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Am J Physiol Renal Physiol 274: F139-F147, 1998;
0363-6127/98 $5.00
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Vol. 274, Issue 1, F139-F147, January 1998

HCOminus 3 absorption in rabbit outer medullary collecting duct: role of luminal carbonic anhydrase

Shuichi Tsuruoka and George J. Schwartz

Departments of Pediatrics and Medicine, University of Rochester School of Medicine, Rochester, New York 14642

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Membrane-bound luminal carbonic anhydrase (CA) IV, by catalyzing the dehydration of carbonic acid into CO2 plus water, facilitates H+ secretion in the renal outer medullary collecting duct from the inner stripe (OMCDi). To examine the role of CA IV on H+ secretion, we measured net HCO-3 transport in perfused OMCDi segments and examined the effect on transport of two extracellular CA inhibitors, benzolamide and F-3500, aminobenzolamide coupled to a nontoxic polymer, polyoxyethylene bis(acetic acid) [synthesized and kindly provided by C. Conroy and T. Maren (C. W. Conroy, G. C. Wynns, and T. H. Maren. Bioorg. Chem. 24: 262-272, 1996)]. These agents would inhibit only the luminal CA enzyme. Dose titration curves for net HCO-3 flux were performed for each drug. Basal HCO-3 absorptive flux was 12 pmol · min-1 · mm-1 in control segments and significantly increased to 16 pmol · min-1 · mm-1 in segments from 3-day acid-treated animals. The concentrations of benzolamide and F-3500 that inhibited HCO-3 absorption by 50% were ~0.1 and ~5 µM, similar to the Ki for CA IV inhibition by these agents (0.2 and 4.0 µM, respectively; T. Maren, C. W. Conroy, G. C. Wynns, and D. R. Godman. J. Pharmacol. Exp. Ther. 280: 98-104, 1997). Adding exogenous CA to the inhibitor in the perfusate nearly restored basal HCO-3 transport, suggesting that cytosolic CA II was not inhibited by these impermeant inhibitors. In OMCDi segments from acidotic rabbits, the concentrations of benzolamide and F-3500 that inhibited HCO-3 absorption by 50% were 50 and 500 µM, respectively, >100 times the Ki for CA IV inhibition and for inhibition of HCO-3 transport in control tubules. Thus, in the OMCDi, doses of extracellular CA inhibitors that inhibited ~50% of CA IV activity also comparably inhibited HCO-3 transport, indicating that H+ secretion depends in part on the availability of luminal CA IV activity. Acidosis substantially decreased the sensitivity of HCO-3 transport to CA inhibition.

inner stripe of outer medulla; kidney; benzolamide; aminobenzolamide; acid-base homeostasis; carbonic anhydrase IV

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

THE KIDNEY PLAYS a major role in regulating acid-base homeostasis, with the final control occurring in the collecting duct. Inhibition of luminal carbonic anhydrase (CA) results in an acid disequilibrium pH in the outer medullary collecting duct (OMCD) (40), indicating that H+ secretion, rather than direct HCO-3 absorption, is the major mechanism of acidification in this segment. Acid excretion there is mediated by H+ secretion via a vacuolar H+-adenosinetriphosphatase (H+-ATPase) and a P-type gastric-like H+-K+-ATPase (1, 2, 43). Under the pathophysiological condition of metabolic acidosis, the cortical collecting duct reverses the polarity of its flux from net secretion of HCO-3 to net proton secretion (3, 27, 35, 41), whereas the OMCD (43) and inner medullary collecting duct (IMCD) (5, 12, 46) increase their H+ secretion rates. Adaptation to acidosis is likely to occur at the level of the H+-secreting cell along the collecting duct (4, 18-20, 44).

Urinary acidification is believed to be facilitated by the enzyme CA, which exists in two isoforms. More than 95% of the activity is located in the cytosol as CA II, whereas up to 5% is membrane bound and corresponds to CA IV (7, 26, 48). CA IV catalyzes the dehydration of intraluminal carbonic acid that results from the secretion of protons into the lumen (11) in the reaction
H<SUP>+</SUP> + HCO<SUP>−</SUP><SUB>3</SUB> &cjs0416; H<SUB>2</SUB>CO<SUB>3</SUB> <AR><R><C>CA</C></R><R><C>⇌</C></R></AR> CO<SUB>2</SUB> + H<SUB>2</SUB>O (1)
Membrane-bound CA IV activity has been detected in the apical membranes of intercalated cells (16, 31), and functional studies have identified luminal CA activity along the inner stripe of rabbit OMCD (OMCDi) (40) and in the initial segment of rat IMCD (45). It is likely that CA IV mediates renal H+ secretion, because, in CA II-deficient patients and mice, inhibition of CA activity diminishes renal acid excretion (6, 39). Moreover, during metabolic acidosis, CA IV mRNA was found to be increased in the renal cortex and outer medulla (47), and sodium dodecyl sulfate (SDS)-resistant hydratase activity (presumably CA IV activity) was increased in the cortex, with the increment in the outer medulla not reaching statistical significance (7).

