Potassium deprivation upregulates expression of ...

Am J Physiol Renal Physiol 279: F532–F543, 2000.

Potassium deprivation upregulates expression of renal basolateral Na⫹-HCO3⫺ cotransporter (NBC-1) HASSANE AMLAL,1 KHALID HABO,1 AND MANOOCHER SOLEIMANI1,2 1 Department of Medicine, University of Cincinnati School of Medicine, and 2Veterans Affairs Medical Center, Cincinnati, Ohio 45267-0585 Received 15 February 2000; accepted in final form 9 May 2000

Amlal, Hassane, Khalid Habo, and Manoocher Soleimani. Potassium deprivation upregulates expression of renal basolateral Na⫹-HCO3⫺ cotransporter (NBC-1). Am J Physiol Renal Physiol 279: F532–F543, 2000.—The purpose of the present experiments was to examine the effect of potassium deprivation on the expression of the renal basolateral Na⫹-HCO3⫺ cotransporter (NBC-1). Rats were placed on a K⫹-free diet for various time intervals and examined. NBC-1 mRNA levels increased by about threefold in the cortex (P ⬍ 0.04) at 72 h of K⫹ deprivation and remained elevated at 21 days. NBC activity increased by ⬃110% in proximal tubule suspensions, with the activity increasing from 0.091 in control to 0.205 pH/min in the K⫹-deprived group (P ⬍ 0.005). The inner stripe of outer medulla and cells of medullary thick ascending limb of Henle (mTAL) showed induction of NBC-1 mRNA and activity in K⫹-deprived rats, with the activity in mTAL increasing from 0.010 in control to 0.133 pH/min in the K⫹-deprived group (P ⬍ 0.004). K⫹ deprivation also increased NBC-1 mRNA levels in the renal papilla (P ⬍ 0.02). We conclude that 1) K⫹ deprivation increases NBC-1 expression and activity in proximal tubule and 2) K⫹ deprivation causes induction of NBC-1 expression and activity in mTAL tubule and inner medulla. We propose that NBC-1 likely mediates enhanced HCO3⫺ reabsorption in proximal tubule, mTAL, and inner medullary collecting duct in K⫹ deprivation and contributes to the maintenance of metabolic alkalosis in this condition. acid-base transporters; proximal tubule; medullary thick ascending limb

of HCO3⫺ is reabsorbed in the kidney proximal tubule (PT) via the luminal Na⫹/H⫹ exchanger NHE3 (reviewed in 44). The exit of HCO3⫺ across the basolateral membrane of PT is via the Na⫹-HCO3⫺ cotransporter (2, 3, 9, 12, 43, 49). A number of acid-base or electrolyte disorders are associated with decreased or increased HCO3⫺ reabsorption in various nephron segments. Alterations in PT acidification in pathological states should ultimately result from changes in the activity of luminal NHE3 and basolateral NBC. Although molecular regulation of NHE3 has been examined and correlated with its functional activity in several pathophysiological states (30, 41), adaptive molecular changes in Na⫹-

HCO3⫺ cotransport have been studied in very few states. K⫹ depletion is known to induce and maintain metabolic alkalosis in rats and humans (16, 23, 26, 28, 36). The maintenance of metabolic alkalosis in K⫹ depletion is predominantly achieved through enhanced HCO3⫺-absorbing ability of renal tubules. In K⫹ deprivation, the luminal Na⫹/H⫹ exchanger NHE3 is upregulated (46) and is likely responsible for enhanced HCO3⫺ reabsorption in the PT (16, 28, 36). Molecular studies indicated that this adaptive regulation is likely a posttranscriptional event (46). In addition to the PT, K⫹ deprivation is also associated with enhanced HCO3⫺ reabsorption in several other nephron segments, including distal convoluted tubules, outer medullary collecting duct, and inner medullary collecting duct (33, 34, 48). A Na⫹-HCO3⫺ cotransporter (NBC-1) was recently cloned from human (14), rat (15), and amphibian kidney (37). A spliced variant of NBC-1 is expressed in pancreas (14). Cloning experiments have identified several other NBC isoforms (NBC-2, kNBC-3, and mNBC-3) (4, 25, 32, 42). NBC-1 is exclusively expressed in the basolateral domain of PT and is the only NBC isoform that is present in this nephron segment. (1, 39). The purpose of these studies was to examine possible functional and molecular adaptive regulation of the basolateral Na⫹-HCO3⫺ cotransporter in kidney PT and other nephron segments in K⫹ deprivation.

THE MAJORITY OF THE FILTERED LOAD

Address for reprint requests and other correspondence: M. Soleimani, Univ. of Cincinnati Medical Center, 231 Bethesda Ave., MSB 5502, Cincinnati, OH 45267-0585 (E-mail: Manoocher. [email protected]). F532

EXPERIMENTAL PROCEDURES

Animal Model Four groups of male Sprague-Dawley rats (125–150g) were used for these experiments. In the first and the second groups, rats were fed either a regular chow (control) or a potassium-deficient (KD) diet (catalog no. 960189; ICN Biochemicals) for up to 21 days. All rats had free access to distilled water. The third and the forth groups were fed control or KD diet for up to 14 days and, in addition, received 0.85% KCl added to their drinking water. Rats were anesthetized by intraperitoneal injection of 50 mg of pentobarbital sodium. Intracardiac blood was obtained for serum K⫹ and HCO3⫺ concentration ([K⫹] and [HCO3⫺], respectively) 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.

0363-6127/00 $5.00 Copyright © 2000 the American Physiological Society

http://www.ajprenal.org

Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (185.158.150.221) on January 3, 2018. Copyright © 2000 American Physiological Society. All rights reserved.

NBC IN K⫹ DEPLETION

Table 1. Composition of experimental solutions Solution Compound

NaCl Choline-HCO3 TMA-Cl KCl K2HPO4 KH2PO4 CaCl2 MgCl2 HEPES

A

B

C

D

25 115 3 0.8 0.2 1 1 10

60 25 55 3 0.8 0.2 1 1 10

60 140 3 0.8 0.2 1 1 10

80 3 0.8 0.2 1 1 10

Concentrations are in mM. TMA, tetraethylammonium. All solutions were adjusted to pH 7.40 with Tris. Solutions A and B were bubbled with 100% O2. Solutions C and D were bubbled with 95% O2 ⫹ 5% CO2. Na⫹ was replaced with TMA.

