Dissociation of sodium-chloride cotransporter expression and blood pressure during chronic high dietary potassium supplementation

Abstract

Dietary potassium (K+) supplementation is associated with a lowering effect in blood pressure (BP), but not all studies agree. Here, we examined the effects of short- and long-term K+ supplementation on BP in mice, whether differences depend on the accompanying anion or the sodium (Na+) intake and molecular alterations in the kidney that may underlie BP changes. Relative to the control diet, BP was higher in mice fed a high NaCl (1.57% Na+) diet for 7 weeks or fed a K+-free diet for 2 weeks. BP was highest on a K+-free/high NaCl diet. Commensurate with increased abundance and phosphorylation of the thiazide sensitive sodium-chloride-cotransporter (NCC) on the K+-free/high NaCl diet, BP returned to normal with thiazides. Three weeks of a high K+ diet (5% K+) increased BP (predominantly during the night) independently of dietary Na+ or anion intake. Conversely, 4 days of KCl feeding reduced BP. Both feeding periods resulted in lower NCC levels but in increased levels of cleaved (active) α and γ subunits of the epithelial Na+ channel ENaC. The elevated BP after chronic K+ feeding was reduced by amiloride but not thiazide. Our results suggest that dietary K+ has an optimal threshold where it may be most effective for cardiovascular health.

Keywords: Epithelial transport of ions and water; Hypertension; Nephrology; Sodium channels.

Figure 1. Project outline.

In the initial cohort (cohort 1), mice were subjected to a chronic dietary regime with blood pressure determined at each dietary stage. Diet key for all figures: NS, normal NaCl (0.3% Na⁺); HS, high NaCl (1.57% Na⁺); NK, normal KCl (1.05% K⁺); +KCl, high KCl (5.25% K⁺); +KCit, high K citrate (5.25% K⁺); 0K, zero K⁺ diet. HCTZ, hydrochlorothiazide. In cohort 2, mice were subjected to either short-term or chronic dietary regime, with blood pressure determined at each period.

Figure 2. Low dietary K⁺ intake increases BP.

Systolic blood pressure (SBP) was recorded by in vivo radio telemetry from individual free-roaming animals. (A) Comparison of fits analysis of curves showed a significant difference (P < 0.0001) across a 24-hour period (indicating significantly higher SBP) between animals fed high-NaCl (HS/NK) diet compared with animals fed control NaCl (NS/NK) diet. A significant difference (P < 0.0001) in curves was also observed after 2-week feeding of a K⁺-deplete diet (NS/0K), with a high-NaCl, K⁺-deplete diet (HS/0K) having the largest difference in SBP relative to control (NS/NK) diet (P < 0.0001). Data are shown as mean ± SEM, n = 5–6 per dietary condition. Dark/light times are shown by lower bar strip. ZT hour 0 = 18:00 hours. (B–D) Quantification of the MESOR value of SBP (representing the SBP averaged across a 24-hour period), 12-hour SBP when animals are in darkness (active phase), and 12-hour light phase. Data are shown as mean ± SEM with individual values shown. Data was analyzed using 2-way ANOVA with Tukey’s multiple-comparison testing. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3. NCC is increased following low dietary K⁺ feeding and is responsible for the increase in BP.

Lysates of total kidney from animals fed control diet (NS/NK) or K⁺-deplete diet (NS/0K) for 2 weeks were assessed by western blotting. (A) Representative immunoblots of total NCC, phosphorylated NCC (pNCC), or proteasome 20s (P20s, loading control). Molecular weight (KDa) is shown on the right. (B and C) NCC and pNCC are significantly increased by the NS/0K diet. (D) Ratio of pNCC/NCC is not significantly different between NS/0K and NS/NK-fed animals. Data are shown as mean ± SEM with individual values shown. ***P < 0.001 by 2-tailed t test. (E) Comparison of fits analysis of curves showed a significant difference (P < 0.0001) in SBP across a 24-hour period between animals fed a high-NaCl, K⁺-deficient (HS/0K) diet compared with animals fed high-NaCl, normal K⁺ (HS/NK) diet. Over a 24-hour period, curves were significantly different (P < 0.0001) between hydrochlorothiazide-treated (HCTZ-treated) animals on a HS/0K diet compared with HS/0K diet alone. Dark/light times are shown by lower bar strip. Time 0 = 18:00 hours. Data are shown as mean ± SEM, n = 5–6 per condition. (F) HCTZ significantly reduces SBP averaged over 8 hours following injection. Data are shown as mean ± SEM with individual values shown. *P < 0.05; ***P < 0.001 by 1-way ANOVA with Dunnett’s multiple-comparison test.

