Activation of the apical Na /H exchanger NHE3 by formate: a basis of enhance - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-22 08:12 |只看该作者 |倒序浏览 |打印

作者：Snezana Petrovic,, Sharon Barone, Alan M. Weinstein, and Manoocher Soleimani,作者单位：1 Department of Medicine, University of Cincinnati, Cincinnati 45267; 2 Veterans Affairs Medical Center, Cincinnati, Ohio 45220; and 3 Department of Physiology, Weill Medical College of Cornell University, New York, New York 10021

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      【摘要】8 u2 X- ?; [5 G- H3 J4 F+ n
   Formate stimulates sodium chloride and fluid reabsorption in kidney proximal tubule; however, the exact cellular mechanism of this effect remains unknown. We hypothesized that the primary target of formate is the apical Na /H exchanger. Here, we demonstrate that formate directly enhances the apical Na /H exchanger (NHE3) activity in mouse kidney proximal tubule. In the absence of CO 2 /HCO 3 -, addition of formate (500 µM) to the bath and lumen of microperfused mouse kidney proximal tubule caused significant intracellular alkalinization, with intracellular pH (pH i ) increasing from baseline levels 7.17 ± 0.01 to 7.55 ± 0.01 ( P < 0.001, n = 14), with a pH of 0.38 ± 0.02. Removal of luminal chloride did not block cell pH alkalinization by formate (baseline pH of 7.26 ± 0.01 to 7.53 ± 0.01 with formate, P < 0.001, n = 10), indicating that the apical Cl - /OH - exchanger was not the primary mediator of the effect of formate on cell pH. However, removal of sodium from the lumen or addition of EIPA completely prevented cell pH alkalinization. Addition of formate to the lumen and bath in the outer medullary collecting duct, which does not express any apical Na /H exchanger, did not cause any cell pH alkalinization. At lower concentrations (50 µM), formate caused significant pH i alkalinization in proximal tubule cells, with pH i increasing from baseline levels 7.15 ± 0.02 to 7.36 ± 0.02 ( P < 0.02, n = 11). Acetate, at 50 µM, had no effect on pH i. Formates effect was observed both in the absence and presence of CO 2 /HCO 3 - in the media. We conclude that formate stimulates the apical Na /H exchanger NHE3 in the kidney proximal tubule. We propose that formate stimulation of chloride reabsorption in the proximal tubule is indirect and is secondary to the activation of apical Na /H exchanger NHE3, which then leads to the stimulation of the apical chloride/base exchanger. 0 i* s, E- d* K$ U* K2 |( k7 P, V( K
      【关键词】 SLCA chloride absorption sodium absorption anion exchange9 M: W# w* |$ @( R
   REABSORPTION OF NACL BY THE proximal tubule ( 3, 5, 44 ) is critical to maintaining extracellular fluid volume. Sodium reabsorption is primarily via Na /H exchange (NHE3), whereas chloride reabsorption is both paracellular (across the tight junction) and transcellular (via anion exchange). The luminal cell membrane anion (Cl - /base) exchanger works in parallel with the Na /H exchanger and is responsible for the bulk of Na and Cl - reabsorption in the proximal tubule ( 2, 3, 5, 44 ). Different isoforms of anion exchangers have been identified in the kidney proximal tubule. These include chloride/formate ( 5 ), chloride/oxalate ( 5 ), Cl - /HCO 3 -, and Cl - /OH - exchangers ( 41, 44 ).0 H6 n6 U7 I( x) ]8 X

