0944: L-sepiapterin treatment reduces renal cortical gene expression associated with oxidative stress and fibrosis in NZM2410 murine lupus nephritis

Background/Purpose: Systemic lupus erythematosus (SLE) is a disease of endothelial (EC) dysfunction. We hypothesize that much of this dysfunction stems from uncoupling of endothelial nitric oxide synthase, which leads to inflammatory and fibrotic states in endothelial cells. In the uncoupled state, eNOS produces superoxide rather than nitric oxide. Sepiapterin (PTC923) increases functional coupling of eNOS homodimers through its in vivo product, BH₄. We have reported that in a murine model of lupus nephritis (NZM2410), PTC923 improves survival and renal pathology. This study was designed to explore potential mechanisms through which this clinical effect is occurring, with a specific focus on pathways of inflammation, oxidative stress, and fibrosis.

Methods: Female NZM2410 mice were aged to 22 weeks, when NZM2410 mice in our colony develop proteinuria. Mice were randomized to receive either vehicle (n=5) or 20 mg/kg/day (n=5) of PTC923 (L-sepiapterin) for 6 weeks. Mice were euthanized, and renal cortex was harvested for single nuclear RNA sequencing (snRNA-seq). Cell-specific gene markers were used to cluster cells, and differential expression of genes (DEG) was determined for each cell type. DEG with p adjusted < 0.05 and log2fold changed > 1.5 were analyzed. Pathway analysis was performed, and CellChat was used to analyze DEGs and hypothesize potential ligand-receptor interactions between cells.

Results: Endothelial cells (ECs) and proximal tubule (PT) segment 1, 2, and 3 cells were differentially increased PTC923 treatment. Inversely, injured PT cells (iPT), injured ECs (iECs), and T cells were reduced in PTC923 treated mice (Figures 1 and 2). ECs from PTC923-treated mice had increases in genes associated with nitric oxide production. Among iECs, Pdgfc and Pdgfd and among ECs, Pdgfc (associated with signaling for fibrosis) were significantly reduced in PTC923-treated mice. An analysis of redox-regulated genes revealed reductions in pathways that enrich for platelet derived growth factor binding transmembrane receptor protein phosphatase activity in PTC923-treated mice. Using CellChat, ligand-receptor interactions between injured ECs and mesangial cells are hypothesized via Pdgfd-Pdgfrb and Pdgfc-Pdgfra. In parallel, Col1a1 and Col3a1 as well as other genes associated with fibrosis were reduced with PTC923 in mesangial cells (Figure 3).

Conclusion: PTC923 therapy in the NZM2410 model of lupus nephritis reduces injured cells and genes associated with inflammation and fibrosis and increases EC expression of genes in pathways associated with ion channels and nitric oxide production. These data suggest that targeting eNOS uncoupling in ECs acts via reducing redox-regulated pathways and increasing nitric oxide production.