Alterations And Non-invasive Diagnostic Value Of Human Gut Microbiome In Non-dialysis Chronic Kidney Disease

Research backgroundChronic kidney disease(CKD)is one of the major public health problems in the world,with significant morbidity,mortality and medical costs.In patients with CKD,the irreversible damage of kidney structure and function can eventually lead to end-stage renal disease(ESRD),which often requires expensive kidney replacement therapy,such as hemodialysis,peritoneal dialysis or kidney transplantation.In addition,patients with ESRD often have life-threatening complications such as acute left heart failure,severe hyperkalemia,gastrointestinal bleeding,and central nervous system dysfunction,with high mortality and poor prognosis.It is estimated that for every 100 patients who receive dialysis,about 10-20 die each year.However,there is currently no clear treatment that can effectively prevent the progression of CKD.Although renal replacement therapy can extend the survival time of CKD patients to some extent,the patients' quality of life is seriously affected,and it imposes a significant financial burden on the patients' families and our countries.Therefore,it is a challenge and urgent need to find new biological diagnostic markers to detect renal function decline earlier and to explore new therapeutic targets in the current research field of CKD diagnosis and treatment.Gut microecosystem is an important part of human body,and the normal gut microbiome is necessary to maintain human health.The gut microbiome is closely related to many important physiological functions of the host,such as nutrition,metabolism and immunity.Science,Nature and other journals have published articles,believing that the gut microbiome is an important metabolic "organ" of the human body.The composition of gut flora "intestinal type" can reflect people's susceptibility to diseases,suggesting that gut microbial markers have potential functions of early warning and diagnosis.At present,it has been reported that gut microbial markers can be used as non-invasive diagnostic tools for early hepatocellular carcinoma,autoimmune hepatitis,tumor and type 2 diabetes,and gut microorganisms are closely related to the occurrence and development of chronic metabolic diseases such as hypertension and obesity.In recent years,with the introduction of the "gut-kidney axis" theory,the role of gut microbiome in CKD has attracted more and more attention.The study found that the feces of ESRD patients on dialysis contained 190 operational taxonomic units(OTUs),including Enterobacteriaceae,Moraxella and Halomonas,significantly different from that of healthy people.The structure and function of the intestinal mucosal barrier are impaired in ESRD patients,and gut microbes may affect the systemic inflammatory response of ESRD patients through microbial community translocation,inflammatory response and immune response.Some metabolites,such as Trimethylamine n-oxide(TMAO),Indoxyl sulfate(IS),.and Para-cresyl sulfate(PCS),may also be associated with gut microorganisms for toxic damage to the kidney.A study of patients with early CKD showed that serum PCS and IS levels can be used as early indicators of renal function decline,and changes in gut microorganisms related to PCS and IS can be detected in patients with early CKD.However,current studies mainly focus on the change characteristics of gut microorganisms and gut metabolite levels in ESRD patients,and have not fully explored the specific change characteristics of gut microbiome in non-dialysis CKD patients and the application value of gut microbial markers in the diagnosis of CKD.In order to clarify the change characteristics of human gut microbiome in non-dialysis CKD and explore the application value of gut microbial markers in the diagnosis of CKD,the following studies were conducted.Research objectiveMiseq sequencing of large clinical samples was used to explore the characteristics of human gut microbiome in non-dialysis chronic kidney disease(CKD)and the characteristics of human gut microbiome in different clinical stages of CKD,and to explore the application value of gut microbial markers as a non-invasive diagnostic tool for CKD.MethodsA total of 520 fecal samples from non-dialysis CKD patients and healthy volunteers in different regions of China were collected.After strict exclusion criteria,DNA extraction and 16SrRNA sequencing were performed on 503 samples.After strict data quality control,sequencing data of 489 fecal samples were finally included for analysis,including 159 CKD samples from Zhengzhou,57 CKD samples from Hangzhou and 273 healthy control samples from Zhengzhou.Fecal samples from Zhengzhou were randomly divided into a discovery phase(110 in the CKD group and 210 in the healthy control group)and a validation phase(49 in the CKD group and 63 in the healthy control group).In the discovery phase,we described and compared the phylogenetic status of the gut microbiome between the CKD group and the healthy control group,identified its key microbial markers,and constructed the CKD classifier model.In the validation phase,we verified the diagnostic efficacy of the CKD classifier by 49 CKD samples and 63 healthy control samples.Finally,the diagnostic efficacy of CKD classifier was independently verified by 57 CKD samples from Hangzhou.In addition,according to the clinical stages of CKD,110 CKD samples from the discovery phase were divided into groups A,B and C(Group A:26 samples of patients with stage 1-2 CKD;Group B:36 samples of patients with stage 3-4 CKD;Group C:48 samples of patients with stage 5 CKD),and the phylogenetic status of gut microbiome of the three groups of CKD samples were described and compared.Results1.The gut microbial diversity of CKD significantly decreased compared with that in the healthy controls.All P values were<0.05,showing statistically significant differences.There was no significant difference in the gut microbial diversity of CKD stage 1-2,CKD stage 3-4 and CKD stage 5.P>0.05,showing no statistically significant differences.2.Twenty-four bacterial genera,including Escherichia shigella,Haemophilus and Klebsiella,were dominant in the CKD,while 9 bacterial genera,including Faecalibacterium,Roseburia and Blautia,were dominant in the healthy controls.3.Based on the analysis of gene prediction function of gut microbiome,50 microbial functions,including ascorbic acid metabolism,tryptophan metabolism,and phenylalanine metabolism,were dominant in the CKD.The 36 microbial functions,including arginine and proline metabolism,starch and sucrose metabolism,and lysine biosynthesis,were dominant in the healthy controls.4.Compared with the gut microbiome of CKD stage 1-2,CKD stage 3-4 and CKD stage 5,Mollicutes and Tenericutes were dominant in the CKD stage 1-2,Parasutterella was dominant in the CKD satge 3-4,and Verrucomicrobia,Blautia and Akkermansia were dominant in CKD stage 5,All P values were<0.05,indicating statistically significant differences.5.Five optimal microbial markers were identified by 5-fold cross-validation of the random forest model,and the CKD classifier constructed based on these microbial markers reached 0.9887 area under the curve(AUC)in the discovery phase,0.9512 AUC in the validation phase,and 0.8986 AUC in the independent diagnosis phase.Conclusions1.The human gut microbiome diversity of non-dialysis CKD decreased,and clinical staging had little effect on the gut microbiome diversity of non-dialysis CKD.2.Compared with healthy people,the community composition,distribution and function of human gut microbiome of non-dialysis CKD have significant changes,and the altered gut microbes can promote the progress of CKD by affecting the metabolism of substances and metabolic pathways of the body.3.Targeted biomarkers of gut microbiome have a strong diagnostic potential for CKD and are expected to become a non-invasive diagnostic tool for CKD.