In the proximal tubule of the rabbit, rat, and mouse kidney, the addition of low concentrations of formate to the luminal and peritubular solutions increased the rate of NaCl absorption ( 1, 2 ). The formate-stimulated NaCl absorption was electroneutral and inhibited in the presence of DIDS (1-3, 5, 39, 44). These results were interpreted to indicate the presence of a chloride/formate exchanger in apical membranes of the proximal tubule ( 5 ). The presence of a Cl - /formate exchanger was demonstrated in luminal membrane vesicles from mammalian kidneys ( 4, 5 ). For intracellular formate to be physiologically relevant in Cl - reabsorption, it must be available in concentrations that can be transported via the apical anion exchanger. It has been proposed that following secretion into the lumen, formate can be converted to formic acid by combining with luminal H that is secreted via apical Na /H exchange ( 5, 44 ). The resultant formic acid diffuses back which, in turn, can provide the proximal tubule cell with a Cl - /base exchange mechanism that, in essence, is driven by recycling of formate across the luminal membrane ( 15, 16 ). Studies evaluating this issue in the kidney proximal tubule, however, found that luminal membrane formic acid permeability is too low to support this mechanism ( 20, 27 ). An alternative mechanism has been proposed which suggests that formate is recycled back by exchanging with cellular OH - ( 5 ).
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Recent molecular studies identified a large, highly conserved family of membrane proteins (designated as SLC26A) many of which have been shown to transport anions ( 8, 11 - 13, 22, 33, 35, 40 ). SLC26A6 (also called CFEX or PAT1), which is a member of this family ( 22 ), was recently shown to be located on the apical membrane of the mouse and rat kidney proximal tubule ( 19 ). Expression studies in oocytes showed that SLC26A6 mediates Cl - /formate exchange. It was proposed that SLC26A6 is the likely apical Cl - /formate exchanger in the kidney proximal tubule ( 19 ). Expression studies in oocytes in our laboratory demonstrated that SLC26A6 (or PAT1) mediates Cl - /OH - and Cl - /HCO 3 - exchange ( 40 ). These results have been confirmed by other investigators ( 14, 46 ). We further demonstrated the presence of apical Cl - /OH - and Cl - /HCO 3 - exchangers in the isolated kidney proximal tubule ( 24 ). Based on immunolocalization studies indicating the expression of SLC26A6 (PAT1) on the apical membrane of mouse proximal tubules, and based on functional studies in in vitro expression systems indicating that SLC26A6 mediates Cl - /HCO 3 - exchange ( 19, 40 ), we hypothesized that SLC26A6 (PAT1) mediates the apical Cl - /HCO 3 - exchange in the kidney proximal tubule ( 24 ).4 P) T: U6 ~! B* g1 k6 s! t

A mathematical model of rat proximal tubule had been developed to examine the stimulatory effect of formate on NaCl reabsorption ( 43 ). This model included luminal membrane Cl - /HCO 3 - and Cl - /formate exchangers, plus a sufficiently high luminal membrane formic acid permeability to allow recycling. Nevertheless, it was observed in model calculations that although activation of Cl - /formate exchange did produce cell swelling, it had little impact on net NaCl reabsorption. In contrast, changes in NHE3 density did modulate NaCl reabsorption. Functional studies indicate that the formate-stimulated Cl - reabsorption in the proximal tubule is blocked in the presence of amiloride analogs in the lumen ( 38 ), suggesting that the presence of NHE3 is essential for the stimulatory effect of formate on Cl - reabsorption. This was interpreted to indicate that there might be a close coupling between the Na /H exchanger NHE3 and Cl - /formate exchanger in the apical membrane of the kidney proximal tubule ( 38 ). An alternative hypothesis, proposed and examined in the current studies, is that formate directly stimulates the apical Na /H exchanger, which then indirectly activates the apical Cl - /base exchanger by increasing its intracellular substrate (base).
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MATERIALS AND METHODS

Animals. Black Swiss mice were used for these experiments. Animals had free access to water and the standard laboratory chow. The use of anesthetics (pentobarbital sodium) and the method of euthanasia (pentobarbital overdose) were according to the institutional guidelines and approved animal protocols.- k$ ?& y1 [% F+ K
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Isolation of proximal tubules and in vitro microperfusion. Mice were killed by intraperitoneal injection of pentobarbital sodium, and kidneys were quickly removed and placed in ice-cold dissection medium ( solution 1, Table 1 ). Thin coronal slices ( 1 mm) were cut and transferred to the dissection chamber. Proximal straight tubules were hand-dissected from the cortical part of the corticomedullary rays with sharpened forceps. Isolated proximal tubules were transferred to a 0.4-ml laminar-flow temperature-controlled specimen chamber mounted on the inverted Zeiss Axiovert S-100 microscope (Carl Zeiss, Thornwood, NY). Experiments were performed at 37°C. In vitro microperfusion of the isolated tubule segments was performed using concentric glass pipettes according to the method of Burg et al. ( 9, 10 ) as previously described at 5 cm water pressure ( 24, 37 ). Solutions used to perfuse and bathe the tubules are listed in Table 1. Solutions were delivered to the specimen chamber in CO 2 - and O 2 -impermeable tubing (Cole Palmer, Chicago, IL) at a rate of 4 ml/min. The chamber was closed by a lid and constantly superfused with 100% O 2 (bicarbonate free) or 95% O 2 -5% CO 2 (bicarbonate buffered) to keep the pH of the bath fluid constant. The chamber pH was frequently checked by a Horiba pH meter (model B 213, Horiba, Japan). Tubules were perfused with 0.15 mg/ml Fast Green Dye (Sigma, St. Louis, MO) at the beginning of each experiment to identify the damaged cells. Tubules were carefully inspected and discarded if damaged cells were found in the tubule wall ( 24, 26, 37 ).( h1 C4 @9 ~3 q4 Q
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Table 1. Chemical composition of solutions used for microperfusion& Q1 {' d6 h, @

