Disruption of prostaglandin E2 receptor EP4 impairs urinary concentration via decreasing aquaporin 2 in renal collecting ducts

The antidiuretic hormone arginine vasopressin is a systemic effector in urinary concentration. However, increasing evidence suggests that other locally produced factors may also play an important role in the regulation of water reabsorption in renal collecting ducts. Recently, prostaglandin E2 (PGE2) receptor EP4 has emerged as a potential therapeutic target for the treatment of nephrogenic diabetes insipidus, but the underlying mechanism is unknown. To evaluate the role of EP4 in regulating water homeostasis, mice with renal tubule-specific knockout of EP4 (Ksp-EP4^−/−) and collecting duct-specific knockout of EP4 (AQP2-EP4^−/−) were generated using the Cre-loxP recombination system. Urine concentrating defect was observed in both Ksp-EP4^−/− and AQP2-EP4^−/− mice. Decreased aquaporin 2 (AQP2) abundance and apical membrane targeting in renal collecting ducts were evident in Ksp-EP4^−/− mice. In vitro studies demonstrated that AQP2 mRNA and protein levels were significantly up-regulated in mouse primary inner medullary collecting duct (IMCD) cells after pharmacological activation or adenovirus-mediated overexpression of EP4 in a cAMP/cAMP-response element binding protein-dependent manner. In addition, EP4 activation or overexpression also increased AQP2 membrane accumulation in a mouse IMCD cell line (IMCD3) stably transfected with the AQP2 gene, mainly through the cAMP/protein kinase A and extracellular signal-regulated kinase pathways. In summary, the EP4 receptor in renal collecting ducts plays an important role in regulating urinary concentration under physiological conditions. The ability of EP4 to promote AQP2 membrane targeting and increase AQP2 abundance makes it a potential therapeutic target for the treatment of clinical disorders including acquired and congenital diabetes insipidus.Urinary concentration is a key process for maintaining body water homeostasis, which is primarily regulated by the antidiuretic hormone arginine vasopressin (AVP). AVP is produced in the hypothalamus and stored in and released from the posterior pituitary, either in response to increased plasma osmolality or decreased blood volume. It binds to its type 2 receptor (V2R) on the basolateral membrane of the principal cells of renal collecting ducts (CDs), triggering the redistribution of aquaporin 2 (AQP2) from intracellular vesicles into the apical membrane. The prolonged activation of V2R can also increase AQP2 expression in CDs, which is essential for urinary concentration (1). AVP thus increases water permeability of the CDs, resulting in enhanced water reabsorption from the tubule lumens and concentrated urine output (1, 2). In some cases, however, urinary concentration is altered independent of AVP, a phenomenon called vasopressin escape, suggesting additional mechanisms may participate in the process of water reabsorption in renal collecting ducts (3–5).Among many identified factors affecting urine output, prostaglandin E2 (PGE2) has been shown to play an important role in urinary concentration. PGE2 is one of the major cyclooxygenated metabolites of arachidonic acid produced in the kidney (6). It exerts various biological functions via its four distinct G protein-coupled receptors, designated EP1–4 (7). Substantial evidence suggests a prominent, yet complex, role for PGE2 in the regulation of water homeostasis. For instance, PGE2 and sulprostone, an EP1/3 agonist, have been found to blunt AVP-induced AQP2 trafficking and urinary concentration (8–11). Results from the EP3 gene knockout mice also suggested a diuretic effect of PGE2, likely via the EP3 receptor (12). Olesen et al. have shown that both G-coupled PGE2 receptor EP2 and EP4 agonists increased AQP2 membrane accumulation and that the EP2 agonist butaprost relieved nephrogenic diabetes insipidus (NDI) symptoms in a rat model of NDI (13). Moreover, a selective EP4 agonist has recently been reported to be able to alleviate symptoms in a mouse model of NDI (14). These results indicate a role for EP2 and EP4 receptors in promoting urinary concentration and suggest the potential use of EP2 and EP4 agonists in the treatment of NDI. However, to date, the underlying mechanism is poorly defined and the precise role of each EP receptor in the regulation of urinary concentration under physiological conditions remains unclear.In the present study, we found a significant increase in renal medullary EP4, but not EP1, EP2, or EP3, expression in wild-type (WT) mice after water deprivation. EP4 renal tubule-specific knockout (Ksp-EP4^−/−) mice and collecting duct-specific knockout (AQP2-EP4^−/−) mice exhibited an impaired urinary concentrating ability, along with decreased AQP2 expression and membrane targeting. The possible mechanisms by which EP4 regulates AQP2 membrane translocation involve both cAMP/protein kinase A (PKA) and extracellular signal-regulated kinase (ERK) pathways, whereas EP4-elicited AQP2 abundance is dependent on the cAMP/cAMP-response element binding protein (CREB) pathway. Our findings highlight an important role for the EP4 receptor in regulating water reabsorption under physiological conditions and support the use of EP4 agonist as a potential therapeutic treatment for NDI.     

