Epidermal growth factor inhibits Na-Pi cotransport in weaned and suckling rats

Mazen Arar1, Hubert K. Zajicek2, Ihsan Elshihabi1
Moshe Levi2
(With the Technical Assistance of Myrna Gonzales and Paul Wilson)

1 Department of Pediatrics, University of Texas Health Science Center at San Antonio, San Antonio 78284; and 2 Department of Internal Medicine, University of Texas Southwestern Medical Center at Dallas and Veterans Affairs Medical Center, Dallas, Texas 75216

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In the present study, we determined the effect of epidermal growth factor (EGF; 10 µg/100 g body wt) on sodium gradient-dependent phosphate transport (Na-Pi cotransport) regulation in suckling (12-day-old) and weaned (24-day-old) rats. Weaned rats had higher proximal tubular brush border membrane vesicle (BBMV) Na-Pi cotransport activity (232 ± 16 in weaned vs. 130 ± 9 pmol · 10 s-1 · mg protein-1 in suckling rats, P < 0.05). Chronic treatment with EGF induced inhibition of BBMV Na-Pi cotransport in both suckling (130 ± 9 vs. 104 ± 7 pmol · 10 s-1 · mg protein-1, P < 0.05) and weaned rats (232 ± 16 vs. 145 ± 9 pmol · 10 s-1 · mg protein-1, P < 0.005). The inhibitory effect was selective for Na-Pi cotransport as there was no inhibition of Na-glucose cotransport. Weaned rats had a higher abundance of BBMV NaPi-2 protein than suckling rats (increase of 54%, P < 0.001) and a twofold increase in NaPi-2 mRNA. The EGF-induced inhibition of Na-Pi transport was paralleled by decreases in NaPi-2 protein abundance in both weaned (decrease of 26%, P < 0.01) and suckling (decrease of 27%, P < 0.01) animals. In contrast, there were no changes in NaPi-2 mRNA abundance. We conclude that proximal tubule BBMV Na-Pi cotransport activity, NaPi-2 protein abundance, and NaPi-2 mRNA abundance are higher in weaned than in suckling rats. EGF inhibits Na-Pi cotransport activity in BBMV isolated from suckling and weaned rats, and this inhibition is mediated via a decrease in NaPi-2 protein abundance, in the absence of a change in NaPi-2 mRNA.

NaPi-2 mRNA; NaPi-2 protein; phosphate transporter maturation

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

EPIDERMAL GROWTH FACTOR (EGF) is a 53-amino acid polypeptide. The kidney is a major site of synthesis of the EGF precursor, prepro-EGF (42). High levels of mRNA for prepro-EGF and EGF immunoreactivity have been localized in the cortical thick ascending limb, distal convoluted tubule, and octopal-shaped intercalated cells of the collecting duct (36, 42). Glomerular mesangial cells and multiple segments of the renal tubule, including the proximal straight tubules, proximal convoluted tubules, cortical collecting ducts, inner medullary collecting ducts, outer medullary collecting ducts, and distal convoluted tubules, express EGF receptors (12). Addition of EGF to embryonic mouse kidney in organ culture stimulates DNA synthesis, renal growth, and distal nephron differentiation (4). EGF causes a decrease in glomerular filtration rate (25), activates the hexose monophosphate shunt, increases Na/H exchanger activity in primary cell cultures from the rat proximal tubule (51), inhibits the hydrosmotic effect of vasopressin and active sodium absorption in the isolated perfused rabbit cortical collecting tubule (13, 53), upregulates amino acid-transport activity in jejunal brush border membrane vesicles (BBMV) (43), and increases absorption of H2O, Na+, Cl-, and glucose from the jejunum (37).

EGF may also play a role in renal growth and development. A role for EGF in renal development is evident by the activation of EGF receptors in late gestation (19) and its effect on growth and development of cultured embryonic kidney rudiments (4).

In renal epithelial cells grown in culture, EGF has also been shown to modulate sodium gradient-dependent phosphate transport (Na-Pi cotransport) activity. In LLC-PK1 cells, a tubular epithelial cell line derived from pig kidney cortex, EGF caused stimulation of Na-Pi cotransport (23). EGF also stimulates Na-Pi cotransport in isolated perfused proximal tubule derived from rabbit kidney (40). In contrast, in opossum kidney (OK) cells, a tubular epithelial cell line derived from the opossum kidney, EGF causes inhibition of Na-Pi cotransport (3, 38). The in vivo effect of EGF on Na-Pi cotransport activity, however, is unknown.

