Department of 1Pediatrics and 2Internal Medicine, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9063
Submitted 8 January 2004 ; accepted in final form 22 April 2004
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ABSTRACT |
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NHE3; adrenalectomy; renal development; microperfusion; cell pH
The levels of both glucocorticoids and thyroid hormone are lower in the first 12 wk of life compared with those of adults (24, 39). The maturational increase in these hormones parallels the maturational increase in NHE3 (36), the predominant Na+/H+ antiporter isoform on the apical membrane of the proximal tubule (15, 40, 41), and Na+/H+ antiporter activity (36). We examined previously the role of glucocorticoids and thyroid hormone in mediating the increase in Na+/H+ antiporter activity (8, 23). In these studies, we either prevented the maturational increase in glucocorticoids or thyroid hormone at a time well before the normal increase during postnatal development. We found that preventing the maturational increase in either thyroid hormone or glucocorticoids suppressed the maturational increase in Na+/H+ antiporter activity and brush-border membrane NHE3 protein abundance without any effect on the basal level of NHE3 mRNA abundance (8, 23). Furthermore, the rate of Na+/H+ antiporter activity in both adrenalectomized and hypothyroid animals was higher than in neonates. These results could be explained if thyroid hormone and glucocorticoids played a compensatory role in the face of deficiency of the other hormone.
The present study was designed to examine the maturation of Na+/H+ antiporter, brush-border membrane vesicle (BBMV) NHE3 protein and renal cortical mRNA abundance in the presence of a combined deficiency of glucocorticoid and thyroid hormones. This is the first study to use a unique animal model of adrenalectomized, hypothyroid neonatal rats. Our results using this model demonstrate that postnatal maturation of Na+/H+ antiporter activity, NHE3 protein, and RNA abundance is completely prevented using this novel model. Furthermore, this model will be useful in the study of other proteins mediated by both thyroid hormone and glucocorticoids.
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METHODS |
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This study was approved by the IACRAC at the University of Texas Southwestern Medical Center and adhered to APSs Guiding Principles in the Care and Use of Animals.
RNA isolation and analysis.
Slices of renal cortex from decapsulated kidneys were homogenized in RNAzol [1:1, phenol-RNAzol stock (4 M guanidine thiocyanate, 25 mM disodium-citrate, pH 7.0), 0.5% sarcosyl] containing 3.6 µl/ml -mercaptoethanol. RNA was extracted in the presence of 3 M NaOAc (pH 4.0) and chloroform, purified using isopropanol precipitation, and washed twice with 80% ethanol (16). RNA was quantitated with a LKB Ultra-spec III spectrophotometer at 260 nm, and 15 µg were fractionated using agarose-formaldehyde gel electrophoresis and transferred to a nylon filter (GeneScreen Plus, New England Nuclear, Boston, MA). The filter was prehybridized at 42°C for 4 h with 5x SSC, 5x Denhardt's solution (Ficoll, BSA, and polyvinylpyrrolidone, each at 1 mg/ml), 0.5% SDS, and 0.5 mg/ml of sheared salmon sperm DNA and then hybridized to double-strand uniformly 32P-labeled cDNA probes (>106 cpm/ml) in the above hybridization solution at 42°C for
16 h. The probes were synthesized by the random hexamer method using 50 to 100 ng of cDNA: NHE3 was the rat 1.2-kb PstI fragment (33) and
-actin was a 1.5-kb EcoRI fragment. The filter was then washed with 2x SSC twice and 0.1% SDS for 5 min at room temperature and then twice at 55°C for 40 min with 0.1x SSC and 1% SDS. NHE3 and
-actin mRNA abundance was quantitated with autoradiography and densitometry.