measurement, and both kidneys were rapidly removed for RNA isolation or functional studies. RNA Isolation and Northern Hybridization Total cellular RNA was extracted from kidney superficial cortex, inner stripe of outer medulla, inner medulla, and tubular suspensions [PT and medullary thick ascending limb of Henle (mTAL)], by the method of Chomczynski and Sacchi (18), quantitated spectrophotometrically, and stored at ⫺80°C. Total RNA samples (30 ␮g/lane) were fractionated on a 1.2% agarose-formaldehyde gel and transferred to Magna NT nylon membranes (MSI) using 10⫻ sodium chloridesodium phosphate-EDTA (SSPE) as transfer buffer. Membranes were cross-linked by ultraviolet light and baked for 1 h. Hybridization was performed according to Church and Gilbert (19). Briefly, membranes were preprehybridized for 1 h in 0.1⫻ SSPE-1% SDS solution at 65°C and then prehybridized for 3 h at 65°C with 0.5 M sodium phosphate buffer, pH 7.2, 7% SDS, 1% BSA, 1 mM EDTA, and 100 ␮g/ml sonicated cDNA. Thereafter, the membranes were hybridized overnight in the above solution with 32P-labeled DNA probe for NBC-1, NBC-2, or kNBC-3. The cDNA probes (25 ng) were labeled with 32P deoxynucleotides by using the RadPrime DNA labeling kit (GIBCO-BRL). The membranes were washed twice in 40 mM sodium phosphate buffer, pH 7.2, 5% SDS, 0.5% BSA, and 1 mM EDTA for 10 min at 65°C, washed four times in 40 mM sodium phosphate buffer, pH 7.2, 1% SDS, and 1 mM EDTA for 10 min at 65°C, exposed to PhosphorImager cassette at room temperature for 24–72 h, and read by PhosphorImager (Molecular Dynamics, Sunnyvale, CA). For NBC-1, the full-length cDNA was used as a specific probe. For NBC-2, the probe was generated by PCR amplification of an EST cDNA fragment (GenBank accession no. AA216661) that encodes nucleotides 8–2808 and is a part of human NBC-2. For kNBC-3, a 700-bp fragment corresponding to nucleotides 900–1600 was used as a specific probe. The respective NBC-1 and kNBC-3 probes recognize appropriate-size mRNA on high-stringency human multipletissue blots (7, 14). NBC-2 cDNA probe recognizes an 8.5-kb mRNA in brain, skeletal muscle, and lymph node tissues on a human multiple-tissue blot (data not shown). Glyceraldehyde-3-phopshate dehydrogenase (GAPDH) cDNA probe was a generous gift from Drs. P. James and J. Lingrel at the University of Cincinnati. Tubular Suspension Preparation PT and mTAL suspensions were prepared as previously described (5, 7, 24). Briefly, kidneys were removed and

F533

bathed in an ice-cold Hanks’ solution consisting of (in mM) 115 NaCl, 3 KCl, 1 Na2HPO4, 1 KH2PO4, 1 MgSO4, 1 CaCl2, 25 NaHCO3, and 10 HEPES, which in addition contained 5 mM glucose, 5 mM leucine and 1 mg/ml BSA, and bubbled with 95% O2-5% CO2 at pH 7.40. The superficial cortex or the inner stripe of outer medulla was dissected and cut into small pieces and then subjected to successive 6-min periods of collagenase digestion (0.40g/l) at 37°C. After each period, the supernatant containing tubule fragments was collected after 1 min of gravity sedimentation and stored on ice. The suspension of PT or mTAL tubules was washed twice by centrifugation (1 min, 50 g) and resuspended in an appropriate solution (solution A, Table 1). Intracellular pH Measurements Intracellular pH (pHi) in PT suspension (or mTAL suspensions) was measured with the use of 2⬘,7⬘-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) as previously described (5, 7). In brief, aliquots of BCECF-loaded tubules were diluted into glass cuvettes containing 2 ml of the experimental medium, and BCECF fluorescence was monitored with the use of a Delta Scan dual-excitation spectrofluorometer (Photon Technology International, Brunswick, NJ) that was equipped with a water-jacketed, temperature-controlled cuvette holder and a magnetic stirrer. Fluorescence intensity was recorded at an emission wavelength of 525 nm and excitation wavelengths of 500 and 450 nm. The fluorescence ratios (F500/F450) were converted into pHi values with the use of calibration curves that were established daily by Triton X-100 methods, as used before (5, 7). Measurement of NBC Activity NBC activity in PT (or mTAL) suspensions was determined as the DIDS1-sensitive, Na⫹ and HCO3⫺-dependent pHi recovery from intracellular acidosis as previously described (6, 14). Briefly, PT suspensions were acidified by the NH4⫹ pulse technique in a Na⫹-free solution (solution A, Table 1). Acidification of mTAL tubular suspensions was achieved by subjecting the cells to two cycles of wash and gentle centrifugation in a Na⫹-free solution (solution B, Table 2). Cells were then allowed to recover from acidosis in the presence of Na⫹ and HCO3⫺ ⫾ DIDS. In both PT and mTAL suspensions, experiments were performed in the presence of 2 mM amiloride (to block the luminal and basolateral Na⫹/H⫹ exchangers) and 50 nM bafilomycin (to inhibit H⫹ATPase; see RESULTS for details). The initial rate of DIDSsensitive, Na⫹-dependent, HCO3⫺-mediated pHi recovery from acidosis (dpHi/dt) was measured as an index of NBC activity. This method has previously been used to determine

Table 2. Composition of experimental solutions Solution Compound

A

Na-gluconate Choline-HCO3 NMG-gluconate Ca-gluconate Mg-gluconate HEPES

115 25 1 1 10

B

C

25 115 1 1 10

60 25 55 1 1 10

D

E

60 140 1 1 10

80 1 1 10

Concentrations are in mM. NMG, N-methyl-D-glucamine. All solutions were adjusted to pH 7.40 with Tris. Solutions A, B, and C were bubbled with 95% O2 ⫹ 5% CO2. Solutions D and E were bubbled with 100% O2. Na⫹ and Cl⫺ were replaced with N-methylD-glucamine and gluconate, respectively.


F534


the activity of NBC in HEK 293 cells transiently transfected with NBC-1 cDNA (6, 14). The initial rates of pHi recovery (dpHi/dt) from acidosis were estimated from the slopes of pHi vs. time (t) and were expressed as pH units per minutes as previously described (4). Buffering Power The intrinsic buffering power (mMH⫹/pH unit) was measured in PT and mTAL tubules by using the NH4⫹ pulse method according to the formula ␤i⫽ ⌬[NH4⫹]i/⌬pHi, and as described (38), where ␤i is buffering power. In both PT and mTAL tubules, 50 nM bafilomycin (to inhibit H⫹-ATPase) and 500 ␮M ouabain (to block NH4⫹ entry into the cells via the Na⫹-K⫹-ATPase pump) (29) were present. In mTAL, 10 mM barium was added to the solution (to block K⫹/NH4⫹ antiport) (5, 8). BCECF-loaded cells (proximal convoluted tubule or mTAL) were initially incubated in Na⫹- and HCO3⫺free solution (solution A, Table 1) and monitored for pHi. At steady-state pHi, addition of 12 mM NH4SCN caused a rapid initial increase in cell pH due to the influx of NH3 and subsequent generation of NH4⫹. This alkalinization was followed by a plateau (no acidification was observed in both proximal convoluted tubule and mTAL tubules). Buffering power was determined in both control and KD rats according to the formula. Materials 32 P-dCTP was purchased from New England Nuclear (Boston, MA). Nitrocellulose filters and other chemicals were purchased from Sigma (St. Louis, MO). RadPrime DNA labeling kit was purchased from GIBCO-BRL. BCECF was from Molecular Probes (Eugene, OR).

