INTRODUCTION

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IN A WHOLE-ANIMAL MODEL, CHRONIC dietary sodium depletion in the rat demonstrated an increase in sodium/hydrogen countertransport activity in brush-border membranes (14). Subsequently, this carrier has been identified in the proximal tubule as the sodium/hydrogen antiporter (NHE3) (1, 3, 8). The possible mechanisms that underlie this change in activity, such as increases in transcription, translation, or activation, were not investigated. Two other recent chronic models investigating the regulation of renal cortical NHE3 activity and expression have been published. In one model, the chronic administration of the glucocorticoid analog dexamethasone resulted in a similar increase in both mRNA and activity of NHE3, suggesting transcriptional regulation of NHE3 (6, 10). In the second model, induction of chronic metabolic acidosis revealed no measurable increase in cortical NHE3 mRNA; however, increases in NHE3 protein abundance were evident (2). In this model it was suggested that NHE3 regulation occurred at the translational or possibly posttranslational level.

Because of the critical role of NHE3 in proximal tubule sodium reabsorption, this study focuses on the adaptive changes in NHE3 mRNA and protein abundance during volume contraction in rabbits. By using diuresis and dietary sodium depletion to create a volume-contracted state, the expression of NHE3 mRNA and protein was quantitated at several time points over 31 days. Results from this study demonstrate that both NHE3 mRNA and protein increase to different degrees in response to volume contraction.

	METHODS

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Animal model. New Zealand White female rabbits (1.5-2.0 kg; HRP, Denver, PA) were housed in cages and given food (Quality Select Basic Blend 1600) and water ad libitum. After an equilibration period, animals were given an intravenous injection of 5 mg/kg furosemide (American Regent Laboratories, Shirley, NY) to create a volume-contracted state, which was maintained by administering a low-salt diet of pearled barley (0.003% sodium, LaCrosse Milling, Cochrane, WI). Animals at 1, 5, 10, and 31 days of volume contraction along with age-matched controls were injected with 50 mg/kg pentobarbital (iv). After confirmation of deep anesthesia, the renal arteries were clamped at the hilum, and the kidneys were removed before cardiac puncture. Slices of kidney cortex were flash-frozen in liquid nitrogen and stored at 70°C for RNA isolation and brush-border membrane preparation. Samples from cardiac puncture were used for determination of plasma renin activity (Mayo Medical Laboratories, Rochester, MN), cortisol, aldosterone, electrolytes, and hematocrit, as well as sodium concentration from urine.

RNAse protection assay. Total RNA was isolated from the kidney cortex by using TRIzol reagent (GIBCO, Grand Island, NY). For in vitro transcription of a -³²P-labeled cRNA probe, a template was constructed by using a 317-bp region of NHE3 cDNA [1181-1498 nucleotides (nt)] (15) cloned into the pGEM-4Z (Promega, Madison, WI) vector (NHE3 cDNA was a gift from Drs. Chung-Ming Tse and Mark Donowitz, The Johns Hopkins University School of Medicine). Transcription of the antisense cRNA probe was performed by using the MaxiScript kit (Ambion, Austin, TX), Sp6 RNA polymerase, and [-³²P]UTP. A 250-bp -actin probe was also synthesized similarly by using a template supplied by the manufacturer. After treatment with RNase-free DNase to destroy the templates, unincorporated nucleotides were removed by precipitating the probe with ammonium acetate and ethanol. A RNase protection assay (RPA) was performed by using an RPA II (Ambion) kit. Labeled cRNA antisense probes (20,000-50,000 cpm/sample, where cpm is counts/min) and 20 µg total RNA in 20 µl hybridization solution were heated to 90°C for 5 min and hybridized overnight at 42°C. After hybridization, the RNA-RNA hybridization mix was treated with 10 U RNase T1 at 37°C for 30 min to degrade the single-strand unhybridized probe. The "protected" RNA-RNA hybrids were precipitated with ethanol and resuspended in loading buffer containing 95% formamide. After heating to 90°C for 5 min, the samples were separated on an 8 M urea-8% polyacrylamide Tris-borate-EDTA denaturing gel. A radiolabeled RNA ladder (Century Marker Template, Ambion) was loaded onto each gel to confirm that the RNA hybrids were of the expected size. After drying, the gel was visualized by autoradiography, and the signals were quantitated by densitometry.

