Contribution of the Na+-K+-2Clminus cotransporter NKCC1 to Clminus secretion in rat OMCD

Susan M. Wall, Michael P. Fischer, Pramod Mehta, Kathryn A. Hassell, and Stanley J. Park

Division of Renal Diseases and Hypertension, University of Texas Medical School at Houston, Houston, Texas 77030


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In rat kidney the "secretory" isoform of the Na+-K+-2Cl- cotransporter (NKCC1) localizes to the basolateral membrane of the alpha -intercalated cell. The purpose of this study was to determine whether rat outer medullary collecting duct (OMCD) secretes Cl- and whether transepithelial Cl- transport occurs, in part, through Cl- uptake across the basolateral membrane mediated by NKCC1 in series with Cl- efflux across the apical membrane. OMCD tubules from rats treated with deoxycorticosterone pivalate were perfused in vitro in symmetrical HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2-buffered solutions. Cl- secretion was observed in this segment, accompanied by a lumen positive transepithelial potential. Bumetanide (100 µM), when added to the bath, reduced Cl- secretion by 78%, although the lumen positive transepithelial potential and fluid flux were unchanged. Bumetanide-sensitive Cl- secretion was dependent on extracellular Na+ and either K+ or NH<UP><SUB>4</SUB><SUP>+</SUP></UP>, consistent with the ion dependency of NKCC1-mediated Cl- transport. In conclusion, OMCD tubules from deoxycorticosterone pivalate-treated rats secrete Cl- into the luminal fluid through NKCC1-mediated Cl- uptake across the basolateral membrane in series with Cl- efflux across the apical membrane. The physiological role of NKCC1-mediated Cl- uptake remains to be determined. However, the role of NKCC1 in the process of fluid secretion could not be demonstrated.

ammonium; type 1 bumetanide-sensitive sodium-potassium-2 chloride cotransporter; type 2 bumetanide-sensitive sodium-potassium-2 chloride cotransporter; fluid flux; chloride-bicarbonate exchange; outer medullary collecting duct


    INTRODUCTION
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INTRODUCTION
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TWO DISTINCT GENES ENCODE the Na+-K+-2Cl- cotransporters, BSC-2 (NKCC1) and BSC-1 (NKCC2). NKCC2, or the "absorptive" isoform, is kidney specific and localizes to the apical membrane of the thick ascending limb (5, 25). In contrast, NKCC1, or the "secretory" isoform, is widely distributed (25). However, the distribution of NKCC1 in the kidney is very species specific (13, 26). Recent cloning of NKCC1 in the mouse has enabled development of antibodies that recognize this cotransporter and has facilitated study of its distribution in various tissues. In rat kidney, immunolocalization studies by Ginns and colleagues (13) have detected the highest levels of expression of the cotransporter along the basolateral membrane of alpha -intercalated cells, with low levels of protein expression in the terminal inner medullary collecting duct (tIMCD). In contrast, in mouse collecting duct (26) NKCC1 expression is highest in the tIMCD, with no expression detected in either the cortical collecting duct (CCD) or the outer medullary collecting duct (OMCD). Thus the physiological role of the cotransporter in the collecting duct is puzzling in view of the species-specific distribution of NKCC1 protein.

The cellular composition of rat OMCD is heterogeneous: 60-64% of cells are principal cells whereas 36-40% are alpha -intercalated cells (16). Although principal cells mediate robust rates of Na+ transport (33), they mediate little Cl- transport (34). Therefore, it remains to be determined whether significant transepithelial movement of Cl- occurs in rat OMCD. If Cl- secretion is observed in rat OMCD, it might occur through anion exchange- and/or NKCC1-mediated Cl- uptake across the basolateral membrane in series with Cl- movement across the apical membrane.

In rabbit OMCD, Cl- is secreted in parallel with H+ secretion (HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption) (39). In this segment, Cl- secretion and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption are eliminated with addition of SITS, an inhibitor of anion exchange (39). Therefore, secretion of Cl- and absorption of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (secretion of H+ equivalents) are mediated by Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange on the basolateral membrane in series with efflux of H+ and Cl- across the apical membrane.

