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Am J Physiol Regul Integr Comp Physiol 283: R1354-R1361, 2002. First published September 5, 2002; doi:10.1152/ajpregu.00475.2002
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Vol. 283, Issue 6, R1354-R1361, December 2002

Transepithelial sulfate transport by avian renal proximal tubule epithelium in primary culture

Paul L. Dudas and J. Larry Renfro

Department of Physiology and Neurobiology, University of Connecticut, Storrs, Connecticut 06269


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The mechanisms and control of transepithelial inorganic sulfate (Si) transport by primary cultures of chick renal proximal tubule monolayers in Ussing chambers were determined. The competitive anion, S2O32- (5 mM), reduced both unidirectional reabsorptive and secretory fluxes and net Si reabsorption with no effect on electrophysiological properties. The carbonic anhydrase (CA) inhibitor ethoxzolamide decreased net Si reabsorption ~45%. CAII protein and activity were detected in isolated chick proximal tubules by immunoblots and biochemical assay, respectively. Cortisol reduced net Si reabsorption up to ~50% in a concentration-dependent manner. Thyroid hormone increased net Si reabsorption threefold in 24 h, and parathyroid hormone (PTH) acutely stimulated net Si reabsorption ~45%. These data indicate that CA participates in avian proximal tubule active transepithelial Si reabsorption, which cortisol directly inhibits and T3 and PTH directly stimulate.

reabsorptive flux; secretory flux; carbonic anhydrase; chicken


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

INORGANIC SULFATE (Si) undergoes bidirectional transport in the renal proximal tubule (7). Thus, it is important to understand the regulation of this ion in a system where the effects on mechanisms in both basolateral and apical membranes can be observed concurrently. In several definitive studies, putative modulators of renal proximal tubule Si transport were administered in vivo followed by isolation of brush-border membrane (BBM) and basolateral membrane (BLM) vesicles and examination of Si transport capacity, renal cortical NaSi-1 mRNA, and protein. The present study is an examination of the direct effects of several of these modulators on transepithelial transport. The preparation of primary monolayer cultures of chick renal proximal tubule cells (PTCs) on contractible collagen substratum yields a highly differentiated, confluent epithelial tissue (52). Using chick PTCs in Ussing chambers allows independent access to basolateral and apical poles of the PTCs (12) and renders the system amenable to examination of direct regulation of active transepithelial Si transport. Additionally, transepithelial electrophysiology can be monitored providing a measure of tissue integrity in response to various treatments. This makes possible the discrimination of nonspecific metabolic effects from more specific physiological changes in transepithelial transport.

The physiological importance of Si has been well established. Sulfated proteoglycans, the largest group of sulfoconjugates in mammals, are required for the maintenance of normal structure and function of bone and cartilage (6, 17). Therefore, normal growth depends, in part, on Si availability. Potter and Shelton (41) showed this to be true in birds as well. Si is also important for hepatic and renal sulfoconjugation reactions involving several exogenous and endogenous compounds including anti-inflammatory drugs, adrenergic blockers and stimulants, and steroids (37). Sulfoconjugation is essential for the biological activity of many endogenous compounds, such as heparin, heparan sulfate, dermatan sulfate, gastrin, and cholecystokinin (37, 39), and is necessary for the biosynthesis of various structural components of membranes and tissues, including sulfated glycosaminoglycans and cerebroside sulfate (20).

In domestic fowl, Si homeostasis is largely maintained by renal proximal tubular reabsorption just as it is in mammals (18, 29). Filtered Si enters the PTC across the BBM via Na+-dependent Si cotransport (55). In the BLM, Si exits the cell by Si-anion exchange transport for which HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> is the most effective counterion (30, 42). In addition to the Na+-dependent process, Si-anion exchange, pH gradient- and electrical gradient-driven concentrative Si transport are present in the chick BBM (46). The latter three processes may be associated with tubular Si secretion. Earlier studies found negligible, if any, tubular secretion in most mammals (3, 5). Brazy and Dennis (7), however, demonstrated facilitated Si transport in the secretory direction in isolated, perfused rabbit proximal tubules, and Frick et al. (15) provided evidence that increased Si secretion may be an adaptive mechanism following Si loading in rats (15).