BBMV isolation. Kidneys were removed and placed in an ice-cold isolation buffer containing 300 mM mannitol, 16 mM HEPES, and 5 mM EGTA titrated to pH 7.4 with Tris. The isolation buffer contained aprotinin (2 µg/ml), leupeptin (2 µg/ml), and phenylmethylsulfonyl fluoride (100 µg/ml). The cortex was homogenized with 20 strokes of a Potter Eljevhem homogenizer at 4°C. BBMV were then isolated by differential centrifugation and magnesium precipitation as described previously (23, 36). The final BBMV fraction was resuspended in isolation buffer. Protein was assayed using the Lowry method with crystalline BSA as the standard (30). There was comparable enrichment of leucine amino peptidase in BBMV from neonatal 10.0 ± 1.6 compared with 13.4 ± 1.2 in 30-day-old rats [n = 7, P = not significant (NS)].
SDS-PAGE and immunoblotting.
Brush-border membrane proteins (40 µg/lane) were denatured and then separated on a 7.5% polyacrylamide gel using SDS-PAGE as previously described (23, 36). The proteins were transferred overnight to a polyvinylidene diflouride membrane at 120140 mA at 4°C. The blot was blocked with fresh Blotto (5% nonfat milk and 0.1% Tween 20 in PBS, pH 7.4) for 1 h followed by incubation with primary antibody to NHE3. NHE3 antibody, a gift from Dr. O. Moe, was a rabbit polyclonal antibody directed against a fusion protein of maltose-binding protein and rat NHE3 amino acids 405831 (2). NHE3 antibody was added at 1:750 dilution and incubated for 16 h at 4°C. The blot was then washed extensively with Blotto. The secondary antibody, horseradish peroxidase-conjugated donkey anti-rabbit immunoglobulin, was added at 1:10,000 dilution and incubated in room temperature for 1 h. The blot was again washed with Blotto, and enhanced chemiluminescence was used to detect bound antibody (Amersham Life Science). The NHE3 protein abundance was quantitated using densitometry. Equal loading of the samples was confirmed using an antibody to -actin at a 1:5,000 dilution (Sigma, St. Louis, MO).
In vitro microperfusion and measurement of intracellular pH. We recently demonstrated that 0.2- to 0.5-mm neonatal and young rat proximal convoluted tubules can be dissected free hand without collagenase and perfused in vitro (23, 36). Isolated segments of neonatal and adult rat proximal convoluted tubules were perfused using concentric glass pipettes using techniques previously described for rabbit proximal tubules (4, 5, 11). Briefly, rat proximal convoluted tubules were dissected from 9-day-old, 30-day-old sham, 30-day-old ADX, 30-day-old ADX-hypothyroid, and 30-day replacement group in Hanks' balanced salt solution containing (in mM) 137 NaCl, 5 KCl, 0.8 MgSO4, 0.33 Na2HPO4, 0.44 KH2PO4, 1 MgCl2, 10 Tris hydrochloride, 0.25 CaCl2, 2 glutamine, 2 heptanoic acid, and 2 lactate at 4°C (pH 7.4). Tubules were transferred to a 0.2-ml chamber in which the bathing solution was preheated to 38°C.
The composition of the solutions used in these experiments is shown in Table 1. The fluorescent dye BCECF was used to determine intracellular pH (pHi) as described previously (1, 4, 5, 33, 37). pHi was measured with a Nikon inverted epifluorescent microscope attached to a PTI Ratiomaster at a rate of 30 measurements per second. A variable diaphragm was placed over the area to be measured. pHi was determined from the ratio of fluorescence (F500/F450) using a nigericin calibration curve as previously described (1, 4, 34).
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Tubules were incubated with an ultrafiltrate-like solution in the lumen and bath (solution B containing 5 mM glucose and 5 mM alanine) for at least 5 min before being loaded with 5 x 106 M BCECF, and tubules had a constant pHi for several minutes before the measurement of transporter activity. dpHi/dt was measured from the slope of change in pHi immediately after a luminal fluid change. Steady-state pHi values were reached within 1 min after a luminal fluid exchange, but pHi was measured for several minutes to ensure a steady-state pHi was achieved.
