Large-conductance Ca2+-activated K+ channels (BKCa) are composed of pore-forming α-subunits and one of four accessory β-subunits. The β1-subunit, found predominantly in smooth muscle, modulates the Ca2+ sensitivity and pharmacological properties of BKCa. BKCa-β1 null mice (Mβ1−/−) are moderately hypertensive, consistent with the role of BKCa in modulating intrinsic vascular tone. Because BKCa are present in various renal cells including the mesangium and cortical collecting ducts, we determined whether fluid or electrolyte excretion was impaired in Mβ1−/−under euvolemic, volume-expanded, or high-salt diet conditions. Under euvolemic conditions, no differences in renal function were found between Mβ1−/− and Mβ1+/+. However, glomerular filtration rate (GFR) and fractional K+excretion were significantly impaired in Mβ1−/− in response to acute volume expansion. In contrast, Mβ1−/−exhibited enhanced Na+ excretion and fractional Na+ excretion responses to acute volume expansion. Differences in renal function between Mβ1+/+ and Mβ1−/− were not observed when chronically treated with a high-salt diet. These observations indicate that the β1-subunit of BKCa contributes to the increased GFR that accompanies an acute salt and volume load and raises the possibility that it is also involved in regulating K+ excretion under these conditions.
- large-conductance, calcium-activated potassium channels
- maxi K channel
- glomerular filtration rate
- volume expansion
- potassium excretion
large-conductance, ca2+ -activated potassium channels (BKCa) are composed of both pore-forming α- and accessory β-subunits. At least four distinct β-subunits, each with a tissue-specific distribution, have been described. When the β1-subunit, found primarily in smooth muscle cells, is expressed with the α-subunit, the voltage and calcium sensitivities of BKCa are enhanced (2). Conversely, BKCa in cerebral artery myocytes from β1 knockout mice (Mβ1−/−) have a reduced open probability at a given voltage and Ca2+ concentration (2). In addition, these mice also have deficient regulation of tone in visceral smooth muscle, such as in the urinary bladder (23). In vascular smooth muscle, a lack of the β1-subunit and the resulting low open probability of BKCa may cause a reduced hyperpolarizing feedback response to contractile agents, resulting in greater vascular tone and generalized hypertension (16). Indeed, the mean arterial pressure (MAP) in Mβ1−/− of the C57BL/6 strain is elevated by ∼20 mmHg (2).
Whereas hypertension can originate from elevated intrinsic vascular tone, MAP is regulated by multiple complex mechanisms that include baroreceptor and renal feedback reflexes, such as pressure-natriuresis and renin release. Indeed, polymorphisms in the human BKCa-β1 have been shown to correlate with baroreflex and arterial pressure regulation (7).
BKCa have been reported in several renal cells, including mesangial cells as well as epithelial cells of the cortical collecting duct (11), proximal tubule (10), and thick ascending limb (20). However, the function of BKCa in these cells in relation to whole animal electrolyte balance has not been determined. In this study, we designed experiments to determine the significance of the β1-subunit with respect to fluid and electrolyte balance. Although the open probability of BKCa is very low under basal conditions, these channels are important mediators of compensatory hyperpolarizing responses after agonist stimulation. Therefore, we examined Mβ1−/−under both euvolemic and volume-expanded conditions, in which a variety of possible influences including increased circulating atrial natriuretic peptide (ANP), stretch, intracellular Ca2+, and increased flow of plasma and filtrate could demand proper function of the renal BKCa.
All experiments were performed under the guidelines of the Institutional Animal Care and Use Committee of the University of Nebraska Medical Center. This study utilized Mβ1−/−mice (with a homogeneous C57BL/6 background) generated by Brenner et al. (2) and C57BL/6 control mice (Mβ1+/+) of both sexes, which were approximately 3 mo of age. Mice received standard chow containing 0.4% NaCl and water ad libitum. Some mice received a high-salt (8% NaCl) diet for 2–3 wk before surgery.
