Screening Of Diabetic Nephropathy Susceptibility Genes And Their Relationship With Gut Microbiota

Diabetic nephropathy(DN),as a major microvascular complication of diabetes,has an increasing incidence rate year by year,which has seriously affected the health level of Chinese people and the development of social economy.However,the understanding of its potential molecular pathogenic mechanism is limited at present.There are approximately 100 trillion different microbes in the human gastrointestinal tract,and the changes of gut microbiota in DN patients and the relationship between microbiota and host genetics are still unclear.Therefore,the genome-wide association study was adopted to explore the DN susceptibility genes from a genetic perspective.In addition,we also explored the gut microbiota imbalance pattern in patients with DN and the influence of genetic variation on gut microbiota.These are conducive to in-depth understanding of the pathogenesis of DN,construction of risk prediction models,accurate diagnosis and treatment.PartⅠ Screening of susceptibility genes for diabetic nephropathyObjective:Genome-wide association studies(GWAS)were used to screen susceptibility genes for diabetic nephropathy in China.Methods:A total of 85 diabetic nephropathy patients(DN group)and 107 patients(DM group)with type 2 diabetes mellitus for more than 10 years without microvascular disease confirmed by renal biopsy in Shanxi Provincial People’s Hospital from September 2019 to September 2022 were included in the genome-wide association study.The results were verified in an independent cohort consisting of 198 patients with DN and 200 patients with DM.Results:Through a two-phase GWAS study of 283 patients with DN and 307 patients with DM,we identified two new genetic susceptibility sites for DN,namely rs251418 in the phosphodiesterase 8B(PDE8B)gene and rs10859525 in the cytokine signal transduction inhibitor protein-2(SOCS2)gene.Risk genotypes at these two sites were closely related to glomerular filtration rate and urinary microalbumin to creatinine ratio.Copy number variation(CNV)analysis showed that a single CNV on chromosome 6 containing human leukocyte antigen complex 5(HCP5),major histocompatibility complex Class Ⅰ chain related molecule A(MICA),and human leukocyte antigen B(HLA-B)was significantly different between the two groups.We also verified previously reported SNPs associated with DN in our study cohort.Conclusion:We found new DN susceptibility genes in Chinese Han population.PartⅡ Study on gut microbiota imbalance pattern in diabetic nephropathyObjective:To explore the changes and imbalance patterns of gut microbiota in patients with diabetic nephropathy,diabetes patients and healthy people,and to find the key flora in the disease.Methods:Fifty patients with diabetic kidney disease confirmed by renal biopsy in Shanxi Provincial People’s Hospital(DN group),50 patients with type 2 diabetes for more than10 years without microvascular disease(DM group),and 50 healthy people matched in age and sex were included.Fecal samples were collected for 16 S r DNA sequencing to identify the structure of the fecal microbiota,and to evaluate the relationship between the relative abundance of gut microbiota and clinical phenotype and pathological indicators.Results:There were significant differences in the richness and diversity of gut microbiota among DN patients,DM patients and healthy control group.DM patients were accurately distinguished from age-and sex-matched healthy controls by g＿Clostridium-XVIII variants(AUC=0.929),and DN patients were accurately distinguished from healthy controls by g＿Gemmiger variants(AUC=0.842).Patients with DN can be accurately distinguished from patients with DM by g＿flavonifractor or g＿Eisenbergiella(AUC of0.909 and 0.886,respectively).The gut microbiota is also closely related to clinical phenotype and pathological indicators.Conclusion:We explored the imbalance pattern and functional changes of gut microbiota in two disease states,and established a classification algorithm model to distinguish the two by gut microbiota,which provides a certain basis for the diagnosis and prediction of diabetic nephropathy through gut microbiota.Part Ⅲ: The relationship between genetic variation of diabetic nephropathy and gut microbiotaObjective:To explore the relationship between genetic variation of diabetic nephropathy and gut microbiota.Methods:Genetic variations associated with gut microbiota were retrieved from the genome-wide association study database,and tested in the DN susceptibility gene screening cohort.Quantitative trait loci annotation of microorganisms were used,and finally 16 S r DNA gene sequencing was used to verify the candidate microorganisms with possible genetic associations.Results:We found 13 loci associated with DN susceptibility,and the genotypes of these loci were closely related to clinical phenotypes,such as TCF7L2 risk genotype was associated with long diabetes course and high diastolic blood pressure,SPECC1 L risk genotype was associated with increased diastolic blood pressure,and ZCWPW2 risk genotype was associated with increased glycosylated hemoglobin.The ZNRF3 risk genotype was associated with an elevated urinary microalbumin-to-creatinine ratio.In addition,the risk genotypes of SPECC1 L and ZNRF3 were associated with Lactococcus,and 16 S r DNA sequencing confirmed that there was indeed a significant difference in the Lactococcus genus between DN and DM patients.Conclusion:Our study demonstrated that the gut microbiota of DN is influenced by host genetics,and the genetic variations controlling gut microbial composition is closely related to DN susceptibility and clinical phenotype.