INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

INORGANIC SULFATE (S_i) 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 S_i transport were administered in vivo followed by isolation of brush-border membrane (BBM) and basolateral membrane (BLM) vesicles and examination of S_i 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 S_i 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 S_i 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 S_i availability. Potter and Shelton (41) showed this to be true in birds as well. S_i 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, S_i homeostasis is largely maintained by renal proximal tubular reabsorption just as it is in mammals (18, 29). Filtered S_i enters the PTC across the BBM via Na⁺-dependent S_i cotransport (55). In the BLM, S_i exits the cell by S_i-anion exchange transport for which HCO is the most effective counterion (30, 42). In addition to the Na⁺-dependent process, S_i-anion exchange, pH gradient- and electrical gradient-driven concentrative S_i transport are present in the chick BBM (46). The latter three processes may be associated with tubular S_i secretion. Earlier studies found negligible, if any, tubular secretion in most mammals (3, 5). Brazy and Dennis (7), however, demonstrated facilitated S_i transport in the secretory direction in isolated, perfused rabbit proximal tubules, and Frick et al. (15) provided evidence that increased S_i secretion may be an adaptive mechanism following S_i loading in rats (15).

Nonsteroidal anti-inflammatory drugs (4), metabolic acidosis (43), dietary S_i deficiency (34), alterations in membrane fluidity (25), dietary K⁺ deficiency (35), and heavy metals (33) alter renal S_i 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 S_i transport. In marine teleosts, cortisol and carbonic anhydrase (CA) activity are known to alter S_i secretory transport (44, 47). Earlier work in the chicken showed that glucocorticoid treatment of intact animals significantly lowered Na⁺:S_i cotransport in isolated BBM and had no significant effect on the other aforementioned S_i transport processes (46).

In the present study, the relevance of several of the above findings to regulation of renal proximal tubule transepithelial S_i transport was investigated. The membrane transport mechanisms for S_i 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 S_i 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 S_i reabsorption in this nephron segment. Additionally, the data indicate that glucocorticoids act directly on the proximal tubule epithelium to decrease active transepithelial S_i reabsorption, whereas T₃ and parathyroid hormone (PTH) act directly to increase active transepithelial S_i 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).

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 S_i transport capacity. Removing the cortisol at this time prevented the hormone's inhibitory effect on S_i 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 S_i 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.

Determination of transepithelial S_i 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 µC_i ³⁵SO (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.

View larger version (19K): [in this window] [in a new window]   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.

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% -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 S_i reabsorption. Figure 1A is a representative plot illustrating active net transepithelial S_i reabsorption by chick PTCs under short-circuited conditions. Unidirectional fluxes were initiated by addition of ³⁵SO at t = 0. Transepithelial reabsorptive flux approached steady state at 1 h, reflecting the time necessary for equilibration of isotopic label and the transportable S_i pool. Secretory flux was ~30% of reabsorptive flux. V_T, TER, and I_PHZ averaged 1.13 ± 0.07 mV (sign relative to luminal side), 29.61 ± 2.06 /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 S_i transport (7), to the basolateral and luminal sides of paired culture mates at t = 0 (Fig. 1B) decreased net transepithelial S_i reabsorption almost to zero. Corresponding electrophysiological data for all flux studies presented below are shown in Table 1.

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View larger version (21K): [in this window] [in a new window]   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 S_i transport. Renfro et al. (47) demonstrated that renal S_i secretion in the marine teleost is enhanced by CA. CA is hypothesized to couple S_i uptake by S_i/OH exchange from interstitium to cell across the BLM to extrusion of S_i by S_i/HCO exchange from cell to lumen across the BBM. An earlier study provided evidence for S_i/HCO₃ exchange in isolated chick BLM and BBM raising the possibility of CA-facilitated S_i transport. Addition of the CA inhibitor ethoxzolamide (100 µM) to basolateral and luminal sides of PTCs in Ussing chambers significantly decreased unidirectional S_i reabsorptive flux (9.23 ± 1.36 to 7.44 ± 1.32 nmol · cm² · h¹) and net transepithelial S_i reabsorption ~45% (5.20 ± 1.52 to 2.86 ± 1.49 nmol · cm² · h¹) (Fig. 2B). Ethoxzolamide inhibition was entirely through inhibition of S_i reabsorption with unidirectional S_i secretory flux remaining unchanged. The drug slightly increased TER. However, I_PHZ and V_T remained unchanged (Table 1).

View larger version (28K): [in this window] [in a new window]   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.

Cortisol effect on S_i transport. Glucocorticoids have been shown to stimulate transepithelial S_i secretion in the proximal tubule of marine teleosts through an increase in BBM S_i/HCO exchange (44), whereas in birds and mammals, such treatment inhibits Na⁺:S_i 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 S_i reabsorptive flux ~30% (8.85 ± 0.43 to 6.48 ± 0.99 nmol · cm² · h¹) and net transepithelial S_i reabsorption ~50% (3.94 ± 0.55 to 1.90 ± 0.70 nmol · cm² · h¹) compared with control tissues from which the cortisol was removed 48 h before and FBS was removed 24 h before examining S_i transport in Ussing chambers. Unidirectional S_i secretory flux was unchanged. I_PHZ and TER were also unaffected by cortisol. However, there was a small but significant increase in V_T (Table 1). These data demonstrate the direct regulation of active transepithelial S_i reabsorption by glucocorticoids in chick PTCs.

