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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 <i>COX2</i>, <i>PTGER2</i>, and <i>PTGER4</i> expression in hPC was quantified. In hPC, stimulation with PGE₂ led to an EP2- and EP4-dependent increase in cyclic adenosine monophosphate (cAMP) and <i>COX2</i>, and induced cellular PGE₂. <i>PTGER4</i> 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. <i>COX2</i> and <i>PTGER2</i> were upregulated by FFSS. Our data thus support an autocrine/paracrine COX2/PGE₂ pathway in hPC linked to concerted EP2 and EP4 signaling.