Expression of urea transporters in the developing rat kidney

Young-Hee Kim1, Dong-Un Kim1, Ki-Hwan Han1, Ju-Young Jung1, Jeff M. Sands2, Mark A. Knepper3, Kirsten M. Madsen4, and Jin Kim1

1 Department of Anatomy, Catholic University Medical College, Seoul, Korea, 137-701; 2 Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322; 3 Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-0951; and 4 Department of Medicine, University of Florida, Gainesville, Florida, 32610-0224


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Urea transport in the kidney is mediated by a family of transporter proteins that includes renal urea transporters (UT-A) and erythrocyte urea transporters (UT-B). Because newborn rats are not capable of producing concentrated urine, we examined the time of expression and the distribution of UT-A and UT-B in the developing rat kidney by light and electron microscopic immunocytochemistry. Kidneys from 16-, 18-, and 20-day-old fetuses, 1-, 4-, 7-, 14-, and 21-day-old pups, and adult animals were studied. In the adult kidney, UT-A was expressed intensely in the inner medullary collecting duct (IMCD) and terminal portion of the short-loop descending thin limb (DTL) and weakly in long-loop DTL in the outer part of the inner medulla. UT-A immunoreactivity was not present in the fetal kidney but was observed in the IMCD and DTL in 1-day-old pups. The intensity of UT-A immunostaining in the IMCD gradually increased during postnatal development. In 4- and 7-day-old pups, UT-A immunoreactivity was present in the DTL at the border between the outer and inner medulla. In 14- and 21-day-old pups, strong UT-A immunostaining was observed in the terminal part of short-loop DTL in the outer medulla, and weak labeling remained in long-loop DTL descending into the outer part of the inner medulla. In the adult kidney, there was intense staining for UT-B in descending vasa recta (DVR) and weak labeling of glomeruli. In the developing kidney, UT-B was first observed in the DVR of a 20-day-old fetus. After birth there was a striking increase in the number of UT-B-positive DVR, in association with the formation of vascular bundles. The intensity of immunostaining remained strong in the outer medulla but gradually decreased in the inner medulla. We conclude that the expression of urea transporters in short-loop DTL and DVR coincides with the development of the ability to produce a concentrated urine.

UT-A; UT-B; immunohistochemistry; urine concentration


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IN MAMMALS, UREA IS THE MAJOR end product of nitrogen metabolism, and its transport in the kidney is important for the formation of a concentrated urine (1, 9, 13, 23). Urea is a small molecule with a molecular mass of 60 Da. It has a low lipid solubility, and, in the absence of specific transporters, it crosses cell membranes slowly by passive diffusion. In the kidney, the movement of urea through cells of the inner medullary collecting duct (IMCD), descending thin limb (DTL) of Henle's loop, and descending vasa recta (DVR) proceeds by "facilitated" transport mediated by specific transport proteins located in the cell membrane (23). Specific urea transporters are also present in other mammalian cells including erythrocytes and hepatocytes (23).

The urea transporter family includes two main groups, renal urea transporters (UT-A) and erythrocyte urea transporters (UT-B), which are encoded by different genes (23). The cDNA of five isoforms of rat UT-A, UT-A1 (26), UT-A2 (27, 39), UT-A3 (11), UT-A4 (11), and UT-A5 (7) have been cloned. They are thought to be variants of mRNA splicing from a single gene. UT-A1 is the longest isoform, and the others are composed of part of UT-A1 (7, 11). The cDNA of UT-B was first cloned from a human bone marrow library (19) and subsequently isolated from a rat inner medullary library by homology screening (3, 35).

UT-A1-A4 are predominantly expressed in the kidney, whereas UT-A5 is expressed in the testes. UT-A1 and UT-A3 are localized in the IMCD (18, 24, 32), and UT-A2 is located in the DTL of Henle's loop (24, 36). The precise segmental distribution of UT-A4 has not been elucidated. UT-B mRNA and protein are expressed in the endothelial cells of the vasa recta (19, 35, 37).

It is known that newborn rats are not capable of concentrating their urine to adult levels (28). Urine osmolality of neonatal rats rises from 300 mosmol/kgH2O at birth to ~2,000 mosmol/kgH2O by 3 wk of age (6, 22, 38). The accumulation of urea in the inner medullary interstitium is important for the generation of a concentrated urine. However, little is known about the expression and distribution of urea transporters in the fetal and neonatal kidney.

