The selective cyclooxygenase-2 inhibitor celecoxib modulates the formation of vasoconstrictor eicosanoids and activates PPARγ. Influence of albumin

Immunocytochemical characterization and profile of eicosanoids formed by rat RMC 85/4 cells. (A) Expression of characteristic cellular markers of mesangial cells. Cells were cultured in RPMI 1640 supplemented with 10% FCS and characterized by immunocytochemistry using specific antibodies against desmin, vimentin, cytokeratin and factor VIII. (B) Detection of COX-1, COX-2 and 12/15-LO mRNA expression by RT-PCR. RNA was reverse-transcribed and COX-1, COX-2 and 12/15-LO amplified by PCR. Representative results obtained from two separate samples (samples 1 and 2) are shown in this ethidium bromide-stained gel; m, size marker (100-base pair DNA ladder). (C) Generation of arachidonic acid metabolites. Eicosanoid levels in supernatants from RMC 85/4 cells growing in serum-free RPMI 1640 medium for 24h were determined by EIA. Results are expressed as mean±SEM of three different experiments with duplicate determinations. 8-iso, 8-epi-PGF_2α.

Effects of the selective COX-2 inhibitor, celecoxib, on eicosanoid formation in rat RMC 85/4 cells. Cells were exposed to vehicle (empty bars) or celecoxib (3μM) (dashed bars) for 16h at 37°C and eicosanoid levels measured in cell supernatants by specific EIAs. Results represent the mean±SEM of 3–6 different experiments with duplicate determinations. *P<0.01 and **P<0.005 versus vehicle.

Effects of the selective COX-2 inhibitor, celecoxib, on renin release by rat RMC 85/4 cells. Cells were exposed to vehicle or celecoxib (3μM) for 16h at 37°C in the absence or presence of albumin (10mg/ml) and renin concentrations in cell supernatants determined by EIA. Results represent the mean±SEM of three different experiments with duplicate measurements. *P<0.025 and **P<0.01 versus vehicle.

Effects of the selective COX-2 inhibitor, celecoxib, on PPARγ in RMC 85/4 cells. Cells were co-transfected with PPARγ-GAL4, luciferase and pCMV-βGal plasmids and treated for 18h with increasing concentrations of celecoxib (3 and 10μM) (A), albumin (1 and 10mg/ml) (C) and combinations of 15d-PGJ₂ (1μM) with celecoxib (3 and 10μM) (B) or albumin (1 and 10mg/ml) (D). Luciferase values were normalized to the level of β-galactosidase activity and results plotted as fold activation relative to untreated cells (vehicle, V) arbitrarily set to a value of 1. Results are the mean±SEM of five different experiments. *P<0.025, **P<0.01 and ***P<0.005 versus vehicle. ^aP<0.01 and ^bP<0.025 versus 15d-PGJ₂.

Effects of the selective COX-2 inhibitor, celecoxib, on α-SMA protein expression in rat RMC 85/4 cells. Cells were exposed to vehicle, 15d-PGJ₂ (1 and 10μM), celecoxib (10μM), albumin (10mg/ml) and combinations of 15d-PGJ₂ (1μM) with either celecoxib (10μM) or albumin (10mg/ml) for 18h at 37°C. Expression of α-SMA was analyzed by Western blot and band intensities determined by scanning densitometry. This figure shows a representative blot and the mean±SEM of four different experiments. *P<0.05, **P<0.020 and ***P<0.01 versus vehicle.