Lanthanum Hydroxide Protects Kidney Through Gut-Metabolite-Kidney Axis in a Rat Model of CKD

Background: Recent evidence suggests alterations in the gut-kidney axis may drive chronic kidney disease (CKD). Results: In the present study, we observed that administration of adenine to rats induced CKD, gut microbial dysbiosis, kidney pathology, and amino acid metabolism. In this model of CKD hyperphosphatemia, lanthanum hydroxide improved kidney function in CKD rats by restoring gut microbial homeostasis, thereby increasing urine ammonium metabolism. These ndings demonstrated that lanthanum hydroxide improves kidney function in a CKD model in mice by restoring homeostasis of the gut-metabolite-kidney axis, which alleviated an amino acid imbalance. Lanthanum hydroxide thus shows therapeutic potential for patients with CKD, through reshaping the composition of gut microbiota. Conclusions: Lanthanum hydroxide plays a kidney protective role through the gut-metabolite-kidney axis in a rat model of chronic kidney disease caused by adenine. constipation inammation of the small intestine of Compared with the group, the inammation of the L, and G group was signicantly improved. No inammatory cell inltration villus shedding was existed. The lamina propria was arranged quite tightly and orderly and closely attached to the mucosal layer. The show that Lanthanum hydroxide the inammation of restoring its and

The intestinal mucosal barrier can prevent harmful microorganisms and toxic metabolites from entering the blood [15]. In patients with chronic kidney disease, the mucosal barrier is destroyed [16]. Vaziri et al. observed in vivo studies in uremic rats that the expression of tight junction proteins (such as claudin 1, occludin, and zonula oc-cludens-1) in the colonic mucosa decreased signi cantly, which indicates that the renal failure of rats The intestinal barrier is obviously damaged [17]. Yang et al. found that in CKD mice, the expression of colonic HSP70 and claudin 1 decreased, while the expression of claudin-2 increased and was accompanied by increased apoptosis [18]. In addition, they also found that although there was no difference in the percentage of regulatory T cells between CKD and control mice, the ratio of cytokines and CX3CRCR1 intermediate / CX3CRCR1 high in the colon of CKD mice was signi cantly increased [18]. The "gut-kidney axis" theory believes that the connection between the kidney and the intestine is twoway, and if one party's function is damaged, it can affect the normal function of the other party through a variety of ways [19][20][21]. Intestinal ora and its metabolites play an important role in it.
Lanthanum is a rare earth element discovered after cerium and yttrium. The metal lanthanum is chemically active and easily soluble in dilute acid [22]. It is easy to oxidize in the air, and the fresh surface will quickly darken when exposed to the air, heating can burn to generate oxides and nitrides. It is heated in hydrogen to generate hydride, which reacts violently in hot water and releases hydrogen. Lanthanum exists in monazite sand and bastnasite. Moreover, the combination of lanthanum preparations with conventional binders can reduce serum phosphate levels instead of increasing serum calcium levels, and has better tolerance and no serious side effects. In recent years, lanthanum preparations have been involved in the treatment of hyperphosphatemia [23]. In this study, we found that Lanthanum hydroxide regulates the homeostasis of intestinal ora, affects amino acid metabolism, increases urinary ammonium circulation and ultimately plays a role in kidney protection in CKD rat models.

Results:
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 re ected 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 re ects 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 signi cant 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 identi ed 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 ora tended to be the K group. The above results demonstrate that lanthanum hydroxide improves the composition of whole intestinal ora 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 ora 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 speci c 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_unidenti ed_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_unidenti led_Actinobacteria, f_Bi dobacteriaceae, g_Bi dobacterium, s_Bi dobacterium_animalis, o_Bi dobateriales, g_Faecalibaculum, p_unidenti ed_Bacteria, g_unidenti ed_Bacteria, o_unidenti ed_Bacteria, g_Candidatus_Saccharimonas, f_unidenti ed_Bacteria, c_unidenti ed_Bacteria and g_Lacihabitams. According to the dominant ora in the three groups, we conducted a PICURES functional analysis and found that the dominant ora 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 signi cantly different from that of the K and G groups ( Figure 2E). Further analysis showed that the differential metabolism of the dominant intestinal ora was mainly concentrated in amino acid metabolism ( Figure 2F). The above results indicate that differences in the dominant intestinal ora in each group will affect amino acid metabolism.

