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Am J Physiol Renal Physiol 274: F687-F692, 1998;
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Vol. 274, Issue 4, F687-F692, April 1998

K+ depletion increases HCO<SUP>−</SUP><SUB>3</SUB> reabsorption in OMCD by activation of colonic H+-K+-ATPase

Suguru Nakamura, Zhaohui Wang, John H. Galla, and Manoocher Soleimani

Department of Medicine, University of Cincinnati School of Medicine, and Veterans Affairs Medical Center, Cincinnati, Ohio 45267

    ABSTRACT
Top
Abstract
Introduction
Materials
Results
Discussion
References

To probe the role of the isoforms of H+-K+-ATPase (HKA) in potassium depletion (KD), rats were placed on a KD diet for 2 wk. Colonic HKA (cHKA) mRNA levels increased ~30-fold in outer medulla, and net HCO<SUP>−</SUP><SUB>3</SUB> flux (JtCO2) in outer medullary collecting duct (OMCD) increased (13.1 pmol · min-1 · mm tubule length-1 in control to 17.7 pmol · min-1 · mm tubule length-1 in KD; P < 0.01). In normal rats, 1 mM ouabain in perfusate had no effect on JtCO2, whereas 10 µM Sch-28080 decreased JtCO2 to 5.1 pmol · min-1 · mm tubule length-1 (P < 0.001). In KD rats, ouabain 1 mM decreased JtCO2 to 6.3 pmol · min-1 · mm tubule length-1 (P < 0.001). Although 10 µM Sch-28080 also decreased JtCO2 to 4.6 pmol · min-1 · mm tubule length-1 (P < 0.001), the inhibitory effects of Sch-28080 and ouabain were not additive. Removal of K+ from perfusate blocked Sch-28080-sensitive JtCO2 in both normal and KD tubules. The data suggest that, in KD, cHKA is induced and mediates increased HCO<SUP>−</SUP><SUB>3</SUB> reabsorption in OMCD, cHKA in vivo is sensitive to both Sch-28080 and ouabain, and cHKA activity is dominant.

acid-base homeostasis; alkalosis; proton secretion; potassium reabsorption; hydrogen-potassium-adenosinetriphosphatase; outer medullary collecting duct

    INTRODUCTION
Top
Abstract
Introduction
Materials
Results
Discussion
References

POTASSIUM DEPLETION (KD) is associated with metabolic alkalosis, but the renal mechanisms that might sustain an elevated serum bicarbonate concentration are incompletely understood. Structurally, the outer medullary collecting duct (OMCD) undergoes striking hypertrophy in KD (30), suggesting that transport in this segment is important for the renal response to KD.

Molecular studies demonstrate the presence of two H+-K+-ATPase (HKA) isoforms, colonic (cHKA) and gastric (gHKA), in collecting duct cells (1, 2, 13, 26), presumed to mediate the exchange of intracellular H+ for luminal potassium at the apical membrane (20, 32, 37, 39). Functional studies do not support a significant role for cHKA under normal conditions. However, cHKA has been suggested to play an important role in potassium conservation, as shown by significant increase in its mRNA or protein abundance in KD (3, 14, 21, 34). However, studies demonstrating a functional role for cHKA in KD are lacking.

Both gHKA and cHKA isoforms show similar patterns of nephron segment distribution in the kidney, with both expressed along the length of collecting duct (1, 2, 8, 18, 38, 39). Functional characterization of HKA isoforms will be essential in determining the role of each isoform in HCO<SUP>−</SUP><SUB>3</SUB> reabsorption or K+ reabsorption, because both isoforms are involved in secretion of H+ in exchange for luminal K+ absorption in collecting duct. Examining the inhibitory profile of each isoform will therefore be critical in distinguishing its contribution to acid-base or electrolyte homeostasis. Studies have shown that gHKA is sensitive to Sch-28080 and omeprazole and insensitive to ouabain (33), whereas cHKA is sensitive to ouabain and insensitive to Sch-28080 and omeprazole (12).