Like rabbit OMCD, rat OMCD absorbs HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and secretes NH<UP><SUB>4</SUB><SUP>+</SUP></UP> (9) However, Cl- transport pathways in this segment and their possible contribution to transepithelial Cl- transport are not understood. The purpose of the present study was to determine whether rat OMCD secretes Cl- and whether transepithelial transport of Cl- is mediated, at least in part, by NKCC1.


    METHODS
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INTRODUCTION
METHODS
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Tubules from the inner stripe of the OMCD were dissected from pathogen-free male Sprague-Dawley rats weighing 65-120 g (Harlan, Indianapolis, IN). Animals were housed in microisolator cages and fed a low-Na+, 0.8% K+ diet (Zeigler Bros., Garners, PA) (41). Rats received 5 mg deoxycorticosterone pivalate (DOCP; CIBA-Geigy Animal Health, Greensboro, NC) by intramuscular injection 5-7 days before death. To induce a rapid diuresis, animals were injected with furosemide (5 mg/100 g body wt ip) 45 min before death by decapitation. This furosemide-induced diuresis reduces the inner medullary axial solute concentration gradient (41) 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). Solution compositions are given in Table 1. The dissection solution was either solution 1 or solution 2 as appropriate, to match the NH4Cl concentration of the perfusate and bath solution used when measurements were performed. To dissect OMCD tubules from the inner stripe of the outer medulla, a cut was made between the inner and outer stripe of the outer medulla using a razor blade, and OMCD tubules were dissected as reported previously (9). Tubules were mounted on concentric glass pipettes and perfused in vitro at 37°C.

                              
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Table 1.   Solution composition

Experiments were performed with symmetrical solutions in the bath and perfusate. Osmolality was measured in all solutions (41). To maintain the desired CO2 concentration in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/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 (41, 42). To maintain pH in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-containing solutions, the bath fluid was constantly bubbled with 95% air-5% CO2. Bath pH was measured continuously during all experiments as described previously (41, 42). Bumetanide and ethoxzolamide were prepared as stock solutions in DMSO. Bumetanide (plus vehicle), ethoxzolamide (plus vehicle), or vehicle alone (DMSO) was added to the bath in all experiments. Vehicle (DMSO) was added to the perfusate such that a final DMSO concentration of 0.04% was always present in both the perfusate and bath.

Because of time-dependent changes in transepithelial potential difference, VT (not shown), measurements in each tubule were made under a single experimental condition. All collections were begun 30 min, and terminated 75 min, after the tubule was warmed. Perfusate samples were collected continuously over this time period. VT was measured continuously; reported VT corresponds to that measured at the midpoint of this time period, or 50 min after the tubule was warmed.

Measurement of transepithelial Cl- flux. Cl- concentration in collected perfusate samples was measured using a continuous-flow fluorometer with an assay developed by Garcia and colleagues (11) that utilizes 6 methoxy-N-(3-sulfopropyl) quinolinium (SPQ; Molecular Probes, Eugene, OR), a Cl--sensitive fluorophore. SPQ was dissolved in water at a 0.20 mM concentration. The reagent was placed in a pasteur pipette and drawn past the injection port and then through stainless steel tubing into a cuvette with a constant-speed withdrawal pump at 55 nl/s. Other details of the fluorometer design have been reported previously (41). Samples were pipetted into the flowing reagent. We have observed that the Cl- assay is linear over a range of at least 0-5.3 nmol Cl- (not shown). By using a 10-nl pipette, differences in Cl- concentration of 2 mM can be detected (2 × coefficient of variation/slope) with this assay (11). Fluorescence of SPQ is not affected by pH, Na+, or HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration over the physiological range (21).

Transepithelial Cl- flux, JCl, was calculated according to the equation
J<SUB>Cl</SUB><IT>=</IT>(C<SUB>o</SUB><IT>−</IT>C<SUB>L</SUB>)<IT>Q/L</IT>
where Co and CL are perfusate and collected fluid Cl- concentration, respectively, Q is flow rate in nanoliters per minute, and L is tubule length.

