1 Department of Pediatrics, 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
NaPi-2 mRNA; NaPi-2 protein; phosphate transporter maturation
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 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.
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
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 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 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.
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
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
, and glucose from the jejunum
(37).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-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.
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.
-actin, were labeled by random priming (Pharmacia) using [
-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
-actin mRNA.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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).
View larger version (18K):
[in a new window]
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.
View larger version (17K):
[in a new window]
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 -actin were
performed. EGF caused similar significant decreases in NaPi-2 and
NaSi-1 protein abundance, whereas EGF caused a slight increase in
-actin abundance (Table 1).
|
|
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 -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
-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,
-actin, and GAPDH probes indicates that EGF treatment is not
associated with a decrease in NaPi-2 mRNA.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
-actin abundance. The reasons for the slight increase in
-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
-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+-3HCO3 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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].
3.
Arar, M.,
M. Baum,
J. Biber,
H. Murer,
and
M. Levi.
Epidermal growth factor inhibits Na-Pi cotransport and mRNA in OK cells.
Am. J. Physiol.
268 (Renal Fluid Electrolyte Physiol. 37):
F309-F314,
1995
4.
Avner, A. D.,
and
W. E. Sweeney, Jr.
Polypeptide growth factors in metanephric growth and segmental nephron differentiation.
Pediatr. Nephrol.
4:
372-377,
1990[Medline].
6.
Baum, M.,
and
R. Quigley.
Prenatal glucocorticoids stimulate neonatal juxtamedullary proximal convoluted tubule acidification.
Am. J. Physiol.
261 (Renal Fluid Electrolyte Physiol. 30):
F746-F752,
1991
7.
Baum, M.
Developmental changes in rabbit juxtamedullary proximal convoluted tubule acidification.
Pediatr. Res.
31:
411-414,
1992[Abstract].
8.
Baum, M.
Neonatal rabbit juxtamedullary proximal convoluted tubule acidification.
J. Clin. Invest.
85:
499-506,
1990[Medline].
9.
Beck, J. C.,
M. S. Lipkowitz,
and
R. G. Abramson.
Characterization of the fetal glucose transporter in rabbit kidney.
J. Clin. Invest.
82:
379-387,
1990.
10.
Beck, J. C.,
M. S. Lipkowitz,
and
R. G. Abramson.
Ontogeny of Na/H antiporter activity in rabbit renal BBM vesicles.
J. Clin. Invest.
87:
2067-2076,
1991[Medline].
11.
Bessey, O. A.,
O. H. Lowry,
and
M. J. Brock.
A method for the rapid determination of alkaline phosphatase with five cubic millimeters of serum.
J. Biol. Chem.
164:
321-329,
1964.
12.
Breyer, M. D.,
R. Redha,
and
J. A. Breyer.
Segmental distribution of epidermal growth factor binding sites in rabbit nephron.
Am. J. Physiol.
259 (Renal Fluid Electrolyte Physiol. 28):
F553-F558,
1990
13.
Breyer, M. D.,
H. R. Jacobson,
and
J. A. Breyer.
Epidermal growth factor inhibits the hydrosmotic effect of vasopressin in the isolated perfused rabbit cortical collecting tubule.
J. Clin. Invest.
82:
1313-1320,
1988[Medline].
14.
Caverzasio, J.,
J. P. Bonjour,
and
H. Fleisch.
Tubular handling of Pi in young growing and adult rats.
Am. J. Physiol.
242 (Renal Fluid Electrolyte Physiol. 11):
F705-F710,
1982[Medline].
15.
Caverzasio, J.,
H. Murer,
H. Fleisch,
and
J. P. Bonjour.
Pi transport in brush border membrane vesicles isolated from renal cortex of young growing and adult rats.
Pflügers Arch.
394:
217-221,
1982[Medline].
16.
Chen, M. L.,
R. S. King,
and
H. J. Armbrecht.
Sodium-dependent phosphate transport in primary cultures of renal tubule cells from young and adult rats.
J. Cell. Physiol.
143:
488-493,
1996.
17.
Chomezynski, P.,
and
N. Sacchi.
Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction.
Anal. Biochem.
162:
156-159,
1987[Medline].
18.
Custer, M.,
M. Lotscher,
J. Biber,
H. Murer,
and
B. Kaissling.
Expression of Na-Pi cotransport in rat kidney: localization by RT-PCR and immunohistochemistry.
Am. J. Physiol.
266 (Renal Fluid Electrolyte Physiol. 35):
F767-F774,
1994
19.
