In rat tIMCD, NH+4 uptake by Na+-K+-ATPase is critical to net acid secretion during chronic hypokalemia

Susan M. Wall, Bradley S. Davis, Kathryn A. Hassell, Pramod Mehta, and Stanley J. Park

University of Texas, Medical School at Houston, Houston, Texas 77030


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The purpose of this study was to determine the magnitude of Na+ pump-mediated NH+4 uptake in the terminal inner medullary collecting duct (tIMCD) at K+ and NH+4 concentrations observed in vivo in the inner medullary interstitium of normal and in K+-restricted rats. Interstitial K+ and NH+4 concentrations in the terminal half of the inner medulla were taken to be 10 and 6 mM in K+-restricted rats, but 30 and 6 mM in K+-replete rats. In tubules from K+-restricted rats, when perfused at a K+ concentration of 10 mM, addition of ouabain to the bath reduced total bicarbonate flux (JtCO2) by 40% and increased intracellular pH (pHi), indicating significant NH+4 uptake by the Na+-K+-ATPase. In tubules from K+-restricted rats, JtCO2 was reduced with increased extracellular K+. At a K+ concentration of 30 mM, ouabain addition neither reduced JtCO2 nor increased pHi in tubules from rats of either treatment group. In conclusion, in the tIMCD from hypokalemic rats, 1) acute changes in extracellular K+ concentration modulate net acid secretion, and 2) Na+ pump-mediated NH+4 uptake should be an important pathway mediating transepithelial net acid secretion in vivo.

collecting duct; sodium-potassium-adenosinetriphosphatase; ouabain; ammonium; potassium


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

OUR LABORATORY has demonstrated previously that in the terminal inner medullary collecting duct (tIMCD) of the rat, NH+4 uptake occurs through the Na+-K+-ATPase (20-22). Across the basolateral membrane, NH+4 uptake with NH3 efflux provides an "NH3 shuttle" with delivery of protons for luminal acidification and the titration of luminal buffers. When the Na+ pump is inhibited by the addition of ouabain to the bath, proton secretion, JtCO2, is reduced and intracellular pH (pHi) is increased. In tubules from deoxycorticosterone (DOC)-treated rats, perfused in vitro in the presence of 3 mM K+ and 6 mM NH+4, JtCO2 was reduced by approximately 70% upon the addition of ouabain to the peritubular fluid (20, 21). However, these studies were performed at very low extracellular K+ concentrations (3-5 mM), which is not typical of interstitial K+ concentration in vivo. Micropuncture studies have shown that in normal, untreated rats, vasa recta K+ concentration, which reflects interstitial concentration in the terminal portion of the inner medulla, is 36 mM (7). Although the interstitial concentration of K+ in the inner medulla in DOC-treated rats in vivo has not been measured directly, most likely it is increased due to medullary K+ recycling (5). Thus, K+ concentration in the interstitium of the inner medulla in DOC-treated rats in vivo is probably an order of magnitude higher than K+ concentrations employed in these previous microperfusion studies (20, 21). Since NH+4 and K+ compete for a common extracellular binding site on the Na+-K+-ATPase (22, 25), increased extracellular K+ concentration reduces NH+4 uptake by the Na+ pump. Therefore, it could be argued that this transport pathway mediates little net acid secretion in vivo.

However, interstitial K+ concentration in the terminal portion of the inner medulla varies widely with changes in systemic K+ homeostasis. In rats ingesting a K+-deficient diet for 1-2 wk, vasa recta K+ concentration is 6.1 mM (7). In contrast, after 1-2 wk of dietary K+ loading, vasa recta K+ concentration is 45-55 mM (2, 6). Since the K+ concentration in the interstitium is reduced with dietary K+ restriction, NH+4 uptake by the Na+ pump in the tIMCD may be more significant in hypokalemic rats than in rats without changes in K+ homeostasis. Thus, in K+ restriction, NH+4 uptake by the Na+ pump might be augmented, since interstitial K+ concentration is reduced.

