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Role of a tyrosine kinase in the CO₂-induced stimulation of HCO₃^– reabsorption by rabbit S2 proximal tubules

Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut

Submitted 29 December 2005 ; accepted in final form 6 March 2006

	ABSTRACT

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A previous study demonstrated that proximal tubule cells regulate HCO₃^– reabsorption by sensing acute changes in basolateral CO₂ concentration, suggesting that there is some sort of CO₂ sensor at or near the basolateral membrane (Zhou Y, Zhao J, Bouyer P, and Boron WF Proc Natl Acad Sci USA 102: 3875–3880, 2005). Here, we hypothesized that an early element in the CO₂ signal-transduction cascade might be either a receptor tyrosine kinase (RTK) or a receptor-associated (or soluble) tyrosine kinase (sTK). In our experiments, we found, first, that basolateral 17.5 µM genistein, a broad-spectrum tyrosine kinase inhibitor, virtually eliminates the CO₂ sensitivity of HCO₃^– absorption rate (J). Second, we found that neither basolateral 250 nM nor basolateral 2 µM PP2, a high-affinity inhibitor for the Src family that also inhibits the Bcr-Abl sTK as well as the Kit RTK, reduces the CO₂-stimulated increase in J. Third, we found that either basolateral 35 nM PD168393, a high-affinity inhibitor of RTKs in the erbB (i.e., EGF receptor) family, or basolateral 10 nM BPIQ-I, which blocks erbB RTKs by competing with ATP, eliminates the CO₂ sensitivity. In conclusion, the transduction of the CO₂ signal requires activation of a tyrosine kinase, perhaps an erbB. The possibilities include the following: 1) a TK is simply permissive for the effect of CO₂ on J; 2) a CO₂ receptor activates an sTK, which would then raise J; 3) a CO₂ receptor transactivates an RTK; and 4) the CO₂ receptor could itself be an RTK.

kidney; out-of-equilibrium solutions; acid-base; volume reabsorption; signal transduction

To study how the PT senses changes in whole body acid-base status, our laboratory developed out-of-equilibrium (OOE) CO₂/HCO₃^– solutions for altering, one at a time, basolateral [CO₂], [HCO₃^–], or [H⁺] (brackets denote concentration) (58, 59). We found that, at least in regard to acute acid-base disturbances, the PT responds not to changes in either basolateral (BL) pH (pH_BL) or intracellular pH (pH_i) but to changes in [CO₂]_BL and [HCO₃^–]_BL (61). In the case of [CO₂]_BL, increases cause HCO₃^– reabsorption to rise, whereas decreases have the opposite effect. Moreover, the data suggest that the PT cell senses [CO₂]_BL directly, utilizing some sort of CO₂ sensor at or near the basolateral membrane. The key questions concern the nature of the CO₂ sensor and the mechanisms by which the cell transduces the CO₂ signal to an increase in HCO₃^– reabsorption.

The past two decades have seen major advances in understanding how organisms sense dissolved gases. For instance, nitric oxide binds to a heme moiety of soluble guanylyl cyclase (45), oxygen binds to a two-component receptor in bacteria, and ethylene binds to a receptor in Arabidopsis thaliana.

The bacterium Rhizobium meliloti senses oxygen using a two-component system, consisting of FixL and FixJ (20). FixL is a transmembrane protein with a COOH-terminal histidine kinase domain and a NH₂-terminal heme-binding domain that blocks the histidine kinase when O₂ binds to the heme. When the [O₂] falls to microaerobic levels, the histidine kinase autophosphorylates a conserved His within its catalytic core, thereby activating FixJ, which, in turn, induces transcription of genes involved in nitrogen fixation (21, 45).

Ethylene acts like a hormone in plants, regulating such events as seed germination, fruit ripening, and leaf senescence (8, 18). In 1993 Chang et al. (13), working on the plant A. thaliana, found that mutations in the ETR1 gene block ethylene signaling. The deduced amino acid sequence of the COOH-terminal half of ETR1 is homologous to both components of the two-component systems. Later work showed that ethylene binds to an NH₂-terminal hydrophobic domain of ETR1 (49) and that the binding involves copper as a cofactor (46). Downstream of ETR1, which is a histidine kinase, the ethylene signaling cascade involves the Raf-like kinase CTR1 (15, 29) and MAPK (37).

Because, like O₂ and ethylene, CO₂ is a small volatile molecule, we entertained the hypothesis that PT cells sense CO₂ using a comparable mechanism. Because mammalian cells do not have histidine kinases, we postulated that the sensor might be either a receptor tyrosine kinase (RTK) or a receptor-associated (i.e., soluble) tyrosine kinase (sTK). Furthermore, previous studies showed that both EGF and TGF-, both of which bind to receptors in the erbB family of RTKs, stimulate HCO₃^– and phosphate reabsorption, with a higher potency for TGF- on the HCO₃^– absorption rate (J) (41, 42). As a first step, in the present study we have examined the effect of tyrosine kinase inhibitors on the CO₂-induced increase in J by the PT cell.

Our approach was to use OOE solutions to vary basolateral [CO₂] from 0 to 20% while keeping basolateral [HCO₃^–] and pH fixed near their physiological values in isolated, perfused rabbit S2 PTs. We found that basolateral 17.5 µM genistein, a broad-spectrum tyrosine kinase inhibitor (1, 24), virtually eliminates the CO₂ sensitivity of J. Basolateral 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidase (PP2), which inhibits the Src family (26) sTK, the Bcr-Abl sTK, as well as the Kit (53) RTK, is without effect at either 250 nM or 2 µM. On the other hand, basolateral 35 nM PD168393, which blocks RTKs in the erbB (i.e., EGF receptor) family by alkylating a cysteine residue in the ATP binding pocket (19), blocks the CO₂-induced increase in J. Moreover, 10 nM basolateral BPIQ-I, which blocks erbB RTKs by competing with ATP (44), also eliminates CO₂ sensitivity. Thus the transduction of the CO₂ signal requires activation of a tyrosine kinase, possibly a member of the erbB family.

	METHODS

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The methods are similar to those in our previous studies (60, 61).

Biological Preparation

According to procedures approved by the Yale Animal Care and Use Committee, we perfused the PTs isolated from "pathogen-free" female rabbits (New Zealand White, Elite, Covance, Denver, PA) using methods similar to those described by Burg et al. (12) and later modified by Baum et al. (5) and by our laboratory (36, 59–61). To summarize, a rabbit weighing 1.4–2.0 kg was euthanized by a single overdose of 3 ml (20 mg) of intravenous pentobarbital sodium. An incision of the abdominal wall exposed the left kidney, which we rapidly removed and cut into coronal slices that we kept in cold (4°C) modified Hanks' solution (solution 1 in Table 1). We hand-dissected a slice to obtain individual midcortical S2 segments of a PT. We cannulated the perfusion end of the tubule using concentric holding, perfusion, and exchange pipettes, and drew the collection end into a holding pipette. We randomized the orientation of the tubule between the two pipettes. However, the extreme proximal portion of the proximal straight tubule was always inside one pipette, the most distal portion of the proximal convoluted tubule was exposed to the bath solution between the two pipettes, and a more proximal portion of the proximal convoluted tubule was inside the second pipette. On the collection end, we used a calibrated collection pipette (volume 55 nl) to obtain samples of fluid. The mean length of perfused tubules in our J/fluid absorption rate (J_V) experiments, as measured with an eyepiece micrometer, was 1.23 ± 0.02 mm (n = 96 tubules). The mean luminal collection rate was 12.1 ± 0.2 nl/min (n = 192 collection periods). We perfused the basolateral side of the tubule (i.e., "bath") at 7 ml/min with a solution at 37°C.

Table 1 lists the compositions of the solutions, all of which were identical to those used in the aforementioned studies (60, 61).

