Nerve Growth Factor Inhibits HCO3minus Absorption in Renal Thick Ascending Limb through Inhibition of Basolateral Membrane Na+/H+ Exchange*

Bruns A. Watts III, Thampi George, and David W. GoodDagger

From the Departments of Medicine and Physiology & Biophysics, University of Texas Medical Branch, Galveston, Texas 77555

    ABSTRACT
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ABSTRACT
INTRODUCTION
REFERENCES

Nerve growth factor (NGF) inhibits transepithelial HCO3- absorption in the rat medullary thick ascending limb (MTAL). To investigate the mechanism of this inhibition, MTALs were perfused in vitro in Na+-free solutions, and apical and basolateral membrane Na+/H+ exchange activities were determined from rates of pHi recovery after lumen or bath Na+ addition. NGF (0.7 nM in the bath) had no effect on apical Na+/H+ exchange activity, but inhibited basolateral Na+/H+ exchange activity by 50%. Inhibition of basolateral Na+/H+ exchange activity with ethylisopropyl amiloride (EIPA) secondarily reduces apical Na+/H+ exchange activity and HCO3- absorption in the MTAL (Good, D. W., George, T., and Watts, B. A., III (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 12525-12529). To determine whether a similar mechanism could explain inhibition of HCO3- absorption by NGF, apical Na+/H+ exchange activity was assessed in physiological solutions (146 mM Na+) by measurement of the initial rate of cell acidification after lumen EIPA addition. Under these conditions, in which basolateral Na+/H+ exchange activity is present, NGF inhibited apical Na+/H+ exchange activity. Inhibition of HCO3- absorption by NGF was eliminated in the presence of bath EIPA or in the absence of bath Na+. Also, NGF blocked inhibition of HCO3- absorption by bath EIPA. We conclude that NGF inhibits basolateral Na+/H+ exchange activity in the MTAL, an effect opposite from the stimulation of Na+/H+ exchange by growth factors in other systems. NGF inhibits transepithelial HCO3- absorption through inhibition of basolateral Na+/H+ exchange, most likely as the result of functional coupling in which primary inhibition of basolateral Na+/H+ exchange activity results secondarily in inhibition of apical Na+/H+ exchange activity. These findings establish a role for basolateral Na+/H+ exchange in the regulation of renal tubule HCO3- absorption.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
REFERENCES

Na+/H+ exchangers mediate the electroneutral exchange of Na+ and H+ across plasma membranes and play key roles in a variety of cell functions, including cell volume regulation, pHi regulation, epithelial sodium reabsorption, and cell growth and proliferation (1-3). At least five mammalian isoforms of Na+/H+ exchange (NHE1-5)1 have been identified, which differ in their tissue distribution and responses to extracellular stimuli (3-5). One of the most prominent features of Na+/H+ exchange is its stimulation by growth factors. This stimulation is rapid, is observed in many different cell types, and occurs with virtually all mitogens (1-6). Furthermore, growth factors stimulate all NHE isoforms examined to date (NHE1, NHE2, and NHE3) (3, 4, 7, 8). The mechanisms involved in growth factor activation of Na+/H+ exchange have been studied extensively because of the close association of increased exchanger activity with cell proliferation and oncogenic transformation (1, 4-11).

The mammalian kidney expresses NHE1-4, and Na+/H+ exchangers are present on both apical and basolateral membranes of tubule epithelial cells. The ubiquitously expressed isoform NHE1 is present on the basolateral membrane of most nephron segments, where it is involved in housekeeping functions such as cell volume and pHi regulation (12-17). NHE4 is also a basolateral isoform localized to collecting ducts and distal tubules, where it may play a specialized role in the control of cell volume (18, 19). NHE3 is localized to the apical membrane of proximal tubule and thick ascending limb cells (20-22), where it mediates the reabsorption of NaCl and NaHCO3 in the proximal tubule and the reabsorption of NaHCO3 in the thick ascending limb (23-27). NHE2 is an apical isoform in the kidney, but its functional significance is unclear (5, 26-29). The kidney is also a site of production of numerous growth factors, including epidermal growth factor, insulin-like growth factor, platelet-derived growth factor, nerve growth factor (NGF), and hepatocyte growth factor (30). Based on their prominent stimulation of Na+/H+ exchange activity in other systems, these factors could influence multiple cellular processes in renal tubules, including urinary acidification, through effects on Na+/H+ exchange activities. At present, however, the effects of locally produced growth factors on Na+/H+ exchangers and their related functions in renal tubules are poorly understood.