The purpose of the present study was to define relative interstitial NH+4 content in normal and K+-restricted rats and to determine the magnitude of NH+4 uptake mediated by the Na+ pump in tIMCD tubules at concentrations of K+ and NH+4 observed in the interstitium of rats during variation in K+ homeostasis.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Animal conditioning. Pathogen-free male Sprague-Dawley rats weighing 65-120 g (Rm. 205G; Harlan, Indianapolis, IN) were used and housed in microisolator cages. To facilitate comparison with previous in vivo studies, the protocol of Dobyan et al. (7) was employed. Rats were fed a diet containing 7.8 g K+/kg food and 0.664 g Na+/kg food (Zeigler Brothers, Garners, PA) (20). Animals were then divided into two treatment groups. Group A rats ate a diet with a normal K+ content (diet no. P1868, 2.3 g K+/kg food, 1.03 g Na+/kg food; ICN Biochemicals, Aurora, OH) for the 3 days prior to death. For the 3 days preceding death, group B rats ate a diet identical to that of group A, but K+ content was restricted. This diet contained 0.0041 g/kg K+ and 1.03 g/kg Na+ (diet no. 960189; ICN Biochemicals). Rats were pair fed.

Urine samples were collected in rats in both treatment groups. Rats were anesthetized with pentobarbital, 5 mg/100 g, by intraperitoneal injection. Urine samples were then collected by bladder puncture. Urine total ammonia concentration was measured with a continuous flow fluorometer (20) using an enzyme assay purchased as a kit (no. 171-A; Sigma Chemical, St. Louis, MO). Urine osmolality was measured using a vapor-pressure osmometer (Wescor, Logan, UT).

Tissue total ammonia content. Total ammonia content of homogenized tissue slides from cortex, outer medulla, and inner medulla was determined as described by Packer and colleagues (16). Rats were anesthetized with an intraperitoneal injection of pentobarbital, 5 mg/100 g body wt. Kidneys were removed and immediately frozen on dry ice. Kidneys were mounted in Tissue-Tek OCT (Sakura Finetek, Torrance, CA). Sagittal sections of the most superficial portions of each kidney were cut using a Reichert Histo Stat Cryostat Microtome (Leica, Deerfield, IL) and then discarded. While the remaining portion of the kidney was still mounted on the cryostat chuck, the cortex, outer medulla, and three inner medullary sections were cut (IM-1, -2, and -3). IM-1 represented the first 25% of the inner medulla, IM-2 represented the second 25%, and IM-3 was the terminal 50% of the inner medulla. Tissue slices were then homogenized in 0.5 ml ice-cold 7% trichloroacetic acid (TCA) using a 1-ml pestle tissue grinder (size O; Thomas Scientific, Swedesboro, NJ). The solution was centrifuged at 1,500 g for 15 min at 4°C. The supernatant was drawn off and frozen. The following day the supernatant sample was thawed and adjusted to near neutral pH by the addition of 0.02 ml of 10 mM Na2HPO4 in 9 N NaOH. A 0.2-ml sample of this buffered supernatant was then analyzed for total ammonium content using a kit (Ammonia, 171-A; Sigma Chemical) following the instructions of the manufacturer. A standard curve was run in parallel with equal volumes of the buffered, neutralized TCA solution adjusted to the same pH. The pellet was resuspended in 0.5 ml 1 N NaOH, shaken overnight at room temperature, and analyzed for total protein using the Bio-Rad protein assay following the instructions of the manufacturer (Bio-Rad Laboratories, Hercules, CA). gamma -Globulin was used as a standard with an equal volume of 1 N NaOH.

tIMCD tubule perfusion in vitro. In perfusion studies, rats were injected with furosemide (5 mg/100 g body wt ip) 45 min before death by decapitation to induce a rapid diuresis (20). Tubules were dissected from the middle third of the IMCD at 11°C (24). tIMCD tubules were mounted on concentric glass pipettes and perfused in vitro at 37°C.

Experiments were performed with identical solutions in the perfusate and bath. Solutions used are given in Table 1. In experiments that employed HCO-3/CO2-buffered solutions (solutions 2 and 3), tubules were dissected in solution 1. With HCO-3-containing solutions in the perfusate and bath, the bath fluid was constantly bubbled with 95% air-5% CO2. The perfusate was passed through jacketed concentric tubing through which 95% air-5% CO2 was blown in a counter-current direction around the perfusate line (20, 24). When tubules were perfused and bathed in HEPES-buffered solutions (solutions 4 and 5), tubules were dissected in solution 4. HEPES-buffered solutions were bubbled continuously with 100% O2. Osmolality was measured in all solutions (20, 24). Bath pH was measured continuously during all experiments (20, 24).

