Hypertension of Kcnmb1 Is Linked to Deficient K Secretion and Aldosteronism

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

P. Richard Grimm, Debra L. Irsik, Deann C. Settles, J. David Holtzclaw, and Steven. C. Sansom1

Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, 985850 Nebraska Medical Center, Omaha, NE 68198-5850

Communicated by Gerhard Giebisch, Yale University School of Medicine, New Haven, CT, May 4, 2009 (received for review February 24, 2009) Mice lacking the ␤1-subunit (gene, Kcnmb1; protein, BK-␤1) of the can stimulate K secretion—directly by high plasma K or indi- large Ca-activated K channel (BK) are hypertensive. This phenotype rectly by K-stimulated aldosterone production. Both BK and is thought to result from diminished BK currents in vascular smooth ROMK knockouts maintain a viable plasma K concentration, muscle where BK-␤1 is an ancillary subunit. However, the ␤1- suggesting that either channel can adequately secrete K in the subunit is also expressed in the renal connecting tubule (CNT), a absence of the other under normal dietary conditions (11, 15). segment of the aldosterone-sensitive distal nephron, where it A recent study showed that the plasma aldosterone concentra- associates with BK and facilitates K secretion. Because of the tion is elevated in BK-␣ knockout mice (16). An elevated plasma correlation between certain forms of hypertension and renal aldosterone concentration may be a compensatory side effect of defects, particularly in the distal nephron, it was determined defective BK-mediated K secretion. whether the hypertension of Kcnmb1؊/؊ has a renal origin. We Potassium adaptation is the enhanced capacity to secrete an found that Kcnmb1؊/؊ are hypertensive, volume expanded, and acute K load after a prolonged elevated K diet (17). The have reduced urinary K and Na clearances. These conditions are mechanism of K adaptation involves both an adrenal (18) and exacerbated when the animals are fed a high K diet (5% K; HK). renal component. In the adrenals, K adaptation includes an Supplementing HK-fed Kcnmb1؊/؊ with eplerenone (mineralocor- increase in aldosterone production per a given increase in plasma ticoid receptor antagonist) corrected the ﬂuid imbalance and more K concentration (19). Renal K adaptation involves increased than 70% of the hypertension. Finally, plasma [aldo] was elevated basolateral membrane Na-K-ATPase activity (20) and increased ؊/؊

