Effect of isolated removal of either basolateral or basolateral CO2 on reabsorption by rabbit S2 proximal tubule

Jinhua Zhao, Yuehan Zhou, and Walter F. Boron

Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520

Submitted 13 January 2003 ; accepted in final form 8 April 2003


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The equilibrium had made it impossible to determine how isolated changes in basolateral CO2 ([CO2]) or concentration ([]), at a fixed basolateral pH, modulate renal or reabsorption. In the present study, we have begun to address this issue by measuring reabsorption (JHCO3) and intracellular pH (pHi) in isolated perfused rabbit S2 proximal tubules exposed to three different basolateral (bath) solutions: 1) equilibrated 5% CO2/22 mM 7.40, 2) an out-of-equilibrium (OOE) solution containing 5% CO2/pH 7.40 but minimal ("pure CO2"), and 3) an OOE solution containing 22 mM 7.40 but minimal CO2 ("pure "). Tubule lumens were constantly perfused with equilibrated 5% CO2/22 mM . Compared with the equilibrated bath solution (JHCO3 = 76.5 ± 7.7 pmol·min1·mm1, pHi = 7.09 ± 0.04), the pure CO2 bath solution increased JHCO3 by ~25% but decreased pHi by 0.19. In contrast, the pure bath solution decreased JHCO3 by 37% but increased pHi by 0.24. Our data are consistent with two competing hypotheses: 1) the isolated removal of basolateral (or CO2) causes a pHi decrease (increase) that in turn raises (lowers) JHCO3; and 2) removal raises JHCO3 by reducing inhibition of basolateral Na/HCO3 cotransport and/or reducing backleak, whereas CO2 removal lowers JHCO3 by reducing stimulation of a CO2 sensor.

bicarbonate; carbon dioxide; intracellular pH; acid-base; volume reabsorption; out-of-equilibrium solutions


THE KIDNEYS, ALONG WITH THE lungs, are one of the two major organ systems that regulate the acid-base balance of the extracellular fluid. A half-century ago, Brazeau and Gilman (6) as well as Dorman et al. (12) showed that acute respiratory acidosis (i.e., an increase in PCO2 that causes a decrease in pH) raises renal reabsorption in whole dogs. Both groups found that isohydric hypercapnia {i.e., a proportional increase in PCO2 and concentration ([]), with no change in pH} raises absolute reabsorption to the same extent as a respiratory acidosis in which PCO2 is elevated to the same degree. They concluded that an increase in CO2, and not the accompanying decrease in blood pH, is the stimulus that elevates reabsorption. However, increasing plasma [] in isohydric hypercapnia also increased the filtered load of and may have had other unintended effects as well. If one expresses reabsorption as the fractional reabsorption of the filtered load, the conclusions of the above studies are quite different: isohydric hypercapnia inhibits reabsorption.

PCO2 also influences reabsorption in the proximal tubule, which is responsible for reabsorbing ~80% of the filtered . Cogan (11) found that acute respiratory alkalosis (lowering plasma PCO2 by 20 mmHg) decreased reabsorption (JHCO3) by ~25% in free-flow micropuncture experiments in rats. Moreover, Sasaki et al. (23) found that although acute metabolic alkalosis (i.e., an increase in basolateral [] and pH with no change in PCO2) inhibits reabsorption, raising PCO2 sufficiently to normalize basolateral pH (i.e., producing isohydric hypercapnia) reverses the inhibition.

The above-mentioned studies clearly demonstrate that an acute increase in PCO2 and/or the accompanying decrease in basolateral pH leads to an increase in JHCO3. Conversely, an acute increase in basolateral [] and/or the accompanying increase in basolateral pH leads to a decrease in proximal reabsorption. However, the equilibrium that interrelates CO2, , and H+ had previously made it impossible to determine the importance of basolateral CO2 per se and of per se, independently of basolateral pH, in controlling proximal reabsorption. In 1995, our laboratory introduced the concept of exploiting the relatively slow equilibrium CO2 + H2O {rightleftarrows} H2CO3 to generate out-of-equilibrium (OOE) solutions (29). The approach is to use a rapid-mixing technique to combine two solutions with different characteristics. With a judicious choice of the above parameters, it is possible to generate OOE solutions with virtually any combination of [CO2], [], and pH, at least within a range of several pH units that encompasses all pH values of pathophysiological interest. In the present study, we exploit this OOE approach to determine the extent to which reabsorption by the S2 segment of the rabbit proximal tubule is affected by the isolated removal of from the basolateral or "bath" solution (keeping bath PCO2 fixed at 5% and bath pH fixed at 7.4), or by the isolated removal of CO2 (keeping bath fixed at 22 mM and bath pH fixed at 7.4). Our results, which are the first to examine an isolated change in CO2 or on any physiological parameter in a vertebrate cell, show that the isolated removal of basolateral stimulates reabsorption, whereas the isolated removal of basolateral CO2 inhibits reabsorption.


