INTRODUCTION

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UNDER NORMAL CONDITIONS, cells of the mammalian renal medullary thick ascending limb (MTAL) of the loop of Henle are exposed to a hypertonic environment. To survive, these cells must have mechanisms that prevent cell shrinkage and/or restore cell volume in a hypertonic melieu (regulatory volume increase, RVI). Consistent with this view, Hebert and Sun (11-13) reported that an arginine vasopressin (AVP)-dependent RVI mechanism was present in the in vitro perfused mouse MTAL and proposed that it was mediated by parallel, basolateral Na⁺/H⁺ and Cl/ (AE) exchangers. More recently, using 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) to monitor intracellular pH (pH_i), Sun et al. (27) showed that an Na⁺/H⁺ exchange was indeed present in the basolateral membrane of the in vitro perfused mouse MTAL. In addition, it was activated by hypertonicity (26) and AVP (27), consistent with its proposed role in the AVP-dependent, RVI response in the mouse MTAL. This basolateral Na⁺/H⁺ exchange is most likely encoded by Na⁺/H⁺ exchanger isoform 1 (5).

Less consistent data have been obtained regarding the presence of a functioning basolateral Cl/ exchanger in MTAL. On the one hand, recent molecular biological studies showed that MTAL cells contain the message of an AE (2, 6). In addition, preliminary studies showed that this exchanger was localized to the basolateral membrane by immunohistochemical techniques (3). Nevertheless, functional studies in MTAL suspensions under isotonic conditions and in the absence of AVP failed to detect any activity of a Cl/ exchange (10, 19). However, due to the relative insensitivity of the tubule suspension technique, it is possible that a low level of activity of a basolateral Cl/ exchange might have been missed. Therefore, in the present study, we reexamined whether a functional Cl/ exchange was present in the basolateral membrane of mouse MTAL in the absence of AVP and under isotonic conditions using the more sensitive, isolated perfused tubule technique. Currently, three genes (AE1, AE2, and AE3) encoding Cl/ exchangers have been identified in mammals (1). In this study, we also examined whether mRNAs from these genes are expressed in the mouse MTAL.

	MATERIALS AND METHODS

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In vitro microperfusion. The basic techniques for in vitro microperfusion of mouse MTAL tubules have been described previously (18, 19, 27). In brief, MTAL segments were dissected from the inner stripe of outer medulla of 20- to 30-day-old CD1 mice and perfused at rates of 15-20 nl/min. The peritubular bath flowed at a rate of 15-25 ml/min, which is sufficient to exchange the bath in ~2 s, and was maintained at 37°C. Control solutions contained (in mM): 140 NaCl, 5.0 KCl, 1.0 CaCl₂, 1.2 MgCl₂, and 3.0 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) for HEPES-buffered solutions and 115 NaCl, 5.0 KCl, 1.0 CaCl₂, 1.2 MgCl₂, and 25 NaHCO₃ for -buffered solutions. Solutions were adjusted to an osmolality of 290 mosoml/kgH₂O and a pH of 7.4 after equilibrating with 100% O₂ for HEPES-buffered solutions or 95% O₂-5% CO₂ for -buffered solutions. Na⁺-free solutions were made by replacing Na⁺ with N-methyl-D-glucamine, and Cl-free solutions were made by replacing Cl with gluconate. Gluconate-containing solutions had an increased total Ca²⁺ (5 mM) to compensate for the complexing of Ca²⁺ by gluconate. Each tubule perfusion experiment was performed in a tubule from one mouse.

pH_i measurement. The techniques for quantitative fluorescence measurement of pH_i using the pH-sensitive dye BCECF (Molecular Probes, Eugene, OR) in mouse MTAL have been described in detail in our previous work (18, 19, 27). Briefly, MTAL tubules were loaded with BCECF by transient exposure (10 min) to the acetoxymethyl ester of BCECF at 37°C. Fluorescence was alternately measured from the output of a photomultiplier tube at excitation wavelengths of 495 and 440 nm (emission wavelength 530 nm) using a dual grating fluorometer (Deltascan; Photon Technology International, South Brunswick, NJ) connected to a inverted microscope (Diaphot 300; Nikon). Background fluorescence (<1% of total) was subtracted from fluorescence intensity at each excitation wavelength to obtain intensities of intracellular fluorescence. The 495 nm-to-440 nm ratio was used as an indicator of pH and was calibrated using high K⁺-nigericin standards (18, 19, 27).

