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Contribution of the Na⁺-K⁺-2Cl^- Cotransporter (NKCC1) to Transepithelial Transport of H⁺, NH₄⁺, K⁺, and Na⁺ in Rat Outer Medullary Collecting Duct

Division of Renal Diseases and Hypertension, University of Texas, Medical School at Houston, Houston, Texas.

Correspondence to: Dr. Susan M. Wall, Division of Renal Diseases and Hypertension, University of Texas, Medical School at Houston, 6431 Fannin, M.S.B. 4.148, Houston, TX 77030. Phone: 713-500-6868; Fax: 713-500-6882; E-mail: susan.m.wall{at}uth.tmc.edu

	Abstract

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ABSTRACT. In rat kidney, the "secretory" isoform of the Na-K-Cl cotransporter, NKCC1 (BSC-2), localizes to the basolateral membrane of the intercalated cell, the acid secreting cell of the outer medullary collecting duct (OMCD). This laboratory has reported that NKCC1 mediates Cl^- uptake across the basolateral membrane in series with Cl^- secretion across the apical membrane in rat OMCD. NKCC1 transports NH₄⁺, K⁺, and Na⁺ as well as Cl^-; therefore, a role for the cotransporter in the process of HCl, NH₄Cl, KCl, and NaCl secretion has been suggested. Thus, it was determined if bumetanide, an inhibitor of NKCC1, alters transepithelial cation transport in rat OMCD. OMCD tubules from deoxycorticosterone pivalate (DOCP)–treated rats were perfused in vitro. Hydration of CO₂, rather than NH₄⁺, provides the principle source of H⁺ for net acid secretion. In HCO₃^-/CO₂-buffered solutions, no effect of bumetanide on net K⁺ flux was detected. Under some conditions, bumetanide addition resulted in a small reduction in secretion of net H⁺ equivalents. Transepithelial Na⁺ flux, J_Na, was -1.5 ± 1.7 pmol/mm per min, values not different from zero. However, with the application of bumetanide to the bath, J_Na was +5.2 ± 1.3 pmol/mm per min (P < 0.05), which indicates net Na⁺ absorption. In conclusion, inhibition of NKCC1 in rat OMCD changes transepithelial movement of Na⁺ and Cl^-. The role of NKCC1 in the secretion of net H⁺ equivalents is small.

	Introduction

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The Na⁺-K⁺-2Cl^- cotransporter is encoded by two distinct genes. The "absorptive isoform," NKCC2 or BSC-1, is kidney-specific and localizes to the apical membrane of the thick ascending limb (6,18). This transporter is crucial in the process of urinary concentration, sodium balance, and the excretion of net acid (6,13). The "secretory isoform," NKCC1 or BSC-2, localizes to the basolateral membrane of secretory epithelia and nonepithelia and serves a variety of functions (12). However, in polarized epithelia, it is most commonly observed to mediate transepithelial movement of Na⁺, Cl^-, and fluid (14). The distribution of NKCC1 in kidney is species-specific. In rat, NKCC1 is most highly expressed on the basolateral membrane of the intercalated cell of the outer medullary collecting duct (OMCD) (12).