Statistics Results are expressed as means ⫾ SE. Statistical significance between experimental groups was determined by paired or unpaired Student’s t-test. P ⬍ 0.05 was considered significant. RESULTS

Serum [K⫹] and [HCO3⫺] in K⫹ Deprivation Similar to the previously published studies from this laboratory (41), rats fed a K⫹-free diet developed significant hypokalemia only at 14 days and after, but not at 2, 3, or 6 days. Serum [K⫹] was not different be-

tween the two experimental groups at 3 days of K⫹ deprivation (5.1 ⫾ 0.3 meq/l in KD vs. 4.8 ⫾ 0.3 meq/l in control, n ⫽ 4, P ⬎ 0.05). Serum [K⫹] was not statistically different between the two groups at 6 days (4.4 ⫾ 0.3 meq/l in KD vs. 4.9 ⫾ 0.4 meq/l in control, n ⫽ 4, P ⬎ 0.05). Serum [K⫹] was significantly lower after 2 wk of a K⫹-free diet (2.9 ⫾ 0.3 meq/l in KD vs. 5.3 ⫾ 0.4 meq/l in control, n ⫽ 4, P ⬍ 0.01). Serum [HCO3⫺] in rats on a K⫹-free diet for 2 wk was not significantly different from control (HCO3⫺ was 27 ⫾ 1.9 meq/l in KD vs. 25 ⫾ 1.7 meq/l in C, n ⫽ 4, P ⬎ 0.05). Effect of K⫹ Deprivation on NBC-1 mRNA Expression in Superficial Cortex The expression of NBC-1 mRNA is predominantly restricted to the PT under normal conditions (1, 39). In the first series of experiments, the effect of K⫹ deprivation on NBC-1 mRNA levels in the superficial cortex of KD animals was studied. As shown in Fig. 1A (top), NBC-1 mRNA levels increased as early as 72 h (by ⬃3-fold, P ⬍ 0.04, n ⫽ 3, Fig. 1B) and remained elevated at 3 wk of K⫹ deprivation. Neither NBC-2 nor NBC-3 mRNA was detected in the cortex or outer medulla of rats on a normal or K-free diet for 14 days. Measurement of NBC Activity in PT Cells Role of Na⫹/H⫹ exchangers and H⫹-ATPase in pHi recovery from acid load in PT cells. To assay NBC activity in PT cells, Na⫹-dependent and -independent H⫹ transporters (NHEs and H⫹-ATPase, respectively) were verified and inhibited in the presence of specific inhibitors. BCECF-loaded PT cells were acidified in a HCO3⫺-free solution by NH4⫹ pulse (EXPERIMENTAL PRO⫹ CEDURES) and then exposed to a Na -containing solution (solution B, Table 1). As shown in Fig. 2A, exposure of PT cells to Na⫹ resulted in a rapid pHi recovery from acidosis, with an initial rate of 0.519 ⫾ 0.041 pH/min (Fig. 2B, n ⫽ 6). This recovery was reduced to 0.046 ⫾ 0.014 pH/min in the presence of 2 mM amiloride (P ⬍ 0.002 vs. no amiloride, n ⫽ 4) and to 0.017 ⫾ 0.006 pH/min in the presence of both 2 mM amiloride (an NHE inhibitor) and 50 nM bafilomycin A1 (H⫹-

Fig. 1. Effect of K⫹ deprivation (KD) on renal basolateral Na⫹-HCO3⫺ cotransporter (NBC-1) mRNA expression in the superficial cortex. A: representative Northern hybridizations of NBC-1 mRNA expression in the superficial cortex of control or K⫹-deprived rats for 2, 3, 6, and 21 days (top). Glyceraldehyde3-phopshate dehydrogenase (GAPDH) mRNA expression is shown as a constitutive control (bottom). B: NBC-1 mRNA-to-GAPDH mRNA ratios (n ⫽ 3 for each bar). NBC is maximally upregulated after 3 days of KD (* P ⬍ 0.04 compared with control, n ⫽ 3 for each group). Thirty micrograms total RNA were loaded per lane.



F535

Fig. 2. Effect of amiloride and/or bafilomycin A1 on intracellular pH (pHi) recovery from acid load in proximal tubule (PT) cells. A: pHi was measured in PT cells from normal rats incubated and acidified in HCO3⫺-free media and then exposed to 60 mM Na⫹ in the absence of any inhibitors (vehicle), or presence of 2 mM amiloride⫹50 nM bafilomycin A1. B: the rate of pHi recovery (dpHi/dt) was significantly blocked by 2 mM amiloride (n ⫽ 4, P ⬍ 0.002, compared with vehicle, n ⫽ 6). Bafilomycin A1 further inhibited the rate of pHi recovery (n ⫽ 4, P ⬍ 0.05) compared with amiloride alone. Each bar is mean ⫾ SE of indicated n runs.

Fig. 3. DIDS-sensitive NBC mediates pHi recovery from acid load in PT cells. PT cells were acid loaded in CO2/HCO3⫺-containing solution and then exposed to 60 mM Na⫹ in the presence of 2 mM amiloride⫹50 nM bafilomycin A1. A: pHi tracing demonstrating the effect of 100 ␮M DIDS on pHi recovery. B: DIDS inhibited dpHi/dt in a dose-dependent manner. ** P ⬍ 0.001 compared with vehicle. * P ⬍ 0.01 compared with 100 ␮M. Each bar is mean ⫾ SE of n ⫽ 3–6 runs.