The purposes of the present study were to determine 1) whether EGF regulates Na-Pi cotransport activity in vivo and 2) whether the effect of EGF to regulate Na-Pi cotransport is dependent on the developmental stage of the animal, i.e., suckling vs. weaned rat.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Animals. Pregnant Sprague-Dawley rats were housed at our institution for 3-4 days before their expected date of delivery. Neonatal rats were cared for by their mothers. Suckling (12-day-old) and weaned (24-day-old) animals received subcutaneous injections of EGF (10 µg/100 g body wt) or vehicle every 12 h for four doses, including a dose 2 h before they were killed, or received only one dose 4 h prior to being killed. The plasma level of EGF was not measured, but such an EGF dose was shown to have a biological effect on intestinal transport (43). Blood and urine samples were collected for measurement of creatinine and phosphate for determination of the fractional excretion of phosphate. Both kidneys were rapidly removed; one half of each kidney was used for brush border membrane (BBM) isolation, and the other half of each kidney was used for RNA isolation. Material from each animal was processed subsequently for 1) transport activity, 2) enzyme activity, 3) protein gels and Western blotting, and 4) RNA gels and Northern blotting. For each BBM and RNA preparation, we pooled kidneys from 2-4 rats (n = 1) and studied at least five samples from each experimental group.

BBM vesicle isolation. Control and EGF-treated rats were killed, and the kidneys were rapidly removed and placed in an ice-cold homogenizing buffer consisting of (in mmol/l) 300 mannitol, 0.5 phenylmethylsulfonyl fluoride, 5 EGTA, and 16 HEPES, pH 7.50, with Tris. The cortex was isolated and homogenized with a Teflon-glass Potter-Eljevhem homogenizer. BBMVs were then isolated by differential centrifugation and magnesium precipitation, as previously described (9, 10, 24, 29, 30). The final BBMV fraction was resuspended at an approximate concentration of 10 mg BBM protein/ml in a buffer containing (in mmol/l) 300 mannitol and 16 HEPES, pH 7.5, with Tris. Protein was measured by the method of Lowry et al. (32) with crystalline BSA as the standard. To minimize the potential day-to-day variation in the BBM isolation procedure, we isolated BBM from the kidneys of control and EGF-treated rats simultaneously each day. Each BBMV sample was aliquoted for simultaneous measurement of enzyme activity, transport activity, and transport protein abundance.

BBM enzyme activity measurements. The purity of each BBMV preparation was determined by measurement of membrane-specific enzyme activity [including alkaline phosphatase), leucine aminopeptidase (BBM-bound)] and Na+-K+-ATPase [basolateral membrane (BLM)-bound], in homogenate and BBM fractions. Alkaline phosphatase activity was measured by a kinetic assay monitoring the production of p-nitrophenyl from p-nitrophenyl phosphate at 405 nm and 37°C (11). Leucine aminopeptidase activity was measured by a kinetic assay that monitored the conversion of L-leucine-p-nitrophenolate at 380 nm and 37°C (11). Na+-K+-ATPase activity was measured by a kinetic assay system coupling ATP hydrolysis to pyruvate kinase and lactate dehydrogenase and monitoring the use of NADH at 340 nm and 37°C (45). Enzyme activities were expressed as picomoles per minute per milligram homogenate or BBM protein. Enrichment (specific activity in BBM fraction/specific activity in homogenate) was determined in each BBM preparation.

BBM transport activity measurements. Transport activity measurements were performed in freshly isolated BBM vesicles by radiotracer uptake before rapid Millipore filtration. All uptake measurements were performed in triplicate, and the uptake was calculated on the basis of specific activity determined in each experiment and expressed as picomoles of solute per time interval per milligram BBM protein. To measure Na+ gradient-dependent 32Pi uptake (Na-Pi cotransport), 10 µl of BBM preloaded with an intravesicular buffer of (in mmol/l) 300 mannitol, 16 HEPES, and 10 Tris, pH 7.50, were vortex mixed at 25°C with 40 µl of an extravesicular uptake buffer of (in mmol/l): 150 NaCl, 25 µmol K2H32PO4, 16 HEPES, and 10 Tris, pH 7.50. Uptake after 10 s (representing initial linear rate) was terminated by an ice-cold stop solution that consisted of (in mmol/l) 100 NaCl, 100 mannitol, 16 HEPES, and 10 Tris, pH 7.50. To determine the Na+-independent (i.e., diffusive) Pi uptake, 150 mmol/l NaCl was replaced with 150 mmol/l choline chloride. To examine whether the effect of EGF was specific for Na-Pi cotransport, we also measured Na+ gradient-dependent glucose transport by a method identical to that of Pi uptake, but in the presence of 25 µmol/l D-[3H]glucose.

