Sp1 trans-activates and is required for maximal aldosterone induction of the αENaC gene in collecting duct cells

The epithelial Na+ channel (ENaC) in the distal nephron constitutes the rate-limiting step for renal sodium reabsorption. Aldosterone increases tubular sodium absorption in large part by increasing alpha ENaC transcription in collecting duct principal cells. We previously reported that Af9 binds to + 78/ + 92 of alpha ENaC and recruits Dot1a to repress basal and aldosterone-sensitive alpha ENaC transcription in mouse inner medullary collecting duct (mIMCD)3 cells. Despite this epigenetic repression, basal alpha ENaC transcription is still evident and physiologically necessary, indicating basal operation of positive regulators. In the present study, we identified Sp1 as one such regulator. Gel shift and antibody competition assays using a + 208/ + 240 probe revealed DNA-Sp1-containing complexes in mIMCD3 cells. Mutation of the + 222/ + 229 element abrogated Sp1 binding in vitro and in promoter-reporter constructs stably expressed in mIMCD3 cells. Compared with the wild-type promoter, an alpha ENaC promoter-luciferase construct with + 222/ + 229 mutations exhibited much lower activity and impaired trans-activation in Sp1 overexpression experiments. Conversely, Sp1 knockdown inhibited endogenous alpha ENaC mRNA and the activity of the wild-type alpha ENaC promoter but not the mutated construct. Aldosterone triggered Sp1 recruitment to the alpha ENaC promoter, which was required for maximal induction of alpha ENaC promoter activity and was blocked by spironolactone. Sequential chromatin immunoprecipitation assays and functional tests of + 78/ + 92 and + 222/ + 229 alpha ENaC promoter mutants indicated that while Sp1, Dot1a, and Af9 co-occupy the alpha ENaC promoter, the Sp1 effects are functionally independent from Dot1a and Af9. In summary, Sp1 binding to a cis-element at + 222/ + 229 represents the first identified constitutive driver of alpha ENaC transcription, and it contributes to maximal aldosterone trans-activation of alpha ENaC.