Department of Anatomy, Cell Death Disease Research Center, The Cathoilc University of Korea;

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1 Articles in PresS. Am J Physiol Renal Physiol (October 5, 2004). doi: /ajprenal Expression of epidermal growth factor in the developing rat kidney Ju-Young Jung, 1 Ji-Hyun Song, 1 Can Li, 2 Chul-Woo Yang, 2 Tae-Chun Kang, 1 Moo-Ho Won, 1 Young-Gil Jeong, 3 Ki-Hwan Han, 4 Kyu-Bok Choi, 5 Seung-Hun Lee, 2 Jin Kim, 4 1 Department of Anatomy, College of Medicine, Hallym University, Chuncheon, Korea; 2 Department of Internal Medicine, College of Medicine, The Catholic University of Korea; 3 Department of Anatomy, College of Medicine, Konyang University, Nonsan, Korea; 4 Department of Anatomy, Cell Death Disease Research Center, The Cathoilc University of Korea; 5 Department of Internal Medicine, College of Medicine, Ewha Women s University, Seoul, Korea; Running title: EGF in developing kidney Address for correspondence: Seung-Hun Lee, M.D. Division of Renal Diseases Department of Internal Medicine The Catholic University of Korea 665, Bupyong-Dong, Bupyong-Gu, Incheon, , Korea Tel : , Fax : henle@korea.com Copyright 2004 by the American Physiological Society.

2 Abstract Epidermal growth factor (EGF) is important in mammalian renal development. In our study we investigated the detailed distribution and the time of the first appearance of EGF in developing rat kidney. Kidneys from 18-(E18) and 20-day-old (E20) fetuses, 1- (P1), 3- (P3), 7- (P7), 14- (P14), and 21-day-old (P21) pups and adults were processed for immunohistochemistry and electronmicroscopy. In adult rat kidney, EGF immunoreactivity was found in distal tubule including thick ascending limb (TAL) and portion 1 of distal convoluted tubule (DCT1), whereas no EGF immunoreactivity was seen in the portion 2 of distal convoluted tubule (DCT2) and connecting tubule. In developing kidney, EGF-positive cells first appeared at P3 and were localized in the middle portion of the differentiating TAL of the corticomedullary junction. By P7 the abundance of EGF expression had dramatically increased in the medullary TAL. Between 14 and 21 days postnatal, EGF immunoreactivity was found in the TAL and the DCT for the first time. However, EGF-positive and EGF-negative cells were in the TAL in developing rat kidney. EGF-positive cells did not differ from negative cells in the expression of sodium transport proteins or in the proliferation rate at P3 and P7. In the TAL, smooth-surfaced cells had strong EGF-immunoreactivity, but no EGF-immunoreactivity was seen in the rough-surfaced cells with well-developed microvilli. Our results suggest that the expression of EGF in developing kidney plays important role in the regulation of growth and differentiation of the loop of Henle during kidney development, and that may act as paracrine mode. Keywords: Development; EGF; Thick Ascending Limb; Proliferation

3 Introduction Epidermal growth factor (EGF) is a 53-amino-acid polypeptide which has been isolated and characterized by Cohen from the mouse salivary gland (6). EGF mrna has been identified in mammalian kidney tissue, and EGF is present in relatively high concentration in urine (22). EGF and EGF mrna levels become also detectable in rodents during the neonatal period. The role of EGF in the developing kidney is unclear. However, a mitogenic role for tubular cells has been proposed. EGF is a potent mitogenic factor for the proximal tubule epithelium, and appears to function as a survival factor in renal cells in the developmentally mature kidney by preventing apoptosis (15). EGF also induces branching of the isolated ureteric bud and stimulates the morphogenesis of the collecting duct (14, 30). It also plays an important role in tubular cystic formation, tubular hyperplasia, and segmental differentiation resulting from the tyrosine kinase activity of EGF-Receptor during renal development (8, 11, 18, 26). Gattone et al. (12) reported that EGF immunoreactivity was first detected in the developing mouse distal nephron at day 6 after birth. A previous study from our laboratory demonstrated that numerous BrdU-positive cells were found in 5HT1A positive thick ascending limb (TAL) of the outer medulla and medullary rays at P3 and P7 (5). The TAL is one of main sites of cell proliferation and differentiation in the developing kidney. However, it is unknown whether the role of EGF is for proliferation during tubule maturation. While it is well established in adult animals that EGF is expressed in both the thick ascending limb and the distal convoluted cells, little is known about the expression and the precise distribution of EGF in the developing rat kidney, and there is no information at all about the ultrastructural localization of expressed EGF in the differentiating loop of Henle. Our study was therefore designed to establish the time of expression and the pattern of distribution of EGF in the developing rat kidney, and to identify which cells are EGF-positive and which EGF-negative in the developing loop of Henle.