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	RESULTS

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Effect of peritubular Cl removal on pH_i in the presence and absence of CO₂/. We have previously demonstrated two types of Na⁺-dependent acid/base transporters in the mouse MTAL (27): 1) Na⁺/H⁺ exchange activity present in both apical and basolateral membranes and 2) an Na⁺-()_n cotransporter that is most likely located on the basolateral membrane (18, 20). Therefore, to avoid the contribution of these Na⁺-dependent acid/base transporters to cell pH_i changes, the effect of peritubular Cl removal on pH_i was assessed in the absence of Na⁺ in all experiments in the present study. Figure 1 shows a representative experiment designed to assess the effect of removal of peritubular Cl on pH_i in the presence of external CO₂/. Removal of apical and basolateral Na⁺ at point a resulted in prompt cell acidification (pH_i changed from 7.20 ± 0.04 to 6.80 ± 0.09, n = 5). This cell acidification was likely due to decreased H⁺ exit via the Na⁺/H⁺ exchanger as well as increased exit via the Na⁺-()_n cotransporter. After pH_i reached a new steady state, removal of basolateral Cl (point b) caused pH_i to rise to 6.95 ± 0.11 (pH = 0.15). This peritubular Cl removal-induced cell alkalization was reversed when peritubular Cl was restored at point c.

View larger version (9K): [in this window] [in a new window]   Fig. 1.   Effect of removal of peritubular Cl on pHi in the presence of external CO2/. Removal of apical (Ap) and peritubular (Bl) Na+ at point a resulted in cell acidification. After pHi reached a steady state, removal of peritubular Cl at point b caused cell alkalization, which was reversed on restoration of peritubular Cl at point c.

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View larger version (6K): [in this window] [in a new window]   Fig. 2.   Effect of removal of peritubular Cl on pHi in the presence (A) and absence (B) of external CO2/ and in the presence of external CO2/ and peritubular DIDS (C). Removal of peritubular Cl (arrow) induced cell alkalization, which was larger in the presence than absence of external CO2/, and was abolished by peritubular DIDS (200 µM).

View this table: [in this window] [in a new window]   Table 1.   Effect of removal of peritubular Cl on the degree and rate of cell alkalinization

Effect of peritubular Cl addition on pH_i. If Na⁺-independent, Cl-coupled transport is present in the basolateral membrane, then addition of peritubular Cl to MTAL tubules depleted of intracellular Cl should induce exit and cell acidification. The results of such an experiment are shown in Fig. 3. To deplete MTAL of intracellular Cl, tubules were initially perfused and bathed in Na⁺- and Cl-free, -buffered solutions for 1 h. As predicted, addition of Cl to the peritubular solution (arrow) in the continued absence of external Na⁺ led to rapid cell acidification (pH = 0.24 ± 0.04, n = 3).

View larger version (9K): [in this window] [in a new window]   Fig. 3.   Effect of addition of Cl to the peritubular solution on pHi in Cl-depleted medullary thick ascending limb (MTAL) in the presence of external CO2/. MTAL tubules were equilibrated in Na+- and Cl-free apical and peritubular solutions for 1 h. Addition of Cl to the peritubular solution (arrow) resulted in cell acidification.

Effect of chemical voltage clamp on cell alkalization induced by peritubular Cl removal. In addition to the presence of a Cl/ exchanger, an alternative explanation for our findings is that the Na⁺-independent, Cl-coupled transport that we observed was mediated indirectly, via changes in transmembrane voltage. Of note, a Cl conductive pathway has been described in the basolateral membrane of mouse MTAL (23); therefore, removal of external Cl might be associated with depolarization of the cell. If so, then cell alkalization could result either from inhibition of exit via a basolateral electrogenic Na⁺-()_n cotransporter or reversal of this transporter or by stimulation of apical H⁺-ATPase (7). Of note, cell alkalinization from entry due to reversal of Na⁺-()_n cotransporter is unlikely to have occurred due to the absence of external Na⁺ in our experiment. Nevertheless, to avoid the confounding effect of alterations in transmembrane voltage on pH_i during peritubular Cl removal, we voltage clamped the membrane with the K⁺ ionophore valinomycin (10 µM) and K⁺ (120 mM). This procedure has been routinely used to clamp membrane voltage in renal tubular cells, including those of the thick ascending limb (20). Addition of valinomycin and high K⁺ to both peritubular and apical solutions caused significant cell alkalization (from 7.25 ± 0.03 to 7.46 ± 0.04, n = 4). The mechanism of this voltage clamp-induced increase in cell pH is unclear but has also been observed in the cortical thick ascending limb (20). In any event and shown in Fig. 4, at the new steady-state pH_i, removal of peritubular Cl at point a led to cell alkalization even in the presence of the voltage clamp. Restoration of peritubular Cl at point b reversed the cell alkalization. As shown in Table 1, imposition of the voltage clamp increased both the magnitude (pH: control 0.15 ± 0.02; voltage clamp 0.37 ± 0.03; P < 0.05) and rate of cell alkalization (J_H⁺: control 4.65 ± 0.22 pmol · min¹ · mm¹; voltage clamp 19.40 ± 1.96 pmol · min¹ · mm¹; P < 0.05; Table 1). These findings are consistent with the presence of a specific Cl/ exchanger and opposite to the predicted response if cell alkalization were the indirect result of a decrease in cell potential after external Cl removal.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Effect of voltage clamp (120 mM K+ and 10 µM valinomycin) on peritubular Cl removal on pHi in the presence of external CO2/. Voltage clamp resulted in cell alkalization. Removal of peritubular Cl at point a resulted in cell alkalization, which was reversed on restoration of peritubular Cl at point b. Degree and rate of cell alkalization induced by removal of Cl were larger in the presence than absence of voltage clamp.