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 nger-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 in ammatory cells. Compared with K group, the small intestinal villi of the jejunum tissue of the M group became shorter, a large number of in ammatory cells were in ltrated in the interstitium, the lamina propria edema was obvious, and the arrangement was sparse. Compared with the M group, the in ammatory symptoms in the jejunum tissue of the L, Z and G group were signi cantly improved. In ammatory cell in ltration 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 nger-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 signi cant increase in the number of goblet cells and no in ltration of in ammatory cells. Compared with the K group, a large number of in ammatory cell in ltrations were existed in the ileum tissue of the M group. At the same time, in ammatory cells tend to migrate to the intestinal cavity. The above results indicate that constipation causes in ammation of the small intestine of mice. Compared with the M group, the in ammation in the ileum tissue of the L, Z and G group mice was signi cantly improved. No in ammatory cell in ltration 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 in ammation 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 in ammatory cell in ltration.
Third, we detected the effect of Lanthanum hydroxide on the cecum of the model group. The cecum is the main component of the rst 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 in ammatory cell in ltration.
There was a small amount of in ammatory cell in ltration in the cecum of the M group, the goblet cells were signifucantly reduced, and the crypts became shallow. These results indicated that constipation causes in ammation of the intestinal tract and a signi cant decrease in goblet cells.
The in ammatory symptoms of the cecum tissue of the L, Z and G groups were signi cantly improved, there was no in ammatory cell in ltration, and the goblet cells were signi cantly 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 signi cantly decreased, and the crypts were signi cantly 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 signi cantly 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 overtting ( 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 identi ed and con rmed ( Figure 5C). The signi cant 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 signi cant 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 in ltration, 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 signi cant. Chronic granulomatous in ammation was observed. Purine deposits were seen in some renal tubules. At the same time, there were white blood cell casts and renal mesenchymal brous 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 in ltration were lighter, and the degree of renal tubule dilatation was signi cantly improved. Kidney interstitial hyperplasia is also effectively controlled, and the glomerular structure is relatively complete.
In summary, lanthanum hydroxide signi cantly reduces serum phosphorus levels, protects the kidneys, and slows down the development of kidney disease.