4 Materials and Methods Animals and Bromo-2 -Deoxy-Uridine Treatment Sprague-Dawley rats were used in all experiment. The kidneys were obtained from 18- (E18) and 20-day-old (E20) fetuses, and 1- (P1), 3- (P3), 7- (P7), 14- (P14), and 21-day-old (P21) pups and adults. The proliferation rate of EGF-labeled cells was measured by administering a single injection of 50ug/g body weight of 5-bromo-2 -deoxy-uridine (BrdU; Boehringer Mannheim GmbH, Mannheim, Germany) to P3 and P7 animals 18 h prior to sacrifice; the kidneys were subsequently preserved for immunohistochemistry. BrdU is a thymidine analog incorporated into DNA during the S phase of the cell cycle and can be subsequently detected in tissue sections with specific antibodies raised against BrdU (29). Tissue preservation The animals were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg body wt). The kidneys were preserved by in vivo perfusion heart or abdominal aorta. The animals were initially perfused with phosphate buffered saline (PBS, osmolality 298 mosm/kg H 2 O, ph 7.4) to wash out the blood. This was followed by perfusion with a periodate lysine-2% paraformaldehyde (PLP) solution for 5 min. After perfusion, the kidneys were removed and cut into 1 2 mm thick slices that were further fixed by immersion in PLP solution overnight at 4 C. Sections for immunoperoxidase pre-embedding staining were cut transversely through the entire kidney on a Vibratome Pelco 101, Sectioning series (Technical Products International, St. Louis, MO) at a thickness of 50 µm and processed for immunohistochemical studies by use of a horseradish peroxidase preembedding technique. Antibodies EGF immunoreactivity was detected using an affinity-purified rabbit polyclonal antibody against

5 EGF (Biosource Technologies, Inc. Vacaville, CA). The TAL was identified by use of rabbit polyclonal antibody against the Na/K/Cl cotransporter (NKCC2, BSC-1; courtesy of Dr. Mark A. Knepper, National Institutes of Health). The antibody labels the apical plasma membrane of thick ascending limb (10). The macula densa of the loop of Henle (MD) were identified by use of rabbit polyoclonal antibody raised against a neuronal nitric oxide synthase (nnos; Sigma, St. Louis, MO). Expression of nnos in the rat kidney has been studied in detail in previous studies (3, 4). The distal convoluted tubule (DCT) was identified by use of thiazide-sensitive Na/Cl cotransporter (TSC; courtesy of Dr. Mark A. Knepper, National Institutes of Health). The antibody labels the apical membrane of all DCT involved DCT1 and DCT2 (17). Calbindin D28k (CaBP; Sigma, St. Louis, MO) was also used for DCT2 and connecting tubule segment (20, 27). For the detection of BrdU, a mouse onoclonal antibody against BrdU (Boehringer Mannheim) was used. Immunolabeling of EGF Fifty-µm vibratome sections were processed for immunohistochemistry using an indirect pre-embedding immunoperoxidase method. All sections were washed with 50 mm NH 4 Cl in PBS three times for 15 min. Before incubation with the primary antibody, the sections were pretreated with PBS containing 1% BSA, 0.05% saponin, and 0.2% gelatin (solution A) for 3 h. The tissue sections were then incubated overnight at 4 C with antibodies to EGF (1: 250) diluted in 1% BSA in PBS (solution B). Control incubations were made using solution B without the primary antibody. After three washes in solution A, the sections were incubated for 2 h in peroxidase-conjugated donkey anti-goat or anti-rabbit IgG Fab fragment (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted 1:100 in solution B. The tissues were then rinsed, first in solution A and then in 0.05 M Tris buffer (ph 7.6). Horseradish peroxidase was detected by incubating the sections in 0.1% 3,3'-diaminobenzidin (DAB) in 0.05 M Tris buffer for 5 min, after which H 2 O 2 was added, to a final concentration of 0.01%, and the incubation was continued for 10 min. After washing with 0.05 M Tris

