Hypertension of Kcnmb1^−/− is linked to deficient K secretion and aldosteronism

Abstract

Mice lacking the β1-subunit (gene, Kcnmb1; protein, BK-β1) of the large Ca-activated K channel (BK) are hypertensive. This phenotype is thought to result from diminished BK currents in vascular smooth muscle where BK-β1 is an ancillary subunit. However, the β1-subunit is also expressed in the renal connecting tubule (CNT), a segment of the aldosterone-sensitive distal nephron, where it associates with BK and facilitates K secretion. Because of the correlation between certain forms of hypertension and renal defects, particularly in the distal nephron, it was determined whether the hypertension of Kcnmb1^−/− has a renal origin. We found that Kcnmb1^−/− are hypertensive, volume expanded, and have reduced urinary K and Na clearances. These conditions are exacerbated when the animals are fed a high K diet (5% K; HK). Supplementing HK-fed Kcnmb1^−/− with eplerenone (mineralocorticoid receptor antagonist) corrected the fluid imbalance and more than 70% of the hypertension. Finally, plasma [aldo] was elevated in Kcnmb1^−/− under basal conditions (control diet, 0.6% K) and increased significantly more than wild type when fed the HK diet. We conclude that the majority of the hypertension of Kcnmb1^−/− is due to aldosteronism, resulting from renal potassium retention and hyperkalemia.

Hypertension, a harbinger of stroke and congestive heart failure, is the most prevalent chronic disorder in the Western Hemisphere. Most cases of hypertension are of unknown origin and therefore categorized as essential hypertension. However, approximately 10% of essential hypertensives are subcategorized as having normokalemic aldosteronism, identified by a normal plasma K concentration despite a normal-to-high plasma aldosterone concentration (1–3). The origins of non-adenomatous forms of normokalemic aldosteronism are not understood, but this phenotype suggests a malfunction of the K-aldosterone feedback control axis.

The large Ca-activated K channel (BK) is considered an important component in the regulation of vascular tone and blood pressure. Like most K-selective channels, BK channels are beset with ancillary partners. BK may contain 1 of 4 different subunits, identified as BK-β1 thru β4 (Kcnmb1-4), which tailor the properties of the BK-α pore (Kcnma1) to the specialized functional requirements of the cell. BK-α/β1 regulates vascular smooth muscle contraction by coupling the outward K currents with Ca sparks—densely localized concentrations of Ca emitted from ryanodyne-sensitive Ca channels near the plasma membrane (4, 5). Consistent with its role to mitigate myogenic tone, mice with deletions of Kcnmb1 (Kcnmb1^−/−) manifest mild but significant hypertension (6–9). However, BK-β1 is not only restricted to smooth muscle. BK-α/β1 also resides in the connecting tubules (CNT) of the mammalian kidney where it is used for K secretion in response to acute volume-expansion (10, 11). This is relevant because long-term hypertension is most often associated with a renal defect.

Hypertension resulting from defective K secretion would manifest most prominently in mice consuming a high K diet. There are 2 K secretory channels—BK and Kcnj1 (ROMK) in the CNT and CCD (12–14) and at least 2 ways that a high K diet can stimulate K secretion—directly by high plasma K or indirectly by K-stimulated aldosterone production. Both BK and ROMK knockouts maintain a viable plasma K concentration, suggesting that either channel can adequately secrete K in the absence of the other under normal dietary conditions (11, 15). A recent study showed that the plasma aldosterone concentration is elevated in BK-α knockout mice (16). An elevated plasma aldosterone concentration may be a compensatory side effect of defective BK-mediated K secretion.

Potassium adaptation is the enhanced capacity to secrete an acute K load after a prolonged elevated K diet (17). The mechanism of K adaptation involves both an adrenal (18) and renal component. In the adrenals, K adaptation includes an increase in aldosterone production per a given increase in plasma K concentration (19). Renal K adaptation involves increased basolateral membrane Na-K-ATPase activity (20) and increased apical membrane K channel activity in the distal nephron (21, 22).

