Renal fluid and electrolyte handling in BKCa-beta 1minus /minus mice

Jennifer L. Pluznick, Peilin Wei, Pamela K. Carmines, and Steven C. Sansom

Department of Physiology and Biophysics, University of Nebraska Medical Center, Omaha, Nebraska 68198-4575


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Large-conductance Ca2+-activated K+ channels (BKCa) are composed of pore-forming alpha -subunits and one of four accessory beta -subunits. The beta 1-subunit, found predominantly in smooth muscle, modulates the Ca2+ sensitivity and pharmacological properties of BKCa. BKCa-beta 1 null mice (Mbeta 1-/-) 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 Mbeta 1-/- under euvolemic, volume-expanded, or high-salt diet conditions. Under euvolemic conditions, no differences in renal function were found between Mbeta 1-/- and Mbeta 1+/+. However, glomerular filtration rate (GFR) and fractional K+ excretion were significantly impaired in Mbeta 1-/- in response to acute volume expansion. In contrast, Mbeta 1-/- exhibited enhanced Na+ excretion and fractional Na+ excretion responses to acute volume expansion. Differences in renal function between Mbeta 1+/+ and Mbeta 1-/- were not observed when chronically treated with a high-salt diet. These observations indicate that the beta 1-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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

LARGE-CONDUCTANCE, CA2+-ACTIVATED potassium channels (BKCa) are composed of both pore-forming alpha - and accessory beta -subunits. At least four distinct beta -subunits, each with a tissue-specific distribution, have been described. When the beta 1-subunit, found primarily in smooth muscle cells, is expressed with the alpha -subunit, the voltage and calcium sensitivities of BKCa are enhanced (2). Conversely, BKCa in cerebral artery myocytes from beta 1 knockout mice (Mbeta 1-/-) 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 beta 1-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 Mbeta 1-/- 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-beta 1 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 beta 1-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 Mbeta 1-/- 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.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 Mbeta 1-/- mice (with a homogeneous C57BL/6 background) generated by Brenner et al. (2) and C57BL/6 control mice (Mbeta 1+/+) 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 procedures. 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.

Statistics. All data are presented as means ± SE. Groups were compared using the unpaired t-test, with P < 0.05 considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Other than the hypertensive phenotype, no other overt physical differences were observed between wild-type mice and Mbeta 1-/-. When animals were on the normal-salt diet, the mean BWs of Mbeta 1+/+ (24 ± 0.5 g, n = 18) and Mbeta 1-/- (25 ± 1.0 g, n = 18) were not significantly different. The kidney weights of Mbeta 1+/+ (0.29 ± 0.01 g, n = 16) and Mbeta 1-/- (0.30 ± 0.02 g, n = 17) were also similar. The high-salt diet did not significantly affect the BWs (Mbeta 1+/+ 24 ± 1.0 g, n = 5; Mbeta 1-/- 24 ± 0.5 g, n = 9) or kidney weights (Mbeta 1+/+ 0.30 ± 0.01 g, n = 5; Mbeta 1-/- 0.32 ± 0.01 g, n = 8) of Mbeta 1+/+ or Mbeta 1-/-. Because Mbeta 1+/+ and Mbeta 1-/- 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.

MAP. 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 Mbeta 1+/+ and Mbeta 1-/- during the equilibration periods under euvolemic, volume-expanded, and high-salt diet conditions. During the euvolemic equilibration periods, the MAP in Mbeta 1-/- was significantly higher than that in Mbeta 1+/+. However, during the volume-expanded equilibration period, the MAP in Mbeta 1+/+ was significantly higher than the Mbeta 1+/+ euvolemic value, whereas the MAP in Mbeta 1-/- was not significantly different from the Mbeta 1-/- euvolemic value. The MAP in neither Mbeta 1+/+ nor Mbeta 1-/- was significantly affected by treatment with a high-salt diet.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Mean arterial pressure in euvolemic, acutely volume-expanded, and chronically volume-expanded mice

