Kidney Week

Abstract: FR-PO083

The mRNA Interactome of Ciliated Renal Cells and Its Modulation by Hypoxia

Session Information

• AKI: Tubules, Metabolism, New Models
  October 26, 2018 | Location: Exhibit Hall, San Diego Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Acute Kidney Injury

• 103 AKI: Mechanisms

Authors

• Kaiser, Rainer, Dept. 2 of Internal Medicine, University of Cologne, Koeln, Germany

Rainer Kaiser,

• Rill, Constantin, Dept. 2 of Internal Medicine, University of Cologne, Koeln, Germany

Constantin Rill,

• Ignarski, Michael, Dept. 2 of Internal Medicine, University of Cologne, Koeln, Germany

Michael Ignarski,

• Atanassov, Ilian, Max Planck Institute for Biology of Aging, Cologne, Germany

Ilian Atanassov,

• Schermer, Bernhard, Dept. 2 of Internal Medicine, University of Cologne, Koeln, Germany

Bernhard Schermer,

• Benzing, Thomas, Dept. 2 of Internal Medicine, University of Cologne, Koeln, Germany

Thomas Benzing,

• Fabretti, Francesca, Dept. 2 of Internal Medicine, University of Cologne, Koeln, Germany

Francesca Fabretti,

• Mueller, Roman-Ulrich, Dept. 2 of Internal Medicine, University of Cologne, Koeln, Germany

Roman-Ulrich Mueller,

Background

RNA-binding proteins (RBPs) are fundamental regulators of cellular biology and affect every step in processing RNA-biology. The scale of their impact has been shown by recent studies that have linked RBPs to a number of human pathologies, ranging from neurological disorders and ischemia reperfusion injury to tumor growth. Despite this central role in cell biology, the global effect of RBPs in the kidney has not been assessed until now. Here, we identify the first set of RBPs in ciliated renal epithelial cells under both hypoxic and normoxic growth conditions.

Methods

Using Oligo(dT) beads to precipitate mRNA-protein complexes on ciliated mIMCD-3 cells, we aimed to identify the kidney-specific mRNA interactome using mass spectrometry. Cells were either exposed to hypoxia or grown under normoxic conditions. In parallel, the proteome of whole cell lysates was identified to assess the abundance of total protein in comparison to RBPs detected using the same conditions. Using TALEN-generated transgenic human cell lines, specific candidates were validated as RNA binding protein with the Polynukleotide (PNK) assay, showing RNA bound directly to the proteins of interest. Localization of the RBPs was analyzed using immunofluorescence.

Results

Our data revealed over 350 significant mRNA interactors and more than 300 candidate RBPs, 84 of which have not been described as RBPs in common data bases. We define these proteins as the renal epithelial cell mRNA interactome. The PNK assay validated all chosen candidates as RBPs and indeed showed direct binding to RNA. Since the whole cell proteome did not show these RBPs to be differentially expressed upon exposure to hypoxia, we hypothesize that the increased detection of RBP candidates in the hypoxic samples is indeed due to differential binding to their target transcripts.

Conclusion

Our data identify the first set of RBPs specific to ciliated renal epithelial cells and show that hypoxia can modulate RNA-binding, adding another regulatory layer to the diverse biology of the kidney. Using established human transgenic cell lines, we are now aiming for functional studies of individual candidates in order to address their impact on hypoxia signaling and their impact of preconditioning-mediated increase of viability both in vitro and in vivo.

Funding

• Private Foundation Support