    METHODS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Biological Preparation

The Yale Animal Care and Use Committee approved the following procedures. Pathogen-free, female New Zealand White rabbits, obtained from Covance (Denver, PA) and weighing 1.4–2.0 kg, were euthanized by a single overdose of 3 ml (~20 mg) of intravenous pentobarbital sodium. The kidneys were then removed, transferred to a dissection dish, sliced into transverse 1- to 1.50-mm-thick sections, and further dissected by hand to yield individual midcortical1 S2 segments2 1.5–1.7 mm in length. We perfused these tubules in a manner similar to that originally described by Burg and co-workers (7), using several modifications described by Baum et al. (1). In particular, we doubly cannulated the tubule (i.e., using holding, perfusion, and exchange pipettes) at the perfusion end of the tubule and singly cannulated the tubule (i.e., using a holding pipette as well as a calibrated collection pipette with a volume of ~55 nl) at the collection end. The collection pipette was coated with cured Sylgard (Sylgard 184 Silicone Elastomer Kit, Dow Corning, Midland, MI) and filled with liquid Sylgard (the silicone elastomer base) at the distal ~50 µm of the pipette. About 80% of the tubules (depending on how they landed on being transferred to the chamber) were cannulated and perfused orthograde. For these, typically 0.1–0.2 mm of the proximal part of the proximal convoluted tubule was held in the perfusion pipette, and typically 0.2–0.3 mm of the proximal straight tubule was held in the collection pipette. The mean length of perfused tubules in our JV/JHCO3 experiments (where JV is the fluid reabsorption rate), as measured with an eyepiece micrometer, was 1.33 ± 0.06 mm (n = 16 tubules), representing the distal end of the proximal convoluted tubule. The mean luminal perfusion rate in our JV/JHCO3 experiments was 13.0 ± 0.6 nl/min. We superfused the basolateral (i.e., bath) side of the tubule with a solution at 37°C and flowing at 7 ml/min.

Solutions and Experimental Protocol

Dissection and luminal perfusion. The compositions of solutions 1–4 (Table 1) were similar to those described by Baum et al. (1), except for changes required in the composition of solution 4 to make it compatible with the OOE solutions (solutions 5 and 6). The tubules were dissected in Hanks' solution (solution 1) at 4°C. Following transfer of the tubule to the chamber filled with cold Hanks' solution, we perfused the tubule lumen with a solution at 37°C buffered to pH 7.40 with 5% CO2/22 mM (solution 2) and delivered by gravity. In experiments whereby we measured JHCO3, this luminal solution also contained dialyzed [3H]methoxyinulin (~30 µCi/ml).


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Table 1. Physiological solutions

 

Warm-up period. After establishing luminal perfusion, we initiated flow of a 37°C bath "warm-up" solution (solution 3) that was buffered to pH 7.40 with 5% CO2/22 mM . After a 20- to 30-min warm-up period, we switched the bath solution from solution 3 to solution 4 (which was identical to our OOE solutions except that it contained both 5% CO2 and 22 mM ), or to solution 5 or 6 (the OOE solutions). In any case, coincident with the switch to the bath solution for the first data-collection period, we used the calibrated collection pipette to remove and discard the fluid that had accumulated in the holding pipette.

Data collection. We allowed the tubule to stabilize in the bath solution for the first data-collection period for 5–8 min. After this period, we again used the calibrated collection pipette to remove and discard the luminal fluid that had accumulated in the holding pipette and then began a series of three (or four) timed and calibrated collections. The first two (or three) were subsequently analyzed for [3H]methoxyinulin for use in the calculation of JV, and the third was analyzed for total CO2 for use in the calculation of JHCO3. We then usually switched to a different bath solution (solution 4, 5, or 6) for the second data-collection period. We repeated the entire procedure of allowing the tubule to stabilize for 5–8 min in the new solution and then performing three (or four) timed and calibrated collections.

OOE solutions: technical considerations. We generated OOE solutions using the approach outlined in a previous paper (29). In brief, we used a dual-syringe pump (model 55–2222, Harvard Apparatus, South Holliston, MA) to drive two 140-ml syringes (Monoject 140 ml, Sherwood Medical Industries, Ballymoney, UK), one containing solution 5A (or 6A) and the other containing solution 5B (or 6B) (Table 1; see Fig. 1, A and B). The A and B solutions each flowed at 3.5 ml/min, for a total flow of 7 ml/min. Separate lengths of Tygon tubing (1/8-in. outer diameter x . inner diameter, Norton Performance Plastics, Akron, OH) which has a relatively low permeability to CO2, carried the outflow of each syringe to one of two inlets of paired computer-actuated five-way valves (Eagle P/N E4–1PP-00–000, Clippard Instrument Laboratory, Cincinnati, OH). In one position, these two valves, which have zero dead space, sent one pair of solutions (e.g., solutions 5A and 5B) toward the chamber and an alternate pair (e.g., solutions 6A and 6B) to a waste recepticle; in the alternate position, the two valves reversed the destinations of the two pairs of solutions. We actually used a series of four such valve pairs, connected to one another in daisy-chain fashion, so that we could switch among up to five bath solutions. All bath solutions, including the ones that were not OOE, were delivered as described above by a pair of syringes driven by a separate dual-syringe pump.



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Fig. 1. Generating out-of-equilibrium (OOE) solutions. A: arrangement of syringes, valve assembly, stainless-steel tubule, mixing T, and mesh. The portions of the tubing not indicated as stainless steel are made of Tygon. The chemical reactions indicate the decay of the OOE solutions as they flow down the tubing after mixing. B: side view of isolated perfused tubule exposed to an OOE solution. C: concentrations of components. Left: compositions of the solutions inside the 4 syringes for generating solutions 5 ("pure CO2") and 6 ("pure "). Middle: compositions of solutions 5 and 6 at the instant of mixing. Right: compositions of solutions 5 and 6 after the solutions have passed the tubule, ~200 ms after mixing.