KD increases the mRNA and protein levels for cHKA but not gHKA in rat kidney (3, 14, 21, 34), with OMCD demonstrating the highest level of enhanced expression (3). To examine the contribution of these HKA isoforms to net HCO<SUP>−</SUP><SUB>3</SUB> reabsorption (JtCO2) in KD, OMCD of normal or KD rats was examined in the presence or absence of Sch-28080 or ouabain in perfusate.

    METHODS AND MATERIALS
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Abstract
Introduction
Materials
Results
Discussion
References

Animal Model

Male Sprague-Dawley rats (175-200 g) were placed on a potassium-deficient diet (27, 34) for 2 wk. Rats were housed two per cage and had free access to food and water. Body weights were recorded at the beginning and at the end of the 2-wk period. Animals were killed by intraperitoneal injection of 50 mg pentobarbital sodium. Aortic blood was obtained at death. Serum K+ concentration was measured by flame photometry. For functional studies, kidneys were used on the same day of death. For Northern hybridizations, kidneys were removed, snap frozen in liquid nitrogen, and stored at -70°C until used.

HCO<SUP>−</SUP><SUB>3</SUB> Transport Measurement

In vitro microperfusion. After removal, both kidneys were decapsulated and sectioned into three to four cross sections per kidney and immediately placed in a petri dish containing dissecting solution. Each section was stripped from the papillary tip to the cortex into smaller wedges and transferred into a second petri dish containing dissecting solution maintained at 14°C under a dissecting microscope. Segments of OMCD were dissected from the zone between the corticomedullary junction and the transition from the thin to the thick ascending limbs of Henle under ×120 magnification (16, 19). Tubules were then transferred to a Lucite chamber containing bathing solution initially at room temperature. One end of the tubule was pulled into an outer holding pipette. Once secure, the inner perfusion pipette was advanced, and the tubule was opened with a slight positive pressure (16, 19). The opposite end of the tubule was then pulled into an outer collecting pipette. The tubules were warmed to 37°C in a temperature-controlled chamber and bathed with solution replaced every 30 min. Perfusion rates were maintained at 1-2 nl/min. Collections were made at 20- to 30-min intervals in a precalibrated constant-bore collection pipette. Two collections (no inhibitor vs. ouabain or Sch-28080 in perfusate) or three collections (no inhibitor vs. ouabain vs. Sch-28080 in perfusate) were made per each tubule. The collected samples were placed in a petri dish under mineral oil (16, 19). The solutions used are shown in Table 1. HCO<SUP>−</SUP><SUB>3</SUB>-containing solutions were bubbled with 5% CO2-95% O2 gas. Bath pH was nominally 7.4 ± 0.05. The osmolarity of the solutions was adjusted to 290 mosM by addition of sucrose. In all perfusions, the sequence of control and inhibitor was varied, and, in some tubules, more than one inhibitor (Sch-28080 vs. ouabain) or more than one perfusate (K containing vs. K free) was used. In addition, the perfusionist was blinded at random to the nature of the perfusate or the inhibitor. For these reasons, the perfusion data for each condition and in each model are presented in bar graph format rather than as paired data.

                              
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  		Table 1.   Composition of solutions

Measurement of tCO2 flux. tCO2 in nanoliter samples from collectate and perfusate was measured by microcalorimetry (Picapnotherm; World Precision Instruments, New Haven, CT). The net flux of tCO2 across the tubule epithelium was calculated as
<IT>J</IT><SUB>tCO<SUB>2</SUB></SUB> = (C<SUB>0</SUB>V<SUB>0</SUB> − C<SUB>1</SUB>V<SUB>1</SUB>)/<IT>L</IT>
where JtCO2 is the net flux of tCO2 (pmol · min-1 · mm tubule length-1), C0 is the concentration of tCO2 in the perfusion fluid (in pmol/nl), C1 is the concentration of tCO2 in the collected fluid (in pmol/nl), V0 is the perfusion rate (in nl/min), V1 is the collection rate (in nl/min) (in the absence of vasopressin, V0 = V1), and L is the length of the tubule (in mm) (16, 19, 20).

