Pendrin regulation is prioritized by anion in high-potassium diets

Abstract

The Cl⁻/${\text{HCO}}_3^{-}$ exchanger pendrin in the kidney maintains acid-base balance and intravascular volume. Pendrin is upregulated in models associated with high circulating aldosterone concentration, such as dietary NaCl restriction or an aldosterone infusion. However, it has not been established if pendrin is similarly regulated by aldosterone with a high-K⁺ diet because the effects of accompanying anions have not been considered. Here, we explored how pendrin is modulated by different dietary potassium salts. Wild-type (WT) and aldosterone synthase (AS) knockout (KO) mice were randomized to control, high-KHCO₃, or high-KCl diets. Dietary KCl and KHCO₃ loading increased aldosterone in WT mice to the same extent but had opposite effects on pendrin abundance. KHCO₃ loading increased pendrin protein and transcript abundance. Conversely, high-KCl diet feeding caused pendrin to decrease within 8 h of switching from the high-KHCO₃ diet, coincident with an increase in plasma Cl⁻ and a decrease in ${\text{HCO}}_3^{-}$. In contrast, switching the high-KCl diet to the high-KHCO₃ diet caused pendrin to increase in WT mice. Experiments in AS KO mice revealed that aldosterone is necessary to optimally upregulate pendrin protein in response to the high-KHCO₃ diet but not to increase pendrin mRNA. We conclude that pendrin is differentially regulated by different dietary potassium salts and that its regulation is prioritized by the dietary anion, providing a mechanism to prevent metabolic alkalosis with high-K⁺ base diets and safeguard against hyperchloremic acidosis with consumption of high-KCl diets.

NEW & NOTEWORTHY Regulation of the Cl⁻/${\text{HCO}}_3^{-}$ exchanger pendrin has been suggested to explain the aldosterone paradox. A high-K⁺ diet has been proposed to downregulate a pendrin-mediated K⁺-sparing NaCl reabsorption pathway to maximize urinary K⁺ excretion. Here, we challenged the hypothesis, revealing that the accompanying anion, not K⁺, drives pendrin expression. Pendrin is downregulated with a high-KCl diet, preventing acidosis, and upregulated with an alkaline-rich high-K⁺ diet, preventing metabolic alkalosis. Pendrin regulation is prioritized for acid-base balance.

INTRODUCTION

K⁺ is critical in the generation and maintenance of the plasma membrane electrical potential (1, 2) and therefore plays a major role in many physiological responses, such as firing action potentials, normal sinus rhythm, muscle contraction, and hormone secretion (3). Long-term control of K⁺ homeostasis is dictated by the aldosterone-sensitive distal nephron (ASDN), which is able to precisely match urinary K⁺ excretion with K⁺ intake, even when dietary K⁺ is varied over an extremely wide range (4). The epithelial Na⁺ channel and K⁺-secretory channels (large-conductance K⁺ and renal outer medullary K⁺ channels; 5) in the ASDN are the key components of the K⁺ excretory machinery but other transporters, including pendrin, have been implicated. The regulation of each component of the machinery is exquisitely orchestrated to adjust K⁺ excretion on demand and thereby maintain serum K⁺ over a very narrow range.

Pendrin, a Cl⁻/${\text{HCO}}_3^{-}$ exchanger (6), is expressed on the apical membrane of type B intercalated cells and non-A, non-B intercalated cells (7) in the aldosterone-sensitive region of the kidney. This exchanger participates in the regulation of acid-base and NaCl balance by mediating ${\text{HCO}}_3^{-}$ secretion and Cl⁻ absorption, which are stimulated in response to either aldosterone or angiotensin II. Although pendrin does not mediate K⁺ transport, it plays a role in K⁺ homeostasis (8–11). Serum K⁺ is much lower and urinary K⁺ is higher in pendrin-null mice than in wild-type mice following dietary restriction of Na⁺ and K⁺ (10). Moreover, since pendrin is an electroneutral exchanger, it is thought to contribute to aldosterone- and angiotensin II-sensitive K⁺-sparing NaCl absorption (12, 13). However, there is evidence that pendrin also augments renal K⁺ excretion when dietary K⁺ is high. Following a diet high in KCl, serum K⁺ is much greater in pendrin-null mice compared with wild-type mice (14). Thus, pendrin-null mice cannot fully regulate K⁺ homeostasis when either dietary K⁺ is very low or high.

Pendrin has been proposed to be regulated differently in high aldosterone states, activated in hypovolemia, and suppressed in hyperkalemia (6, 15). However, the effects of dietary K⁺ and accompanying anions on pendrin abundance are not fully understood. Because dietary ${\text{HCO}}_3^{-}$ loading stimulates pendrin (16–18) and hyperchloremia suppresses it (19, 20), it remains uncertain if the apparent inhibitory response to high dietary KCl is to K⁺ or Cl⁻. It also remains uncertain if the regulation of pendrin in primary aldosteronism is driven by high aldosterone or is secondary to the development of alkalosis (8).

