Abstract: FR-PO246

Quantitative Proteomic Analysis of Induced Renal Tubular Epithelial Cells (iRECs)

Session Information

Category: Cell Biology

  • 202 Apoptosis, Proliferation, Autophagy, Cell Senescence, Cell Transformation

Authors

  • Dittrich, Sebastian, University Hospital Cologne, Cologne, Germany
  • Benzing, Thomas, University Hospital Cologne, Cologne, Germany
  • Lienkamp, Soeren S., University Hospital Freiburg, Freiburg, BW, Germany
  • Kaminski, Michael, University Hospital Freiburg, Freiburg, BW, Germany
  • Rinschen, Markus M., University Hospital Cologne, Cologne, Germany
Background

Reprogramming of differentiated cells into other cell types by forced expression of tissue specific transcription factors has been shown to be an effective tool to generate new cell models. iRECs are a renal epithelial cell line that has been reprogrammed from mouse embryonic fibroblasts (MEFs). While the cell line has been characterized at a genomic and transcriptomic level, proteomic studies still need to be performed to evaluate possible usage as an in vitro model.

Methods

We performed a proteomic mapping of proteins generated from iRECs and original MEFs. The method consists of protein solubilization, tryptic digestion and analysis of peptides by nLC-MS/MS on a quadrupole-orbitrap mass spectrometer.

Results

We mapped the iREC proteome and MEFs and quantified 5315 proteins using label-free quantification. The generated dataset was correlated with mRNA expression (“RNA-seq”) data of microdissected rat kidney tubule segments. Consistent with previous transcriptome analysis, we found that iRECs expressed proteins from different segments of the tubule system, Henle loop and collecting ducts rather than resembling the expression profile of one specific segment. The iRECs had high abundance of mitochondrial proteins. In addition, the most strongly iREC-enriched proteins were the ligand- binding receptors Cubilin (Cubn) and Megalin (Lrp2) and its associated adaptor protein Disabled homolog 2 (Dab2), which mediate reuptake of albumin and other ligands. We furthermore found that iRECs expressed several soluble channels such as sodium bicarbonate transporter-like protein 11 (Slc4a11) which is highly expressed in the thin descending limb of Henle loop. Several subunits of V-type proton ATPase were highly expressed in iRECs as well as the transcription factors JunB and JunD.

Conclusion

Consistent with transcriptomic data, the iREC proteome does not resemble the protein expression profile of one single nephron segment. However, iRECs express several specialized segment specific proteins with high importance in physiological processes as well as for disease models. Especially the surprisingly high protein abundance of ligand-binding receptors Cubilin and Megalin could make this cell type a valuable new in vitro model for elucidating the yet unknown molecular signaling mechanisms of albumin reuptake in the proximal tubule of the nephron.