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Endocrinology Vol. 142, No. 2 710-720
Copyright © 2001 by The Endocrine Society

ARTICLES

Regulation of Phosphate Uptake in Primary Cultured Rabbit Renal Proximal Tubule Cells by Glucocorticoids: Evidence for Nongenomic as Well as Genomic Mechanisms1

Soo-Hyun Park, Mary Taub and Ho-Jae Han

Department of Veterinary Physiology (S.-H.P., H.-J.H.), College of Veterinary Medicine, Hormone Research Center, Chonnam National University, Kwangju 500–757, Korea; and Biochemistry Department (M.T.), School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, New York 14214

Address all correspondence and requests for reprints to: Ho-Jae Han, Department of Veterinary Physiology, College of Veterinary Medicine, Chonnam National University, Kwangju 500–757, Korea. E-mail: hjhan{at}chonnam.chonnam.ac.kr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have investigated the nongenomic as well as the genomic effects of glucocorticoids on phosphate (Pi) uptake in primary rabbit renal proximal tubule cells (PTCs) and have defined the involved signaling pathways. In the present study, cortisol-BSA (cortisol-BSA) (>10-9 M, 30 min) was found to inhibit Pi uptake in a time- and concentration-dependent manner. However, progesterone-BSA (P4-BSA), 17ß-estradiol-BSA (E2-BSA), testosterone-BSA (T4-BSA), aldosterone, P4, E2, and T4 (10-9 M, 1 h) had no effect on Pi uptake. In addition, cortisol-BSA (10-9 M) did not affect either Na+ uptake or {alpha}-methylglucopyranoside ({alpha}-MG) uptake. The cortisol-BSA-induced inhibition of Pi uptake was associated with a decrease in the Vmax for Pi uptake, rather than the Km. The inhibitory effect of cortisol-BSA was not blocked either by actinomycin D (an inhibitor of transcription), cycloheximide (an inhibitor of translation), or classical glucocorticoid receptor antagonists (RU 486 or P4). The cortisol-BSA-induced inhibition of Pi uptake was blocked by two phospholipase C (PLC) inhibitors (neomycin or U73122), and two protein kinase C (PKC) inhibitors (staurosporine or bisindolylmaleimide I) but not by two adenylate cyclase/protein kinase A inhibitors [SQ 22536 (an adenylate cyclase inhibitor) or myristoylated protein kinase A inhibitor amide 14–22]. Furthermore, cortisol-BSA promoted the translocation of PKC from the cytosolic fraction to the membrane fraction, while having no effect on the activity of adenylate cyclase. Our observations may thus be interpreted as indicating that cortisol does indeed inhibit renal Pi uptake via a nongenomic mechanism, which involves the PLC/PKC pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
STEROID hormones are able to produce both early, rapid nongenomic actions, as well as delayed late genomic events in many tissues. Glucocorticoids in particular are known to have pronounced physiological effects on the kidney, including stimulatory effects on the glomerular filtration rate (1), renal gluconeogenesis (2), ammoniagenesis (3), and hydrogen ion secretion (4). In addition, glucocorticoids have been shown to specifically regulate renal phosphate (Pi) transport, as exemplified by the profound decrease in Na+-dependent Pi uptake in rats injected with the synthetic glucocorticoid triamcinolone acetonide (5, 6). Such effects of glucocorticoids on Pi transport are predominantly due to changes in the overall activity of transporters localized to the apical membrane of renal proximal tubule cells (7, 8). A model system that exhibits such inhibitory effects of glucocorticoids on Pi transport is the established Opossum Kidney (OK) cell line, a model of renal proximal tubule cells (9). As the inhibition of Pi uptake caused by glucocorticoids can be blocked by inhibitors of RNA and protein synthesis in this model system, the glucocorticoid-induced decrease in renal Na+-dependent Pi uptake can be explained at least in part by an effect on the level of expression of Na+/Pi cotransporters. Indeed, dexamethasone causes a reduction in renal Na+/Pi cotransporter-2 protein levels in the rat (8), and in renal cortical Na+/Pi cotransporter-6 protein levels in the neonatal rabbit (10). Overall, this evidence suggests that glucocorticoids inhibit renal Pi uptake through their actions at the gene level.

However, a number of lines of evidence suggest the existence of steroid effects that cannot be explained by such classic genetic mechanisms of steroid hormone action (11, 12). The now numerous reports of nongenomic effects of steroids are characterized by a rapid cellular response to a steroid hormone (often within minutes), which does not require protein synthesis, and which may involve distinct membrane receptor systems. Such rapid nongenomic effects of steroids include the activation of protein kinases, the opening of ion channels, the induction of phospholipid turnover, as well as increases in intracellular calcium and cAMP levels. A number of steroid hormones have been reported to elicit such nongenomic effects, including progesterone, 17ß-estradiol, 1,25-dihydroxy vitamin D3, triiodothyronine, aldosterone, and glucocorticoids (12). When considering glucocorticoid hormones in particular, reported nongenomic effects encompass a wide variety of different tissue and cell types, including thrombocytes, endometrial cells, and neurons (13, 14, 15).

In the kidney, evidence for nongenomic effects of the steroid hormone aldosterone has been presented previously (16). In Madin Darby Canine Kidney (MDCK) cells, aldosterone has been observed not only to rapidly activate the Na+/H+ antiport system within seconds (17), but to also rapidly induce a membrane potential-dependent, and Zn2+-sensitive cytoplasmic acidification (18). However, nongenomic effects of glucocorticoids in the kidney have not yet been reported. As steroid hormones conjugated to BSA are considered to be an important and useful tool for studying nongenomic effects mediated through the plasma membrane (12), we have used cortisol-BSA to investigate nongenomic effects of glucocorticoids on Pi uptake in primary rabbit kidney proximal tubule cells (PTCs).