RNA isolation. Total cellular RNA was extracted from kidney (cortex or outer medulla) by the method of Chomczynski and Sacchi (10). The use of outer medulla was for the enrichment of OMCD. In brief, 0.5-1 g of tissue was homogenized at room temperature in 10 ml Tri-Reagent (Molecular Research Center, Cincinnati, OH). RNA was extracted by phenol/chloroform, precipitated by isopropanol (10), and quantitated by spectrophotometry. RNA was stored at -80°C until use.

Northern hybridization. Total RNA samples (30 µg/lane) were fractionated on a 1.2% agarose-formaldehyde gel and transferred to Magna NT nylon membranes (MSI), using 10× sodium chloride-sodium phosphate-EDTA (SSPE) as transfer buffer. Membranes were then cross-linked by ultraviolet light and baked as described (29). Hybridization was performed according to Church and Gilbert (11). Briefly, membranes were preprehybridized for 1 h in 0.1× SSPE/1% SDS solution at 65°C. The membranes were 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 µg/ml sonicated carrier DNA. The cDNA probe was labeled with 32P-labeled deoxynucleotides, using the RadPrime DNA labeling kit (GIBCO-BRL, Life Technologies) and used for overnight hybridization of the membranes as described (10). 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). For cHKA, three PCR products from the rat alpha -subunit cDNA (nucleotides 135-515, 2369-2998, and 3098-3678) were pooled and used as isoform-specific probe. For gHKA, the EcoR V-Pst I fragment from the alpha -subunit, a gift from Dr. Gary Shull, was used as specific probe.

Accuracy of separation of cortical and outer medullary RNA was verified by positive Northern blots for thiazide-sensitive NaCl mRNA in the cortex and not in medulla and by much higher abundance of the apical Na-K-2Cl cotransporter mRNA in the outer medulla.

Materials. [32P]CTP was purchased from NEN (Boston, MA). Nitrocellulose filters, agarose, and other chemicals were purchased from Sigma Chemical (St. Louis, MO). RadPrime DNA labeling kit was purchased from Life Technologies.

Statistical analysis. Data are expressed as means ± SE where appropriate. For statistical analysis of mRNA expression experiments, the PhosphorImager readings were obtained and analyzed by analysis of variance. For functional studies, JtCO2 was considered an approximation of net bicarbonate reabsorption. Analysis of variance and t-test were used where appropriate to determine statistical significance. P < 0.05 was considered statistically significant.

    RESULTS
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Abstract
Introduction
Materials
Results
Discussion
References

Rats fed a KD diet developed significant hypokalemia at 14 days and showed increased kidney weights (Table 2). Body weights increased from 178 ± 4 to 275 ± 5 g in control animals (n = 8) and from 180 ± 4 to 248 ± 5 g in KD animals (n = 8).

                              
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  		Table 2.   Kidney weights and serum [K+]

Levels of cHKA mRNA increased by ~5-fold in the cortex (P < 0.01 vs. normal, n = 4) and by >30-fold in the outer medulla (P < 0.001, n = 4) (Fig. 1). Expression of gHKA remained unchanged.


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  		Fig. 1.   Representative colonic H+-K+-ATPase (cHKA) Northern hybridizations in renal cortex or outer medulla of normal or potassium-depleted (KD) rats. Top: cHKA Northern hybridization. Bottom: ethidium bromide staining of transferred RNA, indicating equal loading of RNA samples. PhosphorImager readings of cHKA mRNA indicated 29.5-fold (22-, 29-, 31-, and 36-fold) increase in outer medulla and 5.8-fold (4- to 6- and 8-fold) increase in cortex of KD animals.