Cybulsky, A. V.,
P. R. Goodyer,
and
A. J. McTavish.
Epidermal growth factor receptor activation in developing rat kidney.
Am. J. Physiol.
267 (Renal Fluid Electrolyte Physiol. 36):
F428-F436,
1994
20.
Dawson, T. P.,
R. Gandhi,
M. Le Hir,
and
B. Kaissling.
Ecto 5'-nucleotidase: localization by light microscopic histochemistry and immunohistochemistry methods in the rat kidney.
J. Histochem. Cytochem.
37:
39-47,
1989[Abstract].
21.
Euzet, S.,
M. Lelievre-Pegorier,
and
C. Merlet-Benichou.
Effect of 3,5,3'-triiodothyronine on maturation of rat renal phosphate transport: kinetic characteristics and phosphate transporter messenger ribonucleic acid and protein abundance.
Endocrinology
137:
3522-3530,
1996[Abstract].
22.
Euzet, S.,
M. Lelievre-Pegorier,
and
C. Merlet-Benichou.
Maturation of rat renal phosphate transport: effect of triiodothyronine.
J. Physiol. (Lond.)
488:
449-457,
1995[Abstract].
23.
Goodyer, P. R.,
Z. Kachra,
C. Bell,
and
R. Rozen.
Renal tubular cells are potential targets for epidermal growth factor.
Am. J. Physiol.
255 (Renal Fluid Electrolyte Physiol. 24):
F1191-F1196,
1988
24.
Haase, W.,
A. Schafer,
H. Murer,
and
R. Kinne.
Studies on the orientation of BBM vesicles.
Biochem. J.
172:
57-62,
1979.
25.
Harris, R. C.
Response of rat inner medullary collecting duct to epidermal growth factor.
Am. J. Physiol.
256 (Renal Fluid Electrolyte Physiol. 25):
F1117-F1124,
1989
26.
Kiebzak, G. M.,
and
B. Sacktor.
Effect of age on renal conservation of phosphate in the rat.
Am. J. Physiol.
251 (Renal Fluid Electrolyte Physiol. 20):
F399-F407,
1986[Medline].
27.
Laemmli, U. K.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature (Lond.)
227:
680-685,
1970[Medline].
28.
Lelievre-Pegorier, M.,
T. Jean,
P. Ripoche,
and
P. Poujeol.
Transport of phosphate, D-glucose, and L-valine in newborn rat kidney brush border.
Am. J. Physiol.
245 (Renal Fluid Electrolyte Physiol. 14):
F367-F373,
1983[Medline].
29.
Levi, M.,
B. M. Baird,
and
P. V. Wilson.
Cholesterol modulates rat renal brush border membrane phosphate transport.
J. Clin. Invest.
85:
231-237,
1990[Medline].
30.
Levi, M.,
D. M. Jameson,
and
B. W. van der Meer.
Role of BBM lipid composition and fluidity in impaired renal Pi transport in aged rat.
Am. J. Physiol.
256 (Renal Fluid Electrolyte Physiol. 25):
F85-F94,
1989
31.
Levi, M.,
M. Lotscher,
V. Sorribas,
M. Custer,
M. Arar,
B. Kaissling,
H. Murer,
and
J. Biber.
Cellular mechanisms of acute and chronic adaptation of rat renal Pi transporter to alterations in dietary Pi.
Am. J. Physiol.
267 (Renal Fluid Electrolyte Physiol. 36):
F900-F908,
1994
32.
Lowry, O. H.,
N. J. Rosebrough,
A. L. Farr,
and
R. J. Randall.
Protein measurements with the Folin phenol reagent.
J. Biol. Chem.
193:
265-275,
1951
33.
Magagnin, S.,
A. Werner,
D. Markovich,
V. Sorribas,
G. Stange,
J. Biber,
and
H. Murer.
Expression cloning of human and rat renal cortex Na/Pi cotransport.
Proc. Natl. Acad. Sci. USA
90:
5979-5983,
1993[Abstract].
35.
Neiberger, R. E.,
M. Barac-Nieto,
and
A. Spitzer.
Renal reabsorption of phosphate during development: transport kinetics in BBMV.
Am. J. Physiol.
257 (Renal Fluid Electrolyte Physiol. 26):
F268-F274,
1989
35a.
Nicholas, K. R.,
and
A. E. Hartmann.
Milk secretion in rat: progressive changes in milk composition during lactation and weaning and the effect of diet.
Comp. Biochem. Physiol.
98A:
535-542,
1991.
36.
Nouwen, E. J.,
and
M. E. De Broe.
EGF and TGFg in the human kidney: identification of octopal cells in the collecting duct.
