Figure 2D). At the same time, after comparison, it was found that the metabolic process of the M group was significantly different from that of the K and G groups (Figure 2E). Further analysis showed that the differential metabolism of the dominant intestinal flora was mainly concentrated in amino acid metabolism (Figure 2F). The above results indicate that differences in the dominant intestinal flora in each group will affect amino acid metabolism.
3.3 Effect of Lanthanum hydroxide on intestinal mucosa of CKD rats.
First, we evaluated the effect of Lanthanum hydroxide on the jejunum of CKD model rats (Figure 3A). Jejunum is the most import place for nutrient absorption in the digestion system, because its structural changes directly affect the digestion and absorption of rats. In the K group, the villi of the jejunum of the mice were tightly arranged, without breaks or missing, and showed finger-like protrusions. There were a large number of tightly arranged absorption cells and a small amount of goblet cells scattered among the absorption cells. The lamina propria were arranged tightly and orderly without inflammatory cells. Compared with K group, the small intestinal villi of the jejunum tissue of the M group became shorter, a large number of inflammatory cells were infiltrated in the interstitium, the lamina propria edema was obvious, and the arrangement was sparse. Compared with the M group, the inflammatory symptoms in the jejunum tissue of the L, Z and G group were significantly improved. Inflammatory cell infiltration was rare in the central chylo duct, the small intestinal villi restored their integrity, and the lamina propria was arranged very tightly and orderly. Compared with the K group, the villi of the jejunum tissue in group LC and CC are loosely arranged with breakage or loss, showing finger-like protrusions. A few number of tightly arranged absorption cells and a large amount of goblet cells scattered among the absorption cells can be seen.
Second, we analyzed the effect of Lanthanum hydroxide on the ileum of model rats. As an important part of small intestine tissue, the ileum has very important digestion and absorption functions. In the K group, the ileum villi were intact and short-tapered. There was no significant increase in the number of goblet cells and no infiltration of inflammatory cells. Compared with the K group, a large number of inflammatory cell infiltrations were existed in the ileum tissue of the M group. At the same time, inflammatory cells tend to migrate to the intestinal cavity. The above results indicate that constipation causes inflammation of the small intestine of mice. Compared with the M group, the inflammation in the ileum tissue of the L, Z and G group mice was significantly improved. No inflammatory cell infiltration or small intestinal villus shedding was existed. The lamina propria was arranged quite tightly and orderly and closely attached to the mucosal layer. The above results show that Lanthanum hydroxide effectively reduce the inflammation of the samll intestine, thereby restoring its digestion and absorption function. Compared with the K group, the intestinal villi in the LC and CC group were intact and there were inflammatory cell infiltration.
Third, we detected the effect of Lanthanum hydroxide on the cecum of the model group. The cecum is the main component of the first mucus layer of the innate immunity of the intestinal tract, and is conductive to the smooth passage of grain. The goblet cells in the cecum secretes mucus proteins, which have lubricating and protective effects. There were many absorption cells and goblet cells in the cecum of mice in the K group, and lamina propria were loosely arranged without inflammatory cell infiltration. There was a small amount of inflammatory cell infiltration in the cecum of the M group, the goblet cells were signifucantly reduced, and the crypts became shallow. These results indicated that constipation causes inflammation of the intestinal tract and a significant decrease in goblet cells.
The inflammatory symptoms of the cecum tissue of the L, Z and G groups were significantly improved, there was no inflammatory cell infiltration, and the goblet cells were significantly increased compared with the M group. Compared with the K group, the goblet cells in the cecum tissue of LC and CC group mice were significantly decreased, and the crypts were significantly shallower. These results demonstrated that Lanthanum hydroxide restore the injured intestinal mucosa.
3.4 Metabolomics analysis reveals that lanthanum hydroxide increases the metabolism of urinary ammonium.
The imbalance of the gut microbiota homeostasis will lead to the disorder of the amino acid metabolism of small molecules. Therefore, we used untargeted metabolomics to explore the impact of changes in gut microbiota on metabolites. Using HPLC-MS/MS, we found 4370 variables in positive ion mode and 732 variables in negative ion model. The total ion current diagram, K, M and treated groups in positive and negative ion mode, indicated that the contours of each group were roughly similar, but the level of metabolites is different. First, we performed mean normalization and logarithmic transformation on all data. Then, based on the QC sample, the small molecule compounds whose relative abundance is lower than 25% of the QC sample are eliminated (Figure 4A). The principal component analysis (PCA) was performed on the sample data of each group. QC samples had a high degree of aggregation, demonstrating high repeatability and stability (Figure 4B-D). The partial least square discriminant analysis (PLS-DA) was further applied to the samples of each group. The groups were clearly separated. WT and M group were clustered on the left and right sides respectively. There were obvious differences in the metabolites. These results shown that the model was successfully. Treated and K group were significantly separated and approached M group. It was consistent with the results of biochemical indicators and pathological changes. It was demonstrated that the model was reliable and didn’t over-fitting (Figure 4E, F).
Based on the above analysis, we screened differential metabolites in the K, M (Figure 5A) and M, administration groups (Figure 5B) according to P < 0.05 and P(corr)> 0.06 (Table 1). There are 47 kinds of differential metabolites in positive and negative ion mode, among which 25 kinds of differential metabolites have been identified and confirmed (Figure 5C). The significant metabolites screened out were imported into MetaboAnalyst 5.0 for metabolic pathway analysis, and 8 related metabolic pathways were found (Figure 5D, E). The main metabolic pathways included urea cycle and arginine biosynthesis (Figure 8a, b). Above results demonstrated that lanthanum hydroxide increase urine ammonium metabolism.
3.5 Lanthanum hydroxide delays the progression of kidney disease and improves kidney function.
In order to evaluate the protective effect of lanthanum hydroxide on the kidneys of CKD rats, we tested the serum phosphorus, creatinine, and urea nitrogen levels 12 weeks after administration. Compared with the control group, the serum phosphorus, creatinine, and urea nitrogen in the M group was increased, and compared with the normal control group, there were significant differences (P<0.01), suggesting adenine The joint 1.2% high-phosphorus diet was successfully modeled (Figure 6A-C). The results of HE staining of rat kidneys showed that compared with K group, the degeneration and necrosis of the proximal convoluted tubule epithelial cells in the renal cortex and the disappearance of the nucleus were observed in the other groups (Figure 6D). The mesenchyme is accompanied by a large number of mononuclear cell infiltration, glomerular necrosis and disappearance, and visible protein casts and obvious expansion of the renal tubules in the renal tubules. Among them, the pathological changes in M group were more significant. Chronic granulomatous inflammation was observed. Purine deposits were seen in some renal tubules. At the same time, there were white blood cell casts and renal mesenchymal fibrous tissue focal hyperplasia lesions in the renal tubules. The pathological results of the lanthanum hydroxide (04g/kg/d, 0.2g/kg/d, 0.1g/kg/d) group showed that compared with the M group, cell deformation and infiltration were lighter, and the degree of renal tubule dilatation was significantly improved. Kidney interstitial hyperplasia is also effectively controlled, and the glomerular structure is relatively complete.
In summary, lanthanum hydroxide significantly reduces serum phosphorus levels, protects the kidneys, and slows down the development of kidney disease.