The present studies were performed to determine the role of BK-β1 in K, Na, and fluid balance and whether the hypertension of Kcnmb1^−/− results from enhanced K adaptation with over-production of aldosterone by the adrenals. Results show that Kcnmb1^−/− are beset with aldosteronism that is exacerbated with a high K diet. The elevated aldo concentration is due to an adrenal gland that is hypersensitive to an elevated plasma K concentration, which is explained by reduction in BK-α/β1-mediated K secretion in the CNT.

Results

K and Na Handling in Kcnmb1^−/−.

Experiments with Kcnmb1^−/− were performed to determine the role of Kcnmb1 in the renal handling of K and Na when consuming a high K diet and to determine whether defective K secretion is the result of reduced eplerenone-sensitive K excretion. As shown in Fig. 1A, the plasma [K] was significantly elevated in Kcnmb1^−/− with a value of 4.39 ± 0.02 mM (n = 11) vs. wild type (WT) (4.08 ± 0.05 mM; n = 7) when mice were on a control diet. When mice were placed on a high K diet (WT-HK and Kcnmb1^−/−-HK), the plasma [K] increased significantly to 4.31 ± 0.09 mM (n = 10) in WT-HK and to 4.67 ± 0.05 mM (n = 11) in Kcnmb1^−/−; however, the plasma [K] was significantly greater in Kcnmb1^−/−-HK than WT-HK. The mineralocorticoid receptor antagonist, eplerenone, but not vehicle, significantly increased the plasma [K] to 5.96 ± 0.04 mM (n = 6) in WT-HK and to 6.00 ± 0.02 mM (n = 6) in Kcnmb1^−/−-HK.

Fig. 1.

Bar plots illustrating the effects of a high K diet (HK) and HK with eplerenone (HK+E) or vehicle (HK+V) on plasma K concentration (A) and K clearance (B) of WT and Kcnmb1^−/−. Values are means ± SEM. *, P < 0.05 vs. WT using unpaired t test. ‡, P < 0.05 vs. control using ANOVA plus SNK test. ¶, P < 0.05 vs., HK using ANOVA plus SNK test. Control group was fed regular mouse chow. § = *, ‡, and ¶.

As shown in Fig. 1B, the differences in K clearance between WT and Kcnmb1^−/− mirrored the differences in plasma K concentrations. On a regular diet, the K clearance of WT was 108.5 ± 2.3 mL/day (n = 7), a value slightly but significantly greater than that of Kcnmb1^−/− (96.5 ± 0.9 mL/day; n = 11). This difference was more pronounced when mice were treated with a high K diet with WT-HK having a K clearance of 760.1 ± 13.7 mL/day (n = 10) and Kcnmb1^−/− -HK having a clearance of 598.0 ± 10.7 mL/day (n = 11). When WT-HK and Kcnmb1^−/−-HK were treated with eplerenone, the K clearances were reduced to values of 401.6 ± 7.1 mL/day (n = 6) and 373.7 ± 4.7 mL/day, respectively (n = 6). Vehicle-treated controls were not significantly different from WT-HK and Kcnmb1^−/−-HK.

Fig. 2 shows the plasma [Na] (A) and Na clearance (B) for WT and Kcnmb1^−/− under conditions of a control and high K diet with and without eplerenone or vehicle treatment. On regular diets, the plasma [Na] was elevated significantly from a value of 136.7 ± 0.6 mM (n = 7) in WT to a value of 141 ± 1.0 mM in Kcnmb1^−/− (n = 11). On a high K diet, the plasma [Na] for WT was 137.5 ± 1.2 mM (n = 10), a value slightly, but significantly less than the value of 140.8 ± 0.8 mM (n = 11) for Kcnmb1^−/−. When the mice on a high K diet were treated with eplerenone, the plasma [Na] decreased to values of 135.7 ± 0.5 mM (n = 6) for WT and 136.4 ± 0.6 mM (n = 6) for Kcnmb1^−/−. When treated with vehicle, Kcnmb1^−/−-HK exhibited a slight but significant increase in plasma [Na] compared to WT-HK.

