We previously demonstrated that there are developmental changes in proximal tubule Na+/H+ exchanger (NHE) activity. There is a maturational increase in postnatal brush-border membrane (BBM) vesicle NHE3 protein abundance and decrease in NHE8 protein abundance. The purpose of this study was to determine whether thyroid hormone plays a role in the rat renal maturational isoform switch from NHE8 to NHE3 and whether thyroid hormone regulates NHE8. Administration of thyroid hormone to neonatal rats, before the normal postnatal increase in serum thyroid hormone levels at 3 wk of age, resulted in a premature increase in NHE3/β-actin BBM protein abundance and mRNA abundance. Thyroid hormone also caused a premature decrease in BBM NHE8/β-actin protein abundance, whereas there was no change in mRNA expression (standardized to 28s). Rats made hypothyroid from birth were studied at 28 days, after the normal maturational increase in thyroid hormone. In these hypothyroid adult rats, the maturational increase in BBM NHE3 protein abundance and NHE3 mRNA expression was prevented. In contrast, the developmental decrease in BBM NHE8 protein abundance was prevented in hypothyroid adults, but mRNA expression was unchanged in hypothyroid rats. To determine whether the effect of thyroid hormone was due to a direct epithelial effect, we studied normal rat kidney cells in culture. We recently showed that this cell line expresses NHE8, but does not express NHE3. Thyroid hormone caused a decrease in surface expression of NHE8, determined by biotinylation, but total cellular abundance remained unchanged. NHE8 activity, measured as the sodium-dependent rate of intracellular pH recovery from an acid load, was less with thyroid treatment than control. In conclusion, thyroid hormone plays a potential role in the developmental isoform change from NHE8 to NHE3 and decreases NHE8 activity.
- proximal tubule
- Na/H exchanger
neonates have lower serum bicarbonate levels due to a lower renal threshold for bicarbonate (18). This is by and large due to lower rates of proximal tubule bicarbonate reabsorption in the neonate compared with the adult (4, 7, 34). In the adult and neonate proximal tubule, the Na+/H+ exchanger (NHE) mediates the majority of luminal proton secretion (4, 32). NHE3 is the predominant NHE on the adult proximal tubule brush-border membrane (40).
We recently found that there was a discrepancy between the NHE activity in the neonatal proximal tubule and NHE3 protein abundance in the rat (35). While there is less NHE activity in the neonatal proximal tubule compared with the adult rat, there was still significant NHE activity despite the fact that neonatal brush-border membrane NHE3 was barely detectable (35). In addition, NHE3-deficient mice have a substantive amount of NHE activity (15). Recently, NHE8, a second proximal tubule NHE expressed on the apical membrane, has been identified (9, 19, 20, 42). Proximal tubule brush-border NHE8 abundance was highest in 7- to 14-day neonates at a time when NHE3 abundance was significantly lower than that of adult rats. We showed that thyroid hormone is a potential factor in the postnatal increase in NHE3 protein abundance (6). In this study, we examined whether the maturational increase in thyroid hormone was a factor in the postnatal maturational decrease in NHE8 and whether thyroid hormone affected NHE8 activity.
Pregnant Sprague-Dawley rats arrived at our institution on day 19 of gestation. Hyperthyroidism was induced in neonatal rats, before the normal maturational increase in thyroid hormone levels (6, 38), by administration of 3,5,3′-triiodothyronine (T3; Sigma, St. Louis, MO) dissolved in 0.01 N NaOH and diluted 1:4 in PBS to a final concentration of 10 μg/ml. Vehicle was 0.01 N NaOH diluted 1:4 in PBS. T3 or vehicle was administered by intraperitoneal injection, daily for 4 days starting on day 4 of life at a dose of 10 μg/100 g body wt. The animals were studied 2 h after the last injection on day 7 of life. This study conformed to the APS's Guiding Principles in the Care and Use of Animals and all protocols were approved by the Institutional Animal Care and Use Committee at the University of Texas Southwestern Medical Center.
Pregnant Sprague-Dawley rats arrived at our institution on day 13 of gestation. To prevent the normal developmental increase in plasma thyroid hormone levels, these pregnant rats and their neonates imbibed water containing propyl-thiouracil (PTU; Sigma) to prevent the maturational increase in thyroid hormone at the time of weaning (3 wk of age) from day 14 of gestation (6, 38, 39). PTU was dissolved in 1 ml of 0.1 N NaOH and then further dissolved in 0.5 l of drinking water to make a final concentration of 0.01% and administered until the time of study. Vehicle was 1 ml of 0.1 N NaOH in 0.5 l of drinking water. The rats were studied at 28 days of age. Hypothyroidism was confirmed by measuring serum T4 using an 125I-labeled T4 RIA kit according to the manufacturer's instructions (Diagnostic Systems Laboratories, Webster, TX).