The purpose of this study was to examine the time of expression and the distribution of UT-A and UT-B in the developing rat kidney by light and electron microscopic immunostaining methods using rabbit polyclonal antibodies to UT-A and UT-B. Because the antibody against UT-A recognizes UT-A1, UT-A2, and UT-A4, it was not possible to distinguish between the different splice variants of UT-A.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and Tissue Preservation

Sprague-Dawley rats were used in all experiments. Prenatal kidneys were obtained from 16-, 18- and 20-day-old fetuses. Postnatal kidneys were obtained the day of birth and from 4-, 7-, 14- and 21-day-old animals. For each age group, animals from two separate litters were used. Kidneys from adult male rats served as a positive reference for the immunohistochemical studies. The kidneys from prenatal and neonatal animals, up to and including those 14 days of age, were preserved by in vivo perfusion through the heart, whereas kidneys from 3-wk-old and adult animals were perfused through the abdominal aorta. The kidneys were initially perfused briefly with PBS to rinse away all blood. This was followed by perfusion with a periodate-lysine-2% paraformaldehyde solution for 10 min. The kidneys were removed and cut into 1- to 2-mm-thick slices that were fixed additionally by immersion in the same fixative for 2 h at room temperature and then overnight at 4°C. Sections of tissue were cut transversely through the entire kidney on a Vibratome (Pelco 101, sectioning series 1000, Technical Products, St. Louis, MO) at a thickness of 50 µm and processed for immunohistochemical studies using the horseradish peroxidase preembedding technique.

Antibodies

To determine the distribution of urea transporters in the developing rat kidney, we used specific rabbit polyclonal antibodies against peptides based on the rat renal urea transporter UT-A (L403; recognizes UT-A1, UT-A2 and UT-A4) (31, 36) and UT-B (33), the human erythrocyte urea transporter. A rabbit polyclonal antibody against aquaporin-1 (AQP1) (30) was used for colocalization with UT-A in the DTL. The antibodies have been characterized in detail in previous studies.

Immunoperoxidase Preembedding Method

Vibratome sections (50 µm thick) were washed with 50 mM NH4Cl in PBS three times for 15 min. Before incubation with the primary antibodies, all tissue sections were incubated for 3 h with PBS containing 1% bovine serum albumin, 0.05% saponin, and 0.2% gelatin (solution A). The tissue sections were then incubated overnight at 4°C in rabbit antisera against UT-A (1:500) and UT-B (1:2,000) in PBS containing 1% bovine serum albumin (solution B). After several washes with solution A, the tissue sections were incubated for 2 h in peroxidase-conjugated donkey anti-rabbit IgG Fab fragment (Jackson ImmunoResearch Laboratories) diluted 1:100 in solution B. The tissues were then rinsed, first in solution A and subsequently in 0.05 M Tris buffer, pH 7.6. For the detection of horseradish peroxidase, the sections were incubated in 0.1% 3,3'-diaminobenzidine in 0.05 M Tris buffer for 5 min, after which H2O2 was added to a final concentration of 0.01% and the incubation was continued for 10 min. After being washed with 0.05 M Tris buffer three times, the sections were dehydrated in a graded series of ethanol and embedded in poly/Bed 812 resin (Polysciences, Warrington, CA). For UT-A and AQP1 double immunostaining, Vibratome sections were labeled with the antibody against UT-A using 3,3'-diaminobenzidine as the chromogen (brown) as described above. The sections were then rinsed with PBS, incubated for 1 h in solution B, and then incubated overnight at 4°C in rabbit antisera against AQP1 (1:500) in solution B. After several washes with 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 B. The tissues were then rinsed in PBS, pH 7.4, and, for the detection of horseradish peroxidase, the sections were incubated in vector SG (blue; Vector Laboratories) in PBS for 5 min, followed by H2O2 incubation for 10 min. After being washed with PBS three times, the sections were dehydrated in a graded series of ethanol and embedded in poly/Bed 812 resin.

For light microscopy, 1-µm sections were cut, the plastic resin was removed by etching with a saturated solution of sodium hydroxide in alcohol, and the sections were stained with hematoxylin. The sections (50 and 1 µm) were examined and photographed with an Olympus photomicroscope equipped with differential-interference contrast optics.