Fig. 2.

Bar plots illustrating the effects of HK and HK+ E or HK+V on mean plasma Na concentration (A), and Na clearance (B) of WT and Kcnmb1^−/−. All symbols are the same as in Fig. 1.

The Na clearance values are shown in Fig. 2B. On a control diet, the Na clearance for WT was 1.87 ± 0.05 mL/day (n = 7), a value slightly, but significantly greater than that of Kcnmb1^−/− (1.69 ± 0.03 mL/day; n = 11). This difference was more pronounced when mice were fed a high K diet; the Na clearance for WT-HK was 3.08 ± 0.04 mL/day (n = 10), a value significantly greater than the value for Kcnmb1^−/−-HK (2.60 ± 0.06 mL/day; n = 11). Eplerenone treatment increased the Na clearance in WT and Kcnmb1^−/− to values of 4.04 ± 0.11 mL/day (n = 6) and 3.62 ± 0.10 mL/day (n = 6), respectively. The Na clearance values in the vehicle-treated groups were not significantly different from the high K diet non-treated group. These results reveal enhanced Na reabsorption in Kcnmb1^−/− that is exaggerated by a high K diet.

Similar to the plasma Na analysis, the plasma osmolality (Fig. S1) was significantly elevated from 316 ± 0.9 mOsm (n = 7) in WT to 321.5 ± 0.8 mOsm (n = 11) in Kcnmb1^−/−. This difference was exaggerated when mice were treated with a high K diet—the plasma osmolality was 317.7 ± 0.6 mOsm (n = 10) in WT-HK and was significantly greater (324.5 ± 0.7 mOsm; n = 11) in Kcnmb1^−/−-HK. Eplerenone reduced the plasma osmolalities of WT-HK and Kcnmb1^−/−-HK to not significantly different values of 314.2 ± 0.7 mOsm (n = 6) and 314.2 ± 0.6 mOsm (n = 6), respectively.

As shown in the Table S1, the plasma creatinine levels were not significantly different when all groups were compared with the ANOVA and SNK test. That WT and Kcnmb1^−/− have the same glomerular filtration rates was shown previously by determining GFR with inulin measurements (23). Therefore, the relative clearance values for Na and K also reflect the fractional excretions for each group.

Extracellular Volume Status.

The results of Fig. 3 show that Kcnmb1^−/− were retaining fluid, especially when treated with a high K diet. Extracellular volume expansion was evaluated by the hematocrit (Hct) in combination with the change in body weight. As shown, the Hct was significantly reduced from a value of 45.8 ± 0.3% (n = 7) in WT to 41.9 ± 0.2% (n = 11) in Kcnmb1^−/−. On a high K diet, the Hct was further reduced from a value of 45.7 ± 0.4% (n = 10) in WT to 37.4 ± 0.4 (n = 11) in Kcnmb1^−/−. When the mice on a high K diet were treated with eplerenone, the Hct in WT and Kcnmb1^−/− were not significantly different with values of 49.7 ± 0.5% (n = 6) and 50.2 ± 0.5% (n = 6), respectively. The Hct values for the vehicle-treated group were not different from the non-treated mice on a high K diet.

Fig. 3.

Bar plots illustrating the effects of HK and HK+ E or HK+V on mean hematocrit (Hct.) of WT and Kcnmb1^−/−. All symbols are the same as in Fig. 1.

