| ObjectivesRenal stones is a common urologic disease.Calcium oxalate(Ca Ox)is the most common component of renal stones.Most renal stones can be removed successfully by surgery.However,we still lack effective methods for preventing the occurrence and recurrence of renal Ca Ox stones.The abnormal oxalate metabolism is the main reason for renal Ca Ox stones.Recent studies found that the gut microbiota alteration was associated with many chronic metabolic diseases,such as obesity and diabetes mellitus.Additionally,short chain fatty acids(SCFAs),produced by gut microbiota,could affect intestinal barrier,inflammation,metabolism of glucose and lipid,and the process of obesity and diabetes mellitus.It was reported that obesity and diabetes mellitus were risk factors for renal Ca Ox stones.These evidences indicated that gut microbiota and metabolites(SCFAs)were involved in the formation of renal Ca Ox stones.Further studies found that gut microbiota may regulate oxalate metabolism in two ways.On the one hand,Oxalobacter formigenes in the gut microbiota could degrade oxalate and reduce the absorption of oxalate from the gut.On the other hand,the gut could also secrete and absorb oxalate by oxalate transporters SLC26.A study found that the culture medium of O.formigenes stimulated oxalate uptake,which indicated that some metabolites from gut microbiota might regulate the expression of intestinal oxalate transporters SLC26.Based on this evidence,we hypothesized that gut microbiota and its metabolites SCFAs might affect the oxalate metabolism and reduce oxalate in urine and renal Ca Ox stones in the kidney by regulating intestinal oxalate transporters.The aim of this study was to explore the effects of gut microbiota and its metabolites SCFAs on the formation of renal Ca Ox stones and the underlying mechanisms by clinical research,animal experiments and cell experiments.Materials and MethodsThe study was divided into the following four sections.1.Part one.The study of the gut microbiota of renal Ca Ox stones patients.We collected the clinical information,feces and urine samples from renal Ca Ox stones patients and non-kidney stone(NS)controls.Gut microbiota was examined by 16S r RNA gene sequence and shotgun metagenomics analysis.The fecal SCFAs and urinary oxalate were determined by gas chromatography-mass spectrometry(GC-MS)and liquid chromatography-mass spectrometry(LC-MS),respectively.2.Part two.The study of the effect of gut microbiota on the formation of renal Ca Ox stones in rats.We used ethylene glycol(EG)to develop renal Ca Ox stones model rats.We also added antibiotics into the drinking water.The intestinal bacterial load was examined by quantitative real-time polymerase chain reaction(q RT-PCR).The renal stones were examined using Hematoxylin Eosin(HE)and Von Kossa(VK)staining of renal tissue.The SCFAs in cecum and urinary oxalate were determined by GC-MS and LC-MS,respectively.We used q RT-PCR and Western blot to determine the expression of intestinal oxalate transporters SLC26A3/6.Gut microbiota was examined by 16S r RNA gene sequence.3.Part three.The study of the effect of SCFAs on the formation of renal Ca Ox stones in rats.We used EG to develop renal Ca Ox stones model rats.We then added SCFAs(acetate,propionate or butyrate)into the drinking water of model rats.We also added antibiotics and SCFAs(acetate,propionate or butyrate)into the drinking water of model rats.The renal stones were examined using HE and VK staining of renal tissue.The SCFAs in cecum and urinary oxalate were determined by GC-MS and LC-MS,respectively.We used q RT-PCR and Western blot to determine the expression of intestinal oxalate transporters SLC26A3/6.Gut microbiota was examined by 16S r RNA gene sequence.4.Part four.The study of the effect of SCFAs on the expression of oxalate transporters in the human intestinal epithelial Caco-2 cells in vitro.Caco-2 cells were exposed to oxalic acid,acetate,propionate and butyrate.The cell viability was examined by cell counting kit-8(CCK-8).The expressions of oxalate transporters SLC26A3/6 were analyzed by q RT-PCR and Western blot.Results1.We recruited 84 NS controls and 69 kidney stone(KS)patients,including 26recurrent stones(RS)patients and 43 occasional stones(OS)patients.There was no statistical difference in age and body mass index(BMI)between the three groups(P>0.05).However,consumption of fat and red meat,intake of milk and fruit,sitting and sleeping time,active smoking,family history of KS,hypertension,and nonalcoholic fatty liver disease were significantly different among NS,OS,and RS groups(P<0.05).Ace and Chao indices at operational taxonomic units(OTUs)level were higher in KS patients than in controls,which indicated that the richness of gut microbiota was higher in KS patients(P<0.01).Principal coordinates analysis(PCo A)showed that the overall microbiota composition of the NS,OS,and RS groups was different,which was confirmed by the Adonis test(R~2=0.031,P=0.002).When