                              
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Table 1.   Solution composition

Measurement of bicarbonate flux. Samples of luminal fluid were collected under oil in calibrated constriction pipettes. Flow rate was determined as described previously (24). Total CO2 concentration (tCO2) was measured in the collected fluid (CL) and perfusate (Co) using a continuous flow fluorometer (18, 20). The CO2 reagent was obtained from a kit (no. 132-A; Sigma) and diluted to 50% strength with water. Bicarbonate flux, JtCO2, was calculated using the equation
<IT>J</IT><SUB>tCO<SUB>2</SUB></SUB> = (C<SUB>o</SUB> − C<SUB><IT>L</IT></SUB>)V<SUB><IT>L</IT></SUB>/<IT>L</IT>
where Co and CL are the perfusate and collected fluid total CO2 concentrations, respectively. VL is the flow rate in nanoliters per minute, and L is the tubule length. It was assumed that in rat tIMCD net fluid transport is negligible in the absence of an imposed osmolality gradient (24). Perfusate total CO2 concentration, Co, was estimated by measuring tCO2 concentration in the collected fluid at very fast flow rates (24).

Measurements were made in two periods. Period 1 began 45-120 min after warming the tubule. Period 2 began with a bath change wherein ouabain (or vehicle) was introduced. This period was begun 2 h, and ended 3 h, after warming the tubule. Ouabain was added to the bath solution 5-10 min prior to beginning a collection.

Measurement of pHi. pHi was measured using the acetoxymethyl ester of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF-AM) using methods described previously by our laboratory (20). pHi was determined by measuring the ratio of emitted light at >530 nm when BCECF was excited alternately at 440 and 495 nm. Measurements were taken either immediately or 4 min after a bath exchange in which ouabain (or vehicle) was introduced (20). Readings were calibrated by measuring fluorescence when the tubule was perfused and bathed in a HEPES-buffered solution containing 120 mM K+ and 14 µM nigericin (solution 6). The pH of this solution was varied between 7.0 and 7.4.

Statistical analysis. In perfused tubule experiments that measured JtCO2, 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 an paired or unpaired two-tailed Student's t-test, with P < 0.05 indicating statistical significance. Data are displayed as means ± SE.

In tissue slice studies, each value of total ammonia content reported reflects either a single measurement or reflects the mean of two measurements made on a tissue sample from a single rat. Protein content on each tissue sample was determined in duplicate. Data shown are the mean ± SE of results from five separate rats. For multiple comparisons, analysis of variance (ANOV) was employed with specific contrasts by the Bonferroni method, with P < 0.05 indicating statistical significance.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of dietary K+ restriction on tissue total ammonia content. To elucidate relative interstitial NH+4 concentration among treatment groups, total ammonia content in renal tissue slices was determined. In the inner medulla tissue, total ammonia concentration reflects NH+4 concentration in all structures of the inner medulla. Nevertheless, it correlates well with interstitial concentration measured by sampling fluid from vasa recta plasma (11, 16). The effect of dietary K+ restriction on tissue total ammonia content is displayed in Figure 1 (Table 2). As shown, in both controls and in K+-restricted rats, an axial gradient for tissue total ammonia content was observed. Ammonia content was lowest in the cortex and outer medulla but increased progressively from the inner/outer medullary junction to the papillary tip. As described previously, however, this axial gradient for tissue total ammonia content was not observed in rats treated with furosemide prior to death (16). At each level along the corticomedullary axis, tissue total ammonia content in K+-restricted rats was the same or greater than in controls.


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Fig. 1.   Effect of K+ restriction on tissue total ammonia content. In kidneys from K+-replete and K+-restricted rats, an axial gradient for total ammonia content was observed. At each level along the corticomedullary axis, K+ restriction did not reduce tissue total ammonia content. In K+-replete rats treated with furosemide, an axial ammonium gradient was not observed. COR, cortex; OM, outer medulla; IM-1, IM-2, and IM-3, the 3 sections of the inner medulla as described in METHODS.