As shown in Fig. S2, WT gained 0.23 ± 0.03 g (n = 7), an amount not significantly different from the weight gained by Kcnmb1^−/− (0.18 ± 0.03 g; n = 11) on a control diet. On a high K diet, WT gained 0.42 ± 0.02 g (n = 10); however, Kcnmb1^−/− gained substantially more weight (3.3 ± 0.3 g; n = 11), consistent with extreme fluid overload. When the mice on a high K diet were treated with eplerenone, WT lost 0.23 ± 0.03 g (n = 6) and Kcnmb1^−/− lost 0.65 ± 0.04 g (n = 6). WT and Kcnmb1^−/− treated with vehicle gained weight by similar amounts as WT-HK and Kcnmb1^−/−-HK. The eplerenone-reversal of the reduced Hct and weight gain in Kcnmb1^−/−-HK demonstrates volume expansion due to aldosteronism.

Mean Arterial Blood Pressure.

As shown in Fig. 4A, Kcnmb1^−/− exhibit mild hypertension with MAP = 137 ± 3 mm Hg (n = 11) while WT were 116 ± 3 mm Hg (n = 7). The MAP of WT was unaffected by eplerenone (114 ± 3 mm Hg; n = 6) or vehicle. The MAP of Kcnmb1^−/− was decreased by eplerenone to a value of 122 ± 2 mm Hg (n = 6) but remained slightly but significantly above WT by 8 mm Hg.

Fig. 4.

Mean arterial blood pressure. (A) Bar plots illustrating the effects of eplerenone (E) or vehicle (V) on mean arterial blood pressure (MAP) of WT and Kcnmb1^−/−. (B) Bar plots showing the effects of a low K diet (LK), HK and HK+ E or HK+V on MAP in WT and Kcnmb1^−/−. All symbols are the same as in Fig. 1.

To determine whether the hypertension of Kcnmb1^−/− is related to a K load, the MAP was determined with mice on a low, normal and high K diet. As shown in Fig. 4B, the MAP of Kcnmb1^−/− on a low K diet was 122 ± 5 mm Hg (n = 10), a value significantly greater than the value of 113 ± 3 mm Hg (n = 7) for WT on a normal K diet but less than the value of 135 ± 5 mm Hg (n = 11) for Kcnmb1^−/− treated with a normal K diet. When Kcnmb1^−/− were treated with a high K diet, the MAP increased significantly to a value of 145 ± 3 mm Hg (n = 11), which was significantly above the MAP of 115 ± 4 mm Hg (n = 10) for WT-HK. The increased MAP of Kcnmb1^−/−-HK was reversed by eplerenone to 122 ± 6 mm Hg (n = 6), a value that was still 9 mm Hg (albeit insignificantly) above the WT-HK value of 113 ± 7 mm Hg (n = 6). The vehicle controls were not different from WT-HK and Kcnmb1^−/−-HK. These results show that the majority of the hypertension of Kcnmb1^−/− is the result of deficient K handling of a dietary K load. The elevated plasma [K] stimulates aldosterone, causing Na retention.

Aldosterone.

High dietary K exaggerated and eplerenone corrected the conditions of volume expansion and hypertension in Kcnmb1^−/−. These results are consistent with primary aldosteronism. The aldosterone (aldo) values for WT and Kcnmb1^−/− on a normal and high K diet are shown in Fig. 5. On a control diet, the plasma [aldo] of Kcnmb1^−/− was 147 ± 4 pg/mL (n = 11), a value significantly greater than the value of 107 ± 3 pg/mL (n = 5) for WT. When treated with a high K diet, the aldo values were 145 ± 8 pg/mL (n = 5) in WT but significantly greater (183 ± 11 pg/mL; n = 5) in Kcnmb1^−/−. When the mice on a high K diet were treated with eplerenone, the plasma [aldo] in WT-HK increased to 187 ± 13 pg/mL (n = 5), a value slightly less than that of Kcnmb1^−/−-HK (216 ± 12 pg/mL; n = 5).

Fig. 5.

Bar plots illustrating the effects of HK and HK+ E or HK+V on plasma [aldo] of WT and Kcnmb1^−/−. All symbols are the same as in Fig. 1.

