INTRODUCTION

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THE NEWBORN INFANT has a rapid maturation in renal tubular reabsorption of sodium. The fractional excretion of sodium is 5-8% in the term sheep fetus and declines to 1% within 24 h of birth (24). A similar pattern has been seen in term neonates; however, this maturation in tubular reabsorption of sodium may be incomplete in preterm human newborns (1). The bulk of filtered sodium is reabsorbed in the proximal tubule, and there is evidence for maturation of the sodium transporters of this segment around the time of birth (13, 15). The mechanisms of this maturation and the hormonal influences controlling it are not well understood.

Glucocorticoid levels increase immediately before birth (22). This rise in glucocorticoid levels may play an important role in the concomitant rise in apical membrane Na⁺/H⁺ exchanger activity seen during the transition from fetal to newborn life (4, 5). We have previously studied the role of glucocorticoids in the maturation of proximal tubule apical membrane Na⁺ transport by giving 132 day fetal sheep (term 145 days) a 48-h intraperitoneal cortisol infusion (14). Plasma cortisol concentrations were increased to levels that are seen at parturition. We saw no significant effects on arterial blood gases, heart rate, or mean arterial blood pressure between controls and their treated twins after 48 h of cortisol infusion, but we found a 61% increase in activity of the proximal tubule Na⁺/H⁺ exchanger. This duplicated most, but not all, of the increase in Na⁺/H⁺ exchanger activity that is seen in vaginally delivered 24-h-old lambs (15). We also saw a fourfold rise in NHE3 mRNA levels (NHE3 is the apical isoform of the Na⁺/H⁺ exchanger and is found in the proximal tubule apical membrane). Despite these findings that Na⁺/H⁺ exchanger activity and mRNA levels increased after cortisol treatment, we found that we did not stimulate increased renal sodium absorption but rather produced a natriuresis.

There are several possible explanations for this failure of cortisol to induce a maturation in renal tubular Na⁺ reabsorption. One possibility is that there was not a fully integrated maturation of proximal tubule Na⁺ and Cl ion transporters; that is, either the apical Cl/base exchanger or basolateral Na⁺-K⁺-ATPase did not mature with our cortisol treatment. Another possibility is that we may have induced a pressure natriuresis (treated fetuses tended to have higher heart rates and mean arterial pressure at 48 h, although this trend was not significant).

Glucocorticoids upregulate Na⁺-K⁺-ATPase directly through transcriptional regulation. In the renal tubule, glucocorticoids may also influence Na⁺-K⁺-ATPase indirectly, by increasing apical Na⁺/H⁺-exchanger activity. This increase in apical Na⁺ transport results in increased intracellular Na⁺ concentration, which in turn may lead to upregulation of Na⁺-K⁺-ATPase. Several studies support such a mechanism playing a role in the developmental maturation of renal Na⁺-K⁺-ATPase activity (21, 30). In guinea pigs, which also complete nephrogenesis before birth, we have shown a marked four- to fivefold increase in renal Na⁺-K⁺-ATPase activity with birth. ₁- And ₁-subunit mRNA increased 1.4- to 2.1-fold and 1.5-fold, respectively. ₁-Subunit protein abundance increased 3.5-fold, whereas ₁-subunit protein abundance increased 2.3-fold with birth (13). The ontogeny of Na⁺-K⁺-ATPase activity in sheep and humans has not been described. A failure of basolateral Na⁺-K⁺-ATPase to become upregulated after cortisol exposure would explain the natriuresis we saw in the fetal sheep studied and suggest a reason for the preterm newborn infant's tendency to waste Na⁺ during the early newborn period.

The purpose of the present study is to determine if a short-term exposure of late gestation but preterm fetal sheep to cortisol results in a coordinated upregulation of proximal tubule Na⁺ transporter function. This is achieved by first describing the normal change in Na⁺-K⁺-ATPase activity that occurs with parturition. We compare renal cortical Na⁺-K⁺-ATPase activity in 140 day gestation fetal lambs to 1-day-old lambs. We chronically instrument twin preterm fetal sheep and expose one twin to cortisol levels normally seen at parturition and then measure Na⁺-K⁺-ATPase activity in both sheep to see if we mimic what is normally seen at parturition. Protein and mRNA levels of the ₁ - and ₁-subunits are measured to look at the origin of changing activity.