GFR. Figure 1 shows the GFR under euvolemic and acutely volume-expanded conditions for Mbeta 1+/+ and Mbeta 1-/-. Under euvolemic conditions, the GFRs in Mbeta 1+/+ and Mbeta 1-/- did not differ significantly. For Mbeta 1+/+, 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 Mbeta 1-/-, GFR (1.4 ± 0.1 ml · min-1 · 100 g BW-1, n = 7) was significantly higher than in euvolemic Mbeta 1-/- (1.0 ± 0.1 ml · min-1 · 100 g BW-1, n = 6; P < 0.05). However, the GFR in volume-expanded Mbeta 1+/+ was significantly higher than the GFR in volume-expanded Mbeta 1-/- (P < 0.02). There was no significant effect of the high-salt diet on the GFR of either genotype (data not shown: Mbeta 1+/+ 1.0 ± 0.1 ml · min-1 · 100 g BW-1, n = 5; Mbeta 1-/- 0.8 ± 0.2 ml · min-1 · 100 g BW-1, n = 9).


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 1.   The effect of acute volume expansion on glomerular filtration rate (GFR) in large-conductance, Ca2+-activated K+ channel beta 1-subunit control (Mbeta 1+/+) and null mice (Mbeta 1-/-). For both Mbeta 1+/+ and Mbeta 1-/-, the euvolemic GFR was significantly less than the volume-expanded GFR (*P < 0.05). However, the GFR in volume-expanded Mbeta 1-/- was significantly lower than the GFR in volume-expanded Mbeta 1+/+ (dagger P < 0.02). BW, body weight.

Na+ handling in Mbeta 1-/-. 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 Mbeta 1+/+ and Mbeta 1-/-. Plasma [Na+] data are shown in Table 2. Under conditions of volume expansion, the UNaV in Mbeta 1+/+ 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 Mbeta 1-/- 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 Mbeta 1+/+ and 0.6 ± 0.2 µeq · min-1 · 100 g BW-1 (n = 5) in Mbeta 1-/-.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 2.   The response of urinary Na+ excretion rate (UNaV) and fractional excretion (FENa) to acute volume expansion in Mbeta 1+/+ and Mbeta 1-/-. A: under volume-expanded conditions, UNaV was significantly elevated compared with euvolemic conditions in both Mbeta 1+/+ (*P < 0.01) and in Mbeta 1-/- (*P < 0.002). No genotypic differences in UNaV were observed between Mbeta 1+/+ and Mbeta 1-/-. B: FENa during euvolemia was similar in Mbeta 1+/+ and Mbeta 1-/-, and there was a significant difference between euvolemic and volume-expanded values of FENa for both Mbeta 1+/+ and Mbeta 1-/- (*P < 0.02, P < 0.001). However, the FENa in volume-expanded Mbeta 1-/- was significantly higher than the FENa in volume-expanded Mbeta 1+/+ (dagger P < 0.04).


                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Plasma Na+ and K+ concentration in euvolemic, acutely volume-expanded, and chronically volume-expanded mice