Brush-border membrane vesicle isolation.
Kidneys were removed and placed in ice-cold PBS. The cortex was quickly dissected and homogenized in ice-cold isolation buffer with 15 strokes of Potter Ejevhem homogenizer maintained at ∼4°C by surrounding the vessel in ice water. The isolation buffer contained 300 mM mannitol, 16 mM HEPES, and 5 mM EGTA titrated to pH 7.4 with Tris containing 1 μl/ml of protease inhibitor cocktail (Sigma) and 100 μg/ml phenyl-methyl-sulfonyl fluoride (Calbiochem). Brush-border membrane vesicles (BBMV) were isolated with magnesium precipitation and differential centrifugation as described previously by our laboratory (6, 9). BBMV were then resuspended with RIPA buffer (150 mM NaCl, 50 mM Tris, 5 mM EDTA, 1% Triton X-100, 0.5% deoxycholate, and 0.1% SDS) containing the same protease inhibitors. The protein concentration was measured by the Bradford method using bovine serum albumin as the standard (11).
SDS-PAGE and immunoblotting.
Brush-border membranes (100 μg of protein/lane) were incubated at 37°C for 15 min and then separated on 7.5% polyacrylamide gel using SDS-PAGE as previously described (6, 9). Proteins were transferred to polyvinylidene diflouride membranes at 350–450 mA at 4°C for 1 h. The blots were then blocked for 1 h with Blotto (0.05% Tween 20 in 5% nonfat milk Tween in PBS) followed by incubation overnight with primary antibody at 4°C. NHE8 and NHE3 antibodies were gifts from Drs. P. Aronson and O. Moe, respectively. The blots were washed several times with PBS containing 0.1% Tween 20 and then incubated for 1 h with secondary antibody, horseradish peroxidase-labeled antirabbit (NHE3) or antimouse (NHE8) at a 1:10,000 dilution and washed again. Bound antibody was detected and measured using enhanced chemiluminescence and densitometry. Equal loading of the samples was confirmed using an antibody to β-actin (Sigma).
cDNA synthesis and real-time PCR.
RNA was isolated from the rat renal cortex and normal rat kidney (NRK) cells with GenEllute mammalian total RNA kit (Sigma). RNA (2 μg) was first treated with DNase (Invitrogen, Carlsbad, CA), and cDNA was synthesized using random primers and dNTP and StrataScript reverse transcriptase (Stratagene, La Jolla, CA). cDNA was synthesized using an annealing temperature of 25°C for 10 min, extension at 42°C for 50 min, and termination at 70°C for 15 min. Success of reverse transcription was validated using primers to GAPDH.
Real-time PCR was performed using an iCycler PCR thermal cycler (Bio-Rad, Hercules, CA) to quantify relative mRNA expression. Primers at 10 μM concentration were mixed with cDNA (1:50 dilution) and SYBR green master mix (Bio-Rad) per the manufacturer's instructions. The PCR cycle was denaturation at 94°C for 30 s, annealing at 61°C for 20 s, and extension at 72°C for 20 s, for 40 cycles. The relative expression of NHE3 and NHE8 was determined by comparing abundance to the housekeeping gene 28S. Relative quantitation was assessed using the method described by Vandesompele et al. (37). Primers were as follows: GAPDH (forward) 5′-CAC CAT GGA GAA GGC-3′ and (reverse) 5′-TGC CAG TGA GCT TCC-3′; NHE3 (forward) 5′-ACT GCT TAA TGA CGC GGT GAC TGT-3′ and (reverse) 5′-AAA GAC GAA GCC AGG CTC GAT GAT-3′; NHE8 (forward) 5′-AAGCCTATTCTTCCGGTGCAGACA-3′ and (reverse) 5′-AGAGAAACAACAGCCACGCTCTCA-3′; and 28S (forward) 5′-TTG AAA ATC CGG GGG AGA G-3′ and (reverse) 5′-ACA TTG TTC CAA CAT GCC AG-3′.
Cell culture and surface NHE8 expression.