	METHODS

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Animals and surgical preparation. Fetal lambs of Dorset and Suffolk mixed breeding were studied. Gestational ages were based on the induced-ovulation technique as described previously (18), and all animals studied were conceived by this technique. Only ewes having radiographically demonstrated twin fetuses were selected for surgery for the cortisol experiments. Anesthesia and surgery of the ewes, fetuses, and lambs were performed as previously described (25, 28). Ewes were not fed for 24 h before surgery, and all animals were anesthetized using a mixture of halothane (1%), oxygen (33%), and nitrous oxide (66%).

In the first series of studies, we studied changes in Na⁺-K⁺-ATPase activity at birth to compare normal maturational changes with the effects of cortisol treatment. For these studies, samples of renal cortex were obtained from anesthetized fetuses at 140 day gestation (term 145 days, n = 6) and anesthetized 1-day-old lambs (n = 6). These samples were snap frozen in liquid nitrogen immediately after removal and then stored at 70°C. After nephrectomy, animals were killed with an intravenous injection of pentobarbital sodium.

In the second series of studies, the effect of cortisol on Na⁺-K⁺-ATPase was studied. From the 10 pairs of fetal sheep we previously studied (14), tissue was available from 9 pairs for further study. These twin fetuses were prepared at 127-day gestation as previously described (14). Briefly, the ewe was not fed for 24 h before surgery and anesthetized as above. Under sterile conditions, the uterus was opened over the fetal hindlimbs. Polyethylene catheters (PE-90) were placed into the fetal femoral veins and arteries bilaterally. Then a small incision was made in the abdominal wall and peritoneal membrane of the fetus. A polyethylene catheter (PE-50) was secured in the peritoneal cavity, and a 3-Fr feeding tube was placed in the fetal bladder for urine collection. An additional catheter was placed in the amniotic cavity for determination of amniotic fluid pressure. After the fetal incisions were closed and the first fetus was returned to the uterus, similar procedures were performed on the second fetus.

At the end of surgery, uterine and maternal abdominal muscles and maternal skin were closed in separate layers. All catheters were exteriorized through a subcutaneous tunnel and placed in a cloth pouch on the ewe's flank. Ampicillin sodium (Wyeth Laboratories) was administered to the ewe intramuscularly before surgery (1 g) and infused into the amniotic cavity after surgery (1 g). After recovery from anesthesia, pregnant ewes were returned to individual pens and allowed free access to food and water. The animals were allowed 72 h to recover from surgery.

All procedures were performed within the regulations of the Animal Welfare Act and the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the University of Iowa Animal Care and Use Committee.

Experimental protocols. The cortisol study began after a 72-h recovery period at 130 day gestation. A priming dose of 2 µCi/ml of [¹⁴C]inulin in 5% dextrose was administered intravenously to both fetuses followed by a continuous infusion at 0.12 µCi/min for later determination of glomerular filtration rate. Urine volume was recorded, and the urine was stored at 70°C for later determination of [¹⁴C]inulin and urinary sodium concentration. Arterial blood was obtained for determination of pH, blood gases (oxygen tension, CO₂ tension), hematocrit, and plasma sodium and cortisol concentrations. All blood samples were replaced with equivalent volumes of maternal blood to avoid any hemodynamic effects of sampling. Mean arterial blood pressure (MABP), heart rate (HR), and amniotic pressure were monitored continuously using the Statham P-23Db pressure transducers and cardiotachometer. The results of these measurements were reported previously (14).

After the first urine collection period, one of each pair of twins was given a continuous intraperitoneal infusion of cortisol for exactly 48 h at a rate of 3 mg/h (1 ml/h). The infusions were carried out with portable peristaltic infusion pumps (Cormed, Middleport, NY), secured on the back of the ewe in pockets of a specially designed jacket that allowed the animal to move freely. The other twin was sham operated and served as a control.

Two hours before completion of the 48-h cortisol infusion, the ewe was brought back to the laboratory, where a priming dose of 2 µCi/ml of [¹⁴C]inulin, followed by a continuous infusion (0.12 µCi/min), was started. Blood and urine samples were obtained. MABP, HR, and amniotic pressure were monitored continuously during this second collection period. The results of these measurements were reported previously (14).