Nonsteroidal anti-inflammatory drugs (4), metabolic acidosis (43), dietary Si deficiency (34), alterations in membrane fluidity (25), dietary K+ deficiency (35), and heavy metals (33) alter renal Si handling in mammals. Also, in mammals, thyroid hormone (T3) (50, 53), glucocorticoids (48), growth hormone (49), vitamin D3 (13), insulin-like growth factor (26), estrogen (26), and progesterone (26) have all been demonstrated to modulate some aspect of proximal tubular Si transport. In marine teleosts, cortisol and carbonic anhydrase (CA) activity are known to alter Si secretory transport (44, 47). Earlier work in the chicken showed that glucocorticoid treatment of intact animals significantly lowered Na+:Si cotransport in isolated BBM and had no significant effect on the other aforementioned Si transport processes (46).

In the present study, the relevance of several of the above findings to regulation of renal proximal tubule transepithelial Si transport was investigated. The membrane transport mechanisms for Si have been relatively well characterized and, in some cases, are known to be hormonally modulated. However, few studies have examined potential regulatory factors at the level of transepithelial transport. Here, chick PTCs were used to investigate the direct effects of putative long-term and acutely-acting modulators on proximal tubule Si transport. We hypothesized that control in the avian system would be similar to known mammalian controls but that the integrated epithelial responses obtainable in Ussing chambers would reflect the presence of additional regulated steps. The data demonstrate the presence of CA in the chick proximal tubule and suggest a role for CA in facilitating Si reabsorption in this nephron segment. Additionally, the data indicate that glucocorticoids act directly on the proximal tubule epithelium to decrease active transepithelial Si reabsorption, whereas T3 and parathyroid hormone (PTH) act directly to increase active transepithelial Si reabsorption.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Kidneys were isolated from six to eight white leghorn chicks (domestic Gallus gallus) at 3-7 days of age for each cell culture preparation. The present study adheres to the "Guiding Principles For Research Involving Animals and Human Beings" as outlined by the American Physiological Society (1).

Solutions and chemicals. HBSS was purchased from Mediatech (Herndon, VA). Krebs-Henseleit buffer was purchased from Sigma Chemical (St. Louis, MO). This medium was supplemented with 4 mM NaHCO3 (pH 7.4). The final plating medium and maintenance medium consisted of DMEM Nutrient Mixture F-12 HAM supplemented with Insulin/Transferrin/Selenium premix (ITS; 5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml selenite), 20 µM ethanolamine, 300 µM L-glutamine, 4.6 µM cortisol, and 10% FBS. The saline solution used for Ussing chamber experiments contained (in mM) 1.1 CaCl2, 4.2 KCl, 0.3 MgCl2, 0.4 MgSO4, 120 NaCl, 0.4 NaH2PO4, 0.5 Na2HPO4, 1.0 glycine, and 25 NaHCO3 (pH 7.4 with 5% CO2-95% O2, 290 mosmol/kgH2O). Additionally, 5.5 mM glucose was added to the saline solution at the start of each experiment (t = 0).

T3, cortisol, and human PTH-(1-34) were purchased from Sigma. DMEM-Ham's F-12 was purchased from Mediatech. FBS and Na2S2O3 were purchased from Fisher Scientific (Pittsburgh, PA). ITS was purchased from Collaborative Biomedical Products (Bedford, MA). Ethoxzolamide was purchased from Aldrich (Sheboygan, WI). Quaternary ammonium sulfonamide (QAS) was kindly supplied by Dr. R. Henry, Auburn University. Corticosterone was purchased from Calbiochem (La Jolla, CA). Percoll was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Coat-A-Count cortisol assay kit was purchased from Diagnostic Products (Los Angeles, CA).

Preparation of chicken PTCs. Chicken renal tubule segments were isolated and dispersed as previously described by Sutterlin and Laverty (52) and modified by Dudas and Renfro (11). Briefly, kidneys were removed; rinsed in HBSS; cleaned of blood vessels, ducts, and connective tissue; and minced. The tissue fragments were incubated in an enzyme solution containing collagenase A (0.13 U/ml) (Roche Applied Science, Indianapolis, IN) and dispase II (0.54 U/ml)(Roche) at 37°C for 10 min. Nephron segments were further dissociated by trituration and filtration through a stainless steel sieve (380 µm). The dissociated tissue was rinsed three times with HBSS, the last rinse containing DNase I (2,161 U/ml) (Roche), and resuspended in a 1:1 Percoll:×2 Krebs-Henseleit buffer. The suspension was centrifuged 17,500 g, and the high-density band consisting of small proximal tubule segments (PTs) was removed, rinsed with HBSS, suspended in culture medium with 10% serum, and plated on native rat-tail collagen as previously described (10). After 6 days, the collagen gels were released, and after ~14 days, the floating collagen gels had been contracted by the epithelial monolayers ~40%. For all experiments, unless otherwise noted, the cortisol supplement was removed from the culture medium 48 h before and FBS was removed from the culture medium 24 h before examining Si transport capacity. Removing the cortisol at this time prevented the hormone's inhibitory effect on Si transport but avoided any detrimental effects associated with insufficient availability [i.e., dedifferentiation, poor attachment (14)]. Because the FBS contained a number of other nutritive components in addition to cortisol (1.2 nM at 10%), it remained in the culture medium 24 h longer.

