Vasopressin-Induced Cell-to-Cell Variability in Aqp2 Expression

Despite identical genomic DNA sequences, cell populations often display variability in gene expression and responses to stimuli. This phenomenon is also observed in mouse cortical collecting duct cells (mpkCCD), where aquaporin-2 (Aqp2) expression varies in response to arginine vasopressin (AVP). To explore the mechanisms underlying vasopressin-mediated cell-to-cell heterogeneity, we engineered an Aqp2 reporter cell line using CRISPR/Cas9 to insert a green fluorescent protein (GFP) cassette at the Aqp2 gene locus in mpkCCD cells. In the absence of dDAVP, an AVP analog, these cells exhibit low or undetectable GFP and Aqp2 expression in all cells. However, dDAVP stimulation (0.1 nM dDAVP for 24hrs or 1nM dDAVP for 48hrs) markedly increased both GFP and Aqp2 expression together in a subset of cells, with expression of both diminishing upon dDAVP removal. This demonstrates that GFP faithfully tracks Aqp2 expression. Interestingly, fewer than 50% of cells express GFP and Aqp2 after stimulation, indicating significant variability. A similar pattern was observed with forskolin, which directly activates adenylyl cyclase to increase cellular cAMP. We then sorted the GFP - cells (Aqp2 - ) and GFP + cells (Aqp2 + ), regrew them, and restimulated them separately with dDAVP. Interestingly, cells originating from GFP - cells give rise to both GFP - cells and GFP + cells, and GFP + cells similarly regenerate both GFP - and GFP + populations in the same proportion. RNA-seq analysis identified several transcription factors linked to GFP (Aqp2) variability, including the pioneer transcription factor Gata3. Immunostaining showed uniform nuclear localization of Gata3 in unstimulated cells. Upon dDAVP stimulation, Aqp2 expression increased in a subpopulation of cells, accompanied by a marked reduction in Gata3 nuclear signal in the same cells, whereas non-expressing cells retained nuclear Gata3 expression. Sequence analysis of Gata3 reveals a highly conserved PKA consensus phosphorylation site (RRXS) at Ser308, located between two zinc fingers, where PKA serves as the primary kinase to induce Aqp2 expression. To test if Gata3 acts as a downstream transcription target of PKA to regulate Aqp2 expression, we analyzed Gata3 distribution in WT and PKA knockout cells with or without dDAVP. In contrast to reduced nuclear Gata3 expression in Aqp2 + upon dDAVP stimulation, PKA knockout cells showed no Aqp2 expression and displayed uniform nuclear Gata3 distribution, regardless of dDAVP treatment. In addition, RNA-seq showed increased expression of cell-cycle related transcripts in the GFP - cells. Flow cytometry analysis of the DNA content confirmed the variability in cell cycle phases, with most GFP + cells in G0/G1, and more GFP - cells in G1/S. Indeed, heterogeneous expression of mki67, a marker for active cell cycle phases, was observed in the unstimulated cells. This heterogeneity was decreased following dDAVP addition. These results suggest that heterogeneity in Aqp2 expression arise partly from variations in Gata3 expression and/or cell cycle-associated genes. This work was funded by NHLBI Division of Intramural Research projects ZIA-HL001285 and ZIA-HL006129 (to M. Knepper) and NIH Clinical Center 2020 Bench-to-Bedside Award. 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.