The epithelial sodium channel (ENaC) is the rate-limiting transport mechanism for Na⁺ reabsorption in the distal nephron and is critical for maintaining sodium homeostasis. In the cortical collecting duct (CCD) ENaC activity is tightly regulated by aldosterone. Its stimulatory effect on ENaC requires the presence of the mineralocorticoid receptor (1) and involves transcriptional regulation of a complex set of genes which are not yet fully characterized.  In particular, a better understanding of the early versus late transcriptional effects of aldosterone is needed (2,3). To address this issue, we used mRNA sequencing (RNAseq) for a comprehensive transcriptome analysis of a highly differentiated and aldosterone sensitive mouse cortical collecting duct (mCCD_cl1) cell line. Cells were grown on permeable supports and exposed to 3 nM aldosterone for 2 h or 24 h. To confirm the hormone’s stimulatory effect on ENaC-mediated transepithelial sodium transport, equivalent short-circuit current (I_SC) measurements were performed as previously described (4). In the 2 h incubation experiments I_SC was continuously recorded in modified Ussing chambers. In the 24 h incubation experiments I_sc was monitored by repeated transepithelial voltage and resistance measurements with two sticks “STX” electrodes and an epithelial volt-ohm-meter. The ENaC-mediated I_SC component (ΔI_SC-ami) was determined by application of 10 µM amiloride. In cells treated with aldosterone for 2 h ΔI_SC-ami was 2.2-fold higher than in controls (27.1±0.9 µA·cm^-2 vs. 12.3±0.8 µA·cm^-2; n=6; mean ± SEM; p<0.001). Similarly, compared to controls ΔI_SC-ami was 3.2-fold higher in cells treated with aldosterone for 24 h (23.1±2.7 µA·cm^-2 vs. 7.2±1.1 µA·cm^-2; n=7; mean ± SEM; p<0.001). Subsequently, mRNA was prepared from cells harvested from each individual permeable support for RNAseq analysis. The mRNA libraries were prepared using the Illumina TruSeq and Stranded mRNA kits and sequenced on the Illumina Hiseq 2500 and Illumina Novaseq 6000 platforms. In cells treated for 2 h or 24 h we identified a total of 94 (66 up- and 28 downregulated) or 453 (347 up- and 106 downregulated) aldosterone-regulated protein coding transcripts, respectively. A set of 49 transcripts was found to be aldosterone-regulated in both the 2 h and 24 h groups.  The well-known aldosterone-regulated serum and glucocorticoid-regulated kinase 1 (Sgk1) was upregulated by a factor of 9.0 (n=6; p<0.001) and 6.4 (n=7; p<0.001) after 2 h and 24 h aldosterone incubation, respectively. The transcript of the α-subunit of ENaC (Scnn1a) was also upregulated after both short-term (1.23-fold; p<0.001) and long-term (1.54-fold; p<0.001) aldosterone treatment. No effect was observed on β- and γ-ENaC transcripts consistent with previous reports. Interestingly, the transcript for the transmembrane serine protease 2 (Tmprss2), which has recently been shown to be involved in proteolytic ENaC activation (5), was upregulated (1.1-fold; p<0.01) after 24 h aldosterone treatment. This work highlights the differential transcriptional effects of acute vs. chronic aldosterone exposure.  Further studies are needed to explore the physiological role and molecular interplay of newly identified early vs. late aldosterone-regulated genes in renal ENaC regulation.