Renal sulfate secretion is carbonic anhydrase dependent in a marine teleost, Pleuronectes americanus

J. Larry Renfro1,4, Thomas H. Maren2,4, Cristina Zeien2,4, and Erik R. Swenson3,4

1 Department of Physiology and Neurobiology, University of Connecticut, Storrs, Connecticut 06269; 2 Department of Pharmacology and Therapeutics, University of Florida Health Science Center, Gainesville, Florida 32610; 3 Department of Veterans Affairs Medical Center and University of Washington, Seattle, Washington 98108; and 4 Mount Desert Island Biological Laboratory, Salsbury Cove, Maine 04672


    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Though chemical assays indicate that carbonic anhydrase (CA) activity is present in marine teleost nephrons, CA inhibitors have no effect on urine pH or bicarbonate excretion, parameters typically CA dependent in almost all vertebrate groups. Because marine teleost renal sulfate secretion is associated with bicarbonate anion exchange, we investigated the effect of CA inhibition on transepithelial sulfate transport by flounder renal tubule primary monolayer cultures (PTC) and on renal sulfate secretion (QSO4) by intact flounder. Both methazolamide and ethoxzolamide (10 µM) inhibited PTC secretory flux by ~50%; reabsorptive sulfate flux, Na-dependent glucose transport, and transepithelial electrical resistance were unaffected. A CA inhibitor restricted to the extracellular space (10 µM polyoxyethylene-aminobenzolamide, 3.7 kDa) had no effect on PTC sulfate transport. Intravenous administration of methazolamide reduced QSO4 almost 40% and had no effect on glomerular filtration rate (GFR), urine flow rate, or Pi excretion rate. Serum pH was significantly reduced 0.2 units, whereas urine pH was unchanged. Together, the in vitro and in vivo results indicate that CA facilitates renal sulfate secretion in the seawater teleost.

anion exchange; sulfate clearance; methazolamide; ethoxzolamide; winter flounder; fish renal function; acid-base balance


    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

THE ROLE OF CARBONIC anhydrase (CA) in the renal function of marine fishes has been uncertain since the early work of Hodler et al. (9), who showed that the typical effects of renal CA inhibition (bicarbonaturia and rise in urine pH), as observed in all vertebrates (except Crocodilia), were not observed in either marine elasmobranch or teleost fishes. It appeared that the enzyme was lacking; however, chemical assays for CA have usually indicated that the enzyme is present in renal tissue (13). Rigorous attempts to assay CA in marine fish were confounded by the presence of hematopoietic tissue in kidneys, but the net result of studies over the years (4, 5, 21, 22) suggests that the enzyme is present in renal cells of marine fish despite failure to demonstrate the typical effects of inhibition with large doses of specific sulfonamide inhibitors.

Maren et al. (13) have noted three patterns of renal acid-base physiology in the vertebrates. Freshwater teleosts follow the pattern of mammals, birds, and amphibia, which have variable urinary pH, sensitive to specific CA inhibitors. Marine elasmobranchs have a fixed, acidic urinary pH that does not respond to CA inhibitors, an observation that led Swenson and Maren (21) to conclude that H+ secretion and CO2 metabolism were not coupled in the shark kidney and that intracellular OH- resulting from substantial tubular H+ secretion could be buffered some other way, perhaps by Cl-/OH- exchange. Marine teleosts can vary urinary pH but also lack a response to CA inhibition, again indicating that renal tubular acid secretion in these kidneys may be sufficient for uncatalyzed bicarbonate reabsorption. Although large doses of a very specific CA inhibitor, methazolamide, were used in this study, there were no apparent alterations in urine composition. The analyses, however, did not include inorganic sulfate.