When grown in a hormonally defined medium, PTCs form confluent monolayers of polarized cells, which retain a number of differentiated transport functions typical of the renal proximal tubule (19). Included among these transport functions are a probenicid sensitive p-aminohippurate transport system, a Na+-dependent sugar transport system, and a Na+-dependent Pi transport system (19, 20, 21). The results of studies concerning these membrane transport systems in PTCs are directly comparable with results obtained with the original renal tissue (22).

The PTCs respond to a number of hormones known to affect renal proximal tubule cells in vivo including insulin (which inhibits phosphoenolpyruvate carboxykinase activity at physiologic concentrations) (23), and PTH (which is stimulatory to adenylate cyclase) (19). The PTCs lack a similar responsiveness to arginine vasopressin and calcitonin, indicating the proximal tubule cell culture preparation is highly purified (19). More recently, we have reported a dose-dependent, biphasic effect of angiotensin II on Na+ uptake by the PTCs, consistent with results obtained with intact renal tissue (24). In addition, we have the growth responsiveness of PTCs to physiologic concentrations of 17ß-estradiol, progesterone, and testosterone (25), and we have presented evidence for the involvement of an estrogen receptor in mediating the growth stimulatory effects of 17ß-estradiol (25). The glucocorticoid hormone hydrocortisone (cortisol) was found to substitute at least in part for 17ß-estradiol in eliciting such growth stimulatory effects (25).

In this report, we present evidence indicating that Pi uptake by the PTCs is inhibited by glucocorticoids, including cortisol and dexamethasone. Two phases of inhibition were observed including an initial phase (up to 1 h), which could not be prevented by actinomycin D and cycloheximide, as well as a second phase (after 4 h), which was prevented by these two drugs. To evaluate whether the initial phase of inhibition of Pi uptake by glucocortiocids occurs by means of a nongenomic mechanism, we have assessed 1) whether cortisol-BSA has effects similar to cortisol and dexamethasone, and in addition 2) whether either the PLC, the PKC, or the cAMP signal transduction pathway is involved in mediating the observed glucocorticoid effects. Our results indicate that cortisol-BSA elicits a rapid, inhibitory effect on Pi uptake that is not prevented by either actinomycin D or cycloheximide (unlike the inhibitory effects of these glucocorticoids after 4 h). In addition, we present evidence for the involvement of PLC/PKC rather than cAMP in mediating the inhibitory effects of cortisol-BSA on Pi uptake observed after 1 h. Thus, we propose an initial nongenomic action of cortisol on Pi uptake in the PTCs, through the PLC/PKC pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
DMEM: Nutrient Mixture F-12 (Ham) (D-MEM/F-12, 1:1), Class IV collagenase and soybean trypsin inhibitor were purchased from Life Technologies, Inc. (Grand Island, NY). Cortisol 3-O-carboxymethyl oxime-BSA (cortisol-BSA) (cortisol : BSA, 26:1), estradiol-6-O-carboxymethyl oxime-BSA (E2-BSA) (estradiol : BSA, 32:1), progesterone-3-O-carboxymethyl oxime-BSA (P4-BSA) (progesterone : BSA, 38:1), testosterone-3-O-carboxymethyl oxime-BSA (T4-BSA) (testosterone : BSA, 29:1), cortisol, estradiol-17ß, progesterone, testosterone, 3-isobutyl-1-methyl-xanthine (IBMX), U 73122, actinomycin D, cycloheximide, and ouabain were from Sigma (St. Louis, MO). SQ 22536 and RU 486 were from Biomol (Plymonth Meeting, PA). Myristoylated protein kinase A inhibitor amide 14–22 (PKI) and bisindolylmaleimide I were purchased from Calbiochem (La Jolla, CA). Antibody to PKC was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). 32Phosphate (32Pi), 22sodium (22Na), and 14C-{alpha}-methylglucopyranoside (14C-{alpha}-MG) were purchased from NEN Life Science Products (Boston, MA), and liquiscint from National Diagnostics (Parsippany, NY). The [3H] cAMP assay system (code TRK 432) was obtained from Amersham International (Buckinghamshire, UK). All other reagents were of the highest purity commercially available.

The culture medium consisted of D-MEM/F-12 supplemented with 15 mM HEPES buffer (pH 7.4), 20 mM sodium bicarbonate. Immediately before the use of the medium two additional growth supplements were added, 5 µg/ml bovine insulin, and 5 µg/ml human transferrin.

Isolation of rabbit renal proximal tubules and culture conditions
Rabbit renal proximal tubule cells in primary culture (PTCs) were prepared by a modification of the method of Chung et al. (19) from the kidneys of male New Zealand White rabbits (1.5–2.0 kg). Kidneys were perfused via the renal artery, first with PBS, and subsequently with D-MEM/F-12 containing 0.5% iron oxide (wt/vol) until the kidney turned gray-black in color. Renal cortical slices were prepared by cutting the renal cortex and then homogenized with four strokes of a sterile glass homogenizer. The homogenate was poured first through a 253 µm and then an 83 µm mesh filter. Tubules and glomeruli on top of the 83 µm filter were transferred into sterile D-MEM/F-12 medium containing a magnetic stirring bar. Glomeruli (containing iron oxide) were removed with a magnetic stirring bar. The remaining proximal tubules were briefly incubated in D-MEM/F-12 containing 60 µg/ml collagenase (Class IV) and 0.025% soybean trypsin inhibitor. The dissociated tubules were then washed by centrifugation, resuspended in D-MEM/F-12 containing the two growth supplements, and transferred into tissue culture dishes. PTCs were maintained at 37 C, in a 5% CO2-humidified environment in D-MEM/F-12 medium containing the two supplements. Medium was changed 1 day after plating and every 2 days thereafter.

