1Department of Anatomy and Cell Death Disease Research Center, Catholic University Medical College, Seoul 137-701, South Korea; and 2Division of Nephrology, University of Maryland, Baltimore, Maryland 21201
Submitted 10 February 2004 ; accepted in final form 22 June 2004
ABSTRACT
Tonicity-responsive enhancer binding protein (TonEBP) is a transcriptional activator of the Rel family. In the renal medulla, TonEBP stimulates genes encoding proteins involved in cellular accumulation of organic osmolytes, the vasopressin-regulated urea transporters (UT-A), and heat shock protein 70. To understand the role of TonEBP in the development of urinary concentrating ability, TonEBP expression during rat kidney development was investigated. In embryonic kidneys, TonEBP immunoreactivity was detected 16 days postcoitus in the cytoplasm of the endothelial cells surrounding the medullary collecting ducts (MCD). By 20 days, TonEBP was detected in most tubular profiles in the medulla, including the loop of Henle and MCD, and interstitial cells. The intensity of TonEBP immunoreactivity was much higher in the vasa recta than the tubules. In addition, immunoreactivity was localized predominantly to the cytoplasm. On postnatal day 1, two major changes were observed. TonEBP immunoreactivity shifted to the nucleus, and the intensity of TonEBP immunoreactivity of the tubules increased dramatically. These changes were associated with an increase in TonEBP and sodium-myo-inositol cotransporter mRNA abundance. Thereafter, TonEBP expression in tubular profiles increased moderately. The adult pattern of TonEBP expression was established at postnatal day 21 coincident with full maturation of the renal medulla. Thus expression of TonEBP in developing kidneys occurred predominantly in the medulla and preceded expression of its target genes, including UT-A. These data suggest that TonEBP contributes to the development of urine-concentrating ability.
urine concentration; sodium-myo-inositol cotransporter
In cultured cells, TonEBP is stimulated when ambient tonicity is raised. The stimulation is accomplished via multiple pathways, including induction (increased protein abundance) (20), nuclear translocation (2, 23), and enhanced transactivation (4, 9). TonEBP stimulates transcription of a number of genes, including the sodium-myo-inositol cotransporter (SMIT), the sodium-chloride-betaine cotransporter (BGT1), aldose reductase (AR), heat shock protein 70 (HSP70), and the vasopressin-regulated urea transporter (UT-A) (13, 14, 22). SMIT and BGT1 actively transport inositol and betaine into the cell. AR catalyzes sorbitol production from glucose. Cellular accumulation of the organic osmolytes such as inositol, betaine, and sorbitol protects cells from the deleterious effects of hypertonicity by reducing cellular ionic strength via osmotic replacement of electrolytes (21). The urea transporters encoded by the UT-A gene are crucial for the countercurrent urea recycling that leads to the generation of high urea concentration in the renal medulla (17). HSP70 prevents cell death caused by high concentrations of urea (15).
In adult rats, expression of TonEBP target genes is much higher in the hypertonic renal medulla than the isotonic cortex (1). This is consistent with the idea that, as in cultured cells, TonEBP is activated by the hypertonicity in the renal medulla and responsible for driving expression of medulla-enriched proteins such as SMIT, BGT1, AR, HSP70, and UT-A. In response to changes in water intake, nuclear distribution of TonEBP changes throughout the kidney, leading to altered expression of SMIT mRNA (1). Thus TonEBP is an important regulator of the renal medulla as it stimulates medulla-specific function (UT-A) as well as protection from hyperosmotic stress (SMIT, BGT1, AR, and HSP70).
Osmolality of the renal medulla is much lower in neonatal rats than in adult rats (18). Osmolality increases dramatically after weaning in parallel with full development of urine-concentrating ability. In this study, we explored TonEBP expression in the developing rat kidney. The data show that expression of TonEBP in the renal medulla is closely associated with the development of urine-concentrating ability.
MATERIALS AND METHODS
Animals and tissue preservation. Spague-Dawley rats were euthanized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg body wt). Animal experiments were performed with the full approval by the Animal Care and Use Committee of the Catholic University of South Korea.
Kidneys from fetuses 16, 18, and 20 days old (postcoitus) and pups up to 7 days old were perfused through the heart with PBS to rinse out the blood. Kidneys from rats 14 days or older were perfused through the abdominal aorta. This was followed by perfusion with 2% paraformaldehyde, 125 mM lysine, and 10 mM periodate, pH 7.4 (PLP solution) for 4 min. The kidneys were removed and cut into sagittal slices of 1- to 2-mm thickness and postfixed overnight in PLP solution at 4°C. For immunohistochemistry, fixed slices were cut along the sagittal plane on a Vibratome (Technical Products, St. Louis, MO) at a thickness of 50 µm. For in situ hybridization, fixed slices were embedded in wax, and 5-µm sections were prepared.