To compensate for osmotic water loss, marine teleosts must ingest seawater. The sulfate concentration of normal seawater is 25 mM, and part of this is absorbed by the gut along with the water and is excreted primarily by the kidneys (16). Flounder plasma inorganic sulfate concentration averages 0.6 mM (18), and renal sulfate clearance ratios (clearance of sulfate/clearance of inulin) may exceed 12 (8). The cellular mechanisms of renal sulfate secretion have been examined in both basolateral and brush border membrane vesicles isolated from two flounder species (19, 20). Sulfate entry, interstitium-to-cell, can be driven by a basolateral membrane pH gradient as either proton symport or hydroxyl exchange. In basolateral membrane vesicles, there is no effect of a Na+ gradient; however, in the intact tubule, sulfate secretion is Na+ gradient dependent, perhaps secondary to apical membrane Na+/H+ exchange. Sulfate exit, cell-to-lumen, is stimulated by counter anion gradients. HCO-3, SCN-, Cl-, and S2O-3 counter gradients can trans-stimulate sulfate transport in flounder brush border membrane vesicles. HCO-3 is the most effective counter anion on this electroneutral exchanger and, in vivo, is formed continuously by CA-catalyzed hydroxylation of CO2 (see Fig. 5). This exchanger is inhibited by disulfonic acid stilbenes but is not sensitive to H+, Na+, or K+ gradients.

In the present study, we sought definitive evidence of renal tubular epithelial CA activity and have examined the effect of CA inhibition on sulfate secretion by winter flounder proximal tubule primary cultures (PTCs) and on renal sulfate clearance in intact animals. The evidence suggests a hitherto unsuspected role of CA in marine fish, that of subserving sulfate secretion by exchange for bicarbonate, which is apparently enzymatically dehydrated (or dehydroxylated) in the secretion process.


    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Animals. Winter flounder, Pleuronectes americanus, were obtained by otter trawl in Frenchman's Bay, Maine, or in Long Island Sound, Connecticut. Animals (250-400 g) were held in flowing seawater (17-19°C) or in Living Stream Units (Toledo) filled with artificial seawater (Utikem) at 12°C. The former were used for renal clearance measurements, the latter for preparation of renal cell cultures.

Renal Clearance Determination. The procedures for determination of renal clearances in winter flounder have been reported (18). Minor modifications are briefly described below. After anesthetization with MS-222 (1:2,000 wt/vol), heparinized polyethylene tubing (PE10) was inserted into the hemal vein near the caudal peduncle. During blood sampling, the initial 50 µl of blood withdrawn was discarded to prevent sample contamination with sulfate from the heparin. Sufficient removal of heparin was confirmed by the fact that the blood clotted on standing (4°C). A polyethylene tube (PE90) was inserted into the urinary bladder. Urine was collected at varying times with a syringe attached to a PE50 tube inserted into the bladder through the PE90 catheter. Animals were maintained in flowing seawater at 15-17°C during clearance determinations. Inulin was injected (150 mg/kg body wt) intramuscularly 24 h before clearance measurements. Plasma and urine inulin concentrations were determined by the indole acetic acid colorimetric method (2), and inorganic sulfate and phosphate were determined after appropriate dilution with ultrapure water (18 MOmega · cm) by anion chromatography (IonPac AG4A-SC; Dionex, Sunnyvale, CA).

Solutions and chemicals. Modified medium 199 with Earle's salts (M199) was purchased from Sigma Chemical (St. Louis, MO). This medium was supplemented with (in mM) 30.0 NaCl, 4.2 NaHCO3, 1.0 L-glutamine, 25.0 HEPES, 14.75 NaOH (pH 7.5, 347 mosmol/kgH2O), and 20 mg/l tetracycline. Modified M199 was also supplemented with 10 µg/ml insulin, 5 µg/ml hydrocortisone, and 10% flounder serum to form the final plating medium. Maintenance medium had 10% bovine serum instead of 10% flounder serum. The Ca- and Mg-free saline solution (CMF) used for removal of extrarenal tissues had the following composition (in mM): 150.0 NaCl, 4.0 KCl, 0.5 NaH2PO4, 4.2 NaHCO3, 25.0 HEPES, 5.5 glucose, 0.3 ethylenediaminetetraacetic acid, 14.75 NaOH (pH 7.5), and 20 mg/l tetracycline. The flounder saline solution used for Ussing chamber experiments contained (in mM) 150.0 NaCl, 4.0 KCl, 2.0 CaCl2, 1.0 MgSO4, 0.4 NaH2PO4, 4.2 NaHCO3, 25.0 HEPES, 5.5 glucose, 1.0 L-glutamine, and 14.75 NaOH (pH 7.5).

L-Glutamine, tetracycline, HEPES, ethylenediaminetetraacetic acid, insulin (26.2 U/mg), hydrocortisone 21-phosphate, trypsin (11,000 U/mg), phloridzin, methazolamide, and ethoxzolamide were purchased from Sigma. Polyethoxyethylene bis(acetic acid) linked to aminobenzolamide, F3500, was synthesized as described previously (12).