Effects of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid on cell alkalization induced by peritubular Cl removal. Stilbenes such as 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) have been shown to inhibit Na⁺-independent Cl/ exchangers in several renal epithelial cells. Therefore, we assessed the effect of peritubular Cl removal on pH_i in the presence of CO₂/ and peritubular DIDS (200 µM) and in the absence of Na⁺ in both the apical and peritubular solutions. Addition of DIDS to the peritubular solution induced a small, but significant, cell acidification (data not shown). As shown in Fig. 2C, 10 min incubation with peritubular DIDS completely prevented cell alkalization after peritubular Cl removal (n = 3). This response contrasted sharply with the prominent cell alkalization induced by peritubular Cl removal in the absence of DIDS (Fig. 2A).

Detection of AE mRNAs in the mouse MTAL. First, mouse kidney was examined by RT-PCR for the expression of mRNAs coding for products of the three known Cl/ exchanger genes, AE1, AE2, and AE3 (positive control). As shown in Fig. 5A, amplification products of predicted sizes for all three AE isoforms were detected in mouse kidney. No PCR products were obtained when the reverse transcriptase was omitted from the RT reaction (data not shown). The identity of the PCR products as specific for each AE isoform was assessed by restriction analysis of the PCR products using enzymes chosen based on the published sequence of each mouse AE isoform. As shown in Fig. 5B, PCR products from the mouse kidney all gave expected restriction fragments and therefore arise from the designated AE mRNAs. Second, the expression of AE mRNAs was examined by RT-PCR in microdissected mouse MTAL. As shown in Fig. 6A, amplification products of the predicted size were detected for AE2 and AE3, but not AE1, in the mouse MTAL. No PCR products were obtained when the reverse transcriptase was omitted from the RT reaction. The identities of AE2 and AE3 were confirmed by restriction analysis of the PCR products, which gave bands of expected size (Fig. 6B). These data indicate that two Cl/ exchanger (AE2 and AE3) mRNAs are expressed in the mouse MTAL.

View larger version (43K): [in this window] [in a new window]   Fig. 5.   Amplification and restriction analysis of Cl/ exchanger (AE) isoform reverse transcription (RT)-polymerase chain reaction (PCR) products from mouse whole kidney homogenate. Each reaction was performed from 1 µg total RNA. PCR products with (B) and without (A) restriction enzyme digestion were electrophoresed on 2% agarose gel and stained with ethidium bromide. Sizes of PCR products of AE1, AE2, and AE3 are indicated at left in A. Restriction enzymes and expected fragment sizes of PCR products are indicated at bottom in B. Molecular standards were from 100-bp ladder from GIBCO-BRL. See text for abbreviations.

View larger version (41K): [in this window] [in a new window]   Fig. 6.   Amplification and restriction analysis of AE isoform RT-PCR products from microdissected mouse MTAL tubules. PCR products with (B) and without (A) restriction enzyme digestion were electrophoresed on 2% agarose gel and stained with ethidium bromide. Results are shown from experiments performed in the presence (+) and absence () of RT. Molecular standards were from 100-bp ladder from GIBCO-BRL. See text for abbreviations.

	DISCUSSION

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In this study, we demonstrate basolateral, Cl-coupled transport in the in vitro perfused mouse MTAL tubule that is Na⁺ independent and DIDS inhibitable (Figs. 1-3). In addition, basolateral transport persisted even when the membrane voltage was chemically clamped with high K⁺ and valinomycin (Fig. 4). These data are consistent with the presence of an Na⁺-independent, Cl/ exchange in the basolateral membrane of the mouse MTAL.

