Pendrin abundance, subcellular distribution, and function are unaffected by either αENaC gene ablation or by increasing ENaC channel activity

Abstract

The intercalated cell Cl^-/HCO₃^- exchanger, pendrin, modulates ENaC subunit abundance and function. Whether ENaC modulates pendrin abundance and function is however unknown. Because αENaC mRNA has been detected in pendrin-positive intercalated cells, we hypothesized that ENaC, or more specifically the αENaC subunit, modulates intercalated cell function. The purpose of this study was therefore to determine if αENaC is expressed at the protein level in pendrin-positive intercalated cells and to determine if αENaC gene ablation or constitutively upregulating ENaC activity changes pendrin abundance, subcellular distribution, and/or function. We observed diffuse, cytoplasmic αENaC label in pendrin-positive intercalated cells from both mice and rats, with much lower label intensity in pendrin-negative, type A intercalated cells. However, while αENaC gene ablation within principal and intercalated cells of the CCD reduced Cl^- absorption, it did not change pendrin abundance or subcellular distribution in aldosterone-treated mice. Further experiments used a mouse model of Liddle's syndrome to explore the effect of increasing ENaC channel activity on pendrin abundance and function. The Liddle's variant did not increase either total or apical plasma membrane pendrin abundance in aldosterone-treated or in NaCl-restricted mice. Similarly, while the Liddle's mutation increased total Cl^- absorption in CCDs from aldosterone-treated mice, it did not significantly affect the change in Cl^- absorption seen with pendrin gene ablation. We conclude that in rats and mice, αENaC localizes to pendrin-positive ICs where its physiological role remains to be determined. While pendrin modulates ENaC abundance, subcellular distribution, and function, ENaC does not have a similar effect on pendrin.

Keywords: Aldosterone; ENaC; Intercalated cells; Pendrin.

Fig. 1

αENaC localizes to mouse intercalated cells. Consecutive sections of the kidneys from aldosterone-treated control (Scnn1a^loxlox) and collecting duct-specific αENaC KO mice (HoxB7; Scnn1α^loxloxcre) were co-immunostained for either αENaC and H⁺-ATPase or for pendrin and H⁺-ATPase. Squares (insets) show high magnifications of type A (IC_A) and type B (IC_B) intercalated cells in the collecting duct (CCD) and connecting tubule (CNT). Scale bars: 25 μm (overviews) and 10 μm (high magnifications). For simplicity, cells labeled as “IC_B” represent both type B and non-A, non-B intercalated cells

Fig. 2

αENaC localizes to mouse and rat intercalated cells. Consecutive kidney cryosections from wild type (WT) rats and mice were co-immunostained for either αENaC and H⁺-ATPase or for pendrin and the H⁺-ATPase. Squares (insets) show type A (IC_A) and type B (IC_B) intercalated cells in the collecting duct (CCD) and connecting tubule (CNT) at high magnification. D, Distal convoluted tubule. Scale bars: 25 μm (overviews) and 10 μm (high magnification)

Fig. 3

αENaC gene ablation does not change renal pendrin immunolabel intensity or subcellular distribution. Panel A shows pendrin immunolabel in a typical CCD and CNT from a male aldosterone-treated floxed αENaC and a male collecting duct-specific αENaC null mouse (HoxB7; Scnn1a^loxloxcre, Treatment #2). Panel B shows cytoplasmic area of pendrin positive cells, pendrin label in the most apical 10% of the cell relative to total label (redistribution ratio) and the label per cell within the cytoplasm of pendrin positive cells of the CCD from both groups (Treatment #2). P = NS, unpaired Student’s t-test

Fig. 4

Constitutively upregulating ENaC does not increase apical plasma membrane pendrin immunolabel in NaCl-restricted mice. A Pendrin gold immunolabel is shown in a representative type B and a non-A, non-B intercalated cell taken from a wild type and a Liddle’s mouse following 7 days of the NaCl-restricted diet (Treatment #3). B Gold immunolabel is shown at higher magnification in the regions marked by the boxes (insets) in A. Arrows show immunogold on the apical plasma membrane, while arrowheads show gold label in the subapical space

Fig. 5

Constitutively upregulating ENaC does not increase pendrin total protein abundance in NaCl-restricted mice. Shown are typical immunoblots and their respective pendrin band density of kidney lysates from NaCl-restricted male (A and B) and female wild type (WT) and Liddle’s (LL) mice (C and D, Treatment #3). *P < 0.05, unpaired Student’s t-test

Fig. 6

Constitutively upregulating ENaC does not increase pendrin protein abundance in kidneys from aldosterone-treated mice. Shown is an immunoblot (A) and its respective pendrin band density (B) of kidney lysates from aldosterone-treated male wild type (WT) and Liddle’s (LL) mice (Treatment #2). P = NS, unpaired Student’s t-test

Fig. 7

Constitutively increasing ENaC activity does not significantly increase pendrin-dependent Cl⁻ absorption in mouse CCD from aldosterone-treated mice. Cl⁻ absorption, J_Cl, was measured in CCDs taken from mice given aldosterone for 5–7 days (Treatment #2) that were homozygous Liddle’s and homozygous pendrin null (ENaC^LL; Slc26a4^−/− or LLKO, n = 7), homozygous for both Liddle’s and wild type pendrin (ENaC^LL; Slc26a4 ^+/+ or LL, n = 6), homozygous wild type ENaC and homozygous pendrin null (ENaC^WTWT; Slc26a4^−/− or KO, n = 11) and mice homozygous for wild ENaC and pendrin (ENaC^WTWT; Slc26a4^+/+ or WT, n = 4). *P < 0.05, by one-way ANOVA with a Holm-Sidak post-test