                              
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Table 2.   Renal tissue total ammonia content

In the inner medullary interstitium, tissue total ammonia content reflects not only true interstitial total ammonia concentration but also total ammonia concentration in other tissue compartments such as luminal fluid. It has been reported previously that urinary ammonium excretion is increased in dietary K+ restriction (19). If so, the increased luminal ammonium concentration of the inner medullary collecting ducts observed in K+ restriction should elevate tissue total ammonia content. Therefore, tissue total ammonia content would overestimate true interstitial concentration relative to controls.

To address this question, urinary total ammonia concentration was measured in both treatment groups. As shown in Table 3, urinary ammonia concentration was lower in rats with dietary K+ restriction than in controls. Therefore, in K+ restriction, the NH+4 concentration of the luminal fluid in the collecting duct does not increase tissue total ammonia relative to controls.

                              
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Table 3.   Urine NH+4 concentration

Urinary ammonium concentration in K+ restriction was 34% of urinary ammonium concentration in controls. Reduced urinary NH+4 concentration observed in K+ restriction may occur in part due to the concentrating defect that has been reported in this treatment group (14) (Table 3).

Interstitial NH+4 and K+ concentrations in K+-restricted and K+-replete controls. To exploit interstitial K+ concentrations reported previously during changes in K+ homeostasis, we have employed the protocol of Dobyan et al. (7). In this study, rats were studied in two treatment groups. In the first group, rats ate a K+-deficient diet for 3 days. In the second group, the rats ate a diet with normal K+ content for the same time period. Vasa recta K+ concentration was measured in the terminal millimeter of the inner medulla in both treatment groups. In K+-restricted rats, vasa recta K+ concentration was 8.6 mM, but 36 mM in K+-replete rats (7).

In K+-replete controls, vasa recta NH+4 concentration is 8.4 mM in rats eating a similar, K+-replete diet for 7 days (8). These samples were taken from the terminal millimeter of the rat inner medulla, or at approximately 80-100% of the distance from the inner/outer medullary junction to the papillary tip. Tubules for perfusion studies were taken at 50-60% of the distance from the inner/outer medullary junction to the papillary tip or from more proximal portions of the inner medulla. Because interstitial total ammonia concentration displays an axial gradient from the cortex to the papillary tip (16), NH+4 concentration at 50% should be slightly lower than at the level sampled by micropuncture. Therefore, interstitial concentration at 50% in control rats was taken to be 6 mM. In K+-restricted rats, interstitial NH+4 concentration has not been measured directly. However, the present tissue slice studies demonstrate that interstitial NH+4 concentration is not reduced in K+ restriction. Whether interstitial NH+4 concentration is increased in K+ restriction remains to be determined. However, in selecting an NH+4 concentration representative of the interstitial concentration in the inner medulla of K+-restricted rats, we chose to err on the side of underestimating, rather than overestimating interstitial NH+4 concentration. Therefore, interstitial NH+4 concentration in K+-restricted rats and in controls was taken to be 6 mM.

In summary, based on these in vivo studies, as well as the tissue slice studies given above, interstitial K+ and NH+4 concentrations in the terminal half of the inner medulla were taken to be 10 and 6 mM, respectively, in K+ restriction. In K+-replete rats, interstitial K+ and  NH+4 concentrations were taken to be 30 and 6 mM, respectively.

Effect of extracellular K+ concentration on JtCO2. With changes in dietary K+ intake, the extracellular environment of the tIMCD is altered. Since a reduction in interstitial K+ concentration is one component of this altered environment, we asked whether acute changes in extracellular K+ concentration modulate JtCO2. Results are shown in Figure 2. In K+-restricted rats, when perfused at an extracellular K+ concentration of 10 mM, baseline JtCO2 was 3.7 ± 0.3 pmol · mm-1 · min-1 (solution 2, n = 8). When separate tubules from K+-restricted rats were perfused under the same conditions but when the K+ concentration was increased to 30 mM, JtCO2 was 1.9 ± 0.4 pmol · mm-1 · min-1 (solution 3, n = 5, P < 0.05). Thus, in tubules from hypokalemic rats, JtCO2 is reduced following an increase in extracellular K+ concentration over the concentration range observed physiologically in the medullary interstitium. In the present study, an effect of extracellular K+ in K+-replete controls could not be demonstrated. JtCO2 was 2.1 ± 0.05 pmol · mm-1 · min-1 at a K+ concentration of 10 mM (solution 2, n = 5) and 2.0 ± 0.3 pmol · mm-1 · min-1 at a K+ concentration of 30 mM [solution 3, n = 5, not significant (NS)].


