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REGULATION OF COLLECTING DUCT ENDOTHELIN‐1 PRODUCTION
Author(s) -
Kohan Donald E.,
Stricklett Peter K.,
Chapman Mark E.,
Strait Kevin A.
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.998.38
Subject(s) - nfat , microbiology and biotechnology , endothelin 1 , endothelins , chemistry , luciferase , calmodulin , calcineurin , biology , transcription factor , transfection , medicine , biochemistry , receptor , enzyme , gene , transplantation
Collecting duct (CD)‐derived endothelin‐1 (ET‐1) is an important autocrine inhibitor of renal Na reabsorption; CD ET‐1 knockout causes marked hypertension. High Na intake augments CD ET‐1 release; the mechanisms involved are unknown. To assess this, primary cultures of rat inner medulla CD (IMCD) were studied. Cells exposed to flow had a 3‐4 fold increase in ET‐1 mRNA and intracellular Ca [Ca]i. Inhibition of calmodulin (CaM) or CaM kinase II, as well as chelation of [Ca]i, markedly reduced ET‐1 release and mRNA levels. Transfection with rat ET‐1 promoter‐luciferase constructs revealed maximal reporter activity, as well as sensitivity to CaM blockade, between the region 1319‐1725 bp 5' to the transcription start site. Incubation of this 406 bp region with IMCD nuclear extracts caused an electrophoretic mobility shift that was prevented by competition with a 125 bp fragment between 1455 and 1576 bp 5' to the transcription start site. This 122 bp region contains two NFAT sites. An antibody to NFAT‐5 caused a supershift, while oligos containing the NFAT consensus site reduced the shift. In vitro footprinting of the ‐1455 and ‐1576 bp region revealed protected areas at the NFAT consensus binding sites. Cyclosporine A (CyA), a calcineurin inhibitor, caused a 60% reduction in IMCD ET‐1 release. In contrast, in rat aortic endothelial cells, ET‐1 promoter‐luciferase activity was maximal in the proximal 500 bp promoter region and was not CaM regulated; further, CyA stimulated ET‐1 production. We propose that Na loading increases CD luminal flow, augmenting [Ca]i, activating CaM and calcineurin, leading to NFAT nuclear translocation and increased ET‐1 gene transcription. This pathway may be unique to CD.