Under euvolemic conditions, FENa (Fig. 2B) was similar in Mbeta 1+/+ (0.15 ± 0.03%, n = 8) and Mbeta 1-/- (0.23 ± 0.07%, n = 5). Both Mbeta 1+/+ and Mbeta 1-/- exhibited higher FENa on volume expansion compared with their respective euvolemic values (P < 0.02; P < 0.001); however, the FENa in volume-expanded Mbeta 1-/- (1.93 ± 0.35%, n = 5) was significantly greater than that in volume-expanded Mbeta 1+/+ (0.83 ± 0.29%, n = 6; P < 0.04). FENa tended to be elevated in mice fed the high-salt diet (data not shown) compared with the normal diet for both Mbeta 1+/+ (0.29 ± 0.09%, n = 5) and Mbeta 1-/- (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 Mbeta 1-/-. Figure 3, A and B, shows the rate of K+ excretion (UKV) and the fractional excretion of K+ (FEK), respectively, for Mbeta 1+/+ and Mbeta 1-/- under euvolemic and acute volume-expanded conditions. For Mbeta 1+/+, 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 Mbeta 1-/-, 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 Mbeta 1+/+ and Mbeta 1-/- did not differ significantly during euvolemia, UKV in volume-expanded Mbeta 1-/- was significantly less than that in volume-expanded Mbeta 1+/+ (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 Mbeta 1+/+ and 0.3 ± 0.1 µeq · min-1 · 100 g BW-1 (n = 9) in Mbeta 1-/-.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 3.   The effect of acute volume expansion on renal K+ handling in Mbeta 1+/+ and Mbeta 1-/-. A: urinary K+ excretion rate (UKV) in Mbeta 1+/+ and Mbeta 1-/- was significantly higher during volume expansion compared with euvolemia (*P < 0.05, P < 0.05). However, the UKV in volume-expanded Mbeta 1-/- was significantly less than the UKV in volume-expanded Mbeta 1+/+ (dagger P < 0.01). B: under euvolemic conditions, urinary fractional K+ excretion (FEK) was similar in Mbeta 1+/+ and Mbeta 1-/-. With volume expansion, the FEK in Mbeta 1+/+ but not in Mbeta 1-/- was significantly higher than the euvolemic value (*P < 0.01).

FEK is shown in Fig. 3B. Under euvolemic conditions, FEK was similar in Mbeta 1+/+ and Mbeta 1-/-. With volume expansion, the FEK in Mbeta 1+/+ was 33 ± 7% (n = 5), a value significantly higher than the FEK for euvolemic Mbeta 1+/+ (13 ± 2%, n = 8; P < 0.01). However, the values of FEK for euvolemic Mbeta 1-/- and volume-expanded Mbeta 1-/- 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: Mbeta 1+/+ 12 ± 3, n = 5; Mbeta 1-/- 10 ± 2%, n = 9).

Plasma [K+] data are shown in Table 2. Although plasma [K+] values tended to be lower in both Mbeta 1+/+ and Mbeta 1-/- under volume-expanded conditions, this decrease achieved statistical significance only in Mbeta 1+/+ (P < 0.05). There were no significant differences in plasma [K+] for either genotype on the high-salt diet (Mbeta 1+/+ 5.1 ± 0.2, n = 5; Mbeta 1-/- 5.1 ± 0.3 mM, n = 9).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Although BKCa are expressed in several types of renal cells (10, 11, 20), the role of BKCa-beta 1 with respect to renal function has not been investigated. The results of this study provide several novel findings related to renal function in BKCa-beta 1 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, beta 1 knockout mice exhibited a depressed GFR and FEK response, and an increased FENa response, compared with Mbeta 1+/+. Therefore, BKCa, in conjunction with its beta 1 auxiliary subunit, may be an important contributor to the maintenance of electrolyte balance during acute volume expansion.

MAP. beta 1 Knockout mice, now studied by several groups of investigators, express moderate but significant hypertension. Brenner et al. (2) have reported that Mbeta 1-/- (C57BL/6 strain) were hypertensive by ~20 mmHg. Using a different Mbeta 1-/- 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, Mbeta 1-/- were hypertensive by ~11 mmHg under euvolemic conditions, whereas the MAP in Mbeta 1-/- and Mbeta 1+/+ was similar when the animals were volume expanded.

Volume handling. 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 Mbeta 1+/+ and Mbeta 1-/- were significantly higher in the volume-expanded groups compared with the euvolemic groups. However, the GFR in volume-expanded Mbeta 1-/- was significantly less than that in volume-expanded Mbeta 1+/+. This was not related to perfusion pressure because the MAPs in Mbeta 1+/+ and Mbeta 1-/- did not differ during volume-expanded conditions. The failure of the GFR in Mbeta 1-/- to appropriately respond to volume expansion implies that the beta 1-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 Mbeta 1-/- 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 beta 1-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 beta 1-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 beta 1, which renders the mesangial BKCa less responsive to ANP.