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Fig. 2.   Effect of extracellular K+ on total bicarbonate flux (JtCO2) in K+-restricted rats. Terminal inner medullary collecting ducts (tIMCD) from K+-restricted rats were perfused in vitro in symmetric, HCO-3/CO2-buffered solutions containing 10 mM K+ and 6 mM NH+4 (solution 2, left bar). Separate tIMCD tubules from rats in the same treatment group were perfused in the same solution but where K+ concentration was increased to 30 mM (solution 3, right bar). JtCO2 was 3.7 ± 0.3 pmol · mm-1 · min-1 (n = 8) in presence of 10 mM K+ but decreased to 1.9 ± 0.4 pmol · mm-1 · min-1 (n = 5, *P < 0.05) in presence of 30 mM K+.

Contribution of Na+-K+-ATPase-mediated NH+4 uptake to JtCO2. Our laboratory has demonstrated previously that addition of ouabain to the peritubular bath increases pHi and reduces JtCO2. This effect of ouabain on pHi and JtCO2 is completely dependent on the presence of NH4Cl. Through a series of ion substitution experiments, we have demonstrated that this effect of ouabain on pHi and JtCO2 is mediated by NH+4 uptake through the Na+-K+-ATPase. K+ and NH+4 are competitive inhibitors for a common binding site on the Na+ pump (22). Since JtCO2 is increased at lower extracellular K+ concentrations in tIMCD tubules from hypokalemic rats, we hypothesized that this occurs at least in part through the increase in NH+4 uptake by the Na+-K+-ATPase, which occurs following a reduction in extracellular K+ concentration.

In the next series of experiments, we tested the contribution of NH+4 uptake through the Na+ pump to JtCO2 in tIMCD tubules at physiological K+ and NH+4 concentrations. tIMCD tubules from K+-restricted rats were perfused in vitro in HCO-3/CO2-buffered solution containing 10 mM KCl and 6 mM NH4Cl (solution 2). The effect of ouabain on JtCO2 was measured as an index of Na+ pump-mediated NH+4 uptake (Figure 3, Table 4). Baseline JtCO2 was 3.9 ± 0.5 but fell to 2.1 ± 0.5 upon the addition of 5 mM ouabain to the bath (n = 5, P < 0.05). This reduction in JtCO2 was not observed when ouabain addition was substituted with a mock bath exchange. Therefore, ouabain-sensitive JtCO2 does not reflect time-dependent changes in flux. Thus, in tIMCD tubules from in K+-restricted rats, a condition associated with reduced interstitial K+ concentration,  NH+4 uptake by the Na+-K+-ATPase, should be an important mediator of transepithelial net acid secretion. However, JtCO2 was not reduced with ouabain addition to the bath when extracellular K+ was increased to 30 mM (Fig. 4B, Table 4). Therefore, in K+-restricted rats, acute changes in extracellular K+ concentration modulate NH+4 uptake through the Na+-K+-ATPase.


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Fig. 3.   Effect of ouabain on JtCO2 in K+-restricted rats. tIMCD tubules from K+-restricted rats were perfused in vitro in symmetric, HCO-3/CO2-buffered solutions containing 10 mM K+ and 6 mM NH+4 (solution 2, A). JtCO2 fell from 3.9 + 0.5 to 2.1 ± 0.5 pmol · mm-1 · min-1 upon addition of 5 mM ouabain to bath (n = 5, P < 0.05). This reduction was not observed in time controls (B).


                              
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Table 4.   Effect of ouabain on JtCO2

The effect of ouabain on K+-replete controls was tested. tIMCD tubules were perfused in the presence of HCO-3/CO2-buffered solutions containing 30 mM KCl and 6 mM NH4Cl (solution 3; Figure 4, Table 4). In K+-replete controls, baseline JtCO2 was 1.9 ± 0.4. Following the addition of ouabain, JtCO2 was 2.4 ± 0.4 pmol · mm-1 · min-1 (n = 5, NS). Therefore, an effect of ouabain on JtCO2 could not be detected in tIMCD tubules from K+-replete controls when perfused at K+ and NH+4 concentrations found in the interstitium of the inner medulla of this treatment group. Because total and ouabain-sensitive JtCO2 are low in K+-replete controls at NH+4 and K+ concentrations expected in vivo (Table 4), the physiological role of NH+4 uptake by the Na+-K+-ATPase in other states of K+ homeostasis, such as in K+-replete controls, remains to be determined.