For electron microscopic observation, Vibratome sections were postfixed with 1% glutaraldehyde and 1% osmium tetroxide in 0.1 M phosphate buffer, pH 7.4, before being dehydrated and embedded in poly/Bed 812 resin. Ultrathin sections were stained with uranyl acetate and lead citrate and photographed with a transmission electron microscope (JEOL 1200EX, Tokyo, Japan).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Expression of UT-A

Adult rat kidney. Light microscopy of 50-µm sections demonstrated strong UT-A immunoreactivity only in the inner stripe of the outer medulla (ISOM) and inner medulla. There was no labeling in the cortex and outer stripe of the outer medulla (Fig. 1). At higher magnification of 1-µm sections, the strongly labeled tubular profiles in the ISOM were identified as the terminal portion of short-loop DTL by the abrupt transition to the unlabeled thick ascending limb (TAL) (Fig. 2A). In the inner medulla, there was intense UT-A immunostaining of the middle and terminal IMCD but only weak or an absence of labeling in the initial IMCD (Fig. 2, B and C). Weak labeling for UT-A was also observed in the DTL of long-loop nephrons close to the border between the outer and inner medulla (Figs. 1 and 2B). To confirm that only the DTL of short-loop nephrons are UT-A positive in the ISOM, electron microscopy was carried out. Strong UT-A labeling was located in the apical and basolateral plasma membrane as well as the cytoplasm of the type I epithelium of the short-loop DTL, which is composed of very flat and noninterdigitating cells without apical microvilli (Fig. 3). There was no labeling of the type II epithelium of the long-loop DTL.


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Fig. 1.   Light micrograph of a 50-µm-thick Vibratome section (A) and a schematic diagram (B) from an adult rat kidney illustrating immunostaining for the renal urea transporter (UT-A). Strong UT-A immunoreactivity was present in the terminal portion of short-loop descending thin limb (DTL; solid arrow; A) in the inner stripe (IS) of outer medulla, and weak labeling was observed in long-loop DTL (open arrows) located at the border between outer and inner medulla (IM). Strong UT-A immunoreactivity was also present in the inner medullary collecting duct (IMCD; arrowheads), where the intensity of the labeling gradually increased toward the papilla tip. Co, cortex; OS, outer stripe of outer medulla. Magnification: ×33.



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Fig. 2.   Light micrographs of 1.5-µm-thick section from IS of outer medulla (A), initial part of IM (B), and middle part of IM (C) of adult rat illustrating immunostaining for UT-A. A: there was strong UT-A immunoreactivity in short-loop DTL(*); note the abrupt transition to the unlabeled thick ascending limb (TAL; curved thick arrows). B: in the initial part of IM, weak UT-A immunoreactivity was present in principal cells (small thin arrows) as well as long-loop DTL (*). IMCDi, initial IMCD. C: there was stronger immunoreativity for UT-A in the middle part of the IMCD (IMCDm). Magnification: ×400 (A-C).



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Fig. 3.   Transmission electron microscopic localization of UT-A in the IS of outer medulla of adult rat kidney. A: strong UT-A immunoreactivity was observed in the apical and basolateral plasma membrane as well as the cytoplasm of type I epithelium (I) of short-loop DTL. In contrast, there was no UT-A immunoreactivity in the type II epithelium (II) of long-loop DTL. AVR, ascending vasa recta. B: curved arrow indicates the abrupt transition from short-loop DTL, labeled with UT-A, to unlabeled TAL. Magnification: ×4,350 (A); ×7,800 (B).

Developing rat kidney. Before birth, there was no UT-A immunoreactivity in the developing uriniferous tubules, including collecting ducts (Fig. 4A). UT-A immunoreactivity was first observed in the terminal part of medullary collecting ducts and in a few DTL in the base of the renal papilla immediately after birth (Fig. 4). During postnatal renal development, the intensity of UT-A immunolabeling gradually increased in both IMCD and DTL (Figs. 4 and 5). Labeling was strong in the terminal IMCD and was observed only in the apical region of IMCD cells throughout development.