Ussing chamber studies. During days 15-29, transepithelial electrical characteristics and Si transport were measured. The tissues were supported by 150-µm nylon mesh and mounted in Ussing chambers as previously described (19). The temperature was maintained at 39°C, and the saline bathing the luminal and basolateral sides of the tissue was continuously gassed (95% O2-5% CO2) and stirred throughout the experiment.

Transepithelial electrical potential (VT) was determined with a pair of reference electrodes connected to the luminal and basolateral compartments via 3 M KCl-2% agar bridges. Current was passed through Ag-AgCl2 electrodes connected to the luminal and basolateral compartments with 3 M KCl-2% agar bridges. Electrical properties were measured with a pair of computer-controlled, high-impedance automatic dual-voltage clamps (DVC 1000; World Precision Instruments, Sarasota, FL). Transepithelial electrical resistance (TER) was determined from the change in VT produced by a 10-µA current pulse and corrected for fluid resistance.

Determination of transepithelial Si fluxes. Tissues were continuously short-circuited during flux determinations, i.e., there were no transepithelial electrical or chemical gradients. Unidirectional tracer fluxes were initiated by the addition of 1.0-2.0 µCi 35SO<UP><SUB>4</SUB><SUP>2−</SUP></UP> (ICN, Costa Mesa, CA) to the appropriate hemichamber at the beginning of each experiment (t = 0). Duplicate 50-µl samples were taken from the unlabeled side every 30 min over a period of 1.5 h and replaced with equal volumes of unlabeled saline (see Fig. 1). The specific activity of the labeled solution was determined at the beginning and end of each experiment.


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  		Fig. 1.   Representative plot of inorganic sulfate (Si) unidirectional and net fluxes vs. time. B to L is basolateral-to-luminal secretory flux; L to B is luminal-to-basolateral reabsorptive flux (shown negative to indicate direction). Net flux is the difference between unidirectional fluxes. A: paired controls. B: Na2S2O3 (5 mM) added to B and L sides at t = 0. Fluxes approached steady state at t = 1 h.

Net flux was calculated as the difference between unidirectional secretory (basolateral to luminal) and reabsorptive (luminal to basolateral) 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 monolayer cultures used in a given experiment were prepared from the same starting tissue at the same time and cultured under identical conditions. For statistical determinations, this is referred to as one preparation. Tissue VT, TER, and phloridzin-sensitive current (IPHZ; Na+-dependent glucose transport) were used to assess tissue integrity and proximal tubule-like function.

CA assay. The paranitrophenol indicator procedure for determining CA activity was adapted from Brion et al. (8). Briefly, PTs were isolated on a Percoll gradient as described above and resuspended in HBSS. The CA assay was conducted at ~0-0.5°C by combination of CO2-saturated HBSS, PT suspension, and buffer/indicator mix [containing (in mM) 5.0 Tris · HCl, 20 imidazole, and 0.4 para-nitrophenol (indicator)] in 400 µl total. To assay intracellular vs. extracellular CA isoforms, the proximal tubule suspensions were preincubated with the membrane-permeable CA inhibitor ethoxzolamide or the membrane-impermeable CA inhibitor QAS, respectively, on ice for 15 min.