At the end of the 48-h cortisol infusion period and after the second urine collection period, the ewe was given spinal-epidural anesthesia, using 10 ml of 1% lidocaine as previously described (24), and the fetuses were delivered by cesarean section. The fetuses were then immediately killed with an intravenous injection of pentobarbital sodium. The kidneys were quickly removed, and portions of renal cortex were excised and placed in liquid nitrogen for later isolation of total RNA and protein. These samples were stored at 70°C until used.

Tissue preparation. Renal cortex was homogenized in ~10 volumes of 250 mM sucrose, 0.1 mM phenylmethylsulfonyl fluoride, and 10 mM Tris · HCl, pH 7.5, using a Tissue Tearor rotor-stator-type tissue homogenizer (Biospec Products) at 30,000 rpm for 30 s. All tissue and homogenates were kept on ice. The homogenates were stored at 20°C until analyzed. We previously showed that freezing does not decrease Na⁺-K⁺-ATPase activity in such homogenates (13), but to control for the effects of freezing, each assay contained samples from each group, and each sample underwent the same number of freeze/thaw cycles. Protein concentrations were determined by the method of Lowry as modified by Peterson (27).

Assay of Na⁺-K⁺-ATPase activity. Ouabain-sensitive Na⁺-K⁺-ATPase activity was determined by the method of Forbush (12). PO₄ generation was assayed colorimetrically after a 10-min incubation of homogenates in (in mM) 120 NaCl, 25 KCl, 4 MgCl₂, 60 Tris · HCl, 4 disodium ATP, and 1 EDTA, pH 7.5, at 37°C with and without 1 mM ouabain. All substrates were at saturating concentrations and so provide an approximation of maximal velocity (V_max), as we have previously demonstrated (13). The method employs a 10-min preincubation of homogenates in SDS, 1.0% (wt/vol) bovine serum albumin, and 17 mM HEPES, pH 7.0, to optimize enzyme activity. Activity was optimal at an SDS concentration of 0.8 mg/ml in preliminary experiments, so this concentration was used for the preincubation step in all experiments. Samples were assayed in triplicate and averaged for each experiment. Experiments were done twice, with the mean values used for comparisons between groups.

Preliminary experiments showed that the rates of ouabain-sensitive Na⁺-K⁺-ATPase specific activity in homogenates remained linear until >30 min under the conditions used. In these preliminary experiments, we measured ouabain-sensitive Na⁺-K⁺-ATPase activity in samples of cortical homegenates (n = 2/group) at 10-min intervals from 0-40 min. In every case, the rate of activity remained linear over the first 40 min (4 time points measured, r² = 0.996). Therefore, measurements of enzyme activity at 30 min represent initial rates of activity, and allow us to estimate V_max under substrate-saturating conditions.

Immunoblotting. Cortical homogenates were diluted in a sample buffer, the final composition of which was 47 mM Tris · HCl, 7.5% glycerol, 1.5% (wt/vol) SDS, 15% 2-mercaptoethanol, and 0.038% (wt/vol) bromophenol blue, pH 6.8. Samples were incubated in a water bath at 37°C for 15 min. Solubilized preparations were then fractionated by SDS-PAGE (7.5% acrylamide) and then transferred to nitrocellulose sheets (Protran, 0.2 µm pore size; Schleicher and Schuell, Keene, NH) by blotting in a Transblot SD, Semi-Dry Transfer Cell (Bio Rad, Hercules, CA) at 15 V for 15 min. The transfer buffer was 25 mM Tris, 192 mM glycine, and 20% methanol, pH 8.2. The nitrocellulose blots were blocked for 2 h or overnight at room temperature in a 5% nonfat milk, Tris-buffered saline (TBS) solution containing 10 mM sodium azide and 0.1% Triton X-100. Two quick rinses in 0.1% Tween 20-TBS (TTBS) followed, and then the blots were washed twice for 10 min each in TTBS at room temperature. The blots were then incubated in primary antibody in TTBS for 2 h at room temperature. The ₁-subunit was probed with a mouse monoclonal anti-sheep Na⁺-K⁺-ATPase ₁-subunit antibody from Affinity Bioreagents (Golden, CO) at a concentration of 1:500. The ₁-subunit was probed using a mouse monoclonal anti-sheep Na⁺-K⁺-ATPase ₁-subunit antibody also from Affinity Bioreagents at a concentration of 1:500. There followed rinses and washes as described above. The blots were then incubated with a 1:6,650 (₁-subunit) or a 1:20,000 (₁-subunit) dilution of rabbit anti-mouse horseradish peroxidase-conjugated antibody (Sigma, St. Louis, MO) in TTBS at room temperature for 1 h. The blots were again rinsed and washed as above. Binding of the secondary antibody was detected by chemiluminescence (Super Signal Substrate; Pierce, Rockford, IL), and blots were then exposed to Fuji RX X-ray film for 2-30 s. All samples were analyzed at least twice on separate blots.