Surgical and clearance procedures were performed as previously described by Wang et al. (30). In brief, mice were anesthetized with Inactin [0.14 mg/g body wt (BW)] and kept at a body temperature near 36°C, using a heat lamp. As required, additional doses of Inactin were used to maintain anesthesia. A tracheostomy was performed using polyethylene (PE)-50 tubing, and the end of the tracheal cannula was exposed to a stream of oxygen-rich air. The left external jugular vein was cannulated with PE-10 tubing for the infusion of fluids, and the bladder was cannulated with PE-50 tubing for urine collection. The right common carotid artery was cannulated with PE-10 tubing for arterial pressure measurements and blood sampling. Arterial pressure was monitored continually and recorded at 5-min intervals. Urine was collected and stored under mineral oil. Physiological saline solution (PSS) containing (in mM) 135 NaCl, 5.0 KCl, 2.0 MgCl2, 1.0 CaCl2, and 10 HEPES as well as 10 μg/ml FITC-inulin was infused at a rate of either 0.4 (euvolemic) or 2.0 ml · h−1 · 25 g BW−1 (volume expanded). Because FITC-inulin is light sensitive, all syringes, tubing, and collection vials were protected from light. The length of the equilibration period was 2 h for the euvolemic treatment and 1 h for the volume-expansion treatment. After an equilibration period, a blood sample (∼20 μl) was taken and urine was collected for a 30-min period. At the end of the period, a larger plasma sample was taken for measurements of plasma Na+ ([Na+]), K+([K+]), and inulin concentrations ([inulin]). Urinary volume was determined gravimetrically, and the [inulin] of the two plasma samples was averaged for calculation of the glomerular filtration rate (GFR).
Measurements of [Na+], [K+], and [FITC-inulin] in urine and plasma.
After the completion of an experiment, urine and plasma samples were stored in the dark at −70°C. [Na+] and [K+] in urine and plasma were measured using an Instrumentation Laboratory 443 Flame Photometer. Plasma samples were run in duplicate. Within 1 wk of the experiment, [FITC-inulin] was measured using a fluorescent microplate reader (Cary Eclipse Fluorescence Spectrophotometer, Varian) as described by Lorenz and Gruenstein (17-19). For each analysis of FITC-inulin samples, a standard curve was generated and used for calculating [FITC-inulin]. All standards and urine samples were run in triplicate; most plasma samples were run in duplicate. Very small blood samples (∼20 μl) were taken to minimize the effect of plasma sampling on blood pressure. Occasionally, a plasma sample was too small to analyze more than once.
All data are presented as means ± SE. Groups were compared using the unpaired t-test, with P < 0.05 considered significant.
Other than the hypertensive phenotype, no other overt physical differences were observed between wild-type mice and Mβ1−/−. When animals were on the normal-salt diet, the mean BWs of Mβ1+/+ (24 ± 0.5 g,n = 18) and Mβ1−/− (25 ± 1.0 g, n = 18) were not significantly different. The kidney weights of Mβ1+/+ (0.29 ± 0.01 g,n = 16) and Mβ1−/− (0.30 ± 0.02 g, n = 17) were also similar. The high-salt diet did not significantly affect the BWs (Mβ1+/+ 24 ± 1.0 g, n = 5; Mβ1−/− 24 ± 0.5 g, n = 9) or kidney weights (Mβ1+/+ 0.30 ± 0.01 g, n = 5; Mβ1−/− 0.32 ± 0.01 g, n = 8) of Mβ1+/+ or Mβ1−/−. Because Mβ1+/+ and Mβ1−/− fed the same diet (normal or high salt) exhibited similar weight gains with age, it is assumed that they ingested equivalent amounts of mice chow.