If the beta 1-subunit plays a role in promoting the vascular response to ANP, Mbeta 1-/- 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 Mbeta 1-/- was not influenced by volume expansion suggests that the beta 1-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 beta 1-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 beta 1-subunit in this process has not been investigated.

Na+ handling in Mbeta 1-/-. Like the GFR, the UNaV was similar in Mbeta 1+/+ and Mbeta 1-/- under euvolemic conditions. However, even under volume-expanded conditions, the UNaV in Mbeta 1-/- was not significantly different from that of Mbeta 1+/+. The fact that the GFR in volume-expanded Mbeta 1-/- 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 Mbeta 1-/-. Indeed, the FENa in Mbeta 1-/- was significantly greater than that in Mbeta 1+/+, indicating that Mbeta 1-/- 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 Mbeta 1-/-. 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 Mbeta 1-/-, the FEK was statistically the same in the euvolemic and volume-expanded groups. Similar to Mbeta 1+/+, the UKV in Mbeta 1-/- was significantly greater in the volume-expanded group compared with the euvolemic group. However, in the volume-expanded condition, the UKV in Mbeta 1-/- was significantly less than the UKV in Mbeta 1+/+. 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 Mbeta 1+/+ and Mbeta 1-/- that were undetectable.

Our data imply that a diminished K+ secretory response to volume expansion in Mbeta 1-/- 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 beta 1-subunit of the BKCa channel as an important component in the mediation of K+ secretion under conditions of volume expansion. However, it is not known whether the beta 1-subunit, either alone or with other beta -subunits, is associated with the BK-alpha in the CCD.

An alternative explanation for the diminished K+ secretory response to volume expansion in Mbeta 1-/- 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 Mbeta 1-/- 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 Mbeta 1+/+ and Mbeta 1-/- had similar responses to the high-salt diet indicates that the loss of the BKCa-beta 1 does not alter the compensatory renal response. In addition, the finding that MAP in Mbeta 1-/- was not increased with the high-salt diet indicates that the hypertension described for Mbeta 1-/- is not salt sensitive.

In conclusion, this study demonstrates the importance of the BKCa-beta 1 in the renal response to volume expansion. BKCa-beta 1 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 beta -subunits associated with BKCa within the kidney.


    ACKNOWLEDGEMENTS

The authors thank Drs. Robert Brenner and Richard Aldrich (Stanford Univ.) for graciously supplying the BKCa-beta 1 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.


    FOOTNOTES

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: ssansom{at}unmc.edu).

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

Received 9 January 2003; accepted in final form 26 February 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Ackermann, U. Cardiac output and renal excretion rates during acute blood volume expansion in rats. Am J Physiol Heart Circ Physiol 234: H21-H27, 1978[Abstract/Free Full Text].

2.   Brenner, R, Perez GJ, Bonev AD, Eckman DM, Kosek JC, Wiler SW, Patterson AJ, Nelson MT, and Aldrich RW. Vasoregulation by the beta 1 subunit of the calcium-activated potassium channel. Nature 407: 870-876, 2000[ISI][Medline].

3.   Cervenka, L, Mitchell KD, and Navar LG. Renal function in mice: effects of volume expansion and angiotensin II. J Am Soc Nephrol 10: 2631-2636, 1999[Abstract/Free Full Text].

4.   Fallet, RW, Bast JP, Fujiwara K, Ishii N, Sansom SC, and Carmines PK. Influence of Ca2+-activated K+ channels on rat renal arteriolar responses to depolarizing agonists. Am J Physiol Renal Physiol 280: F583-F591, 2001[Abstract/Free Full Text].

5.   Field, LJ, Veress AT, Steinhelper ME, Cochrane K, and Sonnenberg H. Kidney function in ANF-transgenic mice: effect of blood volume expansion. Am J Physiol Regul Integr Comp Physiol 260: R1-R5, 1991[Abstract/Free Full Text].

