MOLECULAR MECHANISMS OF EPITHELIAL REGENERATION AFTER OXALATE DAMAGE: THE ROLE OF COX-2.

CASALI, CECILIA I.; ARTUCH, AYELÉN; ERJAVEC, LUCIANA C.; FERNÁNDEZ TOME, MARÍA C.

Salta

Congreso; LV Annual SAIB meeting and XIV PABMB Congress; 2019

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

The renal collecting ducts, which are involved in the urine concentration mechanism, are immersed in an extracellular matrix with the highest body osmolarity. This hyperosmolarity is a key signal for cell differentiation and maturation of the tubular structures, and for the establishment of the urine concentration mechanism. However, hyperosmolarity can induce cell death when there is a great osmolarity change. Renal cells activate adaptive and protective mechanisms to survive in the hyperosmolar environment. One important cell mechanism is the expression of osmoprotective genes such as cyclooxygenase 2 (COX-2). Moreover, renal ducts are exposed to wastes coming from blood filtration that include several nephrotoxic agents and kidney stones. Calcium oxalate stones are the most common type of kidney stone. The crystal aggregates are harmful for epithelial renal cells and tubular structures that could lead to renal kidney disease. It has been described that oxalate induces COX-2 mRNA and protein expression in renal epithelial cells, but the role of this protein is still unknown. The aim of the present work is to evaluate the role of COX-2 in the regeneration mechanism in differentiated renal epithelial cells treated with oxalate. To do that, the renal epithelial cells MDCK were grown in a hyperosmolar environment (NaCl 125 mM, 512 mOsm/Kg H2O) for 72 h in order to get a differentiated epithelium and then subjected to oxalate (Ox) 1.5 mM for 24, 48 and 72 h. After treatment, the cells were harvested, counted and the cell viability was determined by tripam blue assay and MTT assay. Cell morphology was also evaluated. The expression of COX-2 was determined by western blot and PCR. The treatment with Ox decreased the number of cells at 24, 48 and 72 h compared to controls 24, 48 and 72 h, respectively. However, the number of cells recovered after Ox 72h was higher than Ox 48 h and Ox 24 h. Cell viability determined by tripam blue did not change after Ox treatments but the MTT assay showed a decrease at 24, 48 and 72 h compared to controls 24, 48 and 72 h, respectively. The control conditions showed a typical epithelial cobblestone morphology, but the cells treated with Ox for 24 showed a spindle-shaped morphology characteristic of an epithelial mesenchymal transition. After 48 h of Ox the cells started to recover their morphology and after 72 h of Ox the epithelium is almost reestablished. Control conditions showed a high expression of COX-2 protein and the treatment with Ox decreased their expression compared to controls. However, the expression of COX-2 at Ox 72 h is higher than at Ox 48 h. The COX-2 mRNA level is low in control conditions at 24 and 48 h but there is an increase at 72 h. The treatment with Ox increased the levels of COX-2 mRNA with a maximum level at 72 h. The results showed that COX-2 could be involved in the epithelial regeneration mechanisms after the damaged of differentiated renal epithelial cells caused by oxalate.