 

The two outputs of the valve assembly, carrying solutions 5A+5B or 6A+6B to generate OOE solutions, or carrying two identical streams of either solution 4 or solution 7, were carried by separate lengths of Tygon tubing to separate 22-cm lengths of 15-gauge stainless steel tubing, which, in turn, were enclosed within a jacket that was perfused with water by a circulating bath at ~38°C. The downstream ends of the two lengths of stainless steel tubing were connected to 5-cm lengths of Tygon tubing, which carried the now-warmed pair of solutions to the two arms of a polypropylene T ( x x . Tee P/N 6365–77, Cole-Parmer Instrument, Vernon Hills, IL). The base of the T was connected to a length of Tygon tubing stuffed with nylon mesh (35-µm nylon thread, Small Parts, Miami Lakes, FL) to generate turbulence and thereby ensure adequate mixing of the two components of an OOE solution. The opposite end of the tubing was situated at the entrance to the slot of the chamber (see below); the total volume of the system from the point of mixing at the middle of the T to the beginning of the chamber slot was 14 µl. From within the valve assembly to the T, the two members of a pair of pieces of Tygon tubing were of identical length. This precaution ensured that, following a switch in the valve assembly, each member of the solution pair reached the T at the same instant.

OOE solutions: theoretical considerations. Our OOE approach exploits the slow, first equilibrium in the following reaction sequence

(1)

(2)

(3)
Thus we have substantial and individual control over the CO2 concentration ([CO2]). However, because the reaction in Eq. 2 is very rapid, we cannot independently control the H2CO3 concnetration ([H2CO3]), which at any instant should equal . Similarly, because the reaction in Eq. 3 is also very rapid, we cannot independently control concentration ([]), which at any instant should equal K3 .

Figure 1C summarizes the concentrations of CO2, H2CO3, , and as computed from Eqs. 1–3. Take the pure CO2 solution, for example. As indicated by the top half of the left column on Fig. 1C, solution 5A contained 2,210 µM CO2 (which corresponds to a gas mixture of 10% CO2) at a pH of 5.40 (2 pH units below the target pH of 7.40). Because the pK of the overall equilibrium is ~6.1, the [] in solution 5A was 441 µM. The top half of the middle column of Fig. 1C shows, at the idealized instant of perfect mixing, the result of combining solutions 5 and 6 in a 1:1 ratio. This OOE pure CO2 solution contained 1,105 µM CO2 (which corresponds to a gas mixture of 5% CO2) and ~223 µM ; this is ~1% of the value in an equilibrated 5% CO2/22 mM solution. In principle, we could have reduced the contaminating [] even further by lowering the pH of solution 5A below 5.40. The bottom half of Fig. 1C provides a similar analysis for the pure solution. Because solution 6B contained 44 mM total at a pH of 9.40 (2 pH units above the target pH of 7.40), the [CO2] in solution 6B was 19.6 µM (which corresponds to a gas mixture of 0.09% CO2). Thus mixing this solution in a 1:1 ratio with solution 6A initially yielded an OOE pure solution containing nearly 22 mM and 9.8 µM CO2 (which corresponds to a gas mixture of 0.04% CO2); this [CO2] is <1% of the value in an equilibrated 5% CO2/22 mM solution. Again, we could have reduced the contaminating [CO2] even further by raising the pH of solution 6B above 9.40.

Our design criteria for the pure CO2 OOE solution (solution 5) were that solution 5B should be heavily buffered but have a pH just high enough (i.e., 7.55) to drive the pH of the OOE solution to 7.40. This combination of design features yields an OOE solution, as measured with a PCO2 electrode (MI 720, Microelectrodes, Londonderry, NH) at the end of the Tygon tubing that connects to the chamber, that is indistinguishable from 5%. In our experience, attempting to generate a pure CO2 solution by using a solution B with an extremely high pH (e.g., pH 10) yielded an OOE solution with a [CO2] markedly lower than the target value. We presume that rapid mixing of solutions A and B creates short-lived microscopic pockets with a high [CO2] (contributed by solution A) and a high [OH] (contributed by solution B) that allows the uncatalyzed reaction to consume CO2 at a high rate.

Our design criteria for the pure solution (solution 6) were that solution 6A should be heavily buffered but have a pH just low enough (i.e., 6.99) to drive the pH of the OOE solution to 7.40. This approach yields an OOE solution that, when assayed as described in the preceding paragraph, has a PCO2 indistinguishable from zero. Attempts to generate a pure solution by using a solution A with an extremely low pH (e.g., 5.00) yielded an OOE solution with a [CO2] markedly higher than zero. We presume that short-lived microscopic pockets with a high [] (from solution B) and a low pH/high [H+] (from solution A) allowed the reaction to raise [H2CO3] to such high levels that the uncatalyzed reaction H2CO3 -> CO2 + H2O produces CO2 at a high rate.

Because of the considerations discussed in the two previous paragraphs, we suspect that it might be challenging to generate pure CO2 solutions at an extremely alkaline target pH or pure solutions at an extremely acidic target pH.

Finally, it should be noted that we employed OOE solutions only in the bath. Although it is theoretically possible to generate OOE solutions for the lumen, the mixing of the A and B solutions would be extremely challenging, and the relatively slow transit of the fluid down the lumen would permit more extensive equilibration than we observe in the bath.

