Single-Cell Atlas of Pyelonephritis Reveals Divergent Injury and Repair Programs in Proximal Tubule Cells

Background: Pyelonephritis (PN), a bacterial infection of the kidney, affects approximately 1.14 million individuals annually in the United States, resulting in nearly 60,000 hospitalizations and incurring over $2.14 billion in societal costs. PN can lead to acute kidney injury (AKI), chronic kidney disease (CKD), and progression to end-stage renal disease (ESRD). Methods: We performed single-cell RNA sequencing (scRNA-seq, FluentBio) on kidneys harvested from C3H/HeOuJ mice infected with UPEC (CFT073 strain) at 1, 3, 7, and 28 days post-infection. Spatial transcriptomics (10x Genomics Visium) and immunofluorescence (IF) were employed for validation. Results: scRNA-seq analysis identified seven distinct proximal tubule cell (PTC) states across the time course of infection. Two Havcr1+ (Kim1+) populations predominated, expressing canonical injury markers (Havcr1, Agt, Nrg1) alongside metabolic programs for glycolysis and fatty acid oxidation. Notably, we discovered two previously unrecognized Havcr1+ injury-repair subsets: Kim-Hsp60+ PTC 1 and 2, characterized by cell-cycle re-entry, heat-shock responses, elevated reactive oxygen species (ROS), and activation of NF-κB and TNF signaling pathways. Furthermore, we identified stress-adapted transporter PTCs (SAT-PTCs), which maintain solute transport (Slc22a6/8, Slc22a12) despite ongoing inflammation. All injured populations upregulated lipid droplet-associated genes (Plin2 and Ehd1) and secreted antimicrobial peptides (Lyz2 and Ctsb) along with chemokines (Cxcl2 and Cxcl12), linking epithelial injury to immune cell recruitment. Trajectory analysis delineated three distinct fates: classical injury (Havcr1+), dedifferentiation with cycling (Havcr1-PTC-1/2), and stress-adapted solute transport (SAT-PTCs). These PTC states were validates using spatial transcriptomics and IF. Conclusion: Our single-cell atlas of PN revealed previously unrecognized epithelial programs driving antimicrobial defense, metabolic reprogramming, and divergent pathways of injury and repair during ascending UTIs. By defining key PTC subsets that mediate inflammation, transport preservation, or maladaptive fibrosis, our findings provide a mechanistic framework for understanding PN-induced kidney injury and identifying potential therapeutic targets. Funding: NIDDK Support