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Fig. 4.   Light micrographs of 50-µm-thick Vibratome sections from kidneys of an 18-day-old rat fetus (A) and 1 (B)- and 4-day-old (C-E) rat pups illustrating immunoreactivity for UT-A. A: there was no UT-A immunoreactivity in the developing uriniferous tubules, including collecting ducts (star ) of 18-day-old fetus. B: UT-A immunoreactivity first appeared in the terminal part of medullary collecting ducts (star ) and some DTL of Henle's loop (arrows in inset, which is higher magnification of rectangular area) in the base of renal papilla. C: UT-A immunoreactivity was increased in IMCD and DTL of Henle's loop of 4-day-old pup compared with those of 1-day-old pup. D and E: higher magnification of insets D and E in C demonstrating UT-A-positive DTL (*). Magnification: ×100 (A-C); ×400 (D and E).



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Fig. 5.   Light micrographs of 50-µm-thick Vibratome sections from kidneys of 7 (A)-, 14 (B)-, and 21-day-old (C) rat pups illustrating UT-A immunostaining. A: UT-A immunoreactivity was present only in IMCD in the terminal half of renal papilla (arrowhead) and in the DTL of immature loops of Henle (open arrow) located at the border between outer medulla and IM at 7 days after birth. B: UT-A immunoreactivity appeared in the terminal part of DTL of short loop of Henle (arrow) in the IS of outer medulla at 14 days after birth. Note strong immunoreactivity in the DTL of long loop of Henle (open arrow) and IMCD (arrowhead). C: in 21-day-old pups, immunoreactivity for UT-A was increased in the DTL of short loop of Henle (arrow). In contrast, the intensity of immunoreactivity in long-loop DTL (open arrow) was markedly decreased. Magnification: ×20 (A-C).

By postnatal day 7, loops of Henle with the structural characteristics of short-loop nephrons were present at various levels in the renal medulla. UT-A immunolabeling was observed in the terminal part of DTL of immature loops of Henle located in the base of renal papilla, which corresponds to the ISOM of the adult kidney (Fig. 5A). There was no UT-A immunolabeling in the terminal part of the DTL located in the renal papilla, indicating that the UT-A immunoreactivity disappeared as the loops of Henle descended into the papilla. A striking increase in the number of UT-A-labeled DTL and in the intensity of immunostaining was observed in the ISOM in 14-day-old pups (Fig. 5B). The UT-A-positive DTL in the ISOM belonged to short-loop nephrons. There was no labeling of the inner stripe portion of many of the long-loop DTL. However, weak UT-A immunostaining was now present in some of the long-loop DTL in the inner medulla at the base of the papilla, indicating that some immunoreactivity remained in this part of the DTL as the loops descended from the outer to the inner medulla. In the kidney of 21-day-old pups, UT-A was increased in short-loop DTL in the ISOM and decreased in long-loop DTL in the inner medulla, and the pattern of immunoreactivity was similar to that observed in adults (Fig. 5C).

UT-A and AQP1 colocalization. To determine whether UT-A and AQP1 were expressed in the same DTL segments, double-labeling experiments were performed. In adult kidney, AQP1 was expressed in the DTL of both long- and short-loop nephrons. However, there was no AQP1 immunoreactivity in the UT-A-positive part of short-loop DTL in the ISOM. In the inner medulla, however, DTL with weak UT-A immunoreactivity also expressed AQP1 (Fig. 6, A-C). In 4-day-old pups, AQP1 was mainly present in DTL of more mature long-loop nephrons, whereas immature long-loop nephrons located in the area corresponding to the future ISOM had AQP1 only in the proximal part and UT-A in the distal part of the DTL (Fig. 6D). As the DTL of long-loop nephrons descend toward the inner medulla, UT-A immunoreactivity decreased and became restricted to the border between the outer and inner medulla in the adult kidney whereas AQP1 immunoreactivity increased (Fig. 6, E and F).


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Fig. 6.   Light micrographs of 50-µm-thick Vibratome sections from adult rat kidney (A-C) and 4 (D)- and 7-day-old (E and F) rat pups illustrating double immunolabeling for UT-A (brown) and aquaporin-1 (AQP1; blue). B and C: higher magnification of IS of outer medulla and initial part of inner medulla, respectively. Short-loop DTL (*) express UT-A (brown) but not AQP1, whereas long-loop DTL(star ) in the IS of outer medulla express AQP1 (blue) but not UT-A. Note a UT-A-positive cell (arrow) in the AQP1-positive DTL of long-loop nephron located at the border between outer and inner medulla. D: an immature loop of Henle with AQP1 immunoreactivity in proximal part and UT-A immunoreactivity in distal part of the DTL, which is directly connected to the TAL (star ) without ascending thin limb (ATL). Note strong AQP1-positive DTL of longer loop of Henle. E and F: with increasing length of DTL, number of UT-A-positive cells (arrows) is decreased, and cells without either UT-A or AQP1 are present in the terminal part. Many DTL at these ages are directly connected to a TAL (star ) without an ATL being present. Open arrow indicates the junction of DTL and TAL. Note AQP1-positive DTL of longer loop of Henle. Magnification: ×800 (A-C); ×640 (D-F).