The chamber. The chamber (see Fig. 1B), which was machined out of polycarbonate, contained a channel 14-mm long x 2.5-mm wide by 2.5-mm high. The bottom of the slot was formed by a coverslip. About one-fourth of the way (measured from the solution inflow) along this slot was the perfused tubule, with its long axis parallel to the axis of bath flow. The chamber sat atop a heating plate (series 20 chamber and platform with integral TS70B thermistor, Warner Instruments, Hamden, CT) that was clamped to a temperature of 41°C. The combination of the prewarmed solution and the heating plate yielded a bath temperature of 37.5°C (confirmed with a miniature electronic temperature probe Digi-Sense with a type-K thermocouple, Cole Parmer). A length of Tygon tubing from the T (see above) entered the chamber through a port that was colinear with the long axis of the slot. As it terminated at the entrance to the slot, the Tygon tubing had an inner diameter of 1.6 mm, which was modestly less than the width of the chamber slot (2.5 mm) to promote laminar flow along the slot and minimize the formation of dead zones. Such dead zones would have permitted the equilibration of the bath solution within these zones and, to some extent, thwarted our goal of surrounding the tubule with an OOE solution. At the opposite end of the slot, the bath solution was continuously sucked away. The height of the bath solution within the slot was ~1.2 mm.

We calculate that the time required for the newly mixed solution (7 ml/min) to travel from the middle of the T to the tubule (total volume = 14 µl for the tubing + 10 µl for 1/4 of the chamber slot = 24 µl) was ~200 ms. Given an extracellular CO2 concentration ([CO2]o) of 1,105 µM3 for solution 5 at the instant of mixing (see middle column of Fig. 1C, top portion) and a rate constant of 0.08 s1, the reaction CO2 + H2O -> H2CO3 (Eq. 1) would consume CO2 and produce H2CO3 at the rate of ~88 µM/s. Thus after 200 ms (see right column of Fig. 1C, top portion), the preceding reaction would consume 17.7 µM CO2 or ~1.6% of the original 1,105 µM CO2. Simultaneously, the reaction (Eq. 2) would produce 17.7 µM , which represents 0.08% of the present in an equilibrated 5% CO2/22 mM solution. As a result, pH would fall by a trivial amount, [CO2] would fall to 1,087 µM CO2 (which corresponds to a gas mixture of 4.92% CO2), and [] would rise by a trivial amount.

Conversely, given an extracellular concentration () of 22 mM for solution 6 at the instant of mixing, the initial [H2CO3] would be 2.765 µM (see middle column of Fig. 1C, bottom portion). Because the reaction H2CO3 -> CO2 + H2O (Eq. 1) has a rate constant of 32 s1, the preceding reaction would consume H2CO3 and produce CO2 at the rate of ~88 µM s1. Thus, after 200 ms (see right column of Fig. 1C, bottom portion), the preceding reaction would produce 17.7 µM CO2, or ~1.6% of that contained in an equilibrated 5% CO2/22 mM solution. As a result, pH would rise by a trivial amount, [] would fall by a trivial amount, and [CO2] would rise to 27.5 µM (which corresponds to a gas mixture of 0.12% CO2). In a parallel series of experiments, we used a pair of liquid-membrane pH-sensitive electrodes to behave as a double-barreled PCO2 microelectrode (28). We used this combination to monitor PCO2 in the chamber slot at room temperature under conditions mimicking those in the kidney-tubule experiments. While generating a pure solution, we found that the [CO2] in the chamber slot was indistinguishable from zero (not shown). However, this gratifying result was achieved only after employment of the above considerations in chamber design.

Osmolality and pH of solutions. The osmolality of all solutions was measured using a vapor-pressure osmometer (model 5100C, Wescor, Logan, UT). The osmolality was adjusted, if necessary, to 300 ± 2 mosmol/kgH2O by adding either water or NaCl. The pH was adjusted to the value indicated in Table 1 using either HCl or NaOH. All solutions were made using water filtered through a Milli-Q biocel system (Millipore, Bedford, MA).

Measurement of JHCO3 and JV

We measured JHCO3 and JV using an approach similar to that used by McKinney and Burg (16). We assayed total CO2 in aliquots of the perfusate and collected fluid using a WPI NanoFlo device (World Precision Instruments, Sarasota, FL) to measure the -dependent decrease in the fluorescence at 340 nm of NADH as phosphoenolpyruvate carboxylase (from "plant") and malate dehydrogenase (from porcine heart), respectively, catalyzed the following two reactions (13, 26)


We purchased the above reagents as a kit (Diagnostic Kit 132-A, Sigma-Aldrich, St. Louis, MO). Given the fluid collection rate (Vi), the tubule length (L), the concentration of total CO2 in the luminal perfusate ([TCO2]o) and collected luminal fluid ([TCO2]i), and JV (see below), we used the following equation to compute JHCO3

We report JHCO3 in picomoles divided by the product of minutes and millimeters tubule length.