View larger version (25K): [in this window] [in a new window]   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.

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T₃ effect on S_i transport. In mammals, renal S_i reabsorption is modulated by T₃. Elevated plasma T₃ increases Na⁺:S_i cotransport in mouse renal BBM vesicles (53), and decreased plasma T₃ resulted in an approximately twofold decrease in Na⁺:S_i cotransport in rat renal BBM vesicles together with a significant reduction in Na⁺:S_i 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 S_i transport in Ussing chambers resulted in a significant increase in unidirectional S_i reabsorptive flux (7.89 ± 0.55 to 11.47 ± 1.39 nmol · cm² · h¹) and an approximately threefold increase in net transepithelial S_i reabsorption (1.81 ± 1.09 to 5.56 ± 1.06 nmol · cm² · h¹) (Fig. 5). T₃ stimulation was entirely through increased S_i unidirectional reabsorptive flux. I_PHZ, TER, and V_T were unaffected by T₃ (Table 1). These data demonstrate direct T₃ action on active transepithelial S_i 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).

View larger version (22K): [in this window] [in a new window]   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: 109 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 S_i transport. Although in mammals PTH appears to have no effect on the expression or activity of the BBM Na⁺:S_i cotransporter, its dramatic reduction of Na⁺-dependent P_i transport (12, 23, 27) and Na⁺-H⁺ exchange (40) in the BBM might influence other Na⁺-dependent processes, including S_i transport. Addition of PTH (10⁹ M) to the basolateral sides of chick PTCs in Ussing chambers at t = 0 significantly stimulated net transepithelial S_i reabsorption ~45% (3.62 ± 1.52 to 6.55 ± 1.85 nmol · cm² · h¹) (Fig. 5). The increase in net reabsorption was due to a concurrent slight increase in unidirectional S_i reabsorptive flux and a slight decrease in unidirectional S_i secretory flux. I_PHZ was significantly reduced with PTH addition, but TER and V_T 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 S_i transport by inhibiting the unidirectional reabsorptive flux with S₂O in either the bath or perfusate and the unidirectional secretory flux with S₂O to the bath only. A net reabsorption of S_i 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 S_i. These data indicate mediated bidirectional S_i 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 S_i reabsorption. The immunoblot detecting CAII combined with biochemical data indicating intracellular CA activity and strong ethoxzolamide inhibition of S_i transport support the idea of CA-dependent S_i reabsorption. In the mammalian proximal tubule, CA is associated with the BBM, BLM (57), and cytosol (28). The bulk of HCO 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 S_i secretion in primary cultures of marine teleost proximal tubule probably by increasing the availability of OH for a S_i/OH exchanger in the BLM through accelerating dehydroxylation of the HCO entering the cell on a S_i/HCO exchanger in the BBM (47). We tested the effect of ethoxzolamide because S_i/HCO exchange is present in both the BLM and BBM of the chicken proximal tubule and almost certainly functions in transepithelial S_i transport. At 100 µM, the drug inhibited all proximal tubule CA activity and about one-half of net transepithelial S_i reabsorption through inhibition of the unidirectional S_i 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 S_i reabsorption should perhaps involve intracellular CAII dehydroxylation of HCO that enters the cell in exchange for S_i across the BLM by S_i/HCO exchange thus sustaining a local HCO gradient for S_i exit to the interstitium.

Treatment of 3-wk-old chickens with dexamethasone reduces BBM vesicle Na⁺:S_i cotransport (45). Similarly, treatment of rats with the glucocorticoid methylprednisolone increases urinary excretion of S_i and decreases BBM vesicle Na⁺:S_i 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⁺:S_i 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 S_i transport in birds and mammals. The data reported here demonstrate a direct inhibition of proximal tubule epithelium S_i 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 S_i secretory flux. Within this concentration range, there was no effect on unidirectional S_i 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 S_i reabsorption caused mainly by a decrease in unidirectional S_i 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--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 S_i reabsorption ~80% (6.17 to 1.26 nmol · cm² · h¹). I_PHZ increased with corticosterone treatment, indicating a very similar effect to cortisol. Corticosterone treatment tended to increase V_T, and TER remained unchanged.

Treatment of the mammalian proximal tubule with T₃ stimulates Na⁺:S_i 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 S_i transport rather than tissue uptake, demonstrating the effect was entirely through stimulation of the reabsorptive component with no effect on S_i secretory flux.

PTH stimulation of net transepithelial S_i 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 S_i reabsorption and a slight decrease in unidirectional S_i secretion. The majority of previously used methodology examined only S_i 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 I_PHZ (Na⁺:glucose cotransport). Inhibition of this Na⁺-dependent transport process in conjunction with previously demonstrated inhibition of Na⁺:P_i cotransport (12) and likely inhibition of Na⁺-H⁺ exchange [as in the mammal (22)] would facilitate S_i reabsorption through augmentation of the Na⁺ gradient.

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

Perspectives