Regulation of epithelial Na+ channels by adrenal steroids: mineralocorticoid and glucocorticoid effects

Abstract

Epithelial Na+ channels (ENaC) can be regulated by both mineralocorticoid and glucocorticoid hormones. In the mammalian kidney, effects of mineralocorticoids have been extensively studied, but those of glucocorticoids are complicated by metabolism of the hormones and cross-occupancy of mineralocorticoid receptors. Here, we report effects of dexamethasone, a synthetic glucocorticoid, on ENaC in the rat kidney. Infusion of dexamethasone (24 μg/day) for 1 wk increased the abundance of αENaC 2.26 ± 0.04-fold. This was not accompanied by an induction of Na+ currents (I(Na)) measured in isolated split-open collecting ducts. In addition, hormone treatment did not increase the abundance of the cleaved forms of either αENaC or γENaC or the expression of βENaC or γENaC protein at the cell surface. The absence of hypokalemia also indicated the lack of ENaC activation in vivo. Dexamethasone increased the abundance of the Na+ transporters Na+/H+ exchanger 3 (NHE3; 1.36 ± 0.07-fold), Na(+)-K(+)-2Cl(-) cotransporter 2 (NKCC2; 1.49 ± 0.07-fold), and Na-Cl cotransporter (NCC; 1.72 ± 0.08-fold). Surface expression of NHE3 and NCC also increased with dexamethasone treatment. To examine whether glucocorticoids could either augment or inhibit the effects of mineralocorticoids, we infused dexamethasone (60 μg/day) together with aldosterone (12 μg/day). Dexamethasone further increased the abundance of αENaC in the presence of aldosterone, suggesting independent effects of the two hormones on this subunit. However, I(Na) was similar in animals treated with dexamethasone+aldosterone and with aldosterone alone. We conclude that dexamethasone can occupy glucocorticoid receptors in cortical collecting duct and induce the synthesis of αENaC. However, this induction is not sufficient to produce an increase in functional Na+ channels in the apical membrane, implying that the abundance of αENaC is not rate limiting for channel formation in the kidney.

Fig. 1.

Regulation of epithelial Na channel (ENaC) subunits by dexamethasone in the kidney and colon. Rats were infused with dexamethasone (24 μg/day) for 7 days. A: gels for Western blots were loaded with homogenates of whole kidneys (40 μg/lane) or distal colon mucosal epithelial cells (30 μg/lane). Different lanes show extracts from different animals. Blots were probed with anti-αENaC, anti-βENaC, or anti-γENaC antiserum. For the anti-αENaC blots, different portions of the same gel were processed differently to visualize the staining of the full-length (90 kDa) and cleaved (20–30 kDa) forms of the subunit. B: quantitation of effects of dexamethasone on expression of renal ENaC protein. Densitometric data were normalized to means of control measurements and represent means ± SE for 4 animals. Star indicates statistically significant difference from controls (P < 0.05).

Fig. 2.

Whole-cell currents (I_Na, I_tot, I_amil) in principal cells of cortical collecting ducts (CCDs) from rats treated with aldosterone (12 μg/day) or dexamethasone (24 μg/day). Top and middle: current traces for steps to different voltages from 0 mV in the absence (top) and presence (middle) of 10⁻⁵ M amiloride. Bottom: current-voltage relationships. In the case of the aldosterone-treated cell, the amiloride-sensitive current is plotted as the difference in current in the absence and presence of the drug.

Fig. 3.

I_Na in principal cells of CCDs treated with dexamethasone (24 μg/day), aldosterone (12 μg/day), or both [aldosterone 12 μg/day+dexamethasone (60 μg/day)]. I_Na was calculated as the difference in current at a cell voltage of −100 mV without and with amiloride, as shown in Fig. 2. Data are plotted as means ± SE for 22 (aldosterone), 16 (dexamethasone), and 26 (aldosterone+dexamethasone) cells.

Fig. 4.

Effects of dexamethasone on surface expression of ENaC proteins. Kidneys from control and dexamethasone-treated rats were biotinylated, and surface proteins were isolated from 1.25 mg total protein by binding and elution from neutravidin beads. Eluates were analyzed by Western blotting as in Fig. 1. Each lane represents eluate from a different animal. Relative densities of the major bands for dexamethasone-treated/control were 0.74 and 0.56 for βENaC and γENaC, respectively, and were not significantly different from 1.

Fig. 5.

Effects of dexamethasone on expression of Na⁺ transporters in the kidney. A: overall expression. Western blots were loaded with 35 μg protein/lane from kidney homogenates from control and dexamethasone-treated rats. B: surface expression. Western blots were loaded with eluants from 1.25 mg total protein by binding and elution from neutravidin beads. Each lane represents a different animal. Blots were probed with antibodies against Na⁺/H⁺ exchanger 3 (NHE3), Na⁺-K⁺-2Cl⁻ cotransporter (NKCC2), or Na⁺-Cl⁻ cotransporter (NCC). C: quantitation of effects of dexamethasone on expression of Na⁺-transporter protein. Densitometric data were normalized to means of control measurements and represent means ± SE for 4 animals. Stars indicate statistically significant difference from controls (P < 0.05).

Fig. 6.

Regulation of ENaC subunits by dexamethasone in the presence of aldosterone. Rats were infused with aldosterone (12 μg/day) with or without dexamethasone (60 μg/day) for 7 days. A: gels for Western blots were loaded with 40 μg protein/lane. Different lanes represent different animals. Blots were probed with anti-αENaC, anti-βENaC, or anti-γENaC antiserum. For the anti-αENaC blots, different portions of the same gel were processed differently to visualize the staining of the full-length (90 kDa) and cleaved (20–30 kDa) forms of the subunit. B: quantitation of effects of dexamethasone on expression of Na⁺ channel protein in the presence of aldosterone. Densitometric data were normalized to means of measurements with aldosterone alone and represent means ± SE; n = 4 animals for each condition. Stars indicate statistically significant difference from controls (P < 0.05). C: quantification of effects of aldosterone, dexamethasone, and aldosterone+dexamethasone on the expression of αENaC protein. Densitometric data were normalized to means of control measurements and represent means ± SE; n = 4 animals for each condition. Stars indicate statistically significant difference. Values for aldosterone and dexamethasone are significantly different from controls (P < 0.001). Values for aldosterone+dexamethasone are significantly different from aldosterone alone (P = 0.003).

Fig. 7.

Effects of aldosterone and dexamethasone on plasma K. Data represent means ± SE; n = 4 animals for each condition. Stars indicate statistically significant difference from controls (P < 0.05).