We computed JV from Vi, L, and the concentration of [3H]methoxyinulin in the perfusate (Co) and collected luminal fluid (Ci)

We report JV in nanoliters divided by the product of minutes and millimeters tubule length. We accepted the data only if JV was positive and if the computed concentration of reabsorbed NaHCO3 (i.e., the ratio JHCO3/JV) was less than ~160 mM.4

Measurement of Intracellular pH

We calculated intracellular pH (pHi) from the fluorescence excitation ratio of BCECF using the approach outlined previously for osteoclasts (21). We loaded perfused tubules with the dye by exposing them from the bath at room temperature for ~5 min to solution 7 containing 10 µM of BCECF-AM (B-1170, Molecular Probes, Eugene, OR). The microscope was a Zeiss IM-35 inverted microscope, equipped with a x40/numerical aperture 0.85 objective and apparatus for epiillumination. The light source in the fluorescence experiments was a 100-W tungsten halogen lamp. Using a system of dual filter wheels (Ludl Electronic Products, Hawthorne, NY), one wheel carrying chromic filters and the other carrying neutral-density filters, we alternately excited the entire field with light in wavelengths of 495 ± 5 and 440 ± 5 nm (Thermo Oriel, Stratford, CT), using the neutral-density filters to equalize, as nearly as possible, the intensity of incident light at the two wavelengths. The emitted fluorescent light, after passing through a 510-nm long-pass dichroic mirror and a 530-nm long-pass filter in the filter cube beneath the turret, was amplified by an image intensifier (KS-1381 intensifier, Video Scope, Dulles, VA), before it was captured with a charge-coupled-device camera (CCD 72, Dage M.T.I., Michigan City, IN).

To limit both photobleaching of the dye and photodynamic damage of the tubule cells, we limited illumination to ~370 ms for the 490-nm light (producing an I490 image), followed immediately by ~370 ms for the 440-nm light (producing an I440 image). This pair of excitations was repeated at intervals varying from 6 to 20 s; between the 490- and 440-nm excitations, we kept the tubule in the dark by closing the shutter on the filter wheel. Each cycle of excitations had the following protocol.The filter wheel was rotated, under computer control, to put the 490-nm filter in the light path. With the shutter of the filter wheel still closed, we snapped an image to be used for background subtraction and then opened the shutter. After a delay of 100 ms to allow for stabilization of the shutter, we collected and averaged four successive video frames using an image-processing board (DT3155, Data Translation, Marlboro, MA). The shutter was then closed and, after storage of the averaged I490 image, the 440-nm filter was rotated into position and the I440 image was obtained in the same way. Finally, we subtracted the average pixel intensity of the matched background image (see above) from the I490 and I440 images. Software developed in our laboratory, using the Optimas (Media Cybernetics, Silver Spring, MD) platform, controlled all aspects of data acquisition and real-time storage to the hard disk of an Intel-based computer running Microsoft Windows 98SE. This software also allowed us to monitor I490/I440 fluorescence-excitation ratios for selected areas of interest during the experiments. After the experiment, we transferred the data to a CD-ROM for later analysis using Optimas-based software. For the analysis, we outlined two areas of interest, each of which typically represented 9% of the tubule length. In BCECF-loaded tubules, the mean I490 signal was typically ~3,000-fold greater than the I490 signal in tubules not loaded with BCECF. The sum of the I490 values of the pixels in the area of interest was divided by the sum of the corresponding I440 values. The resulting ratio is strongly dependent on pHi but relatively insensitive to other factors, such as dye concentration.

We converted the I490/I440 ratios to pHi values using the high-K+/nigericin technique of Thomas et al. (27), as modified by Boyarsky et al. (4), to permit a one-point calibration at pHi 7.00 for each experiment. At the end of each experiment, we introduced into the bath a pH 7.00 high-K+/nigericin solution (100 mM KCl, 32.2 mM HEPES-free acid, 1 mM CaCl2, 1.2 mM MgSO4, titrated to pH 7.00 with ~32.8 mM N-methyl-D-glucamine) that drives pHi toward 7.00. Potential limitations of this approach are discussed elsewhere (8). The I490/I440 ratios of the entire experiment were normalized by dividing them by the I490/I440 ratio corresponding to pHi 7.00, and we then calculated pHi using the following equation (4)

The values of pK and b were determined from a separate series of experiments in which we exposed the basolateral sides of isolated perfused tubules to a series of solutions containing 10 µM nigericin at 10 different pH values (pH 5.8–8.5 in increments of 0.3 pH units), always including pH 7.00. The plot of (I490/I440)/(I490/I440)pH=7 vs. pHi (not shown), obtained from a total of 79 fluorescence measurements (1 measurement·pH value1·tubule1, not including measurements at pHi 7.00) on a total of 19 tubules, was a typical pH titration curve. The data were fitted by the above equation, which forces the best-fit curve to pass through unity at pHi = 7.00, using a nonlinear least-squares method. The best-fit values, which we used in the above calibration equation for each experiment, were pK = 7.08 ± (SD) 0.02 and b = 1.326 ± (SD) 0.029.

Because the nigericin used in the calibration can stick to the tubing and valves and later dissociate and enter nominally nigericin-free solutions in later experiments (2, 22), at the end of each pHi experiment we routinely flushed ethanol (70% in water) and then water (filtered through a Milli-Q biocel system) through the tubing and valves that carried the nigericin to the chamber, as well as the chamber itself.

Data Analysis

Statistical analyses were performed using the Analysis Toolpack of Microsoft Excel. Two sets of data were considered significantly different if the P value of the paired or unpaired t-test was <0.05. Results are given as means ± SE, with the number of tubules (n) from which it was calculated.


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Control JHCO3 and JV Studies

In the JHCO3/JV experiments described in the next two subsections, we constantly perfused the lumen of the tubule with an equilibrated 5% CO2/22 mM at pH 7.40 (Table 1, solution 2) containing [3H]methoxyinulin. We usually collected luminal samples for data analysis during two periods, each characterized by different bath solutions that were delivered from different pairs of syringes, driven by different syringe pumps. To verify that these two data-collection periods are equivalent, we performed control experiments in which we delivered the standard equilibrated solution (solution 4) for each of the two data-collection periods, first from one pair of syringes driven by one pump, and then from another pair of syringes driven by another pump. As summarized in Fig. 2, we observed no significant difference between the two data-collection periods for either JHCO3 or JV.



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Fig. 2. Control reabsorption (JHCO3; A) and fluid reabsorption rate (JV; B) studies. Each experiment consisted of 2 collection periods. During each, solution 4 (standard; equilibrated 5% CO2/22 mM ) flowed through the bath. However, in the 2 periods this solution was delivered from 2 different pairs of syringes, driven by 2 different syringe pumps, to mimic the protocol used in Fig. 3. Values are means ± SE, with nos. of tubules in parentheses.The differences between the 2 mean values for JHCO3 (P = 0.48) and between the 2 mean values for JV (P = 0.23) were not statistically significant (paired, 2-tailed t-test).

 



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Fig. 3. Effect on JHCO3 (A) and JV (B) of the isolated removal of either bath or bath CO2. In both A and B, the bath solutions were solution 5 (pure CO2 OOE; hatched bars), solution 4 (standard; equilibrated 5% CO2/22 mM ; open bars), and solution 6 (pure OOE; crosshatched bars). Values are means ± SE, with nos. of tubules in parentheses. The difference between each of the hatched and crosshatched bars and the corresponding open bar is statistically significant in unpaired, 2-tailed t-tests (*P < 0.05, **P < 0.01).

 
Effect on JHCO3 and JV of Isolated Removal of Basolateral

A comparison of the open and hatched bars on Fig. 3, A and B, illustrates the effect of using the OOE approach to remove basolateral while holding bath [CO2] fixed at 5% and holding bath pH fixed at 7.40. Compared with those exposed to the equilibrated 5% CO2/22 mM solution at pH 7.40 (open bars), tubules exposed to the corresponding pure CO2 solution (hatched bars) had a mean JHCO3 that was >25% higher (96.5 vs. 76.5 pmol·min1·mm1). JV was ~61% higher (1.03 vs. 0.64 nl·min1·mm1). Viewed differently, the presence of basolateral reduces the reabsorption of both and fluid.

Considering only the data for the equilibrated 5% CO2/22 mM solution at pH 7.40 (open bars in Fig. 3, A and B), our mean JHCO3 value of 76.5 pmol·min1·mm1 is similar to the value of 52.3 pmol·min1·mm1 reported by Baum et al. (1) for midcortical and juxtamedullary proximal convoluted tubules. Similarly, our mean JV value of 0.64 nl·min1·mm1 is similar to the value of 0.72 nl·min1·mm1 reported by Baum et al. and is also similar to the value of ~0.5 nl·min1·mm1 obtained by Quigley et al. (20) on superficial S2 segments of rabbit proximal tubules.

Effect on JHCO3 and JV of Isolated Removal of Basolateral CO2

A comparison of the open and crosshatched bars in Fig. 3, A and B, illustrates the effect of using the OOE approach to remove basolateral CO2 while holding bath [] fixed at 22 mM and holding bath pH fixed at 7.40. Compared with tubules exposed to the equilibrated 5% CO2/22 mM solution at pH 7.40, those exposed to the corresponding pure solution had a JHCO3 that was nearly 40% lower (48.4 vs. 76.5 pmol·min1·mm1). JV was nearly 25% lower (0.49 vs. 0.64 nl·min1·mm1). Viewed differently, adding basolateral CO2 to a pure solution enhances the reabsorption of both and fluid.

Effect on pHi of Isolated Removal of Either Basolateral or CO2

One would expect that tubules exposed to the different bath solutions summarized in Fig. 3 would have different values of pHi. Because such changes in pHi could conceivably affect various solute transporters and thereby alter JHCO3 and/or JV, we examined the effect on pHi of switching the bath solution from 5% CO2/22 mM at pH 7.40 to the corresponding OOE solutions lacking either or CO2. Figure 4A shows an experiment in which we used BCECF to monitor pHi in cells of an isolated perfused S2 segment. Initially, the luminal and bath solutions were buffered with HEPES (Table 1, solution 7). In a total of 38 tubules under these conditions, the mean initial pHi was 7.19 ± 0.03. For two reasons, this value is substantially higher than the values of 6.89–6.92 previously reported by this laboratory for rabbit S3 segments (9, 17). First, mean pHi values steadily increase from the S3 to S2 to S1 segments (14). Second, the luminal solution in the present study contained lactate, which, presumably like lactate in the salamander proximal tubule (24) and acetate in the rabbit S3 segment (18), produces a intracellular alkalinization as it enters the cell with Na+ across the apical membrane and exits with H+ across the basolateral membrane.



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Fig. 4. Effect on intracellular pH (pHi) of the isolated removal of either bath or bath CO2. A: representative experiment. See text for discussion of points a-h. B: data summary. Values are means ± SE, with nos. of tubules in parentheses. The difference between each of the hatched and crosshatched bars and the corresponding open bar is statistically significant in unpaired, 2-tailed t-tests (**P < 0.01).

 

Switching the lumen to 5% CO2/22 mM (solution 2) in Fig. 4A caused an abrupt and sustained decrease in pHi (segment ab), reflecting the influx of CO2 across the apical membrane, the intracellular reaction , and the continuous exit of across the basolateral membrane via the electrogenic Na-HCO3 cotransporter (3). The mean pHi under these conditions was 6.99 ± 0.02 (n = 38).