Isolation of outer medullary collecting duct and in vitro microperfusion. Outer medullary collecting ducts (OMCDs) were isolated from the outer medullary portion of the corticomedullary rays with sharpened forceps. The tubules were obtained below the end of the straight proximal tubule and the beginning of the thin descending limb of Henle's loop, adjacent to the thick ascending limbs of Henle's loop, and microperfused as described above. Initially, tubules were perfused with 0.15 mg/ml of Fast Green Dye (Sigma) to identify the damaged cells as described above. Tubules were inspected and discarded if damaged cells were found in the tubule wall ( 24, 26, 37 ). At least 30 to 40 min were allowed for tubules to equilibrate with the solutions, before any measurements were done.8 o  |+ {6 x$ G0 |2 S; L
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Intracellular pH measurements. Intracellular pH (pH i ) in isolated, microperfused kidney proximal tubule or OMCD was measured using 2',7'-bis-(3-carboxypropyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCPCF-AM) ( 21 ), a close analog of 2',7'-bis(carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM) with improved spectral characteristics, as previously described (24-26, 37). Briefly, after 30-min equilibration in the initial bath and luminal solution, the tubule was perfused with 1 µM BCPCF-AM for 2 min. Ten minutes were allowed for the dye washout. Fluorescent measurements were done on a Zeiss Axiovert S-100 inverted microscope equipped with the Attofluor RatioVision Digital Imaging System (Attofluor, Rockville, MD) as previously described (24-26, 28). An Achroplan x 40/0.8 water objective with 3.6-mm working distance was used. Excitation wavelengths were recorded at 488 and 440 nm, and emission was measured at 520 nm. Digitized images were analyzed by using Attograph software (Attofluor). Intracellular calibration was performed at the end of each experiment by using high- K -nigericin method (24-26, 35-37).8 q7 f! v6 \. m$ q: S
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Experimental maneuvers. For monitoring the baseline pH, stable basal pH i readings were obtained for at least 5 to 10 min before any experimental maneuvers with solution 2 ( Table 1 ). Formate was added at 0.5 mM in the bath and perfusate of the proximal tubule or OMCD. In the experiments in which Cl - and Na dependence of the observed effects of formate on pH i was assessed, the perfusate was switched to a Cl - -free solution ( solution 3, Table 1 ) or Na -free solution ( solution 4, Table 1 ) or NaCl-free solution ( solution 5, Table 1 ), before formate was added. In a separate set of experiments, baseline pH i was also recorded in proximal tubules that were perfused in bicarbonate-buffered solutions ( solution 6, Table 1 )., e* |0 [- v2 [2 n; ?; T6 R, Q0 v
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Buffering capacity. Intrinsic cell buffer capacity ( i ) was measured by using NH 4 Cl prepulse technique according to the established protocols as previously described ( 20, 24, 29, 34, 35 ). Measurement of i was done in solutions without sodium ( solution 4, Table 1 ) with 1 mM DIDS on both bath and luminal side to keep sodium- and chloride-dependent acidification mechanisms inactive. In NH 4 Cl-containing solutions, 20 mM NH 4 Cl replaced equimolar concentration of TMA-Cl. In bicarbonate-free solutions, i was 26.4 ± 4 mM/pH ( n = 7 cells in 2 tubules). In bicarbonate-containing solutions, total buffering capacity ( T ) was estimated as the sum of i and bicarbonate buffer capacity ( HCO 3 - ). HCO 3 - was calculated as 2.3 x [HCO 3 - ] i, where [HCO 3 - ] i is intracellular bicarbonate concentration ( 20, 29 ). The buffer capacity in the presence of bicarbonate was 70.22 ± 8 mM ( n = 7 cells in 2 tubules).
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Materials. BCPCF-AM was purchased from Molecular Probes (Eugene, OR). All the other chemicals were from Sigma. Nigericin was dissolved in ethanol and diluted 1:1,000 for the final concentration of 10 µM. EIPA was dissolved in methanol and diluted 3:1,000 for the final concentration of 500 µM. DIDS was dissolved directly in the solutions used for perfusion.6 [! F9 W$ L" D2 u5 v- [3 \
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Statistics. Results are expressed as means ± SE. Statistical significance between experimental groups was determined by Student's t -test, as required. Significance was asserted if P

