Sodium transport is modulated by p38 kinase-dependent cross-talk between ENaC and Na,K-ATPase in collecting duct principal cells

Abstract

In relation to dietary Na(+) intake and aldosterone levels, collecting duct principal cells are exposed to large variations in Na(+) transport. In these cells, Na(+) crosses the apical membrane via epithelial Na(+) channels (ENaC) and is extruded into the interstitium by Na,K-ATPase. The activity of ENaC and Na,K-ATPase must be highly coordinated to accommodate variations in Na(+) transport and minimize fluctuations in intracellular Na(+) concentration. We hypothesized that, independent of hormonal stimulus, cross-talk between ENaC and Na,K-ATPase coordinates Na(+) transport across apical and basolateral membranes. By varying Na(+) intake in aldosterone-clamped rats and overexpressing γ-ENaC or modulating apical Na(+) availability in cultured mouse collecting duct cells, enhanced apical Na(+) entry invariably led to increased basolateral Na,K-ATPase expression and activity. In cultured collecting duct cells, enhanced apical Na(+) entry increased the basolateral cell surface expression of Na,K-ATPase by inhibiting p38 kinase-mediated endocytosis of Na,K-ATPase. Our results reveal a new role for p38 kinase in mediating cross-talk between apical Na(+) entry via ENaC and its basolateral exit via Na,K-ATPase, which may allow principal cells to maintain intracellular Na(+) concentrations within narrow limits.

Figure 1.

Enhanced apical Na⁺ entry increases Na,K-ATPase activity and expression in isolated rat CCDs. (A) Rats infused with aldosterone for 3 days on a low-Na⁺ diet were subjected to a low-Na⁺ (L) or normal-Na⁺ (N) diet for an additional 4 days. (B) Na,K-ATPase activity measured in isolated rat CCDs. (C) Representative immunoblot showing the Na,K-ATPase α-subunit expression in isolated rat CCDs. α-Tubulin was used as a loading control. (D) Relative densitometric quantification of Na,K-ATPase from immunoblots shown in C. Results are means ± SEM of five independent experiments; *P<0.05.

Figure 2.

Enhanced apical Na⁺ entry increases total and cell surface Na,K-ATPase expression in cultured mouse CD cells. (A–C) γ-ENaC-TetOn-mCCD cells were grown to confluence on filters and treated or not treated with 1.25 µg/ml Dox for 48 hours. (A) Representative immunoblot showing the effect of Dox treatment on total and cell surface expression of γ-ENaC and Na,K-ATPase. GAPDH and E-cadherin were used as loading controls for total and cell-surface proteins, respectively. (B) Relative densitometric quantification of Na,K-ATPase from immunoblots shown in A. (C) Effect of Dox treatment on ouabain-sensitive (ouabain-S; 5×10⁻⁵ M) and benzamil-sensitive (Benza-S; 10⁻⁶ M) transepithelial currents. (D–F) mpkCCD_cl4 cells were grown to confluence on filters and then incubated with apical medium containing low (30 mM) or high (150 mM) Na⁺ for 24 hours. (D) Benzamil-sensitive transepithelial current was increased by 150 mM apical Na⁺ compared with that of 30 mM apical Na⁺. (E) Representative immunoblots showing the effect of increased apical Na⁺ availability on cell surface and total Na,K-ATPase α-subunit expression. GAPDH was used as a loading control. (F) Densitometric quantification of cell surface and total Na,K-ATPase α-subunit expression shown in B. Results are means ± SEM of six independent experiments; *P<0.05, **P<0.01.

Figure 3.

Lysosomal degradation of Na,K-ATPase is involved in cross-talk between ENaC and Na,K-ATPase. (A) Representative immunoblot showing the effect of chloroquine (Chloro, 10⁻⁵ M, 24 hours) on the Na,K-ATPase α-subunit expression in γ-ENaC-TetOn-mCCD cells pretreated or not pretreated with Dox for 24 hours. GAPDH was used as a loading control. (B) Densitometric quantification of immunoblots shown in A. (C) Rat kidney cortices were processed and the Na,K-ATPase α-subunit was detected as described in Concise Methods. Na,K-ATPase labeling was enriched in a distinct population of intracellular organelles in addition to its distribution along the basolateral membrane in rat CCD principal cells. The CCD lumen is denoted as “L.” Two different magnifications are shown. The inset shows a high magnification of a labeled intracellular organelle from a neighboring principal cell. Black arrows show plasma membrane and white arrows show intracellular membranes. (D) Colocalization of Na,K-ATPase and LysoTracker Red in γ-ENaC-TetOn-mCCD cells grown on transparent filters in the presence or absence of chloroquine. Results are means ± SEM of five independent experiments; **P<0.01.