BBM protein SDS gel electrophoresis and Western blot analysis. BBMs were denatured for 2 min at 95°C in 2% SDS, 10% glycerol, 0.5 mM EDTA, and 95 mM Tris-HCl, pH 6.80 (final concentrations), and 40 µg BBM protein per lane were separated on 10% polyacrylamide gels according to the method of Laemmli (27) and electrotransferred onto nitrocellulose paper (52). After blockage with 5% Carnation milk powder with 1% Triton X-100 in Tris-buffered saline (20 mM, pH 7.3), Western blots were performed with antiserum against NaPi-2 (18) and ecto-5'-nucleotidase (20) at dilutions of 1:4,000. Additional Western blots were done with antibodies against NaSi-1 transport protein and beta -actin. Primary antibody binding was visualized using goat anti-rabbit immunoglobulin G (IgG) conjugated to alkaline phosphatase (Bio-Rad, Richmond, CA), developed with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Bio-Rad), and quantified by densitometry. For peptide protection, antigenic peptides were included at a concentration of 100 µg/ml. Prestained molecular weight marker proteins (Bio-Rad) were run in parallel.

Isolation of RNA. Total RNA was isolated by the method of Chomczynski and Sacchi (17). Briefly, kidney cortical slices collected from both suckling and weaned rats were homogenized in 10 ml of RNA isolation buffer [4 M guanidium thiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% sarcosyl, and 0.1 M 2-mercaptoethanol]. Sequentially, 1.0 ml of 2 M sodium acetate, pH 4.0, 10 ml of water-saturated phenol, and 2.0 ml of chloroform-isoamyl alcohol mixture (49:1) were added, with mixing by inversion after the addition of each reagent. The sample was centrifuged at 10,000 g for 20 min at 4°C, and the RNA was partitioned to the aqueous phase. The aqueous phase was mixed with an equal volume of isopropanol and placed at -20°C to precipitate the RNA. Sedimentation of RNA was again performed, and the RNA pellet was dissolved in 0.3 ml of RNA isolation buffer and reprecipitated with equal volume of isopropanol at -20°C. The RNA pellet was sedimented, washed twice in 75% ethanol, and dissolved in 50-200 µl of diethyl pyrocarbonate-treated water at room temperature. Absorbance at 260 and 280 nm was obtained to quantify and assess the purity of the RNA. RNA was stored at -70°C until use for Northern blot analysis.

Formaldehyde agarose gel electrophoresis and Northern blot analysis. After denaturation of RNA samples in formaldehyde, 15 µg total RNA per lane were size-fractionated using 0.66 M formaldehyde and 1% agarose gels (final concn) (Bio-Rad). RNA size standards (GIBCO-BRL, Gaithersburg, MD) were run in parallel. After electrophoresis, the gel was placed onto a vacuum-blotting device (Bio-Rad), and a vacuum of 60 cmH2O was applied for 4 h using 20× standard sodium citrate (SSC) (3 M NaCl and 0.3 M trisodium citrate, pH 7.0) as blotting buffer, which resulted in complete transfer of RNA. The RNA was blotted onto GeneScreen Plus nylon membranes [Du Pont-New England Nuclear (NEN), Boston, MA], and the RNA was immobilized by irradiation with ultraviolet light (UV crosslinker). Prehybridization (4 h at 42°C) and hybridization (18 h at 42°C) of the RNA blots were performed with a buffer (100 µl/cm2) consisting of 5× SSPE (0.75 M NaCl, 50 mM NaH2PO4, and 5 mM EDTA, pH 7.40), 5× Denhardt's solution [0.1% Ficoll 400, 0.1% polyvinylpyrrolidone, 0.1% BSA (fraction V)], 0.1% SDS, 100 µg/ml denatured salmon sperm DNA, and 50% deionized formamide as previously described (31). cDNA probes of NaPi-2 (33), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta -actin, were labeled by random priming (Pharmacia) using [alpha -32P]dCTP (DuPont-NEN). After hybridization, the blots were washed twice for 15 min each time in 2× SSPE, 0.1% SDS at room temperature, twice for 15 min each time in 0.1× SSPE, 0.1% SDS at 37°C, and twice for 15 min each time in 0.1× SSPE, 0.1% SDS at 50°C. Autoradiography was performed at -70°C with DuPont-NEN reflection film using a DuPont intensifying screen (DuPont-NEN). Membranes were stripped (0.1× SSC, 0.1% SDS at 95°C for 5 min) before another hybridization was performed. mRNA levels for NaPi-2 were quantitated by densitometry and normalized to the density of the corresponding GAPDH and beta -actin mRNA.