Transport mechanisms in rat OMCD are poorly understood. This is in part due to difficulty inherent in the dissection and perfusion of the rat OMCD, because of the tendency of the OMCD to adhere to the medullary thick ascending limb (8). Thus, mechanisms of net H⁺, K⁺, Na⁺, and Cl^- uptake across the basolateral membrane in the rat have not been investigated in detail. The rat OMCD generates high rates of apical H⁺ secretion, which results in a lumen-positive potential difference (2,8). In the OMCD, secretion of net H⁺ equivalents is thought to occur across the intercalated cell. Transporters involved in the process of secretion of net H⁺ equivalents in the OMCD are displayed in Figure 1. On the basis of immunolocalization studies in rat and rabbit, it is known that the H⁺-ATPase and Cl^-/HCO₃^- exchange (AE1) are present in intercalated cells at opposite plasma membrane domains (1). Moreover, immunocytochemical studies have demonstrated that apical H⁺-ATPase and basolateral Cl^-/HCO₃^- exchange (AE1) immunoreactivity change in tandem in acid-base disorders such as chronic metabolic acidosis (4,30). How these transporters are thought to participate in net acid secretion is shown in Figure 1A. In the cell, CO₂ is hydrated to form H⁺ and HCO₃^-, a reaction catalyzed by cytosolic carbonic anhydrase (3,33). HCO₃^- leaves the cell across the basolateral membrane through Cl^-/HCO₃^- exchange while H⁺ is secreted into the luminal fluid, where it titrates HCO₃^-. Thus, apical H⁺ and Cl^- secretion occurs in series with basolateral Cl^- and H⁺ uptake (or HCO₃^- exit) across the basolateral membrane, mediated by Cl^-/HCO₃^- exchange (5,37). Studies of rabbit OMCD tubules perfused in vitro have provided functional evidence for this model. In rabbit, J_tCO2 is markedly reduced with inhibition of carbonic anhydrase (24) or with inhibition of basolateral Cl^-/HCO₃^- exchange (37). Similarly, NKCC1-mediated Cl^- uptake across the basolateral membrane in series with Cl^- secretion across the apical membrane could provide a cosecreted anion for H⁺ secretion in rat OMCD (34).

View larger version (20K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Models of transepithelial H+ movement.

The intercalated cell is thought to play a minor role in Na⁺ transport. This hypothesis has followed three lines of evidence. First, expression of Na⁺ transporters such as ENaC, the Na,K-ATPase and Na/H exchange is low in intercalated cells (19,23,25). Second, application of inhibitors of these transporters produces only a small change in intracellular Na⁺ concentration in the intercalated cell (32). Third, in rabbit OMCD, a segment rich in intercalated cells, transepithelial transport of Na⁺ and K⁺ is not different from zero (36). However, net flux of an ion represents the sum of lumen-to-bath and bath-to-lumen flux of that ion. Thus, inhibition of a given ion transporter could unmask significant net flux of that ion. Therefore, the contribution of NKCC1 to transepithelial transport of Na⁺ and K⁺ remains to be determined.

The observation of high levels of expression of a Na⁺ transporter, such as NKCC1, in the intercalated cell was therefore surprising. Because NKCC1 localizes to a cell type thought to mediate H⁺ secretion, instead of Na⁺, and because the rat OMCD is a poorly characterized segment, a comprehensive study of the Na⁺, K⁺, Cl^-, H⁺, and NH₄⁺ transport processes in this segment was needed. This study is the first to examine the role of NKCC1 in transepithelial transport of Na⁺, K⁺, NH₄⁺, and H⁺ in a single study in a native tissue. The purpose of this study was to determine the cosecreted cation, which follows bumetanide-sensitive secretion of Cl^-.

	Materials and Methods

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Tissue Preparation
Collecting ducts from the inner stripe of the outer medulla (OMCD) (7) were dissected from pathogen-free male Sprague Dawley rats weighing 65 to 120 g (Harlan, Indianapolis, IN). Animals were housed in microisolator cages and fed a low Na⁺, 0.8% K⁺ diet (Zeigler Brothers, Garners, PA) (38). Rats received 5 mg of deoxycorticosterone pivalate (DOCP; CIBA-Geigy Animal Health, Greensboro, NC) by intramuscular injection 5 to 7 d before sacrifice. Animals were injected with furosemide (5 mg/100 g body wt intraperitoneally) 45 min before sacrifice by decapitation, to attenuate changes in the extracellular osmolality of the tubule (38).

Coronal slices were cut from the kidneys and placed into a dish containing the chilled experimental solution (11°C). The dissection solution was identical to that used in the lumen and bath in perfusion experiments. To isolate OMCD from the inner stripe (7), a cut was made between the inner and outer stripe of the outer medulla. The OMCD inner stripe and inner medulla were transferred to a second dish for dissection. Tubules were mounted on concentric glass pipettes and perfused in vitro at 37°C.

