INTRODUCTION

Top Abstract Introduction Methods Results Discussion References

IT IS WIDELY ACCEPTED that renal net acid excretion is enhanced by hypokalemia (29). In part, this increase in net acid excretion can be attributed to enhanced ammonium production in the proximal tubule (29). Ammonium is produced through conversion of glutamine to glutamate, a reaction catalyzed by phosphate-dependent glutaminase. Ammoniagenesis is regulated in vivo by dietary K⁺ restriction (24, 29) and in vitro by changes in extracellular K⁺ concentration (23, 27). With hypokalemia, distal delivery of NH⁺₄ is increased, whereas the distal delivery of K⁺ is reduced. This combination augments NH⁺₄ uptake by the Na⁺-K⁺-2Cl cotransporter (BSC-1, NKCC2) on the apical membrane of the thick ascending limb (14), which presumably increases NH⁺₄ concentration in the medullary interstitium. These results suggest that ammonium secretion by the collecting duct is regulated with changes in K⁺ homeostasis.

Numerous studies have elucidated an important role for the H⁺-K⁺-ATPases in K⁺ and acid-base homeostasis. There are at least two and possibly three -isoforms of the H⁺-K⁺-ATPase that localize to the collecting duct (19). In the terminal portion of the rat inner medullary collecting duct (tIMCD), both HK₁ (the "gastric" H⁺-K⁺-ATPase) and HK₂ (the "colonic" H⁺-K⁺-ATPase) have been identified (1, 2). In the kidney, expression of both HK₂ mRNA and protein abundance increase during dietary K⁺ restriction (2, 7, 12, 26). HK₂, in contrast to HK₁, is sensitive to ouabain at high concentrations (24). However, the role of HK₂ in luminal acidification along the collecting duct has not been fully defined. Moreover, it is not known whether HK₂-mediated transport is modulated in the kidney in vivo in response to changes in K⁺ homeostasis.

Although HK₁ mRNA has been detected throughout the collecting duct (1), the regulatory response of HK₁ mRNA, upon changes in K⁺ homeostasis, is apparently confined to the renal cortex (3). Since HK₁ is fully inhibited by Sch-28080 at concentrations below 10 µM, the Sch-28080-sensitive component of J_tCO2 has been taken as an index of H⁺ secretion mediated by the ₁-isoform of the H⁺-K⁺-ATPase (33). However, in the rabbit outer medullary collecting duct (OMCD), total and Sch-28080-sensitive bicarbonate absorption (J_tCO2) are not enhanced with dietary K⁺ restriction (34).

Our laboratory has shown that bicarbonate absorption, J_tCO2, is inhibited by removal of K⁺ from the luminal fluid or by the addition of Sch-28080 to the perfusate (32) in tIMCD tubules from rats with chronic metabolic acidosis. Nevertheless, the specific -H⁺-K⁺-ATPase that mediates Sch-28080-sensitive J_tCO2 in vivo, as well as the conditions in vivo that alter its activity, are not well understood. The purpose of the present study was to determine whether changes in K⁺ homeostasis alter J_tCO2 in the rat tIMCD and to determine whether these changes are mediated through changes in activity of H⁺-K⁺-ATPases previously detected in this segment.

	METHODS

Top Abstract Introduction Methods Results Discussion References

Animal conditioning. Pathogen-free male Sprague-Dawley rats weighing 65-120 g (Rm. 205G; Harlan, Indianapolis, IN) were employed and housed in microisolator cages. Unless otherwise specified, animals were fed a rat chow containing 7.8 g K⁺/kg food, 0.664 g Na⁺/kg food (Zeigler Brothers, Gardners, PA) (30). For group A, 3 days prior to death the diet was changed to one with normal K⁺ content found (2.3 g K⁺/kg food, 1.03 g Na⁺/kg food; ICN Biochemicals, Aurora, OH). For group B, 3 days prior to death, rats were placed on a diet identical to that of group A, but where K⁺ content was restricted. This diet contained 0.0041 g/kg K⁺ and 1.03 g/kg Na⁺ (ICN Biochemicals).

Serum and urine samples were collected in rats in both treatment groups. To do so, rats were anesthetized with pentobarbital 50 mg/kg by intraperitoneal injection. Blood and urine samples were collected by cardiac and bladder puncture, respectively. Serum electrolytes were measured by the Department of Veterinary Medicine, University of Texas, using ion-sensitive electrodes (Vettest; Idex Laboratories, Westbrook, ME). Total CO₂ in serum samples was measured using a kit (Carbon Dioxide Kit 132-A, Sigma Chemical, St. Louis) according to the instructions of the manufacturer. Urine and serum osmolality was measured using a vapor-pressure osmometer (Wescor, Logan, UT).

