Mineralocorticoids Stimulate the Activity and Expression of Renal H⁺,K⁺-ATPases

Abstract

In the renal collecting duct, mineralocorticoids drive Na⁺ reabsorption, K⁺ secretion, and H⁺ secretion through coordinated actions on apical and basolateral transporters. Whether mineralocorticoids act through H⁺,K⁺-ATPases to maintain K⁺ and acid-base homeostasis is unknown. Here, treatment of mice with the mineralocorticoid desoxycorticosterone pivalate (DOCP) resulted in weight gain, a decrease in blood [K⁺] and [Cl⁻], and an increase in blood [Na⁺] and [HCO₃⁻]. DOCP treatment increased the rate of H⁺,K⁺-ATPase–mediated H⁺ secretion in intercalated cells of the inner cortical collecting duct. mRNA expression of the catalytic subunit HKα₁ did not significantly change, whereas HKα₂ mRNA expression dramatically increased in the outer and inner medulla of DOCP-treated mice. A high-K⁺ diet abrogated this increase in renal HKα₂ expression, showing that DOCP-mediated stimulation of HKα₂ expression depends on dietary K⁺ intake. DOCP treatment of mice lacking HKα₁ (HKα₁^−/−) resulted in greater urinary Na⁺ retention than observed in either wild-type mice or mice lacking both HKα₁ and HKα₂ (HKα_1,2^−/−). DOCP-treated HKα_1,2^−/− mice exhibited a lower blood [HCO₃⁻] and less Na⁺ and K⁺ retention than either wild-type or HKα₁^−/− mice. Taken together, these results indicate that H⁺,K⁺-ATPases—especially the HKα₂-containing H⁺,K⁺-ATPases—play an important role in the effects of mineralocorticoids on K⁺, acid-base, and Na⁺ balance.

Mineralocorticoid excess represents the most common endocrine form of hypertension and is poorly responsive to typical anti-hypertensive medications. With improved diagnostic criteria, the prevalence of mineralocorticoid-dependent hypertension is estimated to be as much as approximately 5 to 20% of hypertensive patients.^1–3 A major contributing factor to mineralocorticoid-induced hypertension is increased Na⁺ reabsorption by the kidney. Specifically, mineralocorticoids increase expression and activity of the apical epithelial Na⁺ channel and the basolateral Na⁺,K⁺-ATPase in principal cells of the renal collecting duct to drive net Na⁺ reabsorption.⁴ Mineralocorticoids also stimulate H⁺ secretion by the collecting duct, in part by stimulating the activity of apical H⁺-ATPases,^5,6 but the effect of mineralocorticoids on H⁺,K⁺-ATPase proton transport activity and expression in these segments has not been determined.

The renal H⁺,K⁺-ATPases are known to localize to the apical membrane of intercalated cells in the collecting duct.⁷ H⁺,K⁺-ATPases are composed of a catalytic α subunit and regulatory β subunit, and two different α subunits, HKα₁ and HKα₂, are expressed in the kidney. Only a few studies have investigated the effect of mineralocorticoids on renal H⁺,K⁺-ATPases, and most of these studies focused on acute mineralocorticoid effects (1 to 2 days) and have not directly measured proton secretion.^8–11

In this study, desoxycorticosterone pivalate (DOCP) was used as a model of chronic mineralocorticoid excess.¹² DOCP has long-lasting effects resulting from esterase cleavage in the muscle to the active mineralocorticoid, desoxycorticosterone.^13,14 The timings of DOCP-induced disturbances in body weight, Na⁺, K⁺, and acid-base homeostasis were determined and correlated with renal H⁺,K⁺-ATPase activity and H⁺,K⁺-ATPase α subunit expression. Disturbances in Na⁺, K⁺, Cl⁻, and HCO₃⁻ homeostasis were evident in DOCP-treated wild-type mice after 8 days. DOCP treatment also increased renal H⁺,K⁺-ATPase activity and mRNA expression for HKα₂ by this time point. This study also examined the physiologic role of the HKα₁- and HKα₂-containing H⁺,K⁺-ATPases in mineralocorticoid-induced electrolyte and acid-base disturbances using mice that have disruption of either the gene encoding for HKα₁ (HKα₁^−/−) or both genes encoding for HKα₁ and HKα₂ (HKα_1,2^−/−). These studies show that the H⁺,K⁺-ATPases exert a profound influence on mineralocorticoid-mediated changes in Na⁺, K⁺, and acid-base homeostasis.

