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【摘要】 This study was conducted to test the hypothesis that, during renal development, the Na-K-2Cl cotransporter type 2 (NKCC2) activates the tonicity-responsive enhancer binding protein (TonEBP) transcription factor by creating medullary hypertonicity. TonEBP, in turn, drives the expression of aldose reductase (AR) and urea transporter-A (UT-A). Kidneys from 13- to19-day-old fetuses ( F13 - F19 ), 1- to 21-day-old pups ( P1 - P21 ), and adult mice were examined by immunohistochemistry. NKCC2 was first detected on F14 in differentiating macula densa and thick ascending limb (TAL). TonEBP was first detected on F15 in the medullary collecting duct (MCD) and surrounding endothelial cells. AR was detected in the MCD cells of the renal medulla from F15. UT-A first appeared in the descending thin limb (DTL) on F16 and in the MCD on F18. After birth, NKCC2-positive TALs disappeared gradually from the tip of the renal papilla, becoming completely undetectable in the inner medulla on P21. TonEBP shifted from the cytoplasm to the nucleus in both vascular endothelial cells and MCD cells on P1, and its abundance increased gradually afterward. Immunoreactivity for AR and UT-A in the renal medulla increased markedly after birth. Treatment of neonatal animals with furosemide dramatically reduced expression of TonEBP, AR, and UT-A1. Furosemide also prevented the disappearance of NKCC2-expressing TALs in the papilla. The sequential expression of NKCC2, TonEBP, and its targets AR and UT-A and the reduced expression TonEBP and its targets in response to furosemide treatment support the hypothesis that local hypertonicity produced by the activity of NKCC2 activates TonEBP during development.
【关键词】 urinary concentration hypertonicity thick ascending limb furosemide
THE NA - K -2 CL cotransporter type 2 (NKCC2) is located in the apical membrane of the thick ascending limb (TAL) and macula densa ( 2, 10, 21 ). In concert with the K channel in the apical membrane and the Na-K-ATPase in the basolateral membrane, NKCC2 is involved in the active transport of sodium from the lumen into the basolateral fluid. As a result, salt accumulates in the medullary interstitium. Hypertonicity in the medullary interstitium created by the high concentration of salt is an important local signal for the renal medulla. The hypertonicity stimulates the transcription factor tonicity-responsive enhancer binding protein (TonEBP), which drives several genes important for the function of the renal medulla ( 15 ). TonEBP enhances expression of transporters and biosynthetic enzymes for cellular accumulation of organic osmolytes ( 8 ): Na- myo -inositol cotransporter, Na-Cl-betaine cotransporter, Na-Cl-taurine cotransporter, aldose reductase (AR), which converts glucose into sorbitol, and an esterase that produces glycerophocholine from phosphatidylcholine ( 4 ). Cellular accumulation of the organic osmolytes protects cells from the deleterious effects of hypertonicity. Genetically modified mice with deficiency in TonEBP in the kidney display severe atrophy in the renal medulla because cells fail to adapt to hypertonicity ( 13, 14 ).
Recent studies suggest that TonEBP is a major regulator of urinary concentration. TonEBP directly stimulates transcription of aquaporin-2, indicating that it contributes to the water permeability of the collecting duct independently of vasopressin ( 6 ). In addition, TonEBP stimulates the promoter of urea transporter (UT)-A1 and UT-A3 ( 16 ) that provide urea permeability in the inner medullary collecting duct (IMCD). In the kidneys of transgenic mice expressing an inhibitory form of TonEBP, expression of UT-A1 and UT-A2 is reduced ( 13 ), indicating that UT-A2 in the descending thin limb (DTL) is also a target of TonEBP in addition to UT-A1. Mice deficient in either UT-A1/3 or UT-A2 display reduced urea accumulation in the renal medulla ( 3, 20 ). Thus TonEBP appears to be a key regulator in the countercurrent urea recycling that leads to the massive accumulation of urea in the papilla.
To explore the relationship between the hypertonicity in the renal medulla created by NKCC2 and the response of TonEBP and its target genes in the context of development of urinary concentrating ability, we examined their expression in developing mouse kidneys. We found that the expression of NKCC2 precedes TonEBP, which, in turn, is followed by expression of its target genes AR and UT-A during renal development. These and other data indicate that medullary hypertonicity is an important signal for the medullary accumulation of urea and reabsorption of water in the collecting duct.