Tissue preparation. In perfusion studies, animals were injected with furosemide (5 mg/100 g body wt ip) 45 min before death by decapitation to induce a rapid diuresis. This furosemide-induced diuresis reduces the inner medullary axial solute concentration gradient (30) and attenuates changes in the extracellular osmolality of the tubule.

Coronal slices were cut from the kidneys and placed into a dissection dish containing the chilled experimental solution (11°C). tIMCDs were dissected from the middle third of the IMCD (31). The inner medulla was transferred to a second dissection dish to dissect tIMCD tubules. Tubules were mounted on concentric glass pipettes and perfused in vitro at 37°C.

Most experiments were performed with identical solutions in the perfusate and bath. Tubules were dissected in solution containing (in mM) 144 NaCl, 5 KCl, 6 NH₄Cl, 1 Na₂HPO₄, 2 CaCl₂, 1.2 MgSO₄, 10 mM HEPES, and 5.5 glucose (pH 7.4, solution 1). Tubules were perfused and bathed in solutions of the following composition (in mM): 114 NaCl, 10 KCl, 6 NH₄Cl, 25 NaHCO₃, 0.5 Na₂HPO₄, 2 CaCl₂, 1.2 MgSO₄, and 5.5 glucose (solution 2). In some experiments, the tubule lumen was perfused with a K⁺-free solution containing (in mM) 124 NaCl, 0 KCl, 6 NH₄Cl, 25 NaHCO₃, 0.5 Na₂HPO₄, 2 CaCl₂, 1.2 MgSO₄, and 5.5 glucose (solution 3). Osmolality was measured in all solutions (30, 31). To maintain the desired CO₂ concentration, the perfusate was passed through jacketed concentric tubing through which 95% air-5% CO₂ was blown in a countercurrent direction around the perfusate line (30, 31). To maintain pH in HCO₃-containing solutions, the bath fluid was constantly bubbled with 95% air-5% CO₂. Bath pH was measured continuously during all experiments as described previously (30, 31). In some experiments, Sch-28080 (10 µM) or ouabain (5 mM) was added to the luminal fluid only. Sch-28080 was prepared as a 10 mM stock in ethanol. Ouabain was added directly to the perfusate solution.

All experiments were performed in two periods. Period 1 was begun 45 min, and ended ~75-120 min, after the tubule was warmed. Period 2 began with a perfusate exchange. This period was begun 2 h, and ended 3 h, after the tubule was warmed.

Measurement of bicarbonate flux. Tubule fluid samples were collected under oil in calibrated constriction pipettes. Flow rate was determined as described previously (31). Total CO₂ (tCO₂) concentration was measured in the collected fluid (C_L) and perfusate (C_o) using a continuous flow fluorometer (28, 30). The CO₂ reagent was purchased as a kit (no. 132-A; Sigma, St. Louis, MO) and diluted to 50% strength with water. Using this method, bicarbonate (total CO₂, tCO₂) concentration differences less than 1 mM can be detected using an 8-nl pipette (28, 30). J_tCO2 was calculated according to the equation where C_o and C_L are the perfusate and collected fluid total CO₂ concentration. V_L is the flow rate in nanoliters per minute, and L is the tubule length. Because there is no net water absorption in the rat tIMCD with symmetrical solutions, this equation assumes zero net fluid transport in the absence of an imposed osmolality gradient (31).

To determine the perfusate total CO₂ concentration, C_o was estimated by measuring tCO₂ concentration in the collected fluid at very fast flow rates. Therefore, CO₂ loss was matched in C_o and C_L measurements, allowing the loss terms to cancel (31).

To determine whether ouabain (5 mM) affects the peak height of the CO₂ assay, CO₂ concentration was measured in 25 mM HCO₃ samples in the presence and absence of 5 mM ouabain on 7 separate days (solution 2). The ratio of peak heights in the presence and absence of ouabain was 0.994 ± 0.011 (not significant, n = 7). Therefore, ouabain did not affect the peak height of bicarbonate samples assayed. We have demonstrated previously that 10 µM Sch-28080 does not affect total CO₂ peak height (32).

Statistical analysis. For each experimental condition, two to four measurements were averaged to obtain a single value for each experimental condition. Mean values were used in the statistical analysis. Statistical significance was determined using a paired or unpaired two-tailed Student's t-test, as appropriate, with P < 0.05 indicating statistical significance. Data are displayed as means ± SE.

