Abstract: SA-PO292
Novel Transcriptional Regulators of Tight Junction Biogenesis in Renal Collecting Ducts
Session Information
- Fluid and Electrolytes: Basic - II
November 09, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Fluid and Electrolytes
- 901 Fluid and Electrolytes: Basic
Authors
- Leiz, Janna, Max Delbrueck Center for Molecular Medicine, Berlin, Germany
- Hinze, Christian, Max Delbrueck Center for Molecular Medicine, Berlin, Germany
- Schmidt-Ott, Kai M., Max Delbrueck Center for Molecular Medicine, Berlin, Germany
Background
The renal collecting duct is comprised of a tight epithelial barrier resulting in a strict separation of intraluminal urine and the interstitium. Disruption of this barrier leads to impaired water reabsorption and loss of adequate urine concentration ability. However, the overall transcriptional network controlling this process is only incompletely characterized.
Using an integrated bioinformatics approach, we identified two transcriptional regulators (TRs), Tfap2a and Ncor1, potentially involved in this network. The transcription factor Tfap2a is a trans-regulatory factor implicated in epithelial differentiation and Tfap2a mutations have been associated with renal malformations. Ncor1 is a nuclear corepressor that assists nuclear receptors (such as thyroid hormone receptor) in the downregulation of gene expression. We hypothesize that Tfap2a and Ncor1 play a role in tight junction biogenesis and epithelial barrier formation in the CD.
Methods
Inner medullary collecting duct (IMCD3) cells were engineered to harbour CRISPR/Cas9-induced knock outs (KO) of either Tfap2a or Ncor1. Deregulated genes were identified by mRNA sequencing and confirmed with qPCR.
Results
Tfap2a and Ncor1 show predicted binding to promotors of several critical tight junction components and are highly expressed in IMCD3 cells and CD in mice.
Crispr/Cas9-induced KO of Tfap2a and Ncor1 were confirmed with allele-specific sequencing validating frameshift mutations in the targeted areas. mRNA sequencing revealed a strong impact of Tfap2a and Ncor1 KO on the expression of important tight junction components. For example, two claudins, Cldn4 and Cldn8, showed massive downregulation in comparison to WT clones. Interestingly, the TR grainyhead-like 2 (Grhl2), which we previously identified as a critical regulator of tight junction biogenesis, was also highly downregulated in Tfap2a and Ncor1 KO clones. These findings were confirmed by qPCR. A detailed characterization of the transcriptional network is still ongoing.
Conclusion
Our data support our hypothesis that the candidates Tfap2a and Ncor1 are involved in the transcriptional network regulating tight junction biogenesis and barrier formation in the renal CD. A detailed understanding of the underlying network mechanisms controlling tight junction biogenesis might provide important insights into their potential involvement in kidney disease.