We dissected PTs in Hanks' solution (solution 1) at 4°C. The luminal perfusate always was solution 2, which contained dialyzed [³H]methoxyinulin (MW 7,146, NET-086L, PerkinElmer Life Sciences, Boston, MA) as the volume marker. During a 20- to 30-min warm-up period, solution 3, which contained 2% albumin, flowed through the bath at 37°C. Following this warm-up period, we switched the bath to solution 4, 5, or 6, containing no drugs or DMSO, for the first of two collection periods. During the first 5–8 min of the first collection period, we discarded the first two collected samples before allowing the collected fluid to accumulate in the collection pipette. Subsequently, we began a series of three timed and calibrated collections, two samples for analysis of [³H]methoxyinulin and one for analysis of total CO₂. We then switched to a second bath solution (solution 4, 5, or 6) containing genistein (345834, Calbiochem, La Jolla, CA); PP2 (529573, Calbiochem); PD168393 (513033, Calbiochem); or BPIQ-I (203696, Calbiochem) plus 1:20,000 DMSO (D-5534, Sigma, St. Louis, MO). We then repeated the procedure outlined for the first collection periods. We generated OOE CO₂/HCO3– solutions (solution 5 and 6) by rapidly mixing streams of two dissimilar solutions (i.e., mixing solutions 5A and 5B to yield solution 5 and mixing solutions 6A and 6B to yield solution 6) (58, 61) and delivering the newly mixed solution to the tubule within 200 ms. All solutions had osmolalities of 300 ± 2 mosmol/kgH₂O.

Measurement of J and J_V

Our measurement of J(pmol·min^–1·mm tubule length^–1) and J_V (nl·min^–1·mm^–1) was similar to that used by McKinney and Burg (32) and nearly identical to our previous approach (59–61). We determined total CO₂ in aliquots of the perfusate and collected fluid using a WPI "NanoFlo" device (World Precision Instruments, Sarasota, FL). The one difference from our previous study is that, because Diagnostic Kit 132-A (Sigma-Aldrich) for total CO₂ determinations was no longer commercially available, we generated our own reagents, a total of five components, according to the protocol described by Hall et al. (25) and Krömer et al. (30).

1) Made fresh on the day of analysis, this solution contained (in mM) 10 MgCl₂ 6 H₂O, 1 EDTA, 2 dithiothreitol, 100 Tris·HCl, 15 sodium azide (NaN₃), and 30 NaOH to adjust pH to 7.7. To eliminate any CO₂, we bubbled this solution with 100% O₂ for 30 min at 4°C. On the day of analysis, we added components 2–5 from concentrated stock solutions.

2) Phosphoenol pyruvic acid (151872, MP Biomedicals, Aurora, OH) was added to a final concentration of 2.2 mM from a 220 mM stock solution that also contained (in mM) 100 Tris·HCl and 15 NaN₃, pH 7.7 (stock stored at –20°C).

3) -NADH (10168, MP Biomedicals) was added to a final concentration of 0.32 mM from a 32 mM stock solution that also contained (in mM) 100 Tris·HCl, 15 NaN₃, pH 7.7 (powder stored at –20°C, and the stock solution made fresh).

4) Phosphoenol pyruvate carboxylase (153532, MP Biomedicals) was added to a final concentration of 0.275 U/ml from a 100 U/ml stock solution that also contained (in mM) 6 MgCl₂ 6 H₂O, 1 EDTA, 2 dithiothreitol, 40 Tris·HCl, and 15 NaN₃ as well as 10% glycerol, pH 7.7 (stock stored at –80°C).

5) Malate dehydrogenase (151581, MP Biomedicals) was added to a final concentration of 15.5 U/ml from a 10,000 U/ml stock solution in 70% (NH₄)₂SO₄ (stock stored at 4°C).

We computed J_V and J values from the equations described previously (59).

Data Analysis

The values that we report for J(or J_V) in the first collection period are unnormalized, mean values. The values that we report for J(or J_V) in the second collection period are normalized, mean values computed as described previously (60, 61). Briefly, in each experiment, we divided the J(or J_V) value obtained during the second collection period by the comparable value obtained during the first collection period; the result was a pair of second/first collection period ratios. We then multiplied 1) the second/first ratio for J(or J_V) in a particular experiment by 2) the unnormalized mean J(or J_V) value that we obtained during the first collection periods in a series of experiments following the identical protocol.

