Preliminary Exploration Gene Expression And Clinical Significance On IMN By Single-cell Transcriptome Sequencing

Background:Idiopathic membranous nephropathy(IMN)is a common pathological type of nephrotic syndrome and one of the main causes of end-stage renal disease.The main clinical manifestation of IMN patients is proteinuria,and the clinical outcomes vary greatly.At present,most of the scholars believe that the formation of subepithelial immune complex mediated immune response is the central link of IMN pathogenesis,but the specific pathogenesis has not been clarified.Therefore,in this study,the sc RNA-seq was applied to the renal biopsy tissue of IMN patients to construct gene expression profiles of IMN,analyze the gene changes of various cells in the IMN kidney,find out the active and up-regulated genes and biological pathways in IMN patients,and further expanded the samples to detect the immune indexes and inflammatory factors in serum and urine of patients with IMN,so as to verify the expression changes of these genes clinically,which provides a theoretical basis for further mechanism research and targeted therapy of IMN in the future.Methods:(1)Six patients with newly diagnosed IMN(both kidney PLA2 R expression and serum anti-PLA2 R antibody were positive)were screened from our department,as well as two kidney puncture tissues from healthy donors in the department of organ transplantation.The kidney puncture tissue was prepared by single cell suspension,single cell capture,reverse transcription and PCR amplification to construct the library,and then the single cell transcriptome was sequenced by Illumina Hiseq X Ten sequencing platform.After quality control,dimension reduction cluster analysis,cell annotation and visualization,the kidney gene expression profile of IMN patients was constructed.(2)By comparing the transcriptional profiles of patients with IMN and healthy subjects,Find All Markers was used to identify the differentially expressed genes(DEGs)of each cell cluster between the two groups;GO and KEGG enrichment analysis were performed among the up-regulated DEGs by using cluster Profiler;Cytoscape was used to map the protein network interaction of up-regulated DEGs in renal cells;Cellphone DB was used to analyze the ligand-receptor interaction between cells;Monocle 2 was used to infer the cell trajectory based on the results of cell clustering.(3)By comparing the DEGs,enrichment analysis and protein network interaction analysis of various types of renal cells in IMN patients with massive proteinuria and patients with non-massive proteinuria,we aimed to find the key genes or signal pathways related to proteinuria.(4)44 patients with IMN,29 patients with MCD and 12 healthy subjects in the health examination center were screened in our hospital.The general conditions and clinical examination results of the three groups were collected.The levels of IL17 A,IL8,HSP90α,NF-κB p65 in serum and urine were detected by ELISA;Meanwhile,the paraffin samples of renal biopsy in patients with IMN,MCD and the adjacent tissues of renal cancer in the department of pathology in our hospital for immunohistochemical staining to analysis the expression of IL17 A,IL8,HSP90α,NF-κB p65 in IMN kidney.Results:(1)We successfully established the gene expression profile of single kidney cell in patients with IMN,and obtained the transcriptome data of 30313 cells in 8 subjects.We identified 17 cell clusters,including the traditional known renal innate cells,immune cells and a new epithelial cell population defined by CLDN4,KRT8 and EPCAM gene.(2)(1)Compared with healthy subjects,we identified a large number of DEGs in intrinsic kidney cells of IMN patients.(2)GO enrichment analysis showed that the up-regulated DEGs in most glomerular cells were related to apoptosis,whereas in adhesion junction and oxidative phosphorylation in most tubular epithelial cells.KEGG enrichment analysis showed that the up-regulated DEGs in kidney cells were enriched in IL-17 signaling,TNF signaling,NOD like receptor signaling,MAPK signaling and NF-κB signaling pathway.(3)m RNA interaction network analysis suggested that chemokines such as CCL2,CXCL1,CXCL2,CXCL3,IL6 in endothelial cells may play an important role in the pathogenesis of IMN.(4)Intercellular ligand-receptor analysis showed that mesangial cells communicated widely and actively with other cells,especially with epithelial cells,proximal tubular cells and fibroblasts.(5)Trajectory inference suggested that the pseudo temporal changes of cell state and gene expression in the possible evolution process between pericytes,mesangial cells and podocytes,pericytes and fibroblasts,monocyte and macrophage subtypes.(3)Most renal parenchymal cells of IMN patients in massive proteinuria group were enriched in the regulation of inflammation and immune response,including IL-17 signaling,TNF signaling,MAPK signaling and NOD like receptor signaling pathway,involving FOS,JUN,SOCS3,NFKBIA,CXCL2 etc.Compared with the non-massive proteinuria group in IMN patients,the analysis of kidney m RNA interaction in the massive proteinuria group suggested a central role of UBC,FOS,JUN,IRF1,NFKBIA,DUSP1,CTSD,MMP7.(4)Compared with the healthy control group,the levels of IL17 A,IL8,Hsp90α,NF-κB p65 in serum and renal tissue of IMN increased significantly;the urine IL8 increased,urine Hsp90α reduced,and IL17 A tended to increase but there was no statistical significance.Conclusion:(1)The study successfully constructed a single-cell gene expression profile of IMN patients.(2)The renal intrinsic cells of IMN patients exist disorder of biological processes such as apoptosis,adhesion junction,inflammatory response,immune dysfunction.Chemokines are especially active in endothelial cells.Mesangial cells have extensive communication with other renal intrinsic cells.(3)Massive proteinuria in IMN patients is associated with inflammation and immune disorders,including IL-17 signal,TNF signal,MAPK signal and NOD like receptor signal pathway;UBC,FOS,JUN,IRF1,NFKBIA,DUSP1,CTSD,MMP7 may be related to the excretion of urinary protein and the progression of IMN.(4)The abnormal activity of IL17A/IL8 system and Hsp90α/NF-κB system indicates that IMN patients have inflammatory and immune dysfunction.