Discussion:
With the increasing incidence of chronic kidney disease (CKD), kidney disease has become one of the world's major public health problems [24]. According to the latest global kidney disease health report released by the World Kidney Conference in 2021, 1 out of 10 people in the world suffers from kidney disease [25]. In recent years, more and more evidence has shown that there are disorders of intestinal ora and impaired intestinal barrier function in patients with chronic kidney disease [26][27][28][29][30]. Therefore, more and more scienti c experiments are investigated the mechanism of the intestinal-renal axis in CKD, hoping to delay its progress by regulating the intestinal ora.
Lanthanum is a rare earth element with high phosphorus binding capacity and low oral bioavailability [31]. It forms a highly insoluble compound in the gastrointestinal tract by combining with the phosphorus in the food to form a highly insoluble complex that is excreted from the body and effectively reduces the absorption of phosphorus. A small part of the absorbed lanthanum is mainly excreted through bile, ensuring that the pharmacokinetics of lanthanum in CKD patients and healthy people are similar. Lanthanum carbonate is a new generation of phosphorus binder without aluminum and calcium developed by British Shire Pharmaceutical Company [32]. In 2004, the US FDA approved lanthanum carbonate for the treatment of hyperphosphatemia, which does not cause hypercalcemia. It can be seen that lanthanum plays an important role in reducing phosphate in the blood, so the development of other compounds of lanthanum is one of the directions for the development of innovative drugs for phosphate binders [33][34][35][36]. This article mainly studies the effect of lanthanum hydroxide on adenine-induced CKD and hyperphosphatemia rats.
In this study, we demonstrated that lanthanum hydroxide improved kidney function by promoting urea metabolism through restoration of gut microbial homeostasis in the model mice. Firstly, using 16S ribosomal RNA (rRNA) sequence, we found that lanthanum hydroxide has an effect on the overall structural in microbiota composition. PICURES functional analysis indicates that lanthanum hydroxide affected the amino acid metabolism. In order to explore the effect of lanthanum hydroxide on amino acid metabolism, we conducted a untargeted metabolomics study. Moreover, lanthanum hydroxide increases the metabolism of urinary ammonium by increasing carbamoyl phosphate, asparate, L-Citruline, L-Arginine and reducing glutamate, nitrogen, N-Acetylornithine, N-Acety-L-citrulline. Finally, lanthanum hydroxide plays a role in renal protection (Figure 7).
There are several limitations in this study. Firstly, the relationship between amino acid metabolism and composition of the gut microbiota is not well known. Secondly, intestinal epithelial integrity testing is not comprehensive enough . Therefore, future studies are required to further explore the regulatory role of lanthanum hydroxide in alleviating constipation and metabolites. Wistar rats with similar coat color and body shape were selected as the blank control group, and no drugs were given. All animals were sacri ced on the last day of 12 weeks. On the day before execution, all animals were forbidden to eat, they were allowed to drink freely, and feces were collected in a metabolic cage for 12 hours. Rats were anesthetized with 50mg/kg pentobarbital, blood was collected from the abdominal aorta, and serum was collected by centrifugation. The kidney of each animal was xed in 10% neutral formaldehyde for subsequent histological examination.

LC/MS analysis of serum metabolites
Serum samples were incubated for 10 minutes with pre-chilled methanol in a ratio of 1: 3 to precipitate the proteins. The samples were centrifuged at 12000r/min for 15 minutes at 4℃. The supernatants were analyzed by Thermo Scienti c Dionex UltiMate3000 Rapid Resolution Liquid Chromatography and QExactive mass spectrum. The chromatographic conditions are shown in Table1. The analytes were separated in a XBridge BEH Amide chromatographic column (2.1×100 mm, Waters Co., Milford, MA, USA) using 0.1% formic acid and acetonitrile as mobile phases A and B, respectively. The ow rate was set at 0.4 ml/min, injection volume was 5 µl, and column temperature were set at 25℃. The mass spectrum signals were obtained using the positive and negative ion scanning mode. The ion spray voltage and other speci c MS parameters were shown in Table 2.

Gut microbiota composition
Fecal samples were collected from all mice and immediately stored at -80℃. The V3+V4 region of the 16S rRNA gene was sequenced using Illumina MiSeq (Beijing Novogene Co. Ltd., Beijing, China) and analyzed using the QIIME open platform to determine the gut microbiota pro les.

Statistical analysis
Statistical analysis was performed using the SPSS 13.0 software (SPSS Inc., Chicago, Illinois, USA). The data plots were generated using GraphPad Prism 8.0.1 (GraphPad Software, La Jolla, California, USA). Partial least squares discriminant analysis (OPLS-DA) of SIMCA-P+13.0 (Umetrics, AB, Umeå, Sweden) and Principal Components Analysis (PCA) were used to assess normalized LC-MS spectral data. Variable In uence on Projection (VIP) values were used to identify signi cant variables with VIP values >1.0 and p<0.05. These signi cant variables were used to identify the spectral peaks. Student's t-test was used to analyze differences between two groups of data. The taxonomic rank differential between groups was determined using Student's test (v3.1.2, R programming language). The correlation between genera abundance and mouse behavior was calculated using Spearman correlation coe cients (R language). p<0.05 was considered statistically signi cant. The data are presented as means±SD.