In OMCD from normal rats, JtCO2 was 13.1 ± 0.4 pmol · min-1 · mm tubule length-1 and decreased to 5.1 ± 0.8 pmol · min-1 · mm tubule length-1 (n = 4) with 10 µM Sch-28080 in perfusate (P < 0.001) (Fig. 2). Ouabain (1 mM) in the perfusate had no effect on JtCO2 (11.8 ± 0.6 pmol · min-1 · mm tubule length-1; P > 0.05, n = 4). In KD rats, JtCO2 increased (17.7 ± 0.6 pmol · min-1 · mm tubule length-1) in KD compared with normal tubules (14.4 ± 0.5 pmol · min-1 · mm tubule length-1; P < 0.04, n = 6) (Fig. 3). Ouabain (1 mM) in perfusate decreased JtCO2 (6.3 ± 0.4 pmol · min-1 · mm tubule length-1, n = 8) (Fig. 4). Similarly, Sch-28080 (10 µM) in perfusate decreased JtCO2 (4.6 ± 0.5 pmol · min-1 · mm tubule length-1; n = 6) (Fig. 4), but the inhibitory effect of Sch-28080 was not additive to ouabain (JtCO2, 5.1 ± 0.5 pmol · min-1 · mm tubule length-1; n = 3).


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  		Fig. 2.   Effect of 10 µM Sch-28080 (SCH) or 1 mM ouabain in perfusate on net HCO<SUP>−</SUP><SUB>3</SUB> absorption (JtCO2) in normal rats. Each bar represents mean ± SE. Tubule lengths were 1.07 ± 0.16 mm, and perfusion rates were 1.10 ± 0.02 nl/min. Collectate tCO2 concentrations were as follows: control, 11.7 ± 1.7; SCH, 20.4 ± 0.3; and ouabain, 13.6 ± 1.8 mM.


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  		Fig. 3.   JtCO2 in normal or KD rats. Each bar represents mean ± SE for 6 separate experiments. For normal and KD rats, respectively, tubule lengths were 1.07 ± 0.16 and 1.28 ± 0.10 mm, perfusion rates were 1.10 ± 0.03 and 1.11 ± 0.03 nl/min, and collectate tCO2 concentrations were 13.8 ± 0.8 and 9.1 ± 0.7 mM.


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  		Fig. 4.   Effect of 10 µM SCH or 1 mM ouabain in perfusate on JtCO2 in KD rats. Each bar represents mean ± SE for 6 (control), 8 (ouabain), or 6 (SCH) separate experiments. Tubule lengths were 1.28 ± 0.10 mm, and perfusion rates 1.11 ± 0.02 nl/min. Collectate tCO2 concentrations were control, 7.1 ± 1.0; SCH, 19.8 ± 0.8; and ouabain, 17.5 ± 0.7 mM.

The data thus far suggested a switch from gHKA to cHKA activity in KD. Because cHKA bears ~65% homology at amino acid level to Na+-K+-ATPase (13), cHKA could display different functional characteristics in vivo, such as acceptance of Na+ for K+. Thus JtCO2 was determined in the presence or absence of K+ in perfusate (see Table 1 for composition of K-free solution). Na+ was present in both conditions (Table 1). In normal rats, JtCO2 was 14.7 ± 0.8 pmol · min-1 · mm tubule length-1 in K-containing perfusate and decreased to 9.6 ± 1.2 pmol · min-1 · mm tubule length-1 in the K-free perfusate (P < 0.05; n = 4) (Fig. 5). With 10 µM Sch-28080 in K-free perfusate, JtCO2 did not decrease further [JtCO2, 7.2 ± 0.8 pmol · min-1 · mm tubule length-1 (P > 0.05) vs. K-free perfusate, n = 4] (Fig. 5).


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  		Fig. 5.   Effect of luminal K removal ± SCH on JtCO2 in normal rats. Each bar represents mean ± SE for 4 separate experiments. Tubule lengths were 1.00 ± 0.09 mm, and perfusion rates were 1.15 ± 0.03 nl/min. Collectate tCO2 concentrations were control (K containing), 12.7 ± 0.7; K free, 17.4 ± 0.5, and K free + SCH, 18.7 ± 0.4 mM.