Figure 4.

Enhanced apical Na⁺ entry inhibits cell surface Na,K-ATPase endocytosis and degradation. (A) Confluent γ-ENaC-TetOn-mCCD cells grown on filters were pretreated or not pretreated for 48 hours with Dox before biotinylation of cell surface proteins at 4°C. Biotinylated cell surface proteins were then chased for 2, 6, and 12 hours at 37°C in the presence or absence of chloroquine (Chloro, 10⁻⁵ M). A representative immunoblot showing the effects of Dox and/or chloroquine on cell-surface expression of the Na,K-ATPase α-subunit is shown. E-cadherin was used as a loading control for cell-surface proteins. (B) Nonlinear regression analysis of the Na,K-ATPase α-subunit and E-cadherin internalization rate. The Na,K-ATPase α-subunit and E-cadherin abundance was estimated by densitometric quantification of immunoblots shown in A. Results were expressed as a percentage of densitometric values measured before chase at 37°C (0 hours). (C) Densitometric quantification of the Na,K-ATPase α-subunit abundance after a 12-hour chase at 37°C in the presence and absence of Dox and/or chloroquine. Results were expressed as in B. Results are means ± SEM of seven independent experiments; *P<0.05.

Figure 5.

AMPK is not involved in Na,K-ATPase endocytosis that mediates cross-talk between ENaC and Na,K-ATPase. (A) Representative immunoblots showing the effect of enhanced apical Na⁺ entry on total and phosphorylated AMPK and its downstream target, ACC. GAPDH was used as a loading control. (B) Densitometric quantification of phosphorylated AMPK from immunoblots shown in A. Results are means ± SEM of six independent experiments; *P<0.05. (C and D) Confluent γ-ENaC-TetOn-mCCD cells grown on filters were pretreated or not pretreated with Dox for 48 hours before biotinylation of cell surface proteins. Biotinylated cell surface proteins were then chased for 12 hours at 37°C in the presence or absence of AICAR (1 mM), an activator of AMPK. E-cadherin was used as a loading control.

Figure 6.

Activation of p38 kinase prevents the effect of enhanced apical Na⁺ entry on Na,K-ATPase endocytosis in cross-talk between ENaC and Na,K-ATPase. (A) Representative immunoblots showing the effect of enhanced apical Na⁺ entry on total and phosphorylated p38 kinase and its downstream target, ATF-2. GAPDH was used as a loading control. (B) Densitometric quantification of phosphorylated p38 kinase from immunoblots shown in part A. Results are means ± SEM of seven independent experiments; *P<0.05. (C) Confluent γ-ENaC-TetOn-mCCD cells grown on filters were pretreated or not pretreated with Dox for 48 hours before biotinylation of cell surface proteins. Biotinylated cell surface proteins were then chased for 12 hours at 37°C in the presence or absence of anisomycin (Aniso, 1 µM), an activator of p38 kinase. E-cadherin was used as a loading control. (D) Densitometric quantification of the Na,K-ATPase α-subunit abundance from immunoblots shown in B. The Na,K-ATPase α-subunit abundance after 12-hour chase at 37°C was expressed as a percentage of densitometric values measured before chase (0 hours). Results are means ± SEM of five independent experiments; *P<0.05.

Figure 7.

Inhibition of p38 kinase mimics the effect of enhanced apical Na⁺ entry on Na,K-ATPase endocytosis in cross-talk between ENaC and Na,K-ATPase. (A and C) Confluent γ-ENaC-TetOn-mCCD cells grown on filters were pretreated or not pretreated with Dox for 48 hours before biotinylation of cell surface proteins. Biotinylated cell surface proteins were then chased for 12 hours at 37°C in the presence or absence of PD169316 (PD, 2 µM) or SB203580 (SB, 10 µM), two specific inhibitors of p38 kinase. E-cadherin was used as a loading control. (B and D) Densitometric quantification of the Na,K-ATPase α-subunit abundance from immunoblots shown in A and C, respectively. The Na,K-ATPase α-subunit abundance after 12-hour chase was expressed as a percentage of densitometric values measured before chase (0 hours). Results are means ± SEM of six independent experiments; *P<0.05. Ctl, control.