Membrane transport studies
Pi uptake experiments were conducted as described by the method of Rabito (26). To summarize, the culture medium was removed by aspiration, and the monolayers were gently washed twice with uptake buffer (150 mM NaCl, 1.2 mM MgSO4, 0.1 mM CaCl2, and 10 mM MES/Tris, pH 7.4). After the washing procedure, the monolayers were incubated at 37 C for 30 min in an uptake buffer that contained 1.5 µCi/ml 32Pi and 1 mM unlabeled phosphate. At the end of the incubation period, the monolayers were again washed three times with ice-cold uptake buffer, and the cells were solubilized in 1 ml of 0.1% SDS. To determine the 32Pi incorporated intracellularly, 900 µl of each sample was transferred into scintillation vials containing liquiscint scintillation fluid, and counted in a liquid scintillation counter (LS 6500, Beckman Coulter, Inc., Fullerton, CA). The remainder of each sample was used for protein determination by the Bradford method (27). The radioactive counts in each sample were then normalized with respect to protein and corrected for zero-time uptake. All uptake measurements were made in triplicate. Samples obtained from the Na+ and {alpha}-MG uptake studies were similarly solubilized, and radioactive counts/mg protein determined as described above. Details for 22Na+ and 14C-{alpha}MG uptake studies are otherwise as described by Rindler et al. (28) and Sakhrani et al. (29), respectively.

cAMP assay
Confluent PTC monolayers were preincubated with 100 µM isobutyl methylxanthine (IBMX) for 30 min at 37 C to inhibit the degradation of cAMP. The PTCs were then incubated with 10-9 M cortisol-BSA for 1 h at 37 C in a humidified, 5% CO2/95% air environment. Subsequently, samples were prepared for intracellular cAMP determinations by homogenization in serum free media containing 4 mM EDTA using POLYTRON PT 1200, followed by a 5 min incubation at 100 C. After centrifugation at 3,000 rpm for 5 min, the supernatants were transferred into new tubes and stored at 4 C. These samples were used for cAMP assays, using a [3H] cAMP assay system. Values were expressed as pmol cAMP/mg protein.

Subcellular fractionation of cell lysate
PTCs were grown in D-MEM/F-12 supplemented with 5 µg/ml insulin and 5 µg/ml transferrin. Immediately before their use for subcellular fractionation, the culture medium was removed and the cells were washed twice with ice-cold PBS. The cells were then removed using a cell scraper, harvested by microcentrifugation, and resuspended in 0.2 ml of buffer A (137 mM NaCl, 8.1 mM Na2HPO4, 2.7 mM KCl, 1.5 mM KH2PO4, 0.25 mM EDTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, pH 7.5). Cell lysates were centrifuged at 100,000 x g for 1 h to prepare cytosolic and total particulate fractions. To concentrate the cytosolic fractions, the 100,000 x g supernatants were precipitated with 5 volumes of acetone, incubated at 4 C for 5 min, centrifuged (14,000 rpm for 20 min at 4 C), and the pellet was resuspended in buffer A plus 1% Triton X-100. The particulate fractions containing membrane fraction were washed twice and resuspended in 50 µl of buffer A plus 1% Triton X-100. The quantity of protein in each fraction was quantitated by the Bradford procedure.

SDS-PAGE and immunoblot analysis
Samples equalized with respect to protein were subjected to SDS-PAGE for immunoblot analysis. SDS-PAGE was performed using 8% polyacrylamide gels, followed by transfer to polyvinylidine difluoride membranes for 2 h at 100 V using a Novex (Gronmgen, The Netherlands) wet transfer unit. Immunodetection was performed using the horseradish peroxidase (HRP) method. Briefly, the membrane was blocked overnight with TBS [PBS containing 0.01% (vol/vol) Tween 20] further supplemented with 5% (wt/vol) nonfat dried milk. Blots were then incubated for 2 h with primary antibody in TBS and then for 1 h with HRP-conjugated secondary antibody, before development using an enhanced chemiluminescence kit (ECL, Amersham Pharmacia Biotech, Arlington Heights, IL).

Statistical analysis
Results were expressed as means ± SE. Statistical significance was estimated by ANOVA and by unpaired t test as appropriate. The difference was considered statistically significant when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of cortisol-BSA on Pi uptake
Initially, the effects of cortisol-BSA on Pi uptake by PTCs was determined with treatment times ranging from 0.25 to 4 h, and cortisol-BSA concentrations ranging from 10-11 to 10-5 M. Figure 1AGo shows that cortisol-BSA (>10-9 M) inhibited Pi uptake starting after 30 min, and continuing throughout the remainder of the 4-h incubation period. For example, after a 1-h incubation with 10-9 M cortisol-BSA, the Pi uptake rate was reduced to 174 ± 4 pmol/mg protein·min as compared with 208 ± 3 pmol/mg protein/min in the control condition. The effects of cortisol and dexamethasone on Pi uptake were examined at a concentration where cortisol BSA was inhibitory. At 10-9 M, neither cortisol nor dexamethasone had significant effects after a 1-h incubation. However, ultimately, after 4 h, inhibitory effects of cortisol (10-9 M) and dexamethasone (10-9 M) were observed (Fig. 1Go, B and C). When the cortisol concentration was raised to 10-7 M, however, cortisol inhibited Pi uptake with a similar time course (and to a similar extent) as observed with 10-9 M cortisol-BSA (Fig. 1BGo). BSA alone (10-5 M) had no significant effect over such a 4-h time interval.