The medullary thick ascending limb (MTAL) of the mammalian kidney participates in the regulation of acid-base balance by reabsorbing a sizeable fraction of the HCO3- filtered at the glomerulus (31). The proton secretion required for this HCO3- absorption is mediated virtually completely by apical membrane Na+/H+ exchange (25). Furthermore, the regulation of HCO3- absorption is achieved largely through regulation of this apical exchanger (24, 25, 27, 31-35). The MTAL also contains a basolateral membrane Na+/H+ exchanger that is active at the resting pHi (33, 36). In general, it has been assumed for renal tubules that basolateral membrane Na+/H+ exchange opposes transcellular HCO3- absorption because it diminishes net base efflux (2, 13, 14). Contrary to this view, however, we recently demonstrated that inhibiting basolateral Na+/H+ exchange activity with ethylisopropyl amiloride (EIPA) decreased transepithelial HCO3- absorption in the MTAL (36). This decrease was the result of a functional interaction between the basolateral and apical membrane Na+/H+ exchangers, in which primary inhibition of basolateral Na+/H+ exchange activity secondarily inhibited apical Na+/H+ exchange activity, thereby decreasing HCO3- absorption (36). These studies established that the HCO3- absorption rate in the MTAL is dependent not only on the activity of the apical membrane Na+/H+ exchanger, but also on the activity of the basolateral membrane Na+/H+ exchanger. However, physiological factors that regulate HCO3- absorption through effects on basolateral membrane Na+/H+ exchange activity have not been identified.

In view of their potent stimulation of Na+/H+ exchange activity in other systems, we recently investigated the effects of growth factors on HCO3- absorption by the MTAL. These studies demonstrated that NGF inhibits HCO3- absorption in the MTAL through a tyrosine kinase-dependent signaling mechanism (37). This inhibition was unexpected since it cannot readily be explained by the classical action of growth factors to stimulate Na+/H+ exchange activity. Instead, our results suggested that NGF may inhibit Na+/H+ exchange activity in the MTAL. The purpose of the present study was to examine directly the effects of NGF on apical and basolateral membrane Na+/H+ exchange activities to determine the mechanism by which NGF inhibits HCO3- absorption. Our results demonstrate that, in contrast to the virtually universal stimulation of Na+/H+ exchange by growth factors in other cells, NGF inhibits basolateral membrane Na+/H+ exchange activity in the MTAL. We also demonstrate that the inhibition of basolateral Na+/H+ exchange activity mediates NGF-induced inhibition of transepithelial HCO3- absorption.

    EXPERIMENTAL PROCEDURES

Tubule Perfusion-- MTALs from male Sprague-Dawley rats (60-90 g; Taconic Farms Inc., Germantown, NY) were perfused in vitro as described previously (32, 34, 38). In brief, the tubules were dissected from the inner stripe of the outer medulla, transferred to a bath chamber on the stage of an inverted microscope, and mounted on micropipettes for perfusion at 37 °C. The composition of the perfusion and bath solutions for specific protocols is given below. Experiments were carried out using 7 S NGF (37). Solutions were prepared as described (34, 37).

Measurement of Net HCO3- Absorption-- To measure transepithelial HCO3- absorption rates, tubules were perfused and bathed in control solution that contained 146 mM Na+, 4 mM K+, 122 mM Cl-, 25 mM HCO3-, 2.0 mM Ca2+, 1.5 mM Mg2+, 2.0 mM phosphate, 1.2 mM SO42-, 1.0 mM citrate, 2.0 mM lactate, and 5.5 mM glucose (equilibrated with 95% O2 and 5% CO2, pH 7.45, at 37 °C). Bath solutions also contained 0.2 g/100 ml fatty acid-free bovine albumin. In one series of HCO3- transport experiments (see Fig. 5B), Na+ in the bath solution was replaced completely with N-methyl-D-glucammonium (NMDG+). Experimental agents were added to the bath solutions as described under "Results." The length of the perfused tubule segments ranged from 0.48 to 0.67 mm.

The protocol for study of transepithelial HCO3- absorption was as described (32, 37). The tubules were equilibrated for 20-30 min at 37 °C in the initial perfusion and bath solutions, and the luminal flow rate was adjusted to 1.4-2.0 nl/min/mm. Two or three 10-min tubule fluid samples were then collected for each period (initial, experimental, and recovery). The tubules were allowed to re-equilibrate for 5-15 min after an experimental agent was added to or removed from the bath solution. The absolute rate of HCO3- absorption (JHCO3-, pmol/min/mm) was calculated from the luminal flow rate and the difference between total CO2 concentrations in perfused and collected fluids (32). When repeat measurements were made at the beginning and end of an experiment (initial and recovery periods), the values were averaged. Single tubule values are presented in the figures. Means ± S.E. (n = number of tubules) are presented below.

Measurement of Intracellular pH-- pHi was measured by use of the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and a computer-controlled spectrofluorometer (CM-X, Spex Industries) coupled to the perfusion apparatus as described previously (34, 36, 38). The tubules were perfused in the same manner used for HCO3- transport experiments, except that the lumen and bath solutions were delivered via rapid flow systems that permit complete exchange of the solutions in <2 s (36, 38). Intracellular dye was excited alternately at 500- and 440-nm wavelengths, and emission was monitored at 530 nm using a photon counter. Intracellular dye was calibrated using high K+/nigericin standards at the end of each experiment to convert fluorescence excitation ratios (F500/F440) to pHi values, as described previously (34, 38).