RESULTS
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Effect of formate on apical Na /H exchanger NHE3 activity. To test the possibility that formate directly stimulates the apical Na /H exchanger, mice kidney proximal tubules were isolated and microperfused. The experiments were performed in the absence of CO 2 /HCO 3 - in the media to keep bicarbonate-dependent transporters such as basolateral Na :HCO 3 - cotransporter (NBC1) inactive. As shown in Fig. 1 A (representative tracings), addition of physiological concentrations of formate (0.5 mM) to the bath and lumen (switching from formate-free to formate-containing solution 2, Table 1 ) caused significant intracellular alkalinization in isolated, microperfused mouse kidney proximal tubules. Generation of cell alkalinization was fast and started immediately ( Fig. 1 A ). Removal of sodium from the lumen resulted in a rapid return of the cell pH to baseline levels. The summary of the results is shown in Fig. 1 B. As indicated, the addition of formate increased the pH i from a baseline value of 7.16 ± 0.01 to 7.52 ± 0.02, with a pH of 0.36 ± 0.02 pH units and rate of 0.311 pH units/min ( n = 20 cells in 6 tubules).
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Fig. 1. Effect of formate on basal intracellular pH (pH i ) in isolated, microperfused kidney proximal tubule. A : representative tracings demonstrating rapid generation of cell alkalinization in response to formate addition. B : summary of the results. Addition of formate increased the pH i from a baseline value of 7.16 ± 0.01 to 7.52 ± 0.02, with a pH of 0.36 ± 0.02 pH units and rate of 0.311 pH units/min ( n = 20 cells in 6 tubules).! S: Q% K# j8 _2 I5 H! i

Effect of luminal chloride removal on formate-mediated cell pH alkalinization. Intracellular alkalinization of proximal tubule cells by formate can be either due to increased extrusion of H (via i.e., NHE) or entry of OH - (via i.e., Cl - /OH - exchange working in reverse mode). In the next series of experiments, Cl - was removed from the luminal solution in microperfused kidney proximal tubule cells ( solution 3, Table 1 ). The bath solution was the same as in Fig. 1. As indicated in Fig. 2 A (representative tracings), the addition of formate to the bath and perfusate caused significant pH i alkalinization in proximal tubule cells. The summary of the results is included in Fig. 2 B. As shown, the removal of luminal chloride did not block the cell pH alkalinization by formate (baseline pH of 7.26 ± 0.01 to 7.53 ± 0.01 with formate, n = 11; 3 tubules P " C' Z0 _* w3 L7 u, T- n* v' S6 B
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Fig. 2. Effect of luminal Cl - removal on formate-induced cell pH alkalinization in the isolated, microperfused kidney proximal tubule. A : representative tracings demonstrating generation of cell alkalinization in response to formate addition in the absence of luminal Cl -. B : summary of the results. Removal of Cl - from the lumen did not block the cell pH alkalinization in response to formate. In the absence of luminal chloride, the addition of formate increased baseline pH from 7.26 ± 0.01 to 7.53 ± 0.01 ( n = 11, 3 tubules, P

Effect of luminal sodium removal on formate-mediated cell pH alkalinization. In the next series of experiments, we examined the effect of removal of sodium from the lumen on cell pH alkalinization in response to formate. As demonstrated in representative tracings ( Fig. 3 A ), removal of sodium from the lumen completely prevented the cell alkalinization and indeed was associated with cell acidification. The summary of the results depicting the magnitude of cell acidification in response to formate addition is shown is Fig. 3 B. As observed, in the absence of luminal sodium, the addition of formate resulted in the reduction of cell pH from a baseline level of 7.21 ± 0.03 without formate to 6.98 ± 0.04 pH units with formate ( P

