Digital Spatial Profiling of Individual Glomeruli From Patients With Anti-Neutrophil Cytoplasmic Autoantibody-Associated Glomerulonephritis

Abstract

We previously showed that the rupture of Bowman's capsule (BC) promotes the progression of crescentic glomerulonephritis by enhancing the entry of CD8⁺ T cells into the glomeruli. In the present study, we utilized digital spatial profiling to simultaneously profile the altered abundances of the messenger RNA (mRNA) transcripts and proteins in the glomerular and periglomerular areas of four biopsy samples of anti-neutrophil cytoplasmic autoantibody-associated glomerulonephritis (ANCA-GN) and two biopsy specimens of minimal change disease (MCD) controls. The paraffin-embedded biopsy samples were stained with collagen IV, CD45, and SYTO 13 to distinguish the glomeruli with periglomerular infiltration but intact BC, with focal BC rupture, and with extensive rupture of BC and glomeruli without crescent formation and leukocytic infiltration in ANCA-GN. By assessing multiple discrete glomerular areas, we found that the transcript expression levels of the secreted phosphoprotein-1 and its receptor CD44 were upregulated significantly in the glomeruli with more severe ruptures of BC, and their expression levels correlated positively with the fibrotic markers. We also found that both alternative and classic complement pathways were activated in the glomeruli from patients with ANCA-GN. Furthermore, M1 macrophages were involved mostly in the early stage of BC rupture, while M2 macrophages were involved in the late stage and may contribute to the fibrosis process of the crescents. Finally, loss of glomerular cells in ANCA-GN was likely mediated by apoptosis. Our results show that digital spatial profiling allows the comparative analysis of the mRNA and protein profiles in individual glomeruli affected differently by the disease process and the identification of potential novel mechanisms in ANCA-GN.

Keywords: Bowman’s capsule; CD44; anti-neutrophil cytoplasmic antibody (ANCA); complement; digital spatial profiling (DSP); fibrosis; glomerulonephritis; secreted phosphoprotein 1 (SPP1).

Graphical Abstract

Figure 1

Digital spatial profiling of renal biopsies from patients with ANCA-GN and MCD. (A) Schematic overview of the DSP workflow. (B) Information on the glomeruli included in this analysis. Four patients with ANCA-GN and two patients with MCD were enrolled. The region of interest (ROI) was selected evenly from patients according to the immunofluorescence staining with collagen IV, CD45, and SYTO 13. For the RNA profiles, the glomeruli from MCD (n = 12), from ANCA-GN with crescent formation but intact BC (BCI, n = 7), with segmental BC rupture (SBCR, n = 11), with global BC rupture (GBCR, n = 13), and without crescent formation and leukocyte infiltration (CON, n = 4) were included for analysis. For the protein profiles, glomeruli with the characteristics of CON (n = 6), BCI (n = 7), SBCR (n = 5), and BCR (n = 7) were included for further analysis. (C) Representative images from each group. Scale bar, 25 μm. (D) Split violin plot of the RNA counts by ROIs. Gene counts are shown on the left (orange) and the negative probe counts are displayed on the right (green). (E) Split violin plot of the protein counts by ROIs. ANCA-GN, anti-neutrophil cytoplasmic autoantibody-associated glomerulonephritis; MCD, minimal change disease; DSP, digital spatial profiling; WBCR, without Bowman’s capsule rupture; BCR, Bowman’s capsule rupture; BC, Bowman’s capsule.

Figure 2

The classic complement pathway is involved in the pathogenesis of anti-neutrophil cytoplasmic autoantibody-associated glomerulonephritis (ANCA-GN). (A) Heatmap of the 1,813 detected genes. Cluster analysis indicated seven main clusters marked in different colors (left). (B) Pathway enrichment analysis of cluster 6 [red box in (A)]. (C) Neutrophil degranulation and complement-related pathways are highlighted in green box, and the heatmap of related genes is displayed separately. (D) RNA expression of the complement pathway of lectin-related genes. (E) RNA expression of the alternative complement pathway-related genes. (F) RNA expression of the classic complement pathway-related genes. All data were analyzed by one-way analysis of variance (ANOVA). Compared with the minimal change disease (MCD) group: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Figure 3

SPP1 signaling contributes to the fibrosis pathway in anti-neutrophil cytoplasmic autoantibody-associated glomerulonephritis (ANCA-GN). (A, B, E, F, I) RNA expression of SPP1, CD44, COL1A1, FN, and CD163 with worsening of glomerular injury. (C, D, G, H, J, K) Correlation of the RNA expressions between SPP1 and CD44, TGF-β1, COL1A1, FN, CD163, and CD86. (L) Correlation of the RNA expressions between CD44 and CD163. All data were analyzed by one-way analysis of variance (ANOVA). Compared with the MCD (minimal change disease) group: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Compared with the WBCR (without Bowman’s capsule rupture) group: ^#p < 0.05 and ^##p < 0.01. SPP1, secreted phosphoprotein 1; COL1A1, collagen type I alpha 1 chain; FN, fibronectin 1.

Figure 4

Protein profiles indicating that apoptosis might be the main pathway responsible for the glomerular cell injury in anti-neutrophil cytoplasmic autoantibody-associated glomerulonephritis (ANCA-GN). (A) Heatmap of 45 detected proteins. (B) Correlation analysis of common proteins and RNAs of individual targets generated from digital spatial profiling (DSP) based on all regions of interest (ROIs). (C) Protein expression of CD34 with worsening of the glomerular injury. (D) Protein expression correlation of CD34 and CD45. (E) Transcription expression of VEGFA with worsening of the glomerular injury. (F) Protein expressions of BCL-XL and PARP. (G) Immunofluorescence co-staining of caspase-3 and CD31 in serial renal biopsy specimens of patients with ANCA-GN. Scale bar, 25 μm. The image of collagen IV staining was from the serial kidney section for protein DSP. PAS staining was performed on the same slide after co-staining with caspase-3 and CD31. Compared with the CON (control) group: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Compared with the WBCR (without Bowman’s capsule rupture) group: ^#p < 0.05. VEGFA, vascular endothelial growth factor A; BCL-XL, B-cell lymphoma—extra large; PARP: poly(ADP-ribose) polymerase; PAS, periodic acid–Schiff.

Figure 5

Different types of immune cell infiltration during rupture of the Bowman’s capsule. (A) Heatmap of the cell abundance of immune cell infiltration based on the regions of interest (ROIs) selected from anti-neutrophil cytoplasmic autoantibody-associated glomerulonephritis (ANCA-GN) patients. (B) Average cell abundance of each category of immune cells in different groups. (C) Protein expressions of immune cell markers, including CD45, CD4, CD45RO, CD8, CD20, CD68, CD11C, and CD163. (D) Immunofluorescence co-staining of iNOS and CD206 in serial renal biopsy specimens of patients with ANCA-GN. Scale bar, 25 μm. Intact Bowman’s capsule is indicated by solid circle, while extensive Bowman’s capsule rupture is indicated by the dotted line. The image of collagen IV staining from the serial section for protein digital spatial profiling (DSP) indicated the integrity of the Bowman’s capsule. Periodic acid–Schiff (PAS) staining was performed on the same slide after co-staining with iNOS and CD206. Compared with the CON (control) group: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.