RESULTS

DOCP Caused Disturbances in Na⁺, K⁺, and Acid-Base Homeostasis

A primary goal of this study was to characterize the temporal changes in body weight, Na⁺, K⁺, and acid-base homeostasis during chronic mineralocorticoid excess. Body weight and blood electrolytes were measured over an 8-day time course in untreated (control) mice and those treated with DOCP (1.7 mg; Table 1). Excess body weight gain was apparent in DOCP-treated mice. DOCP treatment caused a considerable increase in body weight by day 4, but control mice exhibited no significant change in body weight over this time period (data not shown). The observed body weight gain in DOCP-treated mice is consistent with the known effect of DOCP to enhance Na⁺ and fluid volume retention. By the fourth day, DOCP treatment resulted in hypernatremia, an effect that started to wane by 8 days. Moreover, DOCP treatment resulted in a reduction in blood [K⁺] by 6 days after DOCP administration. Eight days of DOCP treatment also significantly increased blood [HCO₃⁻] in wild-type mice. The timing and magnitude of blood [HCO₃⁻] increases with DOCP treatment were reflected in a reciprocal decrease in blood [Cl⁻] by approximately 7 mM.

Table 1.

Time course of the physiologic effect of DOCP treatment in wild-type mice

	Control (n)	Day 2 (n)	Day 4 (n)	Day 6 (n)	Day 8 (n)
Change in BW (%)	0 ± 0 (7)	0.06 ± 1.2 (7)	2.0 ± 1.2 (7)	5.4 ± 1.4 (7)a,b	6.1 ± 1.2 (7)a,b
Blood [Na+] (mM)	148 ± 0.41 (13)	150 ± 1.3 (3)	153 ± 1.8 (4)c	153 ± 0.80 (3)c	150 ± 0.41 (3)
Blood [K+] (mM)	3.9 ± 0.08 (14)	3.5 ± 0.12 (4)	3.5 ± 0.17 (4)	2.9 ± 0.21 (3)c	2.8 ± 0.07 (3)b,c
Blood [Cl−] (mM)	118 ± 0.51 (13)	120 ± 1.8 (3)	116 ± 0.95 (4)	117 ± 0.35 (3)	111 ± 0.82 (3)a,b,c,d
Blood [HCO3−] (mM)	18 ± 0.51 (14)	19 ± 0.87 (4)	20 ± 1.2 (4)	20 ± 0.53 (3)	23 ± 2.0 (3)c

BW, body weight. Day signifies days of DOCP treatment.

^aSignificant difference from day 2.

^bSignificant difference from day 4.

^cSignificant difference from control.

^dSignificant difference from day 6.

DOCP Increased H⁺,K⁺-ATPase Activity in the Collecting Duct

The results of the physiology studies showed that DOCP treatment led to the development of a reduced blood [K⁺] and greater blood [HCO₃⁻] within 8 days. One likely mechanism for mineralocorticoid-induced changes in acid-base homeostasis was stimulation of renal H⁺,K⁺-ATPase activity in response to mineralocorticoid-induced K⁺ deficits. Therefore, H⁺,K⁺-ATPase activity was assessed in inner cortical collecting ducts from control and DOCP-treated mice. Individual intercalated cells from microperfused collecting ducts were loaded with a pH-sensitive fluorescent dye. The addition of inhibitors of the Na⁺/H⁺ exchanger and the H⁺-ATPase allowed for the measurement of H⁺,K⁺-ATPase–mediated H⁺ secretion (J_H) in response to an acute intracellular acid load (NH₄Cl). The overall rate of H⁺,K⁺-ATPase–mediated J_H in intercalated cells from control mice was quite slow (approximately 15 H⁺/min; Figure 1). However, a >60% increase in H⁺,K⁺-ATPase–mediated J_H was observed in intercalated cells from DOCP-treated mice.

