Collecting Duct Water Permeability Inhibition by EGF Is Associated with Decreased cAMP, PKA activity and AQP2 Phosphorylation at Ser269

Am J Physiol Renal Physiol. 2024 Jan 11. doi: 10.1152/ajprenal.00197.2023. Online ahead of print.ABSTRACTPrior studies showed that epidermal growth factor (EGF) inhibits vasopressin-stimulated osmotic water permeability in the renal collecting duct. Here, we investigated the underlying mechanism. Using isolated perfused rat inner medullary collecting ducts (IMCDs), we found that addition of EGF to the peritubular bath significantly decreased 1-deamino-8-D-arginine vasopressin (dDAVP)-stimulated water permeability, confirming prior observations. The inhibitory effect of EGF on 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 phosphorylation of AQP2 at S264 and S269. This effect was absent when 8-cpt-cAMP was used to induce AQP2 phosphorylation, suggesting that EGF's inhibitory effect was at 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 PK...

Source: American Journal of Physiology. Renal Physiology - January 11, 2024 Category: Physiology Authors: Chung-Lin Chou Kavee Limbutara Anika R Kao Jevin Z Clark Ellen H Nein Viswanathan Raghuram Mark A Knepper Source Type: research