6 buffer, the sections were dehydrated through a graded series of ethanols and embedded in poly/bed 812 Resin (Polyscience Inc., Warrington, PA). The sections were examined and photographed with a photomicroscope (Olympus, Japan). Preembedding staining of NKCC2 was also performed using same method for doublelabeling for BrdU. Immunolabeling of Bromo-2 -Deoxy-Uridine From flat-embedded 50-µm-thick Vibratome sections of kidney were processed for double-labeling of EGF and BrdU, or NKCC2 and BrdU, in the thick ascending limb of the loop of Henle. Portions of the cortex and outer medulla were excised and glued onto empty blocks of Epon 812. One micron thick sections were cut and treated for 10 to 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 through graded ethanols and rinsed in tap water. To improve DNA accessibility, the sections were treated with 20 µg/ml proteinasek (Fisher Biotech) containing 10 mm ethylenediaminetetraacetate and 10 mm NaCl in 0.05 M Tris buffer (ph 7.8) in a humidified chamber for 5 min at room temperature. This was followed by treatment with 0.2% glycine for 5 min to inhibit the enzyme. After rinsing in tap water for 10 min, the sectionswere incubated for 30 min with H 2 O 2 in methanol, rinsed again in tap water for 10 min, and treated with 0.5% triton X-100 in PBS for 15 min. The sections were then rinsed in PBS three times for 10 min before being treated with 1% BSA for 1 h. The sections were incubated with mouse anti-brdu antibody (1:60) at 4 C. After being washed in PBS, the sections were incubated for 2 h in a mixture of peroxidase-conjugated goat anti-rabbit IgG and peroxidase-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories). Vector SG (Vector Laboratories, Burlingame, CA) was used as the chromogen to detect the peroxidase; this gives a gray-blue color, which is easily distinguished from the brown staining produced by 3,3'-diaminobenzidine used in the pre-embedding procedure for detection of EGF and NKCC2. The sections were washed with distilled water, dehydrated through graded ethanol and xylene, mounted in

7 Canada balsam, and examined by light microscopy. Transmission electron microscopy Electron microscopic observations were made on Vibratome sections 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 using a transmission electron microscope (JEOL 1200EX, Tokyo, Japan). Immunohistochemistry for Identification of the loop of Henle The PLP-fixed kidneys were dehydrated through a graded series of ethanol, and embedded in wax (polyethylene glycol 400 disterate; Polysciences, Inc., Warrington, PA). The wax-embedded tissue was cut serially at 2-µm on a rotary microtome (Leica, Germany), and the sections were dewaxed and rehydrated. The sections were then rinsed with PBS, incubated in normal donkey serum (Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h, and incubated overnight at 4 C with each antibody (NKCC2; 1:200, nnos; 1:10,000, TSC; 1:3000, CaBP D28K; 1:5,000). The sections were washed in PBS, and incubated for 2 h with biotinylated secondary antibody, then with Vectastain ABC reagent for 2 h. The sections were rinsed with PBS, incubated with the peroxidase substrate solution, a mixture of 0.05% 3,3'-diaminobenzidine and 0.01% H 2 O 2, for 2 min at room temperature. The sections were finally rinsed with Tris-HCl buffer, dehydrated through graded ethanols, cleared in xylene, and mounted in Canada balsam for light microscopy examination. Quantitation of BrdU-positive cells TAL cells, identified by NKCC2, were counted in one micron thick (semithin) sections of the cortex and outer medulla from P3 and P7 animals and expressed as percentage of the total number of NKCC2-positive cells containing BrdU-labeled nuclei. In other sections, EGF-positive cells with BrdU-labeled nuclei were counted. The values are presented as mean SD from five animals at P3