In the present study, measurements of MAP were made in anesthetized mice. Although the depth of anesthesia was difficult to determine, a positive correlation between GFR and MAP was observed when MAP was <80 mmHg. Because low perfusion pressure affects autoregulation, we excluded data from further analysis if the average MAP during the collection period was <80 mmHg. It was found that eight mice (of 49) had a MAP of <80 mmHg. Table 1 shows the MAP in Mβ1+/+ and Mβ1−/− during the equilibration periods under euvolemic, volume-expanded, and high-salt diet conditions. During the euvolemic equilibration periods, the MAP in Mβ1−/− was significantly higher than that in Mβ1+/+. However, during the volume-expanded equilibration period, the MAP in Mβ1+/+ was significantly higher than the Mβ1+/+ euvolemic value, whereas the MAP in Mβ1−/− was not significantly different from the Mβ1−/− euvolemic value. The MAP in neither Mβ1+/+ nor Mβ1−/− was significantly affected by treatment with a high-salt diet.
Figure 1 shows the GFR under euvolemic and acutely volume-expanded conditions for Mβ1+/+ and Mβ1−/−. Under euvolemic conditions, the GFRs in Mβ1+/+ and Mβ1−/− did not differ significantly. For Mβ1+/+, GFR was significantly higher under volume-expanded conditions (2.5 ± 0.4 ml · min−1 · 100 g BW−1, n = 6) compared with euvolemic conditions (1.3 ± 0.2 ml · min−1 · 100 g BW−1, n = 8; P < 0.02). Similarly, for volume-expanded Mβ1−/−, GFR (1.4 ± 0.1 ml · min−1 · 100 g BW−1, n = 7) was significantly higher than in euvolemic Mβ1−/− (1.0 ± 0.1 ml · min−1 · 100 g BW−1, n = 6; P < 0.05). However, the GFR in volume-expanded Mβ1+/+ was significantly higher than the GFR in volume-expanded Mβ1−/− (P < 0.02). There was no significant effect of the high-salt diet on the GFR of either genotype (data not shown: Mβ1+/+ 1.0 ± 0.1 ml · min−1 · 100 g BW−1, n = 5; Mβ1−/−0.8 ± 0.2 ml · min−1 · 100 g BW−1, n = 9).
Na+ handling in Mβ1−/−.
Figure 2, A and B, shows the effects of acute and chronic volume expansion on Na+ excretion (UNaV) and fractional excretion of Na+ (FENa) in Mβ1+/+ and Mβ1−/−. Plasma [Na+] data are shown in Table 2. Under conditions of volume expansion, the UNaV in Mβ1+/+ was significantly higher (4.3 ± 1.1 μeq · min−1 · 100 g BW−1, n = 10) than that observed under euvolemic conditions (0.3 ± 0.1 μeq · min−1 · 100 g BW−1, n = 8; P < 0.01). Similarly, the UNaV in Mβ1−/− was significantly higher under volume-expanded conditions (2.7 ± 0.6 μeq · min−1 · 100 g BW−1, n = 9) compared with euvolemic conditions (0.3 ± 0.1 μeq · min−1 · 100 g BW−1, n = 8; P < 0.002). There were no genotypic differences in UNaV under volume-expanded or euvolemic conditions. There was no significant effect of the high-salt diet on UNaV for either genotype (data not shown), with UNaV averaging 0.4 ± 0.1 μeq · min−1 · 100 g BW−1 (n = 5) in Mβ1+/+ and 0.6 ± 0.2 μeq · min−1 · 100 g BW−1 (n = 5) in Mβ1−/−.
Under euvolemic conditions, FENa (Fig. 2 B) was similar in Mβ1+/+ (0.15 ± 0.03%, n= 8) and Mβ1−/− (0.23 ± 0.07%, n= 5). Both Mβ1+/+ and Mβ1−/− exhibited higher FENa on volume expansion compared with their respective euvolemic values (P < 0.02;P < 0.001); however, the FENa in volume-expanded Mβ1−/− (1.93 ± 0.35%,n = 5) was significantly greater than that in volume-expanded Mβ1+/+ (0.83 ± 0.29%,n = 6; P < 0.04). FENatended to be elevated in mice fed the high-salt diet (data not shown) compared with the normal diet for both Mβ1+/+ (0.29 ± 0.09%, n = 5) and Mβ1−/− (1.08 ± 0.63%, n = 9), although the increases were not significant. No significant differences in plasma [Na+] were observed between groups.
K+ handling in Mβ1−/−.
