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Abstract
Background The molecular and cellular heterogeneity of human Lupus Nephritis (LN) at the kidney tissue level makes it challenging to identify key disease drivers and therapeutic targets. Single-cell transcriptomics (scRNA-Seq) has advanced our understanding of LN pathogenesis, but tissue dissociation eliminates all spatial information and several rare cell types (such as podocytes) are under-represented using droplet-based scRNA-Seq protocols.
Methods Using the CosMx Spatial Molecular Imager (Nanostring), we performed spatial transcriptomics on 8 pediatric Class III/IV LN patients and 2 health controls. This platform generated 1000-plex gene expression data at single cell resolution using archived clinical biopsy tissue.
Results After data QC and cell segmentation, we identified a total of 447,892 cells, which were assigned to 33 reference cell types (figure 1A, B). Visualizing spatial relationships provided robust evidence of the accuracy of cell annotation, such as the colocalization of fenestrated glomerular endothelial cells, mesangial cells, and podocytes within glomeruli (figure 1C). Analysis of differential gene expression demonstrated that SLE induced broad transcriptional changes in resident glomerular cells, including markers of both injury response and initiation of tissue repair mechanisms. For example, glomerular endothelial cells downregulate expression of TEK (angiopoietin-1-binding TEK receptor tyrosine kinase), and the angiopoietin-1/TEK signal regulator DUSP1 (Dual Specificity Phosphatase 1), suggesting altered endothelial function and cross-talk with surrounding mesangial matrix. Upregulated genes in LN mesangial cells include: TAGLN, encoding transgrelin, a marker for proliferating mesangial cells; collagen molecules (COL3A1, COL1A2, COL4A1, COL6A); matrix metalloproteinases and inhibitors (MMP14, MMP19, TIMP1); and chemokines (CSF1, CXCL9). In parallel, we demonstrate that individual immune lineages traffic to specific regions in LN kidneys and that transcriptional signatures vary as a function of tissue location.
Conclusions Spatial transcriptomics is a powerful tool to uncover the heterogeneity of LN. The identification of new pathogenic mechanisms may inform the development of new targeted therapies.
(A) UMAP projection of immune and renal cell subsets in LN. (B) Spatial relationships with each dot indicating a single cell. Arrows denote glomeruli and infiltrating tubulointerstitial immune cells. (C) Left: Illustration showing major glomerular cell types. Right: Glomerular cell types are spatially confined to glomerular structures (dashed circle), with podocytes (red) predominantly located peripherally outside mesangial and endothelial cells. Grey dots: unlabeled cells
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