Measurement of intracellular pH in alpha -intercalated cells. Intracellualr pH (pHi) was measured using 2',7'-bis(carboxy-ethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM). Tubules were perfused and bathed for 15 min at 37°C in solution 1. The perfusate was changed to the same solution with the addition of 5 µM BCECF-AM. Tubules were perfused with BCECF for 10 min. The perfusate was then changed to the original solution but with BCECF removed. By using this technique, alpha -intercalated cells are preferentially loaded with BCECF (35). pHi was determined by measuring the ratio of emitted light at >530 nm when BCECF was excited alternatively at 440 and 495 nm. 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. The pH of this solution was varied between 7.0 and 7.8. The other details of pHi measurement in tubules perfused in vitro were performed as described previously by our laboratory (41).

Fluid flux. To measure fluid flux (Jv), changes in raffinose concentration of the luminal fluid were measured, using the assay described by Garvin and Knepper (12). Raffinose concentration in collected fluid samples was measured by using an enzymatic assay in which raffinose is converted to galactonolactone and NADH. This assay was purchased as a kit (Boehringer Mannheim, Mannheim, Germany). Fluorescence of NADH was measured using a continuous-flow ultramicrofluorometer. Samples were collected into the lower chamber (17 nl) of a double-constriction pipette. An enzyme solution was employed that contained 3.5 mg/ml NAD+ in citrate buffer (pH 4.5) and 4.5 U/ml alpha -galactosidase. This enzyme solution was drawn into the pipette until the second chamber (237 nl) was filled. Because the enzymatic conversion of raffinose to NADH and galactonolactone was complete after 6 min at 37°C (data not shown), all samples were incubated for 7-9 min at 37°C and then injected into a flowing stream. The flowing stream was drawn by a constant-withdrawal pump at 166 nl/s and contained 0.3 U/ml beta -galactose dehydrogenase in potassium diphosphate buffer (pH 8.6). By using this assay, raffinose concentration is linear from at least 0 to 203 pmol (not shown).

Fluid absorption was calculated by using the equation
J<SUB>v</SUB><IT>=</IT>(C<SUB>L</SUB><IT>/</IT>C<SUB>o</SUB><IT>−1</IT>)<IT>Q/L</IT>
where CL is the concentration of raffinose in collected fluid, Co is the concentration of raffinose in the perfused fluid, Q is the collection rate in nanoliters per minute, and L is the tubule length.

Measurement of Jv requires use of a volume marker that has a low permeability in rat OMCD. Raffinose permeability (Praf) was therefore measured in rat OMCD tubules. To measure Praf, tubules were bathed in solution 8, which contained 10 mM raffinose. The perfusate solution was the same solution, except that raffinose was replaced with NaCl to match the osmolality of the bath solution. The concentration of raffinose was measured in the collected fluid samples. Samples for raffinose concentration were taken at the same time points as were the Cl- samples. Praf was calculated by using the following equation
P<SUB>raf</SUB><IT>=J</IT><SUB>raf</SUB><IT>/</IT>(C<SUB>raf</SUB><IT>×A</IT><SUB>s</SUB>)
Raffinose flux, Jraf, was calculated as collected raffinose concentration (in mM) times the luminal flow rate (Q; in nl/min) divided by tubule length (L). As is the surface area per unit length of tubule, and Craf is the mean concentration gradient across the epithelium. Praf in three OMCD tubules averaged 1.0 ± 0.5 × 10-6 cm/s. This compares with Praf of 3.0 × 10-6 cm/s reported in rabbit proximal tubule (12). Thus OMCD is relatively impermeable to raffinose, making it a suitable volume marker in this segment.

VT. To measure VT, the solution in the perfusion pipette was connected to an electrometer (model KS-700, World Precision Instruments, New Haven, CT) through an agar bridge saturated with 0.16 M NaCl and a calomel cell as described previously (41). The reference was an agar bridge from the bath to a calomel cell.

Statistics. In all experiments wherein either Cl- or raffinose concentration was assayed, two to three replicate measurements were made in a single tubule. The mean of all measurements made in a single tubule was used in the statistical analysis, where n represents the number of tubules studied. Statistical significance was determined by using a paired or unpaired two-tailed Student's t-test as appropriate. For multiple comparisons, ANOVA was used with specific contrasts by the Bonferroni method. Statistical significance was achieved with P < 0.05. Data are displayed as means ± SE.