Na+/H+ antiporter activity was measured as previously described in adult rat PCT perfused in vivo and neonatal and adult rabbit and rat PCT perfused in vitro (1, 4, 11, 23, 36, 37). Neonatal and adult rat tubules were perfused with an ultrafiltrate-like solution without glucose and amino acids (solution B). Organic solutes were omitted from the luminal solution because sodium-coupled glucose and amino acid transport depolarize the basolateral membrane, which may affect bicarbonate exit, an electrogenic process (1). SITS (1 mM) was present in the bathing solution to inhibit the sodium bicarbonate cotransporter, a major regulator of pHi in proximal convoluted tubules (1, 4). The bathing solution had a bicarbonate concentration of 5 mM and a pH of 6.6 to compensate for the cell alkalinization caused by the addition of bath SITS (1, 4). The bathing solution was exchanged at a rate of at least 5 ml/min. Under these conditions, changes in pHi in response to a change in luminal sodium concentration are a measure of Na+/H+ antiporter activity (4, 15, 36, 37). In the experimental period, luminal sodium was removed (solution C).
Chemicals and RIA kits. Corticosterone, T4, and PTU were obtained from Sigma. Serum corticosterone levels were determined by using corticosterone 3H-RIA Kit (ICN Biomedicals, Costa Mesa, CA). Serum thyroxine was measured using 125I-labeled T4 RIA Kit (Diagnostic Systems Laboratories, Webster, TX).
Statistical analysis. Each experiment was performed at least four times. Data are expressed as means ± SE. Statistical significance was determined using analysis of variance, and statistical significance between the groups was performed using the Student-Newman-Keuls test.
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RESULTS |
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Serum bicarbonate was significantly less in the ADX-hypothyroid group than in the 9- and 30-day-old replacement and sham control groups. The bicarbonate level was likely similar in the 9- and 30-day-old replacement and sham control groups, because the 9-day-old rats were still nursing and rat milk contains significant quantities of bicarbonate (42). The serum bicarbonate level in the ADX-hypothyroid group was significantly less than in the 30-day-old replacement and sham control groups.
Effect of glucocorticoids and thyroid hormone on renal cortical NHE3 mRNA abundance.
To determine the role of a combined deficiency of glucocorticoids and thyroid hormone on the maturation of NHE3 RNA, we measured renal cortical NHE3 RNA abundance in 9-day-old, 30-day-old ADX-hypothyroid, replacement, and sham control rats using Northern blot analysis. The results are shown in Fig. 1. The NHE3 mRNA-to--actin ratio in 9- day-old (0.62 ± 0.08) and in 30-day-old ADX-hypothyroid (1.44 ± 0.22) groups was comparable. Both of these groups had significantly lower NHE3 mRNA/
-actin ratios than that of the 30-day-old replacement (6.46 ± 1.14) group and the 30-day-old sham control (8.54 ± 1.26) groups (P < 0.05). There was no difference in the 30-day-old replacement group and sham-operated group. Thus prevention of a postnatal surge in glucocorticoid and thyroid hormones prevented the maturational increase in renal cortical NHE3 mRNA abundance.
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DISCUSSION |
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The factors that induce the postnatal increase in proximal tubule acidification have been elusive. Prime candidates have been glucocorticoids and thyroid hormone, which increase 25- and
3-fold, respectively, during postnatal development (24, 39) and which are known to affect the Na+/H+ antiporter (NHE3) (8, 23). The promoter of rat NHE3 has been sequenced and characterized (13, 25). Analysis of 1.4 kb of the 5'-flanking promoter region showed multiple DNA sequence elements that are recognized by the glucocorticoid receptors, thyroid hormone receptors, SP1, AP-1, AP-2, and several other transcription factors that may participate in the regulation of the NHE3 gene (13, 25). OKP cells, a cell line with characteristics of proximal tubule, expresses NHE-3. We previously demonstrated that both dexamethasone and thyroid hormone increase NHE3 transcription using nuclear run-on assays (6, 14). Neither thyroid hormone nor dexamethasone affected NHE3 mRNA stability (6, 14). In addition, triiodothyronine did not affect rat NHE3 protein stability (14).