SDS-PAGE and immunoblotting. PTs, chick kidney homogenate (CKH), and chick blood (CB) were placed in Kaman buffer (2.3% SDS, 5% beta -mercaptoethanol, 10% glycerol, 0.5% saturated bromophenol blue, 62.5 mM trizma base, pH 6.8) and vortexed ~20-30 s. Twenty microliters of sample were used for SDS-PAGE (4-12%) and subsequent transfer to polyvinylidene fluoride (PVDF) microporous membrane (Millipore, Bedford, MA). Nonspecific binding was blocked by incubating the PVDF membrane at 4°C overnight in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.3 with HCl) containing 10% nonfat dry milk, 0.01% antifoam-A, 0.02% sodium azide, and 0.05% polyoxyethylene-sorbitan monolaurate (Tween 20). CAII was detected using polyclonal sheep antiserum (antibody dilution, 1/333) raised against human CAII (Accurate Chemical and Scientific, Westbury, NY). Staining of the 29-kDa molecular mass marker (MM) (CAII from bovine erythrocytes) served as a positive control. Incubation with the primary antibody took place for 1 h at room temperature. The PVDF membrane was washed two times in PBS followed by one rinse in phosphate-free buffer [150 mM NaCl, 10 mM trizma-base, 40 mM trizma-HCl (pH 7.5)]. The PVDF membrane was incubated with a 1:5,000 dilution of donkey-anti-sheep IgG alkaline phosphatase conjugate (Sigma) in phosphate-free buffer with 10% nonfat dry milk for 1 h at room temperature. The PVDF membrane was washed three times with phosphate-free buffer and the signals were detected by 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (Sigma) according to the manufacturer's protocol. High-range SDS-PAGE MM marker proteins (Sigma) were run parallel.

Statistics. Experimental results are expressed as means ± SE. Sample means were compared with paired and unpaired one-tailed Student's t-tests. Differences were judged significant if P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Demonstration of active transepithelial Si reabsorption. Figure 1A is a representative plot illustrating active net transepithelial Si reabsorption by chick PTCs under short-circuited conditions. Unidirectional fluxes were initiated by addition of 35SO<UP><SUB>4</SUB><SUP>2−</SUP></UP> at t = 0. Transepithelial reabsorptive flux approached steady state at 1 h, reflecting the time necessary for equilibration of isotopic label and the transportable Si pool. Secretory flux was ~30% of reabsorptive flux. VT, TER, and IPHZ averaged -1.13 ± 0.07 mV (sign relative to luminal side), 29.61 ± 2.06 Omega /cm2, and 10.99 ± 1.14 µA/cm2, respectively. In the example shown, addition of 5 mM Na2S2O3, a specific competitive inhibitor of proximal tubule Si transport (7), to the basolateral and luminal sides of paired culture mates at t = 0 (Fig. 1B) decreased net transepithelial Si reabsorption almost to zero. Corresponding electrophysiological data for all flux studies presented below are shown in Table 1.

                              
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  		Table 1.   Effects of treatments on chick proximal tubule cell monolayer culture transepithelial electrical properties

On average, addition of 5 mM Na2S2O3 to the basolateral and luminal sides of PTCs in Ussing chambers reduced the unidirectional Si reabsorptive flux by ~70% (10.28 ± 1.60 to 3.01 ± 0.70 nmol · cm-2 · h-1) and the Si secretory flux by ~40% (3.33 ± 0.08 to 2.06 ± 0.43 nmol · cm-2 · h-1) resulting in an ~86% decrease in net transepithelial Si reabsorption (6.94 ± 1.58 to 0.95 ± 0.92 nmol · cm-2 · h-1) (Fig. 2A). Inhibition of both unidirectional fluxes by Na2S2O3 was one indication that transport is carrier mediated in both directions. Table 1 shows that Na2S2O3 had no effect on IPHZ, TER, or VT.


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  		Fig. 2.   Effect of Na2S2O3 and ethoxzolamide on transepithelial reabsorptive (L to B), secretory (B to L), and net sulfate (Si) transport by chick proximal tubule cell monolayer cultures. A: 5 mM Na2S2O3 added to both basolateral and luminal sides at t = 0. Each bar is mean + 1 SE of n = 4 preparations. B: 100 µM ethoxzolamide added to both basolateral and luminal sides at t = 0. Electrical properties of these same tissues are shown in Table 1. *Significantly different from paired controls (P < 0.05).

CA modulation of Si transport. Renfro et al. (47) demonstrated that renal Si secretion in the marine teleost is enhanced by CA. CA is hypothesized to couple Si uptake by Si/OH- exchange from interstitium to cell across the BLM to extrusion of Si by Si/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange from cell to lumen across the BBM. An earlier study provided evidence for Si/HCO3- exchange in isolated chick BLM and BBM raising the possibility of CA-facilitated Si transport. Addition of the CA inhibitor ethoxzolamide (100 µM) to basolateral and luminal sides of PTCs in Ussing chambers significantly decreased unidirectional Si reabsorptive flux (9.23 ± 1.36 to 7.44 ± 1.32 nmol · cm-2 · h-1) and net transepithelial Si reabsorption ~45% (5.20 ± 1.52 to 2.86 ± 1.49 nmol · cm-2 · h-1) (Fig. 2B). Ethoxzolamide inhibition was entirely through inhibition of Si reabsorption with unidirectional Si secretory flux remaining unchanged. The drug slightly increased TER. However, IPHZ and VT remained unchanged (Table 1).

Figure 3A shows immunoblots of CAII in isolated chick PTs, CB, and CKH. Polyclonal antibodies raised against human CAII were used for detection under reducing conditions. The 29-kDa MM marker (CAII from bovine erythrocytes) served as a positive control.


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  		Fig. 3.   Immunoblotting and biochemical assay for carbonic anhydrase. A: immunoblotting for carbonic anhydrase II (CAII) in isolated chick proximal tubules (PTs), chick blood (CB), and chick kidney homogenate (CKH). Also shown is the 29-kDa molecular mass marker (MM) (CAII from bovine erythrocytes). A molecular mass band corresponding to CAII expression as detected by a polyclonal antibody raised against human CAII was present in all samples. B: biochemical assay of CA activity in isolated chick PTs. Assay conditions included: No treatment, 100 µM ethoxzolamide (EZ), and 100 µM quaternary ammonium sulfonamide (QAS). Significantly different from controls *P < 0.05 and **P < 0.01.

Biochemical assay of isolated chick PTs confirmed the presence of CA activity. Preincubating the proximal tubule suspension with 100 µM of the moderately lipid soluble CA inhibitor ethoxzolamide reduced enzyme activity from 25.0 ± 1.3 U/mg protein to zero (Fig. 3B). Preincubation with the impermeable CA inhibitor QAS reduced activity to 18.4 ± 1.9 U/mg protein (Fig. 3B). The data support intracellular localization of CA, consistent with the CAII isoform, and indicate a substantial amount of extracellular CA activity (26% of total).

Cortisol effect on Si transport. Glucocorticoids have been shown to stimulate transepithelial Si secretion in the proximal tubule of marine teleosts through an increase in BBM Si/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange (44), whereas in birds and mammals, such treatment inhibits Na+:Si cotransport in BBM (46, 48). Figure 4 shows that cortisol (4.6 µM), present in the chick PTC growth medium from the day of culturing, significantly reduced unidirectional Si reabsorptive flux ~30% (8.85 ± 0.43 to 6.48 ± 0.99 nmol · cm-2 · h-1) and net transepithelial Si reabsorption ~50% (3.94 ± 0.55 to 1.90 ± 0.70 nmol · cm-2 · h-1) compared with control tissues from which the cortisol was removed 48 h before and FBS was removed 24 h before examining Si transport in Ussing chambers. Unidirectional Si secretory flux was unchanged. IPHZ and TER were also unaffected by cortisol. However, there was a small but significant increase in VT (Table 1). These data demonstrate the direct regulation of active transepithelial Si reabsorption by glucocorticoids in chick PTCs.


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  		Fig. 4.   Effect of increasing cortisol concentrations on transepithelial reabsorptive (L to B), secretory (B to L), and net sulfate (Si) transport by chick proximal tubule cell (PTC) monolayer cultures. Each bar is mean + 1 SE of n = 7 preparations for no cortisol and 4.6 µM cortisol and n = 4 for all other concentrations. Electrical properties of these same tissues are shown in Table 1. *Significantly different from paired controls (P < 0.05). aSignificantly different from 0.0046 µM cortisol (P < 0.05). bSignificantly different from 0.46 µM cortisol. Different cortisol concentrations were present in chick PTC growth medium from day of culturing. Control tissues had cortisol removed at t = -48 h and FBS removed at t = -24 h.

The effect of intermediate culture medium cortisol concentrations on chick PTC Si transport is also shown in Fig. 4. Although net transport tended to decrease with continuous exposure of chick PTCs to increasing levels of cortisol, the effects became significant beginning at 0.46 µM (3.94 ± 0.55 to 1.54 ± 1.21 nmol · cm-2 · h-1). The tendency of unidirectional Si secretory flux to increase while unidirectional reabsorptive flux was unchanging caused the decrease in net reabsorption. Comparisons of 0.0046 to 0.46 µM revealed a significantly increased unidirectional secretory flux (4.40 ± 0.49 to 6.95 ± 1.10 nmol · cm-2 · h-1) (Fig. 4). Note that with the increase to 4.6 µM cortisol, both unidirectional fluxes as well as net transepithelial Si reabsorption decreased significantly.

