Urinary single-cell sequence analysis of the urinary macrophage in different outcomes of membranous nephropathy

Abstract

Background: Great progress has been made in the diagnosis and treatment of membranous nephropathy (MN). However, a significant number of patients do not respond to immunosuppressive therapy and eventually progress to end-stage kidney disease. To investigate the mechanism of different outcome of MN, we performed single-cell sequencing to analyze the urine cells of patients with and without complete remission of MN.

Methods: Urine single-cell RNA sequencing was performed on 12 healthy controls (HC) and 15 patients with MN. The patients were divided into a complete remission group (CR, n = 9) and a no remission group (NR, n = 6).

Results: (i) Macrophages were the largest group in urine cells, comprising 48.02%, 68.96% and 20.95% in the HC, CR and NR groups, respectively. (ii) Urinary macrophages expressing FIColin-1 and S100 calcium-binding protein A8 were mainly found in the HC and CR groups, indicating that they were derived from bone marrow and peripheral blood, while the urinary macrophages expressing the regulator of G-protein signaling 1 (RGS1) and HLA-DPA1, mainly found in the NR group, were derived from renal resident macrophages. (iii) In healthy adults, urine macrophages expressed the metallothionein family, indicating that they can regulate anti-inflammatory and proinflammatory functions bidirectionally. In the CR group, the urine macrophages showed strong proinflammatory properties. In the NR group, the urinary macrophages mainly associated with the level of proteinuria and the impaired renal function.

Conclusions: Our study firstly delineated the differences in urinary cell maps between healthy individuals and MN patients with CR or NR outcomes. Not only the origin but also the function of urine macrophages were different in the HC, CR and NR groups.

Keywords: macrophages; membranous nephropathy; proteinuria; urine; urine single-cell sequencing.

Graphical Abstract

Figure 1:

Study design and histopathological findings on the renal biopsy. (A) Granular deposition of IgG along capillary wall (immunofluorescence assay, 400×). (B) Diffuse thickening of the glomerular basement membrane and spike formation (PASM, 400×). (C) Diffuse and irregular thickening of glomerular basement membrane and electron-dense deposits in the subepithelial areas (electron microscopy, 5000×).

Figure 2:

Immunohistochemical staining shows types of inflammatory cells in renal interstitial. (A) CD3; (B) CD20; (C) CD68.

Figure 3:

Single-cell sequencing showed urinary cell types and their distribution in the HC, CR and NR groups. (A) Number and types of total urinary single cells in all groups. (B) The cell markers used to cluster the seven cell subpopulations are shown in a heatmap. (C) tSNE diagram showing the clustering distribution characteristics of all cells in the HC, CR and NR groups. (D) The histogram shows the proportion of different cell types in the HC, CR and NR groups.

Figure 4:

The composition and source of urinary macrophages in patients in the HC, CR and NR groups. (A) Subclustering analysis of the macrophage population obtained three subpopulations (Groups 1, 2 and 3). (B) Different distribution of the macrophages subpopulations in the HC, CR and NR groups. (C) The histogram shows the proportion of the macrophages subpopulations in the HC, CR and NR groups. (D) FeaturePlot shows representative markers of the source of the urine macrophages: S100A8 and FCN1 represent peripheral blood and bone marrow–derived macrophages. RGS1 and C1QC represent the sources of renal resident macrophages. (E) The heatmap shows the source of Groups 1, 2 and 3.

Figure 5:

The functions and characteristics of urinary macrophages in patients in HC, CR and NR groups. (A) Heat map of the top ranked genes highly expressed in three groups of macrophages. Color scheme is based on z-score distribution from –2 (purple) to 2 (yellow). (B and C) Bubble chart and violin plots of the expression of other representative marker genes related to the function across the three groups. (D) GO enrichment shows the biological processes involved in three groups of macrophages. The left side of the circle represents the DEGs of three groups, while the right side represents different biological processes.

Figure 6:

Polarity analysis of urinary macrophages in the CR and NR groups. (A) DEGs analysis on macrophages in the CR and NR groups. (B) Macrophages Macspectrum analysis model was used to analyze the HC, CR and NR groups. (C) GO analysis in the CR and NR groups. (D and E) Gene-set enrichment analysis in the CR and NR groups. (F) Immunohistochemical staining of S100A8/9 in the HC, CR and NR groups.