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Role of anion exchange in JCl. Ion transport pathways along the collecting duct have been studied extensively in DOCP-treated rats (9, 27, 41, 42). Therefore, to explore Cl- transport pathways in rat OMCD, DOCP-treated rats were employed both to facilitate comparison with these previous studies and to stimulate Cl- transport pathways such as anion exchange (17).1

The role of anion exchange in Cl- secretion in rat OMCD was explored by testing the effect of 0.5 mM H2DIDS on pHi and JCl in rat OMCD.2 The effect of H2DIDS on pHi is shown in Fig. 1. pHi increased nearly 0.4 pH units (n = 3, P < 0.05, solution 1) 4 min after the addition of 0.5 mM H2DIDS to the bath solution. However, no change in pHi was noted after the addition of the NKCC1 transport inhibitor bumetanide (100 µM). The effect of H2DIDS on JCl is shown in Fig. 2 (Table 2). Rat OMCD tubules secreted Cl- with a JCl of -11.9 ± 1.7 pmol · mm-1 · min-1 (n = 5, solution 2). In the presence of H2DIDS, JCl was -7.9 ± 1.9 pmol · mm-1 · min-1 [n = 5, P = not significant (NS)]. Although no change in Cl- secretion was detected with the application of H2DIDS, an effect of stilbene inhibitors on JCl cannot be excluded from these data. Nevertheless, these data raise the possibility that along the basolateral membrane, other Cl- transport pathways might participate in the transepithelial transport of Cl-.


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Fig. 1.   Effect of H2DIDS and bumetanide on intracellular pH (pHi). Outer medullary collecting duct (OMCD) tubules from deoxycorticosterone pivalate (DOCP)-treated rats were perfused and bathed in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2-buffered solutions containing 5 mM KCl, but no NH4Cl (solution 1). Baseline pHi was 7.37 ± 0.04 (H2DIDS) and 7.42 ± 0.08 (bumetanide). Four minutes after the addition of 0.5 mM H2DIDS, pHi rose 0.37 ± 0.5 pH units (n = 3, P < 0.05, paired Student's t-test). In separate tubules, 4 min after the addition of 100 µM bumetanide, pHi rose only 0.07 ± 0.03 pH units [n = 3, P = not significant (NS), paired Student's t-test].



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Fig. 2.   Effect of H2DIDS and bumetanide on transepithelia Cl- flux (JCl). OMCD tubules were perfused and bathed in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2-buffered solutions containing 5 mM KCl and 6 mM NH4Cl (solution 2). A: in the absence of inhibitors, JCl was -11.9 + 1.7 pmol · mm-1 · min-1 (n = 5). In separate tubules perfused in the presence of 0.5 mM H2DIDS, a reduction in JCl could not be demonstrated (n = 5, P = NS, unpaired Student's t-test). B: Cl-secretion (-13.5 ± 0.8 pmol · mm-1 · min-1) was attenuated in a dose-dependent fashion with bumetanide. With 10 µM bumetanide, JCl was reduced by 55% (P < 0.05, ANOVA). With 100 µM bumetanide, JCl was reduced by 78% (P < 0.05, ANOVA).


                              
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Table 2.   Chloride flux in OMCD from DOCP-treated rats

Effect of bumetanide on JCl. To determine the role of NKCC1-mediated Cl- uptake in the process of transepithelial transport of Cl-, the effect of bumetanide on JCl was tested. Results are shown in Fig. 2 (Table 2). Cl- secretion (-13.5 ± 0.8 pmol · mm-1 · min-1, n = 5, solution 2) was attenuated in a dose-dependent fashion with bumetanide, an inhibitor of NKCC1, when added to the peritubular bath. JCl was inhibited by 55% with 10 µM bumetanide (P < 0.05) and 78% by 100 µM bumetanide (P < 0.05).