Preparation of flounder PTCs. Flounder renal tubules were isolated and dispersed by cold trypsinization, as previously described by Dickman and Renfro (3) and modified by Gupta and Renfro (7). Briefly, kidneys were perfused, removed, and teased apart in modified M199. Tubules were incubated briefly in CMF with 0.2% trypsin at 22°C. Epithelial cells were released from these isolated tubule fragments by 3 days of cold trypsinization (5°C) in modified M199 containing 0.05% trypsin. Released cells were washed, suspended in culture medium with serum, and plated on native rat-tail collagen. After 4 days, the collagen gels were released, and after ~12 days, the floating collagen gels had been contracted by the epithelial monolayers from the initial 35-mm diameter to 17 mm.

Carbonic anhydrase assay. The micromethod of Maren et al. (14) was used to determine PTC carbonic anhydrase activity. After a 1:10 dilution with distilled water and homogenization, CA activity was estimated as enzyme units per milliliter of cells. Cell volume was estimated from monolayer surface area and cell layer height based on prior electron micrographs (3).

Ussing chamber studies. During days 12-20, transepithelial electrical characteristics and sulfate transport were measured. Tissues, supported by 150 µm nylon mesh, were mounted in Ussing chambers as previously described (7). The temperature was maintained at 21°C, and the fluid inside the chambers was continuously stirred and insufflated with humidified 99% O2-1% CO2.

Transepithelial electrical potential (PD) was determined with Ag/AgCl electrodes connected to the luminal and peritubular compartments with 3 M KCl-2% agar bridges. Electrical properties were determined with a pair of computer-controlled, high-impedance automatic dual voltage clamps (DVC 1000; World Precision Instruments, Sarasota, FL). Electrode asymmetry was corrected at the beginning and end of each experiment and fluid resistance was compensated. Short-circuiting electrodes were connected to the luminal and peritubular solutions with 3 M KCl-2% agar bridges. Transepithelial resistance (TER) was determined from the change in PD produced by a brief 10-µA pulse controlled by the voltage clamps.

Determination of transepithelial sulfate fluxes. Tissues were continuously short-circuited during flux determinations. Unidirectional tracer fluxes were initiated by the addition of 1.0-2.0 µCi 35SO4 to the appropriate hemichamber. Duplicate 50-µl samples were taken from the unlabeled side at least every 30 min over a period up to 1.5 h and replaced with equal volumes of unlabeled flounder saline. The specific activity of the labeled solution was determined at the beginning and end of each experiment. Fluxes were monitored from t = 0 and reached steady state within 1 h. Only the 1-h time points are used for statistical reporting.

Net flux was calculated as the difference between unidirectional secretory (P to L, peritubular-to-luminal) and reabsorptive (L to P, luminal-to-peritubular) fluxes. A single flux experiment was done on culture mates, i.e., both control and treated tissues were paired monolayers from a single preparation. The four monolayer cultures used in a given experiment were prepared from the same starting tissue at the same time and cultured under identical conditions. This is referred to as one preparation for statistical purposes. To determine tissue viability and proximal tubule-like function, we measured the transmural PD, TER, short-circuit current (Isc), and phloridzin (PHZ)-sensitive current (IPHZ, Na-dependent glucose transport).

Statistics. Experimental results are expressed as means ± SE. Paired comparisons of sample means were done with two-tailed Student's t-tests. Differences were judged significant if P < 0.05.


    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

In the presence of flounder saline (1 mM SO2-4), the flux ratio determined from unidirectional 35SO2-4 fluxes in Ussing chambers was approximately 20 to 1 in the secretory direction. Figure 1 is a representative plot of PTC sulfate secretory response. Radiolabeled sulfate was added at t = 0, and transepithelial P to L flux reached steady state at 1 h (Fig. 1A). The addition of 1 mM methazolamide to paired culture mates at t = 0 (Fig. 1B) reduced P to L by one-half and had no effect on L to P flux. Net fluxes calculated from the unidirectional fluxes in paired tissues typically averaged 136 ± 22.3 nmol · cm-2 · h-1. Because control transport rates in primary cultures varied, the effects of methazolamide on secretory (P to L), reabsorptive (L to P), and net fluxes in Fig. 2 are expressed as percentage of inhibition compared with paired controls. These summary data show that, at 10 µM methazolamide, a concentration specific for CA inhibition, secretory sulfate transport was inhibited by approximately one-half. At 1 mM methazolamide, inhibition increased only slightly, i.e., a 100-fold increase in inhibitor concentration produced a further inhibition of only 0.3-fold, which was not a statistically significant change in net flux. Of note is the fact that, even at the highest dosages, nonspecific effects were not apparent. The reabsorptive flux, which is apparently passive in flounder proximal tubule, as no Na-coupled transport occurs (19), is unchanged by CA inhibition. Equally important, Na-dependent IPHZ (3.1 ± 0.70 µA/cm2) and TER (43 ± 2.5 Omega  · cm2) were unaffected by the drug (Fig. 2).