MATERIALS AND METHODS
Animals and tissue preparation. C57BL6 mice were used in all experiments. Animal care and experimental procedures were performed under approval from the Animal Care Committees of the Catholic University of Korea. Prenatal kidneys were obtained from 13-, 14-, 15-, 16-, 18-, and 19-day-old fetuses ( F13 - F19 ), and postnatal kidneys were obtained from 1-, 4-, 7-, 14-, and 21-day-old pups ( P1 - P21 ) and adult animals. Some of the neonatal pups ( P1 ) were subjected to daily subcutaneous injections of furosemide (30 mg/kg body wt in 10% dimethyl sulfoxide, pH 7.0) or vehicle for 7 days (up to P7). For each age group, three or four animals from two separate litters were used. The animals were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg body wt). The kidneys were preserved by in vivo perfusion through the heart. The kidneys were perfused briefly with PBS at an osmolality of 298 mosmol/kgH 2 O (pH 7.4) to rinse away all blood. This was followed by perfusion with a periodate-lysine-2% paraformaldehyde (PLP) solution for 10 min. After perfusion, the kidneys were removed and cut into slices 1-2 mm thick, which were further fixed by immersion in the same fixative overnight at 4°C. Sections of tissue were cut transversely through the entire kidney at a thickness of 50 µm using a vibratome (Pelco 102, series 1000, Technical Products International, St. Louis, MO). They were processed for immunohistochemical studies using a horseradish-peroxidase preembedding technique.
Antibodies. NKCC2 expression was detected using an affinity-purified rabbit polyclonal antibody directed against NKCC2 (courtesy of Dr. Mark A. Knepper, National Institutes of Health, Bethesda, MD). TonEBP expression was detected using a rabbit polyclonal antibody directed against TonEBP ( 15 ). AR expression was detected using an affinity-purified goat polyclonal antibody directed against AR ( 19 ). To determine the distribution of the urea transporters, a rabbit polyclonal antibody directed against a peptide based on rat renal UT-A (courtesy of Dr. Jeff M. Sands, Emory University, Atlanta, GA) was used.
Immunohistochemistry-preembedding procedure. Sections of PLP-fixed tissue were cut transversely through the kidney using a vibratome to a thickness of 50 µm and were processed for immunohistochemistry using an indirect immunoperoxidase method. All sections were washed three times in PBS containing 50 mM NH 4 Cl for 15 min. Before incubation with the primary antibodies, the sections were pretreated with a graded series of ethanol or not pretreated with ethanol, and then incubated for 4 h with PBS containing 1% BSA, 0.05% saponin, and 0.2% gelatin ( solution A ). The tissue sections were then incubated overnight at 4°C with antibodies directed against NKCC2 (1:300), TonEBP (1:3,000), AR (1:30,000), or UT-A (1:4,000) diluted in solution A. After several washes in PBS containing 0.1% BSA, 0.05% saponin, and 0.2% gelatin ( solution B ), the tissue sections were incubated for 2 h in horseradish peroxidase-conjugated donkey anti-rabbit or anti-goat IgG Fab fragment (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted 1:100 in PBS containing 1% BSA ( solution C ). The tissues were then rinsed, first in solution B, and then in 0.05 M Tris buffer (pH 7.6). To detect horseradish peroxidase, the sections were incubated in 0.1% 3,3'-diaminobenzidine (DAB) in 0.05 M Tris buffer for 5 min. Then, H 2 O 2 was added to a final concentration of 0.01% and the incubation was continued for 10 min. The sections were washed three times with 0.05 M Tris buffer, dehydrated in a graded series of ethanol, and embedded in Poly/Bed 812 resin (Polysciences, Warrington, PA). The sections were examined with a light microscope.
