Department of Pediatrics, Louisiana State University Medical Center in New Orleans, New Orleans, Louisiana 70112
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ABSTRACT |
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To assess the
role of distal nephron apical Na channel (ENaC) gene expression in Na
wasting by the immature kidney, ENaC -,
-, and
-subunit mRNA
levels were examined in the rat by RT-PCR. In microdissected nephron
segments, all three ENaC subunit mRNAs were detected in the distal
convoluted tubule, connecting tubule, cortical collecting duct, and
outer medullary collecting duct. The inner medullary collecting duct
and all other nephron segments were consistently negative. The mRNA
levels were quantified in kidneys at different developmental stages by
multiplex RT-PCR with "primer dropping," with endoplasmic
reticulum-specific cyclophilin mRNA as an internal standard. All three
ENaC mRNA levels were low or undetectable on gestational
day 16 and only slightly higher 3 days
before birth. A sharp rise was observed between 3 days before and
1-3 days after birth; the levels at postnatal
days 1-3 were already similar to
those of adult kidneys. The results suggest that ENaC subunit gene
expression is not a limiting factor in the full-term newborn rat
kidney, but low levels of expression may limit distal Na absorption in
more immature kidneys, such as those of very premature human infants.
sodium transport; collecting duct; renal ontogeny; multiplex reverse transcription-polymerase chain reaction; capillary electrophoresis with laser-induced fluorescent detection
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INTRODUCTION |
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PREMATURE INFANTS have a limited ability to regulate Na homeostasis, partly because of renal Na wasting, which may lead to the so-called late hyponatremia of prematurity (1, 23). The renal site of inappropriate Na loss has not been unequivocally determined, but there is indirect evidence to suggest that distal nephron Na reabsorption may be inadequate (2, 19). We previously showed by in vitro microperfusion that rabbit cortical collecting ducts (CCD) have a low Na transport rate during the first 2 wk of life (24), suggesting that the functional characteristics of the CCD may contribute to renal Na wasting by immature kidneys. Furthermore, patch-clamp studies have demonstrated a paucity of functional Na channels in the apical membrane of the CCD of the newborn rabbit (20).
The apical low-conductance, highly selective amiloride-sensitive Na
channel is believed to be the rate-limiting step in CCD Na transport.
This channel, termed ENaC, was recently cloned from rat colon and shown
to consist of at least three subunits, ,
, and
, all of which
were required for full Na conductance in the oocyte expression system.
The subunits share ~35% identity and appear to be products of
different but related genes, which are part of a new gene family (4, 5,
14, 15). The presence of ENaC has been confirmed in kidney, colon,
lung, and salivary and sweat glands (7, 18). Although it is found
predominantly in the distal nephron, the exact extent of ENaC gene
expression along the collecting duct has not been unequivocally
established (6, 7, 25).
The current study was undertaken to investigate the role of ENaC gene expression in the maturation of distal nephron Na transport. When studied in microdissected rat nephron segments by RT-PCR, the three ENaC subunit mRNA species colocalized exclusively to the distal nephron segments from the distal convoluted tubule (DCT) to the outer medullary collecting duct (OMCD). Although relative quantification by multiplex RT-PCR revealed low mRNA levels of all three subunits in late fetal kidney, adult expression levels were already reached within 24-72 h after birth. These findings suggest that the ENaC subunit gene expression is a potentially limiting factor in Na transport only in the very immature kidney.
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METHODS |
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Animals and tissue preparation. Sprague-Dawley rats were used for all experiments. For microdissection of nephron segments, rats weighing 120-160 g were anesthetized with pentobarbital sodium (50 mg/kg ip), and left kidneys were perfused retrograde via the abdominal aorta with dissection solution (see below) containing 2 mg/ml collagenase (Boehringer Mannheim) and 1 mg/ml BSA. Sagittal kidney sections were incubated for 15-30 min at 37°C in the same solution with the addition of 0.2 mg/ml protease (Pronase E, Sigma Chemical) during continuous bubbling with 100% O2. Single nephron segments were dissected freehand in ice-cold dissection solution containing (in mM) 135 NaCl, 5 KCl, 1 Na2HPO4, 3 sodium acetate, 1.2 Na2SO4, 2.5 CaCl2, 1.2 MgSO4, 5 HEPES, 5.5 glucose, and 10 vanadyl ribonucleoside complex (VRC; Life Technologies), a potent RNase inhibitor. VRC was centrifuged and filtered before use to remove precipitates. The pH of the solution was adjusted to 7.4 with NaOH. After dissection, nephron segments were rinsed twice in the same solution without VRC and transferred in 2-µl aliquots to tubes containing 6.6 µl of 2% Triton X-100 (Sigma Chemical) to permeabilize the cells, 1.4 U/µl placental RNase inhibitor (Boehringer Mannheim), and 5 mM dithiothreitol (Sigma Chemical). A total tubule length of 2-3 mm (usually 2 segments) or two to four glomeruli were placed in each tube. The tubes were frozen on dry ice until they were used for RT within 4 h.