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  		Figure 1. Time course of cortisol-BSA (A), cortisol, and dexamethasone (B) on Pi uptake. PTCs were incubated with different concentration of cortisol-BSA (10-11 M ~ 10-5 M), BSA-free cortisol (10-9 M and 10-7 M), or dexamethasone (10-9 M) for various times (0 ~ 4 h), respectively. BSA (10-5 M) was used as a negative control. C indicates the percentage of control of cortisol-BSA (10-9, 10-7 M) and cortisol (10-9, 10-7 M) on Pi uptake at 1 h compared with control. Values are means ± SE of five independent experiments with triplicate dishes. *, P < 0.05 vs. control.

 
To determine the lowest effective dosage at which either cortisol or cortisol-BSA inhibited Pi uptake, PTCs were treated with either cortisol (10-9 to 10-7 M), or cortisol-BSA (10-11 to 10-9 M), and after a 1-h incubation the effect on Pi uptake was measured. Figure 2Go shows that the minimal cortisol dosage that was required to elicit a significant inhibitory effect (10-8 M) on Pi uptake was 100-fold higher than the minimal cortisol-BSA dosage required to elicit a similar effect (10-10 M).



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  		Figure 2. Effective dosage of cortisol and cortisol-BSA on the inhibition of Pi uptake. PTCs were treated with different dosage of cortisol (10-9 to 10-7 M) or cortisol-BSA (10-11 to 10-9 M) for 1 h. Values are means ± SE of five independent experiments with triplicate dishes. *, P < 0.05 vs. control.

 
The possibility was examined that other steroids and steroid-BSA conjugates were similarly inhibitory to Pi uptake. Figure 3Go shows, however, that 10-9 M P4, P4-BSA, E2, E2-BSA, T4, and T4-BSA did not affect Pi uptake after a 1-h incubation. Similarly, the mineralocorticoid aldosterone (10-9 M) at both 1 h and 4 h did not alter Pi uptake compared with the initial control uptake rate (control: 210 ± 4 vs. aldosterone at 1 h: 208 ± 8 and aldosterone at 4 h: 209 ± 5 pmol/mg protein/min, P = NS, n = 4).



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  		Figure 3. Comparison of effect between cortisol and sex steroid hormones (A) and its BSA-conjugated form on Pi uptake. PTCs were treated with cortisol, sex steroid hormone, and its BSA-conjugated form (10-9 M) for 1 h, respectively. Values are means ± SE of five independent experiments with triplicate dishes. *, P < 0.05 vs. control.

 
Cortisol-BSA may inhibit Pi uptake due to a specific effect on a Pi transporter, or as the consequence of a generalized effect on the plasma membrane. However, as shown in Table 1Go, 10-9 M cortisol-BSA did not significantly affect either Na+ uptake or {alpha}-MG uptake by the PTCs. These results thus suggest that glucocorticoids are responsible for eliciting a specific, inhibitory effect on a Pi transport system, rather than on membrane transport in general.


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  		Table 1. Effects of cortisol-BSA on Pi, {alpha}-MG, and Na+ uptake

 
To examine effect of cortisol-BSA on the kinetic properties of such a Pi transport system, the effect of cortisol-BSA on Pi uptake was examined as a function of increasing Pi concentrations (0.0625 to 1 mM). The results of these studies indicated that cortisol-BSA decreased the Vmax of Pi uptake via a Pi transport system (control: 241 ± 9 vs. 10-9 M cortisol-BSA: 189 ± 8 pmol/mg protein/min, P < 0.05), without affecting the Km (control: 0.22 ± 0.04 vs. 10-9 M cortisol-BSA: 0.20 ± 0.03 mM, P = NS) (Fig. 4Go).



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  		Figure 4. Kinetic studies of cortisol-BSA on Pi uptake. PTCs were incubated for 1 h in the presence of 10-9 M cortisol-BSA or vehicle. Then Pi uptake was measured in a buffer containing 32Pi (1.5 µCi/ml) in the presence of unlabeled Pi (0.0625 to 1 mM). Values are means ± SE of three independent experiments with triplicate dishes. *, P < 0.05 vs. control.

 
Nongenomic effect of cortisol-BSA
To ascertain whether the inhibition of Pi uptake caused by either cortisol-BSA or cortisol was dependent upon gene expression, the ability of actinomycin D (10-7 M) and cycloheximide (4 x 10-5 M) to block the inhibitory effects of 10-7 M cortisol and 10-7 M cortisol-BSA on Pi uptake was examined. Initially, the ability of actinomycin D and cycloheximide to block the inhibitory effect of 10-9 M cortisol on Pi uptake was examined. As shown in Fig. 5AGo, the inhibitory effect of 10-9 M cortisol on Pi uptake after 4 h was prevented completely by actinomycin D and cycloheximide (P < 0.05). Cortisol at this concentration only exerted an inhibitory effect on Pi uptake after a 4-h incubation. To assess whether cortisol similarly required such macromolecular synthesis to elicit its early phase of inhibition, a higher cortisol concentration (10-7 M) was used, as well as a shorter (1 h) incubation period with this steroid. Figure 5BGo shows that, under these conditions, actinomycin D and cycloheximide did not block the inhibition of Pi uptake caused by cortisol. However, following a 4-h incubation with 10-7 M cortisol, the 10-7 M cortisol-induced inhibition of Pi uptake was blocked in part (Fig. 5CGo), suggesting two mechanisms of inhibition by 10-7 M cortisol. The inhibition of Pi uptake caused by 10-9 M cortisol-BSA was not prevented by either actinomycin D or cycloheximide, irregardless of incubation time (1 h, Fig. 5BGo; 4 h, Fig. 5CGo). These results suggest that RNA and protein synthesis were not required for the inhibition of Pi uptake observed after a 1-h incubation period with either 10-9 M cortisol-BSA or 10-7 M cortisol, although the synthesis of these macromolecules was required for the full manifestation of the 10-7 M cortisol-mediated inhibition of Pi uptake at 4 h.



