Regulation of cyclooxygenase-2 expression in renal medulla by tonicity in vivo and in vitro

Renal medullary prostaglandins are believed to exert an important functiona l role in antagonizing vasopressin effects in dehydration. Studies were und ertaken to determine the effect of hyperosmolality on cyclooxygenase (COX) isoform expression in the renal medulla. COX-1 and COX-2 mRNA and protein l evels were determined by RT-PCR or Western blotting in Sprague-Dawley rats on varying water intakes, in Brattleboro rats and in Long-Evans controls. O ver a wide range of urinary tonicity, COX-2 expression correlated closely w ith urine osmolality levels (R = 0.872). COX-1 levels did not vary. Immunol ocalization showed that the stimulation of COX-2 expression by dehydration occurred predominantly in the collecting duct. Hypertonicity caused by addi tion of NaCl produced a dose- and time-dependent stimulation of COX-2 expre ssion in mIMCD-K2 cells as well as in MDCK cells. COX-1 was unaffected. In the same cell lines, mannitol, sucrose, and raffinose also had a stimulator y effect. The tonicity-stimulated COX-2 expression in mIMCD-K2 cells was al most completely blocked by a tyrosine kinase inhibitor, genistein at 100 mu M. In MDCK cells transfected with a 2.7-kb COX-2 promoter and lacZ reporte r construct, NaCl induced a twofold increase in beta-galactosidase activity . Using mIMCD-K2 cells, hypertonic NaCl (600 mosmol/kgH(2)O for 24 h) induc ed a 33-fold increase in PGE(2) release determined by enzyme immunoassay, a n effect completely blocked by 3 mu M indomethacin or the COX-2-specific bl ocker N-(2-cyclohexy-4-nitrophenyl)methanesulfonamide (NS-398). We conclude that in inner medulla, COX-2 but not COX-1 is upregulated by hyperosmolali ty.