Double immunolabeling for UT-A and aquaporin-1. Vibratome sections were labeled with anti-UT-A antibody (1:4,000), using DAB as the chromogen (brown), as described above. The sections were rinsed with PBS and then incubated overnight at 4°C in rabbit antibodies directed against aquaporin-1 (AQP1; 1:800; Chemicon, Temecula, CA) in solution A. After several washes in PBS, the tissue sections were incubated for 2 h in horseradish peroxidase-conjugated donkey anti-rabbit IgG Fab fragment (Jackson ImmunoResearch Laboratories) diluted 1:100 in solution C. The tissues were then rinsed in PBS, and the sections were incubated with Vector SG (blue; Vector Laboratories, Burlingame, CA) in PBS for 5 min, followed by incubation with H 2 O 2 for 10 min. The sections were then washed three times with PBS, dehydrated in a graded series of ethanol, and embedded in Poly/Bed 812 resin. The sections were examined with a light microscope.
Statistical analysis. Data are presented as mean ± SD ( n ), where n indicates the number of mice studied. To test for statistically significant differences between two groups, an unpaired Student?s t -test was used. P < 0.05 was considered significant.
RESULTS
To investigate the role of NKCC2 and TonEBP in the development of urinary concentrating ability, we examined the expression of NKCC2, TonEBP, and TonEBP targets AR and UT-A in developing mouse kidneys from F13 through birth up to adulthood, focusing on the temporal relationship among them.
NKCC2. NKCC2 appeared first in the distal anlage on F14 ( Fig. 1, A and B ). From F15 onward, NKCC2 was detected in tubules located in the cortex and medulla, presumably representing the macula densa and TALs ( Fig. 1 C ). During the prenatal period, TALs were present throughout the renal medulla down to the tip of the renal papilla ( Fig. 1 D ). After birth, the NKCC2-positive TALs disappeared gradually from the tip of the renal papilla ( Fig. 1, E - H ). By P21, no TALs were seen in the inner medulla ( Fig. 1 I ).
Fig. 1. Immunostaining of Na-K-2Cl cotransporter (NKCC2) in kidneys from 13-, 14-, 15-, and 18-day-old mouse fetuses ( A - D ) and 1-, 4-, 7-, 14-, and 21-day-old mouse pups ( E - I ). A : on fetal day 13 ( F13 ), there was no immunoreactivity. B - D : Na-K-2Cl cotransporter (NKCC2) was first detected in the distal anlage (arrow) at F14, and its expression gradually increased in the differentiating thick ascending limbs (TALs; arrowheads). Stars denote ureteric buds. Insets : higher magnification views to show apical localization of NKCC2. U, ureter; P, renal pelvis. E - I : after birth, NKCC2-positive TALs disappeared gradually from the tip to the base of the inner medulla (IM). Co, cortex; OM, outer medulla. Scale bars = 0.1 mm.
In the developing rat kidney, NKCC2 appeared first on F18 (data not shown). Thus expression of NKCC2 is expressed several days earlier in the mouse than in the rat during renal development.
TonEBP. TonEBP immunoreactivity was first detected in the renal medulla on F15 ( Fig. 2, A and B ) and gradually increased afterward ( Fig. 2, C and D ). At this stage, TonEBP was intense in the cytoplasm of the vascular endothelial cells surrounding the MCD ( Fig. 3, A and C ), while the level of TonEBP was much lower in the collecting ducts from the cortex ( Fig. 3 B ) or the medulla ( Fig. 3 C ). In the interstitial cells, TonEBP was detected from F18 and increased steadily afterward (not shown).
Fig. 2. Immunostaining of tonicity-enhancer binding protein (TonEBP) from kidneys of mouse fetuses ( A - D ) and pups ( E - I ). A : on F13, there was no immunoreactivity for TonEBP. B : TonEBP immunoreactivity was detected first in the renal medulla on F15. After birth, TonEBP immunoreactivity increased gradually in the IM ( E - I ). Scale bars = 0.1 mm.
Fig. 3. Higher magnification views of TonEBP in developing mouse kidneys. A and C : in fetal kidneys, TonEBP immunoreactivity was intense in the cytoplasm of the endothelial cells of capillary plexus (asterisks) surrounding the medullary collecting ducts (MCD), while moderate in the MCD. In contrast, the immunoreactivity was low in the nuclei of endothelial cells (arrows) and the MCD (arrowheads). B : in the Co of the fetal kidney, immunoreactivity was not detected except for faint labeling in the cortical collecting duct. D : immediately after birth, TonEBP immunoreactivity shifted to the nucleus in the endothelial (arrow) and MCD cells (arrowheads). A, ampulla; S, S-shaped body; CD, collecting duct. Scale bars = 0.03 mm.