The purpose of the present study was to use extracellular CA inhibitors to investigate the role of CA IV in the maintenance of H+ secretion (HCO-3 absorption) at the level of the isolated perfused OMCDi of rabbit kidney. Similarly, a second aim was to examine the role of CA IV in the OMCDi taken from acid-treated animals, since increases in H+ secretion (43) and in CA IV mRNA have been demonstrated in OMCDi during chronic metabolic acidosis (42). We made use of a newly synthesized membrane-impermeant, high-molecular-weight CA inhibitor to inhibit just the luminal enzyme in many of the transport experiments.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Animals

Female New Zealand White rabbits weighing 1.5-3 kg and maintained on normal laboratory chow (Purina lab diet no. 5326; Purina Mills, Richmond, IN) plus free access to tap water were used as normal control rabbits in this study. An additional group of rabbits of comparable weight was acid treated for 3 days by providing 75 mM NH4Cl added to a 7.5% sucrose drinking solution, which yielded an acid-equivalent load of 10-15 meq · kg-1 · day-1, and by limiting food intake to 2% of body weight (7, 33, 35). The animals were killed by intracardial injection of 130 mg pentobarbital sodium after premedication with ketamine (44 mg/kg) and xylazine (5 mg/kg).

Tubule Isolation

Kidneys were removed, and 1- to 2-mm coronal slices were made and transferred to chilled dissection medium containing (in mM) 145 NaCl, 2.5 K2HPO4, 2 CaCl2, 1.2 MgSO4, 5.5 D-glucose, 1 trisodium citrate, 4 sodium lactate, and 6 L-alanine, pH 7.4, 290 ± 2 mosmol/kgH2O (33). From the corticomedullary rays, OMCDi segments were isolated under a dissecting microscope using sharpened forceps (43). Attention was made to obtain the ducts from deep within the OMCDi (below the termination of straight proximal tubule segments and adjacent to the medullary thick ascending limbs of Henle's loop). To maximize the reproducibility of this isolation in such a heterogeneous epithelium (19, 30, 34, 36), relatively short segments (0.5-0.7 mm) were obtained.

In Vitro Microperfusion

In vitro microperfusion was performed by the method of Burg and Green (9), as previously described (33, 35, 41). An isolated OMCDi was rapidly transferred to a 1.2-ml temperature- and environmentally controlled chamber mounted on an inverted microscope and perfused and bathed at 37°C with Burg's solution containing (in mM) 120 NaCl, 25 NaHCO3, 2.5 K2HPO4, 2 CaCl2, 1.2 MgSO4, 5.5 D-glucose, 1 trisodium citrate, 4 sodium lactate, and 6 L-alanine, 290 ± 2 mosmol/kgH2O, and gassed with 94% O2-6% CO2, yielding pH 7.4 at 37°C (33, 35). The specimen chamber was continuously suffused with 94% O2-6% CO2 to maintain pH at 7.4 (37). The collecting end of the segment was sealed into a holding pipette using Sylgard 184 (Dow Corning, Midland, MI). The length of each segment was measured using an eyepiece micrometer. Fourteen-nanoliter samples of tubular fluid were collected under water saturated mineral oil by timed filling of a calibrated volumetric pipette. Collections during each period were made in triplicate.

Bathing solution was exchanged by a peristaltic pump at a rate of 14 ml/h to maintain constant solute concentrations.

Bicarbonate Transport

The concentration of total CO2 (assumed to be equal to that of HCO3) in perfusate (C0) and collected fluid (CL) was measured by microcalorimetry (Picapnotherm; Microanalytical Instrumentation, Mountain View, CA). Because there is no net water absorption in the OMCD (1, 13, 15), the rate of HCO-3 transport (JHCO3) was calculated as JHCO3 = (C0 - CL) × (VL/L), where VL was the rate of collection of tubular fluid (~2 nl/min), L was the tubular length (in mm), and J was in pmol · min-1 · mm tubular length-1. When JHCO3 > 0, there was net HCO-3 absorption equivalent to net H+ secretion. The sensitivity of the Picapnotherm was 10-20 counts/pmol total CO2 (tCO2), so that for samples of 14.5 nl, there were 145-290 counts/mM tCO2. The coefficient of variation for a 20 mM standard measured in quadruplicate was <0.5% (<23 counts/sample of 4,500 counts). This level of sensitivity allowed us to reliably detect HCO-3 differences of 1 mM between perfused and collected fluids. In practice, we perfused tubules at 3-4 nl · min-1 · mm-1, which generally resulted in a difference of ~5 mM between perfused and collected fluids.