8 and P7 after birth. Results Adult rat kidney Expression of EGF In adult rat kidney, EGF immunoreactivity was observed in the cortex and outer medulla (Fig. 1). There was no immunostaining in the inner medulla, and the boundary between the outer and inner medulla was well defined. EGF immunostaining was predominantly observed in the outer and inner stripe of the outer medulla (Fig. 1A, 1C), and was also found in some of the tubular profiles in the cortex (Fig. 1B). Double immunohistochemistry To identify each of the tubular profiles, we used different histochemical techniques: the antibody Na-K-2Cl cotransporter (NKCC2) for TAL, neuronal nitric oxide synthase (nnos) for the MD, thiazide sensitive Na-Cl cotransporter (TSC) for DCT, and calbindin 28k (CaBP) for DCT 2 and connecting tubule (CNT). EGF immunoreactivity was observed in the NKCC2-positive TAL in the outer medulla and cortex (Fig. 2A and 2A ), but there was no immunolabeling in the nnos-positive MD (Fig. 2 B and 2B ). In the cortical labyrinth, EGF-labeling was prominent over region 1 of the TSC-positive DCT, but the labeling did not overlap region 2 of the CaBP-positive DCT or the CNT (Fig. 2C, 2C and 2C ). Electronmicroscopic immunocytochemistry Electronmicroscopy showed the presence of EGF immunoreactivity on the apical plasma membrane and intracellular vesicles, including the golgi complex of DCT (Fig. 3A). In contrast, the distribution of EGF immunolabeling in the medullary TAL was more diffuse than in the DCT cells, being found throughout the cytoplasm, with heavy labeling in perinuclear regions but not in the basolateral

9 membrane (Fig. 3B). In cortical thick ascending limb, smooth surfaced cells (SCC) have strong EGF-immunoreactivity (Fig. 3C). In contrast, weak EGF-immunoreactivity is seen only in a few number of vesicles of the rough surfaced cell (RSC) with well developed microvilli (Fig. 3D). In addition, the intensity of EGF immunolabeling of the TAL was stronger than in the DCT. In developing rat kidney Expression of EGF No EGF-positive cells were found in fetal kidney. At E16, E18, and E20, EGF immunoreactivity was undetectable in any of the tubular segments (Fig 4A). EGF-positive tubular profiles first appeared at P3 in the TAL, between the cortex and medullary regions in middle region of the differentiating loops of Henle. No labeled profiles were found in the outer cortex or the inner medulla (Fig 4B). At this age, EGF-negative cells could be detected among the EGF-positive cells (Fig. 4C). By P7, the pattern of EGF expression was very similar to that observed at P3, but the EGF-positive cells in the loop of Henle were more numerous and more intensely immunoreactive (Fig. 4D). By P14, EGF-positive cells could be found in the outer cortex and had begun appearing in the cortical labyrinth (Fig. 4E). By P21, the distribution of EGF had spread to the inner stripe of the outer medulla with a distribution similar to that seen in adult kidney (Fig. 4F). Double immunohistochemistry To determine whether EGF was expressed in the developing loop of Henle, serial sections were cut and double-labeled. EGF-positive cells were first detected at P3 in the NKCC2-positive medullary TAL, the differentiating loop of Henle (Fig. 5A, 5B). By P7, most of the TAL cells in the outer medulla and the inner cortex had become EGF immunoreactive, with more intense staining than that seen in the P3 kidney (Fig 5D). In contrast, there was no staining in the distal convoluted tubules of

10 outer cortex at this age (figure not shown). By 14 days, EGF-positive cells were found in the initial portion of DCT, near the MD, but the staining was weak compared with that in adult kidney (Fig. 5E). During renal development, the TAL contains both EGF-positive and EGF-negative cells which are clearly separable by light microscopy (Fig 4C). We investigated double immunostaining for Na-K-ATPase and nnos using serial sections cut from a postnatal kidney. Na-K-ATPase immunoreactivity was seen on the basolateral plasma membrane of the loop of Henle. Both EGF-positive and EGF-negative cells were well-stained for Na-K-ATPase, and were indistinguishable (Fig. 5F). EGF was not expressed in the macula densa during renal development (Fig. 4E). Eletronmicroscopic immunocytochemstry EGF immunoreactivity was localized in the apical plasma membrane, the intracellular vesicles, and the golgi complex in the stained semithin sections. EGF-positive vesicles were located in the supranuclear region and were attached to the apical plasma and the nuclear membrane (Fig 6A, 6B). EGF-positive cells in the TAL were smooth-surfaced with few microvilli (Fig 6B), whereas EGF-negative TAL cells had more numerous apical microvilli (Fig. 6C). BrdU-staining (proliferation index) Figure 4C shows that the immunostaining for EGF is heterogenous in the TAL, with both positive and negative immunostaining cells present. We measured the proliferation rates of EGF expressing cells using BrdU staining at P3 and P7, to determine more precisely the differences between the positive and negative cell populations. These observations are relevant because the differentiating TAL of the medullary ray in the inner cortex and in the outer stripe of the outer medulla is a major site for cell proliferation during renal development (5). The number of BrdU-positive nuclei, expressed as a percentage of the total number of EGF-positive

