Kidney Week

Abstract: SA-PO632

Identification of Cell-Specific Transcriptomic Changes in Complement Activation and Regulation in Glomerular Microangiopathy Using Single Nuclei RNA Sequencing

Session Information

• Glomerular Diseases: IgA and Complement-Mediated GN
  November 05, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
  Abstract Time: 10:00 AM - 12:00 PM

Category: Glomerular Diseases

• 1302 Glomerular Diseases: Immunology and Inflammation

Authors

• Estrada, Chelsea C., Northport VA Medical Center, Northport, New York, United States

Chelsea C. Estrada,

• Gujarati, Nehaben A., Stony Brook University Renaissance School of Medicine, Stony Brook, New York, United States

Nehaben A. Gujarati,

• Mallipattu, Sandeep K., Stony Brook University Renaissance School of Medicine, Stony Brook, New York, United States

Sandeep K. Mallipattu,

Background

Krüppel-Like Factor 4 (KLF4), a zinc finger transcription factor, is a key regulator of an anti-inflammatory and anti-thrombotic endothelial cell (EC) phenotype. EC-specific loss of Klf4 exacerbates glomerular microangiopathy and complement activation in three models of endothelial injury (mice with Nos3 deficiency, aging and Shiga-Toxin 2). Glomerular expression of the complement regulatory gene Cd55 was reduced in each of these models. However, the mechanism and cell-specific regulation of complement in thrombotic microangiopathy remains unexplored.

Methods

EC-specific Klf4 knockout mice (Klf4^ΔEC) were generated by crossing Klf4^fl/fl mice with Cdh5-Cre mice. Klf4^ΔEC and Nos3^-/- mice were crossed to generate double knockout mice (DKO). Single nucleus (sn)RNA-seq libraries were prepared from kidney cortex using the 10X Chromium System and raw data was processed with Cell Ranger. Quality control and clustering were performed using the R-package, Seurat. Comparisons were made across four groups and between Klf4^fl/fl and Klf4^ΔEC, and Nos3^-/- and DKO to account for the role of EC-Klf4.

Results

SnRNA-seq generated 26,456 nuclei transcriptomics across four groups of mice (Klf4^fl/fl, Klf4^ΔEC, Nos3^-/- and DKO) and 21 clusters using unsupervised clustering analysis. Enrichment and pathway analyses showed a downregulation of angiogenic pathways across EC clusters in Klf4^fl/fl mice as compared with Klf4^ΔEC as well as in Nos3^-/- mice as compared with DKO. Conversely inflammatory pathways such as STAT3 and NFκB were upregulated in DKO mice compared to Nos3^-/-. A cross-reference of differentially expressed genes (DEGs) in each glomerular cluster with known complement activation genes revealed upregulation of C8a, and pro-thrombotic von Willebrand factor in the EC cluster in the DKO group, with no significant DEGs in the podocyte or mesangial cluster. Across all groups, differential expression of complement regulatory genes in the glomerulus showed upregulation of Cd55 and Cd59 in the podocyte cluster and Cd46 and Cfh in the mesangial cluster without significant basal expression in the EC cluster.

Conclusion

SnRNA sequencing identified other potential mechanisms, such as decreased angiogenic signaling, behing the exacerbated microangiopathy and complement activation upon loss of EC-Klf4.

Funding

• NIDDK Support