The present study was designed to close these gaps. Here, we explored the effect of dietary K⁺ intake on pendrin abundance, determined if this effect is from K⁺ itself or the accompanying anion, and defined the role of aldosterone in the response. We discovered that the accompanying anion rather than K⁺ dictates the effects of high dietary potassium salts on pendrin. Moreover, aldosterone is required for optimal upregulation of pendrin protein with a high-KHCO₃ diet.

MATERIALS AND METHODS

Animal Experiments

All in vivo experiments were approved by the Johns Hopkins University and Emory University Animal Care and Use Committees. Male C57/Bl6J mice (9–12 wk old, The Jackson Laboratory) were used for the in vivo experiments unless otherwise specified. In all experiments, mice were first acclimated to the K⁺ control diet containing 1% K⁺ (Cat. No. 190005, Envigo) as previously described (21). The following dietary protocols were performed to assess the role of K⁺, dietary anion, and aldosterone on pendrin expression: 1) To assess the effect of different high-K⁺ diets on pendrin expression, mice were randomized to three dietary groups and received either K⁺ control diet (Cat. No. 190005, Envigo) or high-K⁺ diets (5% K⁺) as either high-KHCO₃ salt (13 meq ${\text{HCO}}_3^{-}$ per 100 g of diet, Cat. No. 140044, Envigo) or high KCl salt (13 meq Cl⁻ per 100 g of diet, Cat. No. 09075, Envigo) for 4 days in group cages. Diets were matched for caloric content and only differed in potassium salts. Table 1 shows the electrolyte composition of these diets. 2) To test the differential effect of dietary anions independent of K⁺ and aldosterone on pendrin expression, we designed a series of dietary switch protocols. After mice were acclimated to the K⁺ control diet, they were fed a high-K⁺ diet containing either high ${\text{HCO}}_3^{-}$ or high Cl⁻ for 4 days, the dietary anion was then switched, experiments were terminated at different intervals, and the biochemical and physiological responses to dietary anion switch were measured. 3) To test the significance of aldosterone in the pendrin response to a high-KHCO₃ diet, we studied aldosterone synthase (AS) knockout (KO) mice (22). WT and AS KO mice were first acclimated to the K⁺ control diet for 3 days and then randomly assigned to stay on the control diet or receive the high-KHCO₃ diet. After 4 days of dietary intervention, the experiment was terminated in the morning, and the role of aldosterone on biochemical and physiological responses to KHCO₃ was evaluated.

Table 1.

Electrolyte composition of the diets used in the study

Diet	Na+, % g/100 g Diet, mM	K+, % g/100 g Diet, mM	Cl−, % g/100 g Diet, mM	${\mathbf{\text{HCO}}}_3^{-}$, % g/100 g Diet, mM
Control (Cat. No. 190005, Teklad)	0.29 (12.6)	1 (26)	0.9 (2.6)	0 (0)
High KHCO3 (Cat. No. 140044, Teklad)	0.29 (12.6)	5.2 (128)	0.5 (1.3)	8.4 (13)
High KCl (Cat. No. 09075, Teklad)	0.29 (12.6)	5.2 (128)	5.2 (13)	0 (0)

At the conclusion of each dietary protocol, mice were anesthetized in the morning by intraperitoneal ketamine-xylazine injection. After mice reached the surgical plane anesthesia, blood was collected from the carotid artery into heparin tubes. One hundred milliliters of whole blood were used to measure electrolytes as described in Electrolytes and Aldosterone Measurements. The remainder of the collected blood was first centrifuged at 6,000 g for 5 min, and the plasma supernatant was separated and snap frozen for aldosterone measurement. The kidneys were isolated, dissected into the cortex and medulla, and snap frozen until further use. Mice were euthanized by exsanguination.

Electrolytes and Aldosterone Measurements

Blood electrolytes were measured using EC8 cartridges (Cat. No. 03P79-25, Abbott Point of Care) by a handheld iStat machine. The plasma aldosterone concentration was measured using an enzyme immunoassay kit (Cat. No. EIA-5298, DRG) following the manufacturer’s instructions.

Western Blot Analysis

Frozen cortex tissues were homogenized in the sucrose buffer (Table 2) using zirconium beads (Cat. No. Z763799, Sigma-Aldrich) and incubated in detergent buffer (2% SDS, 0.5% Triton X-100, and 0.5% Tween 20 of final volume) rotated for 1 h in the cold room. Protein concentration was measured using a bicinchoninic acid (BCA) assay kit (Cat. No. 23227, Thermo Fisher) following the manufacturer’s instructions. Ten micrograms of total protein were run on a gradient 4–20% SDS-PAGE gel (Cat. No. 5678094, Bio-Rad). Gels were transferred to nitrocellulose membranes using Turbo transfer pack (Cat. No. 1704158, Bio-Rad). Membranes were blocked for 1 h in 5% nonfat powdered milk (Cat. No. A614-1003, Quality Biology) dissolved in Tris-buffered saline-Tween 20 (TBST) buffer. Pendrin antibody (S. M. Wall laboratory; 23) and Gapdh (Cat. No. G8795, Sigma-Aldrich) were diluted in blocking buffer at 1:40,000 and 1:20,000 dilution, respectively, and incubated overnight in the cold room. In the morning, membranes were washed three times in TBST buffer and then incubated in horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit, Cat. No. 111-035-144 for pendrin and goat anti-mouse, Cat. No. 115-035-003 for Gapdh, both from Jackson Laboratories) diluted in blocking buffer at room temperature for 1 h. Membranes were washed three times in TBST and incubated in ECL solution (Cat. No. 32106, Thermo Fisher) for 1 min. Membranes were imaged with an Azure Biosystem Imager within the linear range of the detection system.

