Cell-specific expression of epithelial sodium channel alpha, beta, and gamma subunits in aldosterone-responsive epithelia from the rat: localization by in situ hybridization and immunocytochemistry.

Abstract

Abstract. A highly selective, amiloride-sensitive, epithelial sodium channel from rat colon (rENaC), composed of three homologous subunits termed ~, /3, and 3" rENaC, has been cloned by functional expression and was proposed to mediate electrogenic sodium reabsorption in aldosterone-responsive epithelia. To determine whether rENaC could account for sodium absorption in vivo, we studied the cellular localization of the sodium channel messenger RNA subunits by in situ hybridization and their cellular and subcellular distribution by immunocytochemistry in the kidney, colon, salivary, and sweat glands of the rat. In the kidney, we show that the three subunit mRNAs are specifically co-expressed in the renal distal convoluted tubules (DCT), connecting tubules (CNT), cortical collecting ducts (CCD), and outer medullary collecting ducts (OMCD), but not in the inner medullary collecting ducts (IMCD). We demonstrate co-localization of or, 8, and 3' subunit proteins in the apical membrane of a majority of cells of CCD and OMCD. Our data indicate that c~,/3, and 3' subunit mRNAs and proteins are co-expressed in the distal nephron (excepting IMCD), a localization that correlates with the previously described physiological expression of amiloride-sensitive electrogenic sodium transport. Our data, however, suggest that another sodium transport protein mediates electrogenic amiloride-sensitive sodium reabsorption in IMCD. We also localized rENaC to the surface epithelial cells of the distal colon and to the secretory ducts of the salivary gland and sweat gland, providing further evidence consistent with the hypothesis that the highly selective, amiloride-sensitive sodium channel is physiologically expressed in aldosterone-responsive cells.XLORIDE-sensitive electrogenic transepithelial sodium transport constitutes the rate limiting step for sodium reabsorption in the epithelium of the distal nephron, the distal colon, the ducts of several exocrine glands (for instance the salivary glands and the sweat glands) and the epithelia of the lung (10). In kidney (25) and colon (29), electrogenic sodium transport is upregulated by aldosterone, a mineralocorticoid hormone, allowing the maintenance of sodium balance, blood volume, and blood pressure (30). In the distal nephron of rats submitted to a 1 wk salt restriction, the electrogenic sodium transport is highly efficient at reabsorbing sodium from the urine of collecting ducts, decreasing luminal sodium concentration to levels as low as 1 raM, while the plasma concentration is maintained at 140 raM. To establish such a gradient, the epithelia of the distal nephron expresses an electrogenic sodium transport mediated by an amiloride-sensitive sodium channel located in the apical membrane facing the external compartment (urine), and a ouabain-sensitive sodium pump restricted to the basolateral membrane facing the extracellular compartment (blood). Current physiological evidence indicates that the amiloride-sensitive epithelial sodium channel is mainly expressed in tissues that fall into t...

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“…Rabbit polyclonal anti-␣-rENaC, anti-␤-rENaC, and anti-␥-rENaC antibodies were used at 1:2000 dilution, and mouse monoclonal anti-actin antibodies were used at 1:5000 dilution. The specificity of antibodies against ␣-, ␤-, and ␥-rENaC has previously been demonstrated by the displacement of the immunoprecipitated product with ␣-, ␤-, or ␥-rENaC fusion proteins, respectively (14). The anti-rabbit IgG secondary antibody (Amersham Biosciences) was used at dilution 1:5000, and the signal was developed with the ECL Plus system (Amersham Biosciences).…”

Renal Collecting Duct Epithelial Cells React to Pyelonephritis-Associated Escherichia coli by Activating Distinct TLR4-Dependent and -Independent Inflammatory Pathways

“…3C). They also expressed mRNAs for TLR2 and for the ENaC ␣-subunit and CFTR Cl Ϫ channel, both of which were expressed in principal and intercalated cells (39,40) (Fig. 3C).…”

Increased Apical Targeting of Renal Epithelial Sodium Channel Subunits and Decreased Expression of Type 2 11β-Hydroxysteroid Dehydrogenase in Rats with CCl4-Induced Decompensated Liver Cirrhosis

“…This suggests that hyperaldosteronism is of major importance in the pathogenesis of sodium retention. The epithelial sodium channel (ENaC) is the major sodium transport pathway in the collecting duct (12), and both protein abundance and apical plasma membrane targeting of the ENaC subunits are regulated by hormonal stimulation, e.g., aldosterone (13) and vasopressin (14). We therefore speculated that altered protein abundance and/or apical membrane targeting of ENaC subunits (␣, ␤, and ␥) may account for the increased sodium reabsorption in the collecting duct and sodium retention in liver cirrhosis.…”