11 cells, at P3 and P7, are shown in Table 1. NKCC2 was used to identify the TAL (Fig. 7A, 7B). Among the TAL cells, the largest number of BrdU-positive nuclei were observed in the TAL in the outer medulla at P3 (7.02 %) and P7 (4.46%). In contrast, there were few BrdU-labeled EGF-positive cells in the medullary TAL (between 0.86 and 2.31 %) (Fig. 7C, 7D). Discussion This study was designed to investigate more thoroughly the distribution of the EGF and whether it coincides with proliferation along the loop of Henle in developing rat kidneys. The main result of the study was demonstration of EGF immunoreactivity in the adult rat kidney which was localized to the TAL and DCT1. These results differ from previous studies using rat- (25, 32), mouse- (31, 33) and rabbit kidneys (34). Previous studies have examined the immunoreactivity of EGF in the different segments without demonstrating a difference between DCT1 and DCT2 (25, 31, 32, 33, 34). Dorup (9) reported ultrastructural distinctions between cells in the early and later part of the DCT in rat kidney. Loffing (19) proposed the subdivision of the DCT into DCT1 (sodium/calcium exchanger negative) and DCT2 (sodium/calcium exchanger positive). DCT 2 cells which a transition region between the DCT and the CNT display features of both the DCT 1 and CNT cells. In particular, DCT 2 contains thiazide-sensitive Na-Cl cotransporter (TSC) characteristic of the DCT (19), but also expresses calbindin D28k (20, 27) which is characteristic of the CNT. In addition, intercalated cells are present between the DCT 2 cells (9, 21). In our study, EFG immunoreactivity was found only in the DCT 1 region and not in the DCT 2 region, which expressed both TSC and CaBP. This study confirmed that EGF is produced and secreted from both the TAL and the DCT 1 in adult rat kidney. In developing kidney, EGF was first detected at P3 in the differentiating TAL of the medullary ray of the outer medulla; it had gradually spread to the inner cortex by P7. At P14, EGF first appeared in the distal tubule except for the macula densa. Finally, at P21, EGF immunoreactivity was found in the TAL extending throughout the inner stripe of the outer medulla to the outer cortex and the DCT of the

12 cortical labyrinth. EGF promotes cell proliferation from numerous cell culture studies using kidney cells and those of other organs (1, 13, 24), but the localization of EGF in renal development in vivo has been little studied. Moreover, there is controversy concerning the localization and expression time of EGF during development. Several studies in mice have stated that EGF appeared 3 to 5 days after birth (33), Gattone et al (12) reported the presence of EGF after day 6 and Popliker et al (28) after two weeks. Our study in rats shows that EGF-positive cells can be detected at P3, and are localized in the differentiating TAL of the medullary ray. In developing rat kidney, the ureteric bud and metanephrogenic blastema converge and begin the full development of a new nephron in the nephrogenic zone, from E13 to P7. In this study we found that between P3 and P7, EGF-positive cells appear in the TAL of the corticomedullary junction, one of the main sites of proliferation during renal development. However, developing EGF-negative cells were detected among the EGF-positive cells. Nouwen et al. (25) reported that there were EGF-negative cells among positive ones in the TAL epithelium of the outer stripe in a light microscopy study. Our results, however, demonstrate that EGF-negative cells are found both in the TAL of the outer stripe of the outer medulla and in the cortex. We used three different methods to identify the features of EGF-positive and -negative cells. To confirm the presence of immature EGF-negative cells in the developing kidney we used double staining for EGF and Na-K ATPase 1, which exists in the basolateral membrane of loop of Henle. No difference was found in sodium transport proteins staining between the EGF-positive and -negative cells during renal development. We have previously reported the cell proliferation rate in the developing process of loop of Henle, and the TAL of outer medulla, and have established the importance of that region for cell proliferation during renal development (5). In this study, we did not find a significant difference in the cell proliferation rate of EGF-positive cells of the TAL; in fact, cell proliferation was lower in the EGF stained cells (Table 1). However, electron microscopy confirmed