Table 2.

Buffer recipes

Reagent	Sucrose Buffer	HEENG Buffer
Sucrose	200 mM
HEPES	20 mM	20 mM
CaCl2	0.5 mM
SDS	2%
Triton X-100	0.5%
Tween 20	0.5%
NaCl		125 mM
EDTA		1 mM
EGTA		1 mM
Glycerol		10%

Ubiquitylation Assay

Frozen cortex samples were homogenized in HEENG buffer (Table 2), and cells were homogenized using zirconium beads (Cat. No. Z763799, Sigma-Aldrich), and homogenates were lysed by the addition of 1% Triton X-100 of final volume followed by incubation in the cold room for 1 h on a rotator. Protein concentrations were measured using a BCA assay (Cat. No. 23227, Thermo Fisher). Four hundred micrograms of total protein were incubated in the cold room overnight on the rotator with ubiquitination affinity beads conjugated to a protein containing ubiquitin-binding domains (Cat. No. BK161-S, Cytoskeleton). The same amount of total protein (pooled from samples) was incubated overnight on the rotator with control beads conjugated to a protein containing mutated ubiquitin-binding domains as the nonspecific binding negative control. The next morning, the beads were washed in the wash buffer three times and bound proteins were eluted following the manufacturer’s instructions. For the input control, 10 µg of total protein was run.

Gene Expression

Frozen cortex tissues were homogenized in TRIzol (Cat. No. 15596026, Thermo Fisher) using zirconium beads (Cat. No. Z763799, Sigma-Aldrich), and RNA was extracted using an RNAeasy kit (Cat. No. 74004, Qiagen). The RNA concentration was measured using a nanodrop spectrophotometer, and contaminating genomic DNA was digested by incubation in DNAse I enzyme (Cat. No. 18068015, Thermo Fisher) following the manufacturer’s protocol. cDNA was synthesized from 1 µg of RNA using a high-capacity cDNA reverse transcription kit (Cat. No. 4368814, Thermo Fisher). Quantitative RT-PCR was performed using SYBR green primers for pendrin (forward primer sequence: 5′- TGCCTTCGAGCCTGATGAAG-3′ and reverse primer sequence: 5′- TCACTTTCACCGGGAGTTCG-3′) and Gapdh (forward primer sequence: 5′- TGTAGACCATGTAGTTGAGGTCA-3′ and reverse primer sequence: 5′- AGGTCGGTGTGAACGGATTTG-3′). cDNA was mixed with forward and reverse primers (250 nM) and 2× SYBR powerup master mix (Cat. No. A25918, Thermo Fisher). The reaction was run for 40 cycles. The ΔC_t method (where C_t was threshold cycle) was used to compare C_t values between groups. The amplification specificity of SYBR quantitative PCR primers was verified by the presence of a single peak on the dissociation curve.

Statistical Analyses

To test for statistical significance between three experimental groups, a one-way ANOVA test was performed. To compare statistical significance between two groups, a Student’s t test was used. To compare the interaction of diet and genotype between AS WT and AS KO mice, a two-way ANOVA test was performed. A two-tailed P value of <0.05 was considered significant. All statistical analysis was performed using Graph Pad PRISM (v9.0, La Jolla, CA). Data are presented as means ± SE.

RESULTS

K⁺-Rich Diets Drive Pendrin Regulation via Excess Accompanying Anion

Several studies have raised the possibility that K⁺ downregulates pendrin (9, 11), but the role of the accompanying dietary anion has not been addressed. To test this, mice were acclimated to the control diet (1% K⁺) and then randomized to the control diet or high-K⁺ diets (5% K⁺) that differed in the nature of the potassium salt: 1) high-KHCO₃ diet (13 meq/100 g KHCO₃) or 2) high-KCl diet (13 meq/100 g KCl) for 4 days (Fig. 1A). Mice consumed similar amounts of food on each of the diets (Table 3). After 4 days, mice were euthanized, and pendrin protein and transcript (Fig. 1, B and C), blood electrolytes (Table 3), and aldosterone (Fig. 1D) were measured. Serum K⁺ increased slightly, whereas serum aldosterone increased markedly and to the same extent, with either high-K⁺ diet (Fig. 1D). Hypochloremic metabolic alkalosis was observed in mice that received the high-KHCO₃ diet, whereas no acid-base change was observed in high KCl-fed mice (Fig. 1D). Compared with control mice, pendrin protein and mRNA abundance were higher in mice that consumed the high-KHCO₃ diet relative to controls, whereas pendrin abundance was similar in mice that received the high-KCl and control diets (Fig. 1, B and C). These data reveal that pendrin is not suppressed by dietary K⁺ excess. Instead, the dominant regulatory factor that drives pendrin expression is the counter anion. Because aldosterone increased to the same extent with both high-K⁺ diets, the results also indicate that an elevation of aldosterone itself is not sufficient to increase pendrin.