NRK cells (American Type Culture Collection, Manassas, VA) cultured at 37°C in a 95% air-5% CO2 atmosphere were passaged in a high-glucose (450 mg/dl) DMEM with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 g/ml). Confluent cells were rendered quiescent in a serum-free 1:1 mixture of low-glucose (100 mg/dl) DMEM and Ham's F-12.
Confluent quiescent NRK cells were treated with either thyroid hormone (10−7 M) or vehicle for 24 h in serum-free media. The cells were rinsed with PBS containing 0.1 mM CaCl2 and 1 mM MgCl2 (4°C), and surface proteins were biotinylated by incubating cells with 1.5 mg/ml sulfo-NHS-SS-Biotin in 10 mM triethanolamine (pH 7.4), 2 mM CaCl2, and 150 mM NaCl for 60 min as previously described (10). The plates were washed twice with PBS containing 1 mM MgCl2, 0.1 mM CaCl2, and 100 mM glycine for 20 min at 4°C and subsequently lysed using RIPA buffer with the same protease inhibitors as above. The cell lysates were centrifuged and the supernates were diluted with RIPA buffer. Cell lysates of equivalent amounts of protein (2.5 mg/ml) were incubated overnight using a rotatory shaker with streptavidin-agarose beads at 4°C. The streptavidin-agarose beads were washed with 50 mM Tris·HCl (pH 7.4), 100 mM NaCl, and 5 mM EDTA, then with 50 mM Tris·HCl (pH 7.4) and 500 mM NaCl, and finally with 50 mM Tris·HCl, pH 7.4. The biotinylated proteins were released from the beads by heating to 95°C with 2.5× loading buffer and subjected to immunoblotting with anti-NHE8 antibody (10).
NHE8 activity assay in NRK cells.
NHE-8 activity was measured using the pH-sensitive dye 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) as described previously (5, 29). NRK cells were grown to confluence on glass coverslips and quiescent cells were incubated with either thyroid hormone (10−7 M) or vehicle for 24 h in serum-free media. The cells were incubated with 10 μM BCECF-AM (BCECF-acetoxymethyl ester) for 20 min at 37°C, and intracellular pH (pHi) was determined from the ratio of fluorescence (excitation: 500 and 450 nm, emission: 530 nm) in a computer-controlled spectrofluorometer (QM-8/2003, Photon Technology International, London, Ontario). The 500/450 fluorescence ratio was calibrated to intracellular pH using K+/nigericin (5, 29). Na+/H+ exchange activity was assayed as the initial rate of the Na+-dependent pHi increase after an acid load using nigericin in the absence of CO2/HCO3− as previously shown by our laboratory (42). Briefly, NRK cells were initially incubated in sodium-free solution with nigericin (10 μg/ml) resulting in cell acidification due to the absence of Na+ and the cell-to-bath potassium gradient. The sodium-free solution consisted of (in mM) 115 choline chloride, 5 KCl, 1.54 MgCl2, 1.1 CaCl2, and 30 HEPES. After a steady-state pH was reached (150 s), the nigericin-containing solution was replaced by sodium-free solution containing albumin (1 g/dl). The sodium-containing solution was then added resulting in pH recovery due to reestablishment of sodium-proton exchange. The sodium-containing solution consisted of (in mM) 115 NaCl, 5 KCl, 1.54 MgCl2, 1.1 CaCl2, and 30 HEPES. Both solutions had pH 7.40 and osmolality 295 mosmol/kgH2O.
Effect of T3 on renal cortical NHE8 and NHE3 protein and mRNA abundance in 7-day-old rats.
The serum levels of thyroid hormone are several-fold lower in neonatal rats compared with adult rats (6, 38). At the time of weaning, the serum levels increase and are comparable to that of the mature adult (6, 38). To determine whether thyroid hormone could prematurely affect the maturational brush-border membrane isoform change from NHE8 to NHE3, we administered T3 hormone (10 μg/100 g body wt) daily for 4 days starting on day 4 of life (6). As shown in Fig. 1, 7-day-old neonatal rats receiving thyroid hormone had an ∼50% increase in brush-border membrane protein NHE3 expression and a comparable decrease in NHE8 expression compared with control. The effect of T3 on NHE3 protein abundance was consistent with our previous results when we studied the effect of T3 on 21-day-old rats (6). We also examined the effect of T3 on another brush-border membrane protein, villin. There was no difference in the villin/β-actin ratio between control (0.62 ± 0.05, n = 7) and the hyperthyroid group (0.57 ± 0.05, n = 7). Finally, the effect of thyroid hormone in 7-day-old rats was also assessed in the cortical homogenate to assess the effect on total protein. There was no significant difference in NHE3/β-actin and NHE8/β-actin in the control compared with the hyperthyroid rats as shown in Table 1.