	RESULTS

Top Abstract Introduction Methods Results Discussion References

Effect of changes in K⁺ homeostasis on J_tCO2. Rats ingested a K⁺-restricted diet (0.0041 g K⁺/kg food, 1.03 g Na⁺/kg food, group B) or a normal K⁺ diet (2.3 g K⁺/kg food, group A) for 3 days prior to death. This time period was selected because maximal renal K⁺ conservation following dietary K⁺ restriction is observed at 3 days (21). Moreover, we have observed that if the animals ingest this K⁺-restricted diet for more than 3 days, then tIMCD tubules became very difficult to dissect. Serum and urine electrolytes and urinary pH were measured in both treatment groups (Table 1). As shown, serum K⁺ was lower in the K⁺-restricted group than in controls. Moreover, K⁺-restricted rats developed a mild metabolic alkalosis, increased urinary pH and impaired urinary concentrating ability, as reported previously (22, 29).

1

1

1

1

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Effect of dietary K+ restriction on bicarbonate absorption (JtCO2). In rats eating a normal K+ diet (control), JtCO2 was 2.1 ± 0.3 pmol · mm1 · min1 (n = 6). JtCO2 increased to 5.4 ± 0.7 pmol · mm1 · min1 in rats ingesting a K+-restricted diet (n = 16, P < 0.05).

Effect of Sch-28080 on J_tCO2 in tIMCD tubules from rats with differing states of K⁺ homeostasis. To study possible regulation of H⁺-K⁺-ATPase activity with changes in K⁺ homeostasis, we employed Sch-28080, an inhibitor of the gastric H⁺-K⁺-ATPase. In the stomach, the gastric H⁺-K⁺-ATPase, or HK₁, is fully inhibited with Sch-28080 at concentrations below 10 µM (4). In the kidney, transport sensitive to Sch-28080 at a concentration less than 10 µM has been attributed to the "gastric" isoform of the H⁺-K⁺-ATPase (33). Therefore, J_tCO2 was measured before and following the application of Sch-28080 to the luminal perfusate. The difference between these two values, or the Sch-28080-sensitive component of J_tCO2, was taken to be consistent with activity of the HK₁ isoform of the H⁺-K⁺-ATPase. Results of these experiments are shown in Fig. 2A (Table 2). In K⁺-restricted rats (group B), baseline J_tCO2 was 6.0 ± 1.3 pmol · mm¹ · min¹ but decreased to 3.6 ± 0.7 pmol · mm¹ · min¹ upon the addition of Sch-28080 (10 µM) to the perfusate (n = 8, P < 0.05; series 1). To determine whether the Sch-28080-induced reduction in J_tCO2 is a time-dependent phenomenon, time-control experiments were performed (Fig. 2B; Table 2, series 2). To do so, tIMCD tubules from K⁺-restricted rats were perfused under the same conditions as above in series 1. Baseline J_tCO2 was measured (period 1). In period 2, a mock perfusate exchange was performed, and Sch-28080 vehicle (ethanol) was introduced into the perfusate, without the addition of Sch-28080. As shown, the decline in J_tCO2 observed upon the addition of Sch-28080 to the perfusate was not observed in time controls (Fig. 2B).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Effect of Sch-28080 on JtCO2 in K+-restricted rats. A: after addition of 10 µM Sch-28080, JtCO2 declined from 6.0 ± 1.3 to 3.6 ± 0.7 pmol · mm1 · min1 (n = 8, P < 0.05). B: a time-dependent reduction in JtCO2 was not observed. NS, not significant.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Effect of Sch-28080 on JtCO2 in control rats. No change in JtCO2 was detected following addition of Sch-28080 (10 µM) to the luminal perfusate.