Experiments were performed with symmetric solutions in the bath and perfusate. Solution composition is given in Table 1. Osmolality was measured in all solutions (38). To maintain the desired CO₂ concentration in HCO₃^-/CO₂–buffered solutions, the perfusate was passed through jacketed concentric tubing through which 95% air, 5% CO₂ was blown in a countercurrent direction around the perfusate line (38,41). To maintain pH in HCO₃^--containing solutions, the bath fluid was constantly bubbled with 95% air, 5% CO₂. In HEPES-buffered solutions, bath fluid was bubbled with 100% O₂. Bath pH was measured as described previously (38,41). In some experiments, bumetanide (100 µM) and/or ethoxzolamide (100 µM) were added to the bath fluid only. Bumetanide and ethoxzolamide were prepared as stock solutions, both in dimethyl sulfoxide (DMSO). Inhibitors (plus vehicle) or vehicle alone (DMSO) were added to the bath solution. In all experiments, vehicle (DMSO) was added to the perfusate. Thus a DMSO concentration of 0.04% was always present in the perfusate and bath. When comparing flux in the presence and absence of inhibitors, experimental groups were alternated. Moreover, each experimental protocol was performed in more than one shipment of rats.

Measurement of Bicarbonate, Chloride and Total Ammonia Flux
Tubule fluid samples were collected under oil in calibrated constriction pipettes. Flow rate was determined as described previously (41) and varied between 1 and 4 nl/mm per min. However, when comparing flux between treatment groups, no difference in flow rate was detected between these groups. Total CO₂ concentration was measured in the collected fluid (C_L) and perfusate (C_o) by using a continuous flow fluorometer (35,38). This method can detect bicarbonate (total CO₂) concentration differences of <1 mM with an 8-nl pipette (35,38). Bicarbonate absorption, J_tCO2, was calculated from the luminal flow rate and the total CO₂ (tCO₂) concentration difference measured in the perfusate and collected fluid (38). A sample of the perfusate solution from the perfusate reservoir was placed under water-equilibrated mineral oil. Samples of the perfusate and collected fluid were alternated. Net fluid transport in the OMCD was taken to be zero in the absence of an imposed osmolality gradient (40).

Total ammonia concentration (tAMM) (38,41) and Cl^- concentration (9,40) were measured in collected perfusate samples, as described previously, by using a continuous flow fluorometer. This total ammonia assay can distinguish differences in total ammonia concentration of 0.1 mM using an 11-nl pipette, and the Cl^- assay can distinguish differences of 2 mM with a 10-nl pipette (40). Total ammonia flux (J_tAMM) and net Cl^- flux (J_Cl) determinations were made as described above for tCO₂.

In 18 of 25 tubules perfused and bathed in HCO₃^-/CO₂–buffered solutions containing 6 mM NH₄Cl (solution 2), measurements of tCO₂ and tAMM were made in separate collections from the same tubule. In the remaining experiments (7 of 25), the missing measurement was performed in a second tubule.

Measurement of Na⁺ and K⁺ Flux
The design of the sodiometer and potassiometer are similar to the designs reported by Garvin (10,11). Both involve a K⁺- or Na⁺-sensitive electrode in a continuous-flow apparatus. A model of the apparatus is given in the upper panel of Figure 2. The Na⁺-sensitive and K⁺-sensitive electrodes were purchased from Microelectrodes (MI-420 and MI-442; Bedford, NH). When measuring Na⁺, the flowing stream contained 100 mM MgCl₂. Measurement of K⁺ employed the same apparatus, but with a K⁺-sensitive electrode and 160 mM NaCl was used as the flowing stream. Both electodes are connnected to a voltmeter (197A/1972A; Keithley Instruments, Cleveland, OH). This signal then passes through a bessel filter (900C; Frequency Devices, Haverhill, MA) at a corner frequency of 0.1 Hz. This signal is then relayed to a chart recorder (BD 112; Kipp and Zonen, Bohemia, NY). J_Na and J_K were calculated as described above for J_tAMM, J_tCO2 and J_Cl.

View larger version (22K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Design of sodiometer (upper panel) and typical standard curves (lower panel) generated with the instrument. The pipette volume was 7 nl. Data are given as the mean ± SD of 4 to 5 replicate measurements performed at each sodium concentration.

