PGE₂ EP₃ receptor downregulates COX-2 expression in the medullary thick ascending limb induced by hypertonic NACl

We tested the hypothesis that inhibition of EP₃ receptors enhances cyclooxygenase (COX)-2 expression in the thick ascending limb (TAL) induced by hypertonic stimuli. COX-2 protein expression in the outer medulla increased approximately twofold in mice given free access to 1% NaCl in the drinking water for 3 days. The increase was associated with an approximate threefold elevation in COX-2 mRNA accumulation and an increase in PGE₂ production by isolated medullary (m)TAL tubules from 77.3 ± 8.4 to 165.7 [1] 10.8 pg/mg protein. Moreover, administration of NS-398 abolished the increase in PGE₂ production induced by 1% NaCl. EP₃ receptor mRNA levels also increased approximately twofold in the outer medulla of mice that ingested 1% NaCl. The selective EP₃ receptor antagonist L-798106 increased COX-2 mRNA by twofold in mTAL tubules, and the elevation in COX-2 protein induced by 1% NaCl increased an additional 50% in mice given L-798106. COX-2 mRNA in primary mTAL cells increased twofold in response to media made hypertonic by the addition of NaCl (400 mosmol/kg H2O). L-798106 increased COX-2 mRNA twofold in isotonic media and fourfold in cells exposed to 400 mosmol/kg H2O. PGE₂ production by mTAL cells increased from 79.3 ± 4.6 to 286.7 ± 6.3 pg/mg protein after challenge with 400 mosmol/kg H2O and was inhibited in cells transiently transfected with a lentivirus short hairpin RNA construct targeting exon 5 of COX-2 to silence COX-2. Collectively, the data suggest that local hypertonicity in the mTAL is associated with an increase in COX-2 expression concomitant with elevated EP₃ receptor expression, which limits COX-2 activity in this segment of the nephron.