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Kidney Week

Abstract: FR-PO740

Identification of Cystogenic Signaling Pathways in a Newly Developed, Inducible-Kidney Epithelial Cell Model of Pkd2-Mediated Polycystic Kidney Disease

Session Information

Category: Genetic Diseases of the Kidneys

  • 1001 Genetic Diseases of the Kidneys: Cystic

Authors

  • Jung, Hyun Jun, Physiology Dept, U. of MD School of Medicine, Baltimore, Maryland, United States
  • Coleman, Richard A., Physiology Dept, U. of MD School of Medicine, Baltimore, Maryland, United States
  • Dixon, Eryn E., Physiology Dept, U. of MD School of Medicine, Baltimore, Maryland, United States
  • Outeda, Patricia, Physiology Dept, U. of MD School of Medicine, Baltimore, Maryland, United States
  • Woodward, Owen M., Physiology Dept, U. of MD School of Medicine, Baltimore, Maryland, United States
  • Welling, Paul A., Physiology Dept, U. of MD School of Medicine, Baltimore, Maryland, United States
Background

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disorder. Loss-of-function mutations of Pkd1 and Pkd2 genes cause the disease. However, initiating signaling events, most proximal to the polycystins, have not been precisely identified. Here, we developed in vitro inducible Pkd2 gene knockout (KO) model to explore the immediate consequences of Pkd2 KO and to identify the initiating factors that drive cystogenesis.

Methods

A doxycycline (Dox)-inducible Pkd2 KO renal medulla epithelial cell line was established from Pkd2 flox/flox/Pax8-rtTA/LC1 KO mice crossed with the SV-40 LTA “immorto mouse”. For RNA-Seq, Dox treated cells were compared to isogenetic controls. 4 replicates from two different passages were analyzed. Libraries were constructed from mRNA and sequenced (2×75 bp, paired-end). Sequence reads were aligned on the mouse genome GRCm38p6, and downstream analyses were performed with R packages and multiple bioinformatics tools.

Results

The inner medulla epithelial cell line forms a polarized, electrically-tight, monolayer on filter supports, and as assessed by Western blot analysis, allows complete PC2 KO within 4 days of Dox treatment. Hierarchical clustering analysis of RNA-Seq corroborated renal medullary origin. Differential expression (DE) analysis of RNA-seq showed revealed 3243 genes changed significantly (FDR<0.05) in response to the rapid repression of Pkd2. Chief among them were other Cystic Disease genes, Muc1, Ganab, Pkhd1, Dzip1l, and Dnajb11, but not Pkd1. The other DE genes were significantly enriched in the sonic hedgehog (SHH) signaling pathway and cilia structure components. Evc, Evc2, Gli3, and Prkacb of the SHH signaling pathway were downregulated, implicating suppression of SHH and alter cilia as cytogenetic drivers. In addition to Gli3 of SHH, 108 other transcription factor (TF) genes were significantly changed, including TF associated with cilia (Glis3 and Pax8), Wnt signaling and ER stress.

Conclusion

RNA-Seq analysis of our newly established inducible Pkd2 KO kidney epithelial cell line identified gene networks that are rapidly changed in response to PKD2 repression, providing clues about the proximal cytogenetic signaling events in PKD.

Funding

  • NIDDK Support