Figure 3, A and B, shows the rate of K+ excretion (UKV) and the fractional excretion of K+ (FEK), respectively, for Mβ1+/+ and Mβ1−/− under euvolemic and acute volume-expanded conditions. For Mβ1+/+, the UKV in the euvolemic group was 0.9 ± 0.2 μeq · min−1 · 100 g BW−1 (n = 8), whereas the UKV in the volume-expanded group was significantly higher at 2.7 ± 0.4 μeq · min−1 · 100 g BW−1 (n = 9; P < 0.05). For Mβ1−/−, the UKV under euvolemic conditions was 0.5 ± 0.2 μeq · min−1 · 100 g BW−1 (n = 7), whereas the UKV during volume expansion was significantly greater (1.2 ± 0.1 μeq · min−1 · 100 g BW−1, n = 9; P < 0.05). Although the UKV in Mβ1+/+ and Mβ1−/− did not differ significantly during euvolemia, UKV in volume-expanded Mβ1−/− was significantly less than that in volume-expanded Mβ1+/+(P < 0.01). There was no effect of the high-salt diet on UKV for any treatment group (data not shown), averaging 0.5 ± 0.1 μeq · min−1 · 100 g BW−1 (n = 5) in Mβ1+/+ and 0.3 ± 0.1 μeq · min−1 · 100 g BW−1 (n = 9) in Mβ1−/−.
FEK is shown in Fig. 3 B. Under euvolemic conditions, FEK was similar in Mβ1+/+ and Mβ1−/−. With volume expansion, the FEK in Mβ1+/+ was 33 ± 7% (n = 5), a value significantly higher than the FEK for euvolemic Mβ1+/+ (13 ± 2%, n = 8;P < 0.01). However, the values of FEK for euvolemic Mβ1−/− and volume-expanded Mβ1−/− were not significantly different (12 ± 4,n = 5 and 19 ± 2%, n = 5, respectively). Treatment with the high-salt diet did not alter FEK (data not shown: Mβ1+/+ 12 ± 3,n = 5; Mβ1−/− 10 ± 2%,n = 9).
Plasma [K+] data are shown in Table 2. Although plasma [K+] values tended to be lower in both Mβ1+/+ and Mβ1−/− under volume-expanded conditions, this decrease achieved statistical significance only in Mβ1+/+ (P < 0.05). There were no significant differences in plasma [K+] for either genotype on the high-salt diet (Mβ1+/+ 5.1 ± 0.2,n = 5; Mβ1−/− 5.1 ± 0.3 mM,n = 9).
Although BKCa are expressed in several types of renal cells (10, 11, 20), the role of BKCa-β1 with respect to renal function has not been investigated. The results of this study provide several novel findings related to renal function in BKCa-β1 knockout mice. In the euvolemic conditions of these experiments, no genotype-related differences were found in excretion rates of inulin, Na+, or K+. In contrast, with acute volume expansion, β1 knockout mice exhibited a depressed GFR and FEK response, and an increased FENa response, compared with Mβ1+/+. Therefore, BKCa, in conjunction with its β1 auxiliary subunit, may be an important contributor to the maintenance of electrolyte balance during acute volume expansion.
β1 Knockout mice, now studied by several groups of investigators, express moderate but significant hypertension. Brenner et al. (2) have reported that Mβ1−/− (C57BL/6 strain) were hypertensive by ∼20 mmHg. Using a different Mβ1−/− model (129/SvJ strain), Plüger et al. (24) reported that MAP was elevated by ∼14 mmHg. Both of these measurements were made in conscious mice using arterial catheters. In the anesthetized (C57BL/6) mice in the present study, Mβ1−/− were hypertensive by ∼11 mmHg under euvolemic conditions, whereas the MAP in Mβ1−/− and Mβ1+/+ was similar when the animals were volume expanded.
Because the FITC-inulin method only requires 20 μl of plasma, we were able to obtain accurate GFR measurements while avoiding the hypotensive effects of sampling blood. Hence, the values for GFR in this study correspond well with previously reported values (3, 17,30).
