Kidney Week

Abstract: SA-PO1052

RNA-Seq Reveals the Transcriptome Changes of Mouse Collecting Duct Cells in Response to Urinary Flow and Primary Cilia Sensing

Session Information

• Na+, K+, Cl-
  November 04, 2017 | Location: Hall H, Morial Convention Center
  Abstract Time: 10:00 AM - 10:00 AM

Category: Fluid, Electrolytes, and Acid-Base

• 703 Na+, K+, Cl- Basic

Authors

• Mohammed, Sami Gemal, Radboud University Medical Center, Nijmegen, Netherlands

Sami Gemal Mohammed,

• Arjona, Francisco J., Radboud University Medical Center, Nijmegen, Netherlands

Francisco J. Arjona,

• Bakey, Zeineb Zb, Radboud University Medical Center, Nijmegen, Netherlands

Zeineb Zb Bakey,

• Alkema, Wynand, Radboud University Medical Center, Nijmegen, Netherlands

Wynand Alkema,

• Van hijum, Sacha, Radboud University Medical Center, Nijmegen, Netherlands

Sacha Van hijum,

• Schmidts, Miriam, University College London (UCL), London, United Kingdom

Miriam Schmidts,

• Bindels, René J., Radboud University Medical Center, Nijmegen, Netherlands

René J. Bindels,

• Hoenderop, Joost, Radboud University Medical Center, Nijmegen, Netherlands

Joost Hoenderop,

Background

External cues such as mechanical forces generated by fluid flow play a crucial role in renal physiology. Studies have shown that renal tubular cells respond to mechanical stimuli generated by urinary flow, to regulate the activity and abundance of electrolyte transporters including ion channels. The aim of this study is to reveal the transcriptome changes of tubular epithelia in response to fluid flow and determine the role of primary cilia in this process.

Methods

IMCD3 (inner-medullary collecting duct) cells without cilia were generated using CRISPR/Cas9 technology. Cells were seeded onto µ-slide ibidi chambers and subjected to either static or physiologically relevant fluid flow (~0.7 dyne/cm²) for 3 h at 37 ⁰C. RNA was isolated and prepared for RNA-seq by next generation sequencer. Differentially expressed genes with fluid flow between ciliated and unciliated cells were identified by applying statistical models and validated by RT-qPCR.

Results

The absence of cilia in two independent knockout IMCD3 cell lines (Ift140 KO or Dync2h1 KO) and the presence of cilia in one control cell line was confirmed by immunocytochemistry for the cilia marker ARL13b. RNA-seq analysis of ciliated cells subjected to fluid flow showed upregulation of 1379 genes and repression of 1294 genes compared to static control cells (adjusted p < 0.05). Several known flow-sensitive genes, such as Ptgs2 and Ccl2 were significantly upregulated with fluid flow. Interestingly, fluid flow sensing by primary cilia triggers a transcriptomic response of only 54 genes including genes linked to the regulation of the activity of the epithelial Na⁺ channel ENaC, the activity of aquaporins, tight junction permeability, iron transport, phosphate transport, and bicarbonate handling.

Conclusion

Fluid flow elicits a transcriptomic response in the collecting duct of the kidney. The role of primary cilia in this response is restricted to 54 genes, of which 16 are involved in the primary function of the collecting duct, namely ion (principally Na⁺) and water (re)absorption.

Funding

• Government Support - Non-U.S.