Epidermal Growth Factor Reduces Collecting Duct Water Permeability by Inhibiting Aquaporin-2 Phosphorylation at Ser269

Prior studies showed that epidermal growth factor (EGF) inhibits vasopressin-stimulated osmotic water permeability in the renal collecting duct. Here, we investigated the underlying mechanism of this response. Using the Burg isolated perfused tubule technique in microdissected rat inner medullary collecting ducts (IMCDs), we found that addition of 0.1 μM EGF to the peritubular bath significantly decreased 0.1 nM 1‐deamino‐8‐D‐arginine vasopressin (dDAVP)-stimulated water permeability, confirming prior observations. The inhibitory effect of EGF on dDAVP-stimulated water permeability was associated with a reduction in intracellular cyclic AMP levels and protein kinase A (PKA) activity. Using phospho-specific antibodies and immunoblotting in IMCD suspensions, we showed that EGF significantly reduces dDAVP-stimulated aquaporin-2 (AQP2) phosphorylation at S264 and S269. This effect was absent when membrane-permeable 8-cpt-cAMP (0.1 mM) was used to stimulate AQP2 phosphorylation, suggesting that EGF’s effect was on a pre-cAMP step. Immunofluorescence labeling of microdissected IMCDs showed that EGF significantly reduced apical AQP2 abundance in the presence of dDAVP. To address what protein kinase might be responsible for S269 phosphorylation, we used Bayesian analysis to integrate multiple -omic datasets. Thirteen top ranked protein kinases were subsequently tested by in vitro phosphorylation experiments for their ability to phosphorylate AQP2 peptides using a mass spectrometry readout. The results show that the PKA catalytic-α subunit increased phosphorylation at S256, S264, and S269. None of the other kinases phosphorylated S269. Additionally, PKA inhibitors, H-89 and PKI, strongly inhibited dDAVP-stimulated AQP2 phosphorylation at S269. These results indicate that EGF decreases the water permeability of the collecting duct by inhibiting cAMP production, thereby inhibiting PKA and decreasing AQP2 phosphorylation at S269, a site previously shown to regulate AQP2 endocytosis. This work was funded by NHLBI project ZIA-HL001285 and ZIA-HL006129, M.A.K. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.