Objective: 

The serum- and glucocorticoid-inducible kinase 1 (SGK1) is an important regulator of epithelial ion transport and interferes in several biological processes such as sodium homeostasis, osmoregulation and cell growth. Within the collecting duct activation of SGK1 leads to an elevation of functional ENaC expression and thereby higher sodium currents. NDRG1 (n-myc downstream-regulated gene1) is a protein which specifically becomes phosphorylated at position Thr346/356/366 by activated SGK1. We used the mammalian cell line M-1 to develop an assay to characterize small-molecule SGK1 inhibitors. The SGK1 inhibitor GSK650394 (K4) was used as a prototypic tool compound (IC₅₀ 0.6mmol/l, LC₅₀ 41mmol/l; Selectivity SGK1/AKT >30). The combined functional readout of Na⁺ current together with SGK1 specific phosphorylation was used to exclude false positives.

Methods: 

Confluent M-1 cell monolayers were grown on filters and investigated for the amiloride-dependent short circuit current (DI'_sc) in a continuously perfused Ussing chamber. Cells were pretreated under culture conditions with SGK1 inhibitors at different concentrations. Concentration-response-curves were generated to determine IC₅₀ values for each compound. In parallel, we detected the phosphorylation state of NDRG1 as a measure of SGK1 activity by Western blot and immunostaining against phosphorylated NDRG1 (Thr ³⁴⁶).

Results: 

Under control conditions DI'_sc was 8.64 ± 1.0 mA/cm² (n=9). Inhibitors of SGK1 caused a concentration dependent decrease of amiloride inhibitable I'_sc with IC₅₀ values of 0.8 mM (K4), 0.7 mM (K1) and 2 mM (K7) respectively. Western blot analysis of K4 treated M-1 cells at a concentration of 10 mM for 30 minutes revealed 90.8 % reduction in phosphorylated NDRG1 (Thr ³⁴⁶).

Conclusion: 

The data show that the combined use of transepithelial transport measurement and NDRG1 phosporylation is suitable to investigate SGK1 inhibitors.