in Kcnmb1 under basal conditions (control diet, 0.6% K) and apical membrane K channel activity in the distal nephron (21, 22). PHYSIOLOGY increased signiﬁcantly more than wild type when fed the HK diet. The present studies were performed to determine the role of We conclude that the majority of the hypertension of Kcnmb1؊/؊ BK-␤1 in K, Na, and fluid balance and whether the hypertension Ϫ Ϫ is due to aldosteronism, resulting from renal potassium retention of Kcnmb1 / results from enhanced K adaptation with over- and hyperkalemia. production of aldosterone by the adrenals. Results show that Kcnmb1Ϫ/Ϫ are beset with aldosteronism that is exacerbated adrenal medulla ͉ BK ͉ eplerenone ͉ mineralcorticoid ͉ volume expansion with a high K diet. The elevated aldo concentration is due to an adrenal gland that is hypersensitive to an elevated plasma K ␣ ␤ ypertension, a harbinger of stroke and congestive heart concentration, which is explained by reduction in BK- / 1- Hfailure, is the most prevalent chronic disorder in the West- mediated K secretion in the CNT. ern Hemisphere. Most cases of hypertension are of unknown Results origin and therefore categorized as essential hypertension. How- ؊/؊ Ϫ/Ϫ ever, approximately 10% of essential hypertensives are subcat- K and Na Handling in Kcnmb1 . Experiments with Kcnmb1 egorized as having normokalemic aldosteronism, identified by a were performed to determine the role of Kcnmb1 in the renal normal plasma K concentration despite a normal-to-high plasma handling of K and Na when consuming a high K diet and to aldosterone concentration (1–3). The origins of non- determine whether defective K secretion is the result of reduced eplerenone-sensitive K excretion. As shown in Fig. 1A, the adenomatous forms of normokalemic aldosteronism are not Ϫ/Ϫ understood, but this phenotype suggests a malfunction of the plasma [K] was significantly elevated in Kcnmb1 with a value Ϯ n ϭ Ϯ K-aldosterone feedback control axis. of 4.39 0.02 mM ( 11) vs. wild type (WT) (4.08 0.05 mM; n ϭ 7) when mice were on a control diet. When mice were placed The large Ca-activated K channel (BK) is considered an on a high K diet (WT-HK and Kcnmb1Ϫ/Ϫ-HK), the plasma [K] important component in the regulation of vascular tone and increased significantly to 4.31 Ϯ 0.09 mM (n ϭ 10) in WT-HK blood pressure. Like most K-selective channels, BK channels are and to 4.67 Ϯ 0.05 mM (n ϭ 11) in Kcnmb1Ϫ/Ϫ; however, the beset with ancillary partners. BK may contain 1 of 4 different plasma [K] was significantly greater in Kcnmb1Ϫ/Ϫ-HK than subunits, identified as BK-␤1 thru ␤4(Kcnmb1-4), which tailor WT-HK. The mineralocorticoid receptor antagonist, the properties of the BK-␣ pore (Kcnma1) to the specialized eplerenone, but not vehicle, significantly increased the plasma functional requirements of the cell. BK-␣/␤1 regulates vascular [K] to 5.96 Ϯ 0.04 mM (n ϭ 6) in WT-HK and to 6.00 Ϯ 0.02 mM smooth muscle contraction by coupling the outward K currents (n ϭ 6) in Kcnmb1Ϫ/Ϫ-HK. with Ca sparks—densely localized concentrations of Ca emitted As shown in Fig. 1B, the differences in K clearance between from ryanodyne-sensitive Ca channels near the plasma mem- Ϫ Ϫ WT and Kcnmb1 / mirrored the differences in plasma K brane (4, 5). Consistent with its role to mitigate myogenic tone, concentrations. On a regular diet, the K clearance of WT was mice with deletions of Kcnmb1 (Kcnmb1Ϫ/Ϫ) manifest mild but 108.5 Ϯ 2.3 mL/day (n ϭ 7), a value slightly but significantly significant hypertension (6–9). However, BK-␤1 is not only restricted to smooth muscle. BK-␣/␤1 also resides in the connecting tubules (CNT) of the mammalian kidney where it is used Author contributions: P.R.G., J.D.H., and S.C.S. designed research; P.R.G., D.L.I., D.C.S., and for K secretion in response to acute volume-expansion (10, 11). J.D.H. performed research; P.R.G., D.L.I., D.C.S., and S.C.S. analyzed data; and P.R.G. and This is relevant because long-term hypertension is most often S.C.S. wrote the paper. associated with a renal defect. The authors declare no conﬂict of interest. Hypertension resulting from defective K secretion would Freely available online through the PNAS open access option. manifest most prominently in mice consuming a high K diet. 1To whom correspondence should be addressed. E-mail: [email protected]. There are 2 K secretory channels—BK and Kcnj1 (ROMK) in This article contains supporting information online at www.pnas.org/cgi/content/full/ the CNT and CCD (12–14) and at least 2 ways that a high K diet 0904635106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0904635106 PNAS Early Edition ͉ 1of6 Downloaded by guest on October 2, 2021 A Plasma [Na+] 160 WT Kcnmb1-/-