Fig. 3. Effect of luminal Na (sodium) removal on formate-induced cell pH alkalinization in the isolated, microperfused kidney proximal tubule. A : representative tracings demonstrating that the removal of luminal sodium completely prevents cell alkalinization in response to formate addition. Indeed, there is an unmasking of a cell pH acidification by formate in the absence of luminal sodium. B : summary of the results. In the absence of luminal sodium, the addition of formate resulted in the reduction of cell pH from the baseline level of 7.21 ± 0.03 without formate to 6.98 ± 0.04 pH units with formate ( P * @( ]* y+ y$ X6 Z$ |

Effect of luminal EIPA on formate-induced cell pH alkalinization. The results of the above experiments demonstrate that formate-induced cell alkalinization in isolated, microperfused kidney proximal tubule is dependent on luminal sodium but is independent of luminal Cl -. These results are consistent with the activation of the apical Na /H exchanger by formate. To test this possibility further, the experiments were repeated in the presence of luminal EIPA, a strong inhibitor of NHE3. As demonstrated in Fig. 4 A (representative tracings), the presence of EIPA at 500 µM in Na- and Cl-containing perfusate completely blocked proximal tubule cell alkalinization in response to formate. The summary of the results is included in Fig. 4 B. As evident, the presence of EIPA unmasks cell acidification by formate ( Fig. 4 A ) that was not present in its absence, with baseline pH i decreasing from 7.19 ± 0.03 to 6.96 ± 0.04 ( n = 12 cells in 4 tubules, P 8 o, Q4 r, `# E

Fig. 4. Effect of luminal EIPA on formate-induced cell pH alkalinization in the isolated, microperfused kidney proximal tubule. A : representative tracings demonstrating that the presence of luminal EIPA at 500 µM completely blocks the cell alkalinization and unmasks cell acidification in response to formate. B : summary of the results. In the presence of luminal EIPA, the addition of formate resulted in baseline pH i decreasing from 7.19 ± 0.03 to 6.96 ± 0.04 ( n = 12 cells in 4 tubules, P
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Effect of luminal chloride removal on formate-mediated cell pH acidification in the presence of EIPA. To test whether the generation of intracellular acidosis in response to the addition of formate in the presence of luminal EIPA is via the activation of apical Cl - /OH - exchange, the experiments were repeated in the absence of luminal Cl - ( solution 3, Table 1 ). The bath solution was the same as in Fig. 1. As indicated in Fig. 4, C and D (representative tracings and summary of results), in the presence of EIPA, removal of Cl - from the perfusate did not block cell pH acidification by formate. Baseline pH i dropped from 7.4 ± 0.03 in the absence of formate to 7.18 ± 0.07 with formate ( n = 7 in 3 tubules, P ! H* `# O2 X; a$ y4 |

Effect of formate on pH i in microperfused OMCD. To determine the specificity of formate-induced cell pH alkalinization in the proximal tubule, the effect of formate on pH i was examined in OMCD cells. Accordingly, OMCD was perfused, and cells were loaded with BCECF, as described in MATERIALS AND METHODS. As demonstrated in Fig. 5 A, the addition of 0.5 mM formate to the bath and lumen in the presence of sodium and chloride did not cause any cell pH alkalinization in OMCD cells. Indeed, there was a mild, although not significant, degree of cell pH i acidification on addition of formate. The summary of the results is shown in Fig. 5 B and demonstrates the absence of any significant cell pH alteration by formate in OMCD.

Fig. 5. Effect of formate on baseline pH i in the isolated, microperfused kidney outer medullary collecting duct (OMCD). A : representative tracings demonstrating the absence of generation of cell alkalinization in response to formate addition in OMCD. B : summary of the results. The addition of formate did not cause any significant cell pH alteration in OMCD cells.

Effect of formate on pH i in kidney proximal tubule in the presence of CO 2 /bicarbonate. In the next series of experiments, the effect of formate on pH i was measured in microperfused kidney proximal tubules in the presence of solutions simulating the physiological milieu in the late proximal tubule. Toward this end, proximal tubules were isolated and perfused with a low-bicarbonate (5 mM), high-chloride (140 mM), acidic solution gassed with 5% CO 2 (pH 6.7). The bath solution consisted of 115 mM NaCl, 25 mM NaHCO 3 -, and gassed with 5% CO 2 (pH 7.4). DIDS, at 0.5 mM, was added to the bath to inhibit basolateral Na-HCO 3 - cotransporter NBC1. At steady state, 0.5 mM formate was added to the bath and perfusate. As indicated, the addition of formate caused significant intracellular alkalinization ( Fig. 6 A ), with pH i increasing from baseline levels 7.34 ± 0.02 to 7.50 ± 0.02 ( P

Fig. 6. Effect of formate on basal pH i in kidney proximal tubules in the presence of CO 2 /bicarbonate in the media. A : representative tracings demonstrating cell alkalinization in response to formate addition in the presence of an acidic luminal pH and CO 2 /HCO 3 - in the media. B : summary of the results. In the presence of perfusate and bath solutions simulating late proximal tubule, the addition of formate causes significant intracellular alkalinization, with pH i increasing from baseline levels 7.34 ± 0.02 to 7.50 ± 0.02 ( P ! [. q3 v& Z, Q; F
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Effect of 50 µM formate or acetate on basal pH i in kidney proximal tubules. In the last series of experiments, we examined the effect of a lower, more physiological concentration of formate. Toward this end, proximal tubules were perfused with solutions similar to those in Fig. 1. At baseline pH i, 50 µM formate was added to the bath and perfusate. As demonstrated in a representative tracing in Fig. 7 A, the addition of 50 µM formate caused significant intracellular alkalinization, with pH i increasing from baseline levels 7.15 ± 0.02 to 7.36 ± 0.02 ( P ! f& U& [+ p, F, Z+ W
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Fig. 7. Effect of 50 µM formate or acetate on basal pH i in kidney proximal tubules. A : representative tracings demonstrating cell alkalinization in response to 50 µM formate but not 50 µM acetate in microperfused kidney proximal tubules. B : summary of the results. Fifty micromolar formate added to perfusate and bath causes intracellular alkalinization, with pH i increasing from baseline levels 7.15 ± 0.02 to 7.36 ± 0.02 ( P 4 ~/ M, y8 Y/ Z* _: n

DISCUSSION3 w: J, o+ X# l+ e
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The apical Na /H and Cl - /base exchangers work in parallel and produce net Na and Cl reabsorption in the kidney proximal tubule. This assumption is based on studies demonstrating that the inhibition of either the apical NHE or the Cl/base exchanger inhibits net Na and Cl absorption in the proximal tubule ( 7, 28, 44 ). Inhibition of the apical Na /H exchanger by amiloride analogs inhibits Na and Cl - reabsorption in the kidney proximal tubule. The presence of disulfonic stilbenes, inhibitors of Cl - /base exchanger, inhibits Na and Cl reabsorption ( 23, 44 ). These results support a model of coordinated regulation of Na /H and Cl - /base exchangers in the kidney proximal tubule. According to this model, activation of the apical Na /H exchanger increases the intracellular concentration of base, which in turn is transported via the apical Cl - /base exchanger. The result is net Na and Cl reabsorption. Conversely, the inhibition of the apical Na /H exchanger inhibits the apical Cl - /base exchanger by causing intracellular acidosis and decreasing the intracellular base. The result is reduction in net Na and Cl reabsorption.
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One intriguing and, at the same time, puzzling aspect of the studies on electrolyte absorption in kidney proximal tubule is the interaction of formate with the apical Cl - /base exchanger. Several investigators have studied this issue in detail. In general, it has been convincingly demonstrated that the addition of formate stimulates net Cl - reabsorption in the kidney proximal tubule ( 5 ). This has been interpreted to indicate that formate is transported via the apical Cl - /formate exchanger. It was further suggested that by recycling across the apical membrane, formate can provide the substrate for and activate the Cl - /base exchanger, which can then result in enhanced coordinated absorption of Na and Cl - ( 5, 15, 16, 30, 38 ). With respect to the effect of formate on the apical Cl - /base exchanger, the studies are conflicting. For example, studies examining the apical Cl - /base exchanger by cell pH measurement have been less successful in demonstrating the stimulation of this transporter by formate. Two studies in microperfused kidney proximal tubule have not been consistent in detecting stimulation of apical Cl - /base exchange by formate ( 7, 20 ). These results have raised serious questions in regard to the role of apical Cl - /formate exchange as the mediator of enhanced Na and Cl - reabsorption in the proximal tubule by formate ( 45 ).
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One missing link in the understanding of the electrolyte absorptive processes in the kidney proximal tubule has been the lack of knowledge on the molecular identity of the apical Cl - /base exchanger in the kidney proximal tubule. A new family of anion exchangers has been identified that is referred to as SLC26. One member of this family is SLC26A6 (also known as PAT1 or CFEX) ( 22 ). This exchanger is located on the apical membrane of the kidney proximal tubule and was shown to mediate Cl - /OH -, Cl - /HCO 3 -, Cl - /formate, and Cl - /oxalate exchange, all apical anion exchanger modes described in the kidney proximal tubule ( 14, 19, 40, 46 ). Based on immunolocalization studies indicating the expression of SLC26A6 (PAT1) on the apical membrane of mouse and rat proximal tubules, and based on functional studies in in vitro expression systems indicating that SLC26A6 mediates Cl - /OH - and Cl - /HCO 3 - exchange ( 19, 24, 40 ), we hypothesized that SLC26A6 (PAT1) mediates the apical Cl - /OH - /HCO 3 - exchanger in the kidney proximal tubule under in vivo conditions ( 24 ).