6.   Gebremedhin, D, Kaldunski M, Jacobs ER, Harder DR, and Roman RJ. Coexistence of two types of Ca2+-activated K+ channels in rat renal arterioles. Am J Physiol Renal Fluid Electrolyte Physiol 270: F69-F81, 1996[Abstract/Free Full Text].

7.   Gollasch, M, Tank J, Luft FC, Jordan J, Maass P, Krasko C, Sharma AM, Busjahn A, and Bahring S. The BK channel beta 1 subunit gene is associated with human baroreflex and blood pressure regulation. J Hypertens 20: 927-933, 2002[ISI][Medline].

8.   Hirth, C, Stasch JP, John A, Kazda S, Morich F, Neuser D, and Wohlfeil S. The renal response to acute hypervolemia is caused by atrial natriuretic peptides. J Cardiovasc Pharmacol 8: 268-275, 1986[ISI][Medline].

9.   Holtwick, R, Gotthardt M, Skryabin B, Steinmetz M, Potthast R, Zetsche B, Hammer RE, Herz J, and Kuhn M. Smooth muscle-selective deletion of guanylyl cyclase-A prevents the acute but not chronic effects of ANP on blood pressure. Proc Natl Acad Sci USA 99: 7142-7147, 2002[Abstract/Free Full Text].

10.   Hunter, M, Kawahara K, and Giebisch G. Potassium channels along the nephron. Federation Proc 45: 2723-2726, 1986[ISI][Medline].

11.   Hunter, M, Lopes AG, Boulpaep EL, and Giebisch GH. Single channel recordings of calcium-activated potassium channels in the apical membrane of rabbit cortical collecting tubules. Proc Natl Acad Sci USA 81: 4237-4239, 1984[Abstract].

12.   Iversen, BM, Kvam FI, Matre K, Morkrid L, Horvei G, Bagchus W, Grond J, and Ofstad J. Effect of mesangiolysis on autoregulation of renal blood flow and glomerular filtration rate in rats. Am J Physiol Renal Fluid Electrolyte Physiol 262: F361-F366, 1992[Abstract/Free Full Text].

13.   Kleinman, LI, and Banks RO. Segmental nephron sodium and potassium reabsorption in newborn and adult dogs during saline expansion. Proc Soc Exp Biol Med 173: 231-237, 1983[Abstract].

14.   Kudlacek, PE, Pluznick JL, Green JM, and Sansom SC. The role of hSlo beta  subunits in cGMP-kinase activation of mesangial cell (MC) BKCa channels (Abstract). FASEB J 16: A1174, 2002.

15.   Lincoln, TM, Komalavilas P, and Cornwell TL. Pleiotropic regulation of vascular smooth muscle tone by cyclic GMP-dependent protein kinase. Hypertension 23: 1141-1147, 1994[Abstract].

16.   Lohn, M, Lauterbach B, Haller H, Pongs O, Luft FC, and Gollasch M. beta 1-Subunit of BK channels regulates arterial wall [Ca2+] and diameter in mouse cerebral arteries. J Appl Physiol 91: 1350-1354, 2001[Abstract/Free Full Text].

17.   Lorenz, JN. Considerations for the evaluation of renal function in genetically engineered mice. Curr Opin Nephrol Hypertens 10: 65-69, 2001[ISI][Medline].

18.   Lorenz, JN. A practical guide to evaluating cardiovascular, renal, and pulmonary function in mice. Am J Physiol Regul Integr Comp Physiol 282: R1565-R1582, 2002[Abstract/Free Full Text].

19.   Lorenz, JN, and Gruenstein E. A simple, nonradioactive method for evaluating single-nephron filtration rate using FITC-inulin. Am J Physiol Renal Physiol 276: F172-F177, 1999[Abstract/Free Full Text].