150 ], mM ], + ** * 140 ¶

Plasma [Na Plasma 130

120 control HK HK+E HK+V

B Na+ clearance 5 WT Kcnmb1-/- Ŧ¶ 4 § Ŧ Ŧ Ŧ 3 *Ŧ * 2 * clearance, ml/day +

Na 1

0 control HK HK+E HK+V

Fig. 2. Bar plots illustrating the effects of HK and HKϩ EorHKϩV on mean Fig. 1. Bar plots illustrating the effects of a high K diet (HK) and HK with plasma Na concentration (A), and Na clearance (B) of WT and Kcnmb1Ϫ/Ϫ. All ϩ ϩ eplerenone (HK E) or vehicle (HK V) on plasma K concentration (A) and K symbols are the same as in Fig. 1. Ϫ Ϫ clearance (B) of WT and Kcnmb1 / . Values are means Ϯ SEM. *, P Ͻ 0.05 vs. WT using unpaired t test. ‡, P Ͻ 0.05 vs. control using ANOVA plus SNK test. ¶, P Ͻ 0.05 vs., HK using ANOVA plus SNK test. Control group was fed regular (n ϭ 6) and 3.62 Ϯ 0.10 mL/day (n ϭ 6), respectively. The Na ϭ mouse chow. § *, ‡, and ¶. clearance values in the vehicle-treated groups were not significantly different from the high K diet non-treated group. These Ϫ/Ϫ greater than that of Kcnmb1Ϫ/Ϫ (96.5 Ϯ 0.9 mL/day; n ϭ 11). results reveal enhanced Na reabsorption in Kcnmb1 that is This difference was more pronounced when mice were treated exaggerated by a high K diet. with a high K diet with WT-HK having a K clearance of 760.1 Ϯ Similar to the plasma Na analysis, the plasma osmolality (Fig. Ϫ Ϫ Ϯ ϭ 13.7 mL/day (n ϭ 10) and Kcnmb1 / -HK having a clearance of S1) was significantly elevated from 316 0.9 mOsm (n 7) in Ϫ Ϫ Ϯ ϭ Ϫ/Ϫ 598.0 Ϯ 10.7 mL/day (n ϭ 11). When WT-HK and Kcnmb1 / -HK WT to 321.5 0.8 mOsm (n 11) in Kcnmb1 . This were treated with eplerenone, the K clearances were reduced to difference was exaggerated when mice were treated with a high values of 401.6 Ϯ 7.1 mL/day (n ϭ 6) and 373.7 Ϯ 4.7 mL/day, K diet—the plasma osmolality was 317.7 Ϯ 0.6 mOsm (n ϭ 10) respectively (n ϭ 6). Vehicle-treated controls were not signifi- in WT-HK and was significantly greater (324.5 Ϯ 0.7 mOsm; n ϭ Ϫ Ϫ cantly different from WT-HK and Kcnmb1Ϫ/Ϫ-HK. 11) in Kcnmb1 / -HK. Eplerenone reduced the plasma osmo- Ϫ Ϫ Fig. 2 shows the plasma [Na] (A) and Na clearance (B) for WT lalities of WT-HK and Kcnmb1 / -HK to not significantly and Kcnmb1Ϫ/Ϫ under conditions of a control and high K diet different values of 314.2 Ϯ 0.7 mOsm (n ϭ 6) and 314.2 Ϯ 0.6 with and without eplerenone or vehicle treatment. On regular mOsm (n ϭ 6), respectively. diets, the plasma [Na] was elevated significantly from a value of As shown in the Table S1, the plasma creatinine levels were 136.7 Ϯ 0.6 mM (n ϭ 7) in WT to a value of 141 Ϯ 1.0 mM in not significantly different when all groups were compared with Ϫ Ϫ Kcnmb1Ϫ/Ϫ (n ϭ 11). On a high K diet, the plasma [Na] for WT the ANOVA and SNK test. That WT and Kcnmb1 / have the was 137.5 Ϯ 1.2 mM (n ϭ 10), a value slightly, but significantly same glomerular filtration rates was shown previously by deter- less than the value of 140.8 Ϯ 0.8 mM (n ϭ 11) for Kcnmb1Ϫ/Ϫ. mining GFR with inulin measurements (23). Therefore, the When the mice on a high K diet were treated with eplerenone, relative clearance values for Na and K also reflect the fractional the plasma [Na] decreased to values of 135.7 Ϯ 0.5 mM (n ϭ 6) excretions for each group. for WT and 136.4 Ϯ 0.6 mM (n ϭ 6) for Kcnmb1Ϫ/Ϫ. When treated with vehicle, Kcnmb1Ϫ/Ϫ-HK exhibited a slight but Extracellular Volume Status. The results of Fig. 3 show that Ϫ Ϫ significant increase in plasma [Na] compared to WT-HK. Kcnmb1 / were retaining fluid, especially when treated with a The Na clearance values are shown in Fig. 2B. On a control high K diet. Extracellular volume expansion was evaluated by the diet, the Na clearance for WT was 1.87 Ϯ 0.05 mL/day (n ϭ 7), hematocrit (Hct) in combination with the change in body weight. a value slightly, but significantly greater than that of Kcnmb1Ϫ/Ϫ As shown, the Hct was significantly reduced from a value of (1.69 Ϯ 0.03 mL/day; n ϭ 11). This difference was more 45.8 Ϯ 0.3% (n ϭ 7) in WT to 41.9 Ϯ 0.2% (n ϭ 11) in pronounced when mice were fed a high K diet; the Na clearance Kcnmb1Ϫ/Ϫ. On a high K diet, the Hct was further reduced from for WT-HK was 3.08 Ϯ 0.04 mL/day (n ϭ 10), a value signifi- a value of 45.7 Ϯ 0.4% (n ϭ 10) in WT to 37.4 Ϯ 0.4 (n ϭ 11) cantly greater than the value for Kcnmb1Ϫ/Ϫ-HK (2.60 Ϯ 0.06 in Kcnmb1Ϫ/Ϫ. When the mice on a high K diet were treated with mL/day; n ϭ 11). Eplerenone treatment increased the Na eplerenone, the Hct in WT and Kcnmb1Ϫ/Ϫ were not signifi- clearance in WT and Kcnmb1Ϫ/Ϫ to values of 4.04 Ϯ 0.11 mL/day cantly different with values of 49.7 Ϯ 0.5% (n ϭ 6) and 50.2 Ϯ

2of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0904635106 Grimm et al. Downloaded by guest on October 2, 2021 55 WT Kcnmb1-/- Ŧ¶ Ŧ¶ 50

45 * Ŧ Ŧ 40 * * Hematocrit, % Hematocrit,

30 control HK HK+E HK+V

Fig. 3. Bar plots illustrating the effects of HK and HKϩ EorHKϩV on mean hematocrit (Hct.) of WT and Kcnmb1Ϫ/Ϫ. All symbols are the same as in Fig. 1.