Statistical analysis. All data are expressed as means ± SE. Unpaired t-test was used to compare the results of suckling vs. weaned rats and of control vs. EGF-treated neonatal rats. ANOVA test was used for comparison between multiple groups. Significance was accepted at P < 0.05.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effect of EGF on fractional excretion of phosphate. EGF administered every 12 h for 4 doses resulted in a significant increase in fractional excretion of phosphate [26.5 ± 2.3 in control vs. 39.5 ± 4.4% in EGF-treated suckling rats (P < 0.05), and 0.24 ± 0.02 in control vs. 3.5 ± 0.7% in EGF-treated weaned rats (P < 0.05)].

Effect of EGF on serum phosphate and creatinine. Chronic (36-h) EGF administration induced a small increase in serum phosphate in weaned rats [10.4 ± 0.23 in control vs. 11.2 ± 0.39 in EGF treated (P = 0.22)] and a more significant increase in suckling rats [11.2 ± 0.50 in control vs. 12.7 ± 0.61 in EGF-treated (P = 0.08)]. Chronic EGF treatment causes an increase in serum creatinine of suckling rats [0.57 ± 0.02 in control vs. 0.77 ± 0.06 in EGF-treated (P = 0.003)] and a significant decrease in weaned rats [0.47 ± 0.03 in control vs. 0.36 ± 0.03 in EGF treated (P = 0.03)].

BBM enrichment and enzyme activity. As we have shown previously (29, 30, 39), BBMs isolated from control or EGF-treated rats were 8- to 10-fold enriched, as assessed by the activity of BBM-specific enzymes (leucine aminopeptidase and alkaline phosphatase). Cross contamination with BLM was minimal (less than 1.5-fold), as assessed by the activity of the BLM-specific enzyme Na+-K+-ATPase.

Effect of weaning and EGF on BBM transport activity. Na-Pi cotransport was significantly higher in weaned than in suckling rats [232 ± 16 vs. 130 ± 9 pmol 32Pi · 10 s-1 · mg BBM protein-1 (P < 0.01)] (Fig. 1). Chronic (36-h) EGF treatment of both suckling and weaned rats caused a significant decrease in BBM Na-Pi cotransport activity [130 ± 9 in control vs. 104 ± 7 pmol 32Pi · 10 s-1 · mg BBM protein-1 in EGF-treated suckling rats (P < 0.05) and 232 ± 16 in control vs. 145 ± 9 pmol 32Pi · 10 s-1 · mg BBM protein-1 in EGF-treated weaned rats (P < 0.01)] (Fig. 1). Acute (4-h) EGF treatment of weaned rats had no effect on BBM Na-Pi cotransport activity [248 ± 25 vs. 224 ± 22 pmol 32Pi · 10 s-1 · mg BBM protein-1 in EGF treated (P = NS)]. The effect of EGF on Na-Pi cotransport was specific and selective as EGF had no effect on Na-glucose cotransport in suckling rats [48 ± 6 in control vs. 52 ± 6 pmol [3H]glucose · 10 s-1 · mg BBM protein-1 in EGF treated (P = NS)] and weaned rats [61 ± 7 in control vs. 66 ± 5 pmol [3H]glucose · 10 s-1 · mg BBM protein-1 in EGF treated (P = NS)] (Fig 2).


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Fig. 1.   Effect of epidermal growth factor (EGF) on Na-Pi cotransport. Both suckling (12 day old) and weaned (24 day old) rats were subcutaneously injected with EGF; (10 mg/100 g body wt) or vehicle every 12 h for 4 doses, including a dose 2 h before they were killed. Brush border membrane vesicles (BBMV) were isolated by magnesium precipitation method, and 32Pi uptake was measured by Millipore filtration method; n = 5 individual BBM in each group.


