Modulation of NCC activity by low and high K(+) intake: insights into the signaling pathways involved

Abstract

Modulation of Na(+)-Cl(-) cotransporter (NCC) activity is essential to adjust K(+) excretion in the face of changes in dietary K(+) intake. We used previously characterized genetic mouse models to assess the role of Ste20-related proline-alanine-rich kinase (SPAK) and with-no-lysine kinase (WNK)4 in the modulation of NCC by K(+) diets. SPAK knockin and WNK4 knockout mice were placed on normal-, low-, or high-K(+)-citrate diets for 4 days. The low-K(+) diet decreased and high-K(+) diet increased plasma aldosterone levels, but both diets were associated with increased phosphorylation of NCC (phospho-NCC, Thr(44)/Thr(48)/Thr(53)) and phosphorylation of SPAK/oxidative stress responsive kinase 1 (phospho-SPAK/OSR1, Ser(383)/Ser(325)). The effect of the low-K(+) diet on SPAK phosphorylation persisted in WNK4 knockout and SPAK knockin mice, whereas the effects of ANG II on NCC and SPAK were lost in both mouse colonies. This suggests that for NCC activation by ANG II, integrity of the WNK4/SPAK pathway is required, whereas for the low-K(+) diet, SPAK phosphorylation occurred despite the absence of WNK4, suggesting the involvement of another WNK (WNK1 or WNK3). Additionally, because NCC activation also occurred in SPAK knockin mice, it is possible that loss of SPAK was compensated by OSR1. The positive effect of the high-K(+) diet was observed when the accompanying anion was citrate, whereas the high-KCl diet reduced NCC phosphorylation. However, the effect of the high-K(+)-citrate diet was aldosterone dependent, and neither metabolic alkalosis induced by bicarbonate, nor citrate administration in the absence of K(+) increased NCC phosphorylation, suggesting that it was not due to citrate-induced metabolic alkalosis. Thus, the accompanying anion might modulate the NCC response to the high-K(+) diet.

Keywords: Ste20-related proline-alanine-rich kinase; aldosterone; distal convoluted tubule; with-no-lysine kinase 4.

Fig. 1.

Effects of varying dietary K⁺ on Na⁺-Cl⁻ cotransporter (NCC) expression and phosphorylation. A: specificity of the NCC phospho-antibody used in this work. The antibody recognizes three phosphorylated residues in NCC (Thr⁴⁴/Thr⁴⁸/Thr⁵³) [phosphorylated (p)NCC-3P]. Samples from wild-type (NCC^+/+) and NCC-deficient (NCC^−/−) mice kept on normal- or low-NaCl diets were analyzed by Western blot analysis. No signal was detected in lanes loaded with NCC^−/− samples, and signal intensity was greatly reduced when samples were treated with λ-phosphatase (B). The low signal observed in the phosphatase-treated sample was due to incomplete dephosphorylation. Nonphosphorylated peptide was included in the antibody solution for all blots against phosphorylated epitopes. C: representative Western blot analysis of total kidney protein samples of wild-type mice maintained on diets with normal, low, or high K⁺-citrate content. The solid band observed above the 130-kDa marker band in the NCC blots is a nonspecific band that was not included in the densitometric analyses. D: densitometric analyses were performed on at least two blots per assay, including samples of 6, 8, and 8 mice for the normal-K⁺ (open bars), low-K⁺ (shaded bars), and high-K⁺ (solid bars) diet groups, respectively. The average value in the control group was fixed as 100%, and the effect of the diet was normalized accordingly. Results are expressed as mean percentages ± SE of the normal diet (100%). *P < 0.001 vs. the normal diet.

Fig. 2.

Effect of the high-KCl diet on the expression and phosphorylation of NCC. A: representative Western blot analysis of total kidney protein samples of wild-type mice maintained on diets with normal or high KCl content. B: densitometric analysis of blots shown in A. Results are expressed as mean percentages ± SE of the normal diet (100%). *P < 0.0001 vs. the normal diet.

Fig. 3.

Effect of HCO₃⁻ loading in expression and phosphorylation levels of NCC. A: urine pH of control mice (○) and HCO₃⁻-loaded mice (☐) on the previous day to the beginning of treatment (day 0) and on day 7 of high HCO₃⁻ intake. The mean urine pH for each group is indicated by the horizontal line. B: Western blot analysis of kidney samples from control and HCO₃⁻-loaded mice. C: densitometric analysis of the blot shown in B. No difference in NCC expression or phosphorylation was observed between groups.

Fig. 4.

