66. Protective role of the phospholipase A2-cyclooxygenase 2-prostaglandin E2-EP2/4 receptor axis in the restitution of an oxalate-damaged renal epithelium

SENDYK D.; ERJAVEK L.; PARRA L.; BONINO M.; FLOR S.; LUCANGIOLI S.; FERNANDEZ M.; CASALI L.

Rosario

Congreso; 2023-LIX Reunión de la Sociedad Argentina de Investigaciones Bioquímicas y Biología Molecular- SAIB 2023; 2023

The renal inner medulla is responsible for the hydro-saline equilibrium maintenance through water andelectrolyte excretion in urine. The collecting ducts, involved in the urine concentration, are immersed inan extracellular matrix with the highest body osmolarity and are exposed to wastes coming from bloodfiltration. There are several nephrotoxic agents such as antibiotics, diuretics, antineoplastic andcytostatic agents, and renal stones. Calcium oxalate stones are the most common type of kidneystones. The crystal aggregates are harmful to epithelial renal cells and tubular structures, and thatdamage could lead to the development of chronic kidney disease. Our previous results showed thatrenal differentiated cells treated with oxalate (Ox) for 24 h acquire a spindle-shaped morphologycharacteristic of an epithelial-mesenchymal transition (EMT) and a decrease in cell number. Then, cellsstarted to recover their morphology reaching a restituted epithelium after 72 h of Ox. We also observedthat Ox treatment modulates the expression of mRNA and protein Cyclooxygenase 2 (COX2), and COX2inhibition with 10 μM NS398 prevents epithelial restitution. However, when the inhibition was bypassed byadding 10-6 M PGE2, EMT was not allowed after 24 and 48 h, and cells exhibited a morphologycharacteristic of the renal epithelium (cobblestone). The cPLA2 expression was also modulated by Ox.The aim of the present work is to evaluate the role of the cPLA2-COX2-PGE2-EP axis in the damage andrestitution of a renal differentiated epithelium during the first 24h of Ox treatment. To do that, the renalepithelial cells MDCK were grown in a hyperosmolar environment (512 mOsm/Kg H2O) for 72 h to get adifferentiated epithelium, and then subjected to 1.5 mM Ox for 0, 4, 8, 16, and 24 h. Cell number andmorphology were evaluated. Cell number started to decrease at 4 h, becoming significant at 8h afterOx addition and reaching the lowest value at 24 h. After 8 h, cells began to acquire a spindle-shapedmorphology observing a complete EMT at 24 h. cPLA2 and COX2 mRNA and protein levels wereevaluated. cPLA2 mRNA and protein levels decreased at 4 h of Ox reaching control values at 24 h.However, an increase in COX2 mRNA levels was observed after 4 h of treatment, peaking at 8 h andremaining elevated at 16 and 24 h. Also, COX2 protein level increased after 8 h of Ox reaching controlvalues at 24 h. Also, PGE2 levels were quantified by mass spectrometry, showing an increase after 4 h,reaching their highest value after 8 h of treatment and remaining elevated after 24 h. Finally, theexpression of EP2 and EP4 receptors was assessed by RT-PCR, showing a tendency to increase in thecase of EP2 from 8 h onwards and a peak at 8 h for EP4. These results reinforce the importance of thecPLA2-COX2-PGE2-EP axis in the restitution of damaged differentiated renal epithelium caused byoxalate, highlighting the significance of studying it in the early hours following Ox addition.