0.5% (n ϭ 6), respectively. The Hct values for the vehicle-treated group were not different from the non-treated mice on a high K diet. As shown in Fig. S2, WT gained 0.23 Ϯ 0.03 g (n ϭ 7), an amount not significantly different from the weight gained by Kcnmb1Ϫ/Ϫ (0.18 Ϯ 0.03 g; n ϭ 11) on a control diet. On a high K diet, WT gained 0.42 Ϯ 0.02 g (n ϭ 10); however, Kcnmb1Ϫ/Ϫ gained substantially more weight (3.3 Ϯ 0.3 g; n ϭ 11), consistent with extreme fluid overload. When the mice on a high K diet PHYSIOLOGY Fig. 4. Mean arterial blood pressure. (A) Bar plots illustrating the effects of were treated with eplerenone, WT lost 0.23 Ϯ 0.03 g (n ϭ 6) and Ϫ Ϫ Ϫ Ϫ eplerenone (E) or vehicle (V) on mean arterial blood pressure (MAP) of WT and / Ϯ ϭ / Ϫ Ϫ Kcnmb1 lost 0.65 0.04 g (n 6). WT and Kcnmb1 Kcnmb1 / .(B) Bar plots showing the effects of a low K diet (LK), HK and HKϩ treated with vehicle gained weight by similar amounts as WT-HK EorHKϩV on MAP in WT and Kcnmb1Ϫ/Ϫ. All symbols are the same as in Fig. Ϫ Ϫ and Kcnmb1 / -HK. The eplerenone-reversal of the reduced 1. Hct and weight gain in Kcnmb1Ϫ/Ϫ-HK demonstrates volume expansion due to aldosteronism. ϭ Ϫ/Ϫ (n 5) for WT. When treated with a high K diet, the aldo values Mean Arterial Blood Pressure. As shown in Fig. 4A, Kcnmb1 were 145 Ϯ 8 pg/mL (n ϭ 5) in WT but significantly greater exhibit mild hypertension with MAP ϭ 137 Ϯ 3mmHg(n ϭ 11) Ϫ Ϫ (183 Ϯ 11 pg/mL; n ϭ 5) in Kcnmb1 / . When the mice on a high while WT were 116 Ϯ 3mmHg(n ϭ 7). The MAP of WT was K diet were treated with eplerenone, the plasma [aldo] in unaffected by eplerenone (114 Ϯ 3mmHg;n ϭ 6) or vehicle. Ϫ Ϫ WT-HK increased to 187 Ϯ 13 pg/mL (n ϭ 5), a value slightly The MAP of Kcnmb1 / was decreased by eplerenone to a value Ϫ/Ϫ Ϯ ϭ of 122 Ϯ 2mmHg(n ϭ 6) but remained slightly but significantly less than that of Kcnmb1 -HK (216 12 pg/mL; n 5). above WT by 8 mm Hg. Aldosterone production is often related to the size of the To determine whether the hypertension of Kcnmb1Ϫ/Ϫ is adrenal gland, which hypertrophies with chronic stimulation and related to a K load, the MAP was determined with mice on a low, increased synthesis of aldosterone. As shown in Fig. S3, the mass normal and high K diet. As shown in Fig. 4B, the MAP of of the adrenal glands, normalized to prediet body weight, was Ϫ Ϫ Kcnmb1Ϫ/Ϫ on a low K diet was 122 Ϯ 5mmHg(n ϭ 10), a value slightly, but not significantly, greater in Kcnmb1 / on a control significantly greater than the value of 113 Ϯ 3mmHg(n ϭ 7) diet (0.201 Ϯ 0.008 mg/g BW; n ϭ 11) when compared with WT for WT on a normal K diet but less than the value of 135 Ϯ 5mm (0.192 Ϯ 0.007 mg/g BW; n ϭ 7). When placed on a high K diet, Hg (n ϭ 11) for Kcnmb1Ϫ/Ϫ treated with a normal K diet. When the adrenal mass of Kcnmb1Ϫ/Ϫ was 0.230 Ϯ 0.008 mg/g BW (n ϭ Kcnmb1Ϫ/Ϫ were treated with a high K diet, the MAP increased 11), a value significantly greater than that of WT (0.209 Ϯ 0.004 significantly to a value of 145 Ϯ 3mmHg(n ϭ 11), which was significantly above the MAP of 115 Ϯ 4mmHg(n ϭ 10) for Ϫ/Ϫ WT-HK. The increased MAP of Kcnmb1 -HK was reversed 250 by eplerenone to 122 Ϯ 6mmHg(n ϭ 6), a value that was still WT § Kcnmb1-/- 9 mm Hg (albeit insignificantly) above the WT-HK value of Ŧ Ŧ 113 Ϯ 7mmHg(n ϭ 6). The vehicle controls were not different 200 * Ŧ from WT-HK and Kcnmb1Ϫ/Ϫ-HK. These results show that the Ŧ * Ϫ Ϫ majority of the hypertension of Kcnmb1 / is the result of 150 * Ŧ deficient K handling of a dietary K load. The elevated plasma [K] stimulates aldosterone, causing Na retention. 100

