Role of CREB, CREM and ATF1 Transcription Factors in the Vasopressin-Mediated Increase in AQP2 Abundance in Collecting Duct Cells

In the collecting duct of the nephron, arginine vasopressin (AVP) promotes the upregulation of aquaporin 2 (AQP2)-mediated water reabsorption by two mechanisms: 1) a short-term effect on trafficking of AQP2-containing membrane vesicles to and from the plasma membrane and 2) long-term regulation by increasing AQP2 protein abundance. The second mechanism is largely attributed to increased Aqp2 gene transcription in response to V2R signaling. 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). However, ChIP-seq has not found CREB binding sites within 390 kb of the transcription start site of the Aqp2 gene in mpkCCD cells, suggesting that CREB’s role in regulating Aqp2, if any, might be indirect. Additionally, there are three KID-containing transcription factors, ATF1, CREB and CREM, with all of them being expressed in collecting duct principal cells, which could serve redundant or compensatory roles. In this study, we employed the CRISPR/Cas9 system to delete Atf1, Creb1, and Crem, to investigate if they play causal roles in the upregulation of AQP2 expression in response to the vasopressin analog, dDAVP. By analyzing the effects of these deletions, we aim to clarify the contribution of these transcription factors (if any) in Aqp2 gene expression and to identify Atf1/Creb1/Crem transcriptional targets in mpkCCD cells. Atf1, Creb1 and Crem genes were deleted by CRISPR/Cas9 in WT mpkCCD cells, verified by immunoblotting and Sanger sequencing. AQP2 protein levels were analyzed by immunoblotting with or without dDAVP in Atf1 -KO cells as well as Atf1/Creb1 -KO and Atf1/Creb1/Crem -KO cells. RNA-seq was also performed to measure Aqp2 mRNA abundance and discover differentially expressed genes (DEGs) In WT cells, dDAVP (0.1nM for 72 hours) produced a 15-fold increase in AQP2 band density (P < 0.005), with a similar effect on Atf1 -KO cells as well as Atf1/Creb1 double KO cells. However, in Atf1/Creb1/Crem -KO cells the increase in AQP2 abundance in response to dDAVP was blunted. RNA-seq analysis found only 11 DEGs comparing WT and Atf1 -KO cells in the presence of dDAVP (P adj < 0.05 and |log 2 KO/WT| > 1). When comparing WT and Atf1/Creb1 -KO we found 98 differentially expressed genes. Aqp2 mRNA abundance did not change in these experiments. Comparison between WT and Atf1/Creb1/Crem -KO cells reveals 299 DEGs, with Aqp2 being one of the downregulated genes. Finally, rescue experiments using lentiviral transduction shows that reintroduction of either ATF1, CREB or CREM to the triple-KO cells is sufficient to upregulate AQP2 in the presence of dDAVP. We show that KID-containing transcription factors play a role in the action of dDAVP to increase Aqp2 gene expression. This role is not specific to CREB, as any of these transcription factors is enough for the cells to upregulate AQP2 in the presence of dDAVP. Future studies will assess whether this is a direct effect on Aqp2 gene transcription, or if there is an indirect effect by regulating upstream regulatory elements, or other aspects of cell physiology, such as cell polarity or differentiation. NHLBI, ZIA HL001285 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.