Deletion of renal Nedd4-2 abolishes the effect of high sodium intake (HS) on Kir4.1, ENaC, and NCC and causes hypokalemia during high HS

Abstract

Neural precursor cell expressed developmentally downregulated protein 4-2 (Nedd4-2) regulates the expression of Kir4.1, thiazide-sensitive NaCl cotransporter (NCC), and epithelial Na⁺ channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN), and Nedd4-2 deletion causes salt-sensitive hypertension. We now examined whether Nedd4-2 deletion compromises the effect of high-salt (HS) diet on Kir4.1, NCC, ENaC, and renal K⁺ excretion. Immunoblot analysis showed that HS diet decreased the expression of Kir4.1, Ca²⁺-activated large-conductance K⁺ channel subunit-α (BKα), ENaCβ, ENaCγ, total NCC, and phospho-NCC (at Thr⁵³) in floxed neural precursor cell expressed developmentally downregulated gene 4-like (Nedd4l^fl/fl) mice, whereas these effects were absent in kidney-specific Nedd4-2 knockout (Ks-Nedd4-2 KO) mice. Renal clearance experiments also demonstrated that Nedd4-2 deletion abolished the inhibitory effect of HS diet on hydrochlorothiazide-induced natriuresis. Patch-clamp experiments showed that neither HS diet nor low-salt diet had an effect on Kir4.1/Kir5.1 currents of the distal convoluted tubule in Nedd4-2-deficient mice, whereas we confirmed that HS diet inhibited and low-salt diet increased Kir4.1/Kir5.1 activity in Nedd4l^flox/flox mice. Nedd4-2 deletion increased ENaC currents in the ASDN, and this increase was more robust in the cortical collecting duct than in the distal convoluted tubule. Also, HS-induced inhibition of ENaC currents in the ASDN was absent in Nedd4-2-deficient mice. Renal clearance experiments showed that HS intake for 2 wk increased the basal level of renal K⁺ excretion and caused hypokalemia in Ks-Nedd4-2-KO mice but not in Nedd4l^flox/flox mice. In contrast, plasma Na⁺ concentrations were similar in Nedd4l^flox/flox and Ks-Nedd4-2 KO mice on HS diet. We conclude that Nedd4-2 plays an important role in mediating the inhibitory effect of HS diet on Kir4.1, ENaC, and NCC and is essential for maintaining normal renal K⁺ excretion and plasma K⁺ ranges during long-term HS diet.NEW & NOTEWORTHY The present study suggests that Nedd4-2 is involved in mediating the inhibitory effect of high salt (HS) diet on Kir4.1/kir5.1 in the distal convoluted tubule, NaCl cotransporter function, and epithelial Na⁺ channel activity and that Nedd4-2 plays an essential role in maintaining K⁺ homeostasis in response to a long-term HS diet. This suggests the possibility that HS intake could lead to hypokalemia in subjects lacking proper Nedd4-2 E3 ubiquitin ligase activity in aldosterone-sensitive distal nephron.

Keywords: Kir4.1/Kir5.1; collecting duct; distal convoluted tubule; renal K+ excretion.

Graphical abstract

Figure 1.

Deletion of neural precursor cell expressed developmentally downregulated protein 4-2 (Nedd4-2) abolishes the effect of dietary Na⁺ intake on the basolateral K⁺ channel in the distal convoluted tubule (DCT). A and B: representative single channel recordings showing basolateral K⁺ channel activity in the DCT of wild-type [WT; floxed neural precursor cell expressed developmentally downregulated gene 4-like (Nedd4l^fl/fl)] mice (A) and kidney-specific Nedd2-4 knockout (Ks-Nedd4-2 KO) mice (B) on normal-salt (NS), high-salt (HS), or low-salt (LS) diets for 14 days, respectively. C: scatterplot summarizing the results of experiments in which Kir4.1/Kir5.1 channel activity in the DCT [defined by channel activity (NP_o)] was measured in both male and female WT and Ks-Nedd4-2 KO mice on different Na⁺ diets for 14 days. The experiments were performed in cell-attached patches at a holding potential of 0 mV. The DCT was bathed in a solution containing 140 mM NaCl/5 mM KCl, and the pipette solution contained 145 mM K⁺. Significance was determined by two-way ANOVA.

Figure 2.

