The Contribution Of Monocytes And Glomerular Endothelial Cells To The Formation Of Endocapillary Hypercellularity In Iga Nephropathy

IgA nephropathy (IgAN) is the most commonly reported primary glomerular disease worldwide. It mainly affects young adults, with many progressing to kidney failure over their lifetime. Five histopathological kidney lesions independently predict a poor prognosis in IgAN (MEST-C score). Published case series highlight the ‘endocapillary hypercellularity’ (E1) lesion as potentially reversible with systemic immunosuppression, improving clinical outcomes. E1 is defined by the obliteration of glomerular capillary lumens by cells that appear to be mostly macrophages, likely to be derived from circulating monocytes. Delineating differences in the transcriptomes of glomerular endothelial cells (GEnCs) and monocyte subsets from patients with and without E1 (E0) to model how they interact may allow identification of safer therapeutic targets. Laser capture microdissection (LCMD) and digital spatial profiling (DSP) using a Nanostring GeoMx were evaluated as tools for enriching GEnC transcripts from formalin fixed paraffin embedded sections of tissue. A JavaScript based tool was written to facilitate DSP on GEnCs. Two pilot experiments (n = 2 and n = 9) were conducted to evaluate the efficacy of DSP against LCMD at enriching for GEnC signals, using single cell deconvolution (SCD) against two different kidney cell atlases. Subsequently a larger DSP experiment (n = 38) was conducted to identify differences between E1 and E0 GEnCs, and the transcriptomes of intra-glomerular CD68 positive macrophages were also profiled. Circulating monocyte subsets were then profiled in E1 and E0 using flow cytometry, cell sorting, and next generation sequencing (NGS) (n=11), and interactions between monocytes and GEnCs were modelled using STRING. The JavaScript function allowed good segmentation on GEnCs and intra-glomerular macrophages for DSP, and SCD showed significant enrichment for both cells relative to neighbouring cell types over LCMD. A cluster of biologically relevant genes were found to be up-regulated in E1 GEnCs including CCL3, CX3CR1, CD44, TNFRSF12A and IL-7, compared to E0. Pathway analysis highlighted pathways related to cell adhesion, apoptosis, cell death and inflammation mediated by IL-27 to be up-regulated among E1 GEnCs (p<0.05, FDR<0.05). Lineage analysis performed on intra-glomerular macrophages in E1 indicated derivation from circulating non-classical monocytes, and flow cytometry found this subset to be contracted in the circulation in E1, suggesting they were being sequestered within glomeruli. NGS of nonclassical monocytes found up-regulated pathways and transcripts related to inflammation and chemokine signalling (including CCR7, CXCR1, CXCR4, CXCL8, CCL3, CCL4, CCL20, IL6 and TNF). Non-classical monocyte-GEnC interaction modelling using STRING identified a cluster of 89 interacting transcripts with an enrichment p value of <1.0e-16. Central to this cluster were inflammatory cytokines, chemokine-chemokine receptors, and cell adhesion transcripts, including LIGHT, BAFF-R, IL-6, CD44, TNF, CCL3, CCL4 and CCR7. Pathways most enriched for within the cluster were related to inflammation (NF-κB, TNF and IL-18 signalling) and immune mediated signalling (toll like receptor signalling). 27/89 transcripts in the cluster already have therapies developed to inhibit them, with TNF, IL-6, BAFFR and LIGHT likely representing high yield therapeutic targets to treat those with IgAN who have E1.