Glucose current tended to increase from 0.0046 to 0.046 µM and was statistically significant at 0.46 and 4.6 µM cortisol compared with 0.0046 µM (Table 1). VT followed a similar trend to IPHZ in that compared with control, it was significantly increased at a cortisol concentration of 0.46 µM, and between control and 0.0046 µM, VT remained unchanged. With VT, however, there was a significant increase between 0.0046 and 0.046 µM and also 0.0046 and 0.46 µM (Table 1). At the highest cortisol concentration (4.6 µM), VT was significantly increased compared with control levels (Table 1). TER was unaffected by cortisol.

T3 effect on Si transport. In mammals, renal Si reabsorption is modulated by T3. Elevated plasma T3 increases Na+:Si cotransport in mouse renal BBM vesicles (53), and decreased plasma T3 resulted in an approximately twofold decrease in Na+:Si cotransport in rat renal BBM vesicles together with a significant reduction in Na+:Si cotransporter (NaSi-1) mRNA and BBM NaSi-1 protein levels (50). Addition of 3 µM T3 to the chick PTC growth medium 24 h before examining Si transport in Ussing chambers resulted in a significant increase in unidirectional Si reabsorptive flux (7.89 ± 0.55 to 11.47 ± 1.39 nmol · cm-2 · h-1) and an approximately threefold increase in net transepithelial Si reabsorption (1.81 ± 1.09 to 5.56 ± 1.06 nmol · cm-2 · h-1) (Fig. 5). T3 stimulation was entirely through increased Si unidirectional reabsorptive flux. IPHZ, TER, and VT were unaffected by T3 (Table 1). These data demonstrate direct T3 action on active transepithelial Si reabsorption by chick PTCs. The dosage used in this study greatly exceeds physiological T3 concentrations in the chick (~7-8 nM). T3 was used at this high level because of the expected rapid degradation by peripheral tissues in the chicken, including the kidneys (21) (in vivo half-life of ~3 h) (56), and because the effect of binding proteins present in the FBS was unknown (56).


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  		Fig. 5.   Effect of thyroid hormone (T3) and parathyroid hormone (PTH) on transepithelial reabsorptive (L to B), secretory (B to L), and net sulfate (Si) transport by chick PTC monolayer cultures. A: 3 µM T3 was added to the culture medium at t = -24 h. Each bar is mean + 1 SE of n = 6 preparations. B: 10-9 M PTH added to basolateral sides at t = 0. Each bar is mean + 1 SE of n = 3 preparations. Electrical properties of these same tissues are shown in Table 1. *Significantly different from paired controls (P < 0.05).

PTH effect on Si transport. Although in mammals PTH appears to have no effect on the expression or activity of the BBM Na+:Si cotransporter, its dramatic reduction of Na+-dependent Pi transport (12, 23, 27) and Na+-H+ exchange (40) in the BBM might influence other Na+-dependent processes, including Si transport. Addition of PTH (10-9 M) to the basolateral sides of chick PTCs in Ussing chambers at t = 0 significantly stimulated net transepithelial Si reabsorption ~45% (3.62 ± 1.52 to 6.55 ± 1.85 nmol · cm-2 · h-1) (Fig. 5). The increase in net reabsorption was due to a concurrent slight increase in unidirectional Si reabsorptive flux and a slight decrease in unidirectional Si secretory flux. IPHZ was significantly reduced with PTH addition, but TER and VT remained unaffected at the conclusion of the 1.5-h flux period.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Using isolated, perfused proximal tubule segments from the rabbit, Brazy and Dennis (7) demonstrated bidirectional Si transport by inhibiting the unidirectional reabsorptive flux with S2O<UP><SUB>3</SUB><SUP>2−</SUP></UP> in either the bath or perfusate and the unidirectional secretory flux with S2O<UP><SUB>3</SUB><SUP>2−</SUP></UP> to the bath only. A net reabsorption of Si resulted from the difference in unidirectional fluxes. A similar effect was seen in the present study following exposure of the basolateral and luminal sides of chick PTCs to this inhibitor. The effect was a significant reduction in unidirectional reabsorptive flux, unidirectional secretory flux, and net reabsorption of Si. These data indicate mediated bidirectional Si transport in the avian system with a prevailing net active transepithelial reabsorption in biophysically precise short-circuited conditions and no nonspecific metabolic effects as indicated by the electrophysiological properties.

