Identification of NFAT5 as a transcriptional regulator of the EDN1 gene in collecting duct

Jayalakshmi Lakshmipathi, Will Wheatley, Anil Kumar, Gaelle Mercenne, Aylin R Rodan, Donald E. Kohan

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

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 languageEnglish (US)
Pages (from-to)F481-F487
JournalAmerican journal of physiology. Renal physiology
Volume316
Issue number3
DOIs
StatePublished - Mar 1 2019
Externally publishedYes

Keywords

  • endothelin-1
  • inner medullary collecting duct
  • NFAT5
  • osmolarity
  • promoter

ASJC Scopus subject areas

  • Physiology
  • Urology

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