Abstract: TH-PO0033
Kinase Inhibitor Screening Reveals Novel Regulators of Aqp2 Transcription
Session Information
- Fluid, Electrolyte, and Acid-Base Disorders: Basic Research
October 22, 2026 | Location: Exhibit Hall A, 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 Division of Intramural Research, Bethesda, Maryland, United States
- Chen, Lihe, National Heart Lung and Blood Institute Division of Intramural Research, Bethesda, Maryland, United States
- Knepper, Mark A., National Heart Lung and Blood Institute Division of Intramural Research, Bethesda, Maryland, United States
Group or Team Name
- Epithelial Systems Biology Laboratory
Background
The abundance of the water channel aquaporin-2 (AQP2) is regulated by vasopressin, mainly by increasing Aqp2 gene transcription. Although it is known that protein kinase A (PKA) plays a key role in vasopressin signaling, phosphoproteomic studies have shown that many phosphorylation sites altered in response to vasopressin do not conform to the PKA consensus target sequence. Here, we used a small-molecule screening approach to identify additional protein kinases involved in Aqp2 transcription.
Methods
Aqp2-transcription GFP reporter mpkCCD cells were seeded on 96-well Transwell plates and incubated with desmopressin, a vasopressin analog. Each well was incubated with a different kinase inhibitor (10µM, kinase screening library, Cayman #10505). Confocal imaging and fluorescence quantification were used to identify “hits” that altered GFP abundance. Hits were then tested in mpkCCD cells to assess their effect on AQP2 protein abundance. RNA-seq was performed to evaluate global transcriptomic effects.
Results
After initial screening identified candidate hits, a secondary validation screen was performed to confirm molecules with a reproducible effect. Selected hits were further tested in mpkCCD cells over a range of concentrations by immunoblotting. One of the identified hits is the JNK inhibitor, JNK-IN-8, which increased AQP2 abundance even in the absence of desmopressin. A similar effect was observed by a different JNK inhibitor, CC-930. RNA-seq analysis revealed that treatment with 10µM JNK-IN-8 led to broad transcriptional changes (963 genes with |log2ratio| > 0.58 and adjusted p < 0.05), including downregulation of known JNK-regulated genes, such as Jun and Atf3, as well as upregulation of several vasopressin-responsive genes, such as Aqp2, Adh1 and Arg2. Gene Ontology analysis revealed enrichment of pathways related to steroid biosynthesis, regulation of cell proliferation, apoptosis, TGF-β response and cell junction assembly.
Conclusion
Kinase screening can provide useful new information about cell signaling and at the same time identify new therapeutic strategies. Acquired nephrogenic diabetes insipidus (“vasopressin resistance”) is generally associated with a failure of Aqp2 gene transcription. The finding that JNK inhibition increases Aqp2 gene transcription in the absence of vasopressin points out a possible novel therapeutic direction and a starting point for drug development to optimize therapeutic ratio.
Funding
- Other NIH Support