A major role for the EP4 receptor in regulation of renin.

Prostaglandins have been implicated as paracrine regulators of renin secretion, but the specific pathways and receptor(s) carrying out these functions have not been fully elucidated. To examine the contributions of prostanoid synthetic pathways and receptors to regulation of renin in the intact animal, we used a panel of mice with targeted disruption of several key genes: cyclooxygenase-2 (COX-2), microsomal PGE synthases 1 and 2 (mPGES1, mPGES2), EP2 and EP4 receptors for PGE(2), and the IP receptor for PGI(2). To activate the macula densa signal for renin stimulation, mice were treated with furosemide over 5 days and renin mRNA levels were determined by real-time RT-PCR. At baseline, there were no differences in renin mRNA levels between wild-type and the various strains of mutant mice. Furosemide caused marked stimulation of renin mRNA expression across all groups of wild-type control mice. This response was completely abrogated in the absence of COX-2, but was unaffected in mice lacking mPGES1 or mPGES2. The absence of G(s)/cAMP-linked EP2 receptors had no effect on stimulation of renin by furosemide and there was only a modest, insignificant reduction in renin responses in mice lacking the IP receptor. By contrast, renin stimulation in EP4(-/-) mice was significantly reduced by ∼70% compared with wild-type controls. These data suggest that stimulation of renin by the macula densa mechanism is mediated by PGE(2) through a pathway requiring COX-2 and the EP4 receptor, but not EP2 or IP receptors. Surprisingly, mPGES1 or mPGES2 are not required, suggesting other alternative mechanisms for generating PGE(2) in response to macula densa stimulation.

Hypertension and impaired glycine handling in mice lacking the orphan transporter XT2.

A family of orphan transporters has been discovered that are structurally related to the Na(+)-Cl(-)-dependent neurotransmitter transporters, including the dopamine transporter. One member of this family, the mouse XT2 gene, is predominantly expressed in the kidney and has 95% homology to rat ROSIT (renal osmotic stress-induced Na(+)-Cl(-) organic solute cotransporter). To study the physiological functions of this transporter, we generated XT2-knockout mice by gene targeting. XT2(-/-) mice develop and survive normally with no apparent abnormalities. To attempt to identify potential substrates for XT2, we screened urine from XT2-knockout mice by high-pressure liquid chromatography and mass spectrometry and found significantly elevated concentrations of glycine. To study glycine handling, XT2(+/+) and XT2(-/-) mice were injected with radiolabeled glycine, and urine samples were collected to monitor glycine excretion. After 2 h, XT2(-/-) mice were found to excrete almost twice as much glycine as the XT2(+/+) controls (P = 0.03). To determine whether the absence of the XT2 transporter affected sodium and fluid homeostasis, we measured systolic blood pressure by computerized tail-cuff manometry. Systolic blood pressure was significantly higher in XT2(-/-) mice (127 +/- 3 mmHg) than in wild-type controls (114 +/- 2 mmHg; P < 0.001). This difference in systolic blood pressure was maintained on high and low salt feeding. To examine whether the alteration in blood pressure and the defect in glycine handling were related, we measured systolic blood pressure in the XT2(-/-) mice during dietary glycine supplementation. Glycine loading caused systolic blood pressure to fall in the XT2(-/-) mice from 127 +/- 3 to 115 +/- 3 mmHg (P < 0.001), a level virtually identical to that of the wild-type controls. These data suggest that the XT2 orphan transporter is involved in glycine reabsorption and that the absence of this transporter is sufficient to cause hypertension.

Proinflammatory actions of thromboxane receptors to enhance cellular immune responses.

Metabolism of arachidonic acid by the cyclo-oxygenase (COX) pathway generates a family of prostanoid mediators. Nonsteroidal anti-inflammatory drugs (NSAIDs) act by inhibiting COX, thereby reducing prostanoid synthesis. The efficacy of these agents in reducing inflammation suggests a dominant proinflammatory role for the COX pathway. However, the actions of COX metabolites are complex, and certain prostanoids, such as PGE(2), in some circumstances actually inhibit immune and inflammatory responses. In these studies, we examine the hypothesis that anti-inflammatory actions of NSAIDs may be due, in part, to inhibition of thromboxane A(2) synthesis. To study the immunoregulatory actions of thromboxane A(2), we used mice with a targeted disruption of the gene encoding the thromboxane-prostanoid (TP) receptor. Both mitogen-induced responses and cellular responses to alloantigen were substantially reduced in TP(-/-) spleen cells. Similar attenuation was observed with pharmacological inhibition of TP signaling in wild-type splenocytes, suggesting that reduced responsiveness was not due to subtle developmental abnormalities in the TP-deficient mice. The absence of TP receptors reduced immune-mediated tissue injury following cardiac transplant rejection, an in vivo model of intense inflammation. Taken together, these findings show that thromboxane augments cellular immune responses and inflammatory tissue injury. Specific inhibition of the TP receptor may provide a more precise approach to limit inflammation without some of the untoward effects associated with NSAIDs.