Formate-stimulated Cl - reabsorption in the proximal tubule is absent in the presence of inhibitors of NHE3 ( 38 ), suggesting that the activity of NHE3 is essential for the stimulatory effect of formate on Cl - reabsorption. This was interpreted to indicate that there might be a close coupling between the Na /H exchanger NHE3 and Cl - /formate exchanger in the apical membrane of the kidney proximal tubule. An alternative hypothesis, examined in the present studies, is that formate directly stimulates the apical Na /H exchanger.: ^) g, w1 J, e  L0 q

The most salient feature of the current studies is the stimulation of the apical Na /H exchanger in the proximal tubule by formate. This conclusion is supported by the generation of sodium-dependent, chloride-independent cell pH alkalinization by formate in the kidney proximal tubule ( Figs. 1 - 3 ). The conclusion is confirmed by the absence of cell alkalinization in the presence of EIPA, a strong inhibitor of NHE3 ( Fig. 4 ). Formate had no stimulatory effect on the apical Cl - /base exchanger as determined by cell pH determination ( Fig. 2 ). The activation of NHE3 is rapid and occurs in a matter of seconds ( Figs. 1 and 2 ). The stimulatory effect of formate on apical Na /H exchanger activity was also observed at a lower and more physiological concentration ( Fig. 7 ), consistent with the studies on net chloride and volume absorption in microperfused kidney proximal tubules ( 38 ). The stimulatory effect of apical NHE by formate was unique to this chemical and was not shared by other monocarboxylates. This was specifically tested with acetate added to the bath and perfusate, which showed no significant changes in cell pH ( Fig. 7 ). The stimulatory effect of formate on apical NHE was also observed in the presence of a perfusate simulating late proximal tubule (low-bicarbonate, high-chloride, acidic solution; Fig. 6 ).# y& u7 U$ x) O/ ?8 S
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Although experiments in Figs. 1 - 7 demonstrate that the stimulatory effect of formate on the apical Na /H exchanger can be observed in the presence as well as the absence of bicarbonate, one should notice that the experimental conditions in our studies are not exactly identical to the conditions used by others to assess the effect of formate on net volume reabsorption ( J v ) in microperfused proximal tubule ( 31 ). For example, in the studies demonstrating formate stimulation of volume reabsorption in the proximal tubule ( 31 ), formate failed to stimulate J v when tubules were perfused with a normal HCO 3 -, normal Cl - solution as occurs in the early proximal tubule. Formate only stimulated J v when tubules were perfused with a low-HCO 3 -, high-Cl - luminal solution ( 31 ). This raises the possibility that in addition to apical NHE activation, other factor(s) may contribute to enhanced fluid and chloride reabsorption by formate. A unifying mechanism to explain the results of all published reports on this subject is that formate could be having two distinct effects on the proximal tubule: one is the stimulation of apical Na-H exchange, as clearly demonstrated in the current manuscript. The other could be the stimulation of DIDS-sensitive apical Cl - base exchange, with formate either serving as a substrate or by an indirect mechanism.1 g  K, x/ M4 Y