Kidney Int.
45:
1510-1521,
1994[Medline].
37.
Opelta-Madsen, K.,
J. Harden,
and
D. G. Gall.
Epidermal growth factor upregulates intestinal electrolyte and nutrient transport.
Am. J. Physiol.
260 (Gastrointest. Liver Physiol. 23):
G807-G814,
1991
38.
Pizurki, L.,
R. Rizzoli,
J. Caverzasio,
and
J. P. Bonjour.
Effect of transforming growth factor- and parathyroid hormone-related protein on phosphate transport in renal cell.
Am. J. Physiol.
259 (Renal Fluid Electrolyte Physiol. 28):
F929-F935,
1990
39.
Prabhu, S.,
M. Levi,
V. Dwarakanath,
M. Arar,
J. Biber,
H. Murer,
and
M. Baum.
Effect of glucocorticoids on neonatal rabbit renal cortical sodium-inorganic phosphate messenger RNA and protein abundance.
Pediatr. Res.
41:
20-24,
1997[Abstract].
40.
Quigley, R.,
and
M. Baum.
Effects of epidermal growth factor and transforming growth factor- on rabbit proximal tubule solute transport.
Am. J. Physiol.
266 (Renal Fluid Electrolyte Physiol. 35):
F459-F465,
1994
41.
Quigley, R.,
D. A. Kennerly,
J. N. Sheu,
and
M. Baum.
Stimulation of proximal convoluted tubule phosphate transport by epidermal growth factor: signal transduction.
Am. J. Physiol.
269 (Renal Fluid Electrolyte Physiol. 38):
F339-F344,
1995
42.
Rall, L. B.,
R. J. Crawford,
J. Scott,
G. I. Bell,
J. D. Penschow,
H. D. Niall,
and
J. P. Coghlan.
Mouse prepro-epidermal growth factor synthesis by the kidney and other tissues.
Nature (Lond.)
313:
228-231,
1985[Medline].
43.
Salloum, R. M.,
B. R. Stevens,
G. S. Schults,
and
W. W. Souba.
Regulation of small intestinal glutamine transport by epidermal growth factor.
Surgery
113:
552-559,
1993[Medline].
44.
Schmidt, U.,
and
M. Horster.
Na-K-activated ATPase: activity maturation in rabbit nephron segments dissected in vitro.
Am. J. Physiol.
233 (Renal Fluid Electrolyte Physiol. 2):
F55-F60,
1977[Medline].
45.
Schoner, W.,
C. von Illberg,
and
R. Kramer.
On the mechanism of Na+- and K+-stimulated hydrolysis of adenosine triphosphate.
Eur. J. Biochem.
1:
334-343,
1967[Medline].
46.
Schwartz, G. J.,
and
A. P. Evan.
Development of solute transport in rabbit proximal tubule. I. HCO3 and glucose absorption.
Am. J. Physiol.
245 (Renal Fluid Electrolyte Physiol. 14):
F382-F390,
1983[Medline].
47.
Schwartz, G. J.,
and
A. P. Evan.
Development of solute transport in rabbit proximal tubule. III. Na-K-ATPase activity.
Am. J. Physiol.
246 (Renal Fluid Electrolyte Physiol. 15):
F845-F852,
1984[Medline].
48.
Silverstein, D.,
M. Barac-Nieto,
and
A. Spitzer.
Mechanism of renal phosphate retention during growth.
Kidney Int.
49:
1023-1026,
1996[Medline].
49.
Sorribas, V.,
M. Lotscher,
J. Loffing,
J. Biber,
B. Kaissling,
H. Murer,
and
M. Levi.
Cellular mechanisms of the age-related decrease in renal phosphate reabsorption.
Kidney Int.
50:
855-863,
1996[Medline].
51.
Stanton, R. C.,
and
J. L. Seifter.
Epidermal growth factor rapidly activates the hexose monophosphate shunt in kidney cells.
Am. J. Physiol.
253 (Cell Physiol. 22):
C267-C271,
1988.
52.
Towbin, H.,
T. Staehelin,
and
J. Gordon.
Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
Proc. Natl. Acad. Sci. USA
76:
4350-4354,
1979[Abstract].
53.
Vehaskari, V. M.,
K. S. Hering-Smith,
D. W. Moskowitz,
I. D. Weiner,
and
L. L. Hamm.
Effect of epidermal growth factor on sodium transport in the cortical collecting tubule.
Am. J. Physiol.
256 (Renal Fluid Electrolyte Physiol. 25):
F803-F809,
1989