Maturation of TonEBP expression in developing rat kidney

【摘要】  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.

【关键词】  urine concentration sodium myo inositol cotransporter

TONICITY - RESPONSIVE enhancer binding protein (TonEBP) is a recently identified member of the Rel family of transcriptional activators that include NF- B and nuclear factor of activated T cells (NFAT) ( 12 ). The Rel family is defined based on structural similarity in their DNA binding domains. The DNA binding domain of TonEBP forms a dimer like that of NF- B ( 19 ). On the other hand, it shares 43% of amino acids with the DNA binding domains of NFAT1-NFAT4. Based on this, TonEBP is also called NFAT5 ( 10 ).

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 NH 4 Cl. 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 H 2 O 2 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 50-70 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 10-15 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 H 2 O 2, 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. 1 a ). 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 ).

Fig. 1. Tonicity-responsive enhancer binding protein (TonEBP) expression in adult rat kidney. a : Light micrograph of a 50-µm-thick section illustrating brown immunostaining for TonEBP. Preimmune serum did not stain significantly (not shown) as reported previously ( 1 ). CO, cortex; OS, outer stripe of outer medulla; IS, inner stripe of outer medulla; IM, inner medulla. b and c : Two successive 1-µm-thick plastic sections from OS further immunostained for aquaporin-1 (AQP1; b ) or the 1 -subunit of the Na-K-ATPase ( c ) in blue. PT, proximal tubule; TAL, thick ascending limb of the loop of Henle. The star denotes the collecting duct. d and e : Two successive 1-µm-thick plastic sections from IS further immunostained for AQP1 ( d ) or the 1 -subunit of the Na-K-ATPase ( e ) in blue. VB, vascular bundle. Stars denote descending thin limbs of the loop of Henle. f and g : Two successive 1-µm-thick plastic sections from IM further immunostained for AQP1 ( f ) or CLC-K ( g ) in blue. IMCD, inner medullary collecting duct. Stars and asterisks denote descending and ascending thin limbs of the loop of Henle, respectively. Magnification: x 15 ( a ) and x 200 ( b - g ).

Table 1. Summary of TonEBP expression in developing rat kidneys

Expression of TonEBP in fetal kidneys. TonEBP was detected in the kidney at the fetal age of 16 days (postcoitus) ( Fig. 2 a ). At this stage, TonEBP immunoreactivity was observed only in the renal medulla, where it was localized to the endothelial cells of the capillary plexus surrounding the medullary collecting ducts (MCD) or ureteric buds. At the fetal age of 18 days, TonEBP immunoreactivity was intense in the endothelial cells of the capillary plexus and also detectable in the interstitial cells ( Fig. 2, b and c ). Overall intensity of TonEBP immunoreactivity was higher in the medulla compared with the cortex, where some endothelial cells of the capillary displayed weak TonEBP immunoreactivity ( Table 1 ). In tubular profiles, weak immunoreactivity was seen in the collecting ducts and medullary TAL.

Fig. 2. TonEBP expression in 16-day ( a )- and 18-day-old ( b and c ) fetal kidneys. Light micrographs of 50-µm-thick sections are shown. c : Higher magnification view of the area indicated by the rectangle in b. Asterisks, medullary collecting ducts; arrowheads, endothelial cells; arrows, interstitial cells. Magnification: x 75 ( a and b ) and x 320 ( c ).

At the fetal age of 20 days, the papilla was clearly formed ( Fig. 3 a ). TonEBP immunoreactivity was much higher in the medulla than the cortex. Endothelial cells of the capillary plexus and interstitial cells continued to show prominent immunoreactivity ( Fig. 4 a ). Electron micrographs confirmed the expression of TonEBP in the endothelial cells, which were identified by their nuclei budging into the lumen and by the presence of red blood cells in the lumen ( Fig. 5 a ). Among tubular profiles, only the loop of Henle showed clear TonEBP expression. On the other hand, TonEBP immunoreactivity in the TAL and MCD was barely detectable ( Figs. 4 b and 6, a and b ). Thus the intensity of TonEBP immunoreactivity was much higher in the vasa recta than the tubules except for the loop of Henle. In addition, immunoreactivity was localized predominantly to the cytoplasm in both the endothelial and tubular cells ( Figs. 4 a and 5, a and b ).

Fig. 3. Kidneys from 20-day-old fetuses ( a and f ) and 1 ( b )-, 7 ( c and g )-, 14 ( d )-, and 21-day-old pups ( e ) were immunostained with TonEBP antibody ( a - e ) or preimmune serum ( f and g ). Light micrographs of 50-µm-thick sections are shown. Magnification: x 30 ( a and b ), x 23 ( c - e ), and x 18 ( f and g ).

Fig. 4. TonEBP expression in the renal medulla from a 20-day-old fetus ( a and b ) and 1-day-old pup ( c and d ). Higher magnification views of Fig. 3, a and b, are shown: a and c are from the region of future outer medulla, and b and d are from the tip of the renal papilla. Arrows, endothelial cells of capillary; open arrows, epithelial cells of the loop of Henle; arrowheads, interstitial cells; MCD, medullary collecting duct. Magnification: x 480 ( a and c ) and x 240 ( b and d ).

Fig. 5. Electron micrographs of TonEBP immunostaining in the renal medullas from a 20-day-old fetus ( a and b ) and 1-day-old pup ( c ). VE, vascular endothelial cell; TE, tubular epithelial cell; RBC, red blood cell. Magnification: x 2,500 ( a and c ) and x 6,000 ( b ).