Measurement of Na+/H+ Exchange Activities-- Na+/H+ exchange activities were determined using two previously described methods (34, 36). In the first method (34), Na+/H+ exchange rates (JNa/H, pmol/min/mm) were determined by measuring initial rates of pHi recovery after addition of Na+ to the lumen or bath solutions. The tubules were perfused and bathed initially in HEPES-buffered solution that contained 145 mM Na+, 4 mM K+, 147 mM Cl-, 2.0 mM Ca2+, 1.5 mM Mg2+, 1.0 mM phosphate, 1.0 mM SO42-, 1.0 mM citrate, 2.0 mM lactate, 5.5 mM glucose, and 5 mM HEPES (equilibrated with 100% O2, titrated to pH 7.4). The perfusate also contained furosemide to block Na+-K+-2Cl- cotransport-mediated changes in cell Na+ concentration or volume. After a stable pHi was reached, Na+ was replaced completely with NMDG+ in the lumen and bath solutions. Bilateral Na+ replacement unmasks a background acid loading process that reduces the pHi to 6.5-6.7 (34). Apical Na+/H+ exchange activity was then determined by measurement of the rate of pHi increase after readdition of Na+ to the tubule lumen. H+ flux rates were calculated as (dpHi/dt) × beta i × V, where dpHi/dt is the initial slope of the record of pHi versus time (pH units/min) measured over the first 4 s following an experimental maneuver (34), beta i is the intrinsic intracellular buffering power (mmol/liter·pH unit), and V is cell volume/mm of tubule length (nl/mm). beta i was measured in the presence and absence of NGF as a function of pHi using previously described methods (34). V, determined from inner and outer tubule diameters (12, 24, 34), was 0.30 ± 0.01 nl/mm. NGF had no effect on beta i or V. To determine the pHi dependence of apical membrane Na+/H+ exchange, the Na+-dependent pHi recovery was interrupted at various points along the recovery curve by luminal Na+ removal (NMDG+) plus EIPA, which unmasks background acid loading (34, 39). At the point of interruption, the Na+/H+ exchange rate is calculated as the difference between the net recovery rate and the background acid loading rate (34, 39). This approach permits the Na+/H+ exchange rate to be determined over a range of pHi values, with appropriate corrections for a variable background acid loading rate. Basolateral membrane Na+/H+ exchange rates were determined using similar protocols, i.e. by measuring rates of pHi recovery after readdition of Na+ to only the bath solution. In experiments in which apical or basolateral Na+/H+ exchange activity was measured, EIPA was present on the opposite side of the tubule to eliminate any contribution of the contralateral exchanger to the Na+-induced changes in pHi.

Apical membrane Na+/H+ exchange activity was also assessed using a second method (36). MTALs were perfused and bathed in the control solution used for HCO3- transport experiments, and apical Na+/H+ exchange activity was determined by measuring the initial rate of cell acidification in response to rapid addition of 50 µM EIPA to the tubule lumen. In this analysis, JNa/H = (dpHi/dt) × beta T × V, where dpHi/dt is the initial rate of pHi decrease after lumen EIPA addition, beta T is the sum of beta i and buffering power due to HCO3-/CO2 (computed as 2.3 [HCO3]i) (36), and V is cell volume measured in control solution (0.28 ± 0.02 nl/mm). The basis for this approach is that, prior to EIPA addition, H+ extrusion via the apical Na+/H+ exchanger balances background acid loading to maintain pHi constant (36, 39). When the apical exchanger is inhibited, the initial rate of cell acidification estimates the steady-state rate of apical Na+/H+ exchange that balances background acid loading (36).

Statistical Analysis-- Results are presented as means ± S.E. Differences between means were evaluated using Student's t test for paired or unpaired data, as appropriate. p < 0.05 was considered statistically significant.

    RESULTS

NGF Inhibits HCO3- Absorption-- Previously, we demonstrated that NGF inhibits HCO3- absorption in the MTAL (37). This finding was confirmed in three experiments in the present study (Fig. 1). Addition of 0.7 nM NGF to the bath decreased HCO3- absorption by 30%, from 13.5 ± 0.8 to 9.3 ± 0.6 pmol/min/mm (p < 0.05). The inhibition was observed within 15 min after addition of NGF to the bath and was reversible.


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Fig. 1.   NGF inhibits HCO3- absorption in the MTAL. The absolute rate of HCO3- absorption (JHCO3-) was measured under control conditions and after addition of 0.7 nM NGF to the bath. Data points are average values for single tubules. Lines connect paired measurements made in the same tubule. The p value is for the paired t test. Mean values are given under "Results."