For comparisons of two means, two-tailed paired t-tests were performed using the Analysis Toolpack of Microsoft Excel. For comparisons of more than two means, one-way ANOVA and Dunnett's multiple comparison were performed using KaleidaGraph (Version 4, Synergy Software). Results are given as means ± SE, with the number of tubules (n) from which it was calculated.

	RESULTS

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Evaluation of DMSO

In this study, we added the inhibitors, predissolved in DMSO, to the bath solution at a final DMSO concentration of 1:20,000. Therefore, we first examined the effect of basolateral 1:20,000 DMSO on J and J_V. Figure 1, A and B, summarizes experiments in which DMSO was present in the bath during both collection periods. During the first collection period, with equilibrated 5% CO₂/22 mM HCO3– in the lumen (solution 2) and in the bath (solution 4), the J and J_V values (grey bars) were similar to the historical averages (61). However, during the second collection period, when we switched the bath to an out-of-equilibrium solution (solution 6) containing 20% CO₂, 22 mM HCO3–, and pH 7.40 (filled bars), the 20% CO₂ failed to increase J. Thus we suspected that an extended exposure to DMSO, even at a dilution of 1:20,000, reduces J during the second collection period.

View larger version (17K): [in this window] [in a new window]   Fig. 1. Evaluation of DMSO. A, C, and E: HCO3– reabsorption rate (J). B, D, and F: volume reabsorption rate (JV). Top and bottom: luminal solution was solution 2 throughout the experiment. During the first collection period (left bars), the basolateral solution was always solution 4, with or without 1:20,000 DMSO, as indicated. During the second collection period (right bars), the basolateral solutions always contained 1:20,000 DMSO added to either solution 4 (5% CO2) or solution 6 (20% CO2), as indicated. In the figure, the 2 bars represent the results of paired experiments; the same 11 tubules served as controls (left bars) in C–F. Values are means ± SE, with nos. of tubules in parentheses. As indicated, the difference between left and right bars in C is statistically significant in a 2-tailed unpaired t-test. **P < 0.01.

As a final check, we examined the effect of adding DMSO during the second collection period when the bath contained 5% CO₂ throughout the experiment. Figure 1, E and F, shows that the addition of DMSO had no effect on either J or J_V. Thus, in our remaining experiments, we added DMSO only in the second collection period, where its effects appear to be negligible.

Effects of Basolateral Genistein on Basolateral CO₂ Dependence of J and J_V

To test the hypothesis that the PT's CO₂-sensing mechanism may require a RTK or an sTK, we examined the effects of basolateral genistein (1, 24) on J and J_V. We used OOE solutions to vary basolateral [CO₂] from 0 to 20%, while keeping [HCO3–]_BL fixed at 22 mM and pH_BL fixed at 7.40.

In the first group of experiments, we examined effect of basolateral 7 µM genistein with equilibrated 5% CO₂/22 mM HCO3– in both the lumen (solution 2) and the bath (solution 4). During the first collection period, no drug was present ( in Fig. 2). During the second collection period, we added 7 µM genistein to solution 4 ( in Fig. 2). Although basolateral 7 µM genistein reduced the mean J by 25%, from 55 ± 3 to 41 ± 7 pmol·min^–1·mm^–1 (n = 4 paired experiments), the difference was not statistically significant (P = 0.07, 2-tailed t-test, paired). Then, we increased the concentration of basolateral genistein to 17.5 µM and repeated the above protocol in six paired experiments. Basolateral 17.5 µM genistein reduced the mean J by 45%, from 55 ± 3 to 30 ± 6 pmol·min^–1·mm^–1. In analyzing the effects of basolateral 17.5 µM genistein in basolateral 5% CO₂, we employed a one-way ANOVA for three groups: 1) control data ( in Fig. 2A), 2) 7 µM genistein ( in Fig. 2A), and 3) 17.5 µM genistein (filled pentagon in Fig. 2A). The overall P value was 0.003. Dunnett's multiple comparison shows that although the effect of basolateral 7 µM genistein on J was not statistically significant (P = 0.48, n = 4), the effect of basolateral 17.5 µM genistein was significant (P = 0.0015, n = 6).