Western blot analysis. Brush-border membrane (BBM) was isolated from kidney cortex samples by using a previously established Mg²⁺ aggregation and differential centrifugation technique (7). This technique for BBM isolation was confirmed by assaying for cytosolic (acid phosphatase), basolateral membrane (Na-K-ATPase), and apical membrane (alanyl aminopeptidase) protein markers. The specific activities in each batch of the protein markers obtained from the BBM preparations were similar to those previously published for BBM isolations verifying the technique (7). Protein concentration was determined by the method of Lowry (13), and 150 µg of each sample were then size fractionated by SDS-PAGE on 8% gels. After the samples were electroblotted to a polyvinylidene fluoride Immobilon-P membrane (Millipore, Bedford, MA), the membrane was blocked for 2 h in blocking buffer (TBS with 0.1% Tween 20 and 5% milk protein). The membrane was probed in the same buffer with mouse anti-rabbit NHE3 antibody (a gift from Dr. Daniel Biesmesderfer, Yale Univ. School of Medicine) for 18 h at 4°C with shaking. The membrane was then washed four times for 15 min in blocking buffer and incubated with 1:2,000 dilution of sheep horseradish peroxidase-conjugated anti-mouse IgG (Amersham Life Science, Arlington Heights, IL) for 30 min at room temperature. After being washed four times in blocking buffer, the bound probe was detected with enhanced chemiluminescence (ECL Western Blotting Reagents, Amersham Life Science). NHE3 protein abundance was quantitated by densitometry.

Statistics. Results are expressed as means ± SE. Differences among the groups were assessed by either an F-test or paired Student's t-test with unequal variance, as appropriate. Significance was established as P < 0.05.

	RESULTS

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Animal model characteristics. Urine sodium, plasma renin, and aldosterone levels were measured to assess the rabbits' volume-contracted state. The urine sodium concentration and plasma renin activity for control and treated animals are shown in Figs. 1 and 2, respectively. The urine sodium concentration was significantly less, and plasma rennin activity was greater, in the sodium-restricted animals. Table 1 compares serum potassium, bicarbonate, cortisol, and aldosterone for control and sodium-restricted animals. The data reported in Table 1 are means ± SD of all values comparing experimental and control groups at days 1, 5, 10, and 31. There was no significant difference among time points. There was also no significant difference when sodium-restricted animals are compared with control animals, except that serum aldosterone was significantly higher (P < 0.003) in the volume-contracted group.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Effect of diuresis and chronic dietary sodium restriction on urine sodium concentration. The average urine sodium concentration (mmol/l) in control (n = 4) and volume-contracted rabbits at 1, 5, 10, and 31 days (n = 3 at each time point) is shown. Error bars, SE. *Significant decrease in the average urine sodium concentration of volume-contracted animals compared with controls, P < 0.05.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Effect of diuresis and chronic dietary sodium restriction on plasma renin activity. The average plasma renin activity (ng · ml1 · h1) for control and volume-contracted rabbits at 1, 2, 7, and 31 days and the no. of samples are shown in each bar. Error bars, SE. *P < 0.05.

Effect of volume contraction on NHE3 mRNA abundance. An RPA was chosen to quantitate the mRNA in kidney cortex not only because of the sensitivity of the assay but also to alleviate concern that accurate measurements might not be made if mRNA from the kidney is highly degraded. In Northern analysis, single cleavage of 20% of an mRNA species will result in a 20% signal loss, whereas in an RPA the loss of as little as 1% of the signal would result from such degradation (12). This increase in sensitivity compared with Northern analysis makes an RPA a more appropriate assay for quantitating mRNA in this instance. In Fig. 3, an autoradiograph from a typical RPA indicates the location of -actin and NHE3 bands on an 8 M urea-8% polyacrylamide gel. The detection of -actin in each lane was used to normalize the mRNA loading because it has been determined that the expression of -actin does not change in a volume-contracted state (data not shown). Results of the NHE3 mRNA content in the kidney cortex of volume-contracted animals are shown in Fig. 4. A maximal increase in NHE3 of 172 ± 23% of control was detected at 1 day after volume contraction. The NHE3 mRNA at days 5, 10, and 31 is also significantly increased compared with controls at 153 ± 15, 152 ± 14, and 141 ± 7%, respectively. All of the NHE3 mRNA increases in the volume-contracted animals are significantly higher than in the controls; however, the magnitude of this increase in NHE3 mRNA did not change with time after day 1.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Na+-H+ cotransporter 3 (NHE3) mRNA expression during chronic volume contraction. Results of a typical RNase protection assay indicating NHE3 and -actin bands from 20 µg of total RNA from renal cortex of age-matched and volume contracted rabbits at 1, 5, 10, and 31 days are shown. Isolated RNA was hybridized to a 317-bp NHE3 probe and a 250-bp -actin probe and size fractionated on an 8% polyacrylamide-Tris-borate-EDTA denaturing gel.