NGF Does Not Affect Apical Na+/H+ Exchange Activity, but Inhibits Basolateral Na+/H+ Exchange Activity-- To identify the mechanism by which NGF inhibits HCO3- absorption, we examined directly the effects of NGF on apical and basolateral membrane Na+/H+ exchange activities. Na+/H+ exchange rates were determined from rates of pHi recovery measured after addition of Na+ to the lumen or bath solutions (see "Experimental Procedures"). The results in Fig. 2A show that NGF had no effect on apical membrane Na+/H+ exchange activity over the pHi range 6.7-7.7. The apical exchanger exhibited a sigmoidal dependence on pHi, as described previously (34). A Hill plot of the combined data gave a Vmax of 68 pmol/min/mm, an apparent pK of 7.26, and a Hill coefficient of 1.8, values in close agreement with those obtained previously for MTALs in isosmotic solutions (34). NGF also had no effect on the steady-state pHi reached after lumen Na+ addition (7.74 ± 0.05 without NGF (n = 6) versus 7.76 ± 0.05 with NGF (n = 4); p = not significant). Thus, we found no evidence for direct coupling of the NGF pathway to regulation of apical membrane Na+/H+ exchange activity.


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Fig. 2.   NGF has no effect on apical membrane Na+/H+ exchange activity, but inhibits basolateral membrane Na+/H+ exchange activity. MTALs were studied in the absence (closed circles) and presence (open circles) of 0.7 nM NGF in the bath solution. Na+/H+ exchange rates (JNa/H) were determined at various intracellular pH values from rates of pHi recovery measured after addition of Na+ to lumen or bath solutions (see "Experimental Procedures"). A, apical membrane Na+/H+ exchange activity is not affected by NGF. Data points are from six tubules without NGF and five tubules with NGF. The line is a least-squares fit to the Hill equation; kinetic parameters are given under "Results." B, basolateral membrane Na+/H+ exchange activity is decreased by NGF. Data points are from five tubules without NGF and five tubules with NGF. Note the different scales for JNa/H in the apical and basolateral plots.

The MTAL also contains a basolateral membrane Na+/H+ exchanger that is active at the resting pHi (36). The results in Fig. 2B show that NGF decreased basolateral Na+/H+ exchange activity by at least 50% at all pHi values studied. For data points grouped over the pHi interval 6.9-7.1, NGF decreased the basolateral Na+/H+ exchange rate from 14.8 ± 0.6 to 6.8 ± 0.7 pmol/min/mm (p < 0.001). NGF also decreased the steady-state pHi reached after addition of Na+ to the bath solution (7.33 ± 0.08 without NGF (n = 4) versus 6.99 ± 0.03 with NGF (n = 5); p < 0.005).2 These results demonstrate that NGF inhibits basolateral membrane Na+/H+ exchange activity in the MTAL. Under basal conditions (absence of NGF), basolateral membrane Na+/H+ exchange rates were approximately one-third of apical membrane Na+/H+ exchange rates at comparable pHi (Fig. 2, A and B).

To confirm that the Na+-dependent pHi recovery inhibited by NGF represents basolateral membrane Na+/H+ exchange, we measured initial rates of pHi recovery after bath Na+ addition in the absence and presence of bath EIPA. The results in Fig. 3 show that EIPA blocked pHi recovery nearly completely both in the absence and presence of NGF. With EIPA in the bath, the recovery rate was reduced to 0.7 ± 0.1 pmol/min/mm (n = 3) in the absence of NGF and to 0.6 ± 0.1 pmol/min/mm (n = 3) in the presence of NGF (90% inhibition). These data are consistent with previous results (36) and demonstrate that Na+/H+ exchange is the only Na+-dependent acid extrusion mechanism operating in the basolateral membrane under the conditions of our experiments.


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Fig. 3.   EIPA inhibits basolateral membrane Na+/H+ exchange activity. Tubules were studied in the absence (A) and presence (B) of 0.7 nM NGF in the bath. Basolateral Na+/H+ exchange rates (JNa/H) were determined from rates of pHi recovery after bath Na+ addition in the absence and presence of 50 µM bath EIPA. Data points are values for single tubules. Data points without EIPA are from Fig. 2. Mean rates are given under "Results." The values below the panels are mean pHi ± S.E. at which JNa/H was measured.

