Altered Gut Microbiota In The Progression Of Chronic Kidney Disease And Effects Of Fecal Microbiota Transplantation On Mice With Chronic Renal Failure

BackgroundIt is well known that a complex community of symbiotic microorganisms exists in the human body,colonizing the intestinal,urogenital,respiratory,and other barrier tissues.These microbial communities participate in the development of the body and the formation and maintenance of physiological functions of the human body to maintain homeostasis,so they are called the body's other organs.The human gut contains the largest and most complex microbial community in the human body.A large number of studies have shown that intestinal flora plays an important role in human energy intake,nutritional support,metabolic balance,inflammation,neurohormonal response,immune system development and immune regulation.The chronic kidney disease(CKD)refers to chronic invasive renal injury caused by various causes.Because many metabolic wastes,such as urea and inosine,cannot be completely excreted by the kidney,they accumulate in the body of patients with CKD.These metabolic wastes enter the intestinal cavity through the blood vessels in the intestinal wall,causing changes in the intestinal biochemical environment,leading to changes in the structure and function of intestinal microflora.At the same time,studies have confirmed that changes in the structure and function of intestinal microflora,as well as the destruction of intestinal barrier structure and function will lead to the generation and absorption of certain toxic by-products by the human body,which are prone to cause uremia toxicity,inflammation,malnutrition and other diseases related to uremia.The development of high-throughput molecular biology techniques,such as second-generation sequencing,provides a very good technical support for us to study the structure and function of intestinal flora by using genomics methods.A growing body of research shows that intestinal microecological is not only closely related to intestinal diseases,but also closely related to the development of multiple diseases in many systems,such as obesity,turnor,diabetes,autism and CKD.At present,there is no research has been explored on changes in intestinal flora in the progression of CKD and which intestinal microbial flora is more suitable as biomarkers for noninvasive diagnosis and treatment of CKD.The process of fecal microbiota transplantation(FMT)is as follows:after the standardized treatment of feces of healthy people,the obtained functional microflora can be transplanted into the gastrointestinal tract of patients with dysbacteriosis,and the intestinal microflora can be reconstructed to treat diseases.At present,there have been studies on the application of FMT to gastrointestinal diseases,blood diseases,neuropsychiatric diseases,chronic hepatitis B,metabolic syndrome,drug-resistant bacteria infection and other diseases of the treatment.In 2013,FMT was written into US clinical guidelines for the treatment of recurrent clostridium difficile infections.The clinical efficacy of FMT has been verified in many diseases,but there is no research on the treatment of chronic renal failure by FMT.Based on the above research background and theoretical basis,we designed and conducted this study.This study mainly consisted of two parts.First,through high-throughput sequencing based on 16S rRNA,the structural characteristics of intestinal flora in patients with CKD were studied in order to find out the flora associated with the occurrence and development of chronic renal failure.Then,biomarkers related to chronic renal failure were further explored.Secondly,the mice model of chronic renal failure was established by adenine intragastric administration.The feces of normal mice were treated with fecal bacteria enema to study the effect of FMT on chronic renal failure mice.Then inflammatory cytokines and related signaling pathways related to fecal transplantation were screened.AimsThe aim of the present paper was to determine the differences of gut microbiota between chronic kidney disease and healthy controls(HC)and search better microbial biomarkers associated with CKD,then to explore the therapeutic effect of FMT for chronic renal failure and screen the inflammatory cytokines and signal pathways involved in the treatment of chronic renal failure using FMT,so as to provide some theoretical basis for the study of the mechanism of development of CKD related to gut microbiota and the treatment of CKD by regulating intestinal flora.Methods1.Stool samples from enrolled participants were collected and the V3-V4 region of the 16S rRNA gene was amplified and sequenced on the Illumina Miseq System.2.A variety of biological information analysis methods,such as species components analysis,principal component analysis and linear discriminant analysis,were used to compare the differences of intestinal flora between the patients with CKD and HC.3.LEfSe analysis was used to compare the difference of intestinal flora between patients with different stages of CKD and HC,and the difference of intestinal flora between patients with different stages of CKD.4.ROC curves of five phylotypes with the five highest LDA scores at the genus level in the HC and CKD groups and their area under the ROC curve(AUC)values were obtained to explore the ability of the gut microbiome to differentiate between the HC and CKD groups.5.The data from high-throughput sequencing were analyzed using EXCLE to explore microbial species associated with the progression of CKD and hemodialysis.6.The mouse models of CKD were induced by intragastric administration with adenine.7.FMT were used to treat mice with CKD and stool samples were collected and sequenced on the Illumina Miseq System.8.The effect of FMT was detected by comparing the level of serum creatinine(SCr)and urea nitrogen(BUN)in three group of mice.9.Haematoxylin and Eosin(HE)staining was used to study thepathological changes of mice kidney and colon after FMT.10.The serum of the FMT group and control group with CKD were collected and conduted Chip Test.Chip data analysis software,differential gene GO enrichment analysis,differential