Isolation of RNA and preparation of probe. Total renal cortical cellular RNA was isolated using TriReagent (Molecular Research Center, Cincinnati, OH). RNA was quantitated spectrophotometrically by measuring absorbance at 260 and 320 nm. RNA samples were stored as an ethanol precipitate at 70°C until further analysis.

Na⁺-K⁺-ATPase cDNAs were kindly provided by Dr. J. Lingrel (University of Cincinnati) and were used to make antisense RNA probes. A partial sheep Na⁺-K⁺-ATPase ₁-subunit cDNA (nt 1814-3661, corresponding to the carboxy-terminal half of the protein and 334 bp of the 3'-flanking region) was used to generate antisense RNA probes using D-[³²P]UTP and T7 RNA polymerase. We have previously characterized this probe (13). A partial sheep Na⁺-K⁺-ATPase ₁-subunit cDNA (nt 1-976, corresponding to 514 bp of the 5'-untranslated region and the first 149 amino acids of the protein) was used and cloned into the Pst I site of pGEM-3Z (Promega, Madison, WI). The plasmid was linearized with BamH I and then used to generate an antisense RNA probe using D-[³²P]UTP and SP6 RNA polymerase. The composition and orientation of the insert was confirmed by sequencing analysis (University of Michigan Research Core Facility).

An 18s rRNA probe was used to confirm equal loading and transfer of RNA as well as to normalize counts. The abundance of 18s rRNA does not change with gestational age in the sheep kidney (37). The probe was prepared from an 18s rRNA cDNA clone corresponding to a 80-bp fragment of a highly conserved region of human 18s rRNA (Ambion, Austin, TX). T7 RNA polymerase and D-[³²P]UTP were used to generate an antisense RNA probe.

Northern blot hybridization. Aliquots of 5 µg of RNA were fractionated by formaldehyde-agarose gel electrophoresis. After electrophoresis, RNA was transferred to a 0.45 µm Nytran filter (Schleicher & Schuell). Filters were hybridized and washed, and autoradiographs were made by standard methods (16).

Densitometric analysis of blots. Band intensities were determined with a Hewlett Packard Scan Jet 5P scanner (Greeley, CO) and then analyzed with Sigma Scan 2.0 software (Jandel Scientific, San Rafael, CA.). Background was subtracted from each measurement. To control for variation in band intensities from blot to blot, due to different lengths of immunoblot or autoradiograph exposure time, the most intense band for each blot was set at an arbitrary value of 100%. The other bands on that blot were expressed as a percent of that value. At least two samples from each group were included on each blot. For Northern blots, ratios of these percents of intensity (samples/18s rRNA) were then determined. Autoradiographs of Northern blots were within the linear range of the film, based on preliminary plots of absorbance vs. RNA concentration. All samples were analyzed at least twice on separate blots.

Data analysis. Groups are compared using the Student's t-test. In the cortisol experiments, paired analyses are used. Differences are considered statistically significant at P < 0.05. Values are expressed as means ± SE.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Changes in Na⁺-K⁺-ATPase activity with birth or cortisol infusion. Ouabain-inhibitable Na⁺-K⁺-ATPase specific activity, measured under substrate-saturating conditions, increased by 80 ± 15% within 24 h of birth (P < 0.05, Fig. 1). Cortisol treatment of 132 day gestation preterm fetal sheep resulted in a 122 ± 26% increase in renal cortical ouabain-inhibitable Na⁺-K⁺-ATPase specific activity (Fig. 1). Control animal ouabain-sensitive Na⁺-K⁺-ATPase specific activity was 14.7 ± 2.1 nmol PO₄ · mg protein¹ · min¹, whereas that of their twins that had received a 48-h cortisol infusion was 32.6 ± 3.8 nmol PO₄ · mg protein¹ · min¹ (P < 0.0005).

