Abstract: TH-PO0106
Single-Cell RNA Sequencing (RNA-Seq) Reveals Transcriptionally Distinct Neutrophil Populations During Experimental Pyelonephritis
Session Information
- AKI: Mechanisms - 1
November 06, 2025 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Acute Kidney Injury
- 103 AKI: Mechanisms
Authors
- Sanchez-Orellana, Gamaliel, Nationwide Children's Hospital, Columbus, Ohio, United States
- Wang, Xin, Nationwide Children's Hospital, Columbus, Ohio, United States
- Cotzomi Ortega, Israel, Nationwide Children's Hospital, Columbus, Ohio, United States
- Lopez-Torres, Jeimy M, Nationwide Children's Hospital, Columbus, Ohio, United States
- Sanchez-Zamora, Yuriko I., Nationwide Children's Hospital, Columbus, Ohio, United States
- Becknell, Brian, Nationwide Children's Hospital, Columbus, Ohio, United States
- Ruiz-Rosado, Juan de Dios, Nationwide Children's Hospital, Columbus, Ohio, United States
Group or Team Name
- Ruiz-Rosado Lab.
Background
Pyelonephritis (PN) accounts annually for 1,138,000 cases and nearly 60,000 hospitalizations in the United States. Uropathogenic E. coli (UPEC) is responsible for >80% of PN cases. Despite antibiotic therapy, individuals with PN are at risk of developing acute kidney injury and end stage renal disease. Hence, alternative treatment modalities are desperately needed to alleviate the societal burden imposed by PN. Innate immune phagocytes, particularly neutrophils, play a pivotal in eradicating UPEC; however, the effector functions of these granulocytes during PN remain poorly understood. Here, we studied neutrophil subsets in a mouse model of experimental PN using sc-RNA-seq.
Methods
Kidneys were harvested from female C3H/HeOuj mice transurethrally inoculated with UPEC (CFT073 strain, 1x108) at 0, 3, 7, 28 days post-infection (dpi). Renal CD45+ cells were purified and utilized to generate sc-RNA-seq libraries using the PIP-seq platform (Fluentbio). cDNA libraries were sequenced and FASTQ files were processes through the PIP-seeker software. Annotation and grouping of clusters were performed using the Seurat package. Gene ontology and KEGG pathways were obtained using DAVID Bioinformatic software.
Results
We profiled 59,529 cells, representing 5 major cell types, including neutrophils, macrophages, T- Cells, B-cells, and NK cells. We identified 4 transcriptionally distinct neutrophil subsets (Na, Nb, Nc, and Nd) during PN. At 3 dpi, all neutrophil clusters exhibited a strong mitochondrial response, indicating metabolic adaptations to UPEC-induced PN, characterized by respiratory chain assembly, ATP synthesis, and oxidative stress responses. By 7dpi, a widespread inflammatory response was observed across the neutrophil subsets, characterized by IL-6 and IL-1 signaling, and LPS engagement. By day 28, the shift towards inflammation resolution was evident, with clusters showing a mix of inflammatory and regulatory pathways, highlighting diverse roles in the infection continuum from infection onset to resolution.
Conclusion
Our study highlights the heterogenous response of neutrophil subsets during PN. Studying these cell subsets underscores the potential for targeted therapies to combat UPEC infection while minimizing renal disease.
Funding
- NIDDK Support