gene KEGG pathway enrichment analysis and other analysis methods were used to study the effect of FMT on inflammatory cytokines and related signaling pathways.Results1.The composition of gut microbiota was different in CKD populations compared with healthy populations.In this study,the large majority of the bacteria of fecal samples collected from both CKD and HC groups belonged to the phyla Firmicutes,Bacteroidetes,Proteobacteria and Actinobacteria.The relative abundance of the dominant main phyla Firmicutes,Bacteroidetes,and Proteobacteria between CKD and HC groups significantly changed.The CKD group had a significantly higher abundance of Bacteroidetes and Proteobacteria and lower abundance of Firmicutes compared with the HC group(P=0.002,0.03,and P<0.001 respectively).2.A total of 49 phylotypes were significantly different between the CKD and HC groups;45 phylotypes between the CKD-NonHD and HC groups;70 phylotypes between the CKD-HD and HC groups;26 phylotypes between the CKD-NonHD and CKD-HD groups;13 phylotypes among CKD-stage 2?CKD-stage 3 and CKD-stage 4.3.Out of five phylotypes with the five highest LDA scores at the genus level in the HC and CKD groups,Lachnospira achieved the best performance in the HC group(AUC=0.813;95%CI:0.713-0.912;P<0.001)and Ruminococcus_gnavus was the best in the CKD group(AUC?0.764;95%CI:0.656-0.873;P<0.001).4.At the genus level,the genus Holdemanella,Megamonas and Prevotella2 were not detected in the hemodialysis(HD)samples.The genus Dielma and Scardovia were not detected in all the HC samples.5.Effects of FMT on the composition of gut microbiota of mice with chronic renal failure:compared with the normal mice,the composition of gut microbiota in the control group with CKD was significantly changed,the proportion of phyla Firmicutes was significantly decreased and phyla Bacteroidetes was significantly increased(P=0.007 and P<0.001 respectively);the intestinal micro flora of mice with CKD restored after FMT,compared with the control group with CKD,the proportion of phyla Firmicutes was significantly inecreased and phyla Bacteroidetes was significantly decreased(P<0.001 and P<0.001 respectively).6.The effects of FMT on the biochemical indexes of chronic renal failure mice were as follows:Blood Urea Nitrogen(BUN)levels were significantly increased in the control group of renal failure and the FMT group compared with the normal group(PAB-0.001,PAC=0.001).The BUN level of the FMT group did not change significantly compared with the control group of renal failure(PBc=0.937).The serum creatinine(SCr)level of mice presented the same trend as BUN,that is,the normal group was significantly different from the FMT group and the control group of renal failure(PAB=<0.001,PAC<0.001).SCr levels in the fecal transplant group were lower than those in the control group of renal failure,but the difference was not statistically significant(PBC=0.185).7.The effect of faecal transplantation on the pathology of chronic renal failure mice was as follows.(1)The symptoms of renal histopathological changes in mice included obvious renal lesions and partial glomerular telangiectasia in the control group of renal failure.Renal tubular lesions were manifested as follows:the renal tubular epithelial cells were vesicular degeneration,the cytoplasm was loose and light stained,even vacuoles appeared in the cytoplasm,the tubular lumen of the renal tubules was obviously dilated,and sediments and cell debris were obviously visible in the lumen.There was mild fibrous hyperplasia in the renal interstitium with a small amount of inflammatory cell infiltration.In the fecal transplantation group,some glomerular capillaries were dilated and some glomerular cystic epithelial cells were mildly proliferated.The renal tubular lesions showed mild tubular epithelial cell vesicular degeneration,loose cytoplasm,light staining,obvious dilatation of tubular lumen,and obvious deposits and cell debris in the lumen.There was mild fibrous hyperplasia in the renal interstitium with a small amount of inflammatory cell infiltration.(2)The histopathological changes of the colon in mice were as follows:in the control group of renal failure,the submucosa was loose and edema with enlarged space,and monocytes and neutrophils were distributed.Inflammatory cell infiltration was seen at the bottom of the intestinal mucosa,with focal necrosis of the intestinal mucosa and fibrous hyperplasia.In the fecal fungus transplantation group,the submucosa was loose and edema,and the space was enlarged.Inflammatory cell infiltration was seen at the bottom of the intestinal mucosa,with focal necrosis of the intestinal mucosa and fibrous hyperplasia.8.Nine inflammatory cytokines,including MIP-1 alpha,IL-13,SDF-1,KC,GCSF,BLC,EOTAXIN-2,Lymphotactin and LIX,were highly expressed in the fecal transplantation group,while Leptin and I-TAC were lowly expressed in the fecal transplantation group.9.The pathways for significant enrichment of differentially expressed genes between the FMT group and the control group were Cytokine-cytokine receptor interaction,Chemokine signaling pathway,IL-17 signaling pathway,Rheumatoid arthritis,Jak-STAT signaling pathway,Salmonella infection and Toll-like receptor signaling pathway.Conclusion1.The composition of gut microbiota is different in CKD populations compared with healthy populations.2.The intestinal flora is different between the HC and different stage of CKD groups and CKD-HD group had more differentiated bacteria than CKJD-NonHD group.There were differences in intestinal flora among patients with CKD at different stages.3.Lachnospira and R._gnavuswere are better microbial biomarkers to differentiate between the HC and CKD groups,large-scale prospective studies and control studies with extensive sample panels should be conducted to develop more reliable microbiome biomarkers.4.FMT altered the intestinal flora composition of mice with chronic renal failure,but had no significant effect on BUN and SCr.5.Differential inflammatory cytokines and related signaling pathways related to FMT were screened,which provided a theoretical basis for further study on the mechanism of the effect of FMT on chronic renal failure.