We next assessed the effect of thyroid hormone on NHE8 and NHE3 mRNA in neonatal rats with real-time PCR using 28s as the housekeeping gene. The effect of T3 on NHE3 and NHE8 abundance is shown in Fig. 2. In neonates that received thyroid hormone, relative NHE3 mRNA expression was higher than the control animals. This is consistent with previous studies showing that there is a maturational increase in NHE3 mRNA abundance (35) and that thyroid hormone in 1-mo-old rats increases NHE3 mRNA abundance (6, 13). However, there was no effect of thyroid hormone on NHE8 mRNA abundance.
Effect of hypothyroidism on renal cortical NHE8 and NHE3 protein and mRNA abundance in 4-wk-old rats.
We next examined whether we could prevent the maturational changes in NHE3 and NHE8 by preventing the maturational increase in thyroid hormone. Rats were studied at 28 days of age because adult levels of thyroid hormone, NHE3 mRNA and protein abundance, and NHE8 protein abundance are present by this age (6, 9, 38). Administration of PTU water resulted in serum thyroxine levels that were significantly lower in the hypothyroid group compared with the control group (0.7 ± 0.1 vs. 2.9 ± 0.4 μg/dl; P < 0.001). These values are comparable to those previously measured by our laboratory in neonatal and adult rats, respectively (6). As shown in Fig. 3, prevention of the maturational increase in thyroid hormone resulted in lower levels of NHE3 and higher levels of NHE8 protein abundance in brush-border membranes than were seen in 28-day-old control rats. We also examined the effect of hypothyroidism on villin. There was no effect of hypothyroidism on the villin/β-actin ratio in the control vs. the hypothyroid group (0.91 ± 0.13 vs. 0.99 ± 0.07, n = 7). As shown in Table 1, we also examined the effect of hypothyroidism in cortical homogenate to assess the effect on total protein. There was a significant decrease in NHE3/β-actin in cortical homogenate from hypothyroid 28-day-old rats compared with control rats but no difference in the NHE8/β-actin ratio.
As shown in Fig. 4, the hypothyroid rats had a reduction in NHE3 mRNA but there was no effect on NHE8 mRNA abundance. The effect of hypothyroidism on NHE3 mRNA was at variance with previous studies from our laboratory where we found that hypothyroidism did not affect NHE3 mRNA abundance using Northern blot analysis in 21-day-old rats (6).
Effect of thyroid hormone on NHE8 in vitro.
In the next series of experiments, we examined the effect of thyroid hormone on NRK cells. We recently showed that NRK cells have apical membrane NHE activity mediated by NHE8 and that these cells do not express NHE3 (42). The effects of 10−7 M T3 on NHE8 mRNA and protein abundance are shown in Figs. 5 and 6. NHE8 mRNA abundance was not affected by thyroid hormone. However, as shown in Fig. 6, surface NHE8 protein expression was reduced by thyroid hormone. This was not a nonspecific effect of thyroid hormone as leucine aminopeptidase, a surface protein on NRK cells (31), was not affected (0.61 ± 0.02 in control vs. 0.76 ± 0.08 in the thyroid group, n = 6). There was no effect of thyroid hormone on total cellular NHE8 protein abundance (not shown). Finally, we examined the effect of 10−7 M T3 on NHE activity. As shown in Fig. 7, thyroid hormone resulted in a reduction in NHE activity. Thus thyroid hormone directly decreases apical expression and inhibits NHE8 activity in this cell line.
Thyroid hormone has many actions on the kidney. Hypothyroidism can reduce glomerular filtration rate, renal blood flow, proximal tubule sodium absorption, impair renal acidification, BBMV Na-phosphate transport, NHE activity, and proximal and distal Na-K-ATPase activity (1, 3, 6, 25, 26, 28, 30). Conversely, hyperthyroidism increases proximal tubule sodium absorption, BBMV Na-phosphate transport and NHE activity, as well as proximal and distal tubule Na-K-ATPase activity (1, 6). While many of these alterations in proximal transport can be explained by the thyroid hormones effect on renal hemodynamics (16, 17, 21, 26), thyroid hormone has been shown to have a direct epithelial action to alter proximal tubular transport (13, 14, 24, 41).
