#4851 MOLECULAR FEATURES OF CIRCULATORY B CELLS AND RENAL CELLS AT THE SINGLE-CELL LEVEL IN AUTOANTIBODY-NEGATIVE PRIMARY MEMBRANOUS NEPHROPATHY PATIENTS

Abstract

Abstract Background and Aims Primary membranous nephropathy (PMN) is an organ-specific autoimmune disease that is the most common cause of idiopathic nephrotic syndrome in adults. Furthermore, the incidence of PMN is increasing, especially in young adults and children. The landmark discovery of nephritogenic autoantibodies against podocyte antigens such as M-type phospholipase A2 receptor (PLA2R) and thrombospondin type-1 domain-containing protein 7A (THSD7A) has provided a paradigm shift in diagnosis and treatment of PMN. However, there are still few patients with negative serum and renal tissue autoantibodies. This study aims to explore the molecular features of antibody-negative PMN. Method We sorted CD19+ cells from the peripheral blood of a pediatric patient with seven nephritogenic autoantibodies-negative PMN (NEG) by flow cytometry and performed single-cell transcriptome sequencing (scRNA-seq) and single-cell B cell receptor sequencing (scBCR-seq). Meanwhile, scRNA-seq was performed on renal biopsy tissues from the same patient. In addition, we included the scRNA-seq data of renal from anti-PLA2R antibody positive patients (POS) and healthy controls (CTRL), and the scBCR-seq data of B cell from CTRL for integrative analysis. Results The NEG patient showed typical nephrotic syndrome and was diagnosed as stage Ⅲ membranous nephropathy by renal biopsy. There was no evidence of anti-PLA2R or anti-THSD7A antibodies in peripheral blood and seven nephritogenic autoantibodies such as anti-PLA2R antibodies in renal tissue. After excluding secondary factors, the patient was diagnosed with PMN. Through scRNA-seq of CD19+ cells, we found that the number, characteristic gene, function and clonotype of naÏve B cells and memory B cells in NEG were significantly changed. Expanded CD38+ naÏve B cells in NEG had the molecular characteristics of CD19+CD24+CD27-CD38+, defined as transitional B cells. This group of cells had distinct function features of cell activation, and its up-regulated genes were involved in multiple aspects of the BCR signaling pathway. Pseudotime trajectory analysis suggested CD38+ naÏve B cells were highly enriched in the beginning of B cell differentiation. There was a preference in the use of VJ gene segments of B cells between NEG and CTRL, especially an increase of the IGHV3-23 and IGLV2-14 in NEG. The stronger pairing frequencies, IGLV2-14/IGLJ3 and IGKV2D-29/IGKJ2, were indicated in NEG. We identified 14 distinct kidney cell types by marker genes. Through re-clustering of glomerular parietal epithelial cells (PECs), the patients were clearly distinct from their control counterparts, indicating a major shift in gene expression for this cell type. PECs in NEG showed significant up-regulation of cellular communication network factor-related genes (CCN1, CCN2), phospholipase A and acyltransferase-related genes (PLAAT4, PLAAT3), and septin protein-related genes (SEPTIN2, SEPTIN7), accompanied by significant down-regulation of podocyte-related genes. In addition, there are clearly distinct cellular functions and pseudotime trajectory in PECs from NEG and POS, and genes such as CCN2, PLAAT4, SEPTIN2 might drive the special trajectory of PECs in NEG. For the podocytes, the genes related with extracellular matrix and cell adhesion were significantly enhanced in NEG, which consistented with the functional enrichment analysis of the differentially expressed genes and gene set-based scores. We calculated the gene set-based scores including genes encoding lumen-to-blood sodium transporters. Results indicated the enhanced expression of sodium transporters in distal nephrons of MN patients. More surprisingly, a group of proximal tubule epithelial cells showed significantly higher expression levels of sodium transporters. Among them, the expression of SLC5A12 encoding SMCT2 increased significantly. Conclusion We have systematic revealed the cell-type specific molecular features of PMN patients from circulation to renal tissue. Our research provides valuable evidence for the molecular diagnosis of PMN in children and insights into pathogenic mechanism of classical nephritogenic autoantibody-negative PMN.