NGF Secondarily Inhibits Apical Membrane Na+/H+ Exchange Activity-- Previously in the MTAL, we demonstrated a functional interaction between the basolateral and apical membrane Na+/H+ exchangers, in which primary inhibition of basolateral Na+/H+ exchange activity resulted secondarily in inhibition of apical Na+/H+ exchange activity and thereby HCO3- absorption (36). If a similar mechanism accounts for the inhibition of HCO3- absorption by NGF, then inhibition of apical Na+/H+ exchange activity may not have been observed in the experiments in Fig. 2A because basolateral Na+/H+ exchange activity was blocked due to the absence of bath Na+, thereby preventing NGF from inhibiting the basolateral exchanger and inducing the interaction that secondarily decreases apical Na+/H+ exchange activity. We therefore examined the effect of NGF on apical Na+/H+ exchange using a second protocol in which the basolateral Na+/H+ exchanger was active. MTALs were perfused and bathed with the control solution used for transepithelial HCO3- transport experiments (146 mM Na+ and 25 mM HCO3-, pH 7.4), and apical membrane Na+/H+ exchange activity was determined by measurement of the initial rate of cell acidification after lumen EIPA addition (see "Experimental Procedures"). A typical experiment is shown in Fig. 4A. Addition of 50 µM EIPA to the tubule lumen caused a rapid decrease in pHi due to inhibition of apical membrane Na+/H+ exchange activity (25, 36). Removal of EIPA caused pHi to recover to its initial value (~7.05). NGF was then added to the bath, and the pHi response to lumen EIPA was repeated. NGF reduced the initial rate of cell acidification (Fig. 4A, dashed lines), indicating a decrease in the steady-state rate of apical membrane Na+/H+ exchange (36). Similar results were obtained when the order of the experimental conditions was reversed. For a total of six experiments (Fig. 4B), apical Na+/H+ exchange activity was decreased by 20 ± 4% in the presence of NGF. Thus, NGF inhibits apical membrane Na+/H+ exchange activity in the MTAL, an effect that likely mediates its action to inhibit HCO3- absorption. These findings, together with the results in Fig. 2, are consistent with the view that NGF inhibits apical membrane Na+/H+ exchange activity secondary to inhibition of basolateral membrane Na+/H+ exchange activity, that is, NGF inhibits apical Na+/H+ exchange activity in the experiments in Fig. 4, but not in the experiments in Fig. 2A, because, in the latter protocol, basolateral membrane Na+/H+ exchange was blocked due to the absence of bath Na+, thereby preventing the interaction between basolateral and apical Na+/H+ exchangers that secondarily decreases apical Na+/H+ exchange activity (see "Discussion").


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Fig. 4.   NGF secondarily inhibits apical membrane Na+/H+ exchange activity. MTALs were perfused and bathed in control solution (containing 146 mM Na+ and 25 mM HCO3-, pH 7.4), and apical membrane Na+/H+ exchange activity was assessed from the initial rate of cell acidification after lumen addition of 50 µM EIPA (see "Experimental Procedures"). A, the tracing shows the response of pHi to lumen EIPA addition, first in the absence and then in the presence of 0.7 nM bath NGF. NGF decreased the initial rate of cell acidification (dashed lines), indicating inhibition of the steady-state rate of apical membrane Na+/H+ exchange. B, effect of NGF on apical membrane Na+/H+ exchange activity (JNa/H) in six experiments similar to that in A. Data points are values for single tubules. Lines and the p value are as described in the legend to Fig. 1.

NGF Inhibits HCO3- Absorption through Inhibition of Basolateral Membrane Na+/H+ Exchange-- Transepithelial transport experiments were carried out to test further the hypothesis that the inhibition of HCO3- absorption by NGF was dependent on its action to inhibit basolateral membrane Na+/H+ exchange. If NGF decreases HCO3- absorption through inhibition of basolateral Na+/H+ exchange, then NGF action should be diminished under conditions in which the basolateral exchanger is inhibited. To test this, we examined the effects of NGF on HCO3- absorption in the presence of bath EIPA and in the absence of bath Na+, two conditions under which basolateral Na+/H+ exchange activity is inhibited (36). The results in Fig. 5A show that, in tubules bathed with 1 µM EIPA, addition of NGF to the bath decreased HCO3- absorption only by 9%, from 10.5 ± 0.4 to 9.5 ± 0.4 pmol/min/mm (n = 3; p < 0.025). When compared with the inhibition by NGF in control experiments (Fig. 1), 1 µM bath EIPA inhibited NGF action by >70%. The effect of NGF on HCO3- absorption in tubules studied in a Na+-free bath is shown in Fig. 5B. In these experiments, the tubule lumen was perfused with control solution (containing 146 mM Na+ and 25 mM HCO3-); the bath solution was identical except that Na+ was replaced completely with NMDG+. Under these conditions, addition of NGF to the bath had no effect on HCO3- absorption (11.5 ± 1.0 pmol/min/mm in Na+-free bath versus 11.4 ± 1.0 pmol/min/mm in Na+-free bath + NGF, n = 4; p = not significant). Thus, the effect of NGF to inhibit HCO3- absorption was virtually eliminated under two different conditions in which basolateral membrane Na+/H+ exchange activity was inhibited.3 These findings indicate that inhibition of basolateral membrane Na+/H+ exchange is essential for NGF-induced inhibition of HCO3- absorption.


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Fig. 5.   Inhibition of HCO3- absorption by NGF is eliminated by conditions that inhibit basolateral membrane Na+/H+ exchange. A, effect of NGF (0.7 nM added to the bath) on HCO3- absorption in the presence of bath EIPA. EIPA (1 µM) was present in the bath throughout the experiments. B, effect of NGF on HCO3- absorption in tubules studied in a Na+-free bath. Na+ in the bath was replaced completely with NMDG+ throughout the experiments; the lumen was perfused with control solution containing 146 mM Na+. JHCO3-, data points, lines, and p values are as described in the legend to Fig. 1. Mean values are given under "Results." NS, not significant.

