Serum and glucocorticoid regulated kinase1 (SGK1) is a key component of the pathway that leads to activation of the epithelial sodium channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN). Regulation of ENaC activity is a major determinant of renal Na⁺ absorption and overall body fluid homeostasis and blood pressure (1, 2). Studies from our laboratory have shown that human skin cells express multiple SGK1 isoforms (a-f) that arise from alternative transcriptional start sites and RNA splicing at the SGK1 locus. The aim of this study was to investigate if SGK1 isoforms are also expressed in the ASDN and to assess their potential role in regulating Na⁺ transport. We used the mouse cortical collecting duct cell line mpkCCDcl4 (3) which display aldosterone/sgk1-regulated Na⁺ transport (3, 4). Comparison of multiple mouse sgk1 expressed sequence tags (ESTs) in the mouse EST database (5) with genomic DNA, identified four potential sgk1 isoforms termed sgk1a (original form), 1b, 1c and 1d. Each isoform has a unique amino terminus of varying size but otherwise an identical sequence. Using sequence specific primers, mRNA expression of all four isoforms was confirmed by RT-PCR from purified mpkCCDcl4 cell RNA. Western blotting of protein from mpkCCDcl4 cell lysates overexpressing individually cloned isoforms showed distinct protein bands corresponding to the correct predicted sizes (a~49kDa, b~50kDa, c~48kDa, d~60kDa). Western blot analysis of serum-starved cells exposed to 10 nM aldosterone (Al) or Al plus 1 µM insulin (Ins) showed a time-dependent increase in the endogenous expression levels of multiple sgk1 bands within 1 hr of treatment (n=4). These Al and Al + Ins-induced endogenous sgk1 bands co-migrated with overexpressed sgk1 isoform bands corresponding to sgk1a, 1b, 1c and 1d. However, expression levels of sgk1a and 1b were increased more by Al + Ins treatment compared to sgk1c and 1d. Aldosterone also produced a significant increase in amiloride-sensitive (ENaC-mediated) equivalent short circuit current within 2 hrs of exposure (Con: 6.7 ± 0.1; Al: 12.9 ± 0.8 µA/cm², mean ± sem; p < 0.001, one-way ANOVA, n=3), and current reached a peak after 4-6 hrs exposure. Insulin potentiated the Al response at 2 hrs (Al + Ins: 15.8 ± 0.1 µA/cm², n=3; p < 0.05) and after 6 hrs (Al: 18.3 ± 1.0; Al + Ins: 21.4 ± 0.6 µA/cm², n=3; p < 0.05). Insulin alone had no effect. These data suggest that multiple sgk1 isoforms (1a-1d) are induced by aldosterone which is linked to an increase in ENaC activity. Insulin also potentiated the Al-mediated increase in ENaC activity. Further studies into isoform-specific regulation of Na⁺ transport may provide novel mechanisms and possible therapeutic targets for dysregulated Na⁺ transport such as observed in primary hypertension.