Aldosterone production is often related to the size of the adrenal gland, which hypertrophies with chronic stimulation and increased synthesis of aldosterone. As shown in Fig. S3, the mass of the adrenal glands, normalized to prediet body weight, was slightly, but not significantly, greater in Kcnmb1^−/− on a control diet (0.201 ± 0.008 mg/g BW; n = 11) when compared with WT (0.192 ± 0.007 mg/g BW; n = 7). When placed on a high K diet, the adrenal mass of Kcnmb1^−/− was 0.230 ± 0.008 mg/g BW (n = 11), a value significantly greater than that of WT (0.209 ± 0.004 mg/g; n = 10). These results are consistent with the adrenal gland being the source of the elevated aldosterone in Kcnmb1^−/−-HK.

Adrenal Expression of Kcnmb1.

The BK-α subunit has been localized by immunohistochemistry to the adrenal medulla and adrenal glomerulosa (24), and both types of cells have exhibited BK currents (25, 26). However, the associated BK-β subunit has not been discovered. As shown by RT-PCR (Fig. 6A) and western blot (Fig. 6B), BK-β1 is expressed in mouse adrenal glands of WT but not Kcnmb1^−/−.

Fig. 6.

RT-PCR (A) and western blot (B) determination of BK-β1 expression in mouse adrenal glands. (A) BK-β1 mRNA is expressed at the correct size (561 bp) in WT but not in Kcnmb1^−/− negative control. (B) BK-β1 protein (28 kDa) was expressed in adrenals of WT but not in Kcnmb1^−/− negative control.

As shown in Fig. S4, Kcnmb1 was immunohistochemically localized in the adrenal medulla of WT but not Kcnmb1^−/− negative controls.

Sensitivity of Adrenal Glands to Plasma [K].

The large increase in aldosterone in response to a high K diet in Kcnmb1^−/− suggests an increased sensitivity of the glomerulosa cells to plasma [K]. The secretion of aldo by the adrenal glomerulosa is extremely responsive to small changes in the plasma [K]. The different sensitivities of the glomerulosa cells of WT and Kcnmb1^−/− to K were determined by plotting the plasma K vs. plasma aldosterone concentrations. As shown in Fig. 7, the slope of the relation is 127 pg[aldo]/mM[K] in Kcnmb1^−/− vs. 76 pg[aldo]/mM[K] in WT.

Fig. 7.

Plots of the plasma [K] vs. plasma [aldo] for WT (closed circles) and Kcnmb1^−/− (open circles). The r values (correlation coefficients) were highly significant for WT (P < 0.05) and Kcnmb1^−/− (P < 0.05). The slopes of the relations for plasma K vs. aldo concentrations are 74 and 127 for WT and Kcnmb1^−/−, respectively.

Discussion

The results of this study showed that genetic ablation of BK-β1 results in a hyperaldosterone-hypertension condition that is exacerbated with a high K diet. Evidence supporting this conclusion is the elevated plasma aldo concentration and the eplerenone reversal of hypertension and volume expansion of Kcnmb1^−/−. The data indicate that the aldosteronism of Kcnmb1^−/− is the result of increasing plasma [K] due to diminished aldosterone-induced K secretion as well as increased aldo production per increase in plasma K concentration.

Potassium Secretory Defect of Kcnmb1^−/−.

Adaptation to a high K diet involves both aldo-dependent and -independent increases in K secretory mechanisms. However, it has been difficult to distinguish the individual roles of BK and ROMK channels with respect to secreting K in direct response to elevated K or increased aldosterone. In the CCD (27) and the CNT (28), ROMK is up-regulated in the apical membrane by a high K diet. Aldosterone increases ROMK in the apical membrane of the rat CCD (29). Consistent with the present results, a high K diet induced charybdotoxin-sensitive K secretion in the late distal tubule of the mouse as assessed by micropuncture (30). However, the micropuncture study did not distinguished between the direct effects of high plasma K and aldosterone as the mediator for enhancing BK-mediated K secretion.