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Effect of cortisol on renal cortical Na+-K+-ATPase activity. Renal cortex was obtained from 140 day gestation fetal sheep (14 d F; n = 6), 1-day-old lambs (n = 6), and cortisol-infused and twin control 132 day gestation fetal sheep (n = 9/group). * P < 0.05 vs. 140 day gestation fetal sheep, ** P < 0.0005 vs. twin control fetal sheep.

Na⁺-K⁺-ATPase immunoblotting. We probed blots of cortical homogenates with mouse monoclonal antisera to sheep Na⁺-K⁺-ATPase ₁- and ₁-subunits. Bands of the expected relative molecular mass were seen for each subunit: ₁-subunit at 110 kDa and ₁-subunit at 50-60 kDa (31).

The ₁-subunit was first studied by loading 40 µg protein/lane in immunoblots, conditions under which single bands of the expected size of 110 kDa are clearly resolved. Abundance of the ₁-subunit increased by 19 ± 4% with cortisol treatment (Fig. 2, A and C, control 74 ± 1%, treated 88 ± 3% maximal densitometric signal, P < 0.05). We considered the possibility that this small increase in ₁-subunit abundance was an underestimate, due to measurements made beyond the linear range of the band intensity-protein abundance relationship. This relationship was linear between 1 and 20 µg/lane (Fig. 3, r² = 0.99, 5 concentrations within this range were measured, n = 2). Therefore, the immunoblots were repeated at 5 µg protein/lane. In these blots, bands at 110 kDa were still clearly resolvable; ₁-subunit increased by 16 ± 2% (Fig. 2, B and C, 71 ± 1% in control animals and 82 ± 2% maximal densitometric signal in the cortisol-treated animals, P < 0.05). These results indicate that most of the increase in Na⁺-K⁺-ATPase activity seen with cortisol treatment of fetal sheep was not a result of production of new Na⁺-K⁺-ATPase ₁-subunit, but instead resulted from redistribution or activiation of existing subunits.

View larger version (37K): [in this window] [in a new window]   Fig. 2.   Immunoblot showing effect of cortisol on fetal sheep renal cortical Na+-K+-ATPase 1-subunit protein levels. A: representative blot with 40 µg protein/lane. B: representative blot with 5 µg protein/lane. C: densitometric analysis of blots loaded with either 40 or 5 µg protein/lane (n = 9/group). * P < 0.05 vs. control, ** P < 0.05 vs. control. T, treated; C, control; Mr, molecular mass.

View larger version (11K): [in this window] [in a new window]   Fig. 3.   Plot of immunoblot 1-subunit band intensity vs. µg protein loaded per lane. Curve was fitted using nonlinear regression analysis (Prism software, GraphPad, San Diego, CA); points represent means, with n = 2.

Immunoblots of the ₁-subunit were performed with 40 µg protein loaded per lane. For the ₁-subunit antibody, the band intensity-protein abundance relationship was linear between 10 and 60 µg/lane (r² = 0.98, 4 concentrations within this range were measured, n = 2). ₁-Subunit abundance increased by 39 ± 8% (Fig. 4, control 58 ± 5%, treated twins 81 ± 4% maximal densitometric units, P < 0.005). These results indicate that the increase in Na⁺-K⁺-ATPase activity seen with cortisol treatment was a result of both production of new ₁-subunit protein as well as redistribution or activation of existing ₁-subunits.

View larger version (38K): [in this window] [in a new window]   Fig. 4.   Immunoblot showing effect of cortisol on fetal sheep renal cortical Na+-K+-ATPase 1-subunit protein levels. A: representative blot with 40 µg protein/lane. B: densitometric analysis (n = 9/group). * P < 0.005 vs. control.