Figure 1.

DOCP increases H⁺,K⁺-ATPase-mediated H⁺ secretion (J_H) in ICs of the mouse collecting duct. J_H (H⁺/min) in response to an acute intracellular acid load was measured in ICs of microperfused inner cortical collecting duct from control mice and those treated with DOCP for 8 days using a pH-sensitive dye. Data are presented as mean ± SEM and were analyzed by t test. *P < 0.05 compared with control; n shown as cells (tubules).

Additional experiments were conducted to identify which intercalated cell subtype was responsible for the DOCP-induced increase in H⁺,K⁺-ATPase–mediated J_H. Again, the rate of H⁺,K⁺-ATPase–mediated J_H was measured in intercalated cells of inner cortical collecting ducts from control and DOCP-treated mice. At the end of the experiment, A- and B-type ICs were differentiated by peritubular Cl⁻ removal and return. The characteristic inverse responses of A- and B-type intercalated cells to Cl⁻ removal and return were observed (Table 2). Intrinsic buffering capacity (β_i) was not significantly affected by DOCP treatment. There was no difference in the rate of H⁺, K⁺-ATPase–mediated J_H in A- and B-type intercalated cells (ICs) from control mice (Figure 2). Although the rate of H⁺,K⁺-ATPase–mediated J_H was not affected in B-type ICs, DOCP treatment increased H⁺,K⁺-ATPase–mediated J_H in A-type ICs by approximately 60%.

Table 2.

Characterization of IC subtypes from inner CCD of control and DOCP-treated mice

	A-Type IC		B-Type IC
	Control (n = 48, 7)	DOCP (n = 22, 4)	Control (n = 15, 5)	DOCP (n = 24, 5)
Cl− removal (U/min)	0.52 ± 0.04	0.59 ± 0.04	−0.31 ± 0.06	−0.35 ± 0.03
Cl− return (U/min)	−0.39 ± 0.03	−0.37 ± 0.06	0.37 ± 0.04	0.37 ± 0.03
βi (U/min)	69.1 ± 6.91	86.8 ± 16.6	68.7 ± 10.5	87.7 ± 9.02

U/min, change in pH; β_i, buffering capacity. n = cells, tubules.

Figure 2.

DOCP increases H⁺,K⁺-ATPase-mediated H⁺ secretion (J_H) in A-type ICs of the mouse collecting duct. J_H (H⁺/min) in response to an acute intracellular acid load was measured in ICs of microperfused inner cortical collecting duct from control mice and those treated with DOCP for 8 days using a pH-sensitive dye. A- and B-type ICs were differentiated by response to peritubular Cl⁻ removal/return. Data are presented as mean ± SEM. The effect of DOCP in each cell type was analyzed using a t test. *P < 0.05 compared with control in the same cell type; n shown as cells (tubules).

DOCP Induced Medullary HKα₂ mRNA Expression

The principal mechanism of mineralocorticoid action is through modulation of target gene transcription.¹⁵ Therefore, the next experiments evaluated the effect of DOCP on renal HKα₁ and HKα₂ mRNA expression. Real-time quantitative PCR was used to study changes in steady-state mRNA levels of H⁺, K⁺-ATPase α subunits, HKα₁ and HKα₂, in cortex, outer medulla, and inner medulla of control and DOCP-treated mice. Eight days after DOCP treatment, HKα₁ mRNA expression in all three kidney segments was not significantly altered compared with control (Figure 3A). DOCP treatment had a tendency to increase HKα₁ expression in the inner medulla. DOCP did not affect HKα₂ mRNA expression in the cortex (Figure 3B). However, HKα₂ mRNA levels were increased more than twofold in the outer medulla and as much as fivefold in the inner medulla of DOCP-treated mice.

Figure 3.