Whole kidneys at different developmental stages were harvested for RNA extraction. Fetal kidneys were obtained from timed pregnancies on gestational day 16 (E16) or 19 (E19). The mothers were anesthetized with pentobarbital sodium, and fetal kidneys were quickly removed and snap frozen in liquid nitrogen. Postnatal kidneys were obtained on postnatal days 1-3 (D1-3) and 14 (D14) and from adult rats (120-160 g body wt). The kidneys were quickly removed under anesthesia without perfusion and snap frozen in liquid nitrogen. Tissue was stored atPrimer design.
Rat ENaC (rENaC) -,
-, and
-subunit PCR primer sets were
designed with the aid of computer software (Wisconsin Sequence Analysis
Package, Genetics Computer Group) from published rENaC subunit cDNA
sequences (GenBank) and were selected from the nonhomologous regions on
the basis of product size and optimal annealing temperature (56-59°C). The
-,
-, and
-subunit primers spanned
base pairs 1424-1915 (PCR product size 492 bp), 1295-1685
(391 bp), and 341-610 (270 bp), respectively, and were synthesized
commercially (Integrated DNA Technologies). When tested with rat
genomic DNA (Clontech Laboratories) as a template, the only PCR product
obtained with any of the primers was an occasional
large-molecular-weight (>1,000-bp) DNA, indicating that the primers
spanned an intron. RT-PCR with each primer pair, with rat kidney RNA as
a template, yielded a single DNA product of expected size that was
sequenced to confirm its identity. The DNA sequencing was performed
commercially with an automated sequencer by use of dye deoxy terminator
cycle sequencing chemistry (ACGT). Primers for cDNA of a housekeeping
gene, the endoplasmic reticulum-specific cyclophilin (10), were used to produce an internal standard; these primers (Ambion) spanned base pairs
295-510 and produced the expected 216-bp DNA product. In preliminary experiments, cyclophilin mRNA in the kidney showed little
variation with developmental stage of the kidney (data not shown). The
use of cyclophilin as an internal normalizing standard for quantitative
RT-PCR has been validated (3, 16).
RT-PCR of microdissected nephron segments.
The method of Moriyama et al. (17) with slight modifications was used
for qualitative localization of rENaC subunit mRNAs. The tubule
segments frozen in permeabilizing solution were thawed, and RT was
carried out at 42°C for 50 min in 20 µl of solution containing
11.5 U of avian myeloblastoma virus reverse transcriptase (Stratagene),
0.25 µg of oligo(dT) primer, 50 nmol of each deoxynucleotide triphosphate (dNTP), 50 U of placental RNase inhibitor, 5 mM
MgCl2, and the proprietary buffer
(all from Promega). Avian myeloblastoma virus reverse transcriptase was
then heat inactivated at 95°C for 5 min. The cDNA was stored at
20°C until it was used for PCR.
Isolation and RT of whole kidney RNA.
For quantitative analysis, total RNA was extracted from fetal and
postnatal kidneys. Fetal kidneys from each pregnant animal (usually
from 6 or 7 fetuses) were pooled, and the two kidneys from each newborn
and 14-day-old animal were pooled; single kidneys were taken from adult
rats. Total RNA was isolated with the Ultraspec RNA system (Biotecx
Laboratories) and treated with DNase I (Boehringer Mannheim), 0.25 U/mg
tissue, for 15 min at 37°C. DNase I was removed by
phenol-chloroform-isoamyl alcohol extraction before ethanol precipitation of total RNA with 0.5 M ammonium acetate for 15 min at
20°C. After centrifugation the pellet was washed with 70%
ethanol, air dried, and resuspended in water. RNA concentration was
measured by ultraviolet absorbance at 260 nm, and 100 ng of total RNA
were reverse transcribed as described above for nephron segments,
except the total volume was 10 µl. cDNA was stored at
20°C
until it was used for multiplex PCR.
Multiplex PCR with "primer dropping."