View larger version (16K): [in this window] [in a new window]   Fig. 2. Effects of basolateral 7 or 17.5 µM genistein on the basolateral CO2 concentration ([CO2]BL) dependence of J(A) and JV (B). B: JV. In both A and B, the luminal solution was solution 2 throughout the entire experiment. The basolateral solutions were solutions 5 (0% CO2), 4 (5% CO2), and 6 (20% CO2) during the first collection periods and solutions 5, 4, and 6 plus 7 or 17.5 µM genistein during the second collection periods. Values are means ± SE, with nos. of tubules in parentheses. At 0 and 20% CO2, the statistical comparisons between and filled pentagons at the same [CO2]BL were made using a paired 2-tailed t-test (**P < 0.01). At 5% CO2, the statistical comparison among , , and the filled pentagon at the same [CO2]BL was made using a 1-way ANOVA for 3 groups; Dunnett's multiple comparison indicates that the only significant difference was between and filled pentagon.

As far as J_V is concerned, at a [CO₂]_BL of 5%, the overall P value in a one-way ANOVA was 0.28, and Dunnett's multiple comparison indicates that, relative to the control condition, neither basolateral 7 µM genistein ( in Fig. 2B, P = 0.66) nor basolateral 17.5 µM genistein (filled pentagon in Fig. 2B, P = 0.45) had a significant effect on J_V. In paired, two-tailed t-tests, the effects of basolateral 17.5 µM genistein on J_V were not statistically significant at [CO₂]_BL values of 0% (P = 0.26) or 20% (P = 0.80).

Effects of Basolateral PP2 on Basolateral CO₂ Dependence of J and J_V

The genistein data suggest that the transduction of the basolateral CO₂ signal to an increase in J requires the activity of tyrosine kinase. Our next step was to examine the effect of PP2, a potent and relatively specific inhibitor of the Src family of sTKs (26, 53), on the basolateral CO₂ dependence of J(Fig. 3A). The protocol for this and the remaining series of experiments was somewhat different from that in Fig. 2. As before, we perfused the lumen with solution 2 throughout the entire experiment. However, during the first collection period shown in Fig. 3, we always perfused the bath with solution 4 (equilibrated 5% CO₂/22 mM HCO3–) without any inhibitor ( at [CO₂]_BL = 5%). During the second collection period, we perfused the bath with solution 5 (i.e., 0% CO₂) ± inhibitor, 4 (i.e., 5% CO₂) ± inhibitor, or 6 (i.e., 20% CO₂) ± inhibitor. The control (i.e., drug-free) data at [CO₂]_BL = 0% () are from an earlier study (61), as are 13 of the control points at [CO₂]_BL = 5% () (61). The 5% control data are augmented by 59 points from the current study. Finally, seven of the control (i.e., drug-free) points at [CO₂]_BL = 20% () are from an earlier study (61), augmented by six DMSO points from Fig. 1C in the present study. As described in METHODS, the values of J(or J_V) in the second collection period were normalized to the mean J(or J_V) value computed from 72 experiments during the first collection period.

View larger version (15K): [in this window] [in a new window]   Fig. 3. Effects of basolateral 250 or 2,000 nM 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidase (PP2) on the [CO2]BL dependence of J(A) and JV (B). In both A and B, the luminal solution was solution 2 throughout the entire experiment. The basolateral solutions were solution 4 (5% CO2) during the first collection periods and solutions 5 (0% CO2), 4 (5% CO2), and 6 (20% CO2), to which was sometimes added either 250 or 2,000 nM PP2, during the second collection periods. Values are means ± SE, with nos. of tubules in parentheses. At 0 and 5% CO2, the statistical comparisons were made using a 1-way ANOVA for 3 groups (the 2 groups in this figure and the data for 35 nM PD168393 in Fig. 4). At 20% CO2, the statistical comparisons were made using a 1-way ANOVA for 5 groups (the 3 groups in this figure and the data for 35 nM PD168393 and 10 nM BPIQ-I in Fig. 4). According to Dunnett's multiple comparison, none of the differences in this figure were statistically significant.