F536


ATPase inhibitor) (P ⬍ 0.05 vs. amiloride alone, n ⫽ 4, Fig. 2B). Nadir pHi was not different between the three groups (Fig. 2A). In the presence of 2 mM amiloride and 50 nM bafilomycin, the pHi recovery from acidosis was comparable in the presence (solution B, Table 1) or the absence of Na⫹ (solution A, Table 1, n ⫽ 4, P ⬎ 0.05, data not shown). These results indicate that in the absence of CO2/HCO3⫺ in the media, the pHi recovery from an acid load was completely abolished by 2 mM amiloride and 50 nM bafilomycin. Role of NBC in pHi recovery from acid load in PT cells. To determine NBC activity in PT cells the experiments were repeated in the presence of CO2/HCO3⫺ in the media. In the presence of 2 mM amiloride and 50 nM bafilomycin A1, the switch to a Na⫹-containing solution (solution D, Table 1) resulted in an initial pHi recovery of 0.157 ⫾ 0.013 pH/min (Fig. 3, n ⫽ 6), which was significantly higher than in the absence of bicarbonate (P ⬍ 0.04 vs. Fig. 2). The presence of DIDS inhibited the Na⫹- and HCO3⫺-dependent pHi recovery in a dose-dependent manner (Fig. 3B). The pHi recovery decreased to 0.022 ⫾ 0.008 pH/min in the presence of 300 ␮M DIDS (P ⬍ 0.001 vs. vehicle, n ⫽ 3, Fig. 3B). The DIDS-insensitive pHi recovery from acidosis in the presence of Na⫹ (solution D, Table 1, Fig. 3B) is comparable to the pHi recovery in the absence of Na⫹ and the presence of 2 mM amiloride and 50 nM bafilomycin [solution C, Table 1, dpHi/dt ⫽ 0.022 ⫾ 0.008 pH/min in the presence of Na⫹ (Fig. 3B) vs. 0.017 ⫾ 0.009 in the absence of Na⫹, P ⬎ 0.05, n ⫽ 5]. These results indicate that the presence of Na⫹ is required for the DIDSsensitive HCO3⫺-dependent pHi recovery in PT cells. Effect of K⫹ Deprivation on NBC Activity in PT Cells To examine the effect of K⫹ deprivation on NBC activity, PT suspensions were harvested from rats on a K⫹-free diet for 6 days, loaded with BCECF, and assayed for Na⫹- and HCO3⫺-dependent pHi recovery from intracellular acidosis (see EXPERIMENTAL PROCEDURES). The pHi recovery was significantly increased in KD rats, with dpHi/dt increasing from 0.107 ⫾ 0.017 in control to 0.226 ⫾ 0.021 pH/min in KD rats (P ⬍ 0.02, n ⫽ 6). The NBC-mediated pHi recovery was blocked by 300 ␮M DIDS in control and KD rats. These results are summarized in Fig. 4A and indicate that the DIDSsensitive NBC activity is significantly increased in PT cells of KD rats (Fig. 4A). Taken together, these results demonstrate that enhanced NBC-1 mRNA expression correlates with increased NBC activity in K⫹ deprivation. Cl⫺ Independence of Na⫹-HCO3⫺ Cotransporter in PT Cells The presence of a basolateral Na⫹-dependent Cl⫺/ HCO3⫺ exchanger has been reported in the PT cells (3). To determine whether a possible upregulation of this transporter was contributing to enhanced NBC activity in K⫹ deprivation, the experiments described above (Fig. 4A) were repeated in the absence of Cl⫺. Accordingly, PT cells harvested from control or KD rats were

Fig. 4. Effect of KD on NBC activity in PT cells. PT cells harvested from normal or K⫹-deprived rats were acid loaded in CO2/HCO3⫺containing solution and then exposed to 60 mM Na⫹ in the presence of 2 mM amiloride⫹50 nM bafilomycin A1. A: experiments were performed in the presence of Cl⫺ in the solution. The NBC activity (expressed as DIDS-sensitive dpHi/dt) is increased in KD rats, P ⬍ 0.05. B and C: experiments were performed in the absence of Cl⫺ in the solution. B: pHi time course of PT cells harvested from control or KD rats. C: NBC activity is increased in PT cells of KD rats, P ⬍ 0.04. Each bar is mean ⫾ SE of indicated n ⫽ 5–6 runs. [Cl⫺]o and [HCO3⫺]o, Cl⫺ and HCO3⫺ concentrations.

incubated in a Cl⫺-free solution for 30 min (Cl⫺ replaced by gluconate in solution C, Table 1) and assayed for NBC activity as described above. The results depicted in Fig. 4B show that the Na⫹-and HCO3⫺-dependent pHi recovery from acidosis was increased in PT



F537

Fig. 5. Effect of K⫹ supplement on cortical NBC mRNA expression. A: representative Northern hybridization of NBC-1 mRNA expression in the cortex of control or K⫹-deprived rats for 6 days (top). 28S rRNA expression is shown for control loading (bottom). B: NBC-1 mRNA-to-28S rRNA ratio. Each lane is loaded with 30 ␮g total RNA. Each bar is mean ⫾ SE of n ⫽ 4 rats/ group.

cells of KD rats (dpHi/dt was 0.079 ⫾ 0.015 pH/min in control, n ⫽ 5, and 0.195 ⫾ 0.028 pH/min in KD rats, n ⫽ 6, P ⬍ 0.04). In the presence of 300 ␮M DIDS, dpHi/dt in PT cells of KD rats was reduced to 0.016 ⫾ 0.009 pH/min (n ⫽ 4). The DIDS-sensitive dpHi/dt (NBC activity) was increased from 0.079 ⫾ 0.015 (n ⫽ 5) in control to 0.195 ⫾ 0.028 (n ⫽ 6) in KD rats (P ⬍ 0.04, Fig. 4C). Taken together, the results depicted in Fig. 4, A and C, indicate that enhanced NBC activity (expressed as DIDS-sensitive dpHi/dt) in KD is mediated via a Cl⫺-independent DIDS-sensitive process. Effect of K⫹ Deprivation on Buffering Power in PT Cells The intracellular buffering power was measured according to EXPERIMENTAL PROCEDURES in PT cells of rats on normal or KD diet for 6 days. PT cells were incubated in HCO3/CO2 and Na⫹-free media, loaded with BCECF, and monitored for pHi changes. The buffering power in PT cells was 31 ⫾ 1.27 mM H⫹/pH unit (at pHi ⫽7.13 ⫾ 0.016) in control and 33 ⫾ 1.89 mM H⫹/pH unit (at pHi ⫽ 7.09 ⫾ 0.029) in KD rats (P ⬎ 0.05, n ⫽ 6 for each group). These results indicate that K⫹ deprivation did not alter the intracellular buffering power in PT cells. Effect of K⫹ Supplement on Cortical NBC mRNA Expression To determine whether the KD-mediated NBC mRNA upregulation is specific to the lack of K⫹ in the diet, rats were placed on control or K⫹-free diet and supplemented with 0.85% of KCl in their drinking water.1 Animals were killed after 6 days and examined for the expression of NBC. As shown in Fig. 5, the upregulation of NBC-1 mRNA levels in the KD group was blocked in the presence of KCl in the drinking water (Fig, 5, A and B, P ⬎ 0.05, n ⫽ 4 rats in each group). Effect of K⫹ Deprivation on NBC-1 mRNA Expression in Outer Medulla and mTAL NBC-1 mRNA levels are undetectable in the rat medulla under normal conditions (14). We tested the 1 The concentration of KCl in the drinking water (0.85%) was calculated on the basis of dietary K⫹ intake and the volume of drinking water in control rats.