Aldosterone. High dietary K exaggerated and eplerenone corrected the conditions of volume expansion and hypertension in Plasma [aldo], pg/ml 50 Kcnmb1Ϫ/Ϫ. These results are consistent with primary aldosteronism. The aldosterone (aldo) values for WT and Kcnmb1Ϫ/Ϫ 0 on a normal and high K diet are shown in Fig. 5. On a control control HK HK+E HK+V Ϫ/Ϫ diet, the plasma [aldo] of Kcnmb1 was 147 Ϯ 4 pg/mL (n ϭ Fig. 5. Bar plots illustrating the effects of HK and HKϩ EorHKϩV on plasma 11), a value significantly greater than the value of 107 Ϯ 3 pg/mL [aldo] of WT and Kcnmb1Ϫ/Ϫ. All symbols are the same as in Fig. 1.

Grimm et al. PNAS Early Edition ͉ 3of6 Downloaded by guest on October 2, 2021 ABRT-PCR Western Blot Discussion The results of this study showed that genetic ablation of BK-␤1 WT Kcnmb1-/- WT Kcnmb1-/- 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 Kcnmb1Ϫ/Ϫ. The data indicate that the aldosteronism of Kc- nmb1Ϫ/Ϫ is the result of increasing plasma [K] due to diminished aldosterone-induced K secretion as well as increased aldo pro- BKβ1 duction 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 β-actin K secretory mechanisms. However, it has been difficult to distinguish the individual roles of BK and ROMK channels with ␤ Fig. 6. RT-PCR (A) and western blot (B) determination of BK- 1 expression respect to secreting K in direct response to elevated K or in mouse adrenal glands. (A) BK-␤1 mRNA is expressed at the correct size (561 Ϫ Ϫ increased aldosterone. In the CCD (27) and the CNT (28), bp) in WT but not in Kcnmb1 / negative control. (B) BK-␤1 protein (28 kDa) was expressed in adrenals of WT but not in Kcnmb1Ϫ/Ϫ negative control. 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 ϭ induced charybdotoxin-sensitive K secretion in the late distal mg/g; n 10). These results are consistent with the adrenal gland tubule of the mouse as assessed by micropuncture (30). However, Ϫ/Ϫ being the source of the elevated aldosterone in Kcnmb1 -HK. the micropuncture study did not distinguished between the direct effects of high plasma K and aldosterone as the mediator for ␣ Adrenal Expression of Kcnmb1. The BK- subunit has been local- enhancing BK-mediated K secretion. ized by immunohistochemistry to the adrenal medulla and The present results reveal a diminished K secretory response adrenal glomerulosa (24), and both types of cells have exhibited of Kcnmb1Ϫ/Ϫ to a high K diet. When eplerenone was given to BK currents (25, 26). However, the associated BK-␤ subunit has the mice on high K diets, the K secretory response was not been discovered. As shown by RT-PCR (Fig. 6A) and inhibited substantially more in WT than in Kcnmb1Ϫ/Ϫ. After western blot (Fig. 6B), BK-␤1 is expressed in mouse adrenal eplerenone treatment, the plasma K concentrations increased glands of WT but not Kcnmb1Ϫ/Ϫ. to a similar value in WT and Kcnmb1Ϫ/Ϫ. The K clearances of As shown in Fig. S4, Kcnmb1 was immunohistochemically WT and Kcnmb1Ϫ/Ϫ on high K diets with eplerenone treat- localized in the adrenal medulla of WT but not Kcnmb1Ϫ/Ϫ ment were nearly equivalent but still substantially elevated negative controls. compared with the values of these mice on regular diets. It can be assumed that the remaining K clearance that is not Sensitivity of Adrenal Glands to Plasma [K]. The large increase in eplerenone-sensitive is the high plasma K, eplerenone- aldosterone in response to a high K diet in Kcnmb1Ϫ/Ϫ suggests insensitive K clearance. Therefore, Kcnmb1Ϫ/Ϫ may not have an increased sensitivity of the glomerulosa cells to plasma [K]. a substantial defect in K adaptation by an aldosterone- The secretion of aldo by the adrenal glomerulosa is extremely independent, high plasma K-induced mechanism. responsive to small changes in the plasma [K]. The different When the K clearance values for the mice on high K diets plus sensitivities of the glomerulosa cells of WT and Kcnmb1Ϫ/Ϫ to eplerenone are subtracted from the K clearance values for the K were determined by plotting the plasma K vs. plasma aldo- mice on a high K diet (Fig. 1B), the remainder reveals that Ϫ/Ϫ sterone concentrations. As shown in Fig. 7, the slope of the Kcnmb1 has 37% less eplerenone-sensitive K secretion, Ϫ/Ϫ relation is 127 pg[aldo]/mM[K] in Kcnmb1Ϫ/Ϫ vs. 76 pg[aldo]/ leading to the conclusion that Kcnmb1 has defective renal mM[K] in WT. responsiveness to K-stimulated aldosterone. This result is consistent with a study showing that K excretion was impaired in BK-␣ knockout mice despite very high plasma aldosterone levels (16). Another study reported that aldosterone increased BK- 240 WT mediated K secretion in the colon (31). However, our finding is 220 seemingly in conflict with a recent study that reported that Kcnmb1-/- aldosterone did not increase BK-mediated K secretion in the 200 isolated rabbit cortical collecting ducts (CCD) (32). However, 180 aldosterone would not affect BK in the CCD, which does not 160 express luminal Kcnmb1 (10, 33). Moreover, in the aforemen- tioned study, aldosterone was increased by a low Na diet instead 140 of a high K diet as in the present study. A low Na diet evokes 120 mechanisms that primarily enhance Na reabsorption, whereas a high K diet induces pathways that primarily enhance K secretion.