Expression of UT-B

Adult rat kidney. Light microscopy of 50-µm-thick sections demonstrated that UT-B was expressed in DVR and glomeruli (Fig. 7). UT-B labeling in DVR was pronounced in the ISOM and proximal part of inner medulla but was much weaker in the papillary tip. Electron microscopy revealed strong UT-B immunolabeling in the apical membrane and diffuse staining of the cytoplasm in the continuous endothelial cells of DVR (Fig. 8). There was no UT-B immunoreactivity in the pericytes, embedded in the basement membrane of DVR, or in the fenestrated endothelial cells of ascending vasa recta (Fig. 8).


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Fig. 7.   Light micrograph of a 50-µm-thick Vibratome section (A) of an adult rat kidney and a schematic diagram (B) illustrating UT-B immunoreactivity in glomeruli (G) and descending vasa rectae (DVR) in the IS of outer medulla and upper half of the IM. Magnification: ×33.



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Fig. 8.   Transmission electron microscopic localization of UT-B in the IS of outer medulla of adult rat kidney. A and B: UT-B immunostaining was observed in the apical and basolateral plasma membrane and throughout the cytoplasm of the continuous endothelial cells of DVR. However, there was no immunoreactivity in the fenestrated endothelial cells of AVR. Small arrowheads indicate the fenestrae. C: higher magnification of area indicated by rectangle in B. Note processes (arrows) of pericytes in the basement membrane of UT-B-labeled DVR. Magnification: ×4,350 (A and B); ×7,800 (C).

Developing rat kidney. Before birth, UT-B was weakly expressed in the stage III and IV glomeruli and in some capillary endothelial cells in the medulla (Figs. 9A and 10A).


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Fig. 9.   Light micrographs of 50-µm-thick Vibratome sections from kidney of 20-day-old fetus (A) and 1 (B)-, 4 (C)-, 7 (D)-, and 14-day-old (E) pups illustrating UT-B immunostaining. After birth, there was a striking increase in the number of UT-B-positive DVR, in association with the formation of vascular bundles (B-E). Note glomeruli with weak UT-B immunoreactivity in sections from all age groups. Magnification: ×64 (A); ×33 (B-E).



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Fig. 10.   Light micrographs of 50-µm-thick Vibratome sections illustrating UT-B immunoreactivity in renal papilla from kidneys of 20-day-old fetuses (A), and 1 (B)-, 7 (C)-, and 14-day-old (D) pups. UT-B immunoreactivity of the developing DVR (*) in inner medulla is gradually increased in intensity from 20-day-old fetus to 14-day-old pup. Magnification: ×600 (A-D).

During early postnatal stages (1-, 4-, and 7-day-old pups), UT-B was strongly expressed in DVR, and there was a gradual increase not only in the number of UT-B-labeled DVR but also in the intensity of UT-B immunoreactivity during renal development (Figs. 9 and 10). In 14-day-old pups (Fig. 9), UT-B-positive DVR formed vascular bundles in the ISOM, and in 21-day-old pups the pattern and intensity of UT-B immunostaining was similar to those observed in adult animals. There was no labeling in developing uriniferous tubules including collecting ducts.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study represents the first detailed description of the expression and distribution of urea transporters in the developing kidney. Urea transport in the kidney and the accumulation of urea in the renal medulla are of critical importance for the urinary concentrating process and the regulation of water excretion (1, 13, 14, 23). Newborn rats are unable to produce a concentrated urine but develop this capacity during the first 2-3 wk after birth (4, 22, 34, 38). We therefore determined the time of expression of urea transporters and their pattern of distribution in the developing rat kidney.