Transepithelial Potential Difference
To measure transepithelial potential difference, V_T, the solution in the perfusion pipette was connected to an electrometer (KS-700; World Precision Instruments, New Haven, CT) through an agar bridge saturated with 0.16 M NaCl and a calomel cell (38). The reference was an agar bridge from the bath to a calomel cell.

Statistical Analyses
When measuring t_AMM or t_CO2, one to three replicate measurements were made for each tubule. At least two replicate measurements were made when assaying Cl^-, Na⁺, or K⁺. The flux reported for each tubule represents the mean of all replicate measurements made for that tubule. When only one measurement was made, that value was reported for the tubule. Thus the n reported equals the number of tubules studied. Only one tubule was studied from a given rat. When comparing two groups, statistical significance was determined using an unpaired two-tailed t test. Unless otherwise indicated, comparisons between three or more groups were made using ANOVA with a Dunnett’s post-test. Statistical significance was achieved with a P < 0.05. Unless stated otherwise, data are displayed as the mean ± SEM.

	Results

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Effect of Bumetanide on Net Acid Secretion in OMCD_is
Our laboratory has studied the mechanism of H⁺ and Cl^- secretion in the OMCD of DOCP-treated rats (8,40). Therefore, the role of NKCC1 in transepithelial cation transport was studied in OMCD tubules from DOCP-treated rats to permit correlation with these previous reports. When perfused in HCO₃^-/CO₂–buffered solutions (solution 2; Figure 3), rat OMCD tubules absorb HCO₃^- and secrete ammonium (8) and Cl^- (40), similar to results published previously.

View larger version (28K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Effect of bumetanide, ethoxzolamide, or both on ammonium secretion (JtAMM) and bicarbonate absorption (JtCO2). Left and middle panels: JtAMM and JtCO2 were measured in HCO3/CO2–buffered solutions containing NH4Cl (solution 2, solid bars). In separate tubules, measurements were made with bumetanide (100 µM), ethoxzolamide (100 µM), or both present in the bath solution. Right panel: JtCO2 was measured in HCO3/CO2–buffered solutions that did not contain NH4Cl (solution 1, hatched bars).

By either mechanism, inhibition of NKCC1 should reduce NH₄⁺ secretion. Thus, the effect of bumetanide on J_tAMM was tested. The left panel of Figure 3 shows that J_tAMM was not reduced by addition of bumetanide to the bath in HCO₃^-/CO₂–buffered solutions. However, a small bumetanide-sensitive component of J_tAMM might not be detected due to limitations in the statistical power of the experiment. Thus, the bumetanide-sensitive component of J_tAMM was measured when the conditions of the experiment were changed. The effect of bumetanide on J_tAMM was therefore tested when the catalyzed hydration of CO₂ to HCO₃^- and H⁺ was inhibited. Hydration of CO₂ as a H⁺ source was inhibited either by removal of HCO₃^-/CO₂ from the solutions, through inhibition of the enzyme, carbonic ahydrase, or through both manipulations together. Table 2 and Figure 3 demonstrate that a bumetanide-sensitive component of J_tAMM either was not demonstrated or was small in magnitude (< 1 pmol/mm per min) under each of these conditions. Thus, although a bumetanide-sensitive component of J_tAMM was detected under some conditions, the role of NKCC1 in the transepithelial transport of NH₄⁺ is small.

Hydration of CO₂ as a Proton Source in Rat OMCD
Figure 3 shows that J_tCO2 did not differ significantly in tubules studied in the presence and absence of NH₄Cl in the bath and perfusate. This suggests that NH₄⁺ does not provide a significant H⁺ source in this segment (see appendix, part 1). To determine if the hydration of CO₂ provides an important H⁺ source in rat OMCD, the effect of the carbonic anhydrase inhibitor ethoxzolamide on J_tCO2 and J_tAMM was tested (Figures 3 and 4). Ethoxzolamide addition reduced J_tCO2 >50% either in the presence or absence of NH₄Cl (Figure 3). Thus, J_tCO2 is dependent on activity of cytosolic carbonic anhydrase.