View larger version (16K): [in this window] [in a new window]   Fig. 4. Effects of basolateral 35 nM PD168393 or basolateral 10 nM BPIQ-I on [CO2]BL dependence of J(A) and JV (B). The was moved to the right for legibility. In both A and B, the luminal solution was solution 2 throughout the entire experiment. The basolateral solutions were solution 4 (5% CO2) during the first collection periods and solutions 5 (0% CO2), 4 (5% CO2), and 6 (20% CO2), to which was sometimes added either 35 nM PD168393 or 10 nM BPIQ-I, during the second collection periods. Values are means ± SE, with nos. of tubules in parentheses. At 0 and 5% CO2, the statistical comparisons were made using a 1-way ANOVA for 3 groups (the 2 groups in this figure and the data for 250 nM PP2 in Fig. 3). At 20% CO2, the statistical comparisons were made using a 1-way ANOVA for 5 groups (the 3 groups in this figure and the data for 250 and 2,000 nM PP2 in Fig. 3). According to Dunnett's multiple comparison, the at 5% CO2 is significantly different from the for the J data, and both the and filled diamond at 20% CO2 are significant different from the for J data. *P < 0.05, **P < 0.01.

Our data suggest that the J response of the rabbit S2 PT to basolateral CO₂ does not involve Src. PP2 is also inhibits the Bcr-Abl fusion protein (53), which forms as the Philadelphia chromosome creates a fusion protein of Bcr and Abl (23), which is an sTK, and the Kit family (53) of RTKs. Thus our data also make it unlikely that these kinases are involved in transducing the CO₂ signal.

Effects of Basolateral PD168393 and BPIQ-I on the Basolateral CO₂ dependence of J and J_V

We next examined the effect of PD168393, which is a high-affinity inhibitor (K_i 0.7 nM) (19) of the erbB family of RTKs. The drug covalently reacts with a specific cysteine in the ATP binding pocket (see Table 2) and is thought to be rather specific. Our protocol was the same as in Fig. 3, and the control data in Fig. 4A () are the same as those presented in Fig. 3A. The dependence of J on [CO₂]_BL in the presence of basolateral 35 nM PD168393 is summarized in Fig. 4A (). The statistical analysis of these J data was part of the same J ANOVA that we used to assess the PP2 data in Fig. 3A. Compared with the control condition, PD168393 did not significantly affect J at [CO₂]_BL of 0% (P = 0.998), according to Dunnett's multiple comparison. However, at a [CO₂]_BL of 5%, the drug significantly decreased J from 64 ± 4 to 39 ± 4 pmol·min^–1·mm^–1 (P = 0.011). Moreover, at a [CO₂]_BL of 20%, PD168393 significantly decreased J from 94 ± 2 to 23 ± 3 pmol·min^–1·mm^–1 (P < 0.0001). Thus basolateral 35 nM PD168393 eliminates the PT's J response to basolateral CO₂ in the range 0–20%.

The statistical analysis of the J_V data for PD168393 and BPIQ-I was also part of the same J ANOVA that we used to assess the PP2 data in Fig. 3B. Compared with the control condition with no added inhibitors ( in Fig. 4B), neither PD168393 () nor 10 nM BPIQ-I () produced a significant effect on J_V at any level of [CO₂]_BL according to Dunnett's multiple comparison.

	DISCUSSION

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Use of Out-of-Equilibrium Solutions

Out-of-equilibrium solutions are a powerful tool for performing the experiments outlined in this paper. However, carbonic anhydrase (CA) activity at the extracellular surface of the basolateral membrane would tend to cause a local degradation of the OOE state. Blockade of this activity would independently reduce J and thus be impractical. On the other hand, as we raise [CO₂] from 0 to 5 to 20%, we clearly see major increases in J(e.g., see Fig. 3). Moreover, as shown previously, raising [HCO3–]_BL from 0 to 44 mM causes major decreases in J. Thus any degradation of the OOE state by basolateral CAs must be minor. Nevertheless, to the extent that some degradation of the OOE state does occur, our data underestimate the true magnitude of the response of the tubule to isolated changes in [CO₂]. If a drug added to the PT inhibited all CAs, the result would be an enhancement of the OOE state but a decrease in J. We are unaware of any reports that the drugs used in the present study inhibit CAs.