13 that EGF-positive cells were smooth-surfaced with less well-developed microvilli, whereas EGF-negative cells were rough, with better developed microvilli. Allen and Tisher (2), using scanning electron microscopy, reported the presence of two types of epithelial cells in the thick ascending tubules which were distinguishable by the degree of development of the surface microvilli. Yoshitomi et al. (36) and Tsuruoka et al. (35) reported that these two types of cells have different electrophysiological characteristics; more recently, Nielsen et al. (23) reported that BSC1 immunoreactivity differs between the rough- and smooth-surfaced cells. However, we did not find a difference in the immunoreactivity staining of sodium transport proteins, but observed that the proliferation rate was lower for positive cells than for negative cells. Further research is required to explain the functional significance of these observations. At P14, EGF immunoreactivity appeared in the cortical TAL and the DCT of the cortical labyrinth. We also found that after P21, immunoreactivity dramatically increased to levels similar to those seen in adult animals. It is suggested that EGF in the kidney has a role in differentiation and growth rather than in the cell proliferation as EGF expression is found in kidneys immediately after birth, and expression is increased at P7, at the time when the nephron ends formation and differentiation and maturation begins. The EGF receptor is present in kidney before birth and is lost gradually after birth. However, as stated above, EGF in the developing kidney appears at P3, and EGF produced in the kidney has no effect upon the incipient process of kidney development. It is believed that EGF and TGF participate in the development of kidney by affecting EFG receptors at birth. Apoptosis plays an important role in rat kidney morphogenesis, being mainly present in the nephrogenic zone before birth and in the renal papilla after birth. After birth, apoptosis in the renal papilla participates in the elimination of thick ascending epithelial cells and in the formation of the ascending thin limb epithelium (16). It has been reported that apoptosis is involved in the temporary appearance and subsequent removal of a large number of intercalated cells at early stages of

14 development. Cole et al. (7), having observed the decrease in apoptosis following injection of EGF, suggested that EGF might control apoptosis. Our study suggests the possibility that EGF influences the control of apoptosis as EGF appears at P3, with EGF expression localized to the inner cortex and outer medulla, a region where apoptosis is rarely present. We also observed a sudden increase of EGF expression at P14, approximately at the time when apoptosis ceases. These results suggest that EGF expression in kidney development may be involved not so much in cell proliferation as in the differentiation and maturation of the distal tubule. In summary, our study has demonstrated that EGF immunoreactivity is restricted to the TAL and DCT1 of adult rat kidney, and that there are two cell types in the developing TAL: those which are EGF-positive with smooth surfaces, and those which are EGF-negative with rough surfaces. In addition EGF expression in the developing loop of Henle not coincides with proliferation. Acknowledgments This work was supported by the Korea Science and Engineering Foundation (R ) though the Cell Death Disease Research Center at the Catholic University of Korea.

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17 20. Loffing J, Loffing-Cueni D, Hegyi I, Kaplan MR, Hebert SC, Le Hir M, and Kaissling B. Thiazide treatment of rats provokes apoptosis in distal tubule cells. Kidney Int 50: , Madsen K and Tisher CC. Structural-functional relationships along the distal nephrons. Am J Physiol Renal Fluid Electrolyte Physiol 250: F1-F15, Nexo E, Lamberg SI, and Hollenberg MD. Comparison of a receptor binding assay with a radioimmunoassay for measuring human epidermal growth factor-urogastrone in urine. Scand J Clin Lab Invest 41(6): , Nielsen S, Maunsbach AB, Ecelbarger CA, and Knepper MA. Ultrastructural localization of Na-K-2Cl cotransporter in thick ascending limb and mecula densa of rat kidney. Am J Physiol 275: F885 F893, Norman J, Badie-Dezfooly B, Nord EG, Kurtz I, Schlosser J, Chaudhari A, and Fine LG. EGF-induced mitogenesis in proximal tubular cells: Potentiation by angiotensin II. Am J Physiol 253: F299 F309, Nouwen EJ, Verstrenpen WA, Buyssens N, Zhu MQ, and De Bore ME. Hyperplasia, hypertrophy, and phenotypic alterations in the distal nephron after acute proximal tubular injury in the rat. Lab Invest 70(4): , Orellana SA, Sweeney WE, Neff CD, and Avner ED. Epidermal growth factor receptor expression is abnormal in murine polycystic kidney. Kidney Int 47(2): , Plotkin, MD, Kaplan MR, Verlander JW, Lee WS, Brown D, Poch E, Gullans SR, and Hebert SC. Localization of the thiazide sensitive Na-Cl cotransporter, rtsc1, in the rat kidney. Kidney Int 50: , 1996.