Deletion of neural precursor cell expressed developmentally downregulated protein 4-2 (Nedd4-2) eliminates the effect of dietary Na⁺ intake on the basolateral K⁺ conductance in the distal convoluted tubule (DCT). A: set of traces showing Ba²⁺-sensitive K⁺ currents measured with a step protocol from −60 to 60 mV in the DCT of wild-type [WT; floxed neural precursor cell expressed developmentally downregulated gene 4-like (Nedd4l^fl/fl)] mice and kidney-specific Nedd4-2 knockout (Ks-Nedd4-2 KO) mice on normal-salt (NS), high-salt (HS), and low-salt (LS) diets for 14 days, respectively. B: traces showing Ba²⁺-sensitive K⁺ currents measured with a ramp protocol (from −100 to 100 mV) in Ks-Nedd4-2 KO mice on NS, HS, and LS diets for 14 days, respectively. (A trace from a control mouse on NS is included as a reference.) The DCT was bathed in 140 mM KCl-containing solution, and the pipette solution also contained 140 mM KCl. C: scatterplot summarizing K⁺ currents measured at –60 mV in the DCT of male and female WT and KS-Nedd4-2 KO mice on HS for 7 days (HS-7d), HS for 14 days (HS-14d), NS, and LS for 14 days (LS-14d). Mean values and SEs are shown on the left of each column. Significance was determined by two-way ANOVA.

Figure 3.

Deletion of neural precursor cell expressed developmentally downregulated protein 4-2 (Nedd4-2) abolishes the effect of dietary Na⁺ intake in the distal convoluted tubule (DCT) membrane potential. A: perforated whole cell recordings showing K⁺ current (I_K) reversal potential in the DCT of wild-type [WT; floxed neural precursor cell expressed developmentally downregulated gene 4-like (Nedd4l^fl/fl)] mice on normal-salt (NS), high-salt (HS), and low-salt (LS) diets for 14 days, respectively. B: perforated whole cell recording showing the I_K reversal potential in the DCT of kidney-specific Nedd4-2 knockout (Ks-Nedd4-2 KO) mice on NS, HS, and LS diets for 14 days, respectively. (A trace from a control mouse on NS is included as a reference.) C: scatterplot summarizing the results of experiments in which the I_K reversal potential was measured in both male and female WT and Ks-Nedd4-2 KO mice on different Na⁺ diets for 14 days. Mean values and SEs are shown on the left of each column. For the measurement of I_K reversal potential, the bath solution contained 140 mM NaCl and 5 mM KCl and the pipette solution had 140 mM KCl. Significance was determined by two-way ANOVA.

Figure 4.

High salt (HS) inhibits expression of Kir4.1, large-conductance K⁺ (BK) channels, and epithelial Na⁺ channels (ENaC). A, top: expression of Kir4.1, Kir5.1, and the BK channel α-subunit (BKα) in male wild-type [WT; floxed neural precursor cell expressed developmentally downregulated gene 4-like (Nedd4l^fl/fl)] and kidney-specific neural precursor cell expressed developmentally downregulated protein 4-2 (Nedd4-2) knockout (Ks-Nedd4-2 KO) mice on normal salt (NS) or HS for 14 days. A, bottom: bar graph showing normalized band density of Kir4.1, Kir5.1, and BKα. B, top: expression of ENaCα, ENaCβ, and ENaCγ in male WT and Ks-Nedd4-2 KO mice on NS or HS for 14 days. B, bottom: bar graph showing normalized band density of ENaCα, ENaCβ, and ENaCγ. Significance was determined by two-way ANOVA.

Figure 5.

High salt (HS) inhibits epithelial Na⁺ channels (ENaC) in wild-type [WT; floxed neural precursor cell expressed developmentally downregulated gene 4-like (Nedd4l^fl/fl)] mice but not kidney-specific neural precursor cell expressed developmentally downregulated protein 4-2 (Nedd4-2) knockout (Ks-Nedd4-2 KO) mice. A: set of gap-free recordings showing amiloride-sensitive Na⁺ currents in the late distal convoluted tubule (DCT2)/connecting tubule (CNT) measured at −60 mV with the perforated whole cell patch in WT and Ks-Nedd4-2 KO mice on normal salt (NS) and HS (14 days; HS-14d), respectively. B: scatterplot summarizing the results of experiments in which amiloride (10 µM)-sensitive Na⁺ currents were measured at −60 mV with perforated whole cell recordings in the DCT2/CNT of male WT and Ks-Nedd4-2 KO mice on NS or HS diets for 14 days, respectively. Significance was determined by two-way ANOVA.

Figure 6.