There is an interaction between serum thyroid hormone and glucocorticoid levels. Adrenalectomized rats have an increase in thyroid hormone levels compared with controls (32, 38). Plasma thyrotropin-releasing hormone, TSH, T4, and T3 were elevated a week following adrenalectomy (32) in one study; however, another found elevated T3 levels without any significant change in T4 level, 6 wk following adrenalectomy (38). In contrast, studies in neonatal hypothyroid rats have shown either normal (31) or subnormal basal levels of corticosterone (18), whereas hypothyroid adult rats have normal basal corticosterone levels (38) and they have a subnormal corticosterone response to stress (31).
We previously examined whether administration of dexamethasone would accelerate the maturation of proximal tubule acidification (10, 11). We found that the rate of juxtamedullary proximal convoluted tubule bicarbonate absorption and Na+/H+ antiporter activity in the first 2 days of life in rabbits were one-third that of the adult tubule (4, 10). Administration of 60 µg/kg of dexamethasone to a pregnant doe for 3 days before delivery resulted in an increase in the rates of bicarbonate absorption and Na+/H+ antiporter activity comparable to that in the adult proximal convoluted tubule (11). We also found that there is a fourfold maturational increase in rabbit NHE-3 mRNA and protein abundance during postnatal development (7). Administration of either 6 µg/100 g of dexamethasone to does starting at day 27 of gestation or 10 µg/100 g of dexamethasone to neonates resulted in levels of NHE3 mRNA abundance in neonatal rabbits that were comparable to that of adults (7). In the proximal tubule, NHE1 is located on the basolateral membrane. There was no maturational change in NHE1 mRNA or protein abundance, and neither administration of prenatal nor postnatal dexamethasone affected NHE1 mRNA or protein abundance (7).
We recently examined whether prevention of the postnatal increase in glucocorticoids would affect the maturation of PCT Na+/H+ antiporter activity, BBMV NHE3 protein abundance, and renal cortical NHE3 mRNA abundance (23). In that study, adrenalectomy at 9 days of age, a time before the postnatal increase in glucocorticoids, resulted in a significant attenuation of Na+/H+ exchanger activity at 30 days of age. Nonetheless, Na+/H+ antiporter activity was higher than that of the 9-day-old rats. Corticosterone replacement from day 14 of age in the ADX group restored PCT Na+/H+ antiporter activity. NHE3 mRNA abundance was fivefold lower in 9-day-old than that of 30-day-old control rats. Surprisingly, neonatal adrenalectomy did not prevent the maturational increase in NHE3 mRNA abundance. The 30-day-old ADX mRNA abundance was the same as that of ADX with corticosterone replacement and the sham control groups (23).
Similarly, we studied the role of thyroid hormone in the maturation of proximal tubule Na+/H+ antiporter activity (8). In this study, neonatal rats were made hypothyroid by administrating 0.01% PTU in drinking water from day 14 of gestation until the day of study at 21 days of age. Hyperthyroidism was induced by intraperitoneal injection of triiodothyronine on days 17 to 20 of postnatal life. Although there was a reduction in BBMV Na+/H+ antiporter activity in hypothyroid animals and an increase in BBMV Na+/H+ antiporter activity in hyperthyroid rats compared with euthyroid controls, the effect of thyroid hormone status on Na+/H+ antiporter activity was trivial. Although hyperthyroid rats showed a twofold increase in NHE3 mRNA abundance vs. euthyroid rats, hypothyroid rats had comparable NHE3 mRNA abundance as that of euthyroid rats (8). These studies taken together demonstrate that prevention of the maturational increase in either thyroid hormone or glucocorticoids alone does not affect the postnatal maturational increase in NHE3 mRNA. However, as shown in the present study, prevention of the maturational increase in glucocorticoids and thyroid hormone together prevents the maturational increase in NHE3 mRNA.