Perhaps most notable in the present study was the role of CA in Si reabsorption. The immunoblot detecting CAII combined with biochemical data indicating intracellular CA activity and strong ethoxzolamide inhibition of Si transport support the idea of CA-dependent Si reabsorption. In the mammalian proximal tubule, CA is associated with the BBM, BLM (57), and cytosol (28). The bulk of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption by this segment is CA dependent (31). It is thought that up to 5% of CA activity is probably due to membrane-bound extracellular isoforms with the remainder primarily cytoplasmic (CAII) (9, 36, 51, 58). As noted above, CA modulates renal Si secretion in primary cultures of marine teleost proximal tubule probably by increasing the availability of OH- for a Si/OH- exchanger in the BLM through accelerating dehydroxylation of the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> entering the cell on a Si/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger in the BBM (47). We tested the effect of ethoxzolamide because Si/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange is present in both the BLM and BBM of the chicken proximal tubule and almost certainly functions in transepithelial Si transport. At 100 µM, the drug inhibited all proximal tubule CA activity and about one-half of net transepithelial Si reabsorption through inhibition of the unidirectional Si reabsorptive flux with no effect on secretory flux. Chick proximal tubule CA activity was about one-half that seen in the mammalian proximal tubule (8). However, immunohistochemical work on avian renal CA by others detected the enzyme only in the distal tubule [quail (16), starling (24)]. In the present study, CAII protein and biochemical activity were detected in an enriched population of proximal tubule segments. Either the methods employed or perhaps differences in CA expression in different avian species may explain this discrepancy.

Both immunoblots and the impermeant CA inhibitor indicated the bulk of CA was intracellular. Some inhibition did occur with the impermeable QAS suggesting that about one-fourth of the total CA activity is extracellular. A possible scheme for CA-facilitated Si reabsorption should perhaps involve intracellular CAII dehydroxylation of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> that enters the cell in exchange for Si across the BLM by Si/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange thus sustaining a local HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> gradient for Si exit to the interstitium.

Treatment of 3-wk-old chickens with dexamethasone reduces BBM vesicle Na+:Si cotransport (45). Similarly, treatment of rats with the glucocorticoid methylprednisolone increases urinary excretion of Si and decreases BBM vesicle Na+:Si cotransport, effects that coincide with similar decreases in renal cortical NaSi-1 protein and mRNA levels (48). In a recent review, Markovich (32) reported preliminary data indicating that treatment of rats with dexamethasone reduced BBM vesicle Na+:Si cotransport resulting from downregulation of renal cortical NaSi-1 protein and mRNA levels. The present study with chick PTCs supports these previous vesicle data and indicates a similarity in glucocorticoid effect on transepithelial Si transport in birds and mammals. The data reported here demonstrate a direct inhibition of proximal tubule epithelium Si reabsorption by cortisol. This is consistent with downregulation of NaSi-1 mRNA and protein by glucocorticoids and recent data showing the Nas1 promoter possesses several glucocorticoid response elements that may act in regulating the transcription of the Nas1 gene (32) in mammals.

As culture medium cortisol concentrations increased from no cortisol to 0.46 µM, there appeared to be a progressive increase in unidirectional Si secretory flux. Within this concentration range, there was no effect on unidirectional Si reabsorptive flux. This was unexpected based on previous studies demonstrating a downregulation of the BBM NaSi-1 in response to glucocorticoid treatment (48). Interestingly, with a further 10-fold increase in cortisol concentration to 4.6 µM, there was continued inhibition of net transepithelial Si reabsorption caused mainly by a decrease in unidirectional Si reabsorptive flux. These data supported the presence of mediated secretion indicated by S2O32- inhibition of the secretory flux and also revealed that glucocorticoid-induced inhibition of net reabsorption may result from changes in secretory as well as reabsorptive transport.

Cortisol is present in the plasma of perinatal chicks. However, corticosterone is the principal steroid hormone produced by the adult avian adrenal cortex. Activity of 17-alpha -hydroxylase, an essential enzyme in the cortisol synthetic pathway, declines around hatch and is absent in the adrenals of chickens older than 2 wk (38). Although the birds used in the present study were 5-7 days old, we also tested corticosterone (4.6 µM), instead of cortisol, from the day of culturing. In two preparations, the adult hormone dramatically reduced net transepithelial Si reabsorption ~80% (6.17 to 1.26 nmol · cm-2 · h-1). IPHZ increased with corticosterone treatment, indicating a very similar effect to cortisol. Corticosterone treatment tended to increase VT, and TER remained unchanged.