Our results demonstrate that UT-B immunoreactivity appears in DVR shortly before birth and gradually increases during the first 2-3 wk after birth. It is noteworthy that the UT-B-positive DVR do not form vascular bundles until the animals are 2 wk old. UT-A first appears in the IMCD and in developing long-loop DTL at the base of the renal papilla right after birth. As the UT-A-positive long-loop DTL descend into the renal papilla, UT-A immunoreactivity decreases and is barely detectable in animals after 3 wk of age. In contrast, the expression of UT-A in short-loop DTL does not occur until 2 wk after birth, the time they reach the ISOM, but the level of expression is very strong and remains so in the adult kidney (Fig. 11). UT-A immunoreactivity in the IMCD gradually increased after birth and reached adult levels when the animals were 3 wk of age.


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Fig. 11.   Diagram illustrating changes in UT-A immunostaining in the elongating short and long loops of Henle in the developing rat kidney. Numbers refer to successive generations of loop of Henle. Red, UT-A immunoreactivity in the DTL; blue and green, AQP1 and TAL, respectively. d, Day; OSOM, outer stripe of the outer medulla; ISOM, inner stripe of the outer medulla.

The antibody against UT-A used in this study was raised against a COOH-terminal peptide sequence common to UT-A1, UT-A2, and UT-A4 (31, 36). As reported previously in a detailed study by Wade et al. (36), this antibody labels UT-A1 in the IMCD and UT-A2 in the DTL. The very strong labeling of the IMCD and short-loop DTL in the ISOM observed in our study is similar to the results reported by Wade et al. (36) and also confirms previous observations by Nielsen et al. (18). However, the expression of UT-A2 protein in long-loop DTL has been somewhat controversial. Although UT-A2 mRNA expression was demonstrated in both short-loop DTL in the ISOM and long-loop DTL in the initial inner medulla by RT-PCR of microdissected tubules and by in situ hybridization, previous immunohistochemical studies did not detect any UT-A immunoreactivity in long-loop DTL under normal conditions. The failure to detect UT-A in long-loop DTL is most likely due to the very low abundance of the protein. However, after stimulation of UT-A2 expression by chronic infusion of vasopressin, Wade et al. (36) and Shayakul et al. (25) demonstrated labeling also in long-loop DTL in the initial part of the inner medulla of Brattleboro rats. The high sensitivity of the preembedding method used in our study enabled us to detect UT-A immunoreactivity in long-loop DTL in the base of the renal papilla of normal animals, in a pattern similar to that observed in vasopressin-treated Brattleboro rats (25, 36). The differences in UT-A immunoreactivity along the DTL and the absence of UT-A in the type II epithelium of long-loop DTL in the ISOM, which express high levels of AQP1, are in agreement with results of transport studies demonstrating significant differences in isolated perfused segments of long-loop DTL from chinchilas (2) and hamsters (10). Interestingly, in the developing kidney, the DTL of both long- and short-loop nephrons exhibit strong UT-A immunoreactivity in an area at the base of the renal papilla.

The gradual increase in the expression of UT-A in the IMCD after birth is in agreement with the results of a recent study by Liu et al. (15) demonstrating an increase in urea permeability in the IMCD during postnatal development. Surprisingly, these investigators did not detect UT-A1 mRNA in the IMCD until animals were 14 days of age, although there was an exponential increase in urea permeability from postnatal day 1 until postnatal day 14. These observations raise the possibility that another urea transporter, possibly UT-A4, may be responsible for the UT-A immunoreactivity and urea permeability demonstrated in the neonatal IMCD.

The loops of Henle from juxtamedullary and superficial nephrons descend through the renal medulla at different times during kidney development, from before birth until 2-3 wk after birth (Fig. 11). Loops from juxtamedullary nephrons descend first to form the long loops of Henle, and those from the more superficial nephrons descend subsequently to form the short loops of Henle. Our results demonstrate that UT-A appears in the DTL of both long- and short-loop nephrons when they enter the area corresponding to the future ISOM, located at the border between the outer and inner medulla. As the long loops from juxtamedullary nephrons continue their descent into the inner medulla, UT-A immunoreactivity gradually decreases and finally disappears from long-loop DTL, except for those segments located in the initial part of the inner medulla adjacent to the ISOM. These observations suggest that local factors in the area corresponding to the ISOM play a role in the induction of UT-A2 in DTL in the developing kidney.