View larger version (18K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Effect of HCO3-/CO2 removal and carbonic anhydrase inhibition on JtAMM in outer medullary collecting duct (OMCD). Left panel: OMCD tubules were perfused and bathed in the presence of 5 mM KCl, 6 mM NH4Cl. JtAMM was measured in the presence of HCO3-/CO2 (solution 2) or when the hydration of CO2 was eliminated as a proton source through carbonic anhydrase inhibition (ethoxzolamide, ETH), through HCO3-/CO2 removal (solution 3), or with both manipulations (solution 3 plus ethoxzolamide). Right panel: transepithelial potential difference (VT) measured in tubules given in the left panel. Of the measurements given for VT, 8 of the 12 were taken from previously published data (40).

Effect of Inhibition of NKCC1 and Carbonic Anhydrase on J_Cl
In HCO₃^-/CO₂–buffered solutions, bumetanide inhibits Cl^- secretion (40) by 10.5 pmol/mm per min (solution 2; Figure 5, left panel). However, under identical conditions, an effect of bumetanide on secretion of net H⁺ equivalents, J_H, (solution 2; Figure 5, middle panel) was not detected, despite adequate statistical power to detect a change in J_H of this magnitude (10.5 pmol/mm per min) (see appendix, part 2). The reason for our inability to detect an equivalent change in J_H and J_Cl after the addition of bumetanide was explored. As a positive control, we tested if inhibition of basolateral anion exchange reduced secretion of Cl^- and H⁺/NH₄⁺ to a similar extent, as reported in rabbit OMCD (37). Anion exchange is dependent on the catalyzed hydration of CO₂ to form HCO₃^-; therefore, changes in J_H and J_Cl after inhibition of carbonic anhydrase were measured. As shown, (Figure 5, middle and right panels), ethoxzolamide addition reduced both J_H and J_Cl when studied under identical conditions. Thus, carbonic anhydrase-dependent Cl^- secretion is accompanied by secretion of net H⁺ equivalents (J_H = J_tAMMJ_tCO2) as the cosecreted cations. In contrast, secretion of net H⁺ equivalents cannot account in full as the cosecreted cations that accompany bumetanide-sensitive Cl^- secretion.

View larger version (25K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Effect of bumetanide, ethoxzolamide, or both on JH and JCl. In all experiments shown in this figure, OMCD tubules were perfused and bathed in the presence of 5 mM KCl, 6 mM NH4Cl, 25 mM NaHCO3/CO2 (solution 2). Left panel: Effect of bumetanide on JCl. JCl, measured in the presence of 100 µM bumetanide, was attenuated relative to control. Data displayed in the left panel was taken from previously published work (40). Middle panel: Effect of ethoxzolamide and bumetanide on JH. In separate tubules, JH (JtAMM - JtCO2) was measured in the presence of bumetanide (100 µM), ethoxzolamide (100 µM), both ethoxzolamide and bumetanide, or measured in the absence of inhibitors (control). In the absence of ethoxzolamide, bumetanide did not reduce JH (P = NS) (see appendix, part 6). Right panel: Effect of carbonic anhydrase inhibition on JCl. JCl was attenuated relative to control when measured in the presence of either 100 µM ethoxzolamide or ethoxzolamide plus 100 µM bumetanide (P < 0.05, ANOVA). Baseline JCl (no inhibitors) was similar to JCl reported previously (40) (left panel) measured under the same conditions.

View larger version (12K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. Effect of bumetanide on JK and JNa. Left panel: OMCD tubules were perfused and bathed in HCO3-/CO2–buffered solutions, which did not contain NH4Cl (solution 4). JK was not different from zero either in the presence or the absence of bumetanide (P = NS). Right panel: OMCD tubules were perfused and bathed in solution 5. In the absence of inhibitors, JNa was not different from zero. In the presence of bumetanide (100 mM), Na+ absorption was observed (P < 0.05). Measured luminal flow rates were as follows (in nl/mm per min): JK control, 1.34 ± .14; JK with bumetanide, 1.35 ± .18; JNa control, 1.61 ± .14; JNa with bumetanide, 1.53 ± .13. Tubule lengths were as follows (in mm): JK control, 0.76 ± .06; JK with bumetanide, 0.73 ± .15; JNa control, 0.57 ± .03; JNa with bumetanide, 0.79 ± .07.