Plasma [aldo] (pg/ml) Plasma 100 For example, a low Na diet, but not a high K diet, results in an 80 increase in angiotensin II, which is not increased by a high K diet. 4.04.24.44.64.85.0 Angiotensin II increases ENaC-mediated Na reabsorption in the + Plasma [K ] (mM) CCD (34) but might inhibit or counteract the effects of aldosterone to enhance BK-mediated K secretion. Fig. 7. Plots of the plasma [K] vs. plasma [aldo] for WT (closed circles) and Kcnmb1Ϫ/Ϫ (open circles). The r values (correlation coefﬁcients) were highly ؊/؊ Ϫ/Ϫ signiﬁcant for WT (P Ͻ 0.05) and Kcnmb1Ϫ/Ϫ (P Ͻ 0.05). The slopes of the Hypernatremia and Hyperosmolality of Kcnmb1 . Kcnmb1 ex- relations for plasma K vs. aldo concentrations are 74 and 127 for WT and hibited mild hypernatremia and hyperosmolality. This condition Kcnmb1Ϫ/Ϫ, respectively. was exacerbated by a high K diet and reversed by eplerenone,

4of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0904635106 Grimm et al. Downloaded by guest on October 2, 2021 consistent with primary aldosteronism. The increase in plasma (43–45). In humans, an elevation of 0.2 mM in plasma K Na and osmolality is interesting considering that the plasma concentration increases aldo production by 46% over baseline osmolality is usually tightly regulated by vasopressin secretion in (43). In comparison, the present study reports an increase in steep response to small changes in plasma osmolality. However, plasma [aldo] of 15% over baseline per 0.2 mM increase in subjects with primary aldosteronism are known to have plasma plasma [K]. Thus, the K-stimulated aldo response may be less Na and osmolar concentrations slightly higher than normal (35) sensitive in mice than in humans. as found in the present study. The hypernatremia and hyperos- In the Kcnmb1Ϫ/Ϫ, the plasma [K] vs. plasma [aldo] relation molality is probably the result of resetting the osmostat (36). reveals a K-stimulated aldo response with a slope that is a 72% more than the slope of WT. Therefore, the glomerulosa cell aldo Hypertension of Kcnmb1؊/؊. The mild hypertension of Kcnmb1Ϫ/Ϫ secretion may be more sensitive to plasma [K] in Kcnmb1Ϫ/Ϫ.It has been considered to result from increased vascular reactivity is unclear whether the increase in Kcnmb1Ϫ/Ϫ aldosterone due to a diminished Ca-feedback response of the BK-␣/␤1in production results from the absence of the BK-␤1 subunit in vascular smooth muscle cells (7, 9). However, hypertension is chromaffin cells or is the result of adaptation by glomerulosa often comprised of a combination of increased renal Na reab- cells to chronically elevated plasma K concentrations. sorption and enhanced vascular reactivity. The MAP of Kc- Ϫ Ϫ nmb1 / was elevated by 22 mm Hg, compared with WT. After Perspectives and Significance. That the expression of BK-␣ and eplerenone treatment, MAP of Kcnmb1Ϫ/Ϫ was only 8 mm Hg BK-␤1decreases with aging (49, 50) may partially explain the more than WT. These observations suggest that K-stimulated increased prevalence of hypertension and the dysfunctional aldosteronism may be the primary cause of hypertension, ac- regulation of fluid balance in the aged population. Moreover, the counting for as much 70%, in Kcnmb1Ϫ/Ϫ. importance of the BK-␤1 subunit in blood pressure regulation That the majority of hypertension of Kcnmb1Ϫ/Ϫ is related to has recently been highlighted with the identification of polymor- defective K secretion is evidenced by the finding that MAP is phisms in human KCNMB1. Gain-in-function mutations have nearly normalized when Kcnmb1Ϫ/Ϫ are placed on a low K diet been shown to confer protection against developing hyperten- and exaggerated when placed on a high K diet. The hypertension sion (46, 47). Another BK-␤1 mutation was recently shown to of Kcnmb1Ϫ/Ϫ on a high K diet, which was even more elevated result in a loss-in-function of BK activity in alveolar smooth (by 31 mm Hg) compared to WT on a high K diet, was reduced muscle cells of males resulting in a decrease in forced airway flow by eplerenone to a value that was 8 mM more than WT on a high (48). However, the blood pressure of this asthmatic population K diet. The MAP values for Kcnmb1Ϫ/Ϫ that were either treated is unknown. PHYSIOLOGY with eplerenone or maintained on a low K diet ranged from 5–8 Based on the present results, aldosterone-inhibiting agents mm Hg above WT. Thus, the absence of BK-␤1 in vascular may be beneficial in treating a small minority of essential- smooth