At point b we added equilibrated 5% CO2/22 mM (solution 4) to the bath in the continued presence of luminal . On one hand, we expect this maneuver to cause an abrupt pHi decrease as the basolateral influx of CO2 leads to the intracellular generation of H+ and and to cause a sustained pHi decrease as the elevated intracellular [] () promotes the efflux of through the electrogenic Na-HCO3 cotransporter. On the other hand, based on previous results from this laboratory in rabbit S3 segments (9, 10, 17), we expect the addition of basolateral to cause an increase in pHi for two reasons. First, the increase in bath [] would tend to slow net efflux across the basolateral membrane. Second, basolateral CO2 and/or stimulates apical Na/H exchange and H+ pumping. In our experiments, the alkalinizing tendencies dominated (bc) as the mean pHi increased from 6.97 ± 0.02 to 7.09 ± 0.04 (n = 18; P < 0.0002, paired 2-tailed t-test). The latter value is also summarized by the open bar in Fig. 4B. The absence of a transient, CO2-induced acidification during bc in Fig. 4A probably reflects the limited time resolution of data acquisition.

Removing from the bath in Fig. 4A caused a transient pHi increase due to the basolateral efflux of CO2, followed by a relaxation of pHi (cd). At point d, we switched the bath to the pure CO2 solution (solution 5). The abrupt pHi decrease is the consequence of the CO2 influx and formation of H+ and . We expect the new steady-state pHi to depend on the balance between enhanced efflux (reflecting the increased ) via electrogenic Na-HCO3 cotransport (which would tend to lower pHi) and enhanced H+ extrusion if it is basolateral CO2 that stimulates apical Na/H exchange and H+ pumping. Under the conditions of our study, the acidifying influences dominated (de) as the mean pHi fell from 7.02 ± 0.03 to 6.90 ± 0.03 (n = 17; P < 0.001, paired 2-tailed t-test). The latter condition, with one additional data point, is also summarized by the hatched bar in Fig. 4B. Note that it is under this pure CO2 condition that reabsorption was maximal (see Fig. 3A).

Removing pure CO2 from the bath (Fig. 4A) caused a rebounding pHi increase (ef). At point f, we introduced the pure solution (solution 6) to the bath. We expect the increase in bath [] to inhibit efflux via electrogenic Na-HCO3 cotransport, or even to produce a net influx of . Indeed, we observed a sustained pHi increase (fg) as the mean pHi rose from 7.05 ± 0.06 to 7.33 ± 0.13 (n = 6; P < 0.02, paired 2-tailed t-test). The latter condition, with one additional data point, is also summarized by the crosshatched bar in Fig. 4B. Note that it is under this pure condition that reabsorption was minimal (see Fig. 3A).

Finally, removing the pure solution from the bath causes pHi to drift toward its initial value (gh).


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The pioneering work of others on the impact of acid-base disturbances on renal acid-base transport was necessarily confined by equilibrium, which made it impossible to change just one parameter in the Henderson-Hasselbalch equation. In the present experiments, we have applied OOE technology to renal tubules and have made the first observations of the effects of the isolated basolateral removal of either CO2 or on JHCO3 and pHi. We found that basolateral [CO2] per se and basolateral [] per se are critical parameters for regulating both transepithelial acid-base transport and pHi. The isolated removal of from the basolateral solution, at a fixed [CO2] and pH, raises JHCO3 and lowers pHi. The isolated removal of basolateral CO2 from the basolateral solution, at a fixed [] and pH, produces the opposite effects.

Removing Basolateral Stimulates Reabsorption

As shown in Fig. 5, which is a replot of the relationship between the JHCO3 data in Fig. 3A and the pHi data in Fig. 4B, one way of interpreting the data from the present study is that JHCO3 depends mainly on pHi and that removing basolateral enhances reabsorption by lowering pHi. Although the present data are consistent with this hypothesis, the hypothesis suffers from three weaknesses.



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Fig. 5. Relationship between JHCO3 and pHi. The pHi data are from Fig. 4B, and the JHCO3 data are from Fig. 3A.

 

First, the pHi-dependency model requires that decreasing pHi from 7.1 to 6.9 would increase apical H+ extrusion via the Na/H exchanger and H+ pump (see Fig. 6) by 50%, whereas, at least in the S3 segment of the rabbit proximal tubule, actual data show that acid extrusion is remarkably insensitive to pHi changes in the above pHi range, particularly in the absence of (9, 10).



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Fig. 6. Model of reabsorption by the proximal tubule. Although the CO2 sensor is shown as facing outward from the basolateral membrane, the sensor could face inward from the basolateral membrane, or it could be in the cytosol near the basolateral membrane.

 

Second, the pHi-dependency model requires that decreasing pHi from 7.1 to 6.9 would increase the basolateral efflux of by 50% because changes in basolateral efflux must, in the steady state, parallel changes in apical H+ extrusion. Although the pHi dependence of the electrogenic Na-HCO3 cotransporter (NBC) in the proximal tubule is unknown, one would expect that, if anything, lowering pHi per se would inhibit NBC, just as lowering pHi inhibits exchange (5, 19, 25). For example, lowering pHi from 7.1 to 6.9 at a fixed extracellular [CO2] of 5% would lower the concentration of intracellular , the ostensible substrate for NBC, from ~11 to ~7 mM.