	Discussion

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The secretory isoform of the Na⁺-K⁺-Cl^- cotransporter NKCC1 (BSC-2) is expressed in many tissues and serves a variety of functions, such as intracellular volume regulation and the vectorial transport of Na⁺, K⁺, H⁺, Cl^-, and water (17). However, NKCC1 is most commonly found to mediate net flux of Na⁺ and Cl^- and fluid. This study also demonstrated an effect of inhibition of NKCC1 on net flux of Na⁺ and Cl^- in the rat OMCD.

Net Acid Secretion in Rat OMCD and the Role of NKCC1
In other studies, NKCC1 has been implicated in the process of net acid secretion in the rat OMCD (12,42) for several reasons. First, NKCC1 is expressed in the intercalated cell (12), the acid-secreting cell of the OMCD. Second, NKCC1 transports both NH₄⁺ and Cl^- (42). Finally, NKCC1 protein expression is upregulated during metabolic acidosis (16). In the present study, the contribution of NKCC1 to renal net acid secretion was examined directly.

In both rat and rabbit, Cl^- is fully accountable as the cosecreted anion, which accompanies apical H⁺ secretion in series with anion exchange-mediated net uptake of Cl^- and H⁺ equivalents across the basolateral membrane (37) (see appendix, part 3). The present study did not demonstrate a 1:1 coupling of the bumetanide-sensitive components of J_H and J_Cl. In HCO₃^-/CO₂–buffered solutions, bumetanide reduced J_Cl (40), but it did not change in J_H when studied under identical conditions. Thus, neither NH₄⁺ nor H⁺ movement was found to accompany bumetanide-sensitive Cl^- secretion when studied in the presence of HCO₃^-/CO₂–buffered solutions with intact carbonic anhydrase activity.

Nevertheless, a small bumetanide-sensitive component of net acid secretion was detected under some conditions. NKCC1-mediated Cl^- uptake in series with apical secretion of Cl^- might provide a co-ion for apical H⁺ secretion. Such a mechanism has been proposed in stomach (34), although this interpretation has been debated (26). Soybel et al. (34) have demonstrated that NKCC1 modulates HCl secretion in gastric mucosa of necturus through transport of Cl^-. In kidney, NKCC1 may modulate renal acid secretion through such a mechanism. Cl^- is secreted along the length of the medullary collecting duct (15,37). Moreover, Cl^- excretion changes in tandem with changes in H⁺ excretion, such as during metabolic acidosis (22). However, H⁺ equivalents in rat OMCD cannot account in full as cosecreted cations that accompany bumetanide-sensitive Cl^- secretion. Although NKCC1 mediates NH₄⁺ uptake (42), the present study shows that the role of NKCC1 in the process of secretion of net H⁺ equivalents is small.

In the inner medullary collecting duct (IMCD) and the cortical collecting duct (CCD) from DOCP-treated rats, J_tCO2 is increased at least two- to three-fold when tubules are perfused in the presence of NH₄Cl (21,38). This occurs because NH₄⁺ uptake provides an H⁺ source for bicarbonate absorption and the titration of luminal buffers in these segments. In the rat OMCD, however, NH₄⁺ does not provide an important H⁺ source for luminal acidification. This observation is compatible with mathematical models reported previously, which predict that changes in NH₄⁺ concentration have little impact on J_H in rat OMCD (43). Instead, the model predicts that high rates of J_H can be generated through availability of NH₃ alone (7,8,43). Because of the high permeability of NH₃ relative to that of NH₄⁺ in this segment (7,8), ammonium secretion occurs through active secretion of H⁺ in parallel with the nonionic diffusion of NH₃. Moreover, ammonium secretion, J_tAMM, correlates closely with V_T; therefore, ammonium secretion is largely an electrogenic process.