To minimize problems inherent in the quantification of mRNA with
RT-PCR, primer-dropping multiplex PCR with an internal
standard was employed (26). Preliminary experiments were conducted to determine the optimal number of PCR cycles for rENaC -,
-, and
-subunits, as well as for the internal standard cyclophilin. Figure
1 illustrates the
relationships between cycle numbers and product yield, showing that
below 30 cycles the plateau (saturation) was not reached with any of
the primers. The PCR products, even at these relatively low cycle
numbers, were easily detected by the method used (see below).
Furthermore, adding a single cycle increased the yield approximately
twofold as predicted (data not shown). On the basis of the calculated
slopes and intercepts in Fig. 1, it was estimated and confirmed
experimentally that, with adult rat kidney cDNA as the template,
comparable quantities of DNA were produced with 26 PCR cycles for rENaC
-subunit, 24 cycles for rENaC
-subunit, 28 cycles for rENaC
-subunit, and 20 cycles for cyclophilin. These cycle numbers were
therefore used in all subsequent PCR reactions when all four primer
pairs were included in the same reaction tube (multiplex PCR). To
achieve the intended number of cycles, each primer pair was added
individually during the PCR at its predetermined time point (primer
dropping). DNA polymerase was added at the beginning of the first
extension segment (hot start). The final multiplex PCR reaction volume
was 30 µl, including 3 µl of cDNA, 1.5 U of DNA polymerase
(Taq2000), 1.5 U of
Taq Extender, 5 pmol of each primer, 6 nmol of each dNTP, and the proprietary buffer. After an initial melt at
94°C for 3 min, thermal cycling was carried out for 1 min at
94°C, 1 min at 56°C, and 1 min at 72°C, with a final
extension at 72°C for 7 min.
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Detection and quantification of PCR amplicons by capillary
electrophoresis with laser-induced fluorescence detection.
Capillary electrophoresis with laser-induced fluorescence detection
(CE-LIF) was based on the principle described by Fasco et al. (8).
Before analysis by CE-LIF, each PCR sample was desalted by float
dialysis in deionized, distilled (DD) water for 30 min with use of
0.025-µm VS filters (Millipore). Amplicons were separated and
quantified using a P/ACE 5000 capillary electrophoresis system equipped
with a laser-induced fluorescence detector (argon ion, 488/520 nm
excitation/emission) and System Gold software (Beckman Instruments).
The instrument was operated at constant temperature (25°C) in
reverse polarity with cathode and anode at the inlet and outlet
reservoirs, respectively. Run buffer containing TBE (89 mM
Tris · HCl, 89 mM boric acid, 2 mM EDTA), 0.5%
(wt/vol) hydroxypropylmethylcellulose, and 0.005% (vol/vol) SYBR Green I (Molecular Probes) was used as the catholyte and the anolyte. Before
each run the 100-µm-ID × 27-cm-long coated capillary (DB-1, J & W Scientific) was rinsed with run buffer at high pressure (20 psi) for
1 min. Sample was introduced by electrokinetic injection for 10 s at
3.0 kV. Double-stranded DNA fragments were separated by
electrophoresis at 8.1 kV (300 V/cm) for 8 min while fluorescence was
recorded at 520 nm. The capillary was rinsed with methanol and DD water
for 0.25 min each between samples. A double-stranded DNA standard
containing 100-, 200-, 350-, and 500-bp fragments (Bioventures) in DD
water was analyzed with each run to verify optimal instrument
performance. To quantify mRNA, the amount of each different amplicon
(cyclophilin, 216 bp; rENaC-, 270 bp; rENaC-
, 391 bp; rENaC-
,
492 bp) in each PCR sample was measured by integrating the fluorescence
peak areas. The relative amount of each rENaC subunit mRNA in each
sample was then determined as the ratio of the subunit amplicon peak
area to the amplicon peak area of cyclophilin.
Statistical analysis. Values are means ± SE. The levels of each specific rENaC subunit mRNA, expressed relative to cyclophilin mRNA, were compared between the groups with ANOVA, followed by the Tukey-Kramer multiple comparison test. P < 0.05 was considered significant.
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RESULTS |
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Localization of rENaC subunit mRNA species.
The following microdissected segments were studied by RT-PCR in adult
rats: blood vessels, glomerulus, proximal convoluted tubule (PCT),
proximal straight tubule (PST), thin limbs, medullary and cortical thin
ascending limbs, DCT/CNT, CCD, OMCD, and IMCD. Blood vessels were
dissected as bundles without separation of arteries and veins.