possibility that K⫹ deprivation may induce the expression of NBC-1 mRNA in various medullary segments of rat kidney. The inner stripe of outer medulla was dissected from the kidneys of control and KD animals and utilized for Northern hybridization. Figure 6 demonstrates that NBC-1 mRNA was heavily induced at 2 days of K⫹ deprivation (P ⬍ 0.05, Fig. 6B, n ⫽ 3), further increased at 3 days, reached a maximal plateau at 6 days (P ⬍ 0.04 compared with 3 days, n ⫽ 3 for each, Fig. 6B), and remained elevated at 21 days of K⫹ deprivation. To determine the nephron segment that could be the source of enhanced NBC-1 mRNA in the inner strip of outer medulla, mTAL suspensions were harvested from the inner stripe of outer medulla of both control and KD rats and utilized for Northern hybridization. As shown in Fig. 7, NBC-1 mRNA expression was sharply induced in mTAL cells of rats on K⫹-free diet for 2 wk. It is worth mentioning that the induction of NBC-1 mRNA in the outer medulla of KD rats was prevented when the KD animals were supplemented with 0.85% KCl in their drinking water (data not shown). Effect of K⫹ Deprivation on NBC Activity in mTAL Cells To assay NBC activity in mTAL cells Na-dependent and -independent H⫹ transporters were first determined and inhibited in the presence of specific inhibitors. Inhibition of H⫹-dependent transporters in mTAL cells. mTAL cells express both H⫹-ATPase and NHE3 on their apical membrane (13, 22) and NHE1 on their basolateral membrane (47). In addition, a K⫹/ H⫹ antiport (4, 8), a K⫹-HCO3⫺ cotransport (10, 31), and a Cl⫺/HCO3⫺ exchanger (20) are also expressed in mTAL cells. The experiments were therefore performed in mTAL tubules depleted of Cl⫺ and K⫹ by prolonged incubation (25 min at least) in Cl⫺- and K⫹-free solution (solution A, Table 2) to keep the Cl⫺/HCO3⫺ exchanger and K⫹/H⫹ antiporter inactive. In the presence of CO2/ HCO3, but in the absence of amiloride and bafilomycin A1, exposure of the acid-loaded mTAL cells to the Na⫹-containing solution (solution C, Table 1) resulted in a rapid pHi recovery from intracellular acidosis with an initial rate of 1.198 ⫾ 0.070 pH/min


F538


Fig. 6. Effect of K⫹ deprivation on NBC-1 mRNA expression in the inner stripe of outer medulla. A: representative Northern hybridization of NBC-1 mRNA expression in the inner stripe of outer medulla of control or K⫹-deprived rats for 2, 3, 6, and 21 days (top). GAPDH mRNA expression is shown for control loading (bottom). B: NBC-1 mRNA-to-GAPDH mRNA ratio (n ⫽ 3 for each bar). NBC-1 is maximally upregulated after 6 days of KD (* P ⬍ 0.04 compared with 3 days). Thirty micrograms RNA were loaded per each lane.

(Fig. 8A). This recovery was decreased to 0.106 ⫾ 0.007 pH/min in the presence of 2 mM amiloride and 50 nM bafilomycin A1 (P ⬍ 0.001, Fig. 8, A and B). These results demonstrate the viability of mTAL cells in Na⫹-, K⫹-, and Cl⫺-free medium. To examine whether the residual recovery from acidosis was Na⫹ dependent, mTAL tubules were acid loaded and exposed to a Na⫹-free but HCO3⫺-containing media (solution B, Table 2) that in addition contained 2 mM amiloride and 50 nM bafilomycin A1. As shown in Fig. 7 (A and B), the pHi recovery from acidosis was negligible in the absence of Na⫹, indicating that the residual pHi recovery from acidosis in the presence of 2 mM amiloride and 50 nM bafilomycin (Fig. 8, A and B) was Na⫹ dependent. Lack of NBC activity in mTAL cells under normal conditions. To determine whether the amiloride-resistant pHi recovery from acidosis (Fig. 8) was HCO3⫺ dependent, mTAL tubules were acid loaded in a solution that was buffered with HEPES and was free of Na⫹, K⫹, Cl⫺, and HCO3⫺ (solution D, Table 2). In the absence of HCO3⫺, the switch to a Na⫹-containing solution (solution E, Table 2) that contained 2 mM amiloride and 50 nM bafilomycin A1 resulted in a significant recovery from acidosis, with the pHi recovery of 0.099 ⫾ 0.009 pH/min (Fig. 9A). This HCO3⫺-independent pHi recovery was inhibited in the absence of Na⫹ in the media (solution D, Table 2), with recovery rates of 0.003 ⫾ 0.002 pH/min (Fig. 9, A and B). The Na⫹-

dependent pHi recovery from acidosis was comparable in the presence or absence of HCO3⫺ in the solution (P ⬎ 0.05, Fig. 9C). Taken together, these results indicate that under normal conditions, mTAL cells do not exhibit any Na⫹ dependent HCO3⫺-cotransport activity. This result is consistent with published reports (31). They further correlate with Northern hybridization experiments indicating the absence of NBC-1 mRNA expression in mTAL cells under normal conditions (Fig. 7). Induction of NBC activity in mTAL cells of KD rats. To determine whether induction of NBC-1 mRNA expression correlates with NBC activity, mTAL tubule suspensions were harvested from animals on the control or K⫹-free diet for 14 days. mTAL cells were incubated and acid loaded in a HCO3⫺-containing, Na⫹-, K⫹-, and Cl⫺-free solution (solution B, Table 2) and assayed for DIDS-sensitive, Na⫹- and HCO3⫺-dependent pHi recovery. Amiloride at 2 mM and bafilomycin at 50 nM were present. The results are summarized in Fig. 10 and indicate that in the presence of HCO3⫺, Na⫹-dependent pHi recovery was significantly higher in mTAL cells of KD rats (Fig. 10A), with the recovery rate of 0.227 ⫾ 0.007, n ⫽ 5, vs. 0.110 ⫾ 0.009 pH/min in control rats, n ⫽ 4 (P ⬍ 0.003). The increased pHi recovery in KD animals was completely blocked by 300 ␮M DIDS (the recovery rate was 0.101 ⫾ 0.005 pH/min, n ⫽ 4, P ⬍ 0.001, vs. no DIDS). Figure 10B depicts the NBC activity as DIDS-sensitive

Fig. 7. Effect of K⫹ deprivation on NBC-1 mRNA expression in mTAL cells. A: representative Northern hybridization of NBC-1 mRNA expression in medullary thick ascending limb (mTAL) cells of control or K⫹-deprived rats for 14 days (top). GAPDH mRNA expression is shown for control loading (bottom). B: NBC-1 mRNA-to-GAPDH mRNA ratio. Each lane is loaded with 30 ␮g total RNA, isolated from pooled tissues from 5 rats.



F539

Fig. 8. Na⫹-dependent pHi recovery from acid load in mTAL cells in the presence of CO2/HCO3. mTAL cells harvested from normal rats were incubated and acid loaded in CO2/HCO3⫺-containing solution, in the absence of Na⫹, K⫹, and Cl⫺. A: pHi tracing of cells exposed to 60 mM Na⫹ with vehicle (●, no inhibitors, n ⫽ 5); in the presence of 2 mM amiloride⫹50 nM bafilomycin A1 (■, n ⫽ 4); or in the presence of 2 mM amiloride⫹50 nM bafilomycin A1 but without Na⫹ (Œ, n ⫽ 4). B: dpHi/dt of the pHi tracings. * P ⬍ 0.001 compared with vehicle. ** P ⬍ 0.001 compared with presence of Na⫹ with inhibitors. Each bar is mean ⫾ SE of indicated n runs.

