Prostaglandin E 2 increases proximal tubule fluid reabsorption, and modulates cultured proximal tubule cell responses via EP 1 and EP 4 receptors

Abstract

Renal prostaglandin (PG) E 2 regulates salt and water transport, and affects disease processes via EP 1-4 receptors, but its role in the proximal tubule (PT) is unknown. Our study investigates the effects of PGE 2 on mouse PT fluid reabsorption, and its role in growth, sodium transporter expression, fibrosis, and oxidative stress in a mouse PT cell line (MCT). To determine which PGE 2 EP receptors are expressed in MCT, qPCR for EP 1-4 was performed on cells stimulated for 24 h with PGE 2 or transforming growth factor beta (TGFβ), a known mediator of PT injury in kidney disease. EP 1 and EP 4 were detected in MCT, but EP 2 and EP 3 are not expressed. EP 1 was increased by PGE 2 and TGFβ, but EP 4 was unchanged. To confirm the involvement of EP 1 and EP 4, sulprostone (SLP, EP 1/3 agonist), ONO8711 (EP 1 antagonist), and EP 1 and EP 4 siRNA were used. We first show that PGE 2, SLP, and TGFβ reduced H 3 -thymidine and H 3 -leucine incorporation. The effects on cell-cycle regulators were examined by western blot. PGE 2 increased p27 via EP 1 and EP 4, but TGFβ increased p21; PGE 2 -induced p27 was attenuated by TGFβ. PGE 2 and SLP reduced cyclinE, while TGFβ increased cyclinD1, an effect attenuated by PGE 2 administration. Na-K-ATPase α1 (NaK) was increased by PGE 2 via EP 1 and EP 4. TGFβ had no effect on NaK. Additionally, PGE 2 and TGFβ increased fibronectin levels, reaching 12-fold upon co-stimulation. EP 1 siRNA abrogated PGE 2 -fibronectin. PGE 2 also increased ROS generation, and ONO-8711 blocked PGE 2 -ROS. Finally, PGE 2 significantly increased fluid reabsorption by 31 and 46% in isolated perfused mouse PT from C57BL/6 and FVB mice, respectively, and this was attenuated in FVB-EP 1 null mice. Altogether PGE 2 acting on EP 1 and EP 4 receptors may prove to be important mediators of PT injury, and salt and water transport.