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Fig. 1.   Representative plot of radiolabeled SO2-4 fluxes vs. time. P to L is peritubular-to-luminal secretory flux; L to P is luminal-to-peritubular reabsorptive flux (shown as being negative to indicate direction). Net flux is the difference between unidirectional fluxes. A: paired controls; B: methazolamide (1 mM) added at t = 0. Fluxes reached steady state at t = 1 h.


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Fig. 2.   Dose-response effect of methazolamide on steady-state unidirectional P to L, L to P, and net sulfate fluxes expressed as percentage inhibition compared with paired control fluxes. Phloridzin-sensitive current (IPHZ, µA/cm2) and transepithelial resistance (TER, Omega  · cm2) in same tissues are also shown. Values are means plus 1 SE (vertical line) of 3 preparations. * Significantly different from controls (P < 0.05).

Sulfonamides are, in general, powerful inhibitors of CA; however, they vary greatly in physicochemical characteristics. Methazolamide is moderately water soluble and slightly lipid soluble compared with ethoxzolamide, and as Maren (11) has pointed out, both ionic and lipophilic forces determine the association of a drug with CA. To affirm that sulfonamides other than methazolamide also inhibited sulfate secretion in vitro, we determined the effect of ethoxzolamide on this process in flounder PTCs. Figure 3 shows that 10 µM ethoxzolamide reduced secretory flux to less than one-half that of controls (56% inhibition). It had no significant effect on reabsorptive flux, IPHZ, or TER. Thus ethoxzolamide caused no loss of metabolic or structural integrity.


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Fig. 3.   Dose-response effect of ethoxzolamide on steady-state unidirectional P to L, L to P, and net sulfate fluxes expressed as percentage inhibition compared with paired control fluxes. IPHZ (µA/cm2) and TER (Omega  · cm2) in same tissues are also shown as percentage of paired controls. Values are means plus 1 SE (vertical line) of 3 preparations. * Significantly different from controls (P < 0.05).

A comparison of three sulfonamides at 10 µM, in both peritubular and luminal sides, on net sulfate secretion is shown in Fig. 4. The action of methazolamide and ethoxzolamide (data from Figs. 2 and 3 recalculated as percentage of control) is apparently intracellular as the polymer-linked, high-molecular-mass (3.7 kDa) CA inhibitor, polyoxyethylene-aminobenzolamide, which is restricted to the extracellular space (12), had no effect on sulfate transport (percentage control = 99.6 ± 18.21) by flounder PTCs.


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Fig. 4.   Comparison of inhibitory effects of 3 sulfonamides at 10 µM on steady-state net sulfate secretion by flounder primary monolayer cultures (PTC). Results for ethoxzolamide and methazolamide are data from Figs. 1 and 2, shown here for comparison. The polymer-linked inhibitor was polyoxyethylene-aminobenzolamide, which is restricted to the extracellular space (see RESULTS). Values are means plus 1 SE expressed as percentage of fluxes in paired control tissues (n = 3 preparations). * P < 0.05 and ** P < 0.01 compared with paired controls.

Kinetic assays revealed low levels of CA activity [555 ± 48 (SD) enzyme units/ml] in PTCs. Fresh whole kidney (including hematopoeitic tissue) contained about 2,300 enzyme units/ml, and flounder red blood cells, by comparison, contained about 20,000 U/ml. Prior examination of PTCs with an electron microscope indicates that they consist exclusively of polarized epithelial cells and a few fibroblasts that segregate between the epithelial monolayer and collagen gel (3). No hematopoietic tissue is apparent, although its presence cannot be ruled out. The presence of enzyme in PTCs, coupled with the action of specific CA inhibitors, further supports the notion of CA involvement in sulfate secretion.