Studies have characterized the effect of both glucocorticoid excess and deficiency in adult rats (22, 26, 27, 29). The administration of dexamethasone in both adult and adrenalectomized adult rats stimulated the rate of Na+/H+ antiporter activity in BBMV without altering the affinity of the antiporter for Na+ or H+ (22, 27). Similarly, the increase in NHE3 protein abundance following dexamethasone administration was independent of whether adult rats were adrenalectomized 2 days before the study (29). Adrenalectomized adult rats had comparable Na+/H+ antiporter activity in BBMV as that of the sham control group (22, 26, 27). NHE3 protein abundance was also not found to be different in adrenalectomized and sham control groups (29). In agreement with these studies, use of aminoglutethimide in adult rabbits produced glucocorticoid deficiency but resulted in no change in the basal level of renal cortical NHE3 mRNA abundance (9). At variance with these studies, we found a significant attenuation in Na+/H+ antiporter activity as well as NHE3 protein abundance in BBMV in adrenalectomized neonatal rats. This discrepancy may be explained by the fact that adrenalectomy was performed at 9 days of age in our study, a time before the postnatal increase in glucocorticoid occurs in rats. The duration of glucocorticoid deficiency was also different in the two studies. We made measurements 21 days after adrenalectomy compared with 23 days after adrenalectomy in studies using adult rats described above. Both PCT Na+/H+ antiporter activity and NHE3 protein abundance in adrenalectomized neonatal rats were restored with administration of physiological doses of corticosterone.
Similar to our previous study of the effect of thyroid hormone on neonatal rats (8), the role of thyroid hormone status on Na+/H+ antiporter activity and NHE3 abundance has been studied in adult rats (3, 28). The thyroid hormone status was found to have a greater impact on Na+/H+ antiporter activity in adult rats than we found in our neonatal rats (8, 28). In agreement with our study, NHE3 mRNA abundance was comparable in both hypothyroid and euthyroid adult rats and was higher in hyperthyroid rats (3). However, this study in adult rats failed to show any difference in renal cortical NHE3 protein abundance between hypothyroid and hyperthyroid adult rats (3), in contrast to our finding of a low level of NHE3 protein in hypothyroid vs. hyperthyroid neonatal rats (8). The reason for this discrepancy is not clear but is likely due to the age of the rats.
Our previous study in neonatal adrenalectomized rats showed significant blunting (more than 60%) in postnatal maturation of Na+/H+ antiporter activity in a 30-day-old adrenalectomized group vs. a 30-day-old sham-operated group (23). Once again, we confirmed our previous findings in this experiment that deficiency of glucocorticoids did not totally prevent the maturation of Na+/H+ exchanger activity (23). Hypothyroid status showed a trivial (only 10%) reduction in Na+/H+ antiporter activity in BBMV (in hypothyroid rats) in contrast to euthyroid rats (8). These findings demonstrate that glucocorticoids have a more profound effect on maturation of Na+/H+ antiporter activity than the thyroid hormone. In the present study, Na+/H+ antiporter activity was comparable in the 30-day-old ADX-hypothyroid group and 9-day-old rats. However, the 30-day-old ADX group had a significantly higher level of Na+/H+ antiporter activity than that of the 30-day ADX-hypothyroid group. All these data suggest that the thyroid hormone contributes a small fraction to the maturation of Na+/H+ antiporter activity that persists after complete adrenalectomy.
In summary, the present study shows that maturation of Na+/H+ antiporter activity, BBM NHE3 protein, and renal cortical NHE3 mRNA abundance can be completely prevented by preventing ontogenic rise in glucocorticoid and thyroid hormone levels. Thus both glucocorticoid and thyroid hormones play a combined and interrelated role in maturation of proximal tubule acidification.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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