Figure 4. Chronic high dietary K⁺ feeding elevates BP.

Systolic blood pressure (SBP) was recorded by in vivo telemetry from individual free roaming animals after 3 weeks of dietary intervention. (A) Comparison of fits analysis of curves showed a significant difference across a 24-hour period between animals fed a control diet (NS/NK) compared with animals fed a high-KCl (NS/+KCl, P < 0.0001) or high-KCit (NS/+KCit, P < 0.0001) diet. Dark/light times are shown by lower bar strip. ZT hour 0 = 18:00 hours. Data are shown as mean ± SEM, n = 5–11 per condition. (B) Quantification of the average SBP across a 24-hour period, or stratified by dark/light period. Data are shown as mean ± SEM with individual values shown. Data was analyzed using 2-way ANOVA with Tukey’s multiple-comparison testing. *P < 0.05; **P < 0.01. (C) Comparison of fits analysis of curves showed a significant difference across a 24-hour period between animals fed HS/NK diet compared with animals fed a HS/+KCl (P < 0.0001) or HS/+KCit (P < 0.0001) diets. Dark/light times are shown by lower bar strip. Time 0 = 18:00 hours. Data are shown as mean ± SEM, n = 3–6 per condition. (D) Average SBP across a 24-hour period, or stratified by dark/light period. Data are shown as mean ± SEM with individual values shown. Data were analyzed using 2-way ANOVA with Tukey’s multiple-comparison testing. **P < 0.01.

Figure 5. Chronic high K⁺ feeding reduces NCC.

Western blot analysis of kidney lysates from animals fed various diets for 3 weeks. (A) Representative immunoblots of NCC, phosphorylated NCC (pNCC), and proteasome 20s (P20s, loading control) following normal salt diets. Molecular weight (KDa) of markers is shown on the right. (B and C) NCC and pNCC are significantly decreased by chronic K⁺ supplementation. (D) Ratio of pNCC/NCC is not significantly affected by K⁺ supplementation under NS diet. (E) Representative immunoblots of total NCC, phosphorylated NCC (pNCC), and proteasome 20s (P20s, loading control) following high-Na⁺ diets. (F) NCC is significantly decreased by chronic K⁺ supplementation. (G and H) pNCC and ratio of pNCC/NCC are not significantly changed by chronic K⁺ supplementation. (I) The higher SBP on a NS/+KCl diet is not significantly altered by hydrochlorothiazide (HCTZ) treatment over a 24-hour period. Dark/light times are shown by lower bar strip. ZT Time 0 = 18:00 hours. Data are shown as mean ± SEM, n = 6 per condition. (J) The higher SBP on a NS/+KCl diet is not significantly altered by HCTZ in the initial 8 hours following injection. For graphs, all data are shown as mean ± SEM with individual values shown. Data in B–D and F–H were analyzed by 1-way ANOVA followed by Dunnett’s multiple-comparison test. Data in J were assessed by 2-tailed t test, with the level of significance set as 0.033 to correct for the FDR (76). *P < 0.05; **P < 0.01; ***P < 0.001; ***P < 0.0001.

Figure 6. Increased ENaC expression plays a role in the increased BP following chronic high K⁺ feeding.

Lysates of total kidney from animals fed the various diets were assessed by Western blotting. (A) Representative immunoblots of uncleaved and cleaved (active) αENaC. Molecular weight (KDa) is shown on the right, and the arrowhead represents the specific αENaC band. (B and C) Total αENaC (uncleaved + cleaved) cleaved αENaC were significantly increased by 3 weeks K⁺ supplementation on both a normal or high NaCl intake (NS, HS) and were significantly decreased following 2 weeks K⁺ depleted diet (0K). Data were analyzed using a 1-way ANOVA followed by Dunnett’s multiple-comparison test. *P < 0.05; **P < 0.01; ***P < 0.001. (D) mRNA levels for Scnn1a (encoding αENaC) is significantly higher in whole-kidney samples collected when animals are in dark phase (01:00 hours) compared with when tissue was collected in the light phase (13:00 hours). Data were analyzed by 2-tailed t test. (E) Amiloride significantly reduces SBP over the 8-hour period after injection for animals fed a NS diet supplemented with high KCl (NS/+KCl) for 3 weeks. Data are shown as mean ± SEM with individual values shown (n = 4–6). Data were analyzed using a 1-way ANOVA followed by Dunnett’s multiple-comparison test. *P < 0.05; **P < 0.01. All data are shown as mean ± SEM, with individual values shown.