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  		Figure 5. Effect of inhibitors of RNA (actinomycin D) and protein synthesis (cycloheximide) on cortisol or cortisol-BSA-induced inhibition of Pi uptake. Actinomycin D (10-7 M) and cycloheximide (4 x 10-5 M) were treated to the PTCs for 30 min before the addition of cortisol (10-9 M, 4 h) (A), or cortisol-BSA (10-9 M) or cortisol (10-7 M) were treated for 1 h (B) or 4 h (C). Values are means ± SE of four independent experiments with triplicate dishes. *, P < 0.05 vs. control; **, P < 0.05 vs. cortisol (10-9 M).

 
To evaluate whether these effects of cortisol and cortisol-BSA are mediated by classical glucocorticoid receptors, the effects of the glucocorticoid receptor antagonists RU 486 (10-8 M) or progesterone (10-8 M) on the inhibitory effects of 10-9 M cortisol and 10-9 M cortisol-BSA on Pi uptake were examined. Figure 6Go shows that both of these glucocorticoid receptor antagonists did prevent the cortisol-induced inhibition of Pi uptake (observed after 4 h) but did not prevent the effect of cortisol-BSA (observed after 1 h). These results thus suggest that the initial inhibitory effects of cortisol-BSA (10-9 M, at 1 h) are not mediated via the interaction of cortisol-BSA with a classic glucocorticoid receptor, unlike the inhibition of Pi uptake observed following a chronic, 4 h treatment with glucocorticoids.



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  		Figure 6. Effects of glucocorticoid receptor antagonists (RU 486, progesterone) on cortisol (A) or cortisol-BSA (B)-induced inhibition of Pi uptake. PTCs were incubated with RU 486 (10-8 M) or progesterone (10-8 M) for 30 min before the treatment of 10-9 M cortisol (4 h) or cortisol-BSA (1 h). Values are means ± SE of four independent experiments with triplicate dishes. *, P < 0.05 vs. control; **, P < 0.05 vs. cortisol (10-9 M).

 
Possible involvement of the cAMP/PKA and PLC/PKC pathways
As cAMP has been reported to affect Pi uptake in rabbit renal proximal tubule cells (30), the possible involvement of cAMP in mediating the effect of a 1 h cortisol-BSA treatment was examined. Figure 7AGo shows that the cortisol-BSA-induced inhibition of Pi uptake was not blocked by either an adenylate cyclase inhibitor, SQ 22536 (10-6 M), or a protein kinase A inhibitor, PKI (10-6 M). Furthermore, cortisol-BSA (10-9 M) did not have a significant effect on the intracellular cAMP levels of the PTCs (Fig. 7BGo). These results thus suggest that the adenylate cyclase/protein kinase A pathway is not involved in mediating the acute effects of glucocorticoids on Pi uptake.



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  		Figure 7. A, Effects of cAMP pathway on cortisol-BSA-induced inhibition of Pi uptake. PTCs were incubated with SQ 22536 (10-6 M) or PKI (10-6 M) for 30 min before the treatment of cortisol-BSA. Values are means ± SE of four independent experiments with triplicate dishes. *, P < 0.05 vs. control. B, Effects of 10-9 M cortisol-BSA on cAMP production. PTCs were preincubated to IBMX (10-4 M) for 30 min to prevent the degradation of cAMP into 5'-AMP before exposure to cortisol-BSA (10-9 M) for 1 h. Values are means ± SE of four independent experiments with triplicate dishes. *, P < 0.05 vs. control.

 
To investigate the possible involvement of PLC/PKC in mediating the inhibitory effects of cortisol-BSA on Pi uptake, the influence of several PLC inhibitors [neomycin (10-4 M) and U 73122 (10-6 M)] as well as several PKC inhibitors [staurosporine (10-7 M) and bisindolylmalemide I (10-6 M)] on the cortisol-BSA induced inhibition of Pi uptake was examined. Figure 8Go shows that each of the above PLC and PKC inhibitors blocked the cortisol-BSA-induced inhibition of Pi uptake observed after a 1-h incubation (P < 0.05).



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  		Figure 8. Effects of PLC/PKC pathways on cortisol-BSA-induced inhibition of Pi uptake. PTCs were incubated with neomycin (10-4 M), U 73122 (10-6 M), staurosporine (10-7 M), and bisindolylmaleimide I (10-6 M), for 30 min before the treatment of cortisol-BSA. Values are means ± SE of four independent experiments with triplicate dishes. *, P < 0.05 vs. control, ** P < 0.05 vs. cortisol-BSA.

 
Also consistent with the involvement of PKC are the results of our studies concerning the subcellular localization of PKC. The redistribution of PKC from the soluble (cytosolic) to the insoluble (particulate) compartment is considered to be the first step in PKC activation and was used as a measure of PKC activation in response to cortisol-BSA. PKC was found to be present primarily in the cytosolic fraction of untreated PTCs. Following a 1-h treatment with cortisol-BSA (10-9 M or 10-7 M), there was a loss of PKC from the cytosolic compartment (particularly at 10-7 M cortisol-BSA), and a corresponding increase in the PKC level in the particulate compartment (Fig. 9Go). The lower panel in Fig. 9Go shows that the reduction in cytosolic PKC levels and the increase in particulate PKC levels in cortisol-BSA treated PTCs was statistically significant, as indicated by the results of four independent experiments. These results thus suggest the involvement of the PLC/PKC, rather than the cAMP pathway, in mediating the acute inhibitory effect of cortisol-BSA on Pi uptake.