High K⁺ diet abrogates the increase in medullary HKα₂ expression with DOCP treatment in mice. Real-time PCR was performed to quantify relative mRNA expression for (A) HKα₁ and (B) HKα₂ in cortex, outer medulla, and inner medulla of control mice and those treated with DOCP for 8 days on a normal diet. Relative mRNA expression was also determined for (C) HKα₁ and (D) HKα₂ in cortex, outer medulla, and inner medulla of control and DOCP-treated mice on a high K⁺ (5%) diet. Expression was set relative to β-actin. Fold changes (2^−ΔΔCt) in expression were calculated and set to percentage, with control set at 100%. Data are presented as mean ± SEM, and expression with DOCP treatment was compared with control levels by t test. *P < 0.05 compared with control; n = 7 to 10 for normal diet and n = 4 for high K⁺ diet.

Deficits in K⁺ have long been known to stimulate H⁺,K⁺-ATPase activity and HKα subunit expression. Therefore, the next experiments studied whether the decreases in blood [K⁺] observed in DOCP-treated mice were responsible for the stimulation of HKα₂ mRNA expression. For this experiment, mice were fed a high K⁺ diet and left untreated (control) or given DOCP treatment. Body weight and blood electrolytes were measured 8 days after treatment, and kidneys were collected for analysis of HKα₁ and HKα₂ mRNA expression by real-time PCR. A high K⁺ diet abrogated the effect of DOCP on body weight gain and blood [K⁺], [Cl⁻], and [HCO₃⁻] in mice (Table 3). Similarly, the stimulation of HKα₁ or HKα₂ mRNA expression with DOCP was absent in mice fed a high K⁺ diet (Figure 3, C and D). These results show that stimulation of medullary H⁺, K⁺-ATPase α subunit mRNA expression with DOCP treatment is dependent on dietary K⁺ intake.

Table 3.

Effect of high K⁺ (5%) diet on physiologic response of wild type mice to DOCP treatment

	Control (n = 4)	DOCP (n = 4)
Body weight change (g)	−0.42 ± 0.09	−0.17 ± 0.27
Blood [Na+] (mM)	149 ± 0.99	151 ± 0.88
Blood [K+] (mM)	4.25 ± 0.06	4.30 ± 0.18
Blood [Cl−] (mM)	119 ± 0.45	121 ± 1.30
Blood [HCO3−] (mM)	17.3 ± 0.64	18.2 ± 1.01

HKα-Null Mice Showed Altered Na⁺, K⁺, and Acid-Base Homeostasis with DOCP Treatment

The final set of experiments considered the physiologic function of renal H⁺,K⁺-ATPases in the response to chronic mineralocorticoid excess and specifically characterized the effect of DOCP treatment on the electrolyte and acid-base homeostasis of mice with disruption of the H⁺,K⁺-ATPase HKα₁ subunit (HKα₁^−/−) or both the HKα₁ and HKα₂ subunits (HKα_1,2^−/−). Body weight change (%) and blood electrolytes were first measured in untreated wild-type and knockout mice (Table 4). No appreciable body weight change (%) was observed in untreated mice of any genotype over 8 days (data not shown). Blood [K⁺] was paradoxically greater in the HKα_1,2^−/− compared with wild-type or HKα₁^−/− mice. Blood [Cl⁻] was less in the HKα₁^−/− mice compared with either the wild-type or HKα_1,2^−/− mice.

Table 4.

Differences in blood chemistry of WT and HKα-null mice

	WT (n = 13 to 14)	HKα1−/− (n = 5)	HKα1,2−/− (n = 9)
Blood [Na+] (mM)	148 ± 0.41	149 ± 1.1	148 ± 0.58
Blood [K+] (mM)	3.9 ± 0.08	3.9 ± 0.12	4.4 ± 0.17a
Blood [Cl−] (mM)	118 ± 0.51	114 ± 1.3a	116 ± 0.91
Blood [HCO3−] (mM)	18 ± 0.51	21 ± 1.2	19 ± 0.69

^aSignificant difference from WT.