Inhibitor Specificity

Our previous study (61) demonstrated that PT cells somehow sense acute decreases in [CO₂]_BL and respond by lowering J and somehow sense acute increases in [CO₂]_BL and respond by raising J. In addition, the PT cells change the reabsorption of other solutes (J_Other) in a direction opposite to that of J, thereby minimizing changes in J_V. A key question is how PT cells transduce the CO₂ signal. We hypothesized that an early element in the CO₂ signal-transduction cascade in PT cells is either a receptor tyrosine kinase or a receptor-associated (or soluble) tyrosine kinase.

Genistein is a broad-spectrum tyrosine-kinase inhibitor (24) that blocks both sTKs (e.g., Src family) and RTKs (e.g., erbB family) but exhibits no nonspecific inhibition with other kinases (PKA, PKC) at the concentration we used (24). As showed in Fig. 2A, genistein eliminates the ability of the PT to respond to increases in [CO₂]_BL. These data are consistent with the hypothesis that the CO₂ signal-transduction pathway in the PT cell requires either an sTK or an RTK.

PP2 was developed as a specific inhibitor of the Src family of sTKs (4), but it also inhibits Bcr-Abl (53), which is an sTK, as well as Kit, (53) which is an RTK. Using PTs or PT-like cell lines, others have found that 1–10 µM PP2 can significantly reduce a variety of physiological responses (2, 31, 43, 56). As shown in Fig. 3A, even at a basolateral concentration that is 400-fold greater than its published K_i for Src (26), PP2 did not reduce the CO₂-stimulated increase in J.

PD168393 is a high-affinity, irreversible inhibitor of members of the erbB family of RTKs. It acts by alkylating a cysteine residue [i.e., Cys-773 in human EGF receptor (EGFR)] in the ATP binding pocket (19). Figure 4A shows that, at a concentration 50-fold greater than its published K_i, basolateral PD168393 totally eliminates the response to changes in [CO₂]_BL. Although many authors regard PD168393 as a specific erbB inhibitor, it is in fact impossible to know how specific it is without assaying all tyrosine kinases. A search of human tyrosine kinases reveals a total of eight human tyrosine kinases that have a cysteine residue at a position comparable to Cys-773 in the ATP binding pocket of erbB1 (Table 2). In principle, each of these tyrosine kinases, five RTKs and three sTKs, is a potential target of PD168393. Of these, erbB1 and erbB2 are known to be present in the PT (33, 35), and mRNA transcripts for erbB4 (as well as erbB3) have been reported for the whole kidney (38, 39). TEK or Tie2 (17), the angiopoietin receptor (55), is expressed almost exclusively in endothelial cells as well as certain cancer cells. EphB3 or Hek2 (9), a receptor for the ephrin-B family, plays a key role in neural development but is also expressed in the kidney. ITK plays an important role in T cell activation (28). BLK, which is primarily expressed in hematopoeitic cells (27), is a member of the Src family and thus should have been inhibited by PP2. Finally, JAK3 is expressed primarily in hematopoietic cells, where it interacts with cytokine receptors and plays a role in development and cell activation (54). Thus of the proteins listed in Table 2, erbB1, 2 and 4 as well as EphB3 are prime candidates as targets of PD168393 in our experiments.

BPIQ-I is also a high-affinity inhibitor of EGFR (i.e., erbB1) as well as erbB2 and erbB4. It acts by competing with ATP (44). At a concentration 50-fold greater than its published K_i, basolateral BPIQ-I decreased the CO₂-sensitive component of J by about two-thirds (i.e., decreased total J by 40%) at a [CO₂]_BL of 20% (data not shown). At a concentration 400-fold greater than its published K_i, BPIQ-I blocked virtually 100% of the CO₂-sensitive component of J.

Our observation that both PD-168393 and BPIQ-I block the response to CO₂ increases the odds that, in our experiments, both drugs produce the observed effect by acting on a member of the erbB family.

