Concerted EP2 and EP4 Receptor Signaling Stimulates Autocrine Prostaglandin E ₂ Activation in Human Podocytes

Abstract

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 (PGE ₂) synthesis and upregulated cyclooxygenase 2 (Cox2) are associated with podocyte injury by FFSS. We aimed to elucidate a PGE ₂ autocrine/paracrine pathway in human podocytes (hPC). We developed a modified liquid chromatography tandem mass spectrometry (LC/ESI-MS/MS) protocol to quantify cellular PGE ₂, 15-keto-PGE ₂, and 13,14-dihydro-15-keto-PGE ₂ levels. hPC were treated with PGE ₂ 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 PGE ₂ led to an EP2- and EP4-dependent increase in cyclic adenosine monophosphate (cAMP) and COX2, and induced cellular PGE ₂. PTGER4 was downregulated after PGE ₂ stimulation in hPC. In the corresponding LC/ESI-MS/MS in vivo analysis at the tissue level, increased PGE ₂ and 15-keto-PGE ₂ 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/PGE ₂ pathway in hPC linked to concerted EP2 and EP4 signaling.