Studies in Zebrafish and Rat Models Support Dual Blockade of EP2 and EP4 (Prostaglandin E2 Receptors Type 2 and 4) for Renoprotection in Glomerular Hyperfiltration and Albuminuria.

BACKGROUND: Glomerular hyperfiltration (GH) is an important mechanism in the development of albuminuria in hypertension. Upregulation of COX2 (cyclooxygenase 2) and prostaglandin E2 (PGE2) was linked to podocyte damage in GH. We explored the potential renoprotective effects of either separate or combined pharmacological blockade of EP2 (PGE2 receptor type 2) and EP4 (PGE2 receptor type 4) in GH. METHODS: We conducted in vivo studies in a transgenic zebrafish model (Tg[fabp10a:gc-EGFP]) suitable for analysis of glomerular filtration barrier function and a genetic rat model with GH, albuminuria, and upregulation of PGE2. Similar pharmacological interventions and primary outcome analysis on albuminuria phenotype development were conducted in both model systems. RESULTS: Stimulation of zebrafish embryos with PGE2 induced an albuminuria-like phenotype, thus mimicking the suggested PGE2 effects on glomerular filtration barrier dysfunction. Both separate and combined blockade of EP2 and EP4 reduced albuminuria phenotypes in zebrafish and rat models. A significant correlation between albuminuria and podocyte damage in electron microscopy imaging was identified in the rat model. Dual blockade of both receptors showed a pronounced synergistic suppression of albuminuria. Importantly, this occurred without changes in arterial blood pressure, glomerular filtration rate, or tissue oxygenation in magnetic resonance imaging, while RNA sequencing analysis implicated a potential role of circadian clock genes. CONCLUSIONS: Our findings confirm a role of PGE2 in the development of albuminuria in GH and support the renoprotective potential of combined pharmacological blockade of EP2 and EP4 receptors. These data support further translational research to explore this therapeutic option and a possible role of circadian clock genes.

Tissue lipidomic profiling supports a mechanistic role of the prostaglandin E2 pathway for albuminuria development in glomerular hyperfiltration.

Background: Glomerular hyperfiltration (GH) is an important mechanism in the development of albuminuria in hypertension. The Munich Wistar Frömter (MWF) rat is a non-diabetic model of chronic kidney disease (CKD) with GH due to inherited low nephron number resulting in spontaneous albuminuria and podocyte injury. In MWF rats, we identified prostaglandin (PG) E2 (PGE2) signaling as a potential causative mechanism of albuminuria in GH. Method: For evaluation of the renal PGE2 metabolic pathway, time-course lipidomic analysis of PGE2 and its downstream metabolites 15-keto-PGE2 and 13-14-dihydro-15-keto-PGE2 was conducted in urine, plasma and kidney tissues of MWF rats and albuminuria-resistant spontaneously hypertensive rats (SHR) by liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). Results: Lipidomic analysis revealed no dysregulation of plasma PGs over the time course of albuminuria development, while glomerular levels of PGE2 and 15-keto-PGE2 were significantly elevated in MWF compared to albuminuria-resistant SHR. Overall, averaged PGE2 levels in glomeruli were up to ×150 higher than the corresponding 15-keto-PGE2 levels. Glomerular metabolic ratios of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) were significantly lower, while metabolic ratios of prostaglandin reductases (PTGRs) were significantly higher in MWF rats with manifested albuminuria compared to SHR, respectively. Conclusion: Our data reveal glomerular dysregulation of the PGE2 metabolism in the development of albuminuria in GH, resulting at least partly from reduced PGE2 degradation. This study provides first insights into dynamic changes of the PGE2 pathway that support a role of glomerular PGE2 metabolism and signaling for early albuminuria manifestation in GH.

15-keto-Prostaglandin E2 exhibits bioactive role by modulating glomerular cytoarchitecture through EP2/EP4 receptors.