Figure 1.

Effect of different dietary potassium salts on pendrin protein and RNA expression in the kidney cortex and aldosterone. A: male wild-type (WT) mice were randomly assigned to one of the following three groups that received either 1) control diet, 2) high-KHCO₃ diet, or 3) high-KCl diet as shown in the diagram. The experiment was terminated on the morning of the fourth day. B: high-${\text{HCO}}_3^{-}$ diet increases pendrin protein abundance. B, left: Western blot analysis of pendrin protein across different dietary groups. Gapdh was used as the loading control. B, right: quantification of the Western blot. C: high-KHCO₃ diet increased pendrin mRNA expression. The bar graph shows the results of quantitative RT-PCR analysis on RNA extracted from the cortex of mice subjected to different dietary maneuvers. Gapdh was used as the internal control. D: plasma aldosterone levels were raised in both high dietary K⁺-fed groups to the same level. P < 0.05 was considered statistically significant (*, from control; # from the KCl group). A one-way ANOVA test with Tukey post hoc correction for multiple comparisons was performed. Data are presented as means ± SE; n = 5 mice/group except for the control diet-fed group, where n = 4.

Table 3.

Effect of 4 days of high-K⁺ diet on arterial blood electrolytes

Measurements	Control (n = 4)	High KHCO3 (n = 5)	High KCl (n = 5)
Daily food consumption, g/animal	2.4 ± 0.1	2.2	2.3 ± 0.1
Na+, mM	148 ± 1	150	152 ± 1*
K+, mM	4.1 ± 0.1	4.3 ± 0.3	4.7 ± 0.3
Cl−, mM	117 ± 2	112 ± 1*†	119 ± 1
${\text{HCO}}_3^{-}$, mM	20.2 ± 0.8	26.6 ± 1.6*†	21.7 ± 0.8
Blood urea nitrogen	21 ± 1.1	24.4 ± 2.0	24 ± 2.4

Values are means ± SE. *P < 0.05 between high dietary K⁺-fed groups and the control group; †P < 0.05 between high dietary K⁺-fed groups.

Dietary Cl⁻ Suppresses Pendrin in K⁺-Induced Hyperaldosteronism

Xu et al. (9) showed that consumption of a high dietary KCl diet reduces pendrin expression in a mouse model of aldosterone excess, but it has not been clear if this is due to correction of metabolic alkalosis or an inhibitory effect of K⁺. To test this, we performed an aldosterone-high dietary K⁺ clamp study; mice were first placed on the high-KHCO₃ diet for 4 days to increase aldosterone and activate pendrin. The diet was then switched to a diet with an identical K⁺ content but given as KCl. Mice were initially maintained on the KHCO₃-rich diet. To assess the time course of the pendrin response to substitution of ${\text{HCO}}_3^{-}$ with Cl⁻, the diet was changed at the beginning of the awake active period (night). We then compared the change in pendrin transcript and protein abundance 4 and 8 h following the replacement of the anion from ${\text{HCO}}_3^{-}$ to Cl⁻. Both groups consumed equivalent amounts of food over the treatment period (Fig. 2A). Changing the high-K⁺ diet from KHCO₃ to KCl caused a rapid significant decrease in plasma ${\text{HCO}}_3^{-}$ and an increase in plasma Cl⁻ (Table 4). In fact, after pendrin was activated with the high-KHCO₃ diet, rebound hyperchloremic metabolic acidosis was detected within 4 h of the switch to high KCl, presumably as a consequence of high Cl⁻ delivery in the face of high pendrin protein expression (24) and suppressed acid secretion at the start of the KCl challenge. A decrease in pendrin transcript became detectable within 4 h, whereas a decrease in pendrin protein was detected within 8 h of high-KCl diet consumption (Fig. 2, B and C). Pendrin transcript abundance displayed a steep positive relationship (r² = 0.87, P < 0.05) with plasma ${\text{HCO}}_3^{-}$ and an inverse linear relationship with plasma Cl⁻ (r² = 0.8, P < 0.05; Fig. 2D) but not with K⁺ (r² = 0.02, P = 0.67; not shown). Thus, pendrin is rapidly downregulated when KHCO₃ is replaced with KCl, underscoring the dominant role of the anion over K⁺ or aldosterone in the regulation of pendrin expression.

Figure 2.