To examine further the role of basolateral membrane Na+/H+ exchange in regulation by NGF, we tested whether pretreatment with NGF would alter the inhibition of HCO3- absorption by bath EIPA. Under control conditions, addition of 50 µM EIPA to the bath decreased HCO3- absorption by 40%, from 13.2 ± 1.1 to 8.1 ± 1.1 pmol/min/mm (n = 5; p < 0.001) (Fig. 6A). In contrast, in tubules bathed with NGF, addition of 50 µM EIPA to the bath decreased HCO3- absorption only by 16%, from 10.5 ± 0.3 to 8.8 ± 0.3 pmol/min/mm (n = 4; p < 0.005) (Fig. 6B). Thus, pretreatment with NGF markedly attenuates the inhibition of HCO3- absorption by bath EIPA. These results suggest that NGF and bath EIPA inhibit HCO3- absorption via a common mechanism, namely inhibition of basolateral membrane Na+/H+ exchange activity. When taken together, our results support the conclusion that the inhibition of HCO3- absorption by NGF is mediated through inhibition of basolateral membrane Na+/H+ exchange.


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Fig. 6.   NGF blocks inhibition of HCO3- absorption by bath EIPA. The effect of adding 50 µM EIPA to the bath on HCO3- absorption was examined in the absence (A) and presence (B) of NGF. In B, 0.7 nM NGF was present in the bath throughout the experiments. JHCO3-, data points, lines, and p values are as described in the legend to Fig. 1. Mean values are given under "Results."


    DISCUSSION

In the MTAL of the rat, both apical and basolateral membrane Na+/H+ exchangers play a role in determining the rate of transepithelial HCO3- absorption (25, 31, 36). Recently, we demonstrated that NGF inhibits HCO3- absorption in the MTAL (37). The objective of the present study was to investigate the role of Na+/H+ exchange in this inhibitory response. We found that NGF inhibits basolateral membrane Na+/H+ exchange activity in the MTAL and that this inhibition mediates the NGF-induced inhibition of transepithelial HCO3- absorption. In contrast, apical membrane Na+/H+ exchange activity is not affected directly by NGF, but is inhibited secondary to the inhibition of basolateral membrane Na+/H+ exchange activity. To our knowledge, inhibition of Na+/H+ exchange activity by peptide growth factors has not previously been reported. Thus, our studies identify a novel mechanism by which growth factors may regulate HCO3- absorption in renal tubules, namely inhibition of basolateral membrane Na+/H+ exchange activity.

NGF Inhibits Basolateral Membrane Na+/H+ Exchange Activity in the MTAL-- Stimulation of Na+/H+ exchange by growth factors has been a consistent, virtually universal finding (1, 3, 4). An increase in Na+/H+ exchange activity is believed to be important early in the mitogenic response to stabilize pHi within a range permissive for cell growth (1, 6, 9, 40). In contrast, we found that NGF inhibits basolateral membrane Na+/H+ exchange activity in the MTAL. This inhibition was rapid and was observed under conditions in which the activity of the basolateral exchanger was studied independently of effects of NGF on other transporters, which suggests that the NGF signaling pathway is coupled directly to inhibition of the basolateral exchanger. In addition, the physiological relevance of this inhibition was established by the demonstration that it is necessary for NGF-induced inhibition of HCO3- absorption. Immunocytochemical studies using isoform-specific antibodies (16, 17), studies of NHE transcripts (15, 41), and functional studies of amiloride sensitivity (36) have indicated that basolateral membrane Na+/H+ exchange activity in the MTAL is mediated by NHE1. NHE1 is expressed in virtually all cell types and tissues and is the exchanger isoform whose activation is closely associated with the growth response (1, 4, 6). Our data suggest that NHE1 is inhibited by NGF in the MTAL; however, we cannot rule out the possibility that NGF may inhibit an as yet undetermined exchanger isoform that may co-localize with NHE1 in the basolateral membrane. The inhibition is not the result of a general cellular action of NGF because NGF has been shown to stimulate Na+/H+ exchange activity in other systems (42, 43). At this point, we do not know whether the inhibition of basolateral membrane Na+/H+ exchange activity that we observed represents a general response of the MTAL cells to growth factor stimulation or a specific response to stimulation by NGF.

Although the mechanism by which NGF inhibits basolateral membrane Na+/H+ exchange activity was not identified in our experiments, some insights can be obtained from previous studies of the signaling pathways involved in NGF regulation of HCO3- absorption. The inhibition of HCO3- absorption by NGF was additive to inhibition by cAMP and was unaffected by inhibitors of protein kinase C (37). Thus, the inhibition of basolateral membrane Na+/H+ exchange activity by NGF is unlikely to involve cAMP- or protein kinase C-dependent signaling pathways. In contrast, the inhibition of HCO3- absorption by NGF was eliminated by pretreatment with the tyrosine kinase inhibitor genistein (37). This suggests that tyrosine kinase pathways are involved in the NGF inhibition of Na+/H+ exchange activity. The signal transduction mechanisms by which growth factors activate Na+/H+ exchange (NHE1) in other systems are complex and may include calcium/calmodulin, mitogen-activated protein kinases, phosphatidylinositol 3-kinase, small molecular weight GTPases, and associated regulatory proteins (4, 5, 44-48). It remains to be determined whether the inhibition of Na+/H+ exchange activity by NGF in the MTAL is mediated through these same pathways or through signaling mechanisms distinct from those that mediate exchanger activation.