Transepithelial Voltage

Transepithelial voltage (Vte) was measured using the perfusion pipette as an electrode. The voltage difference between calomel cells connected via 3 M KCl agar bridges to perfusing and bathing solutions was measured with a high-impedance electrometer (World Precision Instruments, New Haven, CT). Collections of tubular fluid were initiated once the Vte had stabilized (30-45 min), and readings were recorded at the conclusion of each collection.

Viability

Evidence of damaged cells and gross leak of perfusate was continually assessed by the inclusion of 0.15 mg/ml FD & C green dye to each perfusate during the study (41). The experiment was discarded if tubular damage was detected.

Experimental Microperfusion Protocols

Net HCO-3 flux was first measured under basal conditions (Burg's solution in lumen and bath), then again after the application of luminal CA inhibitors, and sometimes after the addition of exogenous CA (CA from bovine erythrocytes; Sigma Chemical, St. Louis, MO) to the luminal CA inhibitor. Collections were made 5 min after each maneuver and in triplicate. Two different CA inhibitors were used: the relatively impermeant benzolamide (kindly provided by Dr. T. DuBose) and a high-molecular-weight agent, F-3500 (10), prepared by covalently linking the closely related CA inhibitor, aminobenzolamide, to the nontoxic polymer, polyoxyethylene bis(acetic acid) (3,350 Da, synthesized and kindly provided by Drs. C. Conroy and T. Maren). Each inhibitor was used sequentially in the same tubule at 10-fold higher concentrations until a substantial inhibition of HCO-3 absorption was detected. The Ki at 37°C for F-3500 is 4 µM against CA IV; that of benzolamide is 0.2 µM (10, 24). These sulfonamide inhibitors are ~20-fold more active (lower Ki) against CA II (24). Control experiments were also performed using the 3,350-Da polymer that was not coupled to aminobenzolamide. Once HCO-3 transport was inhibited substantially, 1 or 5 mg/ml (33 or 167 µM, respectively) CA was added to the CA inhibitor, and collections were resumed in the same tubule.

Analysis and Statistics

Data are presented as means ± SE. Paired comparisons for each tubule were analyzed by paired t-test, and comparisons between tubules from control vs. acid-treated animals were analyzed by unpaired t-tests using statistical software (NCSS, Kaysville, UT). Significance was asserted when P < 0.05.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Whole Animal Data

Blood taken from the heart at the time of death confirmed a metabolic acidosis (control, 7.36 ± 0.01 pH units; plasma HCO-3, 23.8 ± 0.3 mM, n = 23; acid, 7.18 ± 0.02 pH units; plasma HCO-3, 15.6 ± 0.8 mM, n = 13; P < 0.01 for both comparisons), and urine taken by postmortem bladder aspiration showed appropriate acidification during acidosis (control, 7.80 ± 0.12 pH units; acid, 4.57 ± 0.08 pH units; P < 0.01).

Benzolamide Studies

OMCD HCO-3 transport in control vs. acid-treated animals. As we have recently shown (43), acid treatment induced H+ secretion (HCO-3 absorption) in isolated perfused OMCDi segments. Control segments absorbed HCO-3 at 12.5 ± 0.2 pmol · min-1 · mm-1 (n = 23) compared with 15.5 ± 0.2 pmol · min-1 · mm-1 (n = 15, P < 0.01) from acid-treated animals. Consistent with the induction of electrogenic H+ secretion by in vivo acidosis (43), there was a more positive Vte in OMCD segments from acid-treated vs. control animals (5.2 ± 0.4 mV vs. 3.8 ± 0.1 mV, P < 0.05).

Benzolamide titration in control tubules. A benzolamide titration of increasing concentration by 10-fold was performed in control tubules (Table 1). In four paired studies, luminal 10-8 M benzolamide reduced HCO-3 transport from 12.9 ± 0.1 to 12.2 ± 0.1 pmol · min-1 · mm-1 (94% of basal rate, Fig. 1), whereas 10-7 M reduced it to 6.0 ± 0.1 pmol · min-1 · mm-1 (46% of basal rate), and 10-6 M reduced it to 0.5 ± 0.1 pmol · min-1 · mm-1 (4% of basal rate, with each change significant at P < 0.05). Associated with the inhibition was the progressively increasing concentration of HCO-3 in collected fluid, despite comparable flow rates, such that, at 10-6 M, the perfused and collected fluids had equal concentrations of HCO-3. With the reduction of electrogenic H+ secretion (43), luminal CA inhibition reduced Vte from a baseline of 3.6 ± 0.1 mV to 3.5 ± 0.1 with 10-8 M, 3.1 ± 0.4 mV with 10-7 M, and 2.5 ± 0.1 mV with 10-6 M (each change significant at P < 0.05). Addition of CA (5 mg/ml or 167 µM) to the luminal fluid containing 10-6 M benzolamide reversed the inhibition of transport and restored it to 11.3 ± 0.2 pmol · min-1 · mm-1 (88% of basal rate but significantly lower, P < 0.01) and restored collected fluid HCO-3 concentration to 20.5 ± 0.2 mM (P < 0.01). Voltage was also increased by luminal CA to 3.3 ± 0.1 mV (P < 0.01 but significantly less than baseline; P < 0.05). The approximate Ki for inhibition of HCO-3 transport by benzolamide was 10-7 M. 