In a separate study, the effect of DOCP treatment on body weight change, blood electrolytes, and urinary and fecal electrolyte excretion was examined in wild-type, HKα₁^−/−, and HKα_1,2^−/− mice on a normal diet. DOCP-induced body weight gain was comparable in wild-type and HKα_1,2^−/− mice but was augmented nearly twofold in HKα₁^−/− mice (Figure 4A). Blood [Na⁺] was similar in mice from all three genotypes (Figure 4B). Although DOCP decreased blood [K⁺] in mice from all three genotypes, HKα_1,2^−/− mice still exhibited greater blood [K⁺] than wild-type or HKα₁^−/− mice (Figure 4C). The effect of DOCP to decrease blood [Cl⁻] (Figure 4D) and increase blood [HCO₃⁻] (Figure 4E) was eliminated in HKα_1,2^−/− mice.

Figure 4.

DOCP treatment differentially affects body weight and blood [HCO₃⁻] in WT and HKα-null mice. All data shown are from the eighth day of DOCP treatment. (A) Body weight change (%) is shown as the percent change in body weight from day 0. Arterial blood samples were collected from the aorta, and (B) blood [Na⁺], (C) [K⁺], (D) [HCO₃⁻], and (E) [Cl⁻] were measured on a clinical blood gas analyzer. Data are presented as mean ± SEM and were analyzed by one-way ANOVA followed by post hoc Student-Newman-Keuls test. †P < 0.05 compared with WT; ‡P < 0.05 compared with HKα₁^−/− mice. n = 10 to 11 WT and n = 5 HKα₁^−/− and HKα_1,2^−/− mice.

To more fully understand the mechanism for the observed differences in body weight gain and blood electrolytes between wild-type and HKα knockout mice, urinary and fecal Na⁺ and K⁺ excretion was measured the day before and 8 days after DOCP treatment. Urine volume doubled by the end of DOCP treatment in both wild-type and HKα_1,2^−/− mice but considerably decreased in HKα₁^−/− mice (Figure 5, A and D). Over the course of DOCP treatment, HKα_1,2^−/− retained significantly less urinary Na⁺ than wild-type or HKα₁^−/− mice (Figure 5B). In comparison with day 8 of DOCP treatment to control urinary Na⁺ retention, HKα₁^−/− mice retained more urinary Na⁺ than wild-type mice at this time point (Figure 5E). After 8 days of DOCP treatment, urinary K⁺ retention was greater in HKα₁^−/− mice than either wild-type or HKα_1,2^−/− mice (Figure 5, C and F).

Figure 5.

DOCP treatment differentially alters urinary Na⁺ and K⁺ retention in WT and HKα-null mice. (A) Urinary volume, (B) Na⁺, and (C) K⁺ retention were measured in WT, HKα₁^−/− and HKα_1,2^−/− mice from the day preceding treatment (control) and over an 8-day period of DOCP treatment. Urinary electrolyte retention (μEq) was calculated as the urinary excretion per day subtracted from dietary intake on that day. (D–F) Comparisons of control urine values versus only day 8 of DOCP treatment are shown. All data were analyzed by a two-way repeated-measure ANOVA with post hoc Student-Newman-Keuls test and are shown as mean ± SEM. P < 0.05 is considered significant. Comparison of genotype within day: ^asignificant difference from WT; ^bsignificant difference from HKα₁^−/− mice. Comparison of genotype over the time course: ^csignificant difference from WT; ^dsignificant difference from HKα₁^−/− mice. ^eSignificant difference from control within the same genotype. WT, n = 10; HKα₁^−/−, n = 5; HKα_1,2^−/−, n = 5.

Analysis of stool samples from wild-type, HKα₁^−/−, and HKα_1,2^−/− mice showed that HKα₁^−/− mice excreted 50% more dry stool weight than either the wild-type or HKα_1,2^−/− mice when treated with DOCP, even though the mice were pair fed (Figure 6A). Fecal Na⁺ excretion significantly decreased in DOCP-treated wild-type and HKα_1,2^−/− mice and was significantly greater in DOCP-treated HKα₁^−/− mice than wild-type or HKα_1,2^−/− mice (Figure 6B). Interestingly, fecal K⁺ loss was evident in both HKα₁^−/− and HKα_1,2^−/− mice in the control period, and this loss increased approximately 50% with DOCP treatment (Figure 6C).

Figure 6.

