BASIC RESEARCH www.jasn.org Intercalated Cell BK-␣/␤4 Channels Modulate Sodium and Potassium Handling During Potassium Adaptation J. David Holtzclaw, P. Richard Grimm, and Steven C. Sansom Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, Nebraska ABSTRACT The large-conductance, calcium-activated potassium (BK) channels help eliminate potassium in mammals consuming potassium-rich diets. In the distal nephron, principal cells contain BK-␣/␤1 channels and intercalated cells contain BK-␣/␤4 channels. We studied whether BK-␤4–deficient mice Ϫ Ϫ (Kcnmb4 / ) have altered renal sodium and potassium clearances compared with wild-type mice when fed a regular or potassium-rich diet for ten days. We did not detect differences in urinary flow or fractional excretions of potassium (FEK) or sodium (FENa) between Kcnmb4-deficient and wild-type mice fed a regular diet. However, a potassium-rich diet led to Ͼ4-fold increases in urinary flows for both groups of mice, although Kcnmb4-deficient mice exhibited less urinary flow, higher plasma potassium concentration, more fluid retention, and significantly lower FEK and FENa than wild-type mice despite similar plasma aldosterone levels. Immunohistochemical analysis revealed increased basolateral Na-K- ATPase in principal cells of all potassium-adapted mice, but expression of Na-K-ATPase in intercalated cells was Ͼ10-fold lower. The size of intercalated cells reduced and luminal volume increased among potassium-adapted wild-type but not Kcnmb4-deficient mice. Paradoxically, this led to increased urinary fluid velocity in potassium-adapted Kcnmb4-deficient mice compared with wild-type mice. Taken to- gether, these data suggest that BK-␣/␤4 channels in intercalated cells reduce cell size, increasing luminal volume to accommodate higher distal flow rates during potassium adaptation. These changes streamline flow across the principal cells, producing gradients more favorable for potassium secretion and less favorable for sodium reabsorption. J Am Soc Nephrol 21: 634–645, 2010. doi: 10.1681/ASN.2009080817 A high-K diet is a natural diuretic,1 causing decreased ate Na and water reabsorption and K secretion, Na and Cl reabsorption in the thick ascending limb and the ICs mediate acid/base transport. Under (TAL) because of medullary recycling and high inter- normal conditions, K secretion by the PCs is me- stitial K levels.2 The decreased Na transport in the diated primarily by the ROMK channel.7 How- medullary TAL disrupts the concentrating mecha- ever, flow-induced K secretion in the distal nism, thereby increasing flow to the distal nephron.2 nephron is mediated by BK.4,8,9 The high deliveries of Na to the connecting tubules BK are a complex of pore-forming ␣ and acces- (CNT) and cortical collecting ducts (CCD) is ex- sory ␤ subunits (BK-␣/␤). The PCs of the CNT changed for K, and the increased flow stimulates K secretion to maximize the amount of K secreted to Na absorbed. The renal outer medullary kidney K chan- Received August 11, 2009. Accepted December 11, 2009. nel (ROMK) and the large conductance, calcium-ac- Published online ahead of print. Publication date available at www.jasn.org. tivated K channels (BK) in the CNT and CCD serve to eliminate K during K adaptation.3–6 Correspondence: Dr. Steven C. Sansom, Department of Cellular and Integrative Physiology, 985850 Nebraska Medical Center, In the distal nephron, the CNT and CCD con- Omaha, NE 68198-5850. Phone: 402-559-2919; Fax: 402-559- sist of two epithelial cell types: principal cells 4438; E-mail: [email protected] (PCs) and intercalated cells (ICs). The PCs medi- Copyright ᮊ 2010 by the American Society of Nephrology 634 ISSN : 1046-6673/2104-634 J Am Soc Nephrol 21: 634–645, 2010 www.jasn.org BASIC RESEARCH contain BK-␣/␤1 and are well equipped with an abundance of significantly lower compared with KA WT (P Ͻ 0.001) and Ϫ Ϫ basolateral Na-K-ATPase to secrete K in K-adapted (KA) con- control Kcnmb4 / (0.39 Ϯ 0.02%, P Ͻ 0.02). The decrease in ditions. However, the preponderance of BK-␣ reside in ICs10,11 Na excretion indicated a large increase in Na reabsorption in along with the ancillary subunit, BK-␤4 (gene: Kcnmb4).12 A the distal nephron. Ϫ Ϫ study has indicated that BK of ICs are regulated by mitogen- As expected, the KA WT and KA Kcnmb4 / exhibited a activated protein kinase to prevent K reabsorption during de- significantly greater fractional excretion of K (FEK) compared Ϫ/Ϫ mand for maximal K secretion.13 It has also been proposed that with controls (Figure 