Kidney Week

Abstract: FR-PO532

CREB and ATF1 Transcription Factors Are Not Required for the Vasopressin-Mediated Increase in AQP2 Abundance in Collecting Duct Cells

Session Information

• Fluid, Electrolyte, Acid-Base Disorders: Basic
  November 03, 2023 | Location: Exhibit Hall, Pennsylvania Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Fluid, Electrolytes, and Acid-Base Disorders

• 1101 Fluid, Electrolyte, and Acid-Base Disorders: Basic

Authors

• Murillo-de-Ozores, Adrian Rafael, National Heart, Lung and Blood Institute, Bethesda, Maryland, United States

Adrian Rafael Murillo-de-Ozores,

• Chen, Lihe, National Heart, Lung and Blood Institute, Bethesda, Maryland, United States

Lihe Chen,

• Knepper, Mark A., National Heart, Lung and Blood Institute, Bethesda, Maryland, United States

Mark A. Knepper,

Background

Aquaporin-2 (AQP2) abundance is regulated by arginine vasopressin (AVP), largely by increasing Aqp2 gene transcription. In both native collecting duct and cultured mpkCCD cells, long-term exposure to AVP increases AQP2 abundance by 10-fold or more. Most review articles and textbook chapters on AQP2 regulation attribute the increase to PKA-mediated activation of the transcription factor CREB, by phosphorylation of S133 at the Kinase Inducible Domain (KID). Two KID-containing transcription factors are strongly expressed in collecting duct principal cells, namely ATF1 and CREB. Here, we investigated their potential roles in the AVP-mediated increase in AQP2 abundance.

Methods

PKA knockout (PKA-KO) and WT mpkCCD cells were utilized. CREB and ATF1 phosphorylation was analyzed by immunoblotting. Atf1 and Creb1 genes were deleted by CRISPR/Cas9 in WT mpkCCD cells and verified by immunoblotting (using a custom mouse ATF1 specific antibody and a commercial CREB antibody) and Sanger sequencing. RNA-seq was performed in Atf1-KO cells and AQP2 protein levels were analyzed by immunoblotting in Atf1-KO cells as well as Atf1/Creb1-KO cells, with or without desmopressin (dDAVP), an AVP analogue.

Results

Basal phosphorylation levels of CREB-S133 and ATF1-S63 were similar between WT and PKA-KO cells, however the dDAVP-mediated increase was blunted in PKA-KO cells. In WT cells, dDAVP (0.1nM for 72 hours) produced a 15-fold increase in AQP2 band density (P < 0.005), whereas it increased AQP2 17-fold in Atf1-KO cells (P < 0.0005) and 14-fold in Atf1/Creb1 double KO cells (P < 0.01). RNA-seq analysis found only 11 differentially expressed genes comparing WT and Atf1-KO cells in the presence of dDAVP (P_adj < 0.05 and |log₂KO/WT| > 1), and Aqp2 mRNA was not affected.

Conclusion

We conclude that CREB and ATF1 are not necessary for the action of dDAVP to increase Aqp2 gene expression, contrary to the conventional belief in the literature. Future studies will assess whether other dDAVP-responsive genes are regulated by KID transcription factors, and what other transcription factors might be modulated by vasopressin-mediated signaling to explain the upregulation of AQP2.

Funding

• Other NIH Support