18 28. Popliker M, Shatz A, Avivi A, Ullrich A, Schiessinger J, and Webb CG. Onset of endogenous synthesis of epidermal growth factor in neonatal mice. Dev Biol 119: 38 44, Robertson H, Wheeler J, and Morley AR. In vivo bromodeoxyuridine incorporation in normal mouse kidney: Immunohistochemical detection and measurement of labeling indices. Histochem J 22: , Sakurai H, and Nigram SK. Transforming growth factor-beta selectively inhibits branching morphogenesis but not tubulogenesis. Am J Physiol Renal Physiol 41(1): F139 F146, Salido EC, Barajas L, Lechago J, Laborde NO, and Fisher DA. Immunocytochemical localization of epidermal growth factor in mouse kidney. J Histochem Cytochem 34(9): , Salido EC, Lakshmanan J, Fisher DA, Shapiro LJ, and Barajas L. Expression of epidermal growth factor in the rat kidney: an immunohistochemical and in situ hybridization study. Histochemistry 96: 65 72, Salido EC, Lakshmanan J, Shapiro LJ, Fisher DA, and Barajas L. Expression of epidermal growth factor in the kidney and submandibular gland during mouse postnatal development. An immunocytochemical and in situ hybridization study. Differentiation 45(1): 38 43, Taira T, Yoshimura A, Lixuka K, Inui K, Oshiden K, Iwasaki S, Ideura T, and Koshikawa S. Expression of epidermal growth factor and its receptor in rabbits with ischaemic acute renal failure. Virchows Arch 427: , 1996.

19 35. Tsuruoka S, Koseki C, Muto S, Tabei K, and Imai M. Axial heterogeneity of potassium transporter across hamster thick ascending limb of Henle's loop. Am J Physiol 267: F121 F129, Yoshitomi K, Koseki C, Taniguchi J, and Imai M. Functional heterogeneity in the hamster medullary thick ascending limb of Henle's loop. Pfugers Arch 408: , a

20 Table 1 Percentage of BrdU-labeled cells in the NKCC2/EGF-positive TAL of loop of Henle in the postnatal rat kidney (n=5) a NKCC2-positive TAL cells (%) EGF-positive cells (%) P3 P * * a Values are means ±SD and expressed as a percentage of NKCC2 / EGF-positive TAL cells in outer medulla. BrdU (5-brom-2 -deoxy-uridine). * P<0.01

21 Figures Fig. 1 Light micrographs illustrating immunostaining for EGF in 50 µm sections in adult rat kidney. The renal cortex, outer and inner medulla at low magnification (A) with the renal cortex (B) and inner stripe of outer medulla (C) at higher magnification. EGF immunoreactivity is localized in the outer medulla and cortex. Note that EGF immunostaining is predominantly located in the outer medulla and as well as in some tubular profiles in the cortex (B). C shows the well-defined border between outer medulla and inner medulla. Co: cortex; OSOM: outer stripe of outer medulla; ISOM: inner stripe of

22 outer medulla; IM: inner medulla. Magnifications: A: 25; B and C: 50. Fig. 2 Light micrographs of serial sections (A, A ; B, B ; C, C, C ) of the outer medulla (A, A ) and cortex (B, B ; C, C, C ) from rat adult kidney, illustrating immunostaining for NKCC2 (A), nnos (B), TSC (C), Calbindin D28K (C'), and EGF (A, B and C '). In the thick ascending limb of the outer medulla, EGF immunoreactivity is seen in the NKCC2-positive thick ascending limb (arrows). B and B : in the macula densa (MD), EGF immunoreactivity is not present in the nnos-positive MD (arrow heads). C, C and C : TSC is expressed in the distal convoluted tubule 1 (D1) and distal convoluted tubule 2 (D2). Calbindin D28K is expressed in the D2 and connecting tubule (CNT). However, EGF labeling is only present in the DCT 1 in the cortical labyrinth. Note that EGF is not expressed in the DCT 2 and CNT (C ). Magnification: A C' 450