Antibodies.
TonEBP expression was detected using the rabbit polyclonal antibody against TonEBP (12). The descending thin limb of the loop of Henle and descending vasa recta in the renal medulla were identified by use of a rabbit polyclonal antibody against aquaporin-1 (AQP1) (Chemicon, Temecula, CA). The thick ascending limb in the outer medulla was identified with the use of a rabbit polyclonal antibody against the 1-subunit of the Na-K-ATPase (UBI, Lake Placid, NY). For identifying the ascending thin limb in the inner medulla, a rabbit polyclonal antibody against the CLC-K chloride channel (Almone Labs, Jerusalem, Israel) was used.
Preembedding immunolabeling for TonEBP. The 50-µm-thick Vibratome sections were processed for immunohistochemistry using an indirect preembedding immunoperoxidase method. The sections were washed three times for 15 min each in PBS containing 50 mM NH4Cl. They were then incubated for 3 h in PBS containing 1% BSA, 0.05% saponin, and 0.2% gelatin (solution A). The tissue sections were incubated overnight at 4°C with the TonEBP antibody diluted 1:5,000 in 1% BSA in PBS (solution B). After several washes with solution A, the tissue sections were incubated for 2 h in a 1:50 dilution of a peroxidase-conjugated goat anti-rabbit IgG Fab fragment (Jackson ImmunoResearch Laboratories) in solution B. The tissues were then rinsed, first in solution A and subsequently in 50 mM Tris·HCl (pH 7.6). For the detection of horseradish peroxidase, the sections were incubated in 0.1% 3,3'-diaminobenzidine in the Tris buffer for 5 min. The reaction was stopped with a 10-min incubation after addition of H2O2 to 0.01%. After being washed in the Tris buffer, the sections were dehydrated in a graded series of ethanol and embedded in Epon 812 (Polysciences, Warrington, CA). The sections were examined and photographed on an Olympus Photomicroscope (Tokyo, Japan) equipped with differential-interference contrast. For electron microscopy, 1-µm sections were cut and examined by light microscopy. Ultrathin sections of 5070 nm were then cut, stained with lead citrate, and photographed with a transmission electron microscope (JEOL 1200EX, Tokyo, Japan).
Postembedding immunolabeling for AQP1, 1-Na-K-ATPase, or CLC-K.
The TonEBP-immunolabeled and -embedded 50-µm-thick Vibratome sections were processed for immunohistochemical identification of the descending thin limb and descending vasa recta, the thick ascending limb, or the ascending thin limb. Portions from the outer and the inner medulla were excised and glued onto empty blocks of Epon 812. The sections from the outer medulla were processed for double immunolabeling with TonEBP and AQP1 or TonEBP and
1-Na-K-ATPase. The sections from the inner medulla were processed for double immunolabeling with TonEBP and AQP1 or TonEBP and CLC-K. Two successive 1-µm-thick sections were cut and treated for 1015 min with a saturated solution of sodium hydroxide, diluted 1:3 in absolute ethanol, to remove the resin. After three brief rinses in absolute ethanol, the sections were hydrated with graded ethanol. After a rinse in tapwater for 10 min, the tissue sections were incubated for 30 min with methanolic H2O2, rinsed in tapwater for 10 min, and incubated with 0.5% Triton X-100 in PBS for 15 min. The sections were rinsed in PBS three times for 10 min and incubated with 10% normal goal serum for 1 h. Tissue sections were incubated with AQP1 (1:500),
1-Na-K-ATPase (1:500), or CLC-K antibody (1:500) overnight at 4°C. After a wash in PBS, the sections were incubated for 2 h with peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories). For detection of peroxidase, Vector SG (Vector Laboratories, Burlingame, CA) was used as the chromogen to produce a grayish blue color, which is easily distinguished from the brown staining produced by 3,3'-diaminobenzidine in the preembedding procedure used for detection of TonEBP. The sections were washed with water, dehydrated, and mounted in Canada balsam.
In situ hybridization. Digoxigenin-labeled sense and antisense riboprobes for TonEBP mRNA were synthesized from the plasmid used for RNAse protection assays (see below) using a DIG RNA Labeling Kit (Boehringer Mannheim, Indianapolis, IN). The wax sections were hybridized to digoxigenin-labeled probes as described previously (1).
RNA isolation and RNAse protection assays.
RNA was isolated from freshly isolated kidneys from fetuses (20 days postcoitus) and 1-, 3-, and 5-day-old pups using TRIzol reagent (Invitrogen, Carlsbad, CA). RNAse protection assays were performed to detect mRNA for TonEBP, SMIT, and -actin as described previously (1).