Finally, it is important to address the mechanism by which removing basolateral lowers pHi. Imagine that we start with equilibrated in the bath. Removing bath should enhance basolateral efflux via NBC, thereby lowering pHi. Note that enhancing the basolateral efflux of is tantamount to enhancing reabsorption. According to this analysis, the fall in pHi is the result of enhanced reabsorption rather than the cause of it.

How then might the removal of basolateral enhance reabsorption? One possibility is that the reduction in basolateral [] slows or even reverses the backflux of through the tight junctions from the bath to the lumen. Another possibility, which is not mutually exclusive, is that the reduction in basolateral [] enhances the net offloading of at the extracellular face of NBC.

Removing Basolateral CO2 Inhibits Reabsorption

Given the relationship between JHCO3 and pHi summarized in Fig. 5, the most tempting explanation for why removing basolateral CO2 inhibits reabsorption by 37% is that the removal of CO2 causes pHi to increase, which in turn inhibits apical H+ extrusion. The present data do not rule out this hypothesis. However, if the hypothesis is true, then we must explain how the pHi increase also inhibits basolateral efflux, because as the cell approaches the new steady state, apical H+ extrusion and basolateral efflux must somehow fall in parallel. We expect that the rise in pHi per se should, if anything, increase the basolateral efflux of . Moreover, at a fixed intracellular [CO2] ([CO2]i), a rise in pHi ought to increase , which should further increase the basolateral efflux of . On the other hand, the decrease in bath [CO2] should lead to a fall in [CO2]i, which would mitigate the rise in and thereby perhaps slow efflux. Thus, for the pHi hypothesis to be correct, removing bath CO2 must indeed lower , and the inhibitory effect on NBC of lowering per se must overwhelm the stimulatory effect of raising pHi per se.

As shown in Fig. 4B, removing bath CO2 caused pHi to rise from 7.09 to 7.33, and we calculate that may have simultaneously fallen from 10.8 to 9.4 mM.5 Even if the Km for intracellular were 10 mM, a decrease in from 10.8 to 9.4 mM would decrease the transport rate by only a trivial amount. An additional complication is that preliminary data indicate that when NBC operates with a Na+: stoichiometry of 1:2, it transports rather than (15). Thus it is likely that NBC in the proximal tubule, which appears to function with Na+: stoichiometry of 1:3, transports one Na+, one , and one . We calculate that the removal of bath CO2 actually causes to rise from 6.7 to 10.1 µM,6 and predict that, if anything, this rise in would stimulate NBC. In other words, for the pHi hypothesis to be correct, the anticipated inhibitory effect of decreasing by 13% would have to outweigh the anticipated stimulatory effects of raising pHi by 0.24 and raising by ~50% and produce a net 37% decrease in electrogenic Na-HCO3 cotransport.

As an alternative hypothesis, we suggest that basolateral CO2 per se may directly enhance the machinery of reabsorption, stimulating in parallel both apical H+ extrusion and basolateral efflux. According to this hypothesis, changes in steady-state pHi caused by removing or adding back CO2 are the consequence, rather than the cause, of altered rates of H+ and transport. This CO2-triggering hypothesis is consistent with earlier observations that the application of basolateral , but not of luminal , causes an increase in both steady-state pHi (17) and H+ extrusion rates (9, 10). If this hypothesis is correct, then it would have been the CO2, and not the , present in the earlier equilibrated solutions that caused proximal tubule steady-state pHi and H+ extrusion rates to increase. Further experiments will be required to distinguish between the pHi hypothesis and the alternative CO2-sensor hypothesis for producing the observed changes in JHCO3.


    DISCLOSURES
 
This work was supported by National Institutes of Health Program Project Grant PO1-DK-17433. J. Zhao was supported by fellowships from the American Heart Association and the American Lung Association.


    ACKNOWLEDGMENTS
 
We thank Dr. Raymond Quigley for advice in measuring reabsorption and fluid reabsorption rates. We are indebted to Duncan Wong for computer assistance.


    FOOTNOTES
 

Address for reprint requests and other correspondence: W. F. Boron, Dept. of Cellular and Molecular Physiology, Yale University School of Medicine, 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.

1 The junction between the proximal straight tubule and proximal convoluted tubule was in the midcortex. Back

2 The dissected length of tubule included the distal-most 1.3–1.4 mm of the proximal convoluted tubule plus the proximal-most 0.2–0.3 mm of proximal straight tubule. Back

3 Assuming 1) a temperature of 37°C and thus a PH2O of 47 mmHg, 2) a barometric pressure of 760 mmHg, 3)5%CO2 in the gas cylinder, and 4) a CO2 solubility of 0.031 mM/mmHg. Thus [CO2] = (760 – 47) x 5% x 0.031 = 1.105 mM = 1,105 µM. Back

4 The reabsorbate of the proximal tubule is very close to isosmotic. Even if the reabsorbate were pure NaHCO3, its [] could be no higher than one-half the osmolality (assuming an osmotic coefficient of unity). Back

5 If we assume that [CO2]i is 5% when 5% CO2 is present in both lumen and bath, but only 2.5% when CO2 in present in the lumen but not the bath, then removing bath CO2 causes to fall from 10.8 to 9.4 mM. Back

6 We assume that the pK of the reaction is 10.3 and thus that . When CO2 is present in the bath, pHi is 7.09 and the computed is 10.8 mM. We calculated that under these conditions, is 6.7 µM. When CO2 is absent from the bath, pHi is 7.33 and we assume that is 9.4 mM (see footnote 5). We calculate that under these conditions, is 10.1 µM. Back


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