muscle accounts for a slight elevation in MAP. However, hypertension patients having normal plasma K and elevated primary aldosteronism, due to the inability to maximally secrete plasma aldosterone levels. Furthermore, such hypertensive pa- K, accounts for the majority of the hypertension in Kcnmb1Ϫ/Ϫ. tients, although a relatively small minority, would exhibit exacerbated hypertension when consuming a K-rich diet. This spe- Role of the Adrenal BK in Regulating Aldosterone Production. We cific phenotype would be clearly different from the well- found that the BK-␤1 subunit is expressed in the adrenal glands described blood pressure lowering effects that a high K diet has of mouse by PCR and western blot. The BK␣ subunit has been on the general population. identified in the mouse adrenal cortex and the adrenal medulla In summary, these results indicate that the majority of the with immunohistochemical staining (37), findings consistent hypertension in Kcnmb1Ϫ/Ϫ is due to aldosteronism resulting with previous patch-clamp studies revealing the presence of BK from renal potassium retention and hyperkalemia. It remains to currents in adrenal glomerulosa cells (25, 26) and the chromaffin be determined whether similar forms of essential hypertension cells of the adrenal medulla (38). in humans results from polymorphisms in BK-␤1 and whether That the BK-␤1 subunit is localized in the adrenal medulla such polymorphisms are the primary cause of hypertension. (Fig. S4) is consistent with studies showing that BK channels of These results may be important for establishing diuretic thera- chromaffin cells modulate the release of catecholamines, which pies in certain individuals with essential hypertension. modulate via adrenergic receptors the release of aldosterone. The role of the BK-␣/␤1 in regulating catecholamine release may Materials and Methods be similar to its role in other tissues, having an effect to Animals. All animals were maintained under the conditions approved by the hyperpolarize the membrane potential and mitigate the Ca- Institutional Animal Care and Use Committee of the University of Nebraska mediated release of catecholamines (39, 40). Catecholamine- Medical Center. Two groups of male mice were used: wild-type mice Ϫ WT Ϫ Ϫ induced elevations in glomerulosa cAMP levels stimulate Ca (C57BL/6) purchased from Charles River and Kcnmb1 / [generated by Bren- ner et al. (7) which were bred in the Animal Care Facility at the University of influx via L-type Ca channels (41, 42), leading to increased aldo Ϫ/Ϫ Ϫ/Ϫ Nebraska Medical Center]. Kcnmb1 were weaned from their mother when production and release. That the adrenal glands of Kcnmb1 -HK 4 weeks old. WT were delivered to UNMC at 4 weeks of age. From 4 weeks until are hypertrophied is consistent with increased enzyme activity assigned to an experimental program (Ϸ8 weeks old), all mice were fed and production of aldosterone, explaining the enhanced control diet. [aldo]. The present study supports the results of Sausbier et al. who Experimental Program. All diets were purchased from Harlan Teklad and were first showed that aldosterone levels were increased in Kc- designed with assistance of a Teklad certiﬁed dietician. WT and Kcnmb1Ϫ/Ϫ nma1Ϫ/Ϫ (37). That study did not determine whether the ele- had free access to water and were fed one of the following diets: control (0.6% vated aldosterone had a role in the increased MAP. However, in Kϩ, 0.32% Naϩ), low Kϩ - LK (0.1% Kϩ, 0.32% Naϩ), or high Kϩ - HK (5.0% Kϩ, ϩ the Sausbier study, the aldo levels on a normal diet were much 0.32% Na ). Additional groups from each strain of mice were given oral doses Ϫ/Ϫ of eplerenone Ϫ E (100 mg/kg BW/day, MR antagonist) in conjunction with greater (2.5-fold) in the Kcnma1 compared with a 40% ϩ Ϫ/Ϫ Ϫ/Ϫ either control or HK diets (E and HK E) or an equal volume of vehicle (V and elevation in aldo in Kcnmb1 . The aldo levels for Kcnma1 HKϩV). Eplerenone was purchased from Tocris Bioscience. 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 Metabolic Cages. Before the experimental program, each animal was placed in Ϫ/Ϫ Kcnmb1 . a mouse metabolic cage (Nalagene) for a 3-day acclimation period. Urine Less than 1-mM changes in plasma K concentration regulate samples were collected several times a day to prevent possible food or fecal aldo release independent of its release induced by ANGII contamination. Food and water intake was measured at the end of each day.

