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GFP reporter system reveals cell-to-cell variability in aquaporin-2 expression
Lihe Chen1, Adrian Rafael Murillo-de-Ozores1, Euijung Park1
1Epithelial Systems Biology Laboratory, Systems Biology Center, National Heart, Lung, and Blood Institute, NIH, Bethesda, Maryland, United States.
American Journal of Physiology. Cell Physiology
|February 23, 2026
Summary
Vasopressin (AVP) regulates aquaporin-2 (Aqp2) gene expression. A new reporter cell line shows Aqp2 expression variability linked to cell cycle, aiding AVP regulation studies.
Area of Science:
- Molecular Biology
- Cell Biology
- Renal Physiology
Background:
- Vasopressin (AVP) is a key regulator of water reabsorption in the kidneys.
- AVP controls the transcription of the aquaporin-2 (Aqp2) gene in collecting duct principal cells.
- Understanding Aqp2 gene regulation is crucial for managing water balance disorders.
Purpose of the Study:
- To develop a reporter cell line for studying Aqp2 gene transcription regulation.
- To investigate the mechanisms underlying variability in Aqp2 expression.
- To identify factors influencing Aqp2 gene transcription.
Main Methods:
- Engineered an Aqp2 reporter cell line using CRISPR/Cas9 in mpkCCD cells.
- Inserted a green fluorescent protein (GFP) cassette at the endogenous Aqp2 gene locus.
- Utilized dDAVP stimulation, flow sorting, cell regrowth, and RNA-seq analysis.
Main Results:
- GFP expression faithfully tracked endogenous Aqp2 expression upon dDAVP stimulation and removal.
- Significant heterogeneity in Aqp2 expression was observed even in clonally-derived cells.
- Aqp2 expression heterogeneity correlated with cell cycle state, with Aqp2-negative cells showing increased cell-cycle transcripts.
Conclusions:
- Aqp2 expression heterogeneity is linked to the cell cycle state of collecting duct principal cells.
- The developed Aqp2 reporter cell line is a valuable tool for studying Aqp2 transcriptional regulation.
- This finding provides new insights into the complex regulation of water transport in the kidney.
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