23 Fig. 3 Transmission electron micrographs illustrating EGF immunoreactivity in adult rat kidney. A: EGF immunoreactivity is present along the apical membrane and vesicles (arrows) in the apical portion of the cytoplasm of the distal convoluted tubule cell (DCT). Note the EGF-positive golgi complex (G). Bar=3µm. B: The intensity of EGF immunoreactivity of the medullary thick ascending

24 limb cell (mtal) is stronger than that of DCT cell in A. S3 is a segment of proximal tubule. Bar = 4µm. C: In the cortical thick ascending limb, smooth surfaced cells (SSC) have EGF-immunoreactivity. In contrast, weak EGF-immunoreactivity is seen only in a few numbers of vesicles of the rough surfaced cells (RSC) with well developed microvilli (arrows). Bar=5µm.

25 Fig. 4 Light micrographs of 50um section of kidney illustrating EGF immunostaining from E20 fetus (A), P3 (B, C), P7 (D), P14 (E), and P21 (F) pups. There is no EGF labeling in any of the tubular profiles in the E20 fetus, but marked expression at P3, in the middle portion of the thick ascending limb. C shows EGF-negative cells among the positive cells. In the P14 pup (E), distinct EGF immunostaining can be seen in the outer cortex; however, the intensity of staining is less than in the P21 pup (F). Magnification: A, B, D F: 30; C: 200.

26

27 Fig. 5 Micrographs of serial sections illustrating the NKCC2 (A, C) and EGF (B, D) in the outer medulla from P3 (A, B), and P7 pups (C, D), and double immunolabeling for EGF (brown) and nnos (blue) (E) or Na-K-ATPase (blue) (F) in the cortex at P14. A and B: The apical staining of NKCC2 in the TAL (*) coincides with weak EGF staining (arrows) at P3. C and D: In a P7 pup, strong EGF labeling overlaps with NKCC2-positive TAL (C, D). E and F: EGF immunoreactivity is not expressed in the macula densa (arrow), whereas it is present in the first part of the initial distal convoluted tubules (D1) in a P14 animal. Magnification: A D: 450.

28 Fig. 6 Transmission electron micrographs illustrating EGF immunoreactivity in a P3 (A) and P7 pup (B, C). EGF-negative cells are present among the EGF-positive cells in the medullary thick ascending limb (A). EGF immunoreactivity is present along the apical membrane and vesicles in the apical portion of the cytoplasm (B). In the thick ascending limb, smooth-surfaced cells (SSC) have strong EGF immunoreactivity. In contrast, no EGF immunoreactivity is seen in the rough-surfaced cells (RSC) with well developed microvilli (arrows). Bar= 5µm.

29 Fig. 7 Micrographs of the outer medulla from P3 (A, C) and P7 pups (B, D), illustrating double-labeling for NKCC2 with BrdU (A, B), and EGF with BrdU (C, D). A and B: Numerous BrdU-positive cells are present in the NKCC2-positive TAL (*) in P3 and P7 pups. C and D: A few BrdU-labeled cells are present among the EGF-positive cells at P3 (C). However, BrdU-labeling is seen in both the EGF-positive cell (arrow) and the EGF-negative cell (arrow head) at P7 (D). Magnification: A D: 430.

30 Fig. 8 Diagram illustrating changes in EGF immunoreactivity in the loop of Henle in the developing rat kidney. Abbreviations: mtal: medullary thick ascending limb; ctal: cortical thick ascending limb; MD: macula densa; G: glomerulus; DCT: distal convoluted tubule; CNT: connecting tubule; CCD: cortical collecting duct; NKCC2: Na-K-2Cl cotransporter; nnos: neuronal nitric oxide synthesis; TSC: thiazide-sensitive Na-Cl cotransporter; CaBP: calbindin-d 28k; P: postnatal day.