Figure 7. Short-term and chronic KCl feeding have opposing effects on BP.

(A and B) Short-term (4 days) high KCl feeding combined with a normal NaCl diet (NS/+KCl) significantly reduces SBP recorded from implanted telemetry probes across 24 hours (n = 4/diet) (A) and over 24 hours as defined by MESOR or during the 12 hours dark or light phase (B), compared with control diet (NS/NK). The magnitude of error from the mean may be less than the smallest size of symbol available.(C and D) SBP and DBP recorded using tail cuff plethysmography during the dark phase are significantly lower during short-term (4 days) high KCl feeding but significantly higher during chronic KCl feeding (3 weeks) compared with control-fed animals (NS/NK). Chronic 0K feeding (2 weeks) significantly increases SBP and DBP compared with NS/NK-fed animals. (E) A single of dose of hydrochlorothiazide (HCTZ) 4 hours before tail cuff recording significantly reduced SBP of NS/NK-fed animals, but not short-term NS/+KCl-fed animals; it had no significant effect on elevated SBP in chronically fed NS/+KCl animals. Data were analyzed by 2-tailed t test between 2 groups (B), using a 1-way ANOVA followed by Dunnett’s multiple-comparison test (3 groups, C and D), or by 2-way ANOVA followed by Tukey’s multiple-comparison testing (E). *P < 0.05; **P < 0.01; ****P < 0.0001. All data are shown as mean ± SEM, with individual values shown.

Figure 8. Potassium and aldosterone are elevated in high-KCl–fed animals.

(A) Plasma [aldosterone] is increased following short-term and chronic KCl feeding. Chronic 0K feeding (2 weeks) significantly reduced [aldosterone]. HS diet reduced [aldosterone] across all groups. Only 2 HS/0K-fed animals had a detectable level of aldosterone, so this condition was not analyzed. Data were analyzed as 2-way ANOVA with Dunnett’s multiple-comparison testing. (B) Urine aldosterone concentrations under the different chronic dietary K⁺ conditions. Data were analyzed by a 2-way ANOVA main effects model, with multiple comparisons with NS/NK group. (C) Urine aldosterone excretion was significantly higher during the 12-hour dark phase (18:00–06:00) compared with the light phase, with the magnitude of increase clearly being greater for high-KCl–fed (+KCl-fed) animals. No clear difference was detected between short-term (4 days) and chronically (3 week) fed animals. Analysis within groups by 2-tailed t test. (D) Plasma [K⁺] under the different short-term or chronic dietary K⁺ conditions. Data are shown as mean ± SEM, with individual values shown. Data were analyzed as 2-way ANOVA with Dunnett’s multiple-comparison testing. (E) Urine aldosterone concentration significantly positively correlates with plasma K⁺ concentration. Best fit analysis, linear regression, with 95% CI limits (dotted lines) displayed. Data are shown as mean ± SEM. (F) The relationship between SBP against plasma [K⁺] fits a second order quadratic equation. Data are from individuals where BP and blood sampling are made from same animal. All individual values considered for best-fit nonlinear regression.*P < 0.05; **P < 0.01; ***P < 0.001. ^##P < 0.01, ^###P < 0.001 versus respective chronic NS condition.

Figure 9. High KCl feeding reduces NCC and increases ENaC.

Lysates of total kidney from animals fed the various diets were assessed by Western blotting. Representative panels (samples run on the same gel but noncontiguous) from representative blots are shown above quantification graphs. (A and B) pNCC (A) and NCC (B) were significantly decreased after short-term and chronic KCl feeding but were increased after chronic K⁺ depletion (0K, 2 weeks). (A and B) Total αENaC (C) and cleaved αENaC (D) were increased following +KCl feeding, but the magnitude of increase in cleaved αENaC after chronic feeding is significantly greater relative to short-term feeding. (E) Total γENaC (full length plus cleaved protein bands) was significantly decreased after short-term and chronic KCl feeding, whereas cleaved γENaC was significantly increased. Data from all individuals plotted with mean ± SEM. Analysis by 1-way ANOVA followed by Tukeys multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 10. Correlation of NCC and ENaC to urine aldosterone and plasma potassium level.