Pendrin expression is rapidly suppressed in the kidney cortex following the dietary switch from a high-KHCO₃ to high-KCl diet. A: male wild-type (WT) mice were given high KHCO₃ for 4 days and then randomly assigned to stay on the diet or receive the high-KCl diet for 4 and 8 h as shown in the diagram of dietary maneuvers. B: high-KCl diet suppressed pendrin protein abundance, which reached statistical significance after 8 h of dietary switch. B, left: Western blot analysis of pendrin protein expression. Gapdh was used as the loading control. B, right: quantification of the Western blot. C: high-KCl diet rapidly suppressed pendrin mRNA expression after 4 h. The bar graph shows the quantitative RT-PCR analysis of pendrin transcript normalized to Gapdh. D: switching the diet from KHCO₃ to KCl elevated plasma Cl⁻ and conversely reduced ${\text{HCO}}_3^{-}$ levels. The change in pendrin mRNA was negatively correlated with the increase in plasma Cl⁻ and positively correlated with plasma ${\text{HCO}}_3^{-}$ levels. Each dot represents the value of a separate mouse. Lines are best fits via linear regression. E: the dietary switch from KHCO₃ to KCl did not change the rate of pendrin ubiquitylation. E, left: Western blot of ubiquitylated pendrin (top) and input (bottom) from the cortex of mice after 4 h of dietary switch. E, right: quantification of ubiquitylated pendrin normalized to total pendrin. *P < 0.05 was considered statistically significant from other groups. Data are presented as means ± SE. For A–D, one-way ANOVA tests with Tukey post hoc correction for multiple comparisons were performed. For E, a two-tailed Student’s t test was performed. n = 5 samples/group except for the results shown in E. IB, immunoblot.

Table 4.

Effect of switching high-KHCO₃ diet to high-KCl diet on arterial blood electrolytes

Measurements	High KHCO3 (n = 5)	High KCl
		4 h (n = 5)	8 h (n = 5)
Na+, mM	152 ± 1	155 ± 1	156 ± 1
K+, mM	4.2 ± 0.2	4.3 ± 0.3	4.9 ± 0.4
Cl−, mM	107 ± 2	123 ± 2*	125 ± 1*
${\text{HCO}}_3^{-}$, mM	26.9 ± 0.8	15.5 ± 1.0*	15.0 ± 0.3*
Blood urea nitrogen	21.8 ± 1.1	18.2 ± 1.2	25.4 ± 1.1

Values are means ± SE. *P < 0.05 between the high KHCO₃-fed group and high KCl-fed group.

Pendrin protein is controlled by the ubiquitin ligase Nedd4-2 (25), which presumably marks pendrin for proteasomal degradation by ubiquitylation. Accordingly, we explored if pendrin ubiquitylation is increased when the high-K⁺ diet is switched from high ${\text{HCO}}_3^{-}$ to high Cl⁻. To detect pendrin ubiquitylation before pendrin protein is downregulated, we examined the response 4 h after the diet was switched from high KHCO₃ to high KCl. Pendrin ubiquitylation was assessed by ubiquitin-binding protein chromatography (26) as the fraction of pendrin that bound to ubiquitin-binding protein-affinity beads and that could be specifically eluted with excess free ubiquitin. As detected in Western blots, ubiquitylated pendrin appeared as a tight smear of protein above the molecular weight of native pendrin in both treatment groups. No difference in the amount of ubiquitylated pendrin protein could be detected between high KHCO₃- and high KCl-fed mice (Fig. 2E). Thus, increased proteasomal degradation is not likely to be responsible for the rapid downregulation of pendrin protein with the high-KHCO₃ to high-KCl diet switch. Given the more rapid changes in pendrin transcript compared with pendrin protein, the data suggest that anion switch affects pendrin through a gene regulatory pathway in the face of a constant protein degradation pathway.

Dietary ${\mathbf{\text{HCO}}}_3^{-}$ Loading Rapidly Increases Pendrin Gene Expression in Mice on a High-K⁺ Diet

Our finding that pendrin is rapidly suppressed when the high-K⁺ diet is switched from high ${\text{HCO}}_3^{-}$ to high Cl⁻ prompted us to ask if there is a reciprocal response when high KCl is changed to high KHCO₃ (Fig. 3A). Switching the high-K⁺ diet from high Cl⁻ to high ${\text{HCO}}_3^{-}$ caused an induction of pendrin transcript within 4 h (Fig. 3B), and pendrin mRNA abundance positively correlated (r² = 0.81, P < 0.05) with the increase in plasma ${\text{HCO}}_3^{-}$ and negatively correlated with the decrease in plasma Cl⁻ (r² = 0.86, P < 0.05; Fig. 3C). The high KCl to high KHCO₃ switch also caused a rapid decrease in plasma K⁺ (Table 5), possibly due to the kaliuretic effects of poorly absorbable anions (27, 28), although urinary K⁺ measurements will be required to confirm this. The increase in pendrin protein expression lagged the increase in pendrin transcript; no increase in pendrin protein abundance could be detected until 24 h after the diet was switched. Thus, the lag between changes in diet and changes in pendrin expression is greater when pendrin expression is activated with the Cl⁻ to ${\text{HCO}}_3^{-}$ switch than when pendrin expression is inhibited with the ${\text{HCO}}_3^{-}$ to Cl⁻ switch (Fig. 3, D and E).

Figure 3.