On P1, two major changes were observed ( Fig. 3 D ). First, TonEBP shifted to the nucleus throughout the papilla, including the endothelial cells of the capillary plexus and the MCD. Second, the intensity of TonEBP immunoreactivity in the tubules, including the MCD, increased dramatically. Subsequently, expression of TonEBP increased gradually ( Fig. 2, E - H ). At P21, as the outer medulla became distinct from the inner medulla ( Fig. 2 I ), the adult pattern of TonEBP expression was observed: the highest level of expression was in the inner medulla, and there was considerable expression in the inner stripe of the outer medulla.
Overall, the ontogeny of TonEBP expression in the mouse is similar to that in the rat ( 5 ), except that the level of expression before birth is considerably higher in the mouse. The nuclear shift and increased expression of TonEBP in the tubules immediate after birth are also observed in the rat ( 5 ).
AR. AR immunoreactivity was first detected in the renal medulla on F15 ( Fig. 4, A and B ) and gradually increased afterward ( Fig. 4, C and D ). At this stage, AR was seen mainly in the developing loop of Henle and the MCD but was not detected in the interstitial cells ( Fig. 5, A and B ). In the interstitial cells of the inner medulla, AR was detected at the terminal part of the renal papilla from P1 and gradually increased afterward ( Fig. 5, C and D ). Expression of AR was predominant in the medulla through development and was confined to the inner medulla by P21 ( Fig. 4, E - I ) except for moderate expression in the cortical collecting duct (not shown). At P21, the expression pattern of AR in the inner medulla was similar to that seen in adult kidneys. In the initial part of the inner medulla, AR immunoreactivity was strong in the ascending thin limb but faint in the IMCD and interstitial cells ( Fig. 5 E ). In contrast, in the middle and terminal part of the inner medulla, AR immunoreactivity was strong in both the IMCD and interstitial cells ( Fig. 5, F and G ).
Fig. 4. Immunostaining of aldose reductase (AR) from kidneys of mouse fetuses ( A - D ) and pups ( E - I ). On F14, there was no immunoreactivity ( A ). AR immunoreactivity was first detected on F15 in the renal papilla ( B ) and increased gradually afterward ( C - I ). Scale bars = 0.1 mm.
Fig. 5. Higher magnification views of AR in developing kidneys. A and B : in fetuses, AR immunoreactivity was detected mainly in the developing loop of Henle and the MCD but was not detected in the interstitial cells. C and D : after birth, AR was also detected in the interstitial cells at the tip of the renal papilla on P1 and gradually increased in intensity during development. E - G : on P21, there was a gradient of AR immunoreactivity in the IM, low in the base ( E ) but higher in the middle ( F ) and the tip ( G ), as in the adult. Note strong AR-positive ascending thin limb (ATL) in the base of the renal papilla. Arrows, interstitial cells; arrowheads, MCD cells; asterisks, loops of Henle. Scale bars = 0.03 mm.
While the pattern of AR expression in the adult rat kidney is almost identical to that in the mouse as described here, AR is not expressed in fetal rat kidneys ( 9 ). Thus expression of AR starts several days earlier during development in the mouse compared with the rat. This correlates with earlier expression of NKCC2 and more vigorous expression of TonEBP in fetal stages (see above and Fig. 9 ).
Fig. 6. Immunostaining of urea transporter-A (UT-A) in kidneys from mouse fetuses ( A - D ) and pups ( E - I ). A and B : no immunoreactivity was seen until E15. C : on E17, UT-A immunoreactivity was detected in the descending thin limb (DTL). C ': higher magnification view of the area indicated by the rectangle in C. D : on F18, UT-A expression appeared in the MCD (arrowhead) in addition to DTL (arrow). E - G : after birth up to P7, UT-A was expressed in the border between the OM and IM (arrows) and in the inner medullary collecting duct (IMCD; arrowheads). H and I : on P14 and P21, UT-A immunoreactivity was strong in DTLs of the terminal portion of short-looped nephrons (open arrows), while moderate in shorter long-looped nephrons (arrows) descending into the innermost part of the inner stripe of the outer medulla (ISOM). OSOM, outer stripe of the outer medulla; IMi, initial part of the inner medulla; IMm, middle part of the inner medulla. Scale bars = 0.1 mm.