1B). However, FEK for KA Kcnmb4 Ϯ BK-␣/␤4 in ICs have a role in flow-mediated K secretion. (93.5 10.8) was significantly attenuated compared with KA Ϯ Ͻ If high flow induces BK-mediated K secretion, then BK-␣/␤4 WT (162.6 10.8%, P 0.001). There was no statistical dif- Ϫ/Ϫ of ICs must have a role. It has been shown that the shear stress ference in FEK between control WT and control Kcnmb4 Ϯ Ϯ ϭ produced by high flow causes a transient increase in intracellular (23.3 1.5% versus 24.0 0.4%, P 0.967). There was no Ca to levels that may activate BK. Indeed, ICs, which protrude into statistical difference in GFR between groups (data not shown). the lumens of the CNT and CCD, are particularly subjected to The plasma Na and K concentrations are shown in Figure 1, C and D, respectively. The plasma Na levels were not signifi- shear stress forces that may elevate intracellular Ca. However, a cantly different among the four treatment groups, with values transient Ca activation of BK would not produce the sustained K varying from 137 Ϯ 1 mM for control WT to 139 Ϯ 2 mM for transport required for long-term K adaptation. Ϫ Ϫ KA Kcnmb4 / (Figure 1C). The plasma K concentrations of That BK-␣/␤4 of ICs may not produce sustained K secre- Ϫ Ϫ control WT and control Kcnmb4 / were not different (Fig- tion is also indicated by the paucity of Na-K-ATPase. K adap- ure 1D). The plasma K concentrations for KA WT was slightly 14 15–17 tation or mineralocorticoid treatment increases the but insignificantly (P ϭ 0.085) increased compared with con- quantity of basolateral Na-K-ATPase of mammalian collecting trol WT (4.33 Ϯ 0.11 versus 4.05 Ϯ 0.05 mM). The plasma K ducts to maintain a favorable electrochemical driving force for concentration of KA Kcnmb4Ϫ/Ϫ (4.75 Ϯ 0.12 mM) was sig- K secretion. However, because ICs have considerably less Na- nificantly greater than Kcnmb4Ϫ/Ϫ control (4.03 Ϯ 0.02 mM; K-ATPase than PCs,18–21 ICs may not have an adequate K P Ͻ 0.001) and KA WT (P Ͻ 0.005). This increase in plasma K ␣ ␤ 22 source to sustain K secretion via BK- / 4. Still, Na-K-AT- concentration reflected the attenuated K secretory response of Pase has not been quantified in ICs in KA conditions. If the KA Kcnmb4Ϫ/Ϫ. BK-␣/␤4 were directly involved in the increased K transport Urinary output and water intake (by drinking) are shown in associated with K adaptation, then it would be expected that Figure 2. As shown in Figure 2A, the urinary output of control the Na-K-ATPase in ICs would increase summarily to PCs.23 Kcnmb4Ϫ/Ϫ (1.01 Ϯ 0.02 ml/d) was not significantly different Ϫ Ϫ To this end, we determined whether Kcnmb4 / have al- from control WT (1.03 Ϯ 0.03 ml/d). The urinary output in- tered renal K and Na excretions compared with wild type (WT) creased in KA WT by nearly 5-fold to 4.94 Ϯ 0.15 ml/d. The under control and KA conditions. Evidence from KA Kc- urinary output increased in KA Kcnmb4Ϫ/Ϫ to 4.32 Ϯ 0.14 nmb4Ϫ/Ϫ indicates that the role of BK-␣/␤4 in ICs is to reduce ml/d; however, this value was significantly attenuated com- cell size, thereby increasing tubular fluid volumes to accom- pared with KA WT. modate the higher distal flow rates of KA mice. By reducing the As shown in Figure 2B, the water consumption of control protrusion of ICs into the lumen and increasing tubular vol- KA (1.93 Ϯ 0.07 ml/d) was not different from control WT ume, flow will be more streamlined across the PCs and a more (1.87 Ϯ 0.19 ml/d). The water consumption of KA WT and KA Ϫ/Ϫ Ϫ/Ϫ a favorable chemical gradient for K secretion and less favorable Kcnmb4 increased substantially; however, KA Kcnmb4 Ϯ gradient for Na reabsorption will be produced. consumed significantly more water (7.70 0.17 ml/d) com- pared with KA WT (6.42 Ϯ 0.14 ml/d). Therefore, consistent with Na retention, KA Kcnmb4Ϫ/Ϫ consumed more water and had less urinary output compared with KA WT. RESULTS Figure 3 shows volume status as related to weight changes (Figure 3A) and hematocrits (Figure 3B) for the four treatment Na, K, and Volume Balance groups. The KA WT gained slightly more weight (0.42 Ϯ Experiments were performed to determine the difference in Na 0.03 g) than control WT (0.22 Ϯ 0.03 g). However, KA Kc- and K handling and volume balance in four groups of mice: nmb4Ϫ/Ϫ gained 5.2 Ϯ 0.2 g, which was significantly greater control diet wild type (control WT), control diet Kcnmb4Ϫ/Ϫ than the weight gain of KA WT (P Ͻ 0.001) and of control (control Kcnmb4Ϫ/Ϫ), K-adapted wild type (KA WT), and K- Kcnmb4Ϫ/Ϫ (0.21 Ϯ 0.02 g; P Ͻ 0.001).