In support of a unique role for formate in the proximal tubule, we observed that formate had no alkalinizing effect on cell pH in OMCD cells ( Fig. 5 ), which do not have an apical Na /H exchanger ( 32, 42 ). Indeed, there was a mild degree of acidosis by formate in OMCD cells. Whether the stimulatory effect of formate on NHE3 is direct or mediated via an intermediary signal/protein remains speculative. Additional studies are underway to examine the effect of inhibitors of exocytosis or various phosphorylation pathways on formate-mediated NHE3 stimulation in the proximal tubule.. @0 [) Y# t! b) s* R
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The removal of sodium or the addition of amiloride analog EIPA in the lumen not only blocked the NHE3-mediated cell alkalinization in response to formate but it also unmasked a cell acidification that is independent of luminal chloride ( Figs. 3 and 4 ). Possibilities such as the inhibition of yet to be determined acid-extruding transporter(s) or the nonionic diffusion of formate (in the form of formic acid) can potentially explain the intracellular acidification in response to the addition of formate. Whether the nonionic diffusion of low levels of formate, 0.5 or 0.05 mM, can cause such a profound acidification ( Fig. 3 ) is doubtful. Given the buffering capacity in proximal tubule cells in the presence or absence of bicarbonate ( MATERIALS AND METHODS ), we suggest that the cell acidification in Figs. 3 and 4 could not be attributed to a weak acid effect alone and most likely reflects alteration of membrane transporters (i.e., apical or basolateral). The identity of these transporters is speculative at present. It should be mentioned that the formate-stimulated NHE3 activation, with subsequent intracellular alkalinization, is a primary event and is independent of intracellular acidification by formate. This is supported by the absence of any initial acidification by formate in the presence of sodium in the lumen ( Figs. 1 and 2 ).

Whether the stimulation of NHE3 by formate is specific to the kidney or is also observed in other epithelia or tissues such as intestine or retina remains speculative. Our preliminary results in nonepithelial Chinese hamster ovary cells transfected with NHE3 cDNA (a generous gift from Dr. J. Orlowski) did not reveal any significant cell alkalinization in response to formate (unpublished observations). These results raise the possibility that a milieu specific to epithelial cells (i.e., proximal tubule or retina) may be essential for the stimulatory effect of formate. Determining the answer to this question is important and may have clinical significance. Accidental exposure to formate-generating chemicals (such as methanol) causes cell swelling in the retina, optic nerve, central nervous system (and possibly other organs) along with decreased visual acuity and blindness. It has been hypothesized that formate inhibits retinal mitochondrial function and increases oxidative stress, leading to cell injury and swelling. We hypothesize that an equally plausible mechanism causing cell swelling in the retina is the activation of the apical Na /H exchanger by formate, which can result in secondary activation of the apical Cl - /base exchanger. This coordinated activation may result in Na and Cl absorption into the retinal cell, thereby causing cell swelling. The apical Na /H exchanger and Cl - /base exchanger have been identified in the apical membranes of the retina ( 17, 18, 47 ). Another organ that develops swelling in response to formate intoxication is the optic nerve, which also has a Na /H exchanger ( 6 ).

In conclusion, formate activates the apical Na /H exchanger NHE3 in the kidney proximal tubule. We propose that formate stimulation of chloride transport in the kidney proximal tubule is indirect and is mediated via the stimulation of the apical Na /H exchanger, which subsequently increases the availability of the substrate (base concentration) for the apical Cl - /base exchanger. Whether the effect of formate on Na /H exchange is isoform specific (i.e., only NHE3) or is also observed with other NHE isoforms (i.e., NHE1 or 2) will remain speculative at the present and will be the subject of future investigations.7 H( s% c1 b: ~: f( T: a3 J

GRANTS

These studies were supported by National Institutes of Health Grants DK-62809 and 54430 (to M. Soleimani) and DK-29857 (to A. Weinstein), a Kidney Foundation of Greater Cincinnati grant (to S. Petrovic), a Merit Review Award, a Cystic Fibrosis Foundation grant, and grants from Dialysis Clinic Incorporated (to M. Soleimani).2 C* u* Y( Y9 H5 w

ACKNOWLEDGMENTS
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The technical assistance of A. Miskini is greatly appreciated.% z3 ^% G+ u, `
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Address for reprint requests and other correspondence: M. Soleimani, Dept. of Medicine, Univ. of Cincinnati, 231 Albert Sabin Way, MSB G259 Cincinnati, OH 45267-0585 (E-mail: Manoocher.Soleimani{at}uc.edu
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