Methazolamide effects on renal sulfate clearance. As noted above, sulfate secretion is HCO-3 dependent in flounder tubule. By contrast, phosphate secretion is thought to be dependent on membrane electrical potential and is not influenced by HCO-3 or other anion gradients, as determined in phosphate-secreting chick renal tubule brush border membrane vesicles and flounder PTCs (1, 10). Inorganic phosphate reabsorption is Na-coupled at the proximal tubule apical membranes and thus sensitive to the plasma membrane Na gradient. Accordingly, sulfate clearance was contrasted with phosphate clearance before and after methazolamide administration.

A bolus of methazolamide was administered intravenously at a dose of 25 mg/kg, an amount that should produce about 200 µM in the extracellular fluid, several thousand-fold higher than its Ki. Plasma sulfate and phosphate concentrations, respectively, averaged 0.50 ± 0.168 and 2.40 ± 0.324 mM before drug infusion and 0.5 ± 0.176 and 2.50 ± 0.253 mM after drug infusion. The aforementioned values and the values shown in Table 1 were obtained in the first hour just before drug infusion and in the first hour after infusion. GFR and urine flow were not significantly changed by the drug. Serum pH fell 0.2 units, which is a highly significant change (probably due to inhibition of the branchial CA and slowing of the acid-base transport systems). The rate of urinary phosphate or sulfate secretion (QPi, QSO4) was calculated as the difference in the quantity excreted and quantity filtered.
<A><AC>Q</AC><AC>˙</AC></A><SC>so</SC><SUB>4</SUB> = (<A><AC>V</AC><AC>˙</AC></A> × U<SUB>SO<SUB>4</SUB></SUB>)  − (GFR × P<SUB>SO<SUB>4</SUB></SUB>)
Where V is urine flow rate, USO4 is urine sulfate concentration, and PSO4 is plasma sulfate concentration. The data in Table 1 show that there was no effect of CA inhibition on phosphate excretion [minus sign (-) indicates net reabsorption]; however, sulfate secretion was inhibited ~40%. As can be seen, sulfate secretion varied considerably; however, in spite of the large individual variability, paired comparisons showed that the response to methazolamide in every case was an immediate drop in the rate of sulfate secretion. This is consistent with the effect predicted by the in vitro experiments.

                              
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Table 1.   Effect of methazolamide on renal function in winter flounder


    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

The present data indicate an important role for CA in renal sulfate secretion by marine fishes. This involvement probably stems from the membrane anion exchangers responsible for secretory transport of sulfate and explains why all previous experiments measuring urinary pH and bicarbonate gave negative results after inhibition of the enzyme.

The effective drug concentrations used here have been shown to be very specific for CA inhibition (11), and it is unlikely that these sulfonamides acted indirectly through some other mechanism to slow sulfate secretion. These drugs were roughly equally effective inhibitors of sulfate secretion at very low concentrations. However, methazolamide and ethoxzolamide are quite different chemically (11). Ethoxzolamide is at least 200× more lipid soluble than methazolamide at pH 7.4, and a higher percentage of the former is bound to plasma proteins. Against red blood cell CA, the inhibitory potency of ethoxzolamide is about 8-fold that of methazolamide (11).

The absence of any effect of the drugs on IPHZ or TER is a sensitive indicator of the integrity of the plasma membrane Na gradient. Any disruption of tubular metabolism would alter both of these parameters by decreasing the plasma membrane Na gradient and by initiating cell swelling. In vivo, methazolamide decreased sulfate secretion by ~40% but had no effect on renal phosphate excretion. The latter is reabsorbed on a Na-coupled transporter and is therefore dependent on the apical membrane Na gradient. Phosphate secretion is membrane-potential dependent at the apical site, another metabolism-dependent process. Thus, of the measurements done in the present study, we show that only renal sulfate secretion and plasma pH were altered by methazolamide.