Consistent with previous studies in rats (8) and mice (3, 5), the GFRs in both Mβ1+/+ and Mβ1−/− were significantly higher in the volume-expanded groups compared with the euvolemic groups. However, the GFR in volume-expanded Mβ1−/− was significantly less than that in volume-expanded Mβ1+/+. This was not related to perfusion pressure because the MAPs in Mβ1+/+ and Mβ1−/− did not differ during volume-expanded conditions. The failure of the GFR in Mβ1−/− to appropriately respond to volume expansion implies that the β1-subunit of BKCa has an important role in mediating the renal response to an increased volume load.
The reason for the attenuated GFR response to volume expansion in Mβ1−/− is not understood. However, a hemodynamic effect is likely because BKCa are present in both renal afferent arterioles (4, 6) and glomerular mesangial cells (25, 27, 28). In afferent renal arterioles, BKCa play a relatively minor role in opposing constriction (4), whereas in mesangial cells BKCa are a major component of the counteractive response to constriction (28). In addition, the β1-subunit is present in human mesangial cells (24a), which are phenotypically similar to smooth muscle and express an abundance of BKCa. When activated by ANP, BKCa have a role in relaxing glomerular mesangial cells, which can contribute to an elevated GFR by increasing the capillary surface area available for filtration (12, 28). This notion is consistent with a recent finding in our laboratory that the β1-subunit is required for PKG activation of mesangial BKCa (14). Therefore, an attenuated GFR response to volume expansion can be explained by the absence of the β1, which renders the mesangial BKCa less responsive to ANP.
If the β1-subunit plays a role in promoting the vascular response to ANP, Mβ1−/− would be expected to exhibit acute hypertension with volume expansion. A recent study by Holtwick et al. (9), using a mouse model with a smooth muscle-selective deletion of guanylyl cyclase, demonstrated that acute vascular volume expansion caused a rapid increase in the blood pressure of these knockout mice. This hypertensive response would be expected if the response to ANP is attenuated; however, in this study we observed no such response in arterial pressure. The fact that arterial pressure in Mβ1−/− was not influenced by volume expansion suggests that the β1-subunit does not have a role in the vascular response to ANP. Whereas BKCa may be the primary effector for cGMP-mediated relaxation of mesangial cells (28), vascular smooth muscle may have a variety of additional cGMP-mediated responses leading to relaxation (15).
Alternatively, it is possible that the absence of the β1-subunit causes the mesangial cells to be less responsive to either a Ca2+ increase or stretch that occurs with volume expansion. Although BKCa have been shown to be stretch activated in some cells (22, 29), the potential role of the β1-subunit in this process has not been investigated.
Na+ handling in Mβ1−/−.
Like the GFR, the UNaV was similar in Mβ1+/+and Mβ1−/− under euvolemic conditions. However, even under volume-expanded conditions, the UNaV in Mβ1−/− was not significantly different from that of Mβ1+/+. The fact that the GFR in volume-expanded Mβ1−/− was attenuated whereas the UNaV approached a normal rate implies that changes in Na+reabsorption account for the majority of the Na+ excretory response to volume expansion in Mβ1−/−. Indeed, the FENa in Mβ1−/− was significantly greater than that in Mβ1+/+, indicating that Mβ1−/− were able to compensate for decreased filtered Na+ by reducing Na+ reabsorption. This is consistent with previous studies showing that volume expansion causes a decrease in distal Na+ reabsorption in addition to its hemodynamic effects (13, 26).
K+ handling in Mβ1−/−.
Consistent with previous studies (1, 3, 26), the UKV and FEK in wild-type mice were substantially greater in the volume-expanded condition. However, in Mβ1−/−, the FEK was statistically the same in the euvolemic and volume-expanded groups. Similar to Mβ1+/+, the UKV in Mβ1−/− was significantly greater in the volume-expanded group compared with the euvolemic group. However, in the volume-expanded condition, the UKV in Mβ1−/− was significantly less than the UKV in Mβ1+/+. Our experimental design (unpaired data) does not permit genotypic comparisons of the changes in UKV from baseline. Therefore, we cannot draw any conclusions about relative increases from baseline. For example, there was a genotypic difference in the volume-expanded but not euvolemic groups with respect to UKV; however, because of the low values and the baseline variability in UKV in the euvolemic groups, it is possible that there may be similar fold-increases in UKV with volume expansion in Mβ1+/+ and Mβ1−/− that were undetectable.