The effect of bumetanide on VT was tested in separate tubules when they were perfused and bathed in the presence of solution 2. As shown in Fig. 3, although bumetanide inhibited JCl, an effect of bumetanide on VT could not be demonstrated. Because of the variability in measured VT, the effect of ethoxzolamide on VT was tested as a positive control (29) to determine our ability to detect changes in VT. Addition to the bath of ethoxzolamide, an inhibitor of carbonic anhydrase, obliterated the lumen positive VT. An effect of bumetanide on membrane potential could not be demonstrated, however, either under baseline conditions (VT = +5 mV) or in the presence of ethoxzolamide (VT = 0). Because bumetanide inhibited JCl without a change in dectect VT, a role of paracellular transport in the bumetanide-sensitive component of Cl- secretion could not be demonstrated.


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Fig. 3.   Effect of bumetanide on transepithelial potential difference (VT). VT was measured in OMCD tubules perfused in the presence of solution 2. Under baseline conditions, a lumen positive potential difference was observed (4.9 ± 1.9 mV, n = 4). Addition of 100 µM bumetanide did not alter VT (4.6 ± 1.5 mV, n = 4, P = NS, unpaired Students t-test). The lumen positive VT was abolished with the addition of the carbonic anhydrase inhibitor ethoxzolamide (100 µM) to the bath solution (0.2 ± 0.1 mV, n = 3). In the presence of ethoxzolamide, VT was unchanged with the addition of bumetanide (100 µM) to the bath (-0.1 ± 0.1 mV, n = 4, P = NS).

Effect of extracellular Na+, K+, and NH<UP><SUB><UP>4</UP></SUB><SUP><UP>+</UP></SUP></UP> on total and bumetanide-sensitive JCl. Because Na+ is a substrate for NKCC1, the effect of Na+ on total and bumetanide-sensitive JCl was tested. Experiments were performed under conditions identical to those in Figs. 2 and 3, except that Na+ was replaced with choline in equal concentration (solution 3, Table 2). In the absence of Na+, no net flux of Cl- was detected either in the presence (-3.6 ± 2.2 pmol · mm-1 · min-1, n = 5) or in the absence of bumetanide (-0.6 ± 2.7 pmol · mm-1 · min-1, n = 5). Thus both total and bumetanide-sensitive JCl are dependent on the presence of Na+ in the bath and perfusate, which eliminates the possibility that Cl- secretion sensitive to bumetanide is mediated by KCl cotransport.

Because K+ is a substrate for NKCC1, the effect of increasing K+ concentration on total and bumetanide-sensitive Cl- secretion was studied. Figure 4 (Table 2) demonstrates that at a K+ concentration of 2 mM (solution 4), JCl was low both in the presence and in the absence of bumetanide. Thus at a K+ concentration of 2 mM, total and bumetanide-sensitive Cl- secretion are very low. However, at a K+ concentration of 20 mM (solution 5, Fig. 4, Table 2), significant Cl- secretion was observed, which was reduced with the application of bumetanide to the bath. Therefore, total and bumetanide-sensitive Cl- secretion are dependent on K+ in the bath and perfusate.


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Fig. 4.   Effect of extracellular K+ and NH<UP><SUB>4</SUB><SUP>+</SUP></UP> on JCl. A: tubules were perfused and bathed in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2-buffered solutions containing 2 mM KCl, but in the absence of NH4Cl (solution 4). JCl was low and not reduced further with the application of bumetanide (100 µM) to the bath (P = NS, unpaired Student's t-test). B: tubules were perfused and bathed in the same solution as described in A, but one in which the K+ concentration was increased to 20 mM (solution 5). Under these conditions, Cl- secretion was observed, which was attenuated with the application of bumetanide (100 µM) to the bath (P < 0.05, unpaired Student's t-test). C: tubules were perfused and bathed in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>/CO2-buffered solutions containing 2 mM KCl, and 6 mM NH4Cl (solution 6). Significant Cl- secretion was observed, which was attenuated with the application of bumetanide (100 µM) to the bath (P < 0.05, unpaired Student's t-test).