compared with NS controls at the genus level,linear discriminant analysis Effect Size(LEf Se)showed that KS patients had fewer SCFAs-producing bacteria in the gut microbiota,like Blautia,Anaerostipes,Coprococcus,Fusobacterium,Ruminococcus and Lachnospiraceae(P<0.05).On the contrary,the relative abundances of Pseudomonas,Staphylococcus,Megamonas,Synechococcus,Acinetobacter,Cetobacterium and Prevotellaceae-UCG-001 were higher in the gut microbiota of KS patients,which were associated with inflammatory diseases(P<0.05).We also predicted the metabolic function of gut microbiota by PICRUSt.LEf Se analysis revealed that the relative abundance of pathways related to inflammation and oxidative stress,such as lipopolysaccharide biosynthesis(ko00540),was higher among KS patients.On the contrary,the relative abundance of pathways associated with SCFAs production(methane metabolism(ko00680)and pathways related to amino acid metabolism(arginine and proline metabolism(ko00330)were higher in NS Controls(P<0.05).Besides,RS patients(18.10μg/m L)presented a higher median urinary oxalate level than OS patients(8.53μg/m L)and NS controls(4.60μg/m L)(P<0.05).We also examined the fecal SCFAs and found that the concentration of acetic acid in the feces was higher in RS patients(53.20μg/mg)than in OS patients(45.63μg/mg)and NS controls(21.44μg/mg)(P<0.05).We also explored the relationship between the fecal SCFAs concentrations and the significantly different metabolic enzymes based on shotgun metagenomics analysis,and found that fecal acetic acid level was positively correlated with some metabolic enzymes higher in RS patients,like propionyl-Co A carboxylase beta chain(PCCB,K01966),which was a key enzyme in pathways where acetic acid was metabolized to oxalic acid.2.Antibiotics reduced intestinal bacteria load.The renal VK staining scores(31582.8 vs.13741.6,P<0.05)and urinary oxalate level(242 vs.202μg/m L,P<0.05)were higher in renal stone model rats with antibiotics than those without antibiotics.When compared with control rats,the relative abundances SCFAs-producing bacteria were lower in rats with antibiotics,such as Lactobacillus,Romboutsia,Ruminococcaceae,Turicibacter and Blautia.Additionally,the level of acetic acid(1.43μg/mg vs 0.80μg/mg,P<0.001),propionic acid(0.64μg/mg vs 0.33μg/mg,P<0.01)and butyric acid(1.07μg/mg vs 0.51μg/mg,P<0.001)in cecal content all decreased in rats with antibiotics.The expression of SLC26A3 in ileum(1.8-fold,P<0.01)and cecum(1.7-fold,P<0.001)increased,while SLC26A6 in ileum(40%,P<0.001)and cecum(50%,P<0.001)decreased in rats with antibiotics.3.(1).After the supplementation of acetate,propionate,or butyrate,the HE(0.8,0.2.0.2 vs.9.2,P<0.05)and VK staining scores(527,516.92 vs.15789,P<0.05)and the level of urinary oxalate(123,128.142 vs.202μg/m L,P<0.05)decreased in renal stone model rats.In the ileum,cecum and colon,the supplementation of acetate,propionate,or butyrate could reduce the expression of oxalate transporters SLC26A3and increased the expression of SLC26A6(P<0.05).The supplementation of acetate,propionate,or butyrate increased the relative abundance of SCFAs-producing bacteria,such as Lachnospiraceae,Ruminococcus,Eubacterium and Prevotellaceae and pathway ko00680(methane metabolism),which is involved in the production of acetic acid(P<0.05).When compared with model rats,the level of propionic acid(0.30μg/mg vs 0.51μg/mg,0.53μg/mg,P<0.05)and butyric acid(0.40μg/mg vs0.65μg/mg,0.71μg/mg,P<0.05)in the content of cecum were higher in rats administrated with propionate and butyrate(P<0.05).(2).The supplementation of acetate,propionate,or butyrate in rats with antibiotics also reduced the renal crystals,urinary oxalate and regulated the intestinal oxalate transporters SLC26A3/6.However,the SCFAs in the content of the cecum did not increase after administration of acetate,propionate,or butyrate.4.After the exposure to oxalic acid,the expression of SLC26A3 increased(1.5-fold,P<0.05)while SLC26A6 decreased(30%,P<0.05)in Caco-2 cells.Propionate(40%,P<0.05)and butyrate(20%,P<0.05)could inhibit the expression of SLC26A3.The level of SLC26A6 in Caco-2 cells increased after the treatment of acetate(1.3-fold,P<0.05)or propionate(1.2-fold,P<0.05).Conclusions1.The disorder of gut microbiota and metabolic pathway of SCFAs were associated with the formation of renal Ca Ox stones.2.Reducing intestinal bacterial load could decrease SCFAs level in intestinal content,regulate the expression of intestinal oxalate transporters and the metabolism of oxalate and promote the formation of renal crystals in rats.3.The supplementation of SCFAs could reduce the formation of renal Ca Ox stones by regulating the expression of intestinal oxalate transporters SLC26A3/6 and oxalate metabolism,which was independent of gut microbiota.4.SCFAs could directly regulate the expression of oxalate transporter SLC26A3/6in intestinal epithelial cells. |
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