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
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ABSTRACT |
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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
(SO4) 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
SO4
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
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INTRODUCTION |
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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. HCO3,
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.
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METHODS |
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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 M · 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.
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RESULTS |
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In the presence of flounder saline (1 mM
SO24), 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
· cm2)
were unaffected by the drug (Fig. 2).
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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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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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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
HCO3 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.
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DISCUSSION |
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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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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.
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ACKNOWLEDGEMENTS |
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We thank Sonda Springer Parker, Jorge Delgado, Chetankuman Patel, and Jason Mills for excellent technical assistance.
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FOOTNOTES |
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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.
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