Pendrin expression is increased in the kidney cortex following a dietary switch from high KCl to high KHCO₃. A: male wild-type (WT) mice were given the high-KCl diet for 4 days and then randomly assigned to stay on the diet or to receive the high-KHCO₃ diet for 4 and 8 h as shown in the diagram of dietary maneuvers. B: high-KHCO₃ diet rapidly increased pendrin transcript after 4 h. The bar graph shows the quantitative RT-PCR analysis of pendrin transcript. Gapdh was used as the internal control. C: switching the diet from KCl to KHCO₃ elevated plasma ${\text{HCO}}_3^{-}$ and conversely reduced Cl⁻ levels. The change in pendrin mRNA was negatively correlated with the increase in plasma Cl⁻ and positively correlated with plasma ${\text{HCO}}_3^{-}$ levels. Each dot represents the value of a separate mouse. Lines are best fits via linear regression. D: pendrin protein abundance did not change after 8 h of the dietary switch from KCl to KHCO₃. D, right: Western blot analysis of pendrin protein abundance. Gapdh was used as the loading control. D, left: quantification of the Western blots. E: 24 h of dietary switch from KCl to KHCO₃ was sufficient to elevate pendrin protein. E, left: Western blot analysis of pendrin protein. Gapdh was used as the loading control. E, right: quantification of the Western blots. P < 0.05 was considered statistically significant (* compared with the KCl group, # compared with 4-h KHCO₃). A one-way ANOVA test with Tukey post hoc correction for multiple comparisons was performed. Data are presented as means ± SE; n = 5 mice/group.

Table 5.

Effect of switching high-KCl to high-KHCO₃ diet on arterial blood electrolytes after 4, 8, 24, and 48 h

Measurements	High KCl (n = 5)	High KHCO3
		4 h (n = 5)	8 h (n = 5)	24 h (n = 5)	48 h (n = 5)
Na+, mM	153 ± 0	151 ± 1	149 ± 0	150 ± 0	150 ± 1
K+, mM	4.9 ± 0.3	3.7 ± 0.1*	3.6 ± 0.1*	3.7 ± 0.1*	3.4 ± 0.1*
Cl−, mM	119 ± 1	109 ± 1*	104 ± 1*	108 ± 1*	111 ± 1*
${\text{HCO}}_3^{-}$, mM	18.9 ± 0.5	24.1 ± 1.3*	29.4 ± 1.5*	27.7 ± 0.8*	24.8 ± 0.4*
Blood urea nitrogen	22.0 ± 0.7	22.2 ± 1.2	21.4 ± 0.9	24.2 ± 0.5	23.8 ± 1.5

Values are means ± SE. *P < 0.05 between the high KCl-fed group and high KHCO₃-fed group.

Dietary ${\mathbf{\text{HCO}}}_3^{-}$ Loading Increases Pendrin Gene Expression but Not Pendrin Protein, Independently of Aldosterone

Aldosterone and the synthetic mineralocorticoid agonist deoxycorticosterone increase the expression of pendrin (8, 9, 29, 30). However, aldosterone administration induces metabolic alkalosis. Therefore, aldosterone may increase pendrin abundance either through a direct effect of aldosterone on pendrin-positive cells or from alkalosis. To determine if aldosterone is required to drive the increase in pendrin expression with consumption of a high-KHCO₃ diet, we explored the effect of this diet on pendrin abundance in mice that do not produce aldosterone, i.e., AS KO mice (22). For these experiments, WT and AS KO littermates were acclimated to the control diet for 3 days and then randomly assigned to the control or high-KHCO₃ diet for an additional 4 days (Fig. 4A). Consumption of high KHCO₃ led to a similar decline in plasma Cl⁻ and an increase in ${\text{HCO}}_3^{-}$ levels in both AS KO and WT mice. However, in AS KO mice, blood K⁺ rose to a similar level as previously observed in these mutant mice following the high-KCl diet (Table 6; 31). In contrast to our observations in WT mice, pendrin protein did not increase in AS KO mice after consumption of the high-KHCO₃ diet (Fig. 4B). In contrast, pendrin mRNA increased to the same extent in AS KO mice as in WT mice following the high-KHCO₃ diet (Fig. 4C). These data suggest that optimal expression of pendrin in response to dietary KHCO₃ loading is governed by two mechanisms involving ${\text{HCO}}_3^{-}$-dependent activation of pendrin gene expression and aldosterone-dependent changes in posttranslational protein stabilization.

Figure 4.

Aldosterone is required for appropriate pendrin response to the high bicarbonate diet. A: male wild-type (WT) and aldosterone synthase (AS) knockout (KO) mice were randomly assigned to receive either K⁺ control or high-KHCO₃ diets for 4 days as shown in the diagram of dietary maneuvers. B: aldosterone deficiency impaired the pendrin protein response in the kidney cortex to a high-KHCO₃ diet. B, left: Western blot analysis of pendrin protein expression in WT and AS KO mice. Gapdh was used as the loading control. B, right: quantification of the Western blots. C: aldosterone was not required for the elevation of pendrin mRNA transcript in the kidney cortex after a high KHCO₃ dietary challenge. The bar graph shows the quantitative RT-PCR analysis of pendrin mRNA in WT and AS KO mice. Gapdh was used as the internal control. *P < 0.05; ****P < 10⁻⁴. A two-way ANOVA test with Tukey post hoc correction for multiple comparisons was performed. Data are presented as means ± SE; n = 4 mice in controls and 5 mice in KHCO₃-treated WT mice and n = 8 AS-KO mice on the control diet and n = 8 AS KO mice on the KHCO₃ diet. ns, not significant.