Regulation of the epithelial exchanger isoforms NHE2 and NHE3 by growth factors has been studied less extensively than regulation of NHE1. However, the available evidence indicates that, like NHE1, NHE2 and NHE3 are activated by growth factors (2, 4) and can support cell proliferation when expressed in NHE-deficient cell lines (7, 8). Furthermore, several growth factors have been shown to stimulate NHE3 activity in the brush-border membrane of intestinal epithelial cells (45). NHE3 is the apical membrane isoform responsible for H+ secretion and HCO3- absorption in the rat MTAL (21, 22, 24, 27, 35, 36). However, at least two observations in the present study indicate that NHE3 activity is not regulated directly by NGF: 1) NGF had no effect on lumen Na+-dependent pHi recovery (Fig. 2A); and 2) NGF had no effect on HCO3- absorption in the absence of bath Na+ or in the presence bath EIPA (Fig. 5). Because apical membrane Na+/H+ exchange mediates HCO3- absorption in the latter conditions (36), any direct coupling of the NGF pathway to stimulation (or inhibition) of the apical exchanger should have been evident as a change in the HCO3- absorption rate. Thus, the stimulation of NHE3 by growth factors in other systems was not observed with NGF in the MTAL, suggesting that growth factor regulation of NHE3 activity also may be cell type-specific. As discussed below, we did find that apical membrane Na+/H+ exchange activity was inhibited by NGF; however, this inhibition occurs secondary to inhibition of basolateral membrane Na+/H+ exchange activity.

Inhibition of Basolateral Membrane Na+/H+ Exchange Activity Is Necessary for NGF-induced Inhibition of HCO3- Absorption-- Several observations support the conclusion that NGF inhibits MTAL HCO3- absorption through inhibition of basolateral membrane Na+/H+ exchange. 1) NGF inhibits basolateral Na+/H+ exchange activity at physiological pHi values. 2) The inhibition of HCO3- absorption by NGF is nearly abolished in tubules bathed with EIPA to decrease basolateral Na+/H+ exchange activity. 3) The inhibition of HCO3- absorption requires Na+ in the bath solution. 4) Pretreatment with NGF blocks inhibition of HCO3- absorption by bath EIPA, an effect mediated through inhibition of basolateral Na+/H+ exchange activity (36). Taken together, these findings indicate that inhibition of basolateral membrane Na+/H+ exchange activity is an essential component in NGF-induced inhibition of HCO3- absorption. Furthermore, they establish that the physiological function of basolateral Na+/H+ exchange in renal tubules is not limited to housekeeping functions such as regulation of pHi and cell volume, but also includes a more specialized role in the regulation of acid secretion and transepithelial NaHCO3 absorption.

Although basolateral membrane Na+/H+ exchange is present in most segments of the nephron, its role in transepithelial HCO3- absorption is not understood. In renal tubules, HCO3- absorption is achieved by secretion of protons across the apical membrane (which titrate lumen HCO3-) and transport of HCO3- across the basolateral membrane (49). It is widely assumed that basolateral membrane Na+/H+ exchange decreases the efficiency of HCO3- absorption by decreasing net basolateral base efflux and/or by increasing cell sodium concentration or pHi and secondarily inhibiting apical proton secretion (2, 13, 14, 50). In contrast to this view, our results demonstrate that inhibiting basolateral membrane Na+/H+ exchange activity with either NGF (present study) or EIPA (36) markedly decreases HCO3- absorption in the MTAL. These studies provide the first identified role for basolateral membrane Na+/H+ exchange in the physiological regulation of renal tubule HCO3- absorption. Furthermore, they demonstrate that, contrary to the generally accepted view that it opposes HCO3- absorption (2, 13, 14), the presence of basolateral membrane Na+/H+ exchange enhances HCO3- absorption in the MTAL.

