Single-Cell Profiling Reveals Transcriptional Signatures and Cell-Cell Crosstalk in Anti-PLA2R Positive Idiopathic Membranous Nephropathy Patients

Abstract

Idiopathic membranous nephropathy (IMN) is an organ-specific autoimmune disease of the kidney glomerulus. It may gradually progress to end-stage renal disease (ESRD) characterized by increased proteinuria, which leads to serious consequences. Although substantial advances have been made in the understanding of the molecular bases of IMN in the last 10 years, certain questions remain largely unanswered. To define the transcriptomic landscape at single-cell resolution, we analyzed kidney samples from 6 patients with anti-PLA2R positive IMN and 2 healthy control subjects using single-cell RNA sequencing. We then identified distinct cell clusters through unsupervised clustering analysis of kidney specimens. Identification of the differentially expressed genes (DEGs) and enrichment analysis as well as the interaction between cells were also performed. Based on transcriptional expression patterns, we identified all previously described cell types in the kidney. The DEGs in most kidney parenchymal cells were primarily enriched in genes involved in the regulation of inflammation and immune response including IL-17 signaling, TNF signaling, NOD-like receptor signaling, and MAPK signaling. Moreover, cell-cell crosstalk highlighted the extensive communication of mesangial cells, which infers great importance in IMN. IMN with massive proteinuria displayed elevated expression of genes participating in inflammatory signaling pathways that may be involved in the pathogenesis of the progression of IMN. Overall, we applied single-cell RNA sequencing to IMN to uncover intercellular interactions, elucidate key pathways underlying the pathogenesis, and identify novel therapeutic targets of anti-PLA2R positive IMN.

Keywords: idiopathic membranous nephropathy; immune response; inflammation; kidney; single-cell RNA sequence.

Figure 1

Cell lineage analysis by comprehensive single-cell RNA-sequencing in anti-PLA2R positive IMN and control subjects. (A) Schematic of the scRNA-seq pipeline. Kidney samples from patients with IMN (n=6) or healthy control subjects (n=2) were collected at the time of clinically indicated renal biopsy or live kidney donation, respectively. Kidney biopsies were enzymatically disaggregated into single-cell suspensions and loaded onto a microfluidic device for cell barcoding, cell lysis, reverse RNA transcription, and then scRNA-seq as well as various other analyses. (B) Seventeen distinct cell clusters were visualized by UMAP plotting, with each cell color-coded for its associated subtypes. The color of the cells represents group origin. (C) UMAP plot of cell clusters from different subjects of IMN patients and control. The color of cells reflected the individual origin. (D) Bar plots of the percent contribution of cell clusters in kidneys from different subjects. Blocks represented different subjects, and block height was in proportion to the number of cells. (E) Heatmap of the top 20 most differentially expressed genes in each cluster to identify mutually exclusive gene sets, which were then used to determine the cell lineage of each cluster. Each column represented a cell cluster, and each row corresponded to a marker gene for the individual cluster. Transcript abundance ranges from low (purple) to high (yellow). (F) Violin plot of selected marker genes that identified the clusters generated by UMAP plotting. It was colored by different cell subtypes. PT, proximal tubule cells; LOH, loop of Henle cells; PC, principal cells; IC, intercalated cells; DT, distal tubule cells; EC, endothelial cells; Pod, podocytes; MC, mesangial cell; DC, dendritic cells; Mac, macrophages; Mono, monocytes; Fib, fibroblasts; Per, pericyte.

Figure 2

DEGs and enrichment analysis in the kidney cells of anti-PLA2R positive IMN and control subjects. (A) Representative DEGs in mesangial cells, endothelial cells, and pericytes comparing the IMN patients to healthy donor control. pct.exp: percentage of cells expressing gene. (B) Representative DEGs in proximal tubule cells, distal tubule cells, loop of Henle cells, principal cells, and intercalated cells between IMN patients and control. (C) Representative DEGs in immune cells between IMN patients and control. (D, E) GO and KEGG enrichment shows the biological processes or signal pathways involved in different kidney cells, respectively. The left side of the circle represents different cell types, while the right side represents different biological processes or signaling pathways. The inner-circle represents gene numbers involved in cells or biological processes and signaling pathways, whereas the outer-circle represents the proportion of each cell type in biological processes and signaling pathways or the proportion of biological processes and signaling pathways in kidney cells.

Figure 3

Possible ligand-receptor interactions between different cell types in the kidney of anti-PLA2R positive IMN patients. (A) Ligand-receptor signaling pathways between cell clusters in the kidney. Cell-cell crosstalk frequency ranges from low (blue) to high (purple). (B) Representative ligand-receptor interactions between mesangial cells and endothelial cells. (C) Representative ligand-receptor interactions between mesangial cells and podocytes. (D) Representative ligand-receptor interactions between mesangial cells and proximal tubule cells. (E) Representative ligand-receptor interactions between mesangial cells and loop of Henle cells. (F) Representative ligand-receptor interactions between mesangial cells and fibroblasts. (G) Representative ligand-receptor interactions between mesangial cells and macrophages. Lines represented interrelations between the mesangial cells and other cells. Lines between the ligand and conjunct receptors were shown. Only IMN patients (n=6) were analyzed.

Figure 4

DEGs involved in biological process and intercellular signaling in the kidney from anti-PLA2R positive IMN patients with massive proteinuria compared to non-massive proteinuria. (A) UMAP plot of cell clusters between massive proteinuria and non-massive proteinuria. The color of cells reflected different groups. (B, C) KEGG and GO enrichment analysis showed that upregulated DEGs were mostly involved in apoptosis, cell adhesion, and regulation of inflammation and the immune response in massive proteinuria patients, compared with non-massive proteinuria subjects.