In rat OMCD, net acid secretion is dependent on H⁺ equivalents derived from the carbonic anhydrase-catalyzed hydration of CO₂, rather than from availability of NH₄⁺. Luminal carbonic anhydrase activity is absent in rat OMCD (8); therefore, the ethoxzolamide-sensitive component of net acid secretion occurs through changes in cytosolic, rather than luminal (membrane-bound) carbonic anhydrase activity. These findings are compatible with immunolocalization and functional studies, which observed that carbonic anhydrase II, a cytosolic carbonic anhydrase, is highly expressed in the intercalated cell of the rat OMCD (20). With inhibition of carbonic anhydrase the hydration of CO₂ is not available as a source of H^+’s for luminal acidification. Thus HCO₃^- absorption, which reflects H⁺ secretion, is reduced. In turn, the NH₃ gradient from the bath to the lumen is reduced, which results in little net diffusion of NH₃ into the luminal fluid.

In conclusion, in HCO₃^-/CO₂–buffered solutions, the effect of bumetanide on secretion of H⁺ equivalents is small. When studied in HCO₃^-/CO₂–buffered solutions with carbonic anhydrase activity intact, the cotransporter serves more as a mediator of transepithelial secretion of Cl^- secretion rather than H⁺. Under these conditions, net H⁺ equivalents cannot account fully as cosecreted cations, which accompany bumetanide-sensitive Cl^- secretion.

Transepithelial Transport of K⁺ and the Role of NKCC1
In inner ear and epididymis robust rates of K⁺ secretion have been observed (27,31). Because of high levels of expression of NKCC1 in these tissues and because NKCC1 mediates uptake of K⁺, the cotransporter has been implicated in the process of K⁺ secretion in these organ systems (27,31). In rat OMCD, no net flux of K⁺ was detected in the absence of inhibitors, similar to previous observations in rabbit OMCD (36). Moreover, in the present study, a role of NKCC1 in transepithelial transport of K⁺ was not demonstrated in this segment.

Transepithelial Transport of Na⁺ and the Role of NKCC1
Studies in rabbit OMCD perfused in vitro in symmetric, HCO₃^-/CO₂–buffered solutions showed no net flux of Na⁺ (36). Similarly, net Na⁺ flux in rat OMCD was not different from zero (see appendix, part 4). However, with bumetanide present in the bath solution, net Na⁺ absorption was detected (see appendix, part 5). One interpretation of these data is that net movement of Na⁺ and Cl^- occurs in part through NKCC1-mediated Na⁺ and Cl^- uptake across the basolateral membrane. However, other interpretations of these data are possible. For example, bumetanide could induce cell shrinkage, which promotes Na⁺ absorption in principal cells. Therefore, further studies are needed to determine the mechanism of bumetanide-sensitive J_Na as well as the physiologic role of NKCC1 in NaCl transport in the rat medullary collecting duct. Whether bumetanide changes net flux of Na⁺ and Cl^- in a 1:1 ratio is beyond the limit of detection of the techniques used.

After a NaCl load, natriuresis and chloruresis are observed in vivo, an effect mediated, at least in part, through the action of atrial natriuretic factor (ANF) on the collecting duct. Previous studies have suggested a role of NKCC1 in ANF-induced NaCl excretion. Rocha and Kudo (12,29) observed both bath-to-lumen and lumen-to-bath flux of Na⁺ and Cl^- in rat initial IMCD, a segment rich in intercalated cells that express NKCC1. With the application of ANF, the lumen-to-bath flux of Na⁺ and Cl^- was reduced, although the bath-to-lumen flux of these ions was increased. The ANF-induced increase in bath-to-lumen flux of Na⁺ and Cl^- was eliminated with the application of furosemide to the bath. These investigators concluded that ANF reduced absorption of Na⁺ and Cl^- by inhibiting apical Na⁺ channels while increasing Na⁺ and Cl^- secretion through stimulation of the Na⁺-K⁺-2Cl^- cotransporter.

In conclusion, NKCC1 mediates or modulates transepithelial transport of Na⁺ in the OMCD of DOCP-treated rats. These data suggest that rat intercalated cells serve important functional roles in addition to secretion of H⁺ equivalents.