The present results reveal a diminished K secretory response of Kcnmb1^−/− to a high K diet. When eplerenone was given to the mice on high K diets, the K secretory response was inhibited substantially more in WT than in Kcnmb1^−/−. After eplerenone treatment, the plasma K concentrations increased to a similar value in WT and Kcnmb1^−/−. The K clearances of WT and Kcnmb1^−/− on high K diets with eplerenone treatment were nearly equivalent but still substantially elevated compared with the values of these mice on regular diets. It can be assumed that the remaining K clearance that is not eplerenone-sensitive is the high plasma K, eplerenone-insensitive K clearance. Therefore, Kcnmb1^−/− may not have a substantial defect in K adaptation by an aldosterone-independent, high plasma K-induced mechanism.

When the K clearance values for the mice on high K diets plus eplerenone are subtracted from the K clearance values for the mice on a high K diet (Fig. 1B), the remainder reveals that Kcnmb1^−/− has 37% less eplerenone-sensitive K secretion, leading to the conclusion that Kcnmb1^−/− has defective renal responsiveness to K-stimulated aldosterone. This result is consistent with a study showing that K excretion was impaired in BK-α knockout mice despite very high plasma aldosterone levels (16). Another study reported that aldosterone increased BK-mediated K secretion in the colon (31). However, our finding is seemingly in conflict with a recent study that reported that aldosterone did not increase BK-mediated K secretion in the isolated rabbit cortical collecting ducts (CCD) (32). However, aldosterone would not affect BK in the CCD, which does not express luminal Kcnmb1 (10, 33). Moreover, in the aforementioned study, aldosterone was increased by a low Na diet instead of a high K diet as in the present study. A low Na diet evokes mechanisms that primarily enhance Na reabsorption, whereas a high K diet induces pathways that primarily enhance K secretion. For example, a low Na diet, but not a high K diet, results in an increase in angiotensin II, which is not increased by a high K diet. Angiotensin II increases ENaC-mediated Na reabsorption in the CCD (34) but might inhibit or counteract the effects of aldosterone to enhance BK-mediated K secretion.

Hypernatremia and Hyperosmolality of Kcnmb1^−/−.

Kcnmb1^−/− exhibited mild hypernatremia and hyperosmolality. This condition was exacerbated by a high K diet and reversed by eplerenone, consistent with primary aldosteronism. The increase in plasma Na and osmolality is interesting considering that the plasma osmolality is usually tightly regulated by vasopressin secretion in steep response to small changes in plasma osmolality. However, subjects with primary aldosteronism are known to have plasma Na and osmolar concentrations slightly higher than normal (35) as found in the present study. The hypernatremia and hyperosmolality is probably the result of resetting the osmostat (36).

Hypertension of Kcnmb1^−/−.

The mild hypertension of Kcnmb1^−/− has been considered to result from increased vascular reactivity due to a diminished Ca-feedback response of the BK-α/β1 in vascular smooth muscle cells (7, 9). However, hypertension is often comprised of a combination of increased renal Na reabsorption and enhanced vascular reactivity. The MAP of Kcnmb1^−/− was elevated by 22 mm Hg, compared with WT. After eplerenone treatment, MAP of Kcnmb1^−/− was only 8 mm Hg more than WT. These observations suggest that K-stimulated aldosteronism may be the primary cause of hypertension, accounting for as much 70%, in Kcnmb1^−/−.

That the majority of hypertension of Kcnmb1^−/− is related to defective K secretion is evidenced by the finding that MAP is nearly normalized when Kcnmb1^−/− are placed on a low K diet and exaggerated when placed on a high K diet. The hypertension of Kcnmb1^−/− on a high K diet, which was even more elevated (by 31 mm Hg) compared to WT on a high K diet, was reduced by eplerenone to a value that was 8 mM more than WT on a high K diet. The MAP values for Kcnmb1^−/− that were either treated with eplerenone or maintained on a low K diet ranged from 5–8 mm Hg above WT. Thus, the absence of BK-β1 in vascular smooth muscle accounts for a slight elevation in MAP. However, primary aldosteronism, due to the inability to maximally secrete K, accounts for the majority of the hypertension in Kcnmb1^−/−.