Table 6.

Effect of the high-KHCO₃ diet on arterial blood electrolytes in wild-type vs. aldosterone synthase knockout mice

Measurements	Wild Type		Aldosterone Synthase Knockout
	Control (n = 4)	High KHCO3 (n = 5)	Control (n = 4)	High KHCO3 (n = 5)
Na+, mM	141 ± 1	143 ± 1	140 ± 1	136.4 ± 1*†
K+, mM	4.1 ± 0.2	3.7 ± 0.2	4.9 ± 0.3	7.7 ± 0.4*†
Cl−, mM	114 ± 1	108 ± 1*	114 ± 1	107 ± 1*
${\text{HCO}}_3^{-}$, mM	22.3 ± 2.1	29.2 ± 1.4*	19.6 ± 1.1	30.5 ± 1.3*
Blood urea nitrogen	25.8 ± 1.31	20 ± 0.8	28 ± 4.14	32.2 ± 3.9†

Values are means ± SE. *P < 0.05 between treatments; †P < 0.05 between genotypes.

DISCUSSION

In an intravascular volume-depleted state, aldosterone increases Na⁺ reabsorption without major changes in K⁺, but in the settings of dietary K⁺ excess or hyperkalemia, aldosterone increases K⁺ excretion without altering Na⁺. This phenomenon, known as the aldosterone paradox, has been a subject of vigorous investigation (32–35). One proposed mechanism involves the differential regulation of pendrin (6); it has been posited that K⁺ suppresses pendrin in hyperaldosterone settings of K⁺ excess (11) to prevent activation of the electroneutral K⁺ sparing Na⁺-Cl⁻ reabsorption pathway, a pathway that is induced by aldosterone and angiotensin II (29, 36, 37) in states of intravascular volume depletion. The present study challenges this hypothesis, revealing that the effects of excess dietary potassium salts on pendrin expression are driven by the anion rather than K⁺.

Previous studies in mouse models (9, 11) and humans with primary aldosteronism (8) were interpreted to indicate that K⁺ suppresses the stimulatory effect of aldosterone on pendrin but did not take into account the effects of the accompanying anion. Because alkalosis stimulates pendrin expression independently of aldosterone (16–18, 38), interpretation of these studies is confounded by the development of alkalosis in primary aldosteronism (39–41) and by correction of alkalosis with Cl⁻ infusion (42). Our study provides a new interpretation of these reports. When all data points from control, high KCl-fed, and high KHOC₃-fed mice were combined, we found no relationship between plasma K⁺ and pendrin mRNA (r² = 0.1, P = 0.06). In fact, we found that a high-K⁺ diet increases, rather than suppresses, pendrin when the accompanying anion is ${\text{HCO}}_3^{-}$ and that switching the diet to high KCl reduces pendrin expression to baseline levels, commensurate with the correction of alkalosis. Together, these findings indicate that correction of alkalosis with Cl⁻, rather than high K⁺, drives the suppression of pendrin in hyperaldosteronism. These data are consistent with other studies showing that pendrin abundance increases with dietary NaCl restriction, which increases aldosterone production, despite the absence of alkalosis (43) and that high NaCl suppresses pendrin, whereas high NaHCO₃ activates it (6).

Pendrin transcript abundance followed a tight, positive correlation with plasma ${\text{HCO}}_3^{-}$ and negative correlation with plasma Cl⁻. Thus, it is not possible from our study to discern the extent to which ${\text{HCO}}_3^{-}$ stimulates pendrin and Cl⁻ inhibits it. However, because consumption of high dietary KCl only suppressed pendrin in mice with hyperaldosteronism and alkalosis and not in control mice, it seems most likely that the acute responses to dietary Cl⁻ are driven by the correction of alkalosis or rebound metabolic acidosis, which is known to strongly repress pendrin expression (6). Within hours of switching the high-KHCO₃ diet to high KCl, pendrin transcript and protein rapidly decayed, indicating that pendrin gene induction is short lived and that pendrin protein is much more liable than typical collecting duct membrane proteins, which have an average half-life of ∼30 h (44). Although we were able to detect pendrin ubiquitylation, we did not observe an increase in pendrin ubiquitylation following the anion switch. It may be that proteasomal degradation of pendrin is too rapid to detect an increase in the ubiquitylated intermediates or that removal of pendrin protein is controlled by a different pathway. Cl⁻ may have longer-term inhibitory effects on pendrin, as Quentin et al. (20) observed that feeding a KCl-rich diet to rats for 6 days suppressed pendrin in the absence of any acid-base abnormalities. Future studies will be required to test if these longer-term responses involve remodeling of the distal nephron and a reduction in the number of pendrin-positive cells or direct effects of Cl⁻ on pendrin gene expression and protein degradation.