There is increasing evidence that urea transporters belonging to the UT-A family are regulated by vasopressin as well as changes in osmolality (17, 21, 23, 25, 36). Recent studies have demonstrated that vasopressin stimulates UT-A2 expression in both long- and short-loop DTL (25, 36). Whether this represents a direct effect of vasopressin on DTL or is secondary to activation of vasopressin receptors in adjacent structures has not been established. There is no evidence so far that vasopressin receptors are expressed in DTL (8, 29). However, V2 vasopressin receptors are expressed in the adjacent TAL (8, 29). Vasopressin is known to stimulate the reabsorption of sodium in the TAL, which leads to increased sodium content in the medullary interstitium and increased interstitial osmolality. Thus it is possible that an increase in interstitial sodium and/or osmolality might play a role in the induction of UT-A2 in the DTL, in particular during development of the renal medulla, when there is a close association between the DTL and the TAL. In this regard, it is noteworthy that at birth all loops of Henle have the structural characteristics of short loops. The DTL continue directly into the TAL, and there are no ascending thin limbs (12). Thus TAL are present throughout the renal papilla in close proximity to the DTL. During the first 2-3 wk of life, cells are deleted by apoptosis from the TAL in the inner medulla and the remaining TAL cells are tranformed into ascending thin limb cells (12). Thus the close association between the DTL and the TAL is lost in the inner medulla. Interestingly, at the same time the expression of UT-A2 decreases or disappears from long-loop DTL in the inner medulla, with the exception of those located close to the ISOM. This observation suggests that the close association with the TAL might be important for the expression of UT-A2.

A recent study by Nakayama et al. (16) demonstrated consensus sequences for the cAMP response element in promoter II of the UT-A2 gene, suggesting that cAMP might play a role in the regulation of UT-A2. In this regard, it is noteworthy that a gradual increase in osmolality has been shown to have a biphasic effect on adenylate cyclase in the papillary collecting duct, increasing adenylate cyclase activity at 800 mosM and inhibiting the activity at 2,000 mosM (5). An increase in osmolality also inhibited cAMP-phosphodiesterase activity (5). Whether changes in osmolality have similar effects on cAMP metabolism in the DTL remains to be established.

The antibody against UT-B used in this study was characterized in detail recently by Timmer et al. (33). As demonstrated by those investigators and confirmed by us, the expression of UT-B in the kidney was restricted to the continuous endothelium of the DVR in the outer and inner medulla, and there was no labeling of the fenestrated endothelium of the ascending vasa recta. These observations are also in agreement with results of previous in situ hybridization and immunohistochemical studies demonstrating expression of UT-B in vasa recta in both outer and inner medulla of the rat kidney (35, 37). The results of these studies indicate that UT-B is responsible for the high urea permeability demonstrated in microperfused DVR (20). The presence of UT-B in the plasma membrane of the DVR allows urea in the medullary interstitium to enter the DVR. When blood leaves the inner medulla via ascending vasa recta, urea can exit these vessels through the UT-B-negative fenestrated endothelium.

Our studies in the developing kidney revealed that UT-B is expressed in the DVR already before birth and gradually increases during the first 2 wk after birth, indicating that countercurrent exchange between ascending vasa recta and DVR can occur already in the neonatal kidney. Interestingly, the formation of vascular bundles coincided with the appearance of UT-A2-positive short-loop DTL in the ISOM and was followed by a striking increase in the expression of UT-A2 and UT-B and a clear delineation of the ISOM. We conclude that the expression of urea transporters in the neonatal rat renal medulla during the first 2 wk after birth coincides with the development of the ability to produce a concentrated urine. The striking increase in UT-A2 and UT-B immunoreactivity 2 wk after birth is closely associated with the descent of short-loop DTL from superficial nephrons and the formation of vascular bundles in the ISOM.


    ACKNOWLEDGEMENTS

We gratefully acknowledge the technical assistance of Hee-Duk Rho and Kyung-A Ryu.


    FOOTNOTES

These studies were supported by Korea Research Foundation Grant KRF-98-005-F00127. They were presented in part at the annual meeting of the American Society of Nephrology held in Miami, FL, in November 1999 and have been published in abstract form (J Am Soc Nephrol 10: 406A, 1999).

Address for reprint requests and other correspondence: J. Kim, Dept. of Anatomy, Catholic University Medical College, 505 Banpo-Dong, Socho-Ku, Seoul 137-701, Korea (E-mail: jinkim{at}cmc.cuk.ac.kr).

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.

10.1152/ajprenal.00246.2001

Received 6 August 2001; accepted in final form 25 October 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Renal Fluid Electrolyte Physiol 282(3):F530-F540