HKα-null mice exhibit altered fecal electrolyte excretion under control and DOCP-treated conditions. (A) Fecal output, (B) Na⁺, and (C) K⁺ excretion were measured in WT, HKα₁^−/−, and HKα_1,2^−/− mice on the day preceding treatment (control) and on day 8 of DOCP treatment. Data are shown as mean ± SEM and were analyzed by two-way repeated-measure ANOVA with post hoc Student-Newman-Keuls test; P < 0.05 is considered significant. ^aSignificant difference from WT; ^bsignificant difference from HKα₁^−/− mice; ^esignificant difference from control within the same genotype. WT, n = 11; HKα₁^−/−, n = 4; HKα_1,2^−/−, n = 5 to 6.

In the context of whole animal physiology, it is important to examine the overall electrolyte balance as the sum of urinary and fecal excretion subtracted from the intake of that electrolyte. Overall Na⁺ and K⁺ balance was not significantly different in mice from any genotype under control conditions (Figure 7). As expected, overall Na⁺ balance increased in wild-type mice with DOCP treatment (Figure 7A). Overall Na⁺ retention was significantly less in HKα_1,2^−/− mice than wild-type or HKα₁^−/− mice on the eighth day of DOCP treatment. Also unlike HKα₁^−/− mice, HKα_1,2^−/− mice exhibited approximately 75% less K⁺ retention than wild-type mice with DOCP treatment (Figure 7B). These results conclusively show that disruption of H⁺,K⁺-ATPases in mice abolishes the effect of DOCP to increase total body Na⁺ and K⁺ retention.

Figure 7.

HKα-null mice display disturbances in overall electrolyte balance with DOCP treatment. Overall (A) Na⁺ and (B) K⁺ balance (μEq) are shown for WT, HKα₁^−/−, and HKα_1,2^−/− mice on the day preceding treatment (control) and on day 8 of DOCP treatment. Overall electrolyte balance was calculated as the amount of the electrolyte excreted in the urine and feces subtracted from dietary intake of that electrolyte. Data are shown as mean ± SEM and were analyzed by two-way repeated-measure ANOVA with post hoc Student-Newman-Keuls test or one-way repeated-measure ANOVA with post hoc Tukey test; P < 0.05 is considered significant. ^aSignificant difference from WT; ^bsignificant difference from HKα₁^−/− mice; ^esignificant difference from control within the same genotype. WT, n = 10; HKα₁^−/−, n = 4; HKα_1,2^−/−, n = 5.

DISCUSSION

In this study, we provided definitive evidence that mineralocorticoids regulate the activity and expression of renal H⁺,K⁺-ATPases. Prolonged exposure to DOCP increased H⁺ secretion in the collecting duct through renal H⁺,K⁺-ATPases. More specifically, the increase in HKα₂ expression with DOCP treatment supports a particularly important role of the HKα₂ H⁺,K⁺-ATPase isoform in mineralocorticoid-induced H⁺ secretion. Whole animal studies in HKα₁^−/− and HKα_1,2^−/− mice subjected to DOCP treatment highlight the significance of H⁺,K⁺-ATPases to mineralocorticoid-mediated disturbances in K⁺ and acid-base homeostasis, as well as Na⁺ homeostasis. The overall deficit in K⁺ retention and lower blood [HCO₃⁻] observed in DOCP-treated HKα_1,2^−/− mice signifies the importance of H⁺,K⁺-ATPases to facilitate K⁺ reabsorption and H⁺ secretion with mineralocorticoid excess. Importantly, the elimination of DOCP-induced Na⁺ retention in HKα_1,2^−/− mice and not in HKα₁^−/− mice indicates that the HKα₂-containing H⁺,K⁺-ATPases indirectly regulate mineralocorticoid-mediated Na⁺ retention, which may present major implications for BP regulation.