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  		Figure 9. Effect of cortisol-BSA on PKC translocation. PTCs were treated for 1 h with cortisol-BSA at either 0, 10-9, or 10-7 M. The PKC protein which was present in either the cytosolic (C) or particulate (P) fraction was then detected by means of Western analysis, as described in Materials and Methods. The arrow indicates the 80-kDa band corresponding to PKC. PKC levels relative to the control were determined by means of scanning densitometry. The lower panel depicts the data expressed as a percentage of the basal value in each fraction and is the mean ± SE of four independent experiments. *, P < 0.05 vs. each control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study with PTCs, we evaluated whether cortisol inhibits Pi uptake in the renal proximal tubule via a nongenomic as well as a genomic mechanism. The classical genomic mechanism of steroid hormone action involves the binding of the steroid hormone to intracellular receptors, followed by migration of the steroid hormone/receptor complex to the nucleus. Once in the nucleus, the activated steroid hormone-receptor complex acts as a transcription factor, modulating the synthesis of specific messenger RNAs (mRNAs), and ultimately the specific proteins that are responsible for the final effects of the steroid hormone (11). The results in this report indicate that Pi uptake in PTCs is inhibited by glucocorticoids by means of a nongenomic as well as a classical genomic mechanism.

Generally, the criteria used for the classification of nongenomic actions of steroid hormones include 1) a rapid time course; 2) insensitivity of the steroid hormone effects to inhibitors of transcription and translation (12); 3) the ability to elicit a rapid steroid effect using steroids coupled to high molecular weight molecules that do not enter the cells; and 4) the involvement of a membrane receptor that is distinct from the classical cytoplasmic receptor for the steroid of interest (11).

Supportive of nongenomic actions of cortisol, were the observed inhibitory effects of 10-9 M cortisol-BSA on Pi uptake, which occurred after a 30-min time interval. Cortisol at a concentration as low as 10-8 M elicited a similar effect. Admittedly, these acute effects of cortisol-BSA (and cortisol) were not observed within a few minutes. Shorter term observations were limited in part by the assay method. Nevertheless, our results do suggest that the mechanisms underlying the 30 min inhibitory effect of 10-9 M cortisol-BSA on Pi uptake, and the 4 h inhibitory effects are distinct. First, actinomycin D and cycloheximide did not prevent the inhibitory effects of 10-9 M cortisol-BSA after a 1-h incubation, although both of these agents prevented the inhibitory effects of cortisol (and cortisol-BSA) after a 4-h incubation. Similarly, the glucocorticoid receptor antagonists RU486 and progesterone did not prevent the inhibitory effects of cortisol-BSA (10-9 M) observed after a 1-h incubation. However, RU 486 and progesterone did block the cortisol-mediated inhibition of Pi uptake following a 4-h incubation.

Nonspecific, nongenomic effects of steroids have been reported to occur at micromolar or even supramicromolar concentrations (11, 31). An example is the effect of 17ß-estradiol and hydrocortisone on intracellular cAMP levels in porcine coronary vascular smooth muscle cells (31). The nonspecific nongenomic steroid effects that occur at very high steroid concentrations have been attributed to effects on the physiochemical properties of the plasma membrane including membrane fluidity (12, 31, 32, 33). However, the glucocorticoid concentrations used in this report were substantially lower (no higher than 10-7 M), and thus are most likely not the consequence of such generalized effects on plasma membrane structure and function. Indeed, the effect of cortisol-BSA appeared to be specific for Pi transport, rather than for either Na+ or {alpha}-MG transport. Furthermore, E2-BSA, P4-BSA, T4-BSA, E2, P4, and T4 did not affect Pi uptake, indicating the specificity of the inhibitory effects of cortisol-BSA on plasma membrane function.

Although, we did not determine whether plasma membrane binding sites for glucocorticoids are indeed present in PTCs, previously specific binding of corticosterone was found to bind specifically to rat kidney plasma membranes was detected; however, the function was not clarified (34). Such observations have not been restricted to the kidney. Indeed, Orchinik et al. (35) reported the presence of a corticosteroid receptor in neuronal membranes obtained from the amphibian Taricha granulosa, which has rapid behavioral responses to corticosterone. The binding was specific, saturable and of a high affinity (Kd = 0.51 nM). Similarly, membrane-binding sites for corticosteroid hormones have been described in liver (36), and pituitary membranes (37).

As cortisol can bind to cytoplasmic mineralocorticoid receptors, as well as cytoplasmic glucocorticoid receptors, the possibility of an interaction of cortisol and cortisol-BSA with a membrane-associated aldosterone receptor cannot be excluded. Indeed, evidence has been presented for the presence of membrane-associated receptors for mineralocorticoids in porcine vascular smooth muscle cells (31), in human mononuclear leukocytes (38), as well as in pig kidney plasma membranes (39). However, unlike the case with cytoplasmic mineralocorticoid receptors, cortisol only affected the nongenomic pathway in vascular smooth muscle cells at very high concentrations (i.e. 10 µM), unlike aldosterone, which was effective at 10-10 M.

