Abstract: SA-PO1032

Gene Expression Changes in Vasopressin-Sensitive mpkCCD Cells after CRISPR/Cas9-Deletion of cAMP-Dependent Protein Kinase (PKA)

Session Information

Category: Fluid, Electrolytes, and Acid-Base

  • 702 Water/Urea/Vasopressin, Organic Solutes

Authors

  • Jung, Hyun Jun, NHLBI/NIH, Bethesda, Maryland, United States
  • Isobe, Kiyoshi, NHLBI/NIH, Bethesda, Maryland, United States
  • Yang, Chin-Rang, NHLBI/NIH, Bethesda, Maryland, United States
  • Burg, Maurice B., NHLBI/NIH, Bethesda, Maryland, United States
  • Raghuram, Viswanathan, NHLBI/NIH, Bethesda, Maryland, United States
  • Knepper, Mark A., NHLBI/NIH, Bethesda, Maryland, United States
Background

Vasopressin regulates collecting duct water permeability in part through stimulation of transcription of the aquaporin-2 gene, Aqp2. Vasopressin signaling occurs via several mediators including β-arrestin, Epac (RapGEF3/4), and cAMP-dependent protein kinase (PKA).

Methods

To address the role of PKA in gene expression in the renal collecting duct, we used genome editing (CRISPR-Cas9) to ablate expression of both PKA catalytic subunits in mouse mpkCCD cells. We carried out transcriptomics (RNA-Seq) and quantitative proteomics (SILAC-based quantitative LC-MS/MS) of in three pairs of PKA-KO vs. control clones. Deletion was confirmed using newly developed isoform-specific PKA antibodies. Cells were grown on permeable supports in the presence of 0.1 nM dDAVP.

Results

The PKA-KO cells were viable and maintained polarity. In RNA-Seq, only 68 out of 10,190 transcripts showed significant abundance decreases based on dual criteria (FDR-corrected P < 0.05 and log2(PKA-KO/Control) < -1). These included several transcripts previously found to be upregulated by vasopressin (Aqp2, Adh1, Akr1b3, Bcat1, Cyfip2, Gsdmc2, Gsdmc4, Gsta4, Pde4b, Tmem45b and Tmprss4). Quantitative proteomics confirmed the deletion of both PKA isoforms, showed that AQP2 protein was undetectable in the PKA-KO cells (confirmed by immunoblotting), and showed decreases in protein abundances of Tmprss4, Baiap2l2, Prom1, C3, Upk1a and Muc4, in parallel with mRNA changes. When either PKA catalytic subunit was transfected into the PKA-KO cells, AQP2 protein expression was rescued (immunofluorescence microscopy and immunoblotting).

Conclusion

We conclude that transcription of the Aqp2 gene requires PKA. PKA-dependent gene transcription is highly selective, involving less than 1% of expressed genes in collecting duct cells.

Funding

  • Other NIH Support