Mechanism of Sch-28080-sensitive J_tCO2. Since total and Sch-28080-sensitive J_tCO2 were increased in rats placed on dietary K⁺ restriction, the mechanism of Sch-28080-sensitive net acid secretion was studied further in rat tIMCD tubules from this treatment group. HK₂ mRNA and protein have been detected in the tIMCD (2, 7). Both H-K₂ mRNA and protein abundance are increased in rat medulla with dietary K⁺ restriction (2, 7, 12, 26). When expressed in Xenopus laevis oocytes and measured in the presence of 1 mM K⁺, rat HK₂ activity fell by 70% upon the application of 1 mM ouabain to the extracellular fluid (8). Therefore, since HK₂ is sensitive to high concentrations of ouabain, we asked whether J_tCO2 is sensitive to luminal ouabain at high concentrations. Results of these experiments are shown in Fig. 4A (Table 2, series 4). In K⁺-restricted rats (group B), baseline J_tCO2 was 5.2 ± 1.1 pmol · mm¹ · min¹ but fell to 4.3 ± 1.0 pmol · mm¹ · min¹ with the addition of 5 mM ouabain to the luminal fluid (n = 4, P < 0.05). Thus J_tCO2 is sensitive to high concentrations of luminal ouabain.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Effect of ouabain on JtCO2 in presence and absence of Sch-28080 in K+-restricted rats. A: after addition of 5 mM ouabain to the luminal perfusate, JtCO2 declined from 5.2 ± 1.1 to 4.3 ± 1.0 pmol · mm1 · min1 (n = 4, P < 0.05). B: in presence of Sch-28080, JtCO2 fell from 4.6 ± 1.4 to 3.4 ± 1.1 (n = 6, P < 0.05) after addition of ouabain to the luminal perfusate. Therefore, the effects of ouabain and Sch-28080 on JtCO2 were additive. Dashed lines indicate that the order of the periods was reversed; in experiments denoted by dashed lines, ouabain was present in the first period and withdrawn in the second period.

1

1

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Effect of a reduction in luminal K+ concentration on the Sch-28080- and ouabain-resistant component of JtCO2. With both ouabain and Sch-28080 present in the luminal perfusate, removal of K+ from the perfusate did not elicit a further reduction in JtCO2. Dashed lines indicate that the order of the periods was reversed. That is, in the first period the luminal K+ was 0. In the second period the luminal K+ concentration was 10 mM.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

It is well recognized that dietary K⁺ depletion increases urinary pH and ammonium excretion (29). The increase in urinary pH has been attributed to enhanced ammoniagenesis, which presumably increases medullary interstitial NH₃ concentration (29). With increased NH₃ in the medullary interstitium, NH₃ secretion into the medullary collecting duct is augmented. Thus luminal buffering is increased, which augments net acid excretion (13, 15). The present study demonstrates that the increase in net acid secretion observed with K⁺ restriction in vivo occurs, in part, as a result of increased apical proton secretion. Moreover, since this study was performed in isolated perfused tIMCDs, it demonstrates a "memory" for the increase in H⁺ secretion observed in response to dietary K⁺ restriction. The mechanism for this increase in proton secretion occurs, in part, through increased H⁺-K⁺-ATPase-mediated transport and is compatible with the recent demonstration of a regulatory response of H⁺-K⁺-ATPase to K⁺ depletion (2, 3, 7, 10, 17, 26).

The role of the H⁺-K⁺-ATPases in acid secretion by the collecting duct, including the tIMCD, has been the subject of much interest over the past decade. HK₁ is exquisitely sensitive to Sch-28080 (4). Therefore, Sch-28080-sensitive J_tCO2 has been taken as a representation of the gastric H⁺-K⁺-ATPase-mediated, or HK₁-mediated, transport (33). In cultured rat and mouse tIMCD cells, Sch-28080 inhibits ATP hydrolysis (18) and K⁺-dependent intracellular pH (pH_i) recovery following NH₄Cl addition and then withdrawal (25). In tIMCD tubules from rats with metabolic acidosis, J_tCO2 is reduced by 50% with either the application of Sch-28080 (10 µM) to the luminal fluid or with removal of luminal K⁺ (32). Collectively, these studies demonstrate that Sch-28080-sensitive transport in the tIMCD is an active process which mediates K⁺-dependent apical proton secretion. HK₁ mRNA has been detected in the tIMCD in some (1), but not in all (6), reports. These previous studies are consistent with the hypothesis that Sch-28080-mediated K⁺/OH/H⁺/HCO₃ flux is mediated by HK₁.

However, with in vivo conditioning, the response of HK₁ mRNA and protein differ from Sch-28080-sensitive transport. In the present study, we demonstrate that in the rat tIMCD, the Sch-28080-sensitive component of J_tCO2 is increased with dietary K⁺ restriction. In tIMCD tubules from K⁺-restricted rats, ~40% of proton secretion is Sch-28080 sensitive. In contrast, in K⁺-replete controls, Sch-28080-sensitive J_tCO2 was not detectable. Thus Sch-28080-sensitive J_tCO2 is upregulated in the tIMCD with hypokalemia. Studies in rat tIMCD cells in suspension and cultured mouse OMCD cells have also demonstrated a role of K⁺ homeostasis in the regulation of Sch-28080-sensitive H⁺/OH/HCO₃ transport (10, 17). In permeabilized rat tIMCD cells, Sch-28080-sensitive ATP hydrolysis was increased with dietary K⁺ restriction in vivo (10). Similarly, cultured mouse OMCD cells, grown in medium with a reduced K⁺ concentration, show increased Sch-28080-sensitive pH_i recovery following NH₄Cl addition and withdrawal relative to cells grown in medium with a higher K⁺ concentration (17).