Fig. 6. TonEBP expression in the renal papillae from a 20-day-old fetus ( a and b ) and 1-day-old pup ( c and d ). Successive 1-µm-thick sections were made from areas shown in Fig. 4, b and d. b And d were further immunostained for the 1 -subunit of the Na-K-ATPase in blue. Arrows denote endothelial cells. Magnification: x 400.

Expression of TonEBP in postnatal kidneys. One day after birth, dramatic changes were observed. First, TonEBP immunoreactivity shifted to the nucleus throughout the papilla, including endothelial cells of the capillary plexus ( Figs. 4 - 6 ), the loop of Henle, interstitial cells, and MCD ( Figs. 4, c and d, and 6, c and d; Table 1 ). Second, except in the TAL, the intensity of TonEBP immunoreactivity of the tubules increased dramatically. Thereafter, the immunoreactivity of the tubular profiles increased slowly over the next 20 days ( Fig. 7, Table 1 ). At 21 days after birth, as the outer medulla became distinct from the inner medulla ( Fig. 3 ), an adult pattern of TonEBP expression was seen: the highest level of expression was in the inner medulla, and there was also considerable expression in the inner stripe of outer medulla. In the cortex, some distal tubule cells as well as endothelial cells of the capillary showed positive TonEBP immunostaining in the nucleus ( Fig. 7 d ).

Fig. 7. TonEBP expression in kidneys from a 14-day-old ( a - c ) and 21-day-old pup ( d - f ). Higher magnification ( x 480) views of Fig. 3, d and e : cortex ( a and d ), inner stripe of outer medulla ( b and e ), and terminal inner medulla ( c and f ). Arrowheads denote endothelial cells. PCT, proximal convoluted tubule; DCT, distal convoluted tubule; DTL, descending thin limb.

TonEBP and SMIT mRNA expression. Expression of TonEBP mRNA was examined by in situ hybridization of the antisense riboprobe. The sense riboprobe did not produce significant hybridization (not shown). At the fetal age of 16 days, little signal was detected by the antisense riboprobe in the kidney ( Fig. 8, a and c ). On the other hand, intense signal was seen in the neuroblast cells of the dorsal root ganglion and the mantle layer of the developing spinal cord ( Fig. 8, a and b ). At the fetal age of 20 days, weak TonEBP mRNA was detected in the TAL and MCD ( Fig. 8, d and e ). Signals in the TAL and MCD increased steadily after birth ( Fig. 9 ). Although TonEBP protein expression was low (see above), TonEBP mRNA expression was high in the TAL of 1- and 7-day-old pups ( Fig. 9, b and e ). Subsequently, TonEBP mRNA expression decreased (not shown), whereas TonEBP protein expression increased moderately in the TAL ( Table 1 ). We suspect a posttranscriptional regulation of TonEBP in the TAL during development: downregulation in younger animals and upregulation in older animals. This might be caused by the changes in interstitial tonicity (see DISCUSSION ).

Fig. 8. Expression of TonEBP mRNA in a 16-day-old ( a - c ) and 20-day-old fetus ( d and e ). In situ hybridization with antisense riboprobe was performed on 4-µm-thick wax sections in transverse planes of posterior abdominal wall ( a ) and fetal kidney ( d ). In a, note the intense signal in the neuroblast cells of the dorsal root ganglion and the mantle layer of developing spinal cord ( box b ). In contrast, no signal is detected in kidney ( box c ). In d, weak signal was detected in TAL and MCD ( box e ). Note that boxes b (dorsal root ganglion and mantle layer of developing spinal cord), c (kidney), and e (renal medulla) are high-magnification views of areas in a and d. Asterisk denotes TAL. Magnification: x 32 ( a ), x 75 ( b and c ), x 40 ( d ), and x 150 ( e ).

Fig. 9. TonEBP mRNA expression in a kidney from 1 ( a and b )-, 5 ( c and d )-, 7 ( e; outer medulla)-, and 21-day-old pups ( e; inner medulla). Note the increase in situ signals in medullary tubular profiles. b and d : High-magnification views of boxed areas in a and c. Asterisks in b and e denote TAL. OMCD and IMCD, outer and inner medullary collecting duct, respectively. Magnification: x 40 ( a ), x 30 ( c ), and x 300 ( b and d - f ).

In view of the dramatic changes in TonEBP expression after birth ( Figs. 4 - 6 ), we quantified the abundance of TonEBP mRNA using an RNAse protection assay ( Fig. 10 ). The abundance of TonEBP mRNA increased slowly from the fetal age of 20 days to the postnatal age of 5 days. The abundance of mRNA for SMIT, a target gene of TonEBP, also increased at a rate equal to that for TonEBP. Thus the nuclear shift of TonEBP at birth did not result in a dramatic boost in transcription of the SMIT gene.

Fig. 10. Abundance of TonEBP and sodium- myo -inositol cotransporter (SMIT) mRNA in the kidneys from 20-day-old fetuses (F20) and 1-day (P1)-, 3-day (P3)-, and 5-day-old pups (P5). RNase protection assays were performed to measure mRNA for SMIT, TonEBP, and -actin ( top ). Radioactivity of SMIT or TonEBP band was divided by the radioactivity of corresponding -actin band to correct for RNA loading. In each experiment, the corrected radioactivity was expressed relative to that of P5. Values are means ± SE; n = 3. * P < 0.05 compared with F20.

DISCUSSION

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/kgH 2 O ( 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/kgH 2 O in rats 3-16 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.

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作者单位：1 Department of Anatomy and Cell Death Disease Research Center, Catholic University Medical College, Seoul 137-70 South Korea; and 2 Division of Nephrology, University of Maryland, Baltimore, Maryland 21201