Abstract: PO0702
An Interplay of Glucose, IL-1β, and PDGF-B Trigger cPLA2 Activation, Prostaglandin Secretion, and Proliferation in Human Mesangial Cells
Session Information
- Diabetic Kidney Disease: Basic - II
November 04, 2021 | Location: On-Demand, Virtual Only
Abstract Time: 10:00 AM - 12:00 PM
Category: Diabetic Kidney Disease
- 601 Diabetic Kidney Disease: Basic
Authors
- Boi, Roberto, Sahlgrenska universitetssjukhuset, Goteborg, Sweden
- Ebefors, Kerstin, Goteborgs universitet Sahlgrenska Akademin, Goteborg, Sweden
- Nystrom, Jenny C., Goteborgs universitet Sahlgrenska Akademin, Goteborg, Sweden
Background
Diabetic kidney disease (DKD) is commonly thought to be originated from diabetic hyperglycemia. DKD development is driven by early glomerular hemodynamic changes and characterized by progressive expansion of the mesangium. A connection between these findings is yet to be elucidated. We speculate that, after hyperglycemia priming, autocrine inflammatory and proliferative stimuli alter mesangial lipid metabolism activating the secretion of vasodilator hormones. Subsequently, this affects glomerular functions. Phospholipase cPLA2 was identified as the central enzyme of the metabolic cascade.
Methods
Human mesangial cells were stimulated with Glucose (30 mM), IL-1β (1 nM), PDGF-B (25 ng/ml). Their synergic counter activation was investigated by western blots and qPCR. Lipidomics was used to analyze lipid variations. Cox-2 induction and prostaglandin secretion were measured via western blot and ELISA. Activation of cPLA2, upstream of Cox-2, was studied using western blot, qPCR, activity assays. ELISA, migration, and proliferation assays were used to evaluate cPLA2 inhibition. Data were validated using the Nephroseq database.
Results
After stimulation with Glucose, NLRP3 and pro-IL-1β were upregulated. IL-1β stimulation increased PDGF-B mRNA levels. In turn, PDGF-B stimulation increased NLRP3 and pro-IL-1β protein levels. Lipidomics analysis after IL-1β and PDGF-B stimulations showed an increase of sphingosine 1 phosphate, a known activator of Cox-2. Cox-2 was induced and prostaglandins secreted accordingly. cPLA2 releases arachidonic acid, the substrate of Cox-2. cPLA2 was upregulated at gene and protein level and activated by phosphorylation. Upregulation of the pathway was confirmed in silico in DKD patients. Since cPLA2 reaction is the rate-limiting step in prostaglandin synthesis, its inhibition with AACOCF3 was studied. Inhibition of cPLA2 reduced migration, proliferation, secretion of prostaglandins in cells treated with IL-1β and PDGF-B.
Conclusion
External stimuli (hyperglycemia from the diabetic environment) and glomerular inflammatory and proliferative stimuli prime DKD early events. The upregulation of cPLA2 was found to be critical in these events. cPLA2 inhibition reduced mesangial secretion of prostaglandins, proliferation, and migration, making it a potential target for therapy.
Funding
- Private Foundation Support