Fig. 7. Double immunostaining for UT-A (brown) and aquaporin-1 (AQP1; blue) in kidneys of mouse fetuses ( A and B ), pups ( C - F ), and adults ( G ). UT-A immunoreactivity in a DTL (filled arrow) was first detected on F16 ( A ). The DTL is shown in higher magnification in the upper right corner. B and C : DTL with AQP1 immunoreactivity in the proximal part and UT-A immunoreactivity (filled arrows) in distal part, respectively, which connected directly to TAL (*) without the ATL. During development from P7 to adult ( D - G ), strong UT-A immunoreactivity in the DTL was present in the terminal portion of short-loop nephrons (open arrows), and weak labeling remained in shorter long-loop nephrons (filled arrows) descending into the innermost part of the ISOM. Scale bars = 0.1 mm.
Fig. 8. Higher magnification views of UT-A in developing kidneys. A : no immunoreactivity for UT-A in the MCD on F17. Arrows indicate UT-A-positive DTL. B : UT-A immunoreactivity in the MCD was first detected on F18. After birth, UT-A immunoreactivity in the MCD increased gradually ( C - E ). Scale bars = 0.03 mm.
Fig. 9. Immunostaining for NKCC2, TonEBP, AR, and UT-A from kidneys of 7-day-old pups treated with vehicle ( A - D ) or furosemide ( E - H ) since birth. In A - H, portions are shown in higher magnification in A '- H ' plus B '' as indicated by different size bars. NKCC2-positive TALs are not seen in the renal papilla of vehicle-treated animals ( A ) but are clearly visible in furosemide-treated animals ( E ). Arrows, interstitial cells; arrowheads, MCD cells.
UT-A. Up to F15, there was no UT-A immunoreactivity in the developing uriniferous tubules, including the collecting ducts ( Fig. 6, A and B ). UT-A immunoreactivity appeared first on F16 in some of the developing DTLs ( Fig. 7 A ). UT-A was detected in the terminal part of the AQP1-positive DTL, which was directly connected to the AQP1-negative TAL. By the P1, many DTLs express high levels of UT-A ( Figs. 6, C - E, and 7, B and C ). In 4- and 7-day-old pups, UT-A immunoreactivity was seen in areas corresponding to the future medullary ray and outer medulla ( Figs. 6, F and G, and 7 D ). From P14 onward, strong UT-A immunoreactivity was seen in the short-looped DTLs that formed bundles in the middle part of the inner stripe of the outer medulla ( Figs. 6, H - I, and 7, E - G ). On the other hand, the intensity of UT-A immunoreactivity in the shorter long-looped DTLs, which were located at the site of the future innermost part of the inner stripe of the outer medulla, was markedly decreased.
In developing MCDs, UT-A was first detected in the papilla on F18 ( Figs. 6 D and 8, A and B ). The level of UT-A expression in the papilla increased dramatically after birth ( Fig. 6, E - I ) showing subcellular distribution seen in adults ( Fig. 8, C - E ). Thus UT-A in the DTL (presumably UT-A2) is expressed a few days ahead of UT-A1 in the MCD.
While the distribution of UT-A in the MCD and of DTLs was quite similar in the mouse and rat, the expression of UT-A started several days earlier in the mouse compared with the rat ( 11 ). This correlates with the earlier expression of NKCC2, TonEBP, and AR (see above), suggesting that hypertonicity in the renal medulla develops earlier in the mouse compared with the rat.
Effects of furosemide on renal expression of NKCC2, TonEBP, AR, and UT-A in neonatal mice. The sequence of expression of NKCC2 and TonEBP shown above suggests that hypertonicity created by the activity of NKCC2 drives the expression of TonEBP. To test this directly, NKCC2 was inhibited in neonatal animals by daily injections of furosemide for 7 days. We noticed that kidneys and bodies ( Fig. 9, Table 1 ) of animals treated with furosemide were considerably smaller. In addition, NKCC2-positive TALs were seen in the entire papillae of furosemide-treated animals ( Fig. 9 E ) while they were not seen in the middle to the terminal portions of vehicle-treated animals ( Fig. 9 A ). It appears that furosemide prevented the growth and development of the kidney. At any rate, expression of TonEBP was dramatically reduced in the papilla, especially in the nuclei of interstitial cells and MCD by the furosemide treatment ( Fig. 9, F, F' and F' ). Similarly, expression of AR and UT-A also decreased considerably ( Fig. 9, G, G', H, and H' ). These data demonstrate that the activity of NKCC2 is required for expression of TonEBP via generation of hypertonicity in the renal medulla.
