Trichostatin A (TSA) disrupts the aldosterone-induced activation of the epithelial Na⁺ channel (ENaC) in mouse cortical collecting duct cells by impairing the control of serum and glucocorticoid-inducible kinase 1 (SGK1)

Aldosterone evokes ENaC-mediated Na⁺ retention in the cortical collecting duct by activating mineralocorticoid receptors (MR) that promote expression of the kinase SGK1 (1). However, the transcriptional activity of the MR seems to be restricted by acetylation (2), a post-translational modification that involves the attachment of acetyl groups to lysine residues. The acetylation status of the MR seems to be determined by lysine deacetylases (KDACs), enzymes that remove acetyl groups from such modified proteins (2). The present study therefore explores the effects of TSA, a broad spectrum KDAC inhibitor, upon the responses to aldosterone in mCCDc11 murine cortical duct cells. Electrometric studies showed that aldosterone (3 nM, 3 h) augments amiloride-sensitive (10 µM) short circuit current (I_Amil) (Fig 1A) whilst Western analysis of protein extracted from these cells showed that aldosterone also increased the abundance of the Thr^346/356/366-phosphorylated form of the protein encoded by n-myc downstream regulated gene 1 (NDRG1) without altering overall NDRG1 abundance. Aldosterone thus promotes NDRG1-Thr^346/356/366 phosphorylation (Fig 1B) and, since this provides a read out of SGK1 activity, the electrometric response to aldosterone is accompanied by activation of this kinase. Aldosterone also increased the abundance of SGK1 protein (Fig 1C) and these findings confirm that this hormone stimulates ENaC-dependent Na⁺ absorption via a mechanism that involves sgk1 gene expression. TSA (1 µM, 2 h pre-incubation) caused a slight (~5%) reduction of basal I_Amil and substantial (~90%) inhibition of the electrometric response to aldosterone (Fig 1A). TSA also abolished the effects of aldosterone on the cellular SGK1 activity (Fig 1B) and abundance (Fig 1C). However, separate experiments (n = 6) showed that TSA did not alter the electrometric response to insulin (20 nM). Since insulin activates SGK1, and ENaC, without evoking gene transcription (3), the effects of TSA upon the response to aldosterone (Fig 1) cannot be attributed to non-specific effects. TSA also increased in the abundance of the hyperacetylated forms of histone 3 (13.2 ± 1.4 fold) and 4 (10.6 ± 3.9 fold) and these effects (n = 4) peaked after 6 - 12 h. Since these proteins are archetypical KDAC substrates, these data confirm that TSA blocks these enzymes under the present conditions. Previous work has shown that a broad spectrum KDAC inhibitor can lower blood pressure in an animal model of hypertension (1) and the present data provide a physiological basis for this by showing that such compounds also block aldosterone-dependent Na⁺ transport by disrupting MR-dependent control over SGK1 gene expression.

Where applicable, experiments conform with Society ethical requirements.