In many cell types, NH<UP><SUB>4</SUB><SUP>+</SUP></UP> substitutes for K+ on NKCC1 (8, 43). Therefore, we asked whether bumetanide-sensitive Cl- secretion persists in rat OMCD when NH<UP><SUB>4</SUB><SUP>+</SUP></UP> is substituted for K+. At a K+ concentration of 2 mM (NH<UP><SUB>4</SUB><SUP>+</SUP></UP> absent), JCl was low in either the presence or the absence of bumetanide (Fig. 4, Table 2). However, with substitution of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> for choline (2 mM K+, 6 mM NH<UP><SUB>4</SUB><SUP>+</SUP></UP>, solution 6), secretion of Cl- was significant (-9.9 ± 1.1 pmol · mm-1 · min-1, n = 5). Moreover, in the presence of 2 mM K+ and 6 mM NH<UP><SUB>4</SUB><SUP>+</SUP></UP> (solution 6), JCl was reduced with the application of bumetanide to the bath solution (P < 0.05). Because bumetanide-sensitive Cl- secretion was observed in the presence of K+ or NH<UP><SUB>4</SUB><SUP>+</SUP></UP>, NKCC1-mediated Cl- uptake occurs when operating as a Na+-NH<UP><SUB>4</SUB><SUP>+</SUP></UP>-2Cl- cotransporter.

Effect of bumetanide on Jv. In other cell types, such as in salivary glands, NKCC1 mediates Cl- secretion, which gives rise to secretion of fluid (28). We therefore tested whether fluid secretion is observed in parallel with Cl- secretion in rat OMCD. Tubules were perfused and bathed in the presence of 5 mM raffinose (solution 7), which was employed as a volume marker. Results are displayed in Table 3. In symmetrical solutions, low levels of fluid secretion were observed in this segment (Jv = -0.042 ± 0.015 nl · mm-1 · min-1, n = 5), which was unchanged with the application of bumetanide to the bath. However, because of the variability in the measurements, we cannot exclude a small effect of bumetanide on Jv.

                              
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Table 3.   Fluid flux in OMCD tubules from DOCP-treated rats

It is possible that Jv is low because of raffinose-induced changes in JCl. Therefore, as a control, JCl was measured in the presence and absence of raffinose. Cl- secretion was similar in the presence (JCl = -10.4 ± 1.4 pmol · mm-1 · min-1, n = 3, solution 7) and in the absence (JCl = -10.6 ± 2.3 pmol · mm-1 · min-1, n = 3, solution 2) of 5 mM raffinose in the perfusate and bath (P = NS). Therefore, our inability to detect significant rates of total and bumetanide-sensitive Jv did not result from a raffinose-induced change in Cl- secretion.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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This study demonstrates that in OMCD tubules perfused in vitro from DOCP-treated rats, Cl- is secreted into the luminal fluid through Cl- uptake across the basolateral membrane, mediated by both NKCC1 and anion exchange, in series with Cl- efflux across the apical membrane. This contribution of NKCC1 to Cl- secretion represents a novel mechanism of transepithelial Cl- transport in rat OMCD.

Significant Cl- secretion was observed in the OMCD in the present in vitro study. In vivo studies suggest that Cl- secretion might occur in rat OMCD. After NaCl stress, Cl- delivery to the base of the collecting duct is greater than Cl- delivery at the level of the superficial distal tubule (18). One explanation for these results is that Cl- secretion occurs along the collecting duct in vivo in one or more nephron segments, which lie between the superficial distal tubule and the base of the collecting duct. The contribution of each segment that falls between these two micropuncture sites to the increment in Cl- delivered to the base of the collecting duct is not known. The present study demonstrates that the OMCD is one segment in this region of the nephron that secretes Cl- in vitro. The pathway(s) that mediates Cl- uptake across the basolateral membrane of the alpha -intercalated cell in rat OMCD were therefore explored in more detail.

Anion exchange mediates Cl- uptake and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exit across the basolateral membrane. Application of H2DIDS led to a marked increase in pHi. Although pHi changes might be due to nonspecific effects of H2DIDS, the most likely explanation for these observations is that H2DIDS prevents Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange from mediating net HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exit. The anion exchanger responsible for the increase in pHi after the addition of H2DIDS to the bath solution is probably AE1, a Na+-independent, Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger, which is stilbene sensitive and highly expressed along the basolateral membrane of the alpha -intercalated cell in rat OMCD (7). Because Cl- secretion was not eliminated with 0.5 mM H2DIDS, other Cl- uptake pathways along the basolateral membrane of rat OMCD might also be important in the process of transepithelial Cl- transport.

