Aldosterone acutely stimulates NCC activity via a SPAK-mediated pathway

Abstract

Hypertension is a leading cause of morbidity and mortality worldwide, and disordered sodium balance has long been implicated in its pathogenesis. Aldosterone is perhaps the key regulator of sodium balance and thus blood pressure. The sodium chloride cotransporter (NCC) in the distal convoluted tubule of the kidney is a major site of sodium reabsorption and plays a key role in blood pressure regulation. Chronic exposure to aldosterone increases NCC protein expression and function. However, more acute effects of aldosterone on NCC are unknown. In our salt-abundant modern society where chronic salt deprivation is rare, understanding the acute effects of aldosterone is critical. Here, we examined the acute effects (12-36 h) of aldosterone on NCC in the rodent kidney and in a mouse distal convoluted tubule cell line. Studies demonstrated that aldosterone acutely stimulated NCC activity and phosphorylation without affecting total NCC abundance or surface expression. This effect was dependent upon the presence of the mineralocorticoid receptor and serum- and glucocorticoid-regulated kinase 1 (SGK1). Furthermore, STE20/SPS-1-related proline/alanine-rich kinase (SPAK) phosphorylation also increased, and gene silencing of SPAK eliminated the effect of aldosterone on NCC activity. Aldosterone administration via a minipump in adrenalectomized rodents confirmed an increase in NCC phosphorylation without a change in NCC total protein. These data indicate that acute aldosterone-induced SPAK-dependent phosphorylation of NCC increases individual transporter activity.

Keywords: NCC; SPAK; WNK4; aldosterone.

Fig. 1.

Effect of aldosterone on sodium chloride cotransporter (NCC) activity. A: mDCT15 cells were treated with 100 nM aldosterone for the indicated times before incubating in uptake medium with ²²Na⁺ and either vehicle or 1 mM metolazone. Radioactive uptake was determined as in materials and methods. Thiazide-sensitive uptake was calculated as the difference in radiotracer uptake between the metolazone-containing and the metolazone-free groups. *P < 0.05; n = 6. B: mDCT15 cells were incubated in aldosterone at the indicated concentrations for 24 h before determination of radiotracer uptake as outlined above. *P < 0.05; n = 4.

Fig. 2.

Effect of mineralocorticoid receptor (MR) antagonism on aldosterone-induced NCC activity. A: mDCT15 cells were lysed and immunoblotted for MR. Kidney cortex is shown as a positive control. A representative blot is shown. B: mDCT15 cells were treated with 100 nM aldosterone (A) for 24 h in the presence or absence of the MR antagonist spironolactone (S) or the GR antagonist RU-486 (R). *P < 0.05; n = 4. C: mDCT15 cells were treated with 100 nM aldosterone (A) or vehicle [V(A)] for 24 h in the presence or absence of the SGK1 antagonist GSK 650394 (I) or vehicle [V(I)]. *P < 0.05; n = 4. #P < 0.05.

Fig. 3.

Effect of aldosterone on NCC surface expression and total abundance. mDCT15 cells were grown to confluence, then treated with 100 nM aldosterone for 24 h before labeling with biotin at 4°C. A: biotinylated (cell surface) proteins were recovered using streptavidin-agarose and immunoblotted for NCC. Representative blot is shown with accompanying densitometry (C = control 0 h, 24C = control 24 h, 24A = aldo 24 h). B: total cell lysate as in A immunoblotted for NCC. Representative blot is shown with accompanying densitometry.

Fig. 4.

Time course of aldosterone's effects on NCC surface expression, total abundance, and NCC phosphorylation. mDCT15 cells were grown to confluence, then treated with 100 nM aldosterone for the indicated times. A: cells were labeled with biotin at 4°C. Biotinylated (cell surface) proteins were recovered using streptavidin-agarose and immunoblotted for NCC. Representative blot is shown with accompanying densitometry (C = control 0 h, 12C = control 12 h, 12A = aldo 12 h, 24C = control 24 h, 24A = aldo 24 h, 36C = control 36 h, 36A = aldo 36 h). B: cell lysate was then immunoblotted for NCC, phospho-NCC (NCC-T53), and actin. Representative immunoblot and densitometry are shown. *P < 0.05; n = 6.

Fig. 5.

Effect of aldosterone on NCC phosphorylation in adrenalectomized rats. Adrenalectomized rats were treated with aldosterone infusion by minipump for 24 h. The resultant kidney lysate was then immunoblotted for NCC, phospho-NCC (NCC-T53), and actin. A: representative immunoblot. B: densitometry of T53-NCC. P < 0.01; n = 10.

Fig. 6.

Effect of aldosterone on STE20/SPS-1-related proline/alanine-rich kinase (SPAK) and oxidative stress-responsive 1 (OSR1) phosphorylation. mDCT15 cells were grown to confluence, then treated with 100 nM aldosterone (A) for 24 h before lysis and immunoblotting for phospho-SPAK/OSR1 (SPAK 373 and OSR325), total SPAK (SPAK), and total OSR1 (OSR1). A: representative immunoblot. B: densitometry for SPAK373. P < 0.05; n = 4. C: densitometry for OSR325. *P < 0.05; n = 4.

Fig. 7.

Effect of SPAK on aldosterone-induced NCC activity. A: mDCT15 cells were infected with short hairpin (sh) RNA specific for SPAK with gene knockdown confirmed via immunoblotting for SPAK (densitometry with representative immunoblot for SPAK and OSR1 shown as inset). C = nontargeting control, shSK = shRNA specific for SPAK. B: mDCT15 or shSPAK-infected cells were treated with either vehicle (mDCT+V, shSK+V) or 100 nM aldosterone (mDCT+A, shSK+A) for 24 h before radioactive uptake determination as in materials and methods. Thiazide-sensitive uptake was calculated as the difference in radiotracer uptake between the metolazone-containing and the metolazone-free groups. *P < 0.01; n = 4.