20.   Lu, L, Markakis D, and Guggino WB. Identification and regulation of whole-cell Cl- and Ca2+-activated K+ currents in cultured medullary thick ascending limb cells. J Membr Biol 135: 181-189, 1993[ISI][Medline].

21.   Lu, M, Wang T, Yan Q, Yang X, Dong K, Knepper MA, Wang W, Giebisch G, Shull GE, and Hebert SC. Absence of small conductance K+ channel (SK) activity in apical membranes of thick ascending limb and cortical collecting duct in ROMK (Bartter's) knockout mice. J Biol Chem 277: 37881-37887, 2002[Abstract/Free Full Text].

22.   Pacha, J, Frindt G, Sackin H, and Palmer LG. Apical maxi K channels in intercalated cells of CCT. Am J Physiol Renal Fluid Electrolyte Physiol 261: F696-F705, 1991[Abstract/Free Full Text].

23.   Petkov, GV, Bonev AD, Heppner TJ, Brenner R, Aldrich RW, and Nelson MT. beta 1-Subunit of the Ca2+-activated K+ channel regulates contractile activity of mouse urinary bladder smooth muscle. J Physiol 537: 443-452, 2001[Abstract/Free Full Text].

24.   Plüger, S, Faulhaber J, Furstenau M, Lohn M, Waldschutz R, Gollasch M, Haller H, Luft FC, Ehmke H, and Pongs O. Mice with disrupted BK channel beta 1 subunit gene feature abnormal Ca2+ spark/STOC coupling and elevated blood pressure. Circ Res 87: E53-E60, 2000[ISI][Medline].

24a.   Pluznick, JL, Kudlacek PE, Padanilam B, and Sansom SC. Identification and localization of BKca-beta 1 subunit in human glomerular mesangial cells (HMC) in culture. FASEB J 17: A1227-A1228, 2003.

25.   Sansom, SC, and Stockand JD. Physiological role of large, Ca2+-activated K+ channels in human glomerular mesangial cells. Clin Exp Pharmacol Physiol 23: 76-82, 1996[ISI][Medline].

26.   Sonnenberg, H. Renal response to blood volume expansion: distal tubular function and urinary excretion. Am J Physiol 223: 916-924, 1972[Free Full Text].

27.   Stockand, JD, and Sansom SC. Mechanism of activation by cGMP-dependent protein kinase of large Ca2+-activated K+ channels in mesangial cells. Am J Physiol Cell Physiol 271: C1669-C1677, 1996[Abstract/Free Full Text].

28.   Stockand, JD, and Sansom SC. Role of large Ca2+-activated K+ channels in regulation of mesangial contraction by nitroprusside and ANP. Am J Physiol Cell Physiol 270: C1773-C1779, 1996[Abstract/Free Full Text].

29.   Taniguchi, J, and Guggino WB. Membrane stretch: a physiological stimulator of Ca2+-activated K+ channels in thick ascending limb. Am J Physiol Renal Fluid Electrolyte Physiol 257: F347-F352, 1989[Abstract/Free Full Text].

30.   Wang, T, Inglis FM, and Kalb RG. Defective fluid and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in proximal tubule of neuronal nitric oxide synthase-knockout mice. Am J Physiol Renal Physiol 279: F518-F524, 2000[Abstract/Free Full Text].

31.   Woda, CB, Bragin A, Kleyman TR, and Satlin LM. Flow-dependent K+ secretion in the cortical collecting duct is mediated by a maxi-K channel. Am J Physiol Renal Physiol 280: F786-F793, 2001[Abstract/Free Full Text].

32.   Zeidel, ML, Kikeri D, Silva P, Burrowes M, and Brenner BM. Atrial natriuretic peptides inhibit conductive sodium uptake by rabbit inner medullary collecting duct cells. J Clin Invest 82: 1067-1074, 1988[ISI][Medline].


Am J Physiol Renal Fluid Electrolyte Physiol 284(6):F1274-F1279
0363-6127/03 $5.00 Copyright © 2003 the American Physiological Society