Role of the Adrenal BK in Regulating Aldosterone Production.

We found that the BK-β1 subunit is expressed in the adrenal glands of mouse by PCR and western blot. The BKα subunit has been identified in the mouse adrenal cortex and the adrenal medulla with immunohistochemical staining (37), findings consistent with previous patch-clamp studies revealing the presence of BK currents in adrenal glomerulosa cells (25, 26) and the chromaffin cells of the adrenal medulla (38).

That the BK-β1 subunit is localized in the adrenal medulla (Fig. S4) is consistent with studies showing that BK channels of chromaffin cells modulate the release of catecholamines, which modulate via adrenergic receptors the release of aldosterone. The role of the BK-α/β1 in regulating catecholamine release may be similar to its role in other tissues, having an effect to hyperpolarize the membrane potential and mitigate the Ca-mediated release of catecholamines (39, 40). Catecholamine-induced elevations in glomerulosa cAMP levels stimulate Ca influx via L-type Ca channels (41, 42), leading to increased aldo production and release. That the adrenal glands of Kcnmb1^−/−-HK are hypertrophied is consistent with increased enzyme activity and production of aldosterone, explaining the enhanced [aldo].

The present study supports the results of Sausbier et al. who first showed that aldosterone levels were increased in Kcnma1^−/− (37). That study did not determine whether the elevated aldosterone had a role in the increased MAP. However, in the Sausbier study, the aldo levels on a normal diet were much greater (2.5-fold) in the Kcnma1^−/− compared with a 40% elevation in aldo in Kcnmb1^−/−. The aldo levels for Kcnma1^−/− on a high K diet were increased by more than 10-fold, a value much greater than the 2.5-fold increase in aldo levels for Kcnmb1^−/−.

Less than 1-mM changes in plasma K concentration regulate aldo release independent of its release induced by ANGII (43–45). In humans, an elevation of 0.2 mM in plasma K concentration increases aldo production by 46% over baseline (43). In comparison, the present study reports an increase in plasma [aldo] of 15% over baseline per 0.2 mM increase in plasma [K]. Thus, the K-stimulated aldo response may be less sensitive in mice than in humans.

In the Kcnmb1^−/−, the plasma [K] vs. plasma [aldo] relation reveals a K-stimulated aldo response with a slope that is a 72% more than the slope of WT. Therefore, the glomerulosa cell aldo secretion may be more sensitive to plasma [K] in Kcnmb1^−/−. It is unclear whether the increase in Kcnmb1^−/− aldosterone production results from the absence of the BK-β1 subunit in chromaffin cells or is the result of adaptation by glomerulosa cells to chronically elevated plasma K concentrations.

Perspectives and Significance.

That the expression of BK-α and BK-β1decreases with aging (49, 50) may partially explain the increased prevalence of hypertension and the dysfunctional regulation of fluid balance in the aged population. Moreover, the importance of the BK-β1 subunit in blood pressure regulation has recently been highlighted with the identification of polymorphisms in human KCNMB1. Gain-in-function mutations have been shown to confer protection against developing hypertension (46, 47). Another BK-β1 mutation was recently shown to result in a loss-in-function of BK activity in alveolar smooth muscle cells of males resulting in a decrease in forced airway flow (48). However, the blood pressure of this asthmatic population is unknown.

Based on the present results, aldosterone-inhibiting agents may be beneficial in treating a small minority of essential-hypertension patients having normal plasma K and elevated plasma aldosterone levels. Furthermore, such hypertensive patients, although a relatively small minority, would exhibit exacerbated hypertension when consuming a K-rich diet. This specific phenotype would be clearly different from the well-described blood pressure lowering effects that a high K diet has on the general population.

