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

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.

Automated summary

The study used digital spatial profiling to simultaneously analyze the mRNA and protein profiles in glomerular and periglomerular areas of ANCA-GN patients and MCD controls. The findings revealed that the severe rupture of Bowman's capsule in ANCA-GN patients led to the upregulation of secreted phosphoprotein-1 and its receptor CD44, which correlated positively with fibrotic markers. Additionally, both alternative and classic complement pathways were activated in ANCA-GN patients, and M1 macrophages were predominantly involved in the early stage of BC rupture while M2 macrophages were involved in the late stage and may contribute to crescent fibrosis. The loss of glomerular cells in ANCA-GN was likely mediated by apoptosis.