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Fig. 4.   Effect of ouabain on JtCO2 in K+-replete rats. A: tIMCD tubules from K+-replete rats were perfused in vitro in symmetric HCO-3/CO2-buffered solutions containing 30 mM KCl and 6 mM NH4Cl (solution 3). JtCO2 was not changed upon addition of 5 mM ouabain to bath. B: similar results were obtained in tubules from K+-restricted rats when perfused under the same conditions. NS, not significant.

Contribution of Na+-K+-ATPase-mediated NH+4 uptake to resting pHi. If NH+4 uptake through the Na+ pump is inhibited, such as through ouabain addition (20, 21), then pHi is increased. Therefore, as an additional test of the physiological significance of this pathway in vivo, we tested whether ouabain addition increases pHi at physiological concentrations of K+ and NH+4 (Table 5). tIMCD tubules from K+-restricted rats were perfused in vitro in HCO-3/CO2-buffered solutions containing 10 mM KCl and 6 mM NH4Cl (solution 2). Baseline pHi was 7.29 + 0.07. pHi rose 0.04 ± 0.01 pHi units 4 min after the addition of ouabain and returned to baseline after ouabain withdrawal (n = 5, P < 0.05). However, at a K+ concentration of 30 mM (solution 3), ouabain addition did not increase pHi in either treatment group (Table 5).

                              
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Table 5.   Effect of ouabain on pHi

At a K+ concentration of 10 mM, ouabain-induced alkalinization was small. Therefore, results were confirmed in HEPES-buffered solutions. We hypothesized that, if this pathway is significant in vivo, then ouabain addition should increase in pHi under physiological conditions in K+-restricted rats, with a smaller effect at a K+ concentration of 30 mM. Results are shown in Figure 5 (Table 5). Tubules from K+-restricted rats were perfused in the presence of 10 mM K+ and 6 mM NH+4 (solution 4). Following ouabain addition to the bath, pHi increased promptly and then plateaued. Four minutes after the addition of ouabain, pHi was 0.08 ± 0.02 pH units above initial values, but returned to baseline following ouabain withdrawal (Table 5). When tIMCD tubules from K+-replete rats were perfused in the presence of 30 mM KCl (solution 5), no change in pHi was detected following ouabain addition.


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Fig. 5.   Effect of ouabain on intracellular pH (pHi). Tubules were perfused in symmetric HCO-3/CO2-free solutions containing NH4Cl. tIMCD tubules from K+-restricted rats were perfused in presence of 10 mM KCl (solution 4). Ouabain was added to bath solution at time 0. pHi rose 0.08 ± 0.02 pH units, 4 min after addition of ouabain. In control rats, when tubules were perfused and bathed in presence of 30 mM KCl (solution 5), no change in pHi was detected following ouabain addition.

Therefore, results of studies in which changes in pHi following ouabain addition were measured as an index of NH+4 uptake by the Na+-K+-ATPase are consistent with the flux studies described above. In tIMCD tubules from K+-restricted rats, when perfused at a K+ concentration observed in the interstitium of this treatment group, considerable NH+4 uptake occurs through the Na+-K+-ATPase. Moreover, in hypokalemia, NH+4 uptake by the Na+ pump is modulated by extracellular K+ concentration. In tubules from K+-replete controls perfused at K+ concentrations observed in vivo in this treatment group, NH+4 uptake through the Na+-K+-ATPase is of lower magnitude.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Since the first report by Skou in 1960 (17), NH+4 transport by the Na+-K+-ATPase has been documented in a number of tissues. However, because of the low NH+4 concentration found in the extracellular fluid of most epithelia, the physiological significance of this pathway has not been established. The present study is the first to demonstrate a physiological role of this transport pathway under conditions observed in vivo. Based previous in vivo studies as well as tissue slice studies reported above, interstitial K+ and NH+4 concentration in K+-restricted and K+-replete controls was estimated. NH+4 uptake by the Na+ pump was measured in tIMCD tubules from rats in the same treatment group at these physiological extracellular NH+4 and K+ concentrations. NH+4 uptake through the Na+ pump in tIMCD tubules from rats with K+ deficiency, when studied at K+ and NH+4 concentrations observed in vivo in hypokalemia, mediates ~40% of net acid secretion.1 These results indicate that NH+4 uptake by the Na+-K+-ATPase should be an important mediator of net acid secretion in vivo during hypokalemia. Since the magnitude of both total and ouabain-sensitive net acid secretion is lower in controls, the physiological significance of this pathway in this treatment group cannot be determined.