In summary, these results indicate that the majority of the hypertension in Kcnmb1^−/− is due to aldosteronism resulting from renal potassium retention and hyperkalemia. It remains to be determined whether similar forms of essential hypertension in humans results from polymorphisms in BK-β1 and whether such polymorphisms are the primary cause of hypertension. These results may be important for establishing diuretic therapies in certain individuals with essential hypertension.

Materials and Methods

Animals.

All animals were maintained under the conditions approved by the Institutional Animal Care and Use Committee of the University of Nebraska Medical Center. Two groups of male mice were used: wild-type mice − WT (C57BL/6) purchased from Charles River and Kcnmb1^−/− [generated by Brenner et al. (7) which were bred in the Animal Care Facility at the University of Nebraska Medical Center]. Kcnmb1^−/− were weaned from their mother when 4 weeks old. WT were delivered to UNMC at 4 weeks of age. From 4 weeks until assigned to an experimental program (≈8 weeks old), all mice were fed control diet.

Experimental Program.

All diets were purchased from Harlan Teklad and were designed with assistance of a Teklad certified dietician. WT and Kcnmb1^−/− had free access to water and were fed one of the following diets: control (0.6% K⁺, 0.32% Na⁺), low K⁺ - LK (0.1% K⁺, 0.32% Na⁺), or high K⁺ - HK (5.0% K⁺, 0.32% Na⁺). Additional groups from each strain of mice were given oral doses of eplerenone − E (100 mg/kg BW/day, MR antagonist) in conjunction with either control or HK diets (E and HK+E) or an equal volume of vehicle (V and HK+V). Eplerenone was purchased from Tocris Bioscience.

Metabolic Cages.

Before the experimental program, each animal was placed in a mouse metabolic cage (Nalagene) for a 3-day acclimation period. Urine samples were collected several times a day to prevent possible food or fecal contamination. Food and water intake was measured at the end of each day. After 3 days, the animals were returned to a group cage and placed on one of the diets for 10 days and then returned to a metabolic cage for another 2 days. Again, urine and feces were collected, volume and mass recorded, and food (experimental diet) and water consumptions measured. The animals next underwent terminal surgery.

Sample Collection, Preparation, and Analysis.

Animals were injected (IP) with Inactin (0.14 mg/g body weight). When unconscious, a tube was inserted in the trachea and catheters were inserted in the bladder (urine collection for creatinine measurements) and carotid artery. Blood samples were collected from the carotid artery for hematocrit, osmolality, [Na], [K], [aldo], and creatinine measurements. Plasma and urine electrolyte concentrations were measured using a flame photometer (Jenway, model PFP 7) while the osmolality of the samples were measured using a freezing point depression osmometer (Advanced Instruments, model 3250). Aldosterone EIA Kits (Cayman Chemical) were used following the manufacturer's protocol to measure plasma aldosterone concentrations. Serum creatinine levels were measured using the QuantiChrom Creatinine Assay Kit (BioAssay Systems) following the manufacturer's protocol.

The adrenal glands and kidneys were harvested, weighed, and then frozen in liquid nitrogen for later protein and RNA isolation. The methods of isolating total RNA and protein and determination of Kcnmb1 expression were performed using protocols, antibodies, and PCR primers as previously described by our lab (51).

Conscience Blood Pressure Measurements.

Conscience blood pressure measurements were made using the CODA-6 tail-cuff system (Kent Scientific) (52) with all mice undergoing at least 3 training sessions to acclimate the animal and prevent stress induced fluctuations in MAP. Blood pressure measurements were recorded before starting an animal on an experimental program and again after 10 days.

Statistics.

Significance between WT and Kcnmb1^−/− for a given treatment was determined by the t test for unpaired data (P < 0.05). Significant differences between treatment groups (control, HK, HK+E, and HK+V) for WT or Kcnmb1^−/− were determined by ANOVA with Student-Newman-Keuls (SNK; P < 0.05 considered significant). To determine whether a correlation existed between the plasma K concentration and the plasma aldosterone concentration the Pearson Correlation was used, with P < 0.05 considered significant.