                              
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  		Table 1.   Benzolamide titration: mean transport data


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  		Fig. 1.   Relationship between net HCO-3 transport (JHCO3) and luminal dose of benzolamide in perfused outer medullary collecting ducts from the inner stripe. After basal rates of transport were established, 4 segments each from control (Ctl) and acid-treated (Acid) rabbits were exposed to 10-fold increasing concentrations of benzolamide. Each line represents an individual tubule experiment.

Benzolamide titration in tubules from acidotic animals. A similar benzolamide titration was performed in OMCD segments from acid-treated animals; basal transport rate was higher than controls by 25% (P < 0.05) (Table 1, Fig. 1). In contrast to what was observed in tubules from control animals, 10-8 M benzolamide had no effect on HCO-3 transport in tubules from acid-treated animals (data not shown). Luminal 10-7 M benzolamide only slightly decreased HCO-3 transport from 16.1 ± 0.1 to 16.0 ± 0.1 pmol · min-1 · mm-1 (99.4% of basal rate, P < 0.05), whereas 10-6 M inhibited transport to 14.4 ± 0.1 pmol · min-1 · mm-1 (89% of basal rate), and 10-5 M reduced it to 4.1 ± 0.2 pmol · min-1 · mm-1 (25% of the basal rate, P < 0.01 for the latter two changes). In keeping with the inhibition of electrogenic H+ secretion, luminal CA inhibition reduced Vte from a baseline of 5.2 ± 0.4 to 5.1 ± 0.3 mV with 10-7 M benzolamide, 4.9 ± 0.3 mV with 10-6 M, and 3.9 ± 0.2 mV with 10-5 M (each change significant at P < 0.05). Collected fluid concentrations under basal conditions were 1 mM below those of control tubules and increased with high concentrations of benzolamide but not >23 mM. Addition of CA (5 mg/ml or 167 µM) to the luminal fluid containing 10-5 M benzolamide reversed the inhibition of transport and restored it to 14.3 ± 0.4 pmol · min-1 · mm-1 (89% of basal but significantly lower, P < 0.01) and restored collected fluid HCO-3 concentration to 18.9 ± 0.2 mM (P < 0.01). Voltage was also increased by luminal CA to 4.9 ± 0.4 mV (P < 0.01 but did not quite reach baseline levels, P < 0.05). The approximate Ki for inhibition of HCO-3 transport in acid-treated OMCDi segments by benzolamide was 5 × 10-5 M.

F-3500 Studies

F-3500 titration in control tubules. An F-3500 titration of increasing concentration by 10-fold was performed in control tubules (Table 2, Fig. 2). In three paired studies, 1 µM F-3500 in the luminal fluid decreased HCO-3 absorption from 12.6 ± 0.2 to 10.7 ± 0.2 pmol · min-1 · mm-1 (85% of basal rate, P < 0.01), whereas 10 µM decreased it to 3.6 ± 0.4 pmol · min-1 · mm-1 (29% of basal rate, P < 0.01), 100 µM decreased it to 2.4 ± 0.1 pmol · min-1 · mm-1 (19% of basal rate, P < 0.01), and 1 mM decreased it to 2.1 ± 0.2 pmol · min-1 · mm-1 (17% of basal rate, P < 0.01). With comparable flow rates, the titration of inhibitor resulted in increasing collected fluid HCO-3 concentrations, which peaked at 1 mM below that of the perfused HCO-3 concentration. The estimated Ki for inhibition of HCO-3 absorption by F-3500 was 5 µM.

                              
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  		Table 2.   F-3500 titration: mean transport data


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  		Fig. 2.   Relationship between JHCO3 and luminal dose of macromolecular carbonic anhydrase inhibitor, F-3500, in perfused outer medullary collecting ducts from the inner stripe. After basal rates of transport were established, 3 segments each from control (Ctl) and acid-treated (Acid) rabbits were exposed to 10-fold increasing concentrations of F-3500. Each line represents an individual tubule experiment.

Consistent with electrogenic H+ secretion mediating HCO-3 absorption in this segment (43), basal Vte was likewise inhibited by F-3500. The basal voltage was 3.8 ± 0.1 mV, which decreased to 3.5 ± 0.1 mV with 1 µM F-3500, to 2.6 ± 0.2 mV with 10 µM, to 2.3 ± 0.2 mV with 100 µM, and to 2.1 ± 0.2 mV with 1 mM F-3500 (all decreases significant, P < 0.05).