	Appendix

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1. With nonbicarbonate buffers, such as NH₄⁺/NH₃, the fall in luminal pH that follows the rate of NH₃ secretion should equal the rate of total ammonia secretion, J_tAMM (21). Moreover, of the total ammonia present in the luminal fluid, only NH₃ secreted across the apical membrane is available to buffer protons. Thus, with the diffusion trapping model, the increase in H⁺ secretion or bicarbonate absorption observed in the presence of NH₄Cl should be equal to the rate of secretion of NH₃ or J_tAMM (21). In contrast, if direct NH₄⁺ uptake is important in transepithelial H⁺ secretion, then the increase in J_tCO2 observed in tubules perfused and bathed in the presence of NH₄⁺ should be much greater than the rate of ammonium secretion, J_tAMM (21,38,39). In the present study, a J_tCO2 of 13.3 ± 1.5 pmol/mm per min was measured (n = 9) with NH₄Cl present (solution 2) and a J_tCO2 of 11.3 ± 1.3 pmol/mm per min (n = 8) was measured with NH₄Cl absent (solution 1). Thus, the absolute value of the difference in J_tCO2 observed in tubules perfused in the presence and absence of ammonia (13.3 - 11.3 = 2.0 pmol/mm per min) was not greater than the absolute value of J_tAMM (-5.6 ± 0.9 pmol/mm per min; n = 9). In contrast, in CCD and tIMCD tubules from DOCP-treated rats, the increase in J_tCO2 observed in the presence of NH₄Cl relative to J_tCO2 measured in the absence of NH₄Cl, is much higher than the increase in J_tCO2 predicted from NH₃-induced changes in luminal buffering (21,38).
   
2. A difference in J_H of 10.5 pmol/mm per min, if present, would be detected with a power of 0.8 with P < 0.05 given the SD observed (Figure 5, middle panel) and the number of experiments performed under each condition (controls, n = 9; with bumetanide, n = 8). Therefore, if bumetanide addition reduces net acid secretion, J_H, and net Cl^- secretion, J_Cl, to a similar extent, a change in J_H should have been detected using the methodology employed.
   
3. The mechanism of Cl^- exit across the apical membrane is not known. Likely it is either a Cl^- channel without a large flux or an electroneutral exchanger. However, we cannot exclude the possibility that the bumetanide-sensitive component of J_Cl is an electrogenic process, but experiments performed had insufficient statistical power to detect these changes in V_T.
   
4. Because low rates of fluid secretion were observed in this segment when studied under similar conditions, net addition of Na⁺ and Cl^- (40) to the luminal fluid may be underestimated. However, a change in J_v with bumetanide has not been detected (40).
   
5. Because the sodiometer weakly detects K⁺ and NH₄⁺ (see Materials and Methods), the bumetanide-sensitive change in J_Na measured might reflect changes in J_tAMM or J_K. A change in collected Na⁺ concentration of 3.5 mM was detected after the application of bumetanide. Given the Na⁺/K⁺ and Na⁺/NH₄⁺ selectivity of this device, a change in collected K⁺ concentration of 21 mM (3.5 mM x 6 = 21 mM) or a change in collected total ammonia concentration of 81 mM (3.5 mM x 23 = 81 mM) would be required to detect a change in ion concentration of 3.5 mM. Figures 3 and 6 show that this does not occur. Moreover, we cannot exclude the possibility that changes in luminal pH produce small changes in the signal detected by the sodiometer (see Materials and Methods). However, in OMCD tubules from DOCP-treated rats perfused in vitro at a bath pH of 7.4, a fall in luminal pH from 7.4 to 6.0 produces a large increase in collected total ammonia concentration (7,8). We did not detect a change in collected total ammonia concentration with the application of bumetanide in HCO₃^-/CO₂–buffered solutions, bumetanide-induced changes in luminal pH are most likely insignificant.
   
6. Because the data violated the assumption of homogeneity of variances, a nonparametric alternative to the ANOVA, the Kruskal-Wallis one-way ANOVA by ranks, was performed. To explore the significant global test among the groups, Mann-Whitney U tests were employed with a Bonferroni correction for multiple tests. Statistical significance was achieved with a P < 0.017.
   

	Acknowledgments

 
This work was supported by National Institutes of Diabetes, Digestive and Kidney Diseases Grant DK 46493 (to S. M. Wall). We thank Drs. David Good, Jeff Sands, Donald Molony, and Robert Lasky for their helpful suggestions.

	References

Top Abstract Introduction Materials and Methods Results Discussion Appendix References

 

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