In KD rats, JtCO2 was 19.1 ± 0.7 pmol · min-1 · mm tubule length-1 in K-containing perfusate and decreased to 10.1 ± 1.1 pmol · min-1 · mm tubule length-1 in K-free perfusate (P < 0.05, n = 4 for each group) (Fig. 6). Sch-28080 (10 µM) in K-free perfusate did not inhibit JtCO2 further (8.5 ± 0.9 pmol · min-1 · mm tubule length-1, P > 0.05, vs. K-free perfusate; n = 4).


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  		Fig. 6.   Effect of luminal K removal ± SCH on JtCO2 in KD rats. Each bar represents mean ± SE for 4 separate experiments. Tubule lengths were 1.00 ± 0.05 mm, and perfusion rates were 1.06 ± 0.03 nl/min. Collectate tCO2 concentrations were control (K containing), 8.3 ± 0.2; K free, 15.6 ± 1.1; and K free + SCH, 16.7 ± 1.1 mM.

    DISCUSSION
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Abstract
Introduction
Materials
Results
Discussion
References

The above studies demonstrate that KD increases the expression of cHKA mRNA in cortex and particularly in the outer medulla (Fig. 1) and is associated with increased HCO<SUP>−</SUP><SUB>3</SUB> reabsorption in the OMCD (Fig. 3). JtCO2 was inhibited by both ouabain and Sch-28080 in KD (Fig. 4) but to only Sch-28080 in normal rats (Fig. 2). In the absence of K+ in the perfusate, the Sch-28080-inhibitable component of JtCO2 was eliminated in either normal or KD tubules (Figs. 5 and 6).

In vitro transfection studies have shown significant ouabain sensitivity but minimal Sch-28080 sensitivity for cHKA (12). In contrast, in vitro studies of gHKA show inhibition by Sch-28080 or omeprazole but not by ouabain (33, 39). However, recent studies (8) show that, in KD, an H+-K+-ATPase activity (as assayed by ATP hydrolysis) is induced in OMCD and cortical collecting duct (CCD), is sensitive to both ouabain and Sch-28080, and replaces a K-dependent ATPase activity in normal rats sensitive to Sch-28080 and not ouabain. Our functional studies showing ouabain-inhibitable HCO<SUP>−</SUP><SUB>3</SUB> reabsorption and a striking increase in cHKA mRNA expression in KD are in complete agreement with these latter findings. In this hypothesis, gHKA activity would be downregulated by posttranscriptional events. Alternatively, gHKA as well as cHKA could acquire ouabain sensitivity in KD. It should be mentioned that evidence for acquisition of Sch-28080 and ouabain sensitivity by cHKA in KD is indirect.

Increased expression of cHKA (Fig. 1) suggests that this isoform is responsible to a large extent for increased HCO<SUP>−</SUP><SUB>3</SUB> reabsorption in OMCD of KD rats. KD is associated with metabolic alkalosis in both rat (24, 27) and human (25) by increasing the reabsorption of HCO<SUP>−</SUP><SUB>3</SUB> in proximal convoluted tubule (PCT) (24) and distal convoluted tubule (DCT) (9). Increased HCO<SUP>−</SUP><SUB>3</SUB> reabsorption in PCT is likely mediated via the Na+/H+ exchanger NHE-3 (27, 28, 34). It should be noted that several of the studies cited employ microperfusion techniques and show accelerated HCO<SUP>−</SUP><SUB>3</SUB> reabsorption independent of load at physiological or increased perfusion rates. The microperfused DCT (9) includes a segment of the CCD (or akin to the CCD) that secretes HCO<SUP>−</SUP><SUB>3</SUB> in exchange for Cl- (16). The majority of HCO<SUP>−</SUP><SUB>3</SUB> reabsorption in the CCD under normal condition is mediated via the bafilomycin-sensitive H+-ATPase (15, 17). However, the nature of the CCD transporter responsible for increased HCO<SUP>−</SUP><SUB>3</SUB> reabsorption in KD rats has not been studied. Whether H+-ATPase activity remains the same in CCD of KD animals or is upregulated remains controversial (15). However, studies have shown that a Sch-28080-sensitive HKA (as assayed by ATP hydrolysis) is increased in CCD of KD rats (15). In view of our functional HCO<SUP>−</SUP><SUB>3</SUB> reabsorption studies (Figs. 4 and 6) demonstrating cHKA sensitivity to both Sch-28080 and ouabain in KD, as well as ATP hydrolysis studies showing induction of a ouabain and Sch-28080 sensitive HKA in KD (8), we suggest that HKA activity in CCD likely represents cHKA and not gHKA. It is therefore likely that increased cHKA in collecting duct in conjunction with increased Na+/H+ exchanger activity in proximal tubule (27) might be responsible for the maintenance of metabolic alkalosis in KD. It is worth mentioning that HKA and H+-ATPase are differentially regulated in certain pathophysiological states, as shown by upregulation of H+-ATPase but not HKA in metabolic acidosis (31).

