Abstract
The inner medullary collecting duct (IMCD) produces very high levels of endothelin-1 (ET-1) that acts as an autocrine inhibitor of IMCD Na+ and water reabsorption. Recent studies suggest that IMCD ET-1 production is enhanced by extracellular hypertonicity as can occur during high salt intake. Although NFAT5 has been implicated in the IMCD ET-1 hypertonicity response, no studies in any cell type have identified NFAT5 as a transcriptional regulator of the EDN1 gene; the current study examined this using a mouse IMCD cell line (IMCD3). Media hypertonicity increased IMCD3 ET-1 mRNA in a dose- and time-dependent manner associated with increased NFAT5 nuclear localization. Knockdown of NFAT5 using small-interfering RNA or by CRISPR/Cas9-mediated targeting of exon 4 of the NFAT5 gene reduced the ET-1 hypertonicity response. Chromatin immunoprecipitation using an NFAT5 antibody pulled down ET-1 promoter regions containing NFAT5 consensus binding sequences. Transfected ET-1 promoter reporter constructs revealed maximal hypertonicity-induced reporter activity in the proximal 1-kb region; mutation of the two NFAT5 consensus-binding sites in this region abolished hypertonicity-induced reporter activity. The 1-kb ET-1 promoter-reporter construct lost hypertonicity responsiveness when transfected in CRISPR/Cas9-induced NFAT5-deficient cells. In summary, these findings represent the first description that NFAT5 is a direct transcriptional regulator of the EDN1 gene in IMCD cells and point to a potentially important mechanism by which body Na+ homeostasis is maintained.
Original language | English (US) |
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Pages (from-to) | F481-F487 |
Journal | American journal of physiology. Renal physiology |
Volume | 316 |
Issue number | 3 |
DOIs | |
State | Published - Mar 1 2019 |
Externally published | Yes |
Keywords
- endothelin-1
- inner medullary collecting duct
- NFAT5
- osmolarity
- promoter
ASJC Scopus subject areas
- Physiology
- Urology