Kidney Week

Abstract: FR-PO282

Mice With a Genomic Deletion of Pkhd1 Exons 3-67 Have Minimal Renal Manifestations but Evidence of Transcriptional Network Changes by snRNA Sequencing

Session Information

• Genetic Diseases of the Kidneys: Cystic - II
  November 04, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
  Abstract Time: 10:00 AM - 12:00 PM

Category: Genetic Diseases of the Kidneys

• 1101 Genetic Diseases of the Kidneys: Cystic

Authors

• Ishimoto, Yu, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland, United States

Yu Ishimoto,

• Menezes, Luis F., National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland, United States

Luis F. Menezes,

• Zhou, Fang, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland, United States

Fang Zhou,

• Yoshida, Teruhiko, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland, United States

Teruhiko Yoshida,

• Outeda, Patricia, University of Maryland School of Medicine, Baltimore, Maryland, United States

Patricia Outeda,

• Watnick, Terry J., University of Maryland School of Medicine, Baltimore, Maryland, United States

Terry J. Watnick,

• Germino, Gregory G., National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland, United States

Gregory G. Germino,

Background

Autosomal recessive polycystic kidney disease (ARPKD) is caused mostly by mutations in PKHD1. We previously generated a mouse with a genomic deletion of exons 3-67 of murine Pkhd1. While Pkhd1^{del3-67/del3-67} kidneys are somewhat enlarged and show no obvious morphological or histological changes. We and others have previously reported kidney cysts in ARPKD models, suggesting that compensatory pathways might prevent cystogenesis. We therefore investigated transcriptomic changes in Pkhd1^{del3-67/del3-67} kidneys and controls, using single-nuclei RNA-sequencing (snRNAseq).

Methods

We sampled kidneys from three male 4-weeks old Pkhd1^{del3-67/del3-67} and three littermate controls. After quality control filtering, we analyzed a total of 83,322 cells, with a median number of ~ 4000 transcripts/cell.

Results

Clusters of cells representing most nephron epithelial cell types had Pkhd1 expression. Grouping similar cell types and re-clustering, we observed no differences between mutant and control samples. Within each cell type, multiple genes distributed across the genome had significant p-value changes but most had minimal fold-change differences, and we found no evidence of significantly dysregulated pathways by gene-set enrichment analysis. Using network analysis, we inferred transcriptional networks and scored genes based on their connectivity. We found evidence that a few pathways were significantly changed in different nephron segments, including some with transcriptional regulator activity. To investigate consistent changes in all nephron segments, we derived a consensus network including only gene-gene interactions present in all segments. Within this network, we identified genes with multiple connections (hubs) that were differentially expressed and showed changed in connectivity between mutants and controls, suggestive of re-wiring. Pathway enrichment analysis of likely re-wired differentially expressed hubs identified ubiquitin-conjugation as a major change in Pkhd1 mutants, consistent with previous results.

Conclusion

Pkhd1^{del3-67/del3-67} kidneys showed no obvious morphological changes or compensatory transcriptional profiles. Network analyses, however, identified possible re-wiring of ubiquitin-conjugation pathway.

Funding

• NIDDK Support