Microperfusion experiments in rabbits have concluded that, in the K-depleted state, H+-K+-ATPases contribute significantly to HCO<SUP>−</SUP><SUB>3</SUB> reabsorption and K+ reabsorption in kidney inner stripe of OMCD (5, 6, 22, 37, 39). Both K+ absorption and H+ secretion were inhibited by omeprazole (5, 6, 22, 37, 39), an inhibitor of gHKA. These results are consistent with increased H+-K+-ATPase activity in rabbits with KD. Based on the inhibitory profile of HKA activity in rabbits with KD (HKA was very sensitive to omeprazole and Sch-28080), it has been proposed that gHKA is the isoform responsible for increased activity (39). However, in view of the above data, as well as ATP hydrolysis experiments (8), molecular studies are needed to determine whether gHKA or cHKA is upregulated in KD rabbits.

In KD, the expression of cHKA mRNA increased by nearly 30-fold, suggesting strongly that this isoform becomes predominantly responsible for K-dependent bicarbonate transport under this condition. This genetic datum is further supported by the functional observations that in only KD is bicarbonate transport inhibited by ouabain, a property of cHKA not shared by gHKA in vitro. Given our conclusion from these genetic and functional data then, the further observation that the effects of Sch-28080 and ouabain were not additive suggests that these inhibitors were acting on the same transporter. The acquisition of Sch-28080 sensitivity by cHKA in vivo in KD is therefore a new finding but confirmatory of that of Buffin-Meyer et al. (8). Because this is at variance with in vitro data (12), further studies will be required.

Whether Sch-28080 and ouabain sensitivity of cHKA is unique to KD or reflects the general in vivo inhibitory profile of cHKA remains to be examined. cHKA mRNA was increased by approximately fourfold in both cortex and medulla of rats on Na-depleted diet for 2 wk (36) with no effect on gHKA. Functional studies in split-perfused CCD intercalated cells by cell pH measurement showed the induction of an HKA activity that is sensitive to ouabain and not to Sch-28080, with no change in baseline gHKA activity sensitive to Sch-28080 but not ouabain (Sch-28080 and ouabain effects were additive; R. Silver, Ref. 26a). These results suggest that cHKA sensitivity to both Sch-28080 and ouabain is likely unique to KD and does not apply to Na depletion. The reason for this difference in cHKA sensitivity to Sch-28080 in KD but not in Na depletion remains speculative. Possibilities such as recruitment of a distinct beta -subunit or alteration in the topology of the assembled cHKA subunits in KD should be considered.

The signal responsible for increased cHKA in KD remains speculative. It has been suggested and widely accepted that cHKA upregulation in KD is for K+ conservation (14, 39). This conclusion has been based on the assumption that, in KD, animals need to minimize their obligatory urinary K+ loss. To accomplish that objective, animals upregulate their cHKA to increase luminal K+ reabsorption (in exchange for H+). Several lines of evidence, however, indicate that this scenario may not reflect the whole story: increased cHKA expression occurs as early as 6 days after KD diet, precedes the onset of hypokalemia (34), and correlates with other electrolyte abnormalities (4) that may be involved in cHKA upregulation as will be discussed.

