Spatial Transcriptomic Profiling Reveals Tubular Injury States and Immune-Epithelial Cross-Talk in Murine Obstructive Nephropathy

Background: Obstructive nephropathy is a major cause of renal disease in children and contributes to progressive kidney dysfunction in adults. The unilateral ureteral obstruction (UUO) model replicates hallmark features such as proximal tubular (PT) injury, macrophage infiltration, and fibrosis. However, spatial mapping of these processes remains limited. Methods: Kidneys from C57BL/6J mice subjected to UUO or sham surgery were collected at 12 hours, 2, 5, and 10 days post-obstruction. Spatial transcriptomic profiling was conducted using the 10x Genomics Visium platform. Cell composition per spot was inferred with SPOTlight and Giotto. PT- and macrophage-enriched spots were analyzed for differential gene expression (Wilcoxon rank-sum, adj. P < 0.005) and pathway enrichment (GSEA). Ligand-receptor interactions were inferred using CytoSignal, which integrates spatial proximity and Gaussian ligand diffusion. Results: UUO kidneys exhibited a trend toward injury-associated PT states (New_PT1/PT2) compared to sham (mean 0.14 vs. 0.08; P = 0.07) (Figure 1). These states showed upregulation of Spp1, Clu, Havcr1, C3, and Lcn2, and downregulation of Miox, Cyp4b1, and Inmt (adj. P < 0.005). Macrophage abundance was elevated in UUO (0.15 vs. 0.01; P = 0.07), with enrichment of Timp1, Col1a1, and Spp1. GSEA identified activation of EMT, oxidative phosphorylation, hypoxia, TNF/NF-κB signaling, and apoptosis (normalized enrichment score >2.0, FDR < 0.05). CytoSignal revealed a significant expansion of contact-mediated signaling via ICAM1 (spot count: 1035 UUO vs. 6 sham; P < 0.01), DLL1/4–NOTCH signaling, and increased ligand diffusion for Il1ra (P = 0.04), Bmp6 (P = 0.065), and Inhbb (P = 0.08) (Figure 2). Conclusion: Spatial transcriptomics identifies injury-associated PT states and localizes immune-epithelial interactions in UUO. We observe PT niches enriched for Spp1 and macrophage-derived profibrotic signals. Expanded ligand-receptor networks in UUO suggest paracrine and juxtacrine mechanisms driving fibrosis and offer potential therapeutic targets.