AIMS: Prostaglandins are important signaling lipids with prostaglandin E2 (PGE2) known to be the most abundant prostaglandin across tissues. In kidney, PGE2 plays an important role in the regulation of kidney homeostasis through its EP receptor signaling. Catabolism of PGE2 yields the metabolic products that are widely considered biologically inactive. Although recent in vitro evidence suggested the ability of 15-keto-PGE2 (a downstream metabolite of PGE2) to activate EP receptors, the question whether 15-keto-PGE2 exhibits physiological roles remains unresolved. MATERIALS AND METHODS: Pharmacological treatment was performed in transgenic zebrafish embryos using 500 µM 15-keto-PGE2 and 20 µM EP receptors antagonists' solutions during zebrafish embryonic development. After the exposure period, the embryos were fixed for confocal microscopy imaging and glomerular morphology analysis. KEY FINDINGS: Here, we show that 15-keto-PGE2 can bind and stabilize EP2 and EP4 receptors on the plasma membrane in the yeast model. Using lipidomic analysis, we demonstrate both PGE2 and 15-keto-PGE2 are present at considerable levels in zebrafish embryos. Our high-resolution image analysis reveals the exogenous treatment with 15-keto-PGE2 perturbs glomerular vascularization during zebrafish development. Specifically, we show that the increased levels of 15-keto-PGE2 cause intercalation defects between podocytes and endothelial cells of glomerular capillaries effectively reducing the surface area of glomerular filtration barrier. Importantly, 15-keto-PGE2-dependent defects can be fully reversed by combined blockade of the EP2 and EP4 receptors. SIGNIFICANCE: Altogether, our results reveal 15-keto-PGE2 to be a biologically active metabolite that modulates the EP receptor signaling in vivo, thus playing a potential role in kidney biology.

Do ß-Blockers Cause Depression?: Systematic Review and Meta-Analysis of Psychiatric Adverse Events During ß-Blocker Therapy.

[Figure: see text].

Concerted EP2 and EP4 Receptor Signaling Stimulates Autocrine Prostaglandin E2 Activation in Human Podocytes.

Glomerular hyperfiltration is an important mechanism in the development of albuminuria. During hyperfiltration, podocytes are exposed to increased fluid flow shear stress (FFSS) in Bowman's space. Elevated Prostaglandin E2 (PGE2) synthesis and upregulated cyclooxygenase 2 (Cox2) are associated with podocyte injury by FFSS. We aimed to elucidate a PGE2 autocrine/paracrine pathway in human podocytes (hPC). We developed a modified liquid chromatography tandem mass spectrometry (LC/ESI-MS/MS) protocol to quantify cellular PGE2, 15-keto-PGE2, and 13,14-dihydro-15-keto-PGE2 levels. hPC were treated with PGE2 with or without separate or combined blockade of prostaglandin E receptors (EP), EP2, and EP4. Furthermore, the effect of FFSS on COX2, PTGER2, and PTGER4 expression in hPC was quantified. In hPC, stimulation with PGE2 led to an EP2- and EP4-dependent increase in cyclic adenosine monophosphate (cAMP) and COX2, and induced cellular PGE2. PTGER4 was downregulated after PGE2 stimulation in hPC. In the corresponding LC/ESI-MS/MS in vivo analysis at the tissue level, increased PGE2 and 15-keto-PGE2 levels were observed in isolated glomeruli obtained from a well-established rat model with glomerular hyperfiltration, the Munich Wistar Frömter rat. COX2 and PTGER2 were upregulated by FFSS. Our data thus support an autocrine/paracrine COX2/PGE2 pathway in hPC linked to concerted EP2 and EP4 signaling.

The Ussing Chamber Assay to Study Drug Metabolism and Transport in the Human Intestine.

The Ussing chamber is an old but still powerful technique originally designed to study the vectorial transport of ions through frog skin. This technique is also used to investigate the transport of chemical agents through the intestinal barrier as well as drug metabolism in enterocytes, both of which are key determinants for the bioavailability of orally administered drugs. More contemporary model systems, such as Caco-2 cell monolayers or stably transfected cells, are more limited in their use compared to the Ussing chamber because of differences in expression rates of transporter proteins and/or metabolizing enzymes. While there are limitations to the Ussing chamber assay, the use of human intestinal tissue remains the best laboratory test for characterizing the transport and metabolism of compounds following oral administration. Detailed in this unit is a step-by-step protocol for preparing human intestinal tissue, for designing Ussing chamber experiments, and for analyzing and interpreting the findings. © 2017 by John Wiley & Sons, Inc.