KD increases urinary chloride excretion in rat and human by decreasing Cl- reabsorption in medullary thick limb and DCTs (23). Recent studies in our laboratory demonstrate that KD decreases the mRNA expression and activity of the apical Na-K-2Cl cotransporter and mRNA expression of the Na-Cl- cotransporter (4). Suppression of these two transporters is an early event (4) and could decrease reabsorption of chloride, sodium, and potassium and increase their delivery to distal nephron segments. But although delivery of both Na+ and Cl- to distal nephron segments is increased, only enhanced urinary Cl- excretion is evident (23), indicating compensatory reabsorption of Na+ in distal segments. The two possibilities for compensatory reabsorption of Na+ in distal segments is via Na+ channel or H+-K+-ATPase (with Na+ substituting for K+). Our functional studies (Fig. 6) indicate that in the absence of K+ but presence of Na+ in perfusate, the cHKA-mediated, Sch-28080-sensitive, and ouabain-sensitive HCO<SUP>−</SUP><SUB>3</SUB> reabsorption in OMCD of KD rats is abolished, strongly suggesting that this transporter exclusively operates on K/H exchange mode and does not accept Na+. The only other possibility for increased Na+ reabsorption in distal nephron would be via Na+ channel. Na+ channel operates electrogenically by exchanging extracellular Na+ for intracellular K+. Indeed, we find that mRNA levels for ROMK (the secretory K+ channels in OMCD) are increased in KD (7). Secretion of K+ in exchange for Na+, however, would exacerbate KD. Increased cHKA expression and activity (Figs. 1, 4, and 6) would then increase reabsorption of the luminal K+ in KD, prevents worsening of hypokalemia, and facilitates Na+ reabsorption indirectly by recycling the potassium.1 Alternative to enhanced Na reabsorption in distal nephron via Na+ channel is the possibility that Na+ excretion is decreased due to enhanced reabsorption in proximal nephron segments (resulting from volume depletion and decreased glomerular filtration rate).

In conclusion, cHKA expression is increased, and a ouabain-sensitive HCO<SUP>−</SUP><SUB>3</SUB> reabsorption is induced in OMCD of rats with KD. cHKA is sensitive to both ouabain and Sch-28080 in KD, is likely responsible for increased HCO<SUP>−</SUP><SUB>3</SUB> reabsorption in OMCD, and thereby plays an important role in the maintenance of metabolic alkalosis in KD.

    ACKNOWLEDGEMENTS

We greatly appreciate the technical assistance of Holli Shumaker.

    FOOTNOTES

These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-46789 and grants from Dialysis Clinic Incorporated (Cincinnati, OH).

1 One has to be cautious with generalization of regulation of cHKA in states associated with increased delivery of Na+ to distal nephron. For example, furosemide-induced delivery of Na+ to distal nephron might be different from KD, since, in the former, the thiazide-sensitive Na-Cl cotransporter is upregulated, which, in turn, blunts the magnitude of the natriuresis, whereas in the latter the Na-Cl cotransporter is downregulated.

Address for reprint requests: M. Soleimani, Division of Nephrology and Hypertension, Dept. of Internal Medicine, Univ. of Cincinnati, 231 Bethesda Ave., MSB 5502, Cincinnati, OH 45267-0585.

Received 23 July 1997; accepted in final form 5 January 1998.

    REFERENCES
Top
Abstract
Introduction
Materials
Results
Discussion
References

1.   Ahn, K. Y., K. K. Kim, and B. C. Kone. Cellular distribution of mRNA encoding the colonic H/K-ATPase a subunit in rat kidney (Abstract). J. Am. Soc. Nephrol. 6: 303, 1995[Medline].

2.   Ahn, K. Y., and B. C. Kone. Expression and cellular localization of mRNA encoding the gastric isoform of the H+-K+-ATPase alpha -subunit. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F99-F109, 1995[Abstract/Free Full Text].

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