Retinoic acids inhibit inducible nitric oxide synthase expression in mesangial cells  Prasun K. Datta, Elias A. Lianos  Kidney International  Volume 56, Issue 2, Pages 486-493 (August 1999) DOI: 10.1046/j.1523-1755.1999.00576.x Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 1 All-trans-retinoic acid (ATRA) and 13-cis-retinoic acid (13-cis-RA) attenuate nitric oxide (NO) production following inducible nitric oxide synthase (iNOS) activation by lipopolysaccharide (LPS) + interferon-γ (IFN-γ) in murine mesangial cells. (A) Concentrations of nitrite (μM) measured by the Griess reaction in mesangial cell (2 × 106 cells per well) culture media after 24 hours of stimulation with LPS + IFN-γ in the absence or presence of ATRA (0.1, 1.0 and 10 μM). For controls, the cells were incubated with DMEM only. In 10 μM ATRA the cells were incubated with DMEM containing 10 μM ATRA. Results are means ±SEM, N = 3. *P < 0.05 compared to LPS + IFN-γ. (B) Concentrations of nitrite (μM) measured by the Griess reaction in mesangial cell (1 × 106 cells per well) culture media after 24 hours of stimulation with LPS + IFN-γ in absence or presence of 13-cis-RA (0.1, 1.0 and 10 μM). Control cells were incubated with DMEM only, while the 10 μM 13-cis-RA cells were incubated with DMEM containing 10 μM 13-cis-RA. Results are means ±SEM, N = 3. *P < 0.05 compared to LPS + IFN-γ. Kidney International 1999 56, 486-493DOI: (10.1046/j.1523-1755.1999.00576.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 2 Inducible nitric oxide synthase (iNOS) enzyme activity is attenuated in lysates prepared from mesangial cells stimulated with lipopolysaccharide (LPS) + interferon-γ (IFN-γ) in the presence of all-trans-retinoic acid (ATRA). iNOS enzyme activity was measured by conversion of L-[14C]arginine to L-[14C]citrulline assay in cell lysates prepared from unstimulated mesangial cells (control), and cells stimulated with LPS + IFN-γ in the absence or presence of 10 μM ATRA for 24 hours. Results are means +SEM, N = 3. *P < 0.001 compared to control; **P < 0.005 compared to LPS + IFN-γ stimulated cells. Kidney International 1999 56, 486-493DOI: (10.1046/j.1523-1755.1999.00576.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 3 ATRA and 13-cis-RA inhibit iNOS protein synthesis in mesangial cells. (A) Western blot analysis of iNOS in protein lysates prepared from mesangial cells after 24 hours of stimulation with LPS + IFN-γ in the absence or presence of ATRA (0.1, 1.0 and 10 μM). Lane 1, control (no additives); lane 2, 10 μM ATRA; lane 3, LPS + IFN-γ alone; lanes 4–6, LPS + IFN-γ in the presence of 0.1, 1.0 and 10 μM ATRA, respectively. Western blot analysis is representative of two separate experiments. (B) Western blot analysis of iNOS in protein lysates prepared from mesangial cells after 24 hours of stimulation with LPS + IFN-γ in the absence or presence of 13-cis-RA (0.1, 1.0 and 10 μM). Lane 1, control (no additives); lane 2, 10 μM 13-cis-RA; lane 3, LPS + IFN-γ alone; lanes 4–6, LPS + IFN-γ in the presence of 0.1, 1.0 and 10 μM 13-cis-RA, respectively. Western blot analysis is representative of two separate experiments. Kidney International 1999 56, 486-493DOI: (10.1046/j.1523-1755.1999.00576.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 4 ATRA inhibits iNOS mRNA expression in mesangial cells. iNOS and GAPDH mRNA expression were assessed by RT-PCR of total RNA prepared from control cells, LPS + IFN-γ stimulated cells and LPS + IFN-γ stimulated cells in the presence of 10 μM ATRA. Lanes 1 and 4, control (no additives); lanes 2 and 5, LPS + IFN-γ alone; lanes 3 and 6, LPS + IFN-γ in the presence of 10 μM ATRA. RT-PCR analysis is representative of two separate experiments. Kidney International 1999 56, 486-493DOI: (10.1046/j.1523-1755.1999.00576.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 5 ATRA reduces nuclear levels of NF-βkgrB subunits. Levels of both subunits of NF-βkgrB p50 (A) and p65 (B) were assessed by immunoblot analysis of nuclear extracts prepared from LPS + IFN-γ stimulated cells and LPS + IFN-γ stimulated cells in the presence of 10 μM ATRA. Lane 1, LPS + IFN-γ alone; lane 2, LPS + IFN-γ in the presence of 10 μM ATRA. Western blot analysis is representative of two separate experiments. Kidney International 1999 56, 486-493DOI: (10.1046/j.1523-1755.1999.00576.x) Copyright © 1999 International Society of Nephrology Terms and Conditions