We demonstrated that JCl is reduced in a dose-dependent fashion with bumetanide. These data are consistent with previous reports of the dose response of bumetanide to rat NKCC1 (14, 28). At a concentration of <10 µM, bumetanide is a relatively specific inhibitor of NKCC1 (15). Therefore, significant Cl- uptake across the basolateral membrane is mediated by NKCC1 because >50% of JCl is inhibited by low concentrations of bumetanide (10 µM) when added to the bath.

In rat, NKCC1 is fully inhibited at a bumetanide concentration of 100 µM (14, 28). However, at this bumetanide concentration, at least partial inhibition of other Cl- transporters, such as Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange (AE1) (46), Cl- channels (19), and KCl cotransport (19), has been observed. Cl- secretion sensitive to 100 µM bumetanide may therefore overestimate the contribution of NKCC1 to total transepithelial Cl- transport in rat OMCD. However, ion substitution experiments demonstrated that the bumetanide-sensitive component of Cl- secretion is dependent on extracellular Na+ and either K+ or NH<UP><SUB>4</SUB><SUP>+</SUP></UP>. This ion dependency is consistent with Cl- uptake mediated by NKCC1 instead of through these other transporters. Although Cl- uptake across the basolateral membrane can be attributed to both anion exchange and NKCC1, the mechanism of Cl- transport across the apical membrane is unknown.

Whether Cl- uptake across the basolateral membrane of the alpha -intercalated cell in rat OMCD is mediated by NKCC1 or NKCC2 cannot be determined directly. In the rat outer medulla, the absorptive isoform of the Na-K-2Cl cotransporter, NKCC2, has been localized to the thick ascending limb by in situ hybridization and immunolocalization studies (5, 25). Although NKCC2 message has also been detected in rat OMCD (45), it is unlikely that the isoform of the cotransporter detected in the present study is NKCC2. First, NKCC2 protein expression has not been detected in rat OMCD (5), although NKCC1 protein has been clearly demonstrated in this segment (13). Therefore, NKCC2 is most probably not as abundant in this segment as NKCC1. Second, bumetanide inhibits Cl- secretion when applied to the bath, consistent with localization of NKCC1 to the basolateral membrane (13).

In the present study it was observed that total and bumetanide-sensitive JCl vary greatly over an extracellular K+ concentration range of 2-20 mM. Because the interstitium of the rat outer medulla is not accessible to micropuncture, interstitial K+ concentration in vivo is not known. However with medullary recycling of K+, interstitial K+ concentration in the outer medulla is expected to be greater than serum values (23), which vary in rat3 between 2 and 7 mM (3, 4), but less than interstitial values in the inner medulla, which range from 6 to 54 mM (2). Thus interstitial K+ concentration in the interstitium of the outer medulla is probably >2 but <50 mM. For NKCC1 the Michaelis-Menten constant (Km)4 for Rb+ (a K+ congener) is 2-15 mM (22, 30, 43). Thus NKCC1-mediated Cl- uptake should vary greatly over a K+ concentration range of 2-50 mM5. If so, changes in K+ concentration in the interstitium of the rat outer medulla in vivo should markedly alter Cl- secretion in the OMCD through changes in NKCC1-mediated Cl- transport.

Although total and bumetanide-sensitive JCl in rat OMCD vary greatly with changes in extracellular Na+ concentration, it is less likely that changes in interstitial Na+ concentration in vivo significantly regulate NKCC1-mediated Cl- uptake. In rat, serum Na+ concentration ranges from 95 to 200 mM (20). Through countercurrent multiplication, interstitial Na+ concentration in rat outer medulla is therefore probably >95 mM, although it has not been measured directly. Because the Km for Na+ reported for mammalian NKCC1 is generally less than 50 mM (22, 30), the Na+ concentration of the rat outer medullary interstitium probably always approaches maximal transport rate conditions for NKCC1. Thus changes in interstitial Na+ concentration over the physiological range expected in vivo probably do not significantly alter NKCC1-mediated Cl- uptake.