Na⫹-dependent, HCO3⫺-mediated pHi recovery and demonstrates a significant induction of NBC activity in mTAL tubules of KD rats (Fig. 10B). In the next series of experiments, the intracellular buffering capacity (␤i) of mTAL cells was measured in control and KD rats for 14 days. The experiments were performed in Na⫹-, K⫹-, Cl⫺-, and HCO3/CO2-free solution (solution D, Table 2). The ␤i was comparable in both groups (49 ⫾ 2.15 mM H⫹/pH unit at pHi ⫽ 9.84 ⫾ 0.021 in control and 45 ⫾ 1.58 mM H⫹/pH unit at pHi ⫽ 9.79 ⫾ 0.032 in KD animals, P ⬎ 0.05). Figure 10C indicates that in the absence of Na⫹ (solution B,

Fig. 9. Functional absence of NBC activity in mTAL cells of normal rats. mTAL cells isolated from normal rats were incubated and acid loaded in a solution lacking HCO3⫺, Na⫹, K⫹, and Cl⫺. A: pHi tracings demonstrating Na⫹-dependent recovery from acidosis in the presence of 2 mM amiloride and 50 nM bafilomycin A1. B: dpHi/dt in the presence (n ⫽ 5) or absence (n ⫽ 3) of Na⫹. C: dpHi/dt of mTAL cells acidified and exposed to 60 mM Na⫹, in the presence of 2 mM amiloride with 50 nM bafilomycin A1, with (n ⫽ 4) or without (n ⫽ 5) CO2/HCO3 in the solution. Each bar is mean ⫾ SE of indicated n runs. NS, not significant.


F540


Fig. 10. Induction of NBC activity in mTAL cells. mTAL cells were incubated and acid loaded in a CO2/HCO3⫺-containing solution that lacked Na⫹, K⫹, and Cl⫺. A: Na⫹-induced pHi recovery in mTAL cells harvested from normal (Control diet) or K⫹-deprived rats in the presence of 2 mM amiloride and 50 nM bafilomycin A1. B: K⫹ deprivation induces a DIDS-sensitive dpHi/dt (NBC activity) in mTAL cells of KD rats, P ⬍ 0.004. C: pHi tracing of mTAL tubules harvested from KD rats (14 days), acidified in and exposed to Na⫹-, K⫹-, and Cl⫺-free solution that contained 25 mM HCO3⫺. Each bar is mean ⫾ SE of n ⫽ 4–5 runs.

Table 2), no pHi recovery was observed in acidified mTAL cells harvested from KD rats, confirming the results in Fig. 10A that the pHi recovery in KD animals is Na⫹ dependent. Taken together, these results indicate that the increase in dpHi/dt in mTAL of KD rats is independent of changes in ␤i, and is mediated via a DIDS-sensitive, Na⫹-dependent HCO3⫺ cotransporter. Effect of K⫹ Deprivation on NBC-1 mRNA Expression in the Papilla of Rat Kidney In the last series of experiments, NBC-1 mRNA expression was examined in the inner medulla of rats that were fed control or K⫹-free diet for 2 wk. The results of these experiments depicted in Fig. 11 demonstrate that NBC-1 mRNA abundance was increased by ⬃3-fold in K⫹ deprivation (P ⬍ 0.02, n ⫽ 3, Fig. 11B). DISCUSSION

The above-mentioned studies demonstrate that K⫹ deprivation increases the mRNA expression and activity of the basolateral Na⫹-HCO3⫺ cotransporter (NBC-1) in PT (Figs. 1 and 4). The time course studies reveal that the expression level of NBC-1 mRNA in the superficial cortex is upregulated at

72 h of K⫹ deprivation (Fig. 1) and precedes the onset of hypokalemia. K⫹ deprivation induces the mRNA expression and activity of NBC-1 in mTAL (Figs. 7 and 10) and enhances the expression of NBC-1 in the inner medulla (Fig. 11). K⫹ depletion enhances HCO3⫺ reabsorption in the PT (16, 17, 23, 26, 28, 36). Functional studies have demonstrated that the activity of the cortical luminal Na⫹ /H⫹ exchanger (NHE3) and the basolateral Na⫹-HCO3⫺ cotransporter is increased in K⫹-depleted animals (41, 46). NHE3 mRNA expression remained unchanged (46), indicating that enhanced NHE3 activity in K⫹ deprivation is likely a posttranslational event (46). Enhanced expression of NBC-1 mRNA (Fig. 1) in the PT, however, indicates that increased NBC activity in K⫹ deprivation (Fig. 4 and Ref. 41) is transcriptionally regulated. The rats were not alkalotic despite significant increase in NBC activity. It has been shown that the generation of metabolic alkalosisin rat is time dependent, with animals demonstrating elevated serum bicarbonate at 4 wk of K⫹ deprivation (41). It is likely that the involvement of other bicarbonate-absorbing transporters (such as apical Na⫹/H⫹ exchanger) is necessary for enhanced vectorial transport of bicarbonate.

Fig. 11. Effect of K⫹ deprivation on NBC-1 mRNA expression in the papilla. Northern hybridization of NBC-1 mRNA expression in inner medulla of control or K⫹deprived rats for 14 days (top). GAPDH mRNA expression is shown for control loading (bottom). B: NBC-1 mRNA-to-GAPDH mRNA ratio (n ⫽ 3 for each bar). Each lane is loaded with 30 ␮g total RNA.



K⫹ deprivation is associated with a sharp induction of NBC-1 in the inner stripe of outer medulla of rat kidney (Fig. 6). This is predominantly due to the induction of NBC-1 in mTAL cells (Figs. 7 and 10). The signal responsible for the induction of NBC-1 in mTAL cells remains unknown. Whether NBC induction indicates enhanced HCO3⫺ reabsorption or reflects a compensatory adaptive regulation remains unknown. HCO3⫺ exit across the basolateral membrane of mTAL cells under normal conditions is predominantly mediated via the K⫹-HCO3⫺ cotransporter and Cl⫺/HCO3⫺ exchanger (10, 20, 31). The K⫹-HCO3⫺ cotransporter has a Michaelis-Menten coeffecient of 70 mM for the intracellular K⫹, and its efflux mode is dependent on a high intracellular [K⫹] (10). It is possible that the reduction of intracellular [K⫹] (if it exceeds the magnitude of extracellular [K⫹] reduction) decreases the outward favorable driving force for the K⫹-HCO3⫺ cotransporter in K⫹ deprivation. Whether NBC-1 induction in K⫹ deprivation is in response to possible downregulation of K⫹-HCO3⫺ cotransport or reflects enhanced HCO3⫺ reabsorption in mTAL cells remains unknown. Additional experiments are needed to evaluate the membrane localization of NBC in mTAL and its role in transepithelial HCO3⫺ reabsorption in K⫹ deprivation. Recent microperfusion studies from our laboratory demonstrated that K⫹ deprivation enhances net HCO3⫺ reabsorption in outer and inner medullary collecting duct of rat kidney (33, 34). Molecular and functional studies indicated that enhanced HCO3⫺ reabsorption in these nephron segments was due to the induction of colonic H⫹-K⫹-ATPase at the luminal membrane (33, 34). Enhanced expression of the basolateral NBC-1 in inner medullary collecting duct cells (Fig. 11) indicates that this transporter works in concert with the luminal colonic H⫹-K⫹-ATPase to increase the transepithelial reabsorption of HCO3⫺ in this nephron segment in K⫹ deprivation. Whether K⫹ deprivation also enhances the expression of NBC-1 in outer medullary collecting duct cells remains speculative. The rat mTAL absorbs up to 15% of the filtered load of HCO3⫺ (21). This is predominantly accomplished via the luminal Na⫹/H⫹ exchanger NHE-3 (21, 22) and H⫹-ATPase (13) and the basolateral K⫹-HCO3⫺ cotansporter and Cl⫺/HCO3⫺ exchanger (10, 20, 31). Our results demonstrate the presence of an amiloride-resistant, Na⫹-dependent pHi recovery (presumably an NHE) in mTAL cells of rat kidney (Figs. 8 and 9). The identity of this transporter and its membrane localization remain speculative. One plausible candidate is NHE4, which is resistant to amiloride and is likely expressed in the basolateral membrane of kidney tubules (11, 35). This transporter does not show adaptive regulation in K⫹ deprivation (see RESULTS). The upregulation of Na⫹-HCO3⫺ cotransport in the PT in K⫹ deprivation is of physiological importance in that it may contribute to acid-base homeostasis by the kidney. In addition to its role in vectorial transport of HCO3⫺ in kidney PT, NBC likely plays an