The cellular mechanisms of renal sulfate secretion recounted in the introduction have been examined in both basolateral and brush border membrane vesicles isolated from two flounder species (17, 19, 20) and are depicted in Fig. 5. The figure shows the proposed role of CA in flounder renal tubule sulfate secretion. As noted above, in the intact tubular epithelium, sulfate secretion is sodium dependent, and in the proposed model, the Na+ electrochemical gradient established by Na+-K+-ATPase drives an assumed Na+/H+ exchanger that establishes a pH gradient across the basolateral membrane favoring continued OH-/SO2-4 exchange. Filtration provides HCO-3 in the tubule lumen, which is exchanged for cellular sulfate. Inside the cell, the conversion of bicarbonate to CO2 and OH- is accelerated by CA, supplying hydroxyl ions that exchange for extracellular sulfate at the basolateral membranes. This process may account for most of the reabsorption of bicarbonate, as such, at the brush border membranes. For example, a typical value for net sulfate secretion by marine teleosts is about 20 µmol · kg-1 · h-1 (19) with a typical GFR of 2 ml · kg-1 · h-1 and urine flow of 0.5 ml · kg-1 · h-1. In winter flounder, plasma and urine [HCO-3] are about 5 and 0.7 mM, respectively, and the amount of HCO-3 reabsorbed would therefore be ~9 µmol · kg-1 · h-1, considerably less than the amount of SO2-4 secreted. The latter can thereby account for the near total removal of HCO-3 from the urine. Catalyzed conversion of reabsorbed HCO-3 would help maintain a chemical driving force for SO2-4 translocation at the apical membrane, as well as contribute to the pH gradient necessary to drive SO2-4 entry at the basolateral membrane. As luminal [HCO-3] is reduced, SO2-4 may continue to exit apically in an electroneutral exchange for luminal anions other than HCO-3 (probably Cl-). The effect of a drug such as methazolamide may therefore be reflected in the amount of sulfate appearing in the urine rather than the urinary [HCO-3] or pH. SO2-4 secretion may be sufficient, even in the absence of CA, to cause reabsorption of most of the filtered bicarbonate, which can probably pass to the interstitium as such at the basolateral membranes. Several such transporters have been found in mammalian proximal tubule (see Ref. 6). There may be a metabolic advantage afforded by CA involvement in sulfate secretion because a lower ATP expenditure would be needed when luminal bicarbonate is available.


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Fig. 5.   Hypothetical model of role of carbonic anhydrase (CA) in marine teleost renal sulfate secretion. The 2HCO-3/SO2-4 and 2OH-/SO2-4 anion exchangers are in the apical and basolateral membranes, respectively (19, 20). Na dependence is indirect and assumed here to be due to luminal Na+/H+ exchanger. Metabolic energy (ATP) is directly needed only for Na-K-ATPase. CA sustains SO2-4 secretion as long as luminal HCO-3 is available for exchange. Once the latter is depleted, Cl- may exchange for SO2-4, and OH- is produced mainly from protolysis of water and Na+/H+ exchange.

Perspectives. One consequence of this transport system is the exchange of a "weak" acid anion for a strong acid anion. Because drinking rate and, therefore, sulfate entry into the animal are likely to be relatively constant, the amount of sulfate secretion is continuous and relatively stable. Thus sulfate secretion may account for the relatively stable acid urine pH of marine teleosts and elasmobranchs. If, as Marshall and Smith (15) contended, bony fishes reinvaded seawater after extensive evolutionary development of the kidney in freshwater, the role of CA in renal acid-base control may have been well established when reinvasion occurred. Thus renal CA may have been a "preadaptation" useful in the development of the sulfate secretory mechanism, which is vital for hypoosmoregulation in seawater.


    ACKNOWLEDGEMENTS

We thank Sonda Springer Parker, Jorge Delgado, Chetankuman Patel, and Jason Mills for excellent technical assistance.


    FOOTNOTES

This work was supported by National Science Foundation Grants IBN9306619 and IBN9604070 (awarded to J. L. Renfro), National Institutes of Health Grant HL-45571 (awarded to E. R. Swenson), and grants from The University of Connecticut Research Foundation (awarded to J. L. Renfro) and The University of Florida Division of Sponsored Research (awarded to T. H. Maren and C. Zeien). J. L. Renfro was also the recipient of a Mount Desert Island Biological Laboratory Salsbury Cove Research Fund Senior Fellowship.

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. §1734 solely to indicate this fact.

Address for reprint requests: J. L. Renfro, Dept. of Physiology and Neurobiology, U-Box 156, Univ. of Connecticut, Storrs, CT 06269.

Received 15 June 1998; accepted in final form 22 October 1998.


    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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Am J Physiol Renal Physiol 276(2):F288-F294
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