Abstract: FR-PO341

Ablation of Transcription Factor HNF-1β Induces Epithelial-Mesenchymal Transition through Twist2 Derepression

Session Information

Category: Chronic Kidney Disease (Non-Dialysis)

  • 308 CKD: Mechanisms of Tubulointerstitial Fibrosis

Authors

  • Chan, Siu Chiu, University of Minnesota, Minneapolis, Minnesota, United States
  • Zhang, Ying, University of Minnesota, Minneapolis, Minnesota, United States
  • Shao, Annie, University of Minnesota, Minneapolis, Minnesota, United States
  • Vrba, Sophia M., University of Minnesota, Minneapolis, Minnesota, United States
  • Avdulov, Svetlana, University of Minnesota, Minneapolis, Minnesota, United States
  • Herrera, Jeremy, University of Minnesota, Minneapolis, Minnesota, United States
  • Farahani, Shayan A., University of Minnesota, Minneapolis, Minnesota, United States
  • Aboudehen, Karam S., University of Minnesota, Minneapolis, Minnesota, United States
  • Igarashi, Peter, University of Minnesota, Minneapolis, Minnesota, United States
Background

Hepatocyte nuclear factor-1β (HNF-1β) is a transcription factor that is essential for normal kidney development and function. Mutations of HNF-1β produce autosomal dominant tubulointerstitial kidney disease (ADTKD) characterized by tubular cysts, renal fibrosis, and progressive decline in kidney function. We have previously shown that HNF-1β regulates a network of cystic disease genes that are down-regulated in HNF-1β mutant kidneys.

Methods

To understand the mechanism whereby mutation of the epithelial-specific transcription factor HNF-1β leads to interstitial fibrosis, we used gene editing with CRISPR/Cas9 to ablate HNF-1β in mIMCD3 renal epithelial cells.

Results

HNF-1β mutant mIMCD3 cells exhibited loss of contact inhibition and adopted a spindle-shaped morphology. Compared with control cells, HNF-1β-deficient cells exhibited EMT features with increased cell migration and higher motility and produced a multilayered epithelium. RNA-seq analysis of HNF-1β-deficient cells and Ingenuity Pathway Analysis (IPA) revealed that fibrosis and epithelial-mesenchymal transition (EMT) pathways were highly activated in HNF-1β-deficient cells. Transcription factors involved in EMT, including TWIST2, SNAIL1, SNAIL2, and ZEB2, were upregulated in HNF-1β mutant cells. Mechanistically, we found that expression of Twist2 was directly repressed by HNF-1β. Concomitant ablation of Twist2 partially rescued the fibroblastic phenotype of HNF-1β mutant cells. Chromatin immunoprecipitation and qRT-PCR analysis of Twist2 mutant cells showed that TWIST2 is an upstream transcriptional activator of Snail2. Immunohistochemistry and RNA in situ hybridization showed that the expression of TWIST and SNAIL, as well as downstream targets TGFβ2 and TGFβ3, was increased in the cyst epithelium of HNF-1β mutant kidneys.

Conclusion

We conclude that ablation of HNF-1β in renal epithelial cells leads to the activation of a transcriptional network that induces EMT and aberrant TGFβ signaling. Targeting this network may inhibit fibrosis in ADTKD and other chronic kidney diseases.

Funding

  • NIDDK Support