Treatment of the mammalian proximal tubule with T3 stimulates Na+:Si cotransport activity in BBM vesicles (53), and reduction in T3 reduces NaSi-1 mRNA and protein levels (50). Beck and Markovich (2) demonstrated that the Nas1 promoter contains several thyroid hormone response elements, and Markovich (32) also provided preliminary data in a recent review indicating Nas1 promoter activity in OK cells is upregulated in the presence of T3. These data suggest that in the mammal, T3 has a direct effect on renal proximal tubule epithelium. The present study in chick PTCs demonstrated the stimulatory effect of the hormone on renal proximal tubule epithelium in an avian system and provided additional evidence that the effect of T3 is indeed direct. Also, for the first time, the effect of T3 was examined on transepithelial Si transport rather than tissue uptake, demonstrating the effect was entirely through stimulation of the reabsorptive component with no effect on Si secretory flux.

PTH stimulation of net transepithelial Si reabsorption was an unexpected result based on earlier findings by others showing no effect of this hormone on proximal tubule NaSi-1 expression in mammals. The stimulation of net transepithelial reabsorption was due to a concurrent slight increase in unidirectional Si reabsorption and a slight decrease in unidirectional Si secretion. The majority of previously used methodology examined only Si uptake into renal epithelial cells or membrane vesicles and would not have revealed an effect on net transepithelial transport. The data also indicate PTH inhibition of IPHZ (Na+:glucose cotransport). Inhibition of this Na+-dependent transport process in conjunction with previously demonstrated inhibition of Na+:Pi cotransport (12) and likely inhibition of Na+-H+ exchange [as in the mammal (22)] would facilitate Si reabsorption through augmentation of the Na+ gradient.

In summary, examination of transepithelial Si transport by primary cultures of chicken proximal tubule epithelium as monolayers in Ussing chambers has provided important evidence for 1) mediated bidirectional Si flux, 2) active transepithelial Si reabsorption, i.e., net transport under short-circuited conditions, 3) the presence in an avian proximal tubule of CAII that subserves Si reabsorption, and 4) a direct action of cortisol on the epithelium to inhibit net transepithelial Si reabsorption and direct action by T3 and PTH to stimulate net transepithelial Si reabsorption.

Perspectives

This study presented the second vertebrate species in which CA activity in the proximal tubule has been associated with Si transport. In the marine winter flounder proximal tubule, CA inhibition strongly inhibits net transepithelial Si secretion, whereas in the chick proximal tubule, CA inhibition strongly inhibited net transepithelial Si reabsorption. Thus, in each case, increased Si excretion ought to result. One possible interpretation is that CA is influencing Si transport through Si/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange at the BBM of the flounder and at the BLM of the bird. Therefore, in the latter case, Si reabsorption is limited by Si exit, cell to interstitium, a fact that raises the prospect of a control point for net renal Si reabsorption in addition to the BBM NaSi-1. This effect provides an interesting perspective on proximal tubule function. All indications at present are that Si control is very similar in birds and mammals. With that assumption, note that metabolic acidosis inhibits NaSi-1 mRNA, NaSi-1 content, BBM Na+:Si cotransport activity (43), and presumably net transepithelial Si reabsorption in the mammalian proximal tubule. This decrease in transporter activity is associated with an increase in proximal tubule CAII and IV mRNA and CA activity (54). The latter presumably facilitates increased acid excretion and concurrent HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption. In this circumstance, if there was no decrease in NaSi-1, the CAII could conceivably increase Si reabsorption; an effect that might counter net acid excretion. Interestingly, glucocorticoids stimulate Si secretion in the flounder and inhibit Si reabsorption in the bird and mammal. However, the relationship of glucocorticoids to CA is not yet clear.


    ACKNOWLEDGEMENTS

The authors gratefully acknowledge the technical assistance of S. Parker, D. Bailey, M. Gleeson, and M. Eckersdorf.


    FOOTNOTES

This work was supported by the University of Connecticut Research Foundation and National Science Foundation Grant 0078093.

Address for reprint requests and other correspondence: J. L. Renfro, Dept. of Physiology and Neurobiology, Univ. of Connecticut, 3107 Horsebarn Hill Rd., U-4156 Storrs, CT 06269-4156 (E-mail: jlrenfro{at}uconnvm.uconn.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.

First published September 5, 2002;10.1152/ajpregu.00475.2002

Received 9 August 2002; accepted in final form 3 September 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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