Grimm et al. PNAS Early Edition ͉ 5of6 Downloaded by guest on October 2, 2021 After 3 days, the animals were returned to a group cage and placed on one of formed using protocols, antibodies, and PCR primers as previously described the diets for 10 days and then returned to a metabolic cage for another 2 days. by our lab (51). Again, urine and feces were collected, volume and mass recorded, and food (experimental diet) and water consumptions measured. The animals next Conscience Blood Pressure Measurements. Conscience blood pressure measure- underwent terminal surgery. ments 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 Sample Collection, Preparation, and Analysis. Animals were injected (IP) with prevent stress induced fluctuations in MAP. Blood pressure measurements Inactin (0.14 mg/g body weight). When unconscious, a tube was inserted in the were recorded before starting an animal on an experimental program and trachea and catheters were inserted in the bladder (urine collection for again after 10 days. creatinine measurements) and carotid artery. Blood samples were collected from the carotid artery for hematocrit, osmolality, [Na], [K], [aldo], and Statistics. Significance between WT and Kcnmb1Ϫ/Ϫ for a given treatment creatinine measurements. Plasma and urine electrolyte concentrations were was determined by the t test for unpaired data (P Ͻ 0.05). Significant measured using a flame photometer (Jenway, model PFP 7) while the osmo- differences between treatment groups (control, HK, HKϩE, and HKϩV) for Ϫ/Ϫ lality of the samples were measured using a freezing point depression os- WT or Kcnmb1 were determined by ANOVA with Student-Newman- Ͻ mometer (Advanced Instruments, model 3250). Aldosterone EIA Kits (Cayman Keuls (SNK; P 0.05 considered significant). To determine whether a Chemical) were used following the manufacturer’s protocol to measure correlation existed between the plasma K concentration and the plasma aldosterone concentration the Pearson Correlation was used, with P Ͻ 0.05 plasma aldosterone concentrations. Serum creatinine levels were measured considered significant. using the QuantiChrom Creatinine Assay Kit (BioAssay Systems) following the manufacturer’s protocol. ACKNOWLEDGMENTS. This work was supported by National Institutes of The adrenal glands and kidneys were harvested, weighed, and then frozen Diabetes and Digestive and Kidney Diseases Grants RO1 DK49461 and RO1 in liquid nitrogen for later protein and RNA isolation. The methods of isolating DK73070 (to S.C.S.), American Heart Association-Heartland Affiliate Fellow- total RNA and protein and determination of Kcnmb1 expression were per- ship 0610059Z (to P.R.G.), and supplemental DK073070–03S1 (D.L.I.).

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