Deletion of neural precursor cell expressed developmentally downregulated protein 4-2 (Nedd4-2) increases epithelial Na⁺ channel (ENaC) activity in the cortical collecting duct (CCD) and abolishes the effect of high salt (HS) on ENaC. A: set of gap-free recordings showing amiloride-sensitive Na⁺ currents in the CCD measured at −60 mV with the perforated whole cell patch in wild-type [WT; floxed neural precursor cell expressed developmentally downregulated gene 4-like (Nedd4l^fl/fl)] and kidney-specific Nedd4-2 knockout (Ks-Nedd4-2 KO) mice on normal salt (NS) and HS (14 days; HS-14d), respectively. B: scatterplot summarizing the results of experiments in which amiloride (10 µM)-sensitive Na⁺ currents were measured at −60 mV with perforated whole cell recordings in the CCD of male WT and Ks-Nedd4-2 KO mice on NS or HS diets for 14 days, respectively. Mean values and SEs are shown on the left of each column. The pipette solution contained 125 mM K-gluconate, 15 mM KCl, 2 mM MgATP, 1 mM EGTA, and 10 mM HEPES (pH 7.4). The bath solution contained 130 mM Na-gluconate, 10 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 2 mM MgCl₂, and 5 mM HEPES (pH 7.4). The addition of amiloride (10 µM) is indicated by an arrow. Significance was determined by two-way ANOVA.

Figure 7.

Deletion of neural precursor cell expressed developmentally downregulated protein 4-2 (Nedd4-2) abolishes the effect of dietary Na⁺ intake on NaCl cotransporter (NCC). A: Western blots showing the expression of phosphorylated (p)NCC and total (t)NCC in male floxed neural precursor cell expressed developmentally downregulated gene 4-like (Nedd4l^fl/fl) and kidney-specific Nedd4-2 knockout (Ks-Nedd4-2 KO) mice on normal salt (NS). B and C: immunoblot (IB) showing the expression of pNCC (at Thr⁵³) and tNCC in male Nedd4l^fl/fl and Ks-Nedd4-2 KO mice on different Na⁺ diets for 14 days, respectively. D: two bar graphs summarizing normalized band density of pNCC and tNCC in control and Ks-Nedd4-2 KO mice on low-salt (LS) and high-salt (HS) diets compared with NS. *Significance determined by one-way ANOVA.

Figure 8.

Deletion of neural precursor cell expressed developmentally downregulated protein 4-2 (Nedd4-2) abolished the effect of high salt (HS) on NaCl cotransporter (NCC) activity/expression. A: hydrochlorothiazide (HCTZ)-induced net renal Na⁺ excretion (E_Na) in floxed neural precursor cell expressed developmentally downregulated gene 4-like (Nedd4l^fl/fl) and kidney-specific Nedd4-2 knockout (Ks-Nedd4-2 KO) mice on low-salt (LS), normal-salt (NS), and HS diets for 14 days. Significance was determined by two-way ANOVA. B: immunoblots showing expression of phosphorylated (p)NCC (at Thr⁵³) and total (t)NCC in male Nedd4l^fl/fl and Ks-Nedd4-2 KO mice on HS plus K⁺ supplement (1 g K-citrate in 100 mL drinking water for 14 days). C: two bar graphs summarizing the above results for control and Nedd4-2 KO mice. *Significance determined by two-way ANOVA.

Figure 9.

Long-term high salt (HS) causes hypokalemia in kidney-specific neural precursor cell expressed developmentally downregulated protein 4-2 (Nedd4-2) knockout (Ks-Nedd4-2 KO) mice. A: scatterplot showing the basal level of renal K⁺ excretion rate in male wild-type [WT; floxed neural precursor cell expressed developmentally downregulated gene 4-like (Nedd4l^fl/fl)] mice and Ks-Nedd4-2 KO mice on normal salt (NS), HS (7 days; HS-7d), and HS (14 days; HS-14d), respectively. Mean values of each group are shown on the left side of column. B: scatterplot showing plasma K⁺ concentration in WT and Ks-Nedd4-2 KO mice on different Na⁺ diets. C: table showing the plasma Na⁺ concentration in WT and Ks-Nedd4-2 KO mice on different Na⁺ diets. *Significant difference compared with the other groups. We used two-way ANOVA to determine significance. LS-14d, low salt (14 days).

Figure 10.

Cell scheme illustrating the role of neural precursor cell expressed developmentally downregulated protein 4-2 (Nedd4-2) in regulating the effect of high salt (HS) intake on Kir4.1/Kir5.1, NaCl cotransporter (NCC), and epithelial Na⁺ channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN) and the role of Kir4.1/Kir5.1, NCC, and ENaC in regulating renal K⁺ excretion of the ASDN during HS. The solid line and dotted line represent enhanced or decreased pathways, respectively. Lines with double arrows indicate unchanged parameters. OSR1, oxidative stress responsive kinase-1; SPAK, STE20 proline-alanine-rich kinase; WNK, with no lysine kinase.