Table 1. Effects of furosemide on body weight and kidney size
DISCUSSION
This is the first report of NKCC2 expression in fetal stages, as previous studies examined only postnatal animals ( 12, 17 ). NKCC2 is detected first on F14, in agreement with expression of its mRNA on F14.5 ( 7 ). By F18, the NKCC2-expressing tubules cover the entire length of the renal medulla and lower part of the cortex. The expression of NKCC2 is followed by that of TonEBP and its target genes AR, UT-A in DTL (presumably representing UT-A2), and UT-A1 in IMCD (see summary in Fig. 10, top ). When the activity of NKCC2 is inhibited in neonatal animals, expression of TonEBP and its target genes is dramatically reduced. These data are consistent with the notion that hypertonicity generated by sodium reabsorption via NKCC2 drives expression and activation of TonEBP in the course of renal development.
Fig. 10. Diagrams summarizing the expression profiles of NKCC2, TonEBP, AR, and UT-A in developing mouse and rat kidneys. Nuclear shift of TonEBP after birth is indicated. VE, vascular endothelial cells; CD, collecting duct cells; lDTL, long-looped descending thin limb; sDTL, short-looped descending thin limb.
While all the NKCC2-/- mice die before weaning due to severe dehydration, renal insufficiency, and electrolyte imbalance, they can be rescued by treatment with indomethacin ( 18 ). Adult NKCC2-/- mice display a phenotype similar to wild-type animals treated with furosemide, i.e., severe polyuria and electrolyte imbalance, except that hydronephrosis is more severe. In fact, they have virtually no medulla, much like TonEBP-/- mice ( 14 ). The medullary atrophy in TonEBP-/- mice is due to the failure of cellular adaptation to hypertonicity. This cannot explain the medullary atrophy in NKCC2-/- animals or wild-type animals treated with furosemide because these animals have limited capacity to generate the medullary hypertonicity. The data in Fig. 9 provide new insight. Furosemide prevents development of thin limbs and the disappearance of the TAL in the papilla, suggesting that the transport activity of NKCC2 is required for the development of the renal medulla. We suggest that medullary hypertonicity and active TonEBP contribute to the process.
The urine concentrating ability is higher in the mouse compared with the rat. This is due to differences in the renal anatomy, including a relatively longer papilla and longer loops of Henle in the mouse ( 1 ). This study adds another factor: earlier expression of NKCC2 and TonEBP during development. We examined development of TonEBP ( 5 ), AR ( 9 ), and UT-A ( 11 ) during renal development in the rat ( Fig. 10, bottom ). The results are similar to what we observed in this study except that the time course is significantly faster in the mouse compared with the rat. NKCC2 is expressed 3-4 days earlier in the mouse. While TonEBP expression is detected first on F15 in both animals, the level of expression during the fetal stage is much higher in the mouse. Expression of AR, UT-A1, and UT-A2 in the mouse starts several days earlier than in the rat. The stronger and earlier expression of TonEBP and UT-A during development is likely a factor contributing to the higher urine concentrating ability of the mouse compared with the rat.
In conclusion, the temporal sequence of expression in NKCC2, TonEBP, and its target genes during kidney development and the effects of furosemide provide strong support for the hypothesis that the hypertonicity produced by NKCC2 is a critical signal in the functional and morphological development of the renal medulla. TonEBP is a major effector carrying out the hypertonicity signal.
GRANTS
This work was supported by the Korea Science and Engineering Foundation (R13-2002-005-01001-0) through the MRC for Cell Death Disease Research Center at The Catholic University of Korea and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-42479.
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作者单位：1 Departments of Anatomy and 2 Internal Medicine, and MRC for Cell Death Disease Research Center, College of Medicine, The Catholic University of Korea, and 3 Department of Anatomy, College of Medicine, Ewha Womans University, Seoul, Korea; and 4 Department of Medicine, University of Maryland, Balti