In vivo studies have shown that in rat tIMCD, net acid secretion is reduced with dietary K+ loading (8), a condition associated with increased vasa recta K+ concentration and decreased NH+4 concentration. In dietary K+ loading, interstitial K+ is increased from values of 36 mM observed in controls to 46-55 mM (2, 6). Interstitial NH+4 concentration is reduced to 6.0 mM in hyperkalemia from values of 8.4 mM reported in the interstitium of controls (8). However, these in vivo studies cannot discern the contribution of long-term, stable adaptation of transporters that mediated net acid secretion. These studies also cannot determine the role of acute changes in the extracellular environment, such as changes in pH or K+ concentration, on changes in net acid secretion that follow changes in K+ homeostasis. The present study demonstrates that in K+-restricted rats, net acid secretion is increased with a reduction in extracellular K+ concentration. Thus, acute changes in extracellular K+ concentration modulate net acid secretion. The increase in net acid secretion observed following a reduction in extracellular K+ concentration can be explained, at least in part, by increased NH+4 uptake through the Na+-K+-ATPase. These results are in keeping with previous observations of our laboratory (21), which examined the effect of changes in extracellular K+ concentration on JtCO2 in t/MCD from DOC-treated rats.

Rates of NH+4 uptake by the Na+ pump can be predicted using the model of Kurtz and Balaban (13). This model incorporates known interstitial K+ and NH+4 concentrations and the Km and Vmax for NH+4 and K+ as substrates of the Na+ pump.2 Using values reported previously in the rat tIMCD, as well as results of the present study, the model predicts that NH+4 uptake by the Na+ pump should fall by ~60% at a NH+4 concentration of 6 mM when K+ concentration is increased from 10 to 30 mM. However, with changes in K+ homeostasis in vivo, stable, long-term adaptation of transport processes has been observed. Our laboratory has shown that with dietary K+ restriction, apical proton secretion is increased, presumably mediated by an H+-K+-ATPase (23). These results are in keeping with observations of the present study, which showed that JtCO2 is low in K+-replete controls relative to JtCO2 observed in K+-restricted rats (4). In hypokalemia, long-term stable adaptation most probably occurs in other transporters, such as the Na+-K+-ATPase. The present study cannot determine whether the Na+-K+-ATPase is up- or downregulated in dietary K+ restriction. Moreover, it cannot determine the role of other cellular changes that might occur with K+ restriction, such as a change in buffering capacity, which could modulate changes in pHi. Nevertheless, the present study demonstrates significant NH+4 uptake by the Na+-K+-ATPase in tIMCD tubules from hypokalemic rats under conditions expected in vivo.

Interstitial NH+4 and K+ concentrations are critical in determining the extent of NH+4 uptake by the Na+ pump. Interstitial NH+4 concentration is modulated by the Na+-K+-2Cl- cotransporter of the thick ascending limb. The Na+-K+-2Cl- cotransporter, NKCC2 or BSC-1, located on the apical membrane of the thick ascending limb, is responsible for the "single effect" of the countercurrent multiplier. In the thick ascending limb, NH+4 replaces K+ on the Na+-K+-2Cl- cotransporter (9, 10). Through this NH+4 transport pathway, an axial gradient for both NH3 and NH+4 is created from the cortex to the papillary tip (11, 16). NH3 concentration is higher in the interstitium than in the collecting duct lumen at the same level (11). This NH3 gradient from the interstitium to the collecting duct lumen facilitates the nonionic diffusion of NH3 into the collecting duct lumen and hence increases renal ammonium excretion (11).