The effect of long-term mineralocorticoid excess to change H⁺,K⁺-ATPase activity and its relation to dietary K⁺ intake has previously been examined by Eiam-Ong et al.¹¹ In contrast to the results described here, 7-day exposure to low, normal, or high aldosterone levels in rats fed diets consisting of low, normal, and high K⁺ content did not significantly affect Schering 28080–sensitive (a gastric or HKα₁-containing H⁺,K⁺-ATPase inhibitor¹⁶) ATPase activity in microdissected cortical and medullary collecting ducts. However, the effect of aldosterone treatment on HKα₂-containing H⁺,K⁺-ATPases was not studied. The observed stimulation of H⁺,K⁺-ATPase activity in A-type ICs of inner cortical collecting ducts from DOCP-treated mice coupled with the dramatic augmentation of HKα₂ mRNA expression in the renal medulla of DOCP-treated mice shows that mineralocorticoids, in fact, do regulate renal H⁺,K⁺-ATPases.

The renal H⁺,K⁺-ATPases have been purported to act as primarily K⁺ reabsorptive mechanisms. Because hypokalemia has been shown to substantially increase medullary HKα₂ expression^17–19 and hypokalemia was quite evident with DOCP treatment, the induction of HKα₂ expression in the outer and inner medulla of DOCP-treated mice could be a result of hypokalemia. Abolishment of DOCP-induced HKα₂ mRNA expression in the medulla by high K⁺ is consistent with this hypothesis.

The increase in H⁺,K⁺-ATPase-mediated H⁺ secretion in the collecting duct and HKα₂ subunit expression in the kidney coincided with the development of a greater blood [HCO₃⁻] in DOCP-treated wild-type mice. The similar time course of these two events suggests that H⁺,K⁺-ATPases are responsible for a significant portion of the increase in blood [HCO₃⁻] with mineralocorticoid excess. Most importantly, DOCP treatment did not significantly increase blood [HCO₃⁻] in HKα_1,2^−/− mice. Taken together with the response of the wild-type mice, these data strongly support the hypothesis that the H⁺,K⁺-ATPases mediate the development of mineralocorticoid-induced alkalosis.

Excessive body weight gain and urinary Na⁺ retention in DOCP-treated HKα₁^−/− mice and its elimination in HKα_1,2^−/− mice shows that the mineralocorticoid-sensitive component of Na⁺ and fluid reabsorption depends directly or indirectly on the HKα₂-containing H⁺,K⁺-ATPases. Precedent for such a conclusion is supported by two separate sets of observations: (1) both the HKα₁- and HKα₂-containing H⁺,K⁺-ATPases have been found to transport Na⁺ on the K⁺ binding site of the transporter,^20–22 and (2) evidence from Spicer et al.²³ supports a dependence of the epithelial Na⁺ channel on the HKα₂-containing H⁺,K⁺-ATPases. Specifically, HKα₂^−/− mice had reduced colonic epithelial Na⁺ channel activity on a normal diet compared with wild-type mice and this effect was exacerbated by dietary Na⁺ restriction. The observation that DOCP treatment affected urinary K⁺ retention in HKα₁^−/− and HKα_1,2^−/− mice, as well as Na⁺, is consistent with an indirect mechanism of H⁺,K⁺-ATPase–mediated Na⁺ reabsorption.

The results of these studies provide evidence for an important role of the H⁺,K⁺-ATPases in normal K⁺ homeostasis and in mineralocorticoid-mediated effects on K⁺, acid-base, and Na⁺ balance. Future investigation into the mechanism(s) by which the renal H⁺,K⁺-ATPases contribute to Na⁺ and fluid balance may very likely shed important light on the pathogenesis of mineralocorticoid hypertension.

CONCISE METHODS

Animals

All animal use protocols were approved by the North Florida/South Georgia Veterans Administration Institutional Animal Care and Use Committee in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. Female C57BL/6J mice were purchased from Jax Labs (Bar Harbor, ME) or bred in house. HKα₁^−/− and HKα_1,2^−/− mice were generous gifts from Dr. Gary Shull. The latter were generated from breeding HKα₁^−/−24 and HKα₂^−/−25 mice and backcrossing onto the C57BL/6J background strain. Genotypes were confirmed by PCR of genomic DNA as described previously.²⁶