Similarly, the evidence presented by Wehling et al. (38) for plasma membrane receptors for aldosterone in human mononuclear leukocytes indicated that dexamethasone, corticosterone, and 18-hydroxyprogesterone were inactive as ligands for this membrane-associated receptor. 10-9 M cortisol did not have an effect on Pi uptake (0.5–1 h), unlike the case with 10-9 M cortisol-BSA. A cortisol concentration of 10-7 M was required to elicit an inhibitory effect on Pi uptake at 0.5–1 h, which was equivalent to that observed with 10-9 M cortisol-BSA. Similarly, 10-9 M progesterone and dexamethasone had no affect on Pi uptake at 0.5- 1 h. Aldosterone (10-9 M) also had no effect on Pi uptake by the PTCs, both at 1 h and 4 h, indicating the involvement of a receptor distinct from the membrane associated aldosterone receptor in vascular smooth muscle cells, and human mononuclear leukocytes.

The lack of an effect of 10-9 M cortisol at early times (0.5–1 h) may possibly be explained by a difference in affinity for cortisol between membrane-associated receptors (mediating the nongenomic effects of cortisol), and cytosolic receptors (mediating genomic effects). Cortisol-BSA was observed to inhibit Pi uptake at substantially lower concentrations than cortisol at early time intervals. This observation may be explained whether plasma membrane-associated glucocorticoid receptors have a higher affinity for cortisol when conjugated to BSA than for free cortisol, the conjugated cortisol being presented to the receptor in a manner more amenable for stereospecific binding than cortisol itself. Indeed in vivo the majority of cortisol is bound to proteins such as BSA and transcortin, which may influence binding to such receptors on the plasma membrane. The possibility of the involvement of another type of steroid receptor (other than a glucocorticoid receptor) in mediating the effects of cortisol-BSA cannot be excluded, although other steroids and steroid-BSA conjugates had no effect at 10-9 M, including P4, P4-BSA, E2, E2-BSA, T4, and T4-BSA.

Although a higher concentration of cortisol (>10-9 M) is required to elicit nongenomic (vs. genomic) effects, cortisol concentrations which elicited an inhibitory effect on Pi uptake after 1 h (10-8 and 5 x 10-8 M) are nevertheless within the physiologic range. The normal level of cortisol in blood ranges from a low at night (in the PM) of 3 µg/dl (or 8.6 x 10-8 M) to a high in the morning (in the AM) of 20 µg/dl (or 5.6 x 10-7 M) (40). As approximately 10% of the total cortisol in blood is free, unbound steroid (41), the molar level of free cortisol actually ranges from 8.6 x 10-9 M to 5.6 x 10-8 M, with a mean of 3.3 x 10-8 M (41). The level of free cortisol may possibly increase above these levels as a consequence of physiologic stress, as well as under pathologic conditions. Therefore, quite clearly under such conditions of stress, cortisol may very well inhibit renal Pi uptake through a nongenomic, as well as a genomic pathway.

We cannot, however, exclude the possibility that lower concentrations of cortisol (below 10-8 M) can indeed inhibit renal Pi uptake through a nongenomic mechanism. Although serum-free culture conditions are being used, the effective cortisol concentration in the medium may decline below the initial concentration (e.g. 10-9 M) because a significant proportion of the cortisol enters the PTCs. Cortisol presumably enters the cytoplasmic compartment of PTCs by passive diffusion through the plasma membrane (due to the small molecular size and lipophilic nature of this steroid), followed either by specific binding to cytoplasmic receptors, and/or by metabolism.

Presumably, cortisol is metabolized intracellularly by the PTCs at least in part by means of 11ß-hydroxysteroid dehydrogenase (11ßHSD). Indeed, 11ßHSD activity has been detected in microdissected rabbit renal proximal tubules, as well as in the collecting duct (42). The reverse oxo-reductase activity has not been observed with either of the 2 renal isoforms, 11ßHSD1 or 11ßHSD2 (43, 44). By inactivating intracellular cortisol in appropriate target cells in the kidney which possess mineralocorticoid receptors, 11ßHSD2 facilitates the specific activation of mineralocorticoid receptors by aldosterone (cortisol and aldosterone bind to such receptors with equal affinity). In the renal proximal tubule (which is devoid of such cytoplasmic mineralocorticoid receptors) 11ßHSD instead acts to modulate cortisol action (under appropriate physiologic conditions) by metabolizing cytoplasmic glucocorticoid, thereby minimizing steroid binding to cytoplasmic glucocorticoid receptors. Presumably then, 10-9 M cortisol-BSA is effective at 30 min, unlike 10-9 M cortisol, because a larger proportion of this conjugate remains unmetabolized, in the extracellular compartment, interacting with membrane-localized receptors.

Both the chronic and the acute inhibition of Pi uptake by cortisol (10-7 M), and cortisol-BSA (10-9 M) is very likely mediated through the regulation of a type II Na+/Pi cotransporter (45). Although three major types of mammalian Na+/Pi cotransporters have been identified by means of expression cloning techniques, only the type I and the type II Na+/Pi cotransporters are localized preferentially in the kidney (almost exclusively in the renal proximal tubule). However a detailed kinetic analysis of these two types of transporters (when expressed in oocytes) indicates that only the type II Na+/Pi cotransporter has transport characteristics typical of the Na+/Pi cotransporter in renal brush border membranes (45).

The level of the renal proximal tubular type II Na+/Pi cotransporter in the apical membrane has previously been shown to be regulated posttranscriptionally in response to a number of changes in the hormonal and nutritional environment. The well documented inhibition of renal proximal tubule Pi transport by PTH has recently been proposed to involve the internalization of Na+/Pi cotransporters by endocytosis, followed by lysosomal degradation, rather than by a decrease in Na+/Pi cotransporter gene expression (45). Similarly, acute increases in renal brush border Na+/Pi cotransporter levels (observed after 2 h of Pi deprivation) have been attributed to a microtubule-dependent translocation of presynthesized Na+/Pi cotransporters to the apical membrane (45). Even the increased levels of Na+/Pi cotransporters observed in rats maintained on a low Pi diet has been attributed to posttranscriptional control mechanisms (i.e. changes in protein- and/or mRNA stability), rather than transcriptional regulation (45).