Although the interstitial concentration of NH+4 during K+ restriction has not been measured directly, it is thought to be increased for several reasons (9). First, with K+ restriction, NH+4 production in the proximal tubule is increased (3, 12, 15, 19). Second, with hypokalemia, distal delivery of NH+4 is increased while distal K+ delivery is reduced. Since NH+4 and K+ compete for a common binding site on the Na+-K+-2Cl- cotransporter (BSC-1 or NKCC2), increased distal delivery of NH+4, relative to K+, increases NH+4 uptake across the apical membrane of the thick ascending limb (9, 10). Finally, the reduction in medullary blood flow observed in hypokalemia might facilitate NH+4 capture in the inner medulla (26). In support of this hypothesis, Jaeger et al. (12) showed that in K+ depletion, ammonium concentration in both late proximal and early distal sites, is increased. Moreover, the difference in NH+4 concentration between the late proximal and the early distal tubule is greater during K+ restriction than in controls. This implies that in K+ restriction there is greater extraction of NH+4 from the luminal fluid of the loop of Henle. The predicted result is increased interstitial NH+4 concentration. However, a recent study by Amlal et al. (1) demonstrated that in K+ restriction, BSC-1/NKCC2 activity and message abundance are reduced by ~50%. Therefore, based on previous studies, the effect of K+ restriction on the concentration of NH+4 in the medullary interstitium is not clear.

In the present study, interstitial NH+4 content in the inner medulla was determined in renal tissue slices. These data provide evidence that in K+ restriction interstitial NH+4 concentration in the inner medulla is not reduced despite a reduction in BSC-1/NKCC2 activity (1). However, these data cannot exclude the possibility that interstitial NH+4 concentration is increased in K+ restriction.3 Thus, although cotransporter activity is reduced in K+ restriction, other physiological changes that occur in hypokalemia are important in modulating interstitial NH+4 concentration. The reduction in BSC-1/NKCC2 activity observed in K+ restriction may be appropriate in that it limits NH+4 excretion and thereby attenuates the metabolic alkalosis.

In conclusion, a ouabain-induced increase in pHi and reduction in JtCO2 were demonstrated in tIMCD tubules from K+-deficient rats at K+ and NH+4 concentrations observed in vivo. This study shows that in K+ depletion, NH+4 uptake by the Na+-K+-ATPase should be an important pathway mediating net acid secretion in the tIMCD in vivo.


    ACKNOWLEDGEMENTS

We thank Drs. Jeff M. Sands, Andrew Kahn, and Thomas D. DuBose, Jr., for critically reading the manuscript. We thank Drs. Randall Packer, David W. Good, and Gerald F. DiBona for helpful suggestions.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-52935 (to S. M. Wall).

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. §1734 solely to indicate this fact.

1 The percent reduction in JtCO2 observed following ouabain addition was calculated by taking the ratio of JtCO2 measured in period 2, relative to JtCO2 measured in period 1. However, in time control experiments, JtCO2 was greater on average in period 2 than in period 1, although these differences did not reach statistical significance (Fig. 3). With a time-dependent increase in JtCO2, the ouabain-sensitive component of JtCO2, calculated as described above, underestimates true ouabain-sensitive JtCO2.

2 The Km values for NH+4 and K+ on the rat Na+-K+-ATPase were taken to be 11.0 and 1.9 mM, respectively (22). The Vmax for NH+4 as a substrate of the Na+ pump was taken to be 20% higher than for K+ (22).

3 Interstitial NH+4 concentration may increase with dietary K+ restriction. In the present study, urinary NH3 concentration was much greater in K+-restricted rats than in controls. If NH3 is secreted from the interstitium to the collecting duct lumen down its concentration gradient, then interstitial NH3 (and hence NH+4) concentration might increase dramatically in K+ restriction. Moreover, tissue slice studies are a composite of NH+4 concentration in all medullary spaces including the collecting duct lumen. Since K+ produces a dilute urine and hence a reduced total ammonia concentration, another structure such as the interstitium must compensate with increased NH+4 content to give a tissue total ammonia content similar to that of controls.

Address for reprint requests and other correspondence: S. M. Wall, Division of Renal Diseases and Hypertension, University of Texas, Medical School at Houston, 6431 Fannin, MSB 4.148, Houston, TX 77030 (E-mail: swall{at}heart.med.uth.tmc.edu).

Received 10 March 1999; accepted in final form 9 July 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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