Expression of 5-LO activating protein (FLAP) enhances albumin permeability in glomerular endothelial...

Abstracts /Prostaglandins & other Lipid Mediators 59 (1999) 1-235 123 EXPRESSION OF 5-LO ACTIVATING PROTEIN (FLAP) ENHANCES ALBUMIN PERME- ABILITY IN GLOMERULAR ENDOTHELIAL CELLS (GEC) Angel Montero, Susumu Uda, Karen A. Munger, José M. Valdivielso and Kamal F. Badr The Center for Glomerulonephritis, Renal Div., Emory U., and the VAMC, Atlanta, GA FLAP is involved in leukotriene synthesis, but does not always co-localize with 5-LO. In fact, we have described enhanced expression of FLAP in glomerular endothelium (which lacks 5LO). Moreover, we have described that FLAP inhibition reduces proteinuria and reverses changes in size permselectivity in glomerulonephritis (Guasch, Zayas and Badr. (1999) Kidney Int. 56:261- 267). Our aim was to study the expression of FLAP in glomerular endothelial cells, as wel1 as the effect of Flap inhibition on glomerular cel1 permeability to albumin. GEC were incubated for 24 hours with TNF or IFNq. RNA and proteins were extracted for analysis of FLAP expression. Permeability was determined in collagen-coated transwell cells by measuring the presence of ‘251-albumin in the lower chamber two hours after adding it to the upper one. Expression of FLAP was detected in GEC by PCR under basal conditions. FLAP protein expression was also detected in control cells and was significantly increased in a dose- dependent manner by incubation with IFN-T (397+26%, lOOng/ml, n=5) or TNF-a (178f17%, lOOng/ml, n=5). Cel1 permeability was increased after 24 hours incubation with IFN-y (100 ng/ ml) (1.26ti.06 VS 1.42ti.06 %, n=6, ~~0.05). Coincubation with the FLAP antagonist BAY Y 1015 (0.1 nM) abolished IFN-y increased cel1 permeability (0.87ti.06 %, n=6, ~~0.05). In light of our previous results, we propose that FLAP is a pathogenic molecule whose expression is enhanced in glomerular endothelium by pro-inflammatory stimuli (IFN and TNFa), and this results in exaggerated protein passage across endothelial cells (likely through increased fenestrae number and/or size). [NIDDK and Veterans Administration Support provided].

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Abstracts /Prostaglandins & other Lipid Mediators 59 (1999) 1-235 123

EXPRESSION OF 5-LO ACTIVATING PROTEIN (FLAP) ENHANCES ALBUMIN PERME- ABILITY IN GLOMERULAR ENDOTHELIAL CELLS (GEC)

Angel Montero, Susumu Uda, Karen A. Munger, José M. Valdivielso and Kamal F. Badr

The Center for Glomerulonephritis, Renal Div., Emory U., and the VAMC, Atlanta, GA

FLAP is involved in leukotriene synthesis, but does not always co-localize with 5-LO. In fact, we have described enhanced expression of FLAP in glomerular endothelium (which lacks 5LO). Moreover, we have described that FLAP inhibition reduces proteinuria and reverses changes in size permselectivity in glomerulonephritis (Guasch, Zayas and Badr. (1999) Kidney Int. 56:261- 267). Our aim was to study the expression of FLAP in glomerular endothelial cells, as wel1 as the effect of Flap inhibition on glomerular cel1 permeability to albumin.

GEC were incubated for 24 hours with TNF or IFNq. RNA and proteins were extracted for analysis of FLAP expression. Permeability was determined in collagen-coated transwell cells by measuring the presence of ‘251-albumin in the lower chamber two hours after adding it to the upper one. Expression of FLAP was detected in GEC by PCR under basal conditions. FLAP protein expression was also detected in control cells and was significantly increased in a dose- dependent manner by incubation with IFN-T (397+26%, lOOng/ml, n=5) or TNF-a (178f17%, lOOng/ml, n=5). Cel1 permeability was increased after 24 hours incubation with IFN-y (100 ng/ ml) (1.26ti.06 VS 1.42ti.06 %, n=6, ~~0.05). Coincubation with the FLAP antagonist BAY Y 1015 (0.1 nM) abolished IFN-y increased cel1 permeability (0.87ti.06 %, n=6, ~~0.05).

In light of our previous results, we propose that FLAP is a pathogenic molecule whose expression is enhanced in glomerular endothelium by pro-inflammatory stimuli (IFN and TNFa), and this results in exaggerated protein passage across endothelial cells (likely through increased fenestrae number and/or size). [NIDDK and Veterans Administration Support provided].