F541

important role in transporting the intracellular HCO3⫺ that is generated in response to enhanced ammoniagenesis in K⫹ depletion (45). In this regard, increased NBC activity in K⫹ deprivation will promote NH4⫹ secretion and Na⫹ retention via the apical Na⫹/H⫹ exchanger (27) and maintain cell pH in a physiological range by extruding the HCO3⫺ load that is originated from both tubular fluid and ammoniagenesis. The mechanism(s) responsible for the upregulation or induction of NBC in various nephron segments in K⫹ deprivation remains unclear. The time course of the effect of K⫹ deprivation demonstrates that enhanced expression of NBC-1 (Figs. 1 and 5) is an early event and precedes the onset of hypokalemia. These results strongly suggest that the signal is likely activated in response to intracellular K⫹ depletion that occurs early in the course of K⫹ deprivation. The identity of this signal(s) remains speculative. K⫹ deprivation is associated with kidney hypertrophy, indicating activation of one or more growth factors (40). It is possible that the growth factor(s) that is activated in K⫹ deprivation is responsible for the enhancement of NBC-1. There is also evidence in support of a role for the pituitary gland in the adaptive regulation of ion transporters in the kidney in K⫹ deprivation. In rats subjected to hypophysectomy, there was blunting of kidney enlargement and colonic H⫹-K⫹-ATPase induction in K⫹ deprivation (46), indicating a possible role for certain pituitary hormones mediating the adaptive changes in this condition. Whether these pituitary factors are also involved in the regulation of NBC by K⫹ deprivation remains to be examined. It is plausible that other hormones (such as ANG II, insulin, adrenal steroids, or catecholamins) could play a role in mediating the effect of K⫹ deprivation on NBC mRNA expression and activity. In conclusion, K⫹ deprivation enhances or induces the expression of the basolateral Na⫹-HCO3⫺ cotransporter (NBC-1) in PT, mTAL, and inner medullary collecting duct. We propose that NBC-1 likely mediates enhanced HCO3⫺ reabsorption in PT, mTAL, and inner medullary collecting duct and contributes to the maintenance of metabolic alkalosis in K⫹ deprivation. These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-46789, DK-52281, and DK-54430 and funds from Dialysis Clinic Incorporated (M. Soleimani). REFERENCES 1. Abuladze N, Lee I, Newman D, Hwang J, Pushkin A, and Kurtz I. Axial heterogeneity of sodium-bicarbonate cotransporter expression in the rabbit proximal tubule. Am J Physiol Renal Physiol 274: F628–F633, 1998. 2. Alpern RJ. Mechanism of basolateral membrane H⫹/OH⫺/ HCO3⫺ transport in the rat proximal convoluted tubule. A sodium-coupled electrogenic process. J Gen Physiol 86: 613–636, 1985. 3. Alpern RJ and Chambers M. Basolateral membrane Cl/HCO3 exchange in the rat proximal convoluted tubule. J Gen Physiol 89: 581, 1987.


F542


4. Amlal H, Burnham CE, and Soleimani M. Characterization of Na⫹/HCO3⫺ cotransporter isoform NBC-3. Am J Physiol Renal Physiol 276: F903–F913, 1999. 5. Amlal H, Paillard M, and Bichara M. NH4⫹ transport pathways in cells of medullary thick ascending limb of rat kidney. J Biol Chem 269: 21962–21971, 1994. 6. Amlal H, Wang Z, Burnham CE, and Soleimani M. Functional characterization of cloned human Na⫹: HCO3⫺ cotransporter. J Biol Chem 273: 16810–16815, 1998. 7. Amlal H, Wang Z, and Soleimani M. Potassium depletion downregulates chloride-absorbing transporters in rat kidney. J Clin Invest 101: 1045–1054, 1998. 8. Attmane-Elakeb A, Boulanger H, Vernimmen C, and Bichara M. Apical location and inhibition by arginine vasopressin of K⫹/H⫹ antiport of the medullary thick ascending limb of rat kidney. J Biol Chem 272: 25668–25677, 1997. 9. Biagi BA and Sohtell M. Electrophysiology of basolateral bicarbonate transport in the rabbit proximal tubule. Am J Physiol Renal Fluid Electrolyte Physiol 250: F267–F272, 1986. 10. Blanchard A, Leviel F, Bichara M, Podevin RA, and Paillard M. Interactions of external and internal K⫹ with K⫹-HCO3⫺ cotransporter of rat medullary thick ascending limb. Am J Physiol Cell Physiol 271: C218–C225, 1996. 11. Bookstein C, Mush M, Depaoli A, Xie Y, Villereal M, Rao M, and Chang A. A unique sodium-hydrogen exchange isoform (NHE-4) of the inner medulla of rat kidney is induced by hyperosmolalilty. J Biol Chem 269: 29704–29709, 1994. 12. Boron WF and Boulpaep EL. Intracellular pH regulation in the renal proximal tubule of the salamander. Basolateral HCO3⫺ transport. J Gen Physiol 81: 53–94, 1983. 13. Brown D, Hirsch S, and Gluck S. Localization of a protonpumping ATPase in rat kidney. J Clin Invest 82: 2114–2126, 1988. 14. Burnham CE, Amlal H, Wang Z, Shull GE, and Soleimani M. Cloning and functional expression of a human kidney Na⫹: HCO3⫺ cotransporter. J Biol Chem 272: 19111–19114, 1997. 15. Burnham CE, Flagella M, Wang Z, Amlal H, Shull GE, and Soleimani M. Cloning, renal distribution, and regulation of the rat Na⫹-HCO3⫺ cotransporter. Am J Physiol Renal Physiol 274: F1119–F1126, 1998. 16. Capasso G, Kinne R, Malnic G, and Giebisch G. Renal bicarbonate reabsorption in the rat. I. Effect of hypokalemia and carbonic anhydrase. J Clin Invest 80: 409–414, 1987. 17. Chan YL, Biagi B, and Giebisch G. Control mechanisms of bicarbonate transport across the rat proximal convoluted tubule. Am J Physiol Renal Fluid Electrolyte Physiol 242: F532–F543, 1982. 18. Chomczynski P and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156–159, 1987. 19. Church GM and Gilbert W. Genomic sequencing. Proc Natl Acad Sci USA 81: 1991–1995, 1984. 20. Eladari D, Blanchard A, Leviel F, Paillard M, Stuart-Tilley AK, Alper SL, and Podevin RA. Functional and molecular characterization of luminal and basolateral Cl⫺/HCO3⫺ exchangers of rat thick limbs. Am J Physiol Renal Physiol 275: F334– F342, 1998. 21. Good DW. The thick ascending limb as a site of renal bicarbonate reabsorption. Semin Nephrol 13: 225–235, 1993. 22. Good DW and Watts BA. Functional roles of apical membrane Na⫹/H⫹ in rat medullary thick ascending limb. Am J Physiol Renal Fluid Electrolyte Physiol 270: F691–F699, 1996. 23. Hernandez RE, Schambelan M, Cogan MG, Colman J, Morris RC, and Sebastian A. Dietary NaCl determines severity of potassium depletion-induced metabolic alkalosis. Kidney Int 31: 1356–1367, 1987. 24. Houillier P, Chambrey R, Achard JM, Froissart M, Poggioli J, and Paillard M. Signaling pathways in the biphasic effect of angiotensin II on apical Na/H antiport activity in proximal tubule. Kidney Int 50: 1496–1505, 1996.