Effect of high citrate intake on NCC phosphorylation. A: Western blot analysis of renal proteins of wild-type mice fed with a high-citrate diet. Citrate was administered in the drinking water. Both groups consumed a similar amount of water (5.16 ± 1.6 ml for the control group vs. 5 ± 1.4 ml for the citrate group). B: results of the densitometric analysis expressed as mean percentages ± SE of control (100%). No significant difference was observed. Three blots per assay were included in the analysis.

Fig. 5.

Effects of low-K⁺ diet on the expression and phosphorylation of Ste20-related proline-alanine-rich kinase (SPAK) and NCC in the renal cortex of with-no-lysine kinase 4 (WNK4)^+/+ and WNK4^−/− mice. A: Western blot analysis of renal cortex protein samples of WNK4^+/+ or WNK4^−/− mice kept on normal- or low-K⁺ diets. Representative blots are shown. Densitometric analyses were performed on at least two blots per assay, including samples of 6 mice/group. For NCC, pNCC, pSPAK2, and the kidney-specific form of pSPAK (pKS-SPAK), the results of these analyses are shown for WNK4^+/+ mice (B) and WNK4^−/− mice (C). For the total SPAK blot, the top lines show densitometric results for KS-SPAK bands. OSR1, oxidative stress response 1 kinase. Results are expressed as mean percentages ± SE of the normal diet (100%). *P < 0.05; #P < 0.005 vs. the normal diet.

Fig. 6.

Characterization of the SPAK Ser³⁸³/OSR1 Ser³²⁵ phospho-antibody. Proteins extracted from mouse kidneys or testes were subjected to Western blot analysis with SPAK- and OSR1-specific antibodies and with a SPAK/OSR1 pSer³⁷⁵/Ser³²⁵ (S-motif) antibody. Labels on the right indicate to which protein each band corresponds according to the size and, in the case of the pSPAK/OSR1 blot, according to comparison with the other blots. FL-SPAK, full-length SPAK.

Fig. 7.

Effects of low-K⁺ diet on the expression and phosphorylation of NCC in SPAK^T243A/T243A mice. A and B: Western blot analyses of total kidney protein samples of SPAK^+/+ [wild type (WT); A] and SPAK^T243A/T243A [knockin (KI); B] mice kept on normal- or low-K⁺ diets. Representative blots are shown. C and D: densitometric analyses of SPAK^+/+ (C) and SPAK^T243A/T243A (D) mice for NCC and pNCC were performed on at least two blots per assay, including samples of seven mice for the normal-K⁺ groups and six mice for the low-K⁺ groups. Results are expressed as mean percentages ± SE of the normal diet (100%). *P < 0.05; #P < 0.005 vs. the normal diet.

Fig. 8.

Effects of ANG II infusion on the expression and phosphorylation of SPAK and NCC in SPAK^T243A/T243A mice. A: Western blot analysis of NCC and pNCC in total kidney protein samples of wild-type SPAK and SPAK^T243A/T243A mice infused with vehicle or ANG II. B: densitometric analyses for NCC and pNCC were performed on at least two blots per assay, including samples of six mice for the wild-type groups and seven mice for the SPAK^T243A/T243A groups. C: Western blot analysis of pSPAK in total kidney protein samples of wild-type SPAK and SPAK^T243A/T243A mice infused with vehicle or ANG II. D: densitometric analysis of pKS-SPAK and pSPAK-2. *P < 0.05 vs. vehicle; #P < 0.05 vs. the wild-type group.

Fig. 9.

Effects of high-K⁺ diet on the expression and phosphorylation of SPAK and NCC in WNK4^−/− mice. A and B: Western blot analyses of total kidney protein samples of WNK4^+/+ (A) or WNK4^−/− mice (B) kept on normal- or high-K⁺ diets. Representative blots are shown. C and D: densitometric analyses were performed on at least two blots per assay, including samples from six different mice per group, and are shown for WNK4^+/+ mice (C) and WNK4^−/− mice (D). Results are expressed as mean percentages ± SE of the normal diet (100%). *P < 0.005; #P < 0.00005 vs. the normal diet.

Fig. 10.

Effects of high-K⁺ diet on NCC and SPAK phosphorylation are blocked by spironolactone. Wild-type mice were kept on normal or high-K⁺ diets. Ethanol-dissolved spironolactone or vehicle (ethanol) was added to the drinking water. The calculated dose was 40 mg·kg⁻¹·day⁻¹. A: Western blot analysis of total kidney protein samples of wild-type mice on normal- or high-K⁺ diets treated with spironolactone. Representative blots are shown. B: Densitometric analyses were performed on at least two blots per assay, including samples from five different mice per group. Results are expressed as mean percentages ± SE of the normal diet (100%). *P < 0.05 vs. the normal diet.