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Fig. 2.   Effect of EGF on Na-glucose cotransport. Both suckling (12 day old) and weaned (24 day old) rats were subcutaneously injected with EGF (10 µg/100 g body wt) or vehicle every 12 h for 4 doses, including a dose 2 h before they were killed. BBMVs were isolated by magnesium precipitation method, and D-[3H]glucose was measured by Millipore filtration method; n = 5 individual BBM in each group. NS, not significant.

Effect of weaning and EGF on renal cortical BBM NaPi-2 protein level. Western blot analysis of BBM proteins isolated from suckling and weaned rats was performed using an antiserum raised against a NH2-terminal peptide of NaPi-2 protein (18). As shown in Fig. 3, BBM NaPi-2 protein abundance is clearly higher in weaned compared with suckling rats. EGF administration causes a significant decrease in BBM NaPi-2 protein abundance in both suckling and weaned rats (Fig. 3). Additional Western blots for the BBM Na-Si transport protein NaSi-1 and beta -actin were performed. EGF caused similar significant decreases in NaPi-2 and NaSi-1 protein abundance, whereas EGF caused a slight increase in beta -actin abundance (Table 1).


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Fig. 3.   Effect of EGF on BBM NaPi-2 protein abundance. Suckling (12 day old) and weaned (24 day old) rats were treated with EGF or vehicle. Renal cortical BBMVs were isolated and Western blot analysis was performed as described. Top: blots were probed with an NaPi-2 antibody. Each lane contained 37.5 mg of BBM protein. Bottom: densitometric data show that EGF causes a decrease in BBM NaPi-2 abundance in both suckling and weaned rats. BBM NaPi-2 abundance is, however, higher in weaned than in suckling rats; n = 8 individual BBM in each group.

                              
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Table 1.   Brush border membrane Western blot densitometry in weaned rats

Effect of weaning and EGF on NaPi-2 mRNA. Northern blot analysis of total RNA isolated from both suckling and weaned rats is shown in Fig. 4. Hybridization with NaPi-2 cDNA probe clearly demonstrates a significant increase of NaPi-2 mRNA abundance in weaned rats. Rehybridization with beta -actin probe indicates that the increase in NaPi-2 mRNA in weaned rats is not due to unequal RNA loading on the gels. The abundance of NaPi-2 mRNA relative to beta -actin mRNA, determined by densitometric analysis, indicates that NaPi-2 mRNA is increased by approximately twofold in weaned rats. Northern blot analysis of total RNA isolated from both control and EGF-treated rats is shown in Fig. 5. Hybridization with NaPi-2, beta -actin, and GAPDH probes indicates that EGF treatment is not associated with a decrease in NaPi-2 mRNA.


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Fig. 4.   Northern blot analysis of total RNA isolated from suckling and weaned rats. After formaldehyde-agarose gel electrophoresis, RNA was transferred to nylon membranes and hybridized sequentially with NaPi-2 and beta -actin probes. mRNA for Na-Pi cotransport and beta -actin were quantitated by densitometry. Weaned rats had a 2-fold increase in NaPi-2 mRNA. Ratios of NaPi-2 to beta -actin are shown as means  ±  SE; n = 5 individual BBMs in each group. P < 0.01.


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Fig. 5.   Northern blot analysis of total RNA isolated from control and EGF-treated weaned rats. After formaldehyde-agarose gel electrophoresis, RNA was transferred to nylon membranes and hybridized sequentially with NaPi-2 and glyceraldehyde-3-phosphate dehydrogenose (GAPDH) cDNA probes. mRNAs for Na-Pi cotransporter and GAPDH were quantitated by densitometry. EGF had no effect on NaPi-2 mRNA. Ratios of NaPi-2 to GAPDH are shown as means ± SE; n = 12 individual BBMs in each group. P = NS.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The present study examined the maturation of the Na-Pi cotransport system and the effect of EGF on Pi transport in both suckling and weaned rats. The data demonstrate that weaned (24 day old) rats had a significant increase in Na-Pi cotransport activity compared with suckling (12 day old) rats. The increase in Na-Pi cotransport activity is paralleled by increased abundance of NaPi-2 protein and NaPi-2 mRNA. EGF administration caused a significant increase in the fractional excretion of Pi (i.e., a decrease in the tubular reabsorption of Pi) and a decrease in BBM Na-Pi cotransport activity, which was paralleled by a significant decrease in NaPi-2 protein abundance with no change in NaPi-2 mRNA.