Our data imply that a diminished K+ secretory response to volume expansion in Mβ1−/− may reflect a role for BKCa to promote K+ efflux from cells of the distal nephron during high volume flow. In support of this notion, Woda et al. (31) have recently shown that flow-mediated K+ secretion in rabbit cortical collecting duct (CCD) is mediated by BKCa. Moreover, Lu et al. (21) provided evidence for an additional K+ secretory channel in the CCD of ROMK (Kir1.1) knockout mice. BKCa, described as stretch activated in the rat and rabbit CCD (22) as well as the rabbit medullary thick ascending limb (29), may be activated by high-volume-induced pressure on the cell membrane. Our data specifically implicate the β1-subunit of the BKCachannel as an important component in the mediation of K+secretion under conditions of volume expansion. However, it is not known whether the β1-subunit, either alone or with other β-subunits, is associated with the BK-α in the CCD.
An alternative explanation for the diminished K+ secretory response to volume expansion in Mβ1−/− is that the compensatory reduction in Na+ reabsorption decreased the driving force for K+ secretion. This would be true if the reduction in Na+ reabsorption resulted in a less negative membrane potential in the CCD, where the predominant amount of K+ is secreted. However, to our knowledge, there is no evidence that ANP (the major hormone responding to volume expansion) affects membrane potential or K+ secretion in the CCD. A study by Zeidel et al. (32) demonstrated that ANP inhibited potential-stimulated Na+ uptake in the inner medullary collecting duct (IMCD); however, little or no K+secretion occurs in the IMCD. In addition, a study using ANP transgenic (overexpressing) mice demonstrated enhanced K+ excretion in response to volume expansion (5). Although it is possible that the diminished K+ secretory response in Mβ1−/− is due to a decrease in Na+reabsorption, for the reasons stated above, a primary defect in K+ secretion is the best explanation for this result.
Effect of a high-salt diet.
Although the high-salt diet did not significantly affect GFR or the rates of Na+ and K+ excretion, it did tend to increase Na+ excretion and decrease K+excretion compared with the normal diet. This result is consistent with the low aldosterone levels expected with a high-salt diet. The fact that Mβ1+/+ and Mβ1−/− had similar responses to the high-salt diet indicates that the loss of the BKCa-β1 does not alter the compensatory renal response. In addition, the finding that MAP in Mβ1−/− was not increased with the high-salt diet indicates that the hypertension described for Mβ1−/− is not salt sensitive.
In conclusion, this study demonstrates the importance of the BKCa-β1 in the renal response to volume expansion. BKCa-β1 may have a role in the glomerulus to mediate an elevated GFR during volume expansion and could potentially be important in the distal nephron for the elevation of K+ secretion associated with high flow rates. To more clearly define these roles, future studies must elucidate the specific pathway(s) involved in the renal responses to volume expansion and the specific expression patterns of the β-subunits associated with BKCa within the kidney.
The authors thank Drs. Robert Brenner and Richard Aldrich (Stanford Univ.) for graciously supplying the BKCa-β1 mice used in this study. We also thank Drs. Tong Wang and Gerhard Giebisch (Yale Univ.) for advice and guidance regarding the surgical procedures for analyzing renal function in mice.
This work was supported by National Institutes of Health Grants RO1-DK-49561 (to S. C. Sansom) and 1T32-HL-0788 (Cardiovascular Research Training Grant; to J. L. Pluznick).
Address for reprint requests and other correspondence: S. C. Sansom, Dept. of Physiology and Biophysics, 984575 Univ. of Nebraska Medical Ctr., Omaha, NE 68198-4575 (E-mail:).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published March 4, 2003;10.1152/ajprenal.00010.2003
- Copyright © 2003 the American Physiological Society