As discussed above, both the mRNA and protein for a Cl/ exchanger are present in MTAL (2, 3, 6). Nevertheless and in contrast to our findings, previous studies performed under isotonic conditions and in the absence of AVP failed to detect significant basolateral Cl-dependent, efflux in mouse (18) and rat (10) MTAL tubule suspensions. There are several potential explanations for these apparently discrepant results. First, it is likely that, under isotonic conditions, the activity of Cl/ exchange that we identified is low, making it difficult to detect with the less sensitive tubule suspension technique. Furthermore, in the suspension studies, MTAL cells were first exposed to media containing /CO₂ and then were abruptly transferred to CO₂/-free media. This procedure results in rapid cell alkalization (mediated by CO₂ exit), followed by a gradual cell acidification (a result of exit). In these studies, the finding that the rate of cell acidification ( exit) was not decreased by reduction or removal of extracellular Cl led the authors to conclude that a basolateral Cl/ exchange was not present. However, it has been shown that the major exit pathways in MTAL are 1) an Na⁺-()_n cotransporter in the mouse MTAL (18) and 2) a K⁺- cotransporter in the rat MTAL (22). Thus the predominant mechanism of cell acidification in CO₂/-free media is not Cl/ exchange, and, therefore, a small decrease in the rate of cell acidification after Cl removal could have been missed. In contrast and in the present study, the mouse Na⁺-()_n cotransporter was inhibited because both apical Na⁺ entry (via the Na⁺-K⁺-2Cl cotransporter) and entry (via the Na⁺/H⁺ exchanger) were inhibited by the removal of extracellular Na⁺. Therefore, Cl/ exchange was the only remaining mechanism for movement, and it was readily detected.

As shown in Fig. 4, the extent and rate of cell alkalization induced by peritubular Cl removal were greater in the presence than in the absence of the voltage clamp. Although the mechanism of this increase was not investigated in the current study, studies in other cell types have demonstrated that the activities of AE2 and AE3, two AE isoforms expressed in the mouse MTAL (Figs. 5 and 6 and see below), are sensitive to pH_i (stimulation by alkaline pH_i and inhibition by acidic pH_i; see Refs. 14, 17, 21). Because pH_i was higher (pH_i = 7.46 vs. 6.80, clamp vs. no clamp) under the conditions of the voltage clamp, the greater rate of cell alkalization in clamped tubules is consistent with increased activity of the Cl/ exchanger due to the initial, more alkaline pH_i.

Recent molecular cloning studies have shown that AE in mammalian tissues are encoded by a family that includes at least three genes, AE1, AE2, and AE3 (1). In this study, we showed that AE2 and AE3, but not AE1, mRNAs were expressed in the mouse MTAL (Figs. 5 and 6). The absence of AE1 mRNA in the MTAL is consistent with previous immunohistochemical studies showing that AE1 was present only in the basolateral membrane of intercalated cells of cortical collecting duct; no MTAL staining was demonstrated (8). Although both AE2 and AE3 mRNAs are both expressed in the mouse MTAL, basolateral Cl/ exchanger in this tubule segment is likely encoded mainly, if not exclusively, by AE2. In a preliminary study, Alper et al. (3) found that AE2 protein was abundantly distributed in the basolateral membrane of MTAL. In contrast, there was minimal, if any, AE3 present in MTAL, and it was confined to the intracellular compartment (4). It has been shown that recombinant AE2 could be activated by hypertonicity when expressed in Xenopus oocytes (15). In addition, this heterologous AE2 expression conferred a hypertonic RVI response on Xenopus oocytes that lack intrinsic volume regulatory mechanisms (16). In this, the presence of AE2 in the basolateral membrane of MTAL would be consistent with the participation of a basolateral Cl/ exchanger in the hypertonic RVI response (12).

In a manner analogous to MTAL, recent studies indicate that AE2 is expressed by inner medullary collecting duct (IMCD) cells where it also localizes to the basolateral membrane (3). Consistently, Star (24) demonstrated that Na⁺-independent, Cl/ exchange is present in the basolateral membrane of the in vitro perfused rat IMCD. Like MTAL, IMCD cells exist in a hypertonic environment in vivo. In addition, Sun and Hebert (25) observed an RVI response in isolated, perfused IMCD tubules exposed to external hypertonicity. In fact, the hypertonic RVI responses in MTAL and IMCD have similar characteristics, including 1) a requirement for AVP and 2) operation of parallel Na⁺/H⁺ and Cl/ exchangers located in the basolateral membrane. Taken together, these results suggest a general role for basolateral AE2 as a mediator of the hypertonic RVI response in renal medullary epithelial cells.

In summary, the present study demonstrates that a potentially functional, Na⁺-independent, Cl/ exchanger is present in the basolateral membrane of mouse MTAL under isotonic conditions and in the absence of AVP. In the mouse MTAL, this exchanger is most likely encoded by the AE2 gene. Under isotonic conditions in the intact kidney, activity of this transporter may be low or absent. We propose, however, that it is activated by hypertonicity and/or AVP when, in conjunction with a basolateral Na⁺/H⁺ exchanger, it facilitates salt entry during the AVP-dependent, hypertonic RVI response. Further studies are required to determine the precise effects of hypertonicity and AVP on this basolateral Cl/ exchanger and to fully characterize its role in ion transport and cell volume regulation in this nephron segment.