25. Ishibashi K, Sasaki S, and Marumo F. Molecular cloning of a new sodium bicarbonate cotransporter cDNA from human retina. Biochem Biophys Res Commun 246: 535–538, 1998. 26. Jones JW, Sebastian A, Hutler HN, Schambelan M, Sutton JM, and Biglieri ED. Systemic and renal acid-base effects of chronic dietary potassium depletion in humans. Kidney Int 21: 402–410, 1982. 27. Kinsella JL and Aronson PS. Interaction of NH4⫹ and Li⫹ with the renal microvillus membrane Na⫹/H⫹ exchanger. Am J Physiol Cell Physiol 241: C220–C226, 1981. 28. Kunau, RT Jr, Frick A, Rector FC Jr, and Seldin DW. Micropuncture study of the proximal tubular factors responsible for the maintenance of alkalosis during potassium deficiency in the rat. Clin Sci (Lond) 34: 223–231, 1968. 29. Kurtz I and Balaban RS. Ammonium as a substrate for Na⫹K⫹-ATPase in rabbit proximal tubules. Am J Physiol Renal Fluid Electrolyte Physiol 19: F497–F502, 1986. 30. Laghmani K, Borensztein P, Ambu ¨ hl P, Froissart M, Bichara M, Moe OW, Alpern RJ, and Paillard M. Chronic metabolic acidosis enhances NHE-3 protein abundance and transport activity in the rat thick ascending limb by increasing NHE-3 mRNA. J Clin Invest 99: 24–30, 1997. 31. Leviel F, Borensztein P, Houllier P, Paillard M, and Bichara M. Electroneutral K⫹/HCO3⫺ cotransport in cells of medullary thick ascending limb of rat kidney. J Clin Invest 90: 869–878, 1992. 32. Pushkin A, Abuladze N, Lee I, Newman D, Hwang J, and Kurtz I. Cloning, tissue distribution, genomic organization, and functional characterization of NBC3, a new member of the sodium bicarbonate cotransporter family. J Biol Chem 274: 16569– 16575, 1999. 33. Nakamura S, Amlal H, Galla JH, and Soleimani M. Colonic H⫹-K⫹-ATPase is induced and mediates increased HCO3⫺ reabsorption in inner medullary collecting duct in potassium depletion. Kidney Int 54: 1233–1239, 1998. 34. Nakamura S, Wang Z, Galla JH, and Soleimani M. K⫹ depletion increases HCO3⫺ reabsorption in OMCD by activation of colonic H⫹-K⫹-ATPase. Am J Physiol Renal Physiol 274: F687–F692, 1998. 35. Pizzonia JH, Biemesderfer D, Abualfa A, Wu M, Exner M, Isenring P, Igarashi P, and Aronson PS. Immunochemical characterization of Na⫹/H⫹ exchanger isoform NHE4. Am J Physiol Renal Physiol 275: F510–F517, 1998. 36. Rector, FC Jr, Bloomer HA, and Seldin DW. Effect of potassium deficiency on the reabsorption of bicarbonate in the proximal tubule of the rat kidney. J Clin Invest 43: 1976– 1982, 1964. 37. Romero MF, Hediger MA, Boulpaep EL, and Boron WF. Expression cloning and characterization of a renal electrogenic Na⫹: HCO3⫺ cotransporter. Nature 387: 409–413, 1997. 38. Roos A and Boron WF. Intracellular pH. Physiol Rev 61: 296–434, 1981. 39. Schmitt BM, Biemesderfer D, Romero MF, Boulpaep EL, and Boron WF. Immunolocalization of the electrogenic Na⫹HCO3⫺ cotransporter in mammalian and amphibian kidney. Am J Physiol Renal Physiol 276: F27–F36, 1999. 40. Schrader GA, Prickett CO, and Salmon WD. Symptomatology and pathology of potassium and magnesium deficiencies in the rat. J Nutr 14: 85–110, 1935. 41. Soleimani M, Bergman JA, Hosford MA, and McKinney ⫽ TD. Potassium depletion increases Na⫹:CO3 :HCO3⫺ cotransport in rat renal cortex. J Clin Invest 86: 1076–1083, 1990. 42. Soleimani M and Burnham CE. Physiologic and molecular aspects of the Na:HCO3⫺ cotransporter in health and disease processes. Kidney Int 57: 371–384, 2000. 43. Soleimani M, Grassl SM, and Aronson PS. Stoichiometry of Na⫹-HCO3⫺ cotransport in basolateral membrane vesicles isolated from rabbit renal cortex. J Clin Invest 79: 1276–1280, 1987. 44. Soleimani M and Singh G. Physiologic and molecular aspects of the Na⫹/H⫹ exchangers in health and disease processes. J Invest Med 43: 419–430, 1995.


NBC IN K⫹ DEPLETION 45. Tannen RL. Relationship of renal ammonia production and potassium homeostasis. Kidney Int 11: 453–456, 1977. 46. Wang Z, Baird N, Shumaker H, and Soleimani M. Potassium depletion and acid-base transporters in rat kidney: differential effect of hypophysectomy. Am J Physiol Renal Physiol 272: F736–F743, 1997. 47. Watts BA and Good DW. Apical membrane Na⫹/H⫹ exchange in rat medullary thick ascending limb. pHi-dependence and inhibition by hyperosmolality. J Biol Chem 269: 20250–20255, 1994.

F543

48. Wingo CS and Armitage FE. Rubidium absorption and proton secretion by rabbit outer medullary collecting duct via H-KATPase. Am J Physiol Renal Fluid Electrolyte Physiol 263: F849–F857, 1992. 49. Yoshitomi K, Burckhardt BC, and Fromter E. Rheogenic sodium-bicarbonate cotransport in the peritubular cell membrane of rat renal proximal tubule. Pflu¨gers Arch 405: 360–366, 1985.