Although Cl- transporters such as AE1 clearly participate in transepithelial secretion of net H+ equivalents, the role of NKCC1 in renal physiology is not known. The Na-K-2Cl cotransporter has been implicated in a number of cell functions, including the secretion of HCl (38) and KCl (32). However, its primary physiological function is in volume regulation and the vectorial transport of water and NaCl (25). Therefore, rat alpha -intercalated cells, which express high levels of NKCC1 relative to other cells in rat kidney, may serve physiological functions other than secretion of H+ equivalents.

In vivo studies have demonstrated secretion of NaCl and fluid along rat IMCD (36, 40). However, more recent studies of Wallace and co-workers (44) have reported secretion of fluid in vitro in rat initial IMCD (44), a segment that contains alpha -intercalated cells and expresses NKCC1 (13). Fluid secretion, determined by measuring luminal diameter over a 5- to 12-h period in tubules with sealed ends, was attenuated with the addition of bumetanide to the bath (44). In the present study, very low levels of fluid secretion were observed in rat OMCD, with a Jv similar to that reported previously in rat CCD (1) and rat tIMCD (42). Low levels of fluid secretion observed in rat OMCD in the present study are compatible with observations of Wallace et al. (44) in initial IMCD. However, in rat OMCD no change in Jv was detected with inhibition of NKCC1. Therefore, under the conditions of the present study a role of NKCC1 in fluid secretion or absorption in OMCD tubules from DOCP-treated rats could not be demonstrated.

Previous studies have suggested a role of the Na-K-2Cl cotransporter in NaCl secretion in the rat IMCD (31). The natriuresis and chloruresis observed in rats given a NaCl load occurs in part through atrial natriuretic factor (ANF) (10, 37, 40). In rat initial IMCD, Rocha and Kudo (31) observed that with the application of ANF to the bath fluid, bath-to-lumen flux of Na+ and Cl- is increased, whereas the lumen-to-bath flux of these ions is reduced. This ANF-induced change in Na+ and Cl- secretion was fully inhibited with low concentrations of furosemide when added to the bath solution. The authors concluded that ANF reduced Na+ and Cl- absorption through inhibition of apical Na+ channels while stimulating Na+ and Cl- secretion mediated by the Na-K-2Cl cotransporter. The possible role of NKCC1 in mediating NaCl excretion after NaCl stress will require further study.

In conclusion, rat OMCD secretes Cl-. Cl- secretion in this segment occurs in part through Cl- uptake across the basolateral membrane, mediated by NKCC1, in series with Cl- efflux across the apical membrane. The physiological role of this transport process remains to be determined. However, under the conditions of the present in vitro study, a role of NKCC1 in fluid secretion or absorption was not demonstrated in this segment. The cosecreted cation or counterion, which accompanies bumetanide-sensitive Cl- secretion, remains to be established.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-46493 (to S. M. Wall).


    FOOTNOTES

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

1 An effect of DOCP on JCl could not be detected in rat OMCD. JCl was -2.9 ± 1.1 pmol · mm-1 · min-1 (n = 5) in untreated controls and -7.1 ± 1.6 pmol · mm-1 · min-1 in tubules from DOCP-treated rats (solution 2, n = 7, P = 0.075, unpaired Student's t-test).

2 At an H2DIDS concentration of 0.5 mM, 66% of Na+-independent anion exchange is inhibited along the basolateral membrane of intercalated cells in rat CCD (6).

4 The Km, or apparent affinity, of a transport protein reflects the substrate concentration needed to achieve half the maximal transport rate (Vmax).

5 For mouse NKCC1, our laboratory has reported a Km for K+ of 4.6 mM (43). Assuming that NKCC1 follows Michaelis-Menton kinetics, at an extracellular K+ concentration of 2 mM, NKCC1-mediated Cl- uptake should operate at ~28% of Vmax. If a perfusate flow rate of 2 nl · mm-1 · min-1 is employed, the predicted change in perfusate Cl- concentration with the application of bumetanide should be 0.8 mM, which is beyond the limit of detection of this assay (11).

3 Interstitial ion concentration in cortex is taken to be equivalent to serum levels. In inner medulla, interstitial ion concentrations are assumed to reflect values measured in vasa recta plasma at the same level along the corticomedullary axis (24).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 5 October 2000; accepted in final form 20 December 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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