Effects of cortisol infusion on renal cortical Na⁺-K⁺-ATPase subunit mRNA levels. We used Northern blot analysis of total cortical RNA to provide a measure of proximal tubule Na⁺-K⁺-ATPase ₁- and ₁-subunit mRNA abundance. Data were normalized to 18s rRNA abundance. There were no significant differences between the groups in 18s rRNA abundance for any of the Northern blots presented in this paper. Blots for the ₁-subunit showed a band of the expected size of 3,700 bp, as reported previously (26). ₁-Subunit mRNA abundance (₁-subunit/18s rRNA) increased by 58 ± 8% (Fig. 5, control fetuses 0.66 ± 0.04, treated fetuses 1.04 ± 0.05, P < 0.0005). The ₁-subunit abundance (₁-subunit/18s rRNA) showed a similar 72 ± 27% increase (Fig. 6, control fetuses 0.71 ± 0.13, treated fetuses 1.22 ± 0.20, P < 0.005). The results from these Northern blots suggest that transcriptional regulation or increased stability of message may account for the increased subunit protein abundances detected on immunoblot. Nevertheless, such transcriptional changes cannot account for the greater than doubling of Na⁺-K⁺-ATPase activity seen with cortisol treatment. Clearly a combination of transcriptional, translational, and posttranslational events may result from the short cortisol infusion regimen we used.

View larger version (36K): [in this window] [in a new window]   Fig. 5.   Northern blot showing effect of cortisol on fetal sheep renal cortical Na+-K+-ATPase 1-subunit mRNA levels. A: representative blot probed for 1-subunit mRNA. B: representative blot stripped and reprobed for 18s rRNA. C: densitometric analysis of 1-subunit mRNA abundance, normalized to 18s rRNA abundance (n = 9/group). * P < 0.0005 vs. control.

View larger version (35K): [in this window] [in a new window]   Fig. 6.   Northern blot showing effect of cortisol on fetal sheep renal cortical Na+-K+-ATPase 1-subunit mRNA levels. A: representative blot probed for 1-subunit mRNA. B: representative blot stripped and reprobed for 18s rRNA. C: densitometric analysis of 1-subunit mRNA abundance, normalized to 18s rRNA abundance (n = 9/group). * P < 0.005 vs. control.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In this study, we showed that treating 132 day gestation fetal sheep with 48 h of intraperitoneal cortisol, attaining plasma cortisol levels comparable to those seen at parturition, mimics the postnatal maturation in Na⁺-K⁺-ATPase activity seen in 1-day-old lambs (15). Therefore, we have been able to induce a full maturation in Na⁺ pump activity with short-term physiological levels of cortisol. These cortisol levels are similar to those seen during the process of birth. Previous studies of glucocorticoid treatment of immature animals have been inconsistent in the effects on renal tubular salt handling. Some studies have demonstrated natriuresis with glucocorticoid administration (29, 34), whereas others have shown increased sodium absorption (11, 32, 33). It is likely that the variable effects observed by others depend on the species studied, when during gestation or development the steroids are given, as well as the type and amount of steroid used. Glucocorticoids are frequently given to preterm human fetuses and newborns, and so it is important to consider these variable effects.

In previous studies, we learned from the animals described in METHODS that a 48-h infusion of physiological doses of cortisol into 132 day gestation fetal sheep induces a maturation in the activity of the proximal tubule Na⁺/H⁺ exchanger through what appears to be a transcriptional effect (14). Despite this maturation in the ion transporter, which accounts for proximal tubule apical membrane Na⁺ transport, a natriuresis occurred. As we discussed previously (14), a variety of factors could explain this observation, which has also been described by others in premature fetal lambs after short-term exposure to glucocorticoids (29, 33). It seems most likely that there was not a full maturation in renal tubular function after cortisol treatment due to the absence of a fully integrated maturation in all apical and basolateral Na⁺ transporting sites along the nephron. This absence of a complete response may have been due either to an inability of the premature tubule to respond fully to the cortisol stimulus or to other developmentally regulated hormonal influences in conjunction with increased cortisol levels that are necessary for a fully integrated response to occur. Such other hormonal influences to consider are those of catecholamines, angiotensin II, or thyroid hormones. Glucorticoids and thyroid hormones act synergistically to induce lung functional maturation (23), supporting the possibility that a fully integrated maturation in renal tubular function depends on multiple hormonal influences.

The purpose of this study was to determine the extent to which cortisol treatment induced a coordinated maturation in renal cortical Na⁺ transporters; an increase in proximal tubule apical Na⁺/H⁺ exchanger activity would need to be coupled to an increase in basolateral Na⁺-K⁺-ATPase activity for an increase in transcellular NaCl flux to occur, because Na⁺-K⁺-ATPase provides the driving force for this transcellular flux. Increases in intracellular sodium concentration, as would be mediated by increased Na⁺/H⁺ exchanger activity, have been shown to increase Na⁺-K⁺-ATPase subunit gene expression in adult rats (20, 36). Alternatively, cortisol may have increased Na⁺-K⁺-ATPase activity directly; a glucocorticoid responsive element has been identified in the 5'-flanking region of the ₁-subunit in rats (35). In vivo experiments have suggested that glucocorticoids can regulate the transcription of both Na⁺-K⁺-ATPase subunit genes in the rat renal cortex but that this effect may be limited to differentiating renal cortical cells (7). Our results suggest the possibility of a mixed pattern of Na⁺-K⁺-ATPase upregulation, with transcriptional, posttranscriptional, and posttranslational effects.

Both Na⁺-K⁺-ATPase ₁- and ₁-subunit protein (19-39%) and mRNA (58-72%) levels increased with cortisol treatment, but not to the degree that the enzyme activity did. Immunoblotting is a semiquantitative technique, and so it is possible that our method underestimated the actual increase in protein abundance occurring with treatment. We show, however, that the antiserum used has a linear relationship when the amount of protein is plotted against densitometric signal intensity. The amount of protein that we used was within this range. These considerations notwithstanding, neither subunit protein nor mRNA levels increased to the same degree as did Na⁺ pump activity, suggesting posttranscriptional upregulation. A similar pattern of greatly increasing Na⁺-K⁺-ATPase activity accompanied by lesser increases in subunit mRNA has been described in the guinea pig at the time of birth (13). Posttranslational upregulation of Na⁺-K⁺-ATPase could have resulted from activation of already existing Na⁺ pumps, either by dephosphorylation (6) or recruitment of latent pools of Na⁺-K⁺-ATPase to sites where they become activated (3). We cannot exclude the possibility that non-₁- and/or non-₁-isoforms could account for the apparent incongruities seen. When described, the alternative isoforms have been found in much lower amounts than the ₁- and ₁-isoforms; mRNA analysis of rat kidney found that ₁-, ₂-, and ₃-isoforms contributed ~70, ~20, and ~10%, respectively, whereas ₁- and ₂-isoforms made up ~95 and ~5% (9). Protein was not detected in this analysis. Another group did find ₃-isoform protein in rat and human nephron collecting tubules (2). ₁- But not ₂-isoform protein was detected in adult rat renal cortex in another recent study (10). It is unclear whether alternative isoforms are present in fetal or newborn sheep kidney.

From the results of this study we learned that we can induce a physiologically relevant maturation of Na⁺-K⁺-ATPase with physiological doses of glucocorticoids in 132 day fetal sheep. We mimicked the increase in cortisol levels seen at parturition in sheep (and humans) and were able to show increased enzyme activity as well as subunit protein and mRNA that reflect the changes in Na⁺-K⁺-ATPase seen at birth (13). Some of the increased activity noted was likely due to increased protein abundance, but the remainder appears to have resulted from posttranscriptional and posttranslational influences. Nevertheless, despite this maturation in proximal tubule Na⁺ transporters, a natriuresis occurred. Cortisol may activate natriuretic mechanisms such as increasing medullary blood flow, changes in direct distal sodium reabsorption mediated by factors intrinsic to the distal tubular cells, or other hormonal factors such as insulin-like growth factor-1, which masked a maturation in proximal tubule ion transporter function. Cortisol infusion alone may not completely mimic the normal maturational process involving other factors affecting sodium reabsorption such as angiotensin II and atrial natriuretic factor. Cortisol may fail to induce the maturation of hydraulic conductivity or the decrease in the size in the luminal openings of the paracellular channels that occurs in the kidney (17, 19).

Perspectives

An understanding of the mechanisms of the maturation in renal tubular function at birth may have manifold benefits. Prevention of salt wasting by induction of tubular maturation in preterm infants may help prevent growth and developmental delays. Glucocorticoids are routinely administered to pregnant women in preterm labor, as well as to premature newborn infants to induce pulmonary maturation. Understanding the scope of the effects of these drugs on extrapulmonary sites may improve both safety and efficacy of the drug regimens.