View larger version (11K): [in this window] [in a new window]   Fig. 4.   Using -actin to normalize for all measurements, the average NHE3 mRNA is shown as a percentage of the age-matched control (n = 6 at each time point). Error bars, SE. *Significant increase in NHE3 mRNA in the renal cortex of volume-contracted animals compared with controls, P < 0.05.

Effect of volume contraction on NHE3 protein abundance. BBM were prepared from kidney cortex, and NHE3 protein abundance was assessed by Western blot analysis. As shown in Figs. 5 and 6, the NHE3 protein abundance progressively increased over time in the volume-contracted rabbits. A typical blot (Fig. 5) demonstrates that, as the volume-contracted time is increased, the rabbits respond by increasing the amount of NHE3 in the BBM. No significant increase in NHE3 abundance was observed at day 1 of volume contraction; however, after 5 days of volume contraction protein abundance did significantly increase (P < 0.05) compared with controls (190 ± 59%). This increase climbed to 307 ± 72% at 10 days and 427 ± 41% at 31 days after volume contraction. The stepwise increase in NHE3 protein at 5, 10, and 31 days reflects not only a significant increase from controls but also significant increases at each successive measurement compared with the previous time period.

View larger version (8K): [in this window] [in a new window]   Fig. 5.   NHE3 protein expression during chronic volume contraction. Typical Western blot of 150 µg of cortical brush border from age-matched control and volume-contracted rabbits at 1, 5, 10, and 31 days is shown. By using anti-NHE3 antibody, bands were detected between the 80- and 110-kDa markers.

View larger version (11K): [in this window] [in a new window]   Fig. 6.   The average NHE3 protein abundance is shown as a percentage of the age-matched control (n = 6 at each time point). Error bars, SE. Exp, experimental.*Significant increase in NHE3 protein abundance from the brush border of volume-contracted animals compared with controls, P < 0.05.

	DISCUSSION

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This study tested the hypothesis that chronic extracellular volume depression would have a significant effect on NHE3 mRNA and protein content in the renal cortex. NHE3 is the isoform that is heavily expressed on proximal tubule brush border and is responsible for a majority of proximal tubule sodium reabsorption (1). Significant increases in expression of this protein would probably reflect adaptive changes in proximal tubule sodium-hydrogen exchange that have previously been described in salt-depleted rats (14).

Using the volume-contracted rabbit as our model, we demonstrated a significant increase in NHE3 mRNA by day 1. This increase was maintained throughout the 31-day experimental period. Additionally, levels of proximal tubule BBM NHE3 protein steadily increased, starting at day 5 and continuing through day 31. Mechanisms causing this increase are as yet unknown.

Glucocorticoids have been shown to increase NHE3 mRNA and NHE3 activity, at least in part, via glucocorticoid-response elements within the NHE3 promoter region (5, 9, 11). However, in our experiments there was no detectable increase in serum cortisol.

Acidosis also has an effect on proximal tubular NHE3. In rats, chronic acidosis increased NHE3 protein levels in proximal tubule BBM, but increased NHE3 mRNA was not detected (2). In an opossum kidney cell line, however, acid incubation increased NHE3 mRNA and protein levels (4). However, we could not detect a change in acid-base status in our animals, as determined by there being no significant difference in serum bicarbonate between control and experimental animals.

We have demonstrated that chronic volume contraction in the rabbit significantly increases NHE3 mRNA (~150% for at least 31 days) and NHE3 protein abundance in the apical surface of the proximal tubule (>400% at 31 days). Further experiments are necessary to clarify the cellular mechanisms involved in this process and the signals informing the proximal tubule cell of the presence of chronic volume contraction.