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 (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.
Highlights
Podocytes are terminally differentiated epithelial cells that form the third layer of the glomerular filter with their interdigitating foot processes [1]
In differentiated human podocytes (hPC) (Figure S3), which encode for EP1, EP2, and EP4, respectively
As only EP2 and EP4 are reported to mediate an increase in intracellular cyclic adenosine monophosphate (cAMP), we investigated the effect of pharmacological inhibition of EP2 and EP4 signaling on Prostaglandin E2 (PGE2) -stimulated intracellular cAMP levels in hPC
Summary
Podocytes are terminally differentiated epithelial cells that form the third layer of the glomerular filter with their interdigitating foot processes [1] Their high degree of differentiation permits podocytes to accomplish their highly specialized functions. Cells 2020, 9, 1256 the single nephron and concomitantly in higher ultrafiltrate flow in Bowman’s space [2,3,4] (reviewed in [5]). This causes increased fluid flow shear stress (FFSS) and contributes to podocyte damage [3, 6]. Perturbation of the glomerular filtration barrier contributes to proteinuria, glomerulosclerosis, and alteration in GFR, and promotes the gradual decline in renal function as observed in chronic kidney disease (CKD) (reviewed in [7,8,9])
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