Statistical anlaysis. Students t-test was performed using Excel software (Microsoft, Redmond, WA).
RESULTS
Localization of TonEBP expression in adult rat kidney.
We had previously reported localization of TonEBP in adult rat kidney with an emphasis on changes induced by water diuresis and antidiuresis (1). In this study, localization of TonEBP was revisited with various tubular markers. Overall intensity of TonEBP immunoreactivity increased along the corticomedullary axis (Fig. 1a). In the outer stripe of the outer medulla, TonEBP staining was strong in the collecting ducts. TonEBP was also detectable in the thick ascending limb (TAL; strong immunoreactivity of the 1-subunit of the Na-K-ATPase) and the straight part of the proximal tubule (AQP1-positive brush borders) (Fig. 1, b and c). TonEBP expression was high in all cells in the inner stripe of the outer medulla (Fig. 1, d and e) and the inner medulla (Fig. 1, f and g) including the ascending (CLC-K positive) and descending thin limbs and endothelial cells in the vascular bundles (AQP1 positive). Except for those in the glomeruli (see below), all endothelial cells throughout the kidney displayed TonEBP immunoreactivity with increasing intensity along the corticomedullary axis (Table 1).
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Previous work has shown that TonEBP expression is detected early in mouse development (11). At the fetal age of 11 days, abundant expression of TonEBP is seen in eyes, brain, and spinal cord. TonEBP expression is maintained throughout the fetal stages and into adulthood in many organs including the brain and heart. This study examined TonEBP expression in the developing rat kidney. The data show that TonEBP expression is detected as early as the fetal age of 16 days in endothelial cells surrounding the MCD. Tubular expression is first detectable at the fetal age of 18 days. TonEBP expression increases steadily through birth until postnatal day 21, when the adult pattern of expression is established coincidently with the full maturation of the renal medulla (7) and urine-concentrating ability (18, 24).
It is of interest to speculate whether TonEBP expression in the fetal kidney is due to hypertonic stimulation. Because neonatal mammals including rats are not capable of concentrating urine (3), it has been believed that the hyperosmolality in the renal medulla is established after birth. Accumulated data, however, raise the possibility that the fetal kidney medulla is hyperosmotic. 1) In mice, the Na-K-2Cl cotransporter (NKCC2) is expressed in the developing loop of Henle as early as 14.5 days of fetal age (5). If functional NKCC2 is expressed in developing rat kidneys from the fetal age of 15 days onward, the osmolality in the medullary interstitium might be high enough to stimulate expression of the TonEBP gene on fetal day 16 as observed. In cultured cells, TonEBP induction reaches the maximum at a modest hypertonicity of 450 mosmol/kgH2O (20), which is lower than most part of the renal medulla in adult rat kidneys. 2) Direct measurements using vapor pressure osmometry showed that the osmolality of the renal medulla was
600 mosmol/kgH2O in rats 316 days old (18). While this is much lower than in adults, it is high enough to stimulate TonEBP. Because the medulla is already hyperosmotic on postnatal day 3, it is likely that the medulla is hyperosmotic in fetal kidneys a few days earlier. Thus it is plausible that hypertonicity is a signal for the expression of TonEBP in the fetal kidney. The dramatic nuclear shift of TonEBP after birth might also be due to a rise in tonicity.
During kidney development, TonEBP expression is largely limited to the medulla. In addition, the timing of TonEBP expression either precedes or coincides with that of its target genes. AR and UT-A are not expressed until postnatal day 1 (6, 8). The level of AR and UT-A expression rises slowly over the next 3 wk in parallel with the expression of TonEBP. SMIT mRNA is expressed in the fetal kidney, but the level of expression parallels that of TonEBP through birth and the early postnatal stage (Fig 10). These data are consistent with the view that TonEBP stimulates expression of its target genes during development. If TonEBP is indeed driving the expression of UT-A in neonatal kidneys, it can be stated that hypertonicity is required for generation of the high urea concentrations that account for more than two-thirds of medullary osmolality during antidiuresis (16). In other words, TonEBP is an important part of the urine-concentrating mechanism in addition to its widely known role in protecting renal medullary cells from the deleterious effects of hyperosmolality. This can be tested directly once genetically modified mice with a deficiency in TonEBP expression in the kidney are available.
GRANTS
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-42479 to H. M. Kwon and Korea Research Foundation Grant KRF-99-042-F00072 to J.-H. Cha. S.-H. Park was supported by the Postdoctoral Fellowship Program of the Korea Science and Engineering Foundation.
FOOTNOTES
Address for reprint requests and other correspondence: H. Moo Kwon, Nephrology, N3W143, 22 South Greene St., Baltimore, MD 21201 (E-mail: mkwon{at}medicine.umaryland.edu)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES