Construction And Trans-species Analysis Of Single-cell Transcriptome Atlas Of Macaca Kidney

Backgrounds The development of high-throughput sequencing technique facilitates in-depth research on mechanism of diseases.It has been realized that the acknowledge of diseases should be refined from tissue level to single-cell level and single-cell RNA sequencing(sc RNA-seq)emerges at a historic moment.The universal utilization of experimental animals in life science provides a promising platform for investigating underlying cellular and molecular mechanism of development and disease,and screening drugs.Rodents have been widely applied to study the etiology and pathology of numerous diseases due to their advantages in small body size,easy-feeding and propagating rapidly.However,rodent based disease models fail to copy characteristics of clinical patients well.For example,although mouse or rat model of UUO(unilateral ureteral obstruction)has been demonstrated to mimic the key features of renal interstitium fibrosis and utilized for study of role for EMT(epithelial-mesenchymal transition)in renal fibrosis,it is inconsistent with the fact that progressive destruction of functional nephrons contributes to renal fibrosis in chronic kidney disease.NHPs(Non-Human Primates)show high similarity to human in anatomical structure,genetics,physiological metabolism,etcetera.Approximately 98.5% of NHPs genes are homogenous to human genes.Therefore,NHPs show superiority over other mammals and are the optimal animal models for human major diseases research and preclinical drug screening.Currently,many studies have been conducted to construct single-cell transcriptome atlas of human and mouse kidney via sc RNAseq successfully.Using the advantages of 10× genomics,this study established the single-cell transcriptome atlas of cynomolgus macaque kidney and made trans-species analysis on gene expression differences among human,cynomolgus macaque and mouse kidney,with the aim of exploring the cell distribution and characteristics of normal kidney in NHPs and providing novel insight into the choice of animal models for designing kidney associated diseases.Methods1.The kidney single cell suspension was separated from cynomolgus macaque normal kidney tissues by enzyme digestion.A qualified c DNA library that prepared with single cell suspension which meet the criteria for the machine was tested for sequencing.The software including Fastq,Cell Ranger and Seurat were used for quality control,dimension reduction,cell clustering analysis and differential gene expression analysis of kidney single cell sequencing data.2.Single cell RNA sequencing data of human and mouse kidney were obtained from NCBI database.We performed data integration,quality control,batch effects elimination,dimension reduction and cell clustering analysis using Seurat,Harmony and other R packages.3.We transferred the cynomolgus macaque and mouse gene names into human homologous gene names with the ENSEMBL R package bioma Rt and eliminated genes with no gene names and retained identical names between the human and cynomolgus macaque database as well as the human and mouse database.We used average expression of these homologous genes for comparison among human,cynomolgus macaque and mouse cell types and to calculate the correlation coefficients between human and cynomolgus macaque,human and mouse.4.The software including pheatmap and Seurat were used for investigating the expression pattern differences of GWAS(Genome-Wide Associated Studies,GWAS)identified complex traits kidney disease related genes in human,cynomolgus macaque and mouse.Meanwhile,we compared the differential expression of target genes that related to hypertension treatment(ACE、ACE2、MME、ECE1、MAS1、AGTR1 and AGTR2)cross-species.Results1.We identified 37 cluster cells from cynomolgus macaque normal kidney tissues,including proximal tubule cells 1-13,intercalated cells 1-3,endothelial cells 1-4,principle cells,T/NK cells,myofibroblasts 1-2,macrophages 1-2,Loop of Henle,B cells and erythrocytes.2.We identified 28 cluster cells from integrated mouse normal kidney tissues,including proximal tubule cells 1-11,distal tubule cells 1-3,intercalated cells 1-2,endothelial cells 1-3,principle cells,T/NK cells,myofibroblasts 1-2,macrophages,Loop of Henle,B cells and podocytes.3.We identified 32 cluster cells from integrated human normal kidney tissues,including proximal tubule cells 1-20,T/NK cells 1-5,neutrophils,Loop of Henle,endothelial cells,B cells,intercalated cells,principle cells and plasma cells4.The AE(average expression)of homologous genes between cynomolgus macaque kidney cells and human kidney cells showed higher correlation,the Pearson correlation coefficient was 0.806,whereas the AE of homologous genes between mouse kidney cells and human kidney cells showed lower correlation,the Pearson correlation coefficient was 0.778.5.High correlations were observed between cynomolgus macaque and human proximal tubule cells,endothelium,and T cells,the Pearson correlation coefficients were 0.838,0.824 and 0.836,respectively.Similarly,we observed high correlations between mouse and human intercalated cells,principle cells,Loop of Henle and B cells,the Pearson correlation coefficients were 0.749,0.794,0.748 and 0.815,respectively6.Many genes that associated with renal diseases were specifically expressed in single cell type,but the expression of some genes also showed differences across species.The expression pattern of blood pressure associated genes in human and cynomolgus macaque kidney cells showed high similarity.7.Therapeutic genes related to hypertension also showed correlations and differences across species.ConclusionsUsing 10× Genomics single-cell RNA sequencing(sc RNA-seq)technique,we constructed the single-cell transcriptome atlas of non-human primate normal kidney and integrated sc RNA-seq data of human and mouse normal kidney.Meanwhile,we analyzed the correlations and differences of the cellular composition and gene expression across species and investigated the gene expression patterns of renal disease associated genes in cynomolgus macaque,human and mouse kidney cells.The single cell description of non-human primate kidney provides the baseline for the further studies that related to kidney disease and contributes to the choice of experimental animal models in kidney disease studies.