Data availability_GPR39_MK

G protein-coupled receptor 39 (GPR39) is an orphan receptor that is highly expressed in renal collecting duct principal cells. GPR39 activation <i>in vivo</i> leads to reduced urinary concentration capacity. In this study, we utilized mpkCCD cells, a model of principal cells in the collecting duct, to examine the cell biological effects of GPR39 activation. Pharmacological activation of GPR39 with the synthetic agonist cpd1324 impaired vasopressin-mediated aquaporin-2 (AQP2) apical trafficking and reduced total AQP2 expression following long-term treatment, consistent with its known <i>in vivo</i> role. These effects were absent in GPR39 knockout cells. In addition, GPR39 activation altered apical membrane morphology, disrupted the tight junction network, and reduced cortical F-actin expression, suggesting a shift toward a dedifferentiated phenotype. GPR39 activation also increased glycolytic ATP production while reducing mitochondrial ATP output without affecting proliferation. RNA-seq analysis of acutely treated mpkCCD cells revealed upregulation of inflammatory and dedifferentiation-associated gene programs, including cytokines. These findings indicate that the role of GPR39 in principal cells goes beyond AQP2 regulation and imply that GPR39 functions as a negative regulator of epithelial differentiation, perhaps acting to coordinate metabolic and inflammatory responses to stress.