However, a number of reports have indicated that glucocorticoids do indeed affect renal proximal tubule transport activities by changes in the level of expression of transporter genes. In the rat, dexamethasone has been reported to cause an increase in the levels of the mRNA encoding the basolateral Na+:HCO3- cotransporter type I (NBC-1) (46), while inducing a decrease in the abundance of the Na+/Pi cotransporter-2 mRNA (47). Similar changes in the activities of these two transporters have been reported following dexamethasone treatment (46, 47, 48). A similar reduction in the level of the Na+/Pi cotransport protein in renal brush border membranes has been reported (47). In addition, increased levels of mRNA for the Na+/H+ antiport system type 3 (NHE-3) and enhanced NHE-3 activity has been reported in the renal proximal tubule (49). Indeed, evidence has been presented for the coordinate regulation of these renal transport systems in a number of pathophysiologic states (46).

Although direct evidence for transcriptional regulation of the type II Na+/Pi cotransporter by glucocortiocids has not been demonstrated, in this study we have presented evidence indicating that chronic regulation of Pi transport in our primary PTC cultures by glucocorticoids is prevented by actinomycin D and cycloheximide. Similarly, the glucocorticoid-mediated inhibition of Pi uptake reported previously in primary cultured chick renal cells was found to be inhibited by actinomycin D and cycloheximide, indicating a genetic mechanism. Also similar to our primary PTC culture system, the inhibitory effects of glucocorticoid treatment on Pi uptake in the OK cell line (9), and in neonatal rabbit renal brush border membranes are associated with a reduction in the Vmax value for Pi uptake, rather than the Km value. However, unlike this report, these previously published inhibitory effects of glucocorticoids on Pi uptake were most likely mediated through cytoplasmic glucocorticoid receptors, ultimately acting at the gene level, and resulting in a reduction in the number of Na+/Pi cotransporters.

In this report, the acute inhibition of Pi uptake caused by cortisol-BSA (after 1 h) was presumably mediated via membrane associated glucocorticoid receptors. Possible explanations for the observed reduction in the Vmax for Pi uptake in the cortisol-BSA treated PTCs include a decrease in the efficiency of the transporter, an alteration in the processing of the transporter, as well as an alteration affecting the internalization of Na+/Pi cotransporters in the apical membrane, and their subsequent lysosomal degradation and/or retrieval back to the apical membrane. Ultimately, such changes would result in a reduction in the level of incorporation of Na+/Pi cotransporters into the apical membrane.

Protein phosphorylation events may affect either transporter efficiency or protein processing. Indeed, both the cAMP and the PKC signaling pathways have previously shown to be involved in mediating the nongenomic actions of glucocorticoids. One example is the case of PC12 cells, in which glucocorticoids blocked the inhibitory effect of nicotine on calcium influx via PKC (14). Another example is the nongenomic effect of dexamethasone on actin assembly in human endometrial cells, which is cAMP mediated (50). Although the activation of the cAMP pathway has been reported to cause an inhibition of the renal Na+/Pi cotransport system (39), our results indicate that cAMP was not involved as a mediator in the cortisol-BSA-induced inhibition of Pi uptake in the PTCs.

The evidence in the present study suggests instead the involvement of the PLC/PKC pathway in mediating the inhibition of Pi uptake caused by 10-9 M cortisol-BSA, and 10-7 M cortisol after an acute 1-h incubation. Not only did inhibitors of PLC and PKC block this effect of cortisol-BSA, but in addition cortisol-BSA significantly increased membrane-bound PKC activity. The redistribution of PKC from the soluble to the insoluble subcellular compartment is considered to be the first step in PKC activation. The activation of PKC involves not only the interaction of diacylglycerol (DAG) with the PKC molecule but may also involve the interaction of PKC with the membrane lipid bilayer, calcium, and other lipid mediators (51). Although the reversible activation of PKC is a typical model for enzyme activation by second messengers, the more prolonged activation of PKC has been reported previously. Hypotheses to explain prolonged activation of PKC include the intercalation of the PKC molecule into a specific membrane structure that becomes constitutively active, as well as the anchoring of PKC to specific cellular sites by PKC-binding proteins (51). Whether membrane associated cortisol-BSA plays a direct role in the prolonged activation of PKC has not been determined in this study.

Although our results are unique in proposing the involvement of PKC in mediating nongenomic effects of glucocorticoids on renal Pi uptake, the involvement of PKC in mediating the effects of glucocorticoids on Pi uptake in the renal proximal tubule has been previously indicated in studies with OK cells (52). Dousa (53) has similarly reported that PLC/PKC activation is involved in the inhibition of renal Na+/Pi cotransport, albeit by a genomic mechanism. The activation of a particular PKC isozyme, PKC-{epsilon}, by dexamethasone, has been reported in thymocytes, although in this case as well, a genomic rather than a nongenomic mechanism was inferred (54). Thus, intracellular signaling pathways such as cAMP and PKC have also been proposed to modulate the long-term actions of such steroids at the gene level (31). One such proposed mechanism is through the activation of transcriptional coactivators such as the cAMP regulatory element binding protein (31). Further studies will be required to examine the interaction between such nongenomic and genomic effects, to identify the putative membrane receptor, and other factors mediating rapidly elicited inhibition of Pi uptake by cortisol and cortisol-BSA.


   Footnotes
 
1 This work was supported by grants awarded to Dr. H. J. Han from Korea Science and Engineering Foundation (KOSEF 971-0605-036–2, HRC 1998G0301). Back

Received August 3, 2000.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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