EGF has been shown to modulate membrane transport in both the kidney and the gastrointestinal tract. It upregulates jejunal BBM amino acid transport activity (43) and also increases jejunal absorption of H2O, Na+, Cl-, and glucose (37). EGF stimulates Pi transport in LLC-PK1 cells (23) and in isolated perfused rabbit proximal tubules (40). On the other hand, we have demonstrated that EGF inhibits Na-Pi cotransport in OK cells (3). The in vivo effect of EGF, however, is not known. The present study was therefore designed to examine the in vivo effect of EGF on Na-Pi cotransport in the rat. These experiments indeed demonstrate that EGF-treated rats have a higher fractional excretion of Pi, which is paralleled with a decrease in Na-Pi cotransport activity in BBMVs isolated from both suckling and weaned rats. Furthermore, the EGF-induced inhibition of BBM Na-Pi cotransport activity is mediated by decreased expression of NaPi-2 protein at the level of the proximal tubular apical BBM, in the absence of a decrease in renal cortical NaPi-2 mRNA abundance. As shown in Table 1, EGF causes significant decreases in NaPi-2 and NaSi-1 protein abundance but causes a slight increase in beta -actin abundance. The reasons for the slight increase in beta -actin abundance are not certain but may include slightly higher loading with EGF samples, which would then make the changes in NaPi-2 more significant, or slightly higher BBM purification from EGF-treated animals, which would also make the changes in NaPi-2 even more significant. A true effect of EGF to cause an increase in beta -actin protein abundance is also possible.

Although the in vivo inhibitory effect of EGF on Na-Pi cotransport in the rat is similar to what we have reported in the OK cell (3), the cellular mechanisms seem to be somewhat different. In OK cells, the inhibition of Na-Pi cotransport by EGF was associated with a decrease in Na-Pi mRNA (3), whereas in the rat EGF does not cause a change in Na-Pi mRNA levels. Although the reasons are not known at the present time, it may be related to differences in EGF-activated signaling mechanisms (41), which eventually modulate NaPi-2 protein expression at the level of the apical BBM by transcriptional or posttranscriptional mechanisms.

In the present study, we have demonstrated that 24-day-old weaned rats have a higher BBM Na-Pi cotransport activity compared with that of 12-day-old suckling rats. It has been previously shown that proximal tubular volume reabsorption is lower in the neonate than in the adult (1, 6, 46). The lower rate of volume reabsorption in the neonate is associated with a lower rate of net tubular glucose (6, 9, 46) and bicarbonate (6, 46) transport activity and decreases in BBM Na+-glucose cotransport (9), Na+/H+ antiport (7, 8, 10), and H+-ATPase (7) activities, as well as decreases in BLM Na+-K+-ATPase (1, 2, 44, 47) and Na+-3HCO-3 symporter (8) activities.

Previously, it was shown that 21-day-old rats have a higher Na-Pi cotransport activity compared with that of 14-day-old rats (28). In this study, however, kinetic analysis of Na-Pi cotransport revealed that the higher uptake rate of Pi observed in the 21-day-old rats was mediated by a decrease in the Km for Pi rather than a change in Vmax, which suggested that an increase in the affinity rather than an increase in the number of Na-Pi cotransporters mediated the enhanced Pi uptake in 21-day-old rats. Our study, on the other hand, indicates that the higher Na-Pi cotransport in 24-day-old rats is mediated by an increased number of Na-Pi cotransporters, which is, in turn, mediated by increased abundance of NaPi-2 mRNA. At the present time, we cannot explain the difference in our study versus the earlier study. A difference in dietary Pi intake between suckling and weaned rats could well explain these differences (31); however, an earlier study clearly demonstrated that, in contrast to the adult animals, in the neonate the Vmax of Na-Pi cotransport activity is not modulated by a low-Pi diet (35). The phosphate content of rat milk was not measured; but in a previous study it was found that the inorganic phosphorus content of rat milk is 100 mg/100 ml (0.1%) at day 15 of lactation (35a). Since the solid content of rat milk at the same period of lactation is 24%, the inorganic phosphorus is 0.4% of total solid weight. Weaned rats received regular chow with a phosphate content of 0.65%. Serum phosphate level was slightly higher in suckling compared with weaned rats (11.2 ± 0.5 vs. 10.4 ± 0.23 mg/dl, P = 0.24), and chronic administration of EGF only caused a small and nonsignificant increase in serum Pi concentration. Although an increase in serum Pi (due to increased gastrointestinal absorption or increased bone release) could cause a secondary decrease in renal Pi transport, it is quite unlikely that such a small increase in serum Pi would account for the changes in Na-Pi transport that we have seen. In addition, we have also seen a direct effect of EGF, in the absence of other systemic factors, cause a decrease in OK cell Na-Pi transport (3).