Model

Previous work from our laboratory showed that simultaneously adding CO₂ and HCO3– to the basolateral, but not the luminal, side of the S3 segment of the rabbit PT triggers a fourfold increase in total H⁺ extrusion (14). The results of our most recent study (61) indicate that this increase is due to basolateral CO₂. The data in the present study suggest that the tubule's response to altered [CO₂]_BL requires one of a small group of tyrosine kinases (see Table 2). Figure 5 summarizes several potential mechanisms by which the PT cell might transduce the CO₂ signal. One possibility (Fig. 5, 1) is that an RTK is simply permissive for the effect of CO₂ on J. Thus blocking the RTK would eliminate the response to CO₂ even though the RTK would not be downstream from the CO₂ receptor. Second (Fig. 5, 2), a CO₂ receptor could activate an PP2-insensitive sTK, which would then raise J. Third (Fig. 5, 3), a CO₂ receptor could transactivate an RTK (57), which would, in turn, raise J. In a variant of this third pathway, the CO₂ receptor could trigger a metalloproteinase to release an EGF-like proligand, which would, in turn, activate EGFR (40). In yet another variant of this third pathway, CO₂ could block the ability of a receptor protein tyrosine phosphatase to transinactivate an sTK or RTK. Finally (Fig. 5, 4), the CO₂ receptor could itself be an RTK. Clearly, additional experiments would be required to establish the molecular identity of the tyrosine kinase involved in the response to CO₂.

View larger version (58K): [in this window] [in a new window]   Fig. 5. Model of how basolateral CO2 might stimulate HCO3– reabsorption by the proximal tubule. On the basis of inhibitor studies, a receptor tyrosine kinase (RTK), an early element in a signal transduction cascade, could be 1) permissive, 2) activated by the CO2 receptor, 3) transactivated by the CO2 receptor, or 4) the CO2 receptor itself. sTK, receptor-associated (or soluble) tyrosine kinase; NHE3, type 3 Na-H exchanger; NBCe1-A, Na-HCO3– cotransporter.

View larger version (9K): [in this window] [in a new window]   Fig. 6. Effects of basolateral drugs on the calculated reabsorption of other solutes (JOther). A: drug-free control experiments. B: basolateral PP2 experiments. C: basolateral PD-168393 or BPIQ-I experiments. For each panel, the J data (points connected by solid lines) are taken from Figs. 3 or 4, whereas the Jother values (points connected by dashed lines) were calculated from the corresponding J and JV values using the equation (2 x J+ Jother)/JV = 300 mosmol/kgH2O.

Finally, in Fig. 6C are the J data obtained in the presence of 35 nM PD168393 (3 connected by solid lines) and 10 nM BPIQ-I (adjacent ), respectively, replotted from Fig. 4A. Also shown are the corresponding J_Other values ( connected by dashed lines and the adjacent ). For PD168393, the J_Other value at 0% CO₂ is not significantly different from that at 5%, whereas the J_Other value at 20% CO₂ is significantly different from that at 5%. For BPIQ-I, the J_Other value at 20% CO₂ () is similar to the PD168393 data at 0 and 5% CO₂. Thus with blockade of the CO₂ response, the trend is for J_Other to be relatively stable as [CO₂]_BL rises. In other words, the effective tyrosine kinase inhibitors not only blocked the J response to CO₂, they also tended to block the J_Other response. These results are consistent with the hypothesis that the tyrosine kinase targeted by PD168393 and BPIQ-I are relatively early in the signal-transduction cascade, that is, before the bifurcation to stimulate J and inhibit J_Other.

It is interesting to speculate that CO₂ sensors similar to that in the PT may be present in other cells that perform large amounts of acid-base transport, as in the choroid plexus, ciliary body, stomach, pancreatic ducts, intestines, and male genital tract. Thus understanding how the PT transduces the CO₂ signal may provide important clues about how such cells regulate acid-base transport.

	GRANTS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was funded by National Institute of Diabetes and Digestive and Kidney Diseases Program Project Grant PO1-DK-17433. P. Bouyer was supported by a National Kidney Foundation fellowship.

	ACKNOWLEDGMENTS

 
We thank Duncan Wong for computer support.

	FOOTNOTES

 

Address for reprint requests and other correspondence: W. F. Boron, Dept. of Cellular and Molecular Physiology, Yale Univ. School of Medicine, 333 Cedar St., New Haven, CT 06520 (e-mail: walter.boron{at}yale.edu)

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.

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