Previous studies showed that the maturational increase in thyroid hormone likely is a significant factor affecting proximal tubule postnatal development. In neonatal rats, hypothyroidism prevents the maturational increase in NHE3 and reduces BBMV NHE activity (6). Thyroid hormone plays an important role in the developmental increase in mitochondrial oxidative enzymes (39). There is a maturational increase in both active transcellular and passive paracellular proximal tubule NaCl transport in the rabbit. The maturational increase in thyroid hormone has a role in increasing not only transcellular NaCl transport mediated by parallel Na+/H+ and Cl−/base exchangers but also in increasing the paracellular chloride permeability and mediating some of the developmental changes that occur in the paracellular pathway in the rabbit (8, 33, 36).
Most pertinent to this study are studies examining the effect of thyroid hormone on NHE activity. In adult rats, hypothyroidism has been shown to decrease and administration of thyroid hormone to increase BBMV NHE activity (22, 23). Prevention of the maturational increase in thyroid hormone resulted in a decrease in rat NHE activity and brush-border NHE3 protein abundance but did not affect NHE3 mRNA abundance when rats were studied at 21 days of age using Northern blot analysis (6). Previous studies in adult rats showed that hypothyroidism does not affect NHE3 mRNA abundance using dot blots (2). These studies are at variance with our current result showing lower NHE3 mRNA expression in hypothyroid animals studied at 28 days of age. The reason for this is unclear but may be due to the greater sensitivity of real-time PCR. Administration of thyroid hormone to young rats studied at 21 days of age resulted in an increase in NHE activity, NHE3 protein, and mRNA abundance (6). The stimulation in NHE activity by thyroid hormone is seen in vitro in OK cells, a proximal tubule cell line which expresses NHE3 (13, 41) where the increase in NHE3 by thyroid hormone is due to an increase in transcription (13). The NHE3 promotor has several partial thyroid response elements that are consistent with transcriptional regulation by thyroid hormone; however, similar studies have not been performed for NHE8 (12).
We previously demonstrated a disjoint between NHE activity and the abundance of NHE3 in neonatal brush-border membranes (35). This prompted us to examine whether NHE8 could potentially be a factor mediating NHE activity in neonatal rats. NHE8 is localized on the brush-border membrane of proximal tubules (19, 20, 42). NHE8 has EIPA-sensitive NHE activity (42).
While NHE3 mRNA and protein abundance was far less in 1- and 2-wk-old neonates than after weaning (9, 35), brush-border membrane NHE8 protein abundance was highest in neonates at that age and decreased in adulthood (9). However, there was no maturational change in NHE8 mRNA abundance. Thus, there is a developmental brush-border membrane NHE isoform change during postnatal development. Interestingly, while brush-border membrane NHE8 protein abundance was higher in 1- and 2-wk-old neonates than adults, total homogenate NHE8 was higher in adults (9). We also found a discordance in the effect of thyroid hormone in 7-day-old rats and hypothyroidism in 28-day-old rats compared with their respective controls on brush-border membrane NHE8 compared with the cortical homogenate. While brush-border membrane NHE8 was affected by the thyroid hormone status, there was no effect in NHE8 protein abundance from the cortical homogenate.
This study examined the effect of thyroid hormone on the postnatal development of NHE3 and NHE8 to determine whether the postnatal increase in thyroid hormone was a potential factor mediating the change from NHE8 to NHE3 on the apical membrane. Administration of thyroid hormone before the normal developmental increase resulted in a precocious increase in brush-border membrane NHE3 and decrease in NHE8 protein abundance, although there was no effect on NHE3 and NHE8 in the cortical homogenate. Prevention of the postnatal increase in thyroid hormone resulted in higher brush-border NHE8 and lower brush-border NHE3 protein abundance than controls. These observations are consistent with thyroid hormone playing a role in the NHE isoform change on the brush-border membrane. Furthermore, we directly demonstrate that NHE8 surface expression is reduced by thyroid hormone in vitro as is NHE activity. This is the first demonstration that NHE8 activity is regulated. Whether other factors play a role in the regulation of NHE8 and changes in postnatal expression is yet to be determined.
This work was supported by National Institutes of Health Grant DK-41612.
We thank Dr. R. Quigley for reviewing this manuscript.
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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