Nevertheless, following K⁺ restriction, it is not clear whether HK₁ is the sole transporter responsible for the observed increase in Sch-28080-sensitive J_tCO2, ATP hydrolysis, and pH_i recovery following an acid load. In the medulla, HK₁ mRNA and protein abundance are not increased by dietary K⁺ restriction (3, 7, 12). Enhanced HK₁ activity could occur through posttranslational regulation, or through trafficking of HK₁ between the plasma membrane and the cytoplasm. In the stomach, regulation of HK₁-mediated H⁺ secretion occurs through trafficking of HK₁ protein between the plasma membrane and the intracellular vesicles (9). In the kidney, it is possible that the increase in H⁺-K⁺-ATPase activity observed following dietary K⁺ restriction occurs through increased insertion of HK₁ into the apical membrane. Alternatively, the Sch-28080-sensitive component of J_tCO2 could represent activity of a transporter other than HK₁.

In the kidney, Sch-28080 inhibits transporters other than HK₁ (5, 16), although Sch-28080 (10 µM) does not inhibit the H⁺-ATPase in the rat tIMCD (32). The present study cannot exclude the possibility that Sch-28080 inhibits K⁺/OH/H⁺ transporters other than HK₁. H⁺-K⁺-ATPases other than HK₁ have been defined that also show sensitivity to Sch-28080. ATP1AL1, a gene encoding a polypeptide of P-type K⁺-dependent ATPases, is sensitive to both ouabain and Sch-28080 when expressed in COS cells (16). The mechanism of the Sch-28080- and ouabain-sensitive components of J_tCO2 observed in the present study, however, cannot be attributed to ATP1AL1, since the Sch-28080- and ouabain-sensitive components of J_tCO2 are additive. This additivity cannot be explained by only partial inhibition of the transporter by these agents when administered separately at concentrations employed in the present study. Moreover, ATP1AL1 has not been detected in the collecting duct (16).

It is well established that along the collecting duct, HK₂ mRNA and protein abundance is upregulated by chronic hypokalemia (2, 7, 12, 26). Moreover, splice variants of HK₂ have been identified (20). Both HK_2a and HK_2b display a similar tissue distribution, and both are upregulated by dietary K⁺ restriction. HK_2a expression increases from the more proximal (cortical collecting duct) to the more distal (tIMCD) segments of the collecting duct (2). Thus HK_2a is highly expressed in the rat tIMCD. However, participation of either HK_2a or the splice variant, HK_2b, in luminal acidification has not been established unequivocally in the tIMCD. The present study demonstrates that a component of net acid secretion is sensitive to ouabain present in the luminal fluid at high concentrations, consistent with a role for HK₂ in mediating net acid secretion (7, 26). Although HK₂ mRNA and protein abundance are increased by hypokalemia, it is not known whether changes in extracellular K⁺ concentration in vivo or in vitro modulate activity of HK₂. The present study demonstrates a small component of J_tCO2 sensitive to ouabain in the luminal fluid. However, since the ouabain-induced change in tCO₂ concentration measured in collected samples approaches the limit of detectability of the fluorometer (28, 30), it was not possible in the present study to determine whether ouabain-sensitive J_tCO2 is reduced in K⁺-replete rats. Whether the component of J_tCO2 sensitive to luminal ouabain is regulated in vivo by K⁺ restriction remains to be established, therefore.

Although a component of J_tCO2 sensitive to ouabain was observed, consistent with a contribution to J_tCO2 by HK₂, this ouabain-sensitive component of J_tCO2 is quite small. The observation that the ouabain-sensitive component of J_tCO2 is small cannot be explained by only partial inhibition of HK₂ by ouabain at a concentration of 5 mM.

In conclusion, net acid secretion in the rat tIMCD is upregulated by dietary K⁺ restriction. This increase in H⁺ secretion is mediated through transport processes sensitive to both Sch-28080 (10 µM) and ouabain (5 mM). The Sch-28080- and ouabain-sensitive components of J_tCO2 appear to be distinct. These observations could be the result of enhanced bicarbonate absorption mediated through the combined response of both HK₁ and HK₂ to dietary K⁺ restriction.