Ascending and descending thin limbs were not separated. Because it was
not possible to identify a clear transition from DCT to CNT, these
segments were treated as one; the dissected DCT/CNT usually included a
branch at the distal end. Inner and outer stripes of OMCD were not
separated. Because controversy exists regarding the presence of ENaC in
IMCD (6, 7, 25), the papilla was carefully excised and the dissected
papillary IMCDs were considered separately from the more proximal
initial IMCDs, which were dissected from the proximal one-third of the total IMCD length. At least four different specimens of each nephron segment were studied (Fig. 2).
All primer pairs produced a single DNA band of the expected size. All
segments were positive for cyclophilin. rENaC -,
-, and
-subunit mRNAs colocalized consistently to the following nephron
segments: DCT/CNT, CCD, and OMCD. The initial and papillary IMCDs, as
well as all other nephron segments and blood vessels (not shown), were
negative for all subunits, except an occasional (<10% of specimens)
very faint band with a single subunit.
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rENaC subunit mRNA levels during development.
ENaC -,
-, and
-subunit mRNA levels were determined in kidneys
at five different stages of maturation: E16, E19, D1-3, D14, and
adult. Figure 3 shows a typical CE-LIF
recording from each age group. The peaks of all three rENaC subunits
from the fetal E16 and E19 kidneys were small compared with those from the newborn kidneys (D1-3). There was no further increase during the postnatal period after D1-3. There appeared to be no
differences in the developmental patterns between the subunits, except
the rENaC
-subunit was not detected in any of the samples at E16. For statistical analysis, each specific rENaC subunit mRNA peak area
was normalized to the cyclophilin peak area obtained from the same
multiplex PCR reaction. Figure 4
illustrates the relative abundances of the rENaC
-subunit mRNA.
Although the mRNA was detected in all samples, the levels appeared very
low in the prenatal samples; however, the difference did not reach
statistical significance. The rENaC
-subunit mRNA levels are shown
in Fig. 5. At E16 there was no detectable
mRNA, and at E19 the level was still significantly lower than at
D1-3 or D14. The rENaC
-subunit mRNA is illustrated in Fig.
6. The level was measurable but very low at
E16 and significantly lower at both prenatal ages than at any of the
postnatal ages. All three rENaC subunit mRNA levels in the immediate
newborn period (D1-3) were already comparable to the levels in
adult kidneys. At D14 the levels were numerically higher than in
adults, but only the
-subunit mRNA reached a statistically
significant higher value. When the rENaC subunit mRNA abundances were
analyzed without normalization to cyclophilin, as the peak area of
specific mRNA per microgram of total RNA, a similar developmental
pattern for all subunits was observed (data not shown).
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DISCUSSION |
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Distal nephron Na reabsorption is critical for Na homeostasis and the
target for regulation by mineralocorticoids. The rate-limiting step in
transepithelial Na transport is believed to be the entry of Na into the
cell via the apical Na channel, ENaC. The present studies were
undertaken to investigate the role of ENaC gene expression in the
developmental regulation of distal nephron Na transport in the rat. The
main findings were 1) colocalization
of rENaC -,
-, and
-subunit mRNAs exclusively to the distal
nephron, 2) sharp developmental
increase in the mRNAs of all subunits between 72 h before birth and
24-72 h after birth, and 3) no
further increase in the subunit mRNA levels after the first 1-3
days of life.
The presence of ENaC in distal nephron segments, beginning with DCT, is consistent with electrophysiological studies documenting electrogenic Na transport in these segments (for review see Ref. 9). Whether the distribution of ENaC extends to the most distal nephron segment, the IMCD, is controversial. We were unable to detect rENaC mRNAs in the initial one-third or in the terminal (papillary) two-thirds of the IMCD. Others found ENaC mRNA along the IMCD by in situ RT-PCR in the mouse (6) and by Northern analysis in the rat (25). In agreement with our results, Duc et al. (7) did not detect the message beyond the OMCD by in situ hybridization. The reason for the discrepancies is not clear. Another, less selective, cGMP-gated cation channel has been described in the IMCD (6, 12, 21); hence, the presence of ENaC is not required to explain the Na transport in this segment. In support of the cGMP-gated channel mediating Na transport in the IMCD is the ability of atrial natriuretic peptide, via cGMP generation, to block the cation channel in IMCD cells (13).