F-3500 titration in tubules from acidotic animals. In tubules taken from acid-treated animals, the basal rate of HCO-3 absorption was higher, and the degree of inhibition of HCO-3 absorption by F-3500 was less than that observed for control tubules (Table 2, Fig. 2), as was seen for benzolamide. In one segment, 1 µM F-3500 decreased HCO-3 absorption to 99% of basal rate (data not shown), whereas 10 µM decreased HCO-3 absorption in two segments from 15.9 ± 0.3 to 15.5 pmol · min-1 · mm-1 (97% of basal rate). In three segments, 100 µM F-3500 decreased HCO-3 absorption from 15.9 ± 0.3 to 10.8 ± 0.1 pmol · min-1 · mm-1 (68% of basal, P < 0.01), and 1 mM decreased it to 2.7 ± 0.1 pmol · min-1 · mm-1 (17% of basal rate, P < 0.01). At comparable flow rates, collected fluid HCO-3 concentrations increased with increasing dose of inhibitor. The estimated Ki for inhibiting HCO-3 absorption by F-3500 was 500 µM.

Vte decreased from a baseline value of 4.9 ± 0.2 to 3.9 ± 0.1 mV with 100 µM F-3500 and to 2.8 ± 0.4 mV with 1 mM inhibitor (P < 0.05 for both comparisons).

In tubules from both control and acid-treated animals, the inhibition at 1 mM (presumed to inhibit 99.2% of CA IV activity) (24) reduced the HCO-3 flux to ~17% of the basal rate. Interestingly, the residual (uninhibited) flux from acid-treated animals was still 29% greater than that from control animals, proportional to the increase in net HCO-3 absorptive flux observed in tubules from acid-treated animals noted above and previously (43).

Effect of F-3500 and exogenous CA on HCO-3 transport. In these experiments, we attempted to determine whether exogenous CA could reverse the effects of luminal CA IV inhibition and thus determine better a role for this membrane-bound enzyme in mediating transepithelial H+ secretion (HCO-3 absorption). In 10 OMCDi segments, the basal rate of net HCO-3 transport was 12.1 ± 0.3 pmol · min-1 · mm-1 (Table 3), and this was decreased to 3.8 ± 0.1 pmol · min-1 · mm-1 (31% of basal rate, P < 0.01), whereas collected fluid HCO-3 concentration was increased 2.5 mM to 23.1 ± 0.1 mM by 10 µM F-3500. Concomitant with the inhibition of electrogenic H+ secretion, Vte decreased from a baseline of 3.4 ± 0.2 to 1.9 ± 0.2 mV (P < 0.01). The addition of 1 mg/ml (33 µM) of exogenous CA (to the luminal fluid containing the CA inhibitor) restored HCO-3 flux to 9.4 ± 0.3 pmol · min-1 · mm-1, decreased collected fluid HCO-3 concentration to 21.0 ± 0.2 mM, and increased voltage to 2.6 ± 0.2 mV. When 5 mg/ml (167 µM) CA was added in the presence of the inhibitor, reversal of CA inhibition was nearly complete: flux was 11.2 ± 0.3 pmol · min-1 · mm-1 (93% of basal), collected fluid HCO-3 concentration reached basal levels, and voltage increased to 2.8 ± 0.2 mV. However, both the flux and voltage in the presence of CA + F-3500 were still significantly less than basal values (P < 0.05).

                              
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  		Table 3.   F-3500 inhibition of HCO-3 transport: effect of luminal carbonic anhydrase

Similar experiments addressed the role of CA in mediating HCO-3 transport in eight OMCDi tubules from acid-treated rabbits (Table 3). The basal rate of HCO-3 transport was 15.0 ± 0.3 pmol · min-1 · mm-1, and Vte averaged 4.8 ± 0.3 mV. Compared with control tubules, the inhibition of HCO-3 transport and voltage by 10 µM F-3500 was quite modest in OMCDi segments from acid-treated animals [flux, 14.6 ± 0.3 pmol · min-1 · mm-1 (P < 0.01) but 97% of basal rate; collected fluid HCO-3 concentration, 19.1 ± 0.3 mM; voltage, 4.1 ± 0.3 mV (P = NS)]. Increasing F-3500 to 100 µM reduced HCO-3 flux to 10.5 ± 0.2 pmol · min-1 · mm-1 (70% of basal rate, P < 0.01), increased collected fluid HCO-3 concentration by 1.5 mM (P < 0.05), and reduced voltage to 2.7 ± 0.3 mV (P < 0.01). Adding 5 mg/ml (167 µM) of exogenous CA (to the luminal fluid containing the CA inhibitor) restored the HCO-3 flux to 14.5 ± 0.3 pmol · min-1 · mm-1 (97% of basal rate), reduced collected fluid HCO-3 to basal levels, and restored the voltage to 3.2 ± 0.3 mV. However, both the flux and voltage in the presence of CA + F-3500 were still significantly less than basal values (P < 0.05).

