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Molecular determinants of sodium transport: role of a new aldosterone induced gene

Final Activity Report Summary - ALSOSTERONE-FELLOW (Molecular determinants of sodium transport: role of a new aldosterone induced gene)

Aldosterone plays a major role in Na absorption and thus in the control of volemia and blood pressure. It exerts its effects through the mineralocorticoid receptor which belongs to the nuclear receptor superfamily and acts as a transcription factor to modulate transcription of target specific genes. These induced proteins then stimulate sodium reabsorption by increasing both its luminal entry into epithelial cells through the rate limiting amiloride-sensitive sodium channel ENaC and its active extrusion into the blood by the basolateral Na/K-ATPase.

We identified a novel protein, NDRG2 (N-myc Downstream Regulated Gene 2) whose expression was very early stimulated by aldosterone, both in an established cell line (RCCD2) and in the kidney and colon of rats. NDRG2 belonged to a family of highly conserved genes with mostly unknown functions. Aldosterone induction of NDRG2 was transient in epithelia (0.5-1h). In contrast, steady-state changes in NDRG2 expression were associated to altered cell proliferation and differentiation in pancreas, liver or neuronal tumors.

To progress in the understanding of its role in kidney cells, RCCD2 were stably transfected with NDRG2 that yielded a three-fold overexpression of the protein over several passages. When grown on plastic, transfected cells showed significantly higher density of domes than parental RCCD2. When grown on filters, NDRG2 transfected cells had similar proliferation rate (cell count) and cell cycle (FACS) than RCCD2 but also had a two-fold reduction in apoptosis. At confluence, the transepithelial resistance of NDRG2 overexpressing cells was enhanced to 6 000-8 000 ohms.cm2 (as compared to 5 000 in RCCD2 control cell line).

Western blot showed that NDRG2 expression was associated to an increase in expression of the alpha subunit of the epithelial sodium channel ENaC, with no change in beta or gamma subunits, or in serum and glucocorticoid kinase1 nor the ubiquitin ligase Nedd4-2. In these experiments, the 2 isoforms of NDRG2 (complete coding sequence or with a 14 aa deletion in the N-ter) gave comparable results. We also used the doxycyclin (Dox)-sensitive tet-on system introduced in RCCD2 to overexpress NDRG2 transiently. 48 h after transfection, NDRG2 protein levels were increased by 10 to 20 folds in Dox-treated cells, together with a three-fold reduction in ERK phosphorylation and two to three-fold increase in the sole alpha ENaC expression.

Altogether, these results showed that the early aldosterone-induced protein NDRG2, when overexpressed in CD cells, enhanced the differentiation status of cells, reduced apoptosis and led to higher alpha ENaC expression. Therefore NDRG2 appeared to be a novel element in the cascade of events that controlled ENaC expression.