Aldosterone and tight junctions: modulation of claudin-4 phosphorylation in renal collecting duct cells

Aldosterone classically modulates Na transport in tight epithelia such as the renal collecting duct (CD) through the transcellular route, but it is not known whether the hormone could also affect paracellular permeability. Such permeability is controlled by tight junctions (TJ) that form a size- and charge-selective barrier. Among TJ proteins, claudin-4 has been highlighted as a key element to control paracellular charge selectivity. In RCCD2 CD cells grown on filters, we have identified novel early aldosterone effects on TJ. Endogenous claudin-4 abundance and cellular localization were unaltered by aldosterone. However, the hormone promoted rapid (within 15-20 min) and transient phosphorylation of endogenous claudin-4 on threonine residues, without affecting tyrosine or serine; this event was fully developed at 10 nM aldosterone and appeared specific for aldosterone (because it is not observed after dexamethasone treatment and it depends on mineralocorticoid receptor occupancy). Within the same delay, aldosterone also promoted an increased apical-to-basal passage of 125I (a substitute for 36Cl), whereas 22Na passage was unaffected; paracellular permeability to [3H]mannitol was also reduced. Later on (45 min), a fall in transepithelial resistance was observed. These data indicate that aldosterone modulates TJ properties in renal epithelial cells.     

(NDRG2) stimulates amiloride-sensitive Na+ currents in Xenopus laevis oocytes and fisher rat thyroid cells

Regulation of the epithelial sodium channel (ENaC) is highly complex and may involve several aldosterone-induced regulatory proteins. The N-Myc downstream-regulated gene 2 (NDRG2) has been identified as an early aldosterone-induced gene. Therefore, we hypothesized that NDRG2 may affect ENaC function. To test this hypothesis we measured the amiloride-sensitive (2 microm) whole cell current (DeltaI(ami)) in Xenopus laevis oocytes expressing ENaC alone or co-expressing ENaC and NDRG2. Co-expression of NDRG2 significantly increased DeltaI(ami) in some, but not, all batches of oocytes tested. An inhibitory effect of NDRG2 was never observed. Using a chemiluminescence assay we demonstrated that the NDRG2-induced increase in ENaC currents was accompanied by a similar increase in channel surface expression. The stimulatory effect of NDRG2 was preserved in oocytes maintained in a low sodium bath solution to prevent sodium feedback inhibition. These findings suggest that the stimulatory effect of NDRG2 is independent of sodium feedback regulation. Furthermore, the stimulatory effect of NDRG2 on ENaC was at least in part additive to that of Sgk1. A short isoform of NDRG2 also stimulated DeltaI(ami). Overexpression of NDRG2 and ENaC in Fisher rat thyroid cells confirmed the stimulatory effect of NDRG2 on ENaC-mediated short-circuit current (I(SC-ami)). In addition, small interference RNA against NDRG2 largely reduced I(SC-ami) in Fisher rat thyroid cells. Our results indicate that NDRG2 is a likely candidate to contribute to aldosterone-mediated ENaC regulation.