The ENaC mRNA species were quantified in whole kidney RNA, because reproducible quantification of mRNA in single nephron segments is problematic, especially in the fetus and the newborn animal. The measured mRNA levels, therefore, reflect the total of all ENaC-positive distal nephron segments. We believe that the use of multiplex RT-PCR, with all templates coamplified in the same PCR reaction tube with the internal standard, reduced the inherent variability of PCR quantification. The primer-dropping method was used to avoid the plateau effect with any of the primer sets that otherwise might mask quantitative differences (26). Moreover, the sensitivity of the CE-LIF minimized the number of PCR cycles required, reducing the potential for nonlinear amplification.
Rat kidney ENaC subunit mRNA levels are clearly developmentally
regulated, as shown in Figs. 3-5. In the mature animal, ENaC -,
-, and
-subunit genes may be differentially regulated (18, 25).
In contrast, we found the developmental profiles for all three subunits
in the current study to be similar (although the values for the
-subunit did not reach statistical significance). The mRNA levels in
the fetal kidney were undetectable or low at E16 and only slightly
higher 3 days before birth, at E19. A sharp increase appears to occur
during the last 2 prenatal days and/or the first day(s) of
life. The signal for this increase is unknown. Further studies are
necessary to determine whether it is related to the physiological
changes occurring during the transition from intrauterine to
extrauterine life, such as increase in renal blood flow and glomerular
filtration rate. Another important finding was that during the first
24-72 h of life, the rENaC mRNA levels were already as high as
those in mature kidneys. They remained at the levels of mature kidneys
(or higher) at 14 days of age, suggesting that the genes were not just
transiently turned on during the perinatal period.
Na transport rate in the newborn rat distal nephron has not been directly measured; however, in the newborn rabbit CCD, we demonstrated very low active Na transport rates by in vitro microperfusion during the first 2 wk of life (24), and Satlin and Palmer (20) were unable to find many open Na channels by patch clamping. Rat and rabbit kidneys are relatively immature at birth, with nephrogenesis continuing well into the postnatal life. If it is assumed that distal nephron Na transport characteristics in the newborn rat and rabbit are similar, our results suggest that lack of ENaC gene expression is not responsible for the inefficient Na reabsorption. Possible underlying mechanisms include impaired translation of the mRNAs, impaired incorporation of synthesized proteins into the apical membrane, and effects of various regulatory pathways. The current study did not address the question of ENaC subunit protein synthesis. In the mature kidney, regulation of the channel activity may occur independently of changes in mRNA levels (18). ENaC regulatory mechanisms may involve recycling of channel subunits between the apical membrane and the subapical compartment (11) or regulatory degradation of the subunits via the ubiquitination pathway (22). Further studies are needed to determine whether the immature kidney lacks mechanisms for inserting ENaC proteins into the apical membrane and/or retaining inserted channels in the membrane. It is also possible that the channel is present in the apical membrane in the closed state, hence, undetectable by patch clamping, due to absence of proregulatory elements or presence of inhibitory factors.
Detailed information on the developmental pattern of ENaC gene expression in the human kidney is not available. Human kidneys are relatively well developed at birth, with cessation of new nephron formation at about gestational week 36. Only premature infants born before gestational week 35 appear to be at risk of developing renal Na wasting (1). It is conceivable that, at that stage of maturation, human kidneys resemble late prenatal rat kidneys and that low levels of ENaC gene transcription limit renal Na conservation. If, on the other hand, the described perinatal surge in mRNA levels is induced by birth regardless of gestational age, the premature human kidney may resemble the newborn rat kidney with adequate ENaC mRNA levels; a discrepancy between collecting duct Na transport and ENaC expression similar to that postulated for newborn rat and rabbit would then have to be assumed to be present.
In summary, we have shown that the ENaC subunit gene expression in the rat kidney is localized to the distal nephron and is developmentally regulated, with a sharp rise in all subunit mRNAs from low prenatal levels to adult levels during the perinatal period. The low levels of expression in late fetal life may be of importance in the development of renal Na wasting and hyponatremia in premature infants.
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ACKNOWLEDGEMENTS |
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The authors are grateful to Drs. Susan DiGiovanni and Mark Knepper for advice about RT-PCR methodology.
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FOOTNOTES |
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This work was supported by the Department of Pediatrics, Louisiana State University Medical Center in New Orleans.
Address for reprint requests: V. M. Vehaskari, Dept. of Pediatrics, LSU Medical Center, Box T8-1, 1542 Tulane Ave., New Orleans, LA 70112.
Received 8 December 1997; accepted in final form 16 February 1998.
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