(A–C) Best fit analysis for short-term and chronically fed animals. (A) pNCC significantly negatively correlates with urine [aldosterone]. The correlation for short-term feeding (5 days, R² = 0.839) and chronically fed animals (3 weeks, R² = 0.110) did not significantly differ for slope or y intercept. (B) Cleaved αENaC significantly positively correlates with urine [aldo]. Correlation for short-term feeding (R² = 0.652) and chronic feeding (R² = 0.258) tended to differ for slope (P = 0.0546) with a significantly different y intercept (P = 0.0469). (C) Cleaved γENaC significantly positively correlates with urine [aldo]. The correlation for short-term feeding (R² = 0.669) and chronic feeding (R² = 0.516) significantly differed (P < 0.01). (D–I) Best fit analysis with 95% CI (dotted lines) for whole data set. (D and E) NCC and pNCC significantly negatively correlate with plasma [K⁺]. (F and G)Total αENaC and cleaved αENaC significantly positively correlate with plasma [K⁺]. (H and I) Total γENaC significantly negatively correlates with plasma [K⁺], while cleaved γENaC significantly positively correlates. Each dot represents protein abundance (arbitrary units), and urine analysis from the same animal. Data are from all animals in cohort 1 and 2 combined, with the exception of γENaC data, which are only from cohort 2.

Figure 11. High KCl feeding promotes kidney remodeling.

(A and B) After chronic NS/+KCl feeding, parvalbumin (A) was significantly reduced, whereas calbindin D28 (B) was significantly increased after both short-term and chronic NS/+KCl feeding. (C) The H⁺-ATPase B1 subunit (HATPase) was significantly reduced following chronic K⁺ depletion (0K, 2 weeks). Representative panels (samples run on the same gel but noncontiguous) from representative blots are shown above quantification graphs. (D) Representative images of kidney sections labelled with proliferating cell nuclear antigen (PCNA, green) alongside NCC (red). White arrowheads highlight PCNA positive nuclei. Boxed area highlights a DCT that is expanded in the right panel. Scale bar: 50 μm. (E) Quantification of PCNA staining from imaged whole kidney and from DCT cells. (F) A significantly increased number of PCNA⁺ cells are observed in whole kidney and the DCT after 4 days of NS/+KCl feeding. Data were analyzed using a 1-way ANOVA followed by Dunnett’s multiple-comparison test. *P < 0.05; **P < 0.01. Data are shown as mean ± SEM.

Figure 12. Large-scale proteomic profiling of short-term and long-term effects of high KCl intake in kidney cortex.

Kidney cortex homogenates were examined using LC-MS/MS–based quantitative proteomics. (A) Volcano plot of the protein quantification where the primary axis shows the log₂ (mean peptide abundance ratio), while the secondary axis designates the −log₁₀(P value). After short-term feeding of NS/+KCl, 105 proteins were significantly decreased relative to NS/NK diet and 147 were significantly increased. After chronic feeding, 163 proteins were significantly reduced and 479 significantly increased in abundance. (B) IPA of the significantly changed proteins (equivalent human gene name shown) highlighted that many more proteins associated to the transport of ions were changed in abundance during chronic NS/+KCl feeding.

Figure 13. Proteomic profiling identifies proteins differentially regulated following short-term or chronic KCl feeding.

(A) Each dot represents a protein significantly altered in abundance in at least 1 of the dietary conditions. Pearson’s correlation coefficient for all proteins, R = 0.4. Proteins with correlating changes in abundance are shown by blue dots, proteins increased in abundance during short-term but decreased during chronic feeding are represented by red dots, and proteins decreased in abundance during short-term but increased during chronic feeding are represented by green dots. Proteins of interest (corresponding mouse gene name shown) are highlighted. (B) Proteins that were significantly altered in abundance after short-term and chronic NS/+KCl feeding were mapped to various nephrotoxicity pathways using IPA. (C) After chronic NS/+KCl feeding, a greater number of proteins (corresponding human gene name shown) associated to kidney damage were observed.