The mechanism by which the NGF-induced inhibition of basolateral membrane Na+/H+ exchange results in inhibition of HCO3- absorption likely involves interaction between the basolateral and apical membrane Na+/H+ exchangers. In the MTAL, changes in the rate of HCO3- absorption are mediated through changes in apical membrane Na+/H+ exchange activity (24, 25, 31, 34, 35). In previous studies in which EIPA was used to inhibit basolateral Na+/H+ exchange activity, we demonstrated that the basolateral and apical membrane Na+/H+ exchangers are functionally coupled, wherein primary inhibition of basolateral Na+/H+ exchange activity results secondarily in inhibition of apical Na+/H+ exchange activity (36). The evidence presented in this report that 1) NGF primarily inhibits basolateral membrane Na+/H+ exchange activity, 2) inhibition of basolateral Na+/H+ exchange activity is required for inhibition of HCO3- absorption, and 3) apical membrane Na+/H+ exchange activity is inhibited by NGF only under conditions in which basolateral Na+/H+ exchange activity is inhibited supports the view that NGF inhibits HCO3- absorption via this coupling mechanism. Thus, our data are consistent with NGF inhibiting HCO3- absorption in the MTAL through the following events. 1) Interaction of NGF with its membrane receptor activates signaling mechanisms coupled to the inhibition of basolateral membrane Na+/H+ exchange. 2) Inhibition of basolateral Na+/H+ exchange activity results secondarily in inhibition of apical Na+/H+ exchange activity due to coupling between the exchangers. 3) The decrease in apical membrane Na+/H+ exchange activity diminishes luminal acid secretion, thereby decreasing net HCO3- absorption (25, 31, 36). This mechanism can account for our observations that apical membrane Na+/H+ exchange activity is inhibited by NGF when studied by lumen EIPA addition in physiological (146 mM Na+) solutions (Fig. 4), but not when studied by bilateral Na+ removal and lumen Na+ readdition (Fig. 2A): in the latter protocol, basolateral membrane Na+/H+ exchange is blocked due to the absence of bath Na+, which prevents NGF from inhibiting the basolateral exchanger and inducing the basolateral exchanger-dependent interaction that secondarily decreases apical Na+/H+ exchange activity. Important goals for future studies will be to identify the signaling pathway that couples NGF to the inhibition of basolateral Na+/H+ exchange activity and the mechanism of interaction between the basolateral and apical membrane Na+/H+ exchangers. Our results indicate, however, that communication between the exchangers is activated in the MTAL by growth factors and thus represents a physiologically relevant pathway of transport regulation.

NGF and its receptors are expressed in the kidney (30, 51-54), but their roles in the regulation of renal function remain to be determined. Our results demonstrating that NGF inhibits HCO3- absorption in the MTAL through inhibition of basolateral membrane Na+/H+ exchange activity establish directly that NGF can influence the function of renal tubules. The change in HCO3- absorption induced by NGF (30-40%) is similar to that observed with other regulatory factors, such as chronic metabolic acidosis and alkalosis, dietary sodium intake, and angiotensin II (31, 55). These findings suggest that NGF may be involved in the control of urinary acidification, as discussed previously (37). In addition, other stimuli that regulate HCO3- absorption in the MTAL, including angiotensin II, vasopressin, changes in osmolality, and aldosterone (24, 31, 32, 55), have been shown to alter basolateral membrane Na+/H+ exchange activity in renal cells (12, 33, 56-58). Thus, an important goal for future investigations will be to determine whether basolateral membrane Na+/H+ exchange may be an effector that mediates the regulation of HCO3- absorption by a wide variety of physiological stimuli.

    ACKNOWLEDGEMENT

We thank L. Reuss for critical reading of the manuscript.

    FOOTNOTES

* This work was supported by NIDDK Grant DK-38217 from the National Institutes of Health and a research fellowship from the National Kidney Foundation (to B. A. W.), with funds contributed in part by the National Kidney Foundation of Southeast Texas.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.

Dagger To whom correspondence should be addressed: 4.200 John Sealy Annex 0562, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0562. Tel.: 409-772-2472; Fax: 409-772-5451.

2 In our analysis, the pHi range over which Na+/H+ exchange activities are measured is bracketed on the low end by the pHi reached after bilateral Na+ replacement and on the high end by the pHi achieved after lumen or bath Na+ readdition (34). Because the basolateral Na+/H+ exchange rate is less than the apical Na+/H+ exchange rate, the steady-state pHi reached after bath Na+ addition is less than that reached after lumen Na+ addition. This limits the pHi range over which the basolateral exchange rate is determined, which precludes kinetic analysis of the mechanism by which NGF inhibits exchange activity. Our data show clearly, however, that NGF inhibits basolateral membrane Na+/H+ exchange activity at the resting pHi normally measured in MTAL segments (7.0-7.1) (25, 36).

3 The small residual inhibition of HCO3- absorption by NGF in the presence of bath EIPA likely is due to incomplete inhibition of the basolateral Na+/H+ exchanger by 1 µM EIPA at physiological Na+ concentrations (36) rather than to an exchanger-independent regulatory mechanism. 1 µM EIPA was used in this experiment because this concentration has a negligible effect on pHi (36); thus, the lack of effect of NGF on HCO3- absorption could not be attributed to an EIPA-induced cell acidification.

    ABBREVIATIONS

The abbreviations used are: NHE, Na+/H+ exchanger; NGF, nerve growth factor; MTAL, medullary thick ascending limb; EIPA, ethylisopropyl amiloride; NMDG+, N-methyl-D-glucammonium.

    REFERENCES
TOP
ABSTRACT
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