Molecular cloning and characterization of chicken prostaglandin E receptor subtypes 2 and 4 (EP2 and EP4)

Prostaglandin E₂ (PGE₂) is an important chemical mediator responsible for regulation of many vital physiological processes. Four receptor subtypes have been identified to mediate its biological actions. Among these subtypes, prostaglandin E receptor subtypes 2 and 4 (EP₂ and EP₄), both coupled to cAMP-protein kinase A (cAMP-PKA) signaling pathway, are proposed to play crucial roles under both physiological and pathological conditions. Though both receptors were extensively studied in mammals, little is known about their functionality and expression in non-mammalian species including chicken. In present study, the full-length cDNAs for chicken EP₂ and EP₄ receptors were first cloned from adult chicken ovary and testis, respectively. Chicken EP₂ is 356 amino acids in length and shows high amino acid identity to that of human (61%), mouse (63%), and rat (61%). On the other hand, the full-length cDNA of EP₄ gene encodes a precursor of 475 amino acids with a high degree of amino acid identity to that of mammals, including human (87%), mouse (86%), rat (84%), dog (85%), and cattle (83%), and a comparatively lower sequence identity to zebrafish (52%). RT-PCR assays revealed that EP₂ mRNA was expressed in all tissues examined including the oviduct, while EP₄ expression was detected only in a few tissues. Using the pGL3-CRE-luciferase reporter system, we also demonstrated that PGE₂ could induce luciferase activity in DF-1 cells expressing EP₂ and EP₄ in dose-dependent manners (EC₅₀: <1 nM), confirming that both receptors could be activated by PGE₂ and functionally coupled to the cAMP-PKA signaling pathway. Together, our study establishes a molecular basis to understand the physiological roles of PGE₂ in target tissues of chicken.     

A role for extracellular copper in modulating prostaglandin E2 (PGE2) action: facilitation of PGE2 stimulation of the release of gonadotropin-releasing hormone (LHRH) from median eminence explants

Copper is present in high concentrations in axonal terminals of hypothalamic neurons (1). We have previously postulated that copper is released from axonal terminals of hypothalamic neurons (2). In this study, we addressed the question: Does extracellular copper facilitate PGE2 action, specifically, the stimulation of LHRH release? The median eminence area (MEA) of male rats was incubated under in vitro conditions, and LHRH release into the medium was quantified by RIA. When MEA were incubated for 5 min with 100 microM copper and then for 15 min with 50 microM PGE2 (Cu/PGE2), Cu/PGE2-stimulated release of LHRH was significantly (P less than 0.001) greater (2 times) than the sum of copper- and PGE2-stimulated release. When MEA were first incubated for 15 min with PGE2 and then for 5 min with copper (PGE2/Cu), PGE2/Cu-stimulated release equalled the sum of PGE2- and copper-stimulated release of LHRH. Thus, copper facilitates PGE2 stimulation of LHRH release from the MEA, and the characteristics of this facilitation are consistent with an enhancement of PGE2 binding to its receptor. These results support the proposition that extracellular copper can serve as a modulator of PGE2 action.