Polyoxyethylene bis(Acetic Acid) Polymer: Effect of Polymer and Exogenous CA on HCO-3 Transport in Control Tubules

To ensure that the polymer, polyoxyethylene bis(acetic acid), not linked to aminobenzolamide, was not toxic to the OMCD and to examine the effect of exogenous luminal CA on HCO-3 transport, we examined six OMCDi segments using 10 µM polymer, followed by the addition of exogenous CA at 1 mg/ml (33 µM; Table 4, Fig. 3). The basal flux was 12.9 ± 0.4 pmol · min-1 · mm-1, with a collected fluid HCO-3 concentration of 20.1 ± 0.3 mM and Vte of 3.3 ± 0.2 mV. Addition of polymer had no effect on flux (12.9 ± 0.5 pmol · min-1 · mm-1), HCO-3 concentration, or voltage (3.1 ± 0.2 mV). Addition of exogenous CA to the perfused polymer also had no effect on flux (12.8 ± 0.4 pmol · min-1 · mm-1), collected HCO-3 concentration, or voltage (3.2 ± 0.2 mV). These studies ruled out a toxic effect of the polymer to which had been coupled aminobenzolamide. These data also showed that exogenous CA failed to stimulate HCO-3 absorption, indicating that endogenous luminal CA was more than adequate to support the observed rate of transport.

                              
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  		Table 4.   Polymer effect on HCO-3 transport: effect of luminal carbonic anhydrase


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  		Fig. 3.   Relationship between JHCO3 and luminal dose of the polymer comprising the macromolecule of F-3500, polyoxyethylene bis(acetic acid), in perfused outer medullary collecting ducts from the inner stripe. After basal rates of transport were established, 6 segments were exposed to the polymer and then to the polymer + exogenous carbonic anhydrase (1 mg/ml or 33 µM). Each line represents an individual tubule experiment.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

In the lumen of the proximal tubule (8, 29, 31), membrane-bound CA IV catalyzes the dehydration of carbonic acid (formed from the reaction between filtered HCO-3 and secreted H+) into CO2 + water, thereby favoring further H+ secretion (HCO-3 absorption). Inhibition of this enzyme with a dextran-bound CA inhibitor reduced HCO-3 reabsorption by 80% (17). These data suggested that inhibition of the membrane-bound CA would cause an acid disequilibrium pH to be generated, the opposing gradient of which would secondarily slow down the rate of H+ secretion that mediates HCO-3 reabsorption. In the proximal tubule, inhibition of luminal CA reduced net HCO-3 reabsorption to the same extent as did a permeant CA inhibitor, but only the former caused an acid disequilibrium pH because H+ secretion was not substantially inhibited (17). Thus a functional luminal CA is necessary for optimal HCO-3 reabsorption in the proximal tubule.

In the OMCDi, there is functional evidence that membrane-bound carbonic anhydrase (CA IV) is present on the luminal membrane of the OMCDi (30, 40) to facilitate H+ secretion. There are no studies reported to test the role of luminal CA on mediating H+ secretion (HCO-3 absorption) in this segment. Previous studies (15, 40) using isolated perfused OMCD segments have shown that permeant CA inhibitors such as acetazolamide can markedly inhibit net HCO-3 absorption. However, in one study in the outer stripe of the outer medullary collecting duct (OMCDo), CA inhibition had little effect on HCO-3 transport (28), despite use of high concentrations of the more potent and lipid-soluble ethoxzolamide. Presumably, these inhibitors affected both membrane-bound and cytosolic CA. It was not possible to reconcile these findings with others showing sensitivity of HCO-3 transport to CA inhibitors and with studies identifying CA in the cytosol and lumen of the OMCD. Furthermore, none of the studies reported the use of an impermeant CA inhibitor to separate the roles of luminal and cytosolic CA. Our studies represent the first to formally establish a role for luminal CA in mediating H+ secretion (HCO-3 absorption) in the rabbit OMCDi.

In mice genetically deficient in cytosolic CA II, administration of a CA inhibitor increased urinary HCO-3 excretion (6), indicating a major role for CA IV in mediating HCO-3 reabsorption. However, a titration of the luminal enzyme to elicit a Ki for HCO-3 transport has not been performed. Benzolamide is a relatively impermeant CA inhibitor (17, 22, 23) that enters the cell to a lesser extent than does acetazolamide; yet, benzolamide can still not be rigorously excluded from inhibiting cytosolic CA as well (14).