There were no short-term effects on Na-Pi transport after a 4-h treatment with EGF. The discrepancy between the acute effect in these experiments and the reported EGF-induced acute increase of Na-Pi transport in isolated perfused rabbit proximal tubule cannot be explained (40).

It is interesting that EGF causes an increase in serum creatinine of suckling rat and an opposite effect in the weaned rat. However, we only measured creatinine and Pi in spot urine at the time the rats were killed to calculate fractional excretion of phosphate and correlate it with BBM Na-Pi transport activity. We did not attempt to measure glomerular filtration rate (GFR) because this was not the aim of this study. We should note that our previous study in OK cells demonstrates a direct effect of EGF to inhibit Na-Pi transport independently of changes in hemodynamics or GFR.

Recent studies have shown that thyroid hormone may play an important role in the maturation of the Na-Pi cotransporter, as there is an increase in thyroid hormone level in 21-day-old vs. 14-day-old rats and administration of thyroid hormone to 14-day-old rats results in markedly increased Na-Pi cotransport activity and parallel increases in Na-Pi protein and mRNA abundance (21, 22). These results are in agreement with the findings of the current study.

Interestingly, although there is an initial maturational increase in Na-Pi cotransport activity, later there is a gradual age-related decrease in Na-Pi cotransport activity (14, 15, 16, 26, 30). Recent studies indicate that this biphasic regulation of Na-Pi cotransport activity may be mediated by complex cellular mechanisms. One study found that the increase in Na-Pi cotransport activity in 3-wk-old rats, when compared with that in rats older than 12 wk, may be mediated by unique mRNA transcripts able to encode for a Na-Pi protein homologous to, but distinct from, NaPi-2 (48). On the other hand, in a recent study from our laboratory we reported that compared with rats 3-6 mo. old, in rats older than 12 mo the decrease in Na-Pi cotransport activity was associated with parallel decreases in NaPi-2 protein and NaPi-2 mRNA abundance (49).

In summary, we have demonstrated that Na-Pi cotransport activity is higher in weaned than in suckling rats and that it is mediated by higher NaPi-2 protein and mRNA abundance. EGF inhibits Na-Pi cotransport activity in both the suckling and weaned rats. Although EGF causes a decrease in NaPi-2 protein abundance, there is no change in NaPi-2 mRNA abundance, which suggests that the inhibitory effect of EGF on Na-Pi cotransport may be mediated by posttranscriptional mechanisms.

    ACKNOWLEDGEMENTS

The authors thank Drs. Jurg Biber and Heini Murer for providing Na-Pi probes, Myrna Gonzales and Paul Wilson for technical assistance, and Molly West, Jill Fauss, and Teresa Autrey for secretarial assistance.

    FOOTNOTES

These studies were supported by the Dept. of Pediatrics at the Univ. of Texas Health Science Center at San Antonio and by the Medical Research Service of the Department of Veterans Affairs (Merit Review) and by the National Kidney Foundation. H. K. Zajicek was supported by National Institutes of Health Research Service Award 1F-32-DK-09689-01.

Address for reprint requests: M. Arar, 7703 Floyd Curl Dr., San Antonio, TX 78284-7813.

Received 24 September 1997; accepted in final form 18 September 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Aperia, A., and L. Larsson. Induced development of proximal tubular Na-K-ATPase, basolateral cell membrane and fluid reabsorption. Acta. Physiol. Scand. 121: 133-141, 1984[Medline].

2.   Aperia, A., L. Larsson, and R. Zetterstrom. Hormonal induction of Na-K-ATPase in developing proximal tubular cells. Am. J. Physiol. 241 (Renal Fluid Electrolyte Physiol. 10): F356-F360, 1981[Medline].

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