Dartmouth Hitchcock Medical Center Lebanon, NH, United States
Angelique Cortez1, Lindsay Mendyka1, Fred Kolling2 and Sladjana Skopelja-Gardner1, 1Dartmouth Hitchcock Medical Center, Lebanon, NH, 2Geisel School of Medicine at Dartmouth, Lebanon, NH
Background/Purpose: Sensitivity to ultraviolet (UV) light affects ~ 80% of systemic lupus erythematosus (SLE) patients. UV light exposure of lupus skin leads to local and systemic inflammation and can trigger lupus nephritis flares. Cellular and molecular mechanisms of UV light-triggered kidney injury are unknown. We use single-cell RNA-sequencing analysis to identify renal cell subsets damaged by skin exposure to UV light and how neutrophils mediate the injury.
Methods: B6 female mice (3-mo) were exposed to UVB light (1x500mJ/cm2). Cardiac perfusion with Dynabeads for glomerular isolation was performed on day 2 after UV (peak inflammation). Glomeruli were digested (Collagenase IV, Pronase I, DNase I) following initial kidney processing in Collagenase I and scRNA-seq performed by 10X Genomics (Illumina NextSeq2000). Kidney neutrophils were enriched by Ly6G magnetic beads and sorted for >90% purity. Single Cell clustering and differential gene expression (DGE) analyses were performed using Seurat 4.1.1 R package. Gene and phenotype ontology analyses on kidney neutrophils were performed.
Results: Through scRNA-seq analysis, we examined the cellular composition of UV and non-UV exposed kidney cells to define cellular subsets sensitive to UV. Visualization using uniform manifold approximation and projection (UMAP) revealed 14 distinct kidney cell types. Skin exposure to UV light led to the reduction in podocytes and distal tubular cells (DTCs), while an expansion in endothelial and stromal cells was observed. DGE analysis showed that podocytes in UV-exposed mice upregulated injury markers podoplanin (Pdpm), Vimentin (Vim) and Stat3, and downregulated the genes important for podocyte integrity and function (Magi2, Datch1, Prkg1). DTCs upregulated injury markers Umod and Osteopontin (Spp1). UV light triggered type I interferon response genes were upregulated in podocytes, DTCs, and stromal cells. Podocyte and DTC injury was accompanied by an increase in kidney neutrophils, cells we previously showed mediate renal injury after skin exposure to UV. RNA-seq analyses revealed an activated (CD11bhighIfitm6high) CD177high neutrophil cluster present only in the kidneys of UV exposed mice. Concurrent upregulation of CD177 ligand Pecam-1 in renal endothelial cells provides a candidate mechanism for neutrophil recruitment from UV exposed skin. Of relevance to lupus, ontology analysis showed the neutrophils recruited to the kidney are transcriptionally similar to the neutrophils in SLE and patients with glomerulonephritis. Neutrophil degranulation was the most highly upregulated pathway in the CD177high neutrophil cluster.
Conclusion: Using single cell analysis, we captured renal cellular heterogeneity across UV and non-UV conditions and identified podocytes and DTCs as primary targets of UV light-triggered injury in the kidney. Concurrent transcriptional analysis of renal neutrophils identified Pecam-1 – CD177 as the likely adhesion mechanism involving Pecam-1 on renal endothelial cells and Cd177 on kidney recruited neutrophils. Transcriptional similarity of kidney-recruited neutrophils and those in SLE and LN patients supports the neutrophil-mediated skin-kidney pathogenic axis in lupus.
Disclosures: A. Cortez, None; L. Mendyka, None; F. Kolling, None; S. Skopelja-Gardner, None.