The high-molecular-weight CA inhibitor F-3500 was especially prepared to inhibit solely the luminal enzyme CA IV (10, 24). Rigorous testing has shown that the agent is not actively taken up by the kidney, does not cross cell membranes, and is excluded from the intracellular fluid (24). Because F-3500 has residual contamination by unreacted free aminobenzolamide up to a maximum of 0.2%, one must calculate whether cytosolic CA could be inhibited by the free drug. At concentrations of F-3500 that inhibited most of HCO-3 absorption in control tubules (10 µM), the maximal free drug concentration would be 0.02 µM, not nearly sufficient to fully inhibit cytosolic CA II.

The residual HCO-3 absorptive flux in OMCD segments from control animals averaged 0.5 ± 0.1 pmol · min-1 · mm-1 at a benzolamide concentration of 10-6 M and comprised <5% of the basal H+ secretion rate. The titration of benzolamide concentration vs. HCO-3 absorptive rate indicated that the Ki for reducing transport by 50% was ~0.1 µM. This concentration of benzolamide would be expected to inhibit ~50% of luminal CA of the tubule (22-24), and, therefore, the substantial inhibition might reflect inhibition of cytosolic CA II as well. If the concentration of 1 µM benzolamide were achieved in the cytosol, it would be expected to inhibit 95% of cytosolic CA II. On the other hand, the rapid reversal of inhibition with CA perfusion (see RESULTS and below) tends to rule out substantial cytosolic CA inhibition. The residual HCO-3 absorptive flux in OMCD segments from control animals that were treated with 1 mM F-3500 was 2.1 ± 0.2 pmol · min-1 · mm-1, and this was considered the membrane-bound CA IV uncatalyzed H+ secretory rate. This rate was 16% of the basal rate, similar to the 22% observed in proximal tubules perfused with a macromolecular CA inhibitor (17). The Ki for reducing by 50% the transport with F-3500 was ~5 µM, similar to the Ki for inhibiting CA IV (24), indicating that approximately one-half of the luminal membrane activity was inhibited. Thus H+ secretion (HCO-3 absorption) was extremely sensitive to luminal CA inhibition within the concentration range of the Ki.

The rationale for the perfusion of CA simultaneously with CA inhibitor was to confirm that the depression of transport could be reversed by competing for binding to the luminal enzyme. In addition, this experiment allowed us to examine the extent of cytosolic CA inhibition, which would not be reversible by perfusing with exogenous CA. Indeed, perfusion with 1 µM benzolamide and 1 mg/ml (33 µM) CA restored HCO-3 absorption to 11.3 ± 0.2 pmol · min-1 · mm-1 (88% of basal rate), indicating nearly complete reversibility. It also meant that little cytosolic CA could have been inhibited by benzolamide. Similarly, with the large CA inhibitor, F-3500, at 10 µM, perfusion with 5 mg/ml (167 µM) CA restored HCO-3 absorption to 11.2 ± 0.3 pmol · min-1 · mm-1 (93% of basal rate), indicating near complete reversibility of this inhibitor. It is likely that little cytosolic CA II was inhibited by free aminobenzolamide in view of the high percentage of basal rate achieved. On the other hand, the failure to completely restore HCO-3 absorption might indicate residual cytosolic CA II inhibition by permeant (benzolamide) or unreacted (aminobenzolamide) CA inhibitor.

The secretion of protons by OMCD segments taken from acid-treated animals was relatively resistant to CA inhibition. As noted previously (43), OMCDi segments from acid-treated rabbits absorbed HCO-3 at a 30% higher basal rate than did segments from control animals. With benzolamide, the Ki for inhibiting HCO-3 transport was increased from 0.1 to 50 µM in acid-treated tubules, whereas, with F-3500, the Ki was increased from 5 to 500 µM. From the F-3500 titration, the residual flux was 2.7 ± 0.1 pmol · min-1 · mm-1 or 17% of the basal flux, and this was considered the uncatalyzed H+ secretion (HCO-3 absorption) rate. The residual flux after 10-5 M benzolamide was 4.1 ± 0.2 pmol · min-1 · mm-1 or 25% of the basal flux in a separate set of tubules; the near identity of these percentages suggests that ~20% of the basal flux is uncatalyzed by luminal CA IV. When CA was perfused simultaneously with benzolamide, HCO-3 absorptive flux was restored to 14.3 ± 0.4 pmol · min-1 · mm-1 (89% of basal rate); with F-3500, the flux was restored to 14.5 ± 0.3 pmol · min-1 · mm-1 (97% of basal rate). These studies suggest that there was little inhibition of cytosolic CA II, despite the high concentrations of inibitors used. The nearly complete reversibility also ruled out a toxic effect of the agents on tubule function. Whether the CA inhibitors could have directly inhibited the luminal H+-ATPase was not formally tested; however, a previous study of proton pumping in rat renal cortical endocytotic vesicles (32) showed that ethoxzolamide did not inhibit proton pumping into endocytotic vesicles. This finding would suggest that the CA inhibitors did not directly inhibit the luminal H+-ATPase.