Consumption of a KHCO₃ load caused a rapid increase in pendrin mRNA independently of aldosterone, which was coincident with the development of alkalosis. This finding is in line with previous reports showing that pendrin is upregulated in response to an alkaline load (16, 45), although it has been reported that chronic consumption (7 days) of high KHCO₃ in rats does not affect pendrin expression (20), which is in contrast to the stimulatory effect of high NaHCO₃ chronic consumption (16, 18, 46). At the molecular level, the rapid increase in pendrin mRNA may be explained by a ${\text{HCO}}_3^{-}$-induced increase in pendrin transcript stability or an increase in pendrin transcription or a combination of both. It has been shown that exposing human embryonic kidney cells to an alkaline medium (pH 7.6) increases pendrin promoter activity, independent of aldosterone (17, 38), which raises the possibility that an alkaline-activated transcription factor induces pendrin gene expression. Deyev et al. (47) reported that the physiological response of the kidney to an alkaline load requires the insulin receptor-related receptor INSRR as an extracellular alkali sensor. INSRR is specifically expressed in pendrin-positive intercalated cells (48–50), raising the possibility that a signaling network emanating from the receptor activates the pendrin gene.

Although ${\text{HCO}}_3^{-}$ drives the increase in pendrin transcript, it is not sufficient to increase pendrin protein since aldosterone is also required. Indeed, when challenged with an increased dietary KHCO₃ load, AS KO mice increase pendrin RNA to the same extent as WT mice but display a blunted increase in pendrin protein. These observations suggest that aldosterone-induced or -repressed genes stabilize pendrin protein rather than through a direct effect on pendrin transcription. Future studies will be required to test if aldosterone must be elevated above basal levels for this downstream pathway to become activated. Nevertheless, these observations are consistent with previous studies documenting the requirement of the mineralocorticoid receptor (MR) for the induction of pendrin protein expression in hyperaldosteronism (15, 51) despite the absence of classic mineralocorticoid response elements in the pendrin gene promoter (17, 38). Moreover, Pham et al. (51) found that ablation of MR in intercalated cells reduced the abundance of pendrin protein and apical localization. The synergizing effects of aldosterone and ${\text{HCO}}_3^{-}$ on pendrin protein stability and pendrin gene expression would maximize pendrin expression to optimize clearance of excess KHCO₃ with increased consumption of an alkaline-ash load, common with vegetarian diets. This would also explain why aldosterone, itself, is not sufficient to increase pendrin (19).

The discovery that the dietary anion determines the effects of high K⁺ and aldosterone on pendrin provides a new interpretation of MR signaling in the intercalated cell. Shibata et al. (11) proposed that dietary K⁺ excess inhibits aldosterone signaling in intercalated cells by increasing phosphorylation of MR. Our study indicates that the inhibitory effects of a high-K⁺ diet on pendrin are mediated by Cl⁻ rather than K⁺. Further studies will be required to determine if the Cl⁻ inhibitory effect is driven by MR phosphorylation. Regardless of the mechanism, the ability of Cl⁻ to shut off pendrin activation may provide a means to safeguard against hyperchloremic acidosis with consumption of high-KCl diets.

Although dietary K⁺ excess does not directly regulate pendrin, K⁺ deficiency might. Pendrin is required to limit urinary K⁺ losses when dietary K⁺ is restricted (10). People with Pendred syndrome are susceptible to the development of hypokalemia (52, 53), and recent studies in pendrin-null mice indicated that the pendrin-null kidney cannot fully conserve K⁺. The association of metabolic alkalosis with hypokalemia makes it difficult, however, to interpret these observations toward the direct stimulatory effect of low K⁺ on pendrin.

In summary, dietary K⁺ excess does not suppress pendrin. The accompanying anion drives the effects of dietary potassium salts on pendrin. Ingestion of an alkaline-rich, high-K⁺ diet drives pendrin expression to prevent metabolic alkalosis. In contrast, pendrin is rapidly downregulated to prevent hyperchloremic acidosis with the consumption of a high-KCl diet.

DATA AVAILABILITY

Data will be made available upon reasonable request.

GRANTS

This work was supported by a Ben J. Lipps Fellowship from the American Society of Nephrology, Fondation Leducq (Potassium in Hypertension Grant; to P.A.W.), and National Institute of Diabetes and Digestive and Kidney Diseases Grants DK054231 (to P.A.W.), DK093501 (to P.A.W.), DK110375 (to P.A.W.), and DK110375 (to S.M.W.).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

E.T., L.A.-Q., and P.A.W. conceived and designed research; E.T., T.D.P., L.A.-Q., and R.G. performed experiments; E.T., L.A.-Q., R.G., S.M.W., and P.A.W. analyzed data; E.T., L.A.-Q., S.M.W., and P.A.W. interpreted results of experiments; E.T. and T.D.P. prepared figures; E.T. drafted manuscript; E.T., L.A.-Q., S.M.W., and P.A.W. edited and revised manuscript; E.T., T.D.P., L.A.-Q., R.G., S.M.W., and P.A.W. approved final version of manuscript.