Characterization of early aldosterone-induced RNAs identified in A6 kidney epithelia

Abstract

 The early aldosterone-induced increase in Na reabsorption across tight epithelia is characterized by a transcription-dependent activation of epithelial Na channels (ENaC) and pumps (Na,K-ATPase). In order to contribute towards the identification of transcriptionally regulated mediators of this process, we first tested mRNAs of proteins previously suggested to be involved. Epithelia were treated for 1 h with 10^–6 M aldosterone, a concentration which produces a maximal transport response and at which both mineralo- and glucocorticoid receptors are occupied. Northern blot analysis showed no change in mRNAs of trimeric G protein alpha subunits, calmodulin, and mitochondrial energy metabolism proteins, whereas Na,K-ATPase α1 and β1 subunit mRNAs were slightly increased (1.2- to 1.4-fold). In a second approach, we visualized 5000 cDNA bands generated from A6 RNAs by differential display polymerase chain reaction (PCR). After 1 h of aldosterone treatment, ≈0.5% of these appeared to be regulated. Four cDNA fragments corresponding to early adrenal-steroid-upregulated RNAs (ASURs) were cloned and for two of them cDNAs containing entire coding sequences were isolated by library screening. ASUR4 is the Xenopus laevis homologue of human E16 and rat TA1, a membrane protein structurally related to yeast and prokaryotic permeases, and ASUR5 is the A transcript of Xenopus K-ras2. The rapid inductions of the four ASURs correspond to direct transcriptional effects since they were not inhibited by cycloheximide but were blocked by actinomycin D. The K_1/2 values were similar or slightly below those reported for stimulation of Na transport. These characteristics of RNA accumulation and their time courses suggest a possible role of one of these induced RNAs in the mediation of the early effect of aldosterone on Na transport.