Chronic metabolic acidosis has been shown to result in a threefold increase in CA IV mRNA in the kidney cortex and outer medulla (47) and a more than twofold increase in SDS-resistant hydratase activity (presumably CA IV) in the cortex, particularly in the proximal convoluted tubule (7); the 138% increase in the OMCD activity failed to reach statistical significance (7). It is unlikely that such increases in luminal CA activity could mediate a >50-fold decrease in sensitivity of HCO-3 transport to CA inhibition in OMCDi segments taken from acid-treated rabbits.

Studies using the polymer without the sulfonamide also showed no toxic effect, confirming that the depression of HCO-3 absorption was not due to tubular damage by the polymer. Because of the stability of HCO-3 transport in the presence of the polymer (see Fig. 3), plus the previous findings of stable HCO-3 absorption after a 3-h incubation (43), time-control studies without inhibitors were not performed. In addition, we failed to show that exogenous CA further stimulated HCO-3 absorption (see Table 4), indicating adequacy of the endogenous luminal membrane-bound CA IV activity. Similar findings for HCO-3 transport were previously observed in the OMCDo, which does not express luminal CA IV (40); however, exogenous CA eliminated the observed disequilibrium pH. These findings would suggest that the disequilibrium pH was not large enough to inhibit HCO-3 absorption in the OMCDo. But in the OMCDi, where luminal CA IV has been detected functionally, the amount of enzyme is adequate to limit any fall in luminal pH due to H+ secretion (40), thereby reducing the opposing pH gradient. These and other studies suggest that luminal CA plays an important role in mediating HCO-3 absorption in segments with high intrinsic rates of H+ secretion, including the S1 and S2 proximal tubules and OMCDi.

Carbonic anhydrase inhibitors cause metabolic acidosis at doses that maximally inhibit renal carbonic anhydrase (21, 22). It is known (21, 25) that renal bicarbonaturia induced by acetazolamide is markedly reduced during acidosis. Indeed, the greater the acidosis, the less the diuretic effect of CA inhibition. The mechanism for this renal resistance has not been determined, but it has not been attributed to decreased filtered load of HCO-3. Maren (21) originally suggested that the participation of CA in renal acidification is diminished during metabolic acidosis, but the observed increases in CA activity (7) would suggest a bigger, not smaller, role for CA during acidosis. Further studies are needed to characterize luminal CA IV and its sensitivity to inhibition before and during chronic metabolic acidosis. Alternatively, the pumps secreting protons in tubules from acidotic animals may be more resistant than controls to the disequilibrium pH induced by the inhibitors. A formal examination of proton pumping as a function of luminal pH in the perfused OMCDi is required to investigate this alternative explanation.

In summary, we have used nonpermeant CA inhibitors to show, for the first time, that inhibition of luminal CA at a concentration similar to that of the Ki resulted in a major reduction in HCO-3 absorption in isolated perfused OMCDi segments. These findings would suggest that luminal CA IV, which comprises <5% of total kidney CA activity (7, 26, 48), might indeed be rate limiting when renal acidification is increased. Perhaps the modest increase observed in CA IV activity (7) reflects an appropriate adaptation to chronic metabolic acidosis. This would be in contrast to the situation for CA II, which is present in major excess in the kidney and requires a >99% inhibition for an effect on renal acid-base transport to be observed (22). In this case, small increases in CA II mRNA and activity (7, 38) might not be physiologically important in the adaptation to chronic metabolic acidosis. We have also shown for the first time a resistance of HCO-3 transport to CA inhibition in tubules taken from rabbits with chronic metabolic acidosis: the Ki for reducing HCO-3 absorption by 50% increased by a factor of 100 for F-3500 and by a factor of 500 for benzolamide. The mechanism for this apparent resistance to CA inhibition is not entirely clear but may involve changes in conformation, secondary structure, or glycosylation, which could affect substrate binding, alter membrane characteristics, or change the interactions with specific cofactors. Any of these possibilities could protect the enzyme from inhibition.

    ACKNOWLEDGEMENTS

We are grateful for the technical assistance of A. Kittelberger and J. Toner. We thank Drs. M. Flessner and A. Weinstein for helpful discussions and for reading the manuscript.

    FOOTNOTES

S. Tsuruoka was supported by a postdoctoral fellowship award from the American Heart Association New York State Affiliate. G. J. Schwartz was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-50603.

Address for reprint requests: G. J. Schwartz, Div. of Pediatric Nephrology, Depts. of Pediatrics and Medicine, PO Box 777, University of Rochester School of Medicine, 601 Elmwood Ave., Rochester, NY 14642.

Received 11 June 1997; accepted in final form 22 September 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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