Mice were fed normal laboratory chow, given free access to water, and given an intramuscular injection of 1.7 mg DOCP (Percorten V; Novartis Pharmaceuticals). For perfusion and expression studies, mice were killed with Na⁺ pentobarbital (120 mg/kg, intraperitoneally), followed by cervical dislocation. For time course experiments, another group of animals were anesthetized with 3 to 4% isoflurane, and aortic blood was collected into a heparinized syringe for immediate analysis of electrolytes and blood gases (Nova pHOx Plus analyzer, Nova Biomedical). For high K⁺ diet studies, mice were given a normal, powdered diet (TD99131, Teklad) supplemented with KCl to equal 5% K⁺, which was made as a gel. One half of the mice were treated with DOCP. Blood and tissues were collected as in the time course experiments. For urinalysis experiments, mice were housed in metabolic cages for 13 days and fed a normal gel diet with free access to a water bottle. On day 5, mice were treated with DOCP as above. Urine and feces were collected daily. Urine electrolytes were measured on a clinical analyzer (Nova 16 analyzer; Nova Biomedical), and fecal electrolytes were measured on a digital flame photometer (Model 2655-00; Cole-Parmer).

Tubule Perfusion and pH_i Recovery

Inner cortical collecting ducts were hand dissected at 4 °C, transferred to a mounting chamber, and perfused as described previously.²⁶ The ratiometric, pH-sensitive dye, 3′,6′-bis(Acetyloxy)-5(or 6)-[[(acetyloxy)methoxy]carbonyl]-3-oxo-spiro[isobenzofuran-1(3H),9′-[9H]xanthene]-2′,7′-dipropanoic acid 2′,7′-bis[(acetyloxy)methyl] ester (15 μM; Invitrogen), was added to the luminal perfusate for 10 minutes, and fluorescence measurements were performed after dye de-esterification. Tubules were equilibrated with 100 nM bafilomycin A₁ in the luminal perfusate and 10 μM EIPA in the peritubular solution for 20 min, and inhibitors were present throughout the remainder of the experiment. Cells were acid loaded by a 3-minute, peritubular exposure to 40 mM NH₄Cl, followed by NH₄Cl removal. Ratiometric intensity measurements (I_490/440) were made in individual, well-defined ICs and converted to pH_i using linear regression, based on a high K⁺/nigericin calibration curve. Recovery rates were calculated from the linear portion of the pH_i recovery phase, starting with the lowest pH_i achieved. The intrinsic buffering capacity was calculated from the formula β_i = ΔH_i/ΔpH_i, where ΔH_i is the change in the calculated [NH₄⁺]_i and ΔpH_i is the change in pH_i during the acid-loaded phase. Acid secretion rates (J_H) were calculated from the formula J_H = β_i × ΔpH_i (U/min) and expressed as [H⁺]/min (H⁺/min). A- and B-type ICs were differentiated using peritubular chloride removal and return in HCO₃⁻-containing solutions as described previously.^26–28

Analysis of mRNA

RNA was recovered from cortex, medulla, and inner medulla with TRIzol reagent (Invitrogen). RNA was treated with DNase 1 (Ambion DNA Free) and converted to cDNA using SuperScript III (Invitrogen). For real-time quantitative PCR, 20 ng cDNA was used to quantify mRNA expression with TaqMan Gene Expression (Applied Biosystems) primer/probe sets for HKα₁ (Atp4a, Mm00444423_m1), HKα₂ (Atp12a, Mm0131809_m1), and β-actin (Actb, Mm00607939_s1). Cycle threshold values were normalized to β-actin, and relative expression (control set to 100%) was calculated by the ΔΔCt method.²⁹

Statistical Analysis

All data are represented as mean ± SEM. Statistics were performed with Origin 8 and Sigma Stat 3.1, and all graphs/plots were made with Origin 8. The effects of time and treatment or time and genotype were analyzed by a two-way repeated-measure ANOVA with post hoc Student-Newman-Keuls test. The effect of treatment duration or genotype was analyzed by one-way ANOVA with a post hoc Student-Newman-Keuls test. An unpaired t test was used to compare differences between control and treated groups. P < 0.05 was considered significant.

DISCLOSURES

None.