Aldosterone-mediated regulation of ENaC alpha, beta, and gamma subunit proteins in rat kidney - PubMed (original) (raw)

Aldosterone-mediated regulation of ENaC alpha, beta, and gamma subunit proteins in rat kidney

S Masilamani et al. J Clin Invest. 1999 Oct.

Abstract

Aldosterone stimulates sodium transport in the renal collecting duct by activating the epithelial sodium channel (ENaC). To investigate the basis of this effect, we have developed a novel set of rabbit polyclonal antibodies to the 3 subunits of ENaC and have determined the abundance and distribution of ENaC subunits in the principal cells of the rat renal collecting duct. Elevated circulating aldosterone (due to either dietary NaCl restriction or aldosterone infusion) markedly increased the abundance of alphaENaC protein without increasing the abundance of the beta and gamma subunits. Thus, alphaENaC is selectively induced by aldosterone. In addition, immunofluorescence immunolocalization showed a striking redistribution in ENaC labeling to the apical region of the collecting duct principal cells. Finally, aldosterone induced a shift in molecular weight of gammaENaC from 85 kDa to 70 kDa, consistent with physiological proteolytic clipping of the extracellular loop as postulated previously. Thus, at the protein level, the response of ENaC to aldosterone stimulation is heterogenous, with both quantitative and qualitative changes that can explain observed increases in ENaC-mediated sodium transport.

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Figures

Figure 1

Figure 1

Characterization of ENaC subunit antibodies. (a) Immunoblots of rat renal cortical proteins obtained by differential centrifugation. Blots were probed with the 3 ENaC subunit antibodies and with the antibodies preadsorbed with 1 mg of the respective immunizing peptides. Differential centrifugation was carried out as described (6), yielding membrane fractions (17,000 g and 200,000 g pellets) and a cytosolic fraction (200,000 g supernatant). (b) Immunofluorescence localization of ENaC subunits in rat renal cortical collecting duct. Three sections are shown, each double-labeled with 1 of 3 rabbit antibodies to ENaC subunits (left-hand panels) and a chicken anti–aquaporin-2 (right-hand panels). Arrows point to cells lacking aquaporin-2, i.e., intercalated cells. All tissue sections were from sodium-restricted rats. Scale bars: 10 μm.

Figure 2

Figure 2

Effect of dietary NaCl restriction for 10 days. (a) Semiquantitative immunoblots for ENaC subunits and other major renal sodium transporters. For each blot, each lane was loaded with a homogenate from a different rat (n = 6 for both control and sodium-restricted rats). Preliminary Coomassie-stained gels demonstrated equality of loading among lanes. Asterisks mark statistically significant increases. (b) Deglycosylation with PNGase. SDS-solubilized samples from control rats and NaCl-restricted rats were incubated with PNGase F (200 U/40 μg protein; New England Biolabs Inc., Beverly, Massachusetts, USA) or vehicle.

Figure 3

Figure 3

Semiquantitative immunoblots showing effect of aldosterone infusion for 10 days on the abundance and molecular weight of each ENaC subunit in whole kidneys of rat. For each blot, each lane was loaded with 30 μg cortical homogenate from a different rat.

Figure 4

Figure 4

Immunofluorescence localization of ENaC subunit proteins in control rats and rats undergoing dietary NaCl restriction. (a) Comparison of the 3 subunits. Left-hand panels are from rats on control diet; right-hand panels are from salt-restricted rats. Scale bars: 10 μm. (b) γENaC labeling in multiple rat renal cortical collecting ducts. Left-hand panels are from rats on control diet; right-hand panels are from salt-restricted rats. Scale bars: 10 μm.

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