J Am Soc Nephrol 12: 1410–1421, 2001 Cell Proliferation in the Loop of Henle in the Developing Rat Kidney JUNG-HO CHA,* YOUNG-HEE KIM,* JU-YOUNG JUNG,* KI-HWAN HAN,* KIRSTEN M. MADSEN,† and JIN KIM* † *Department of Anatomy, Catholic University Medical College, Seoul, Korea; and Division of Nephrology, Hypertension and Transplantation, University of Florida, Gainesville, Florida. Abstract. In the developing rat kidney, there is no separation of receptor, 5-HT1A, and aquaporin-1, respectively. Proliferating the medulla into an outer and inner zone. At the time of birth, cells were identified with an antibody against BrdU. BrdU- ascending limbs with immature distal tubule epithelium are positive cells in descending and ascending limbs of the loop of present throughout the renal medulla, all loops of Henle re- Henle were counted and expressed as percentages of the total semble the short loop of adult animals, and there are no number of aquaporin-1–positive and 5-HT1A–positive cells in ascending thin limbs. It was demonstrated previously that the different segments. In the developing kidney, numerous immature thick ascending limbs in the renal papilla are trans- BrdU-positive nuclei were observed in the nephrogenic zone. formed into ascending thin limbs by apoptotic deletion of cells Outside of this location, BrdU-positive tubule cells were most and transformation of the remaining cells into a thin squamous prevalent in medullary rays in the inner cortex and in the outer epithelium. However, it is not known whether this is the only medulla. BrdU-labeled cells were rare in the papillary portion source of ascending thin limb cells or whether cell proliferation of the loop of Henle and were not observed in the lower half of occurs in the segment undergoing transformation. This study the papilla after3dofage. BrdU-labeled nuclei were not was designed to address these questions and to identify sites of observed in segments undergoing transformation or in newly cell proliferation in the loop of Henle. Rat pups, 1, 3, 5, 7, and formed ascending thin limb epithelium. It was concluded that 14 d old, received a single injection of 5-bromo-2'-deoxy- the growth zone for the loop of Henle is located around the uridine (BrdU) 18 h before preservation of kidneys for immu- corticomedullary junction, and the ascending thin limb is nohistochemistry. Thick ascending and descending limbs were mainly, if not exclusively, derived from cells of the thick identified by labeling with antibodies against the serotonin ascending limb. The mammalian kidney is composed of two embryologically Two types of nephrons, juxtamedullary and superficial, can distinct tissues: the nephrons, which are derived from the be distinguished in the mature kidney (6,7). Juxtamedullary metanephric blastema, and the collecting duct system, which is nephrons have long loops of Henle that descend into the inner derived from the ureteric bud (1,2). Anatomically, the nephron medulla and often reach the tip of the papilla. They have a long includes the glomerulus, the proximal and distal convoluted descending thin limb and an ascending thin limb that continues tubules, and the loop of Henle. into the thick ascending limb. The abrupt transition from the The loop of Henle is formed as an outgrowth from the squamous epithelium of the ascending thin limb to the cuboidal proximal and distal tubule anlage of the nephron, close to the epithelium of the thick ascending limb constitutes the border vascular pole of the glomerulus. Concomitant with the growth between the inner and outer medulla. In contrast, superficial and development of the renal papilla, the loops of Henle grow nephrons have short loops of Henle that terminate in the outer and descend from the glomerulus toward the tip of the papilla medulla close to the border between the outer and inner me- (3–5). Although numerous mitoses have been reported in the dulla, and they do not have an ascending thin limb. The anlage of the loop of Henle (4), the site or sites of cell transition between the descending thin limb and the thick proliferation during the growth and development of the loop ascending limb epithelium is located in the descending limb have not been established. just before the bend of loop. In the developing rat kidney, before and at the time of birth, there is no separation of the medulla into an outer and inner Received October 19, 2000. Accepted December 19, 2000. zone. Thick ascending limbs are present throughout the renal Dr. Jeff M. Sands served as guest editor and supervised the review and final papilla, which therefore has the structural composition charac- disposition of this manuscript. teristic of the mature inner stripe of the outer medulla (3–5,8). Correspondence to Dr. Kirsten M. Madsen, Division of Nephrology, Box Thus, all loops of Henle have the structural configuration of the 100224, University of Florida, Gainesville, FL 32610-0224. Phone: 352-846- 1665; Fax: 352-392-3581; E-mail: [email protected] short-looped nephrons of the adult kidney, and there are no 1046-6673/1207-1410 ascending thin limbs. However, during the first 2 wk of life, the Journal of the American Society of Nephrology cuboidal epithelium of the thick ascending limb in the renal Copyright © 2001 by the American Society of Nephrology papilla is gradually transformed into the ascending thin limb by J Am Soc Nephrol 12: 1410–1421, 2001 Cell Proliferation in Loop of Henle 1411 a process that starts just before the bend of the loop and (11). The proximal tubule and descending thin limb of the loop of proceeds toward the outer medulla (4,8). Henle were identified by use of an affinity-purified rabbit polyclonal A recent study from our laboratory demonstrated that the antibody raised against a synthetic peptide corresponding to the ter- transformation of the ascending limb epithelium occurs by minal 22 amino acids of rat aquaporin-1 (AQP1). The antibody deletion of thick ascending limb cells by apoptosis, followed recognizes AQP1 in the rat kidney and has been characterized in detail in previous studies (12). For the detection of BrdU, a mouse mono- by a structural transformation of remaining thick ascending clonal antibody against BrdU (Boehringer Mannheim) was used. limb cells into the squamous epithelium of the ascending thin limb (8). However, it is not known whether this transformation is accompanied by cell proliferation in the newly formed Preembedding Immunolabeling for 5-HT1A Receptor ␮ epithelium. Neither is it known whether the thick ascending Fifty- m Vibratome sections were processed for immunohisto- limb epithelium is the only source of ascending thin limb cells chemistry by use of an indirect preembedding immunoperoxidase method. All sections were washed with 50 mM NH Cl in PBS, three or whether cell proliferation occurs in the adjacent descending 4 times for 15 min. Before incubation with the primary antibody, the thin limb, followed by migration of cells into the ascending sections were pretreated with a graded series of ethanol for antigen limb. Furthermore, our previous study does not rule out the retrieval and incubated for 2 h with PBS containing 1% bovine serum presence of a putative as-yet-undetected stem cell that might albumin (BSA), 0.05% saponin, and 0.2% gelatin (solution A). The also give rise to ascending thin limb cells. tissue sections then were incubated overnight at 4°C with the antibody The purpose of the present study was to identify the site(s) against the 5-HT1A receptor, diluted 1:50 in 1% BSA-PBS (solution of cell proliferation in the loop of Henle and to establish B). After several washes with solution A, the tissue sections were whether there is evidence of cell proliferation in the epithelium incubated for2hinperoxidase-conjugated goat anti-rabbit IgG, Fab undergoing transformation, in the newly formed ascending thin fragment (Jackson ImmunoResearch Laboratories, West Grove, PA), limb epithelium, or in the adjacent descending thin limb at the diluted 1:50 in solution B. The tissues then were rinsed, first in transition between the two epithelia. solution A and subsequently in 0.05 M tris(hydroxymethyl)amin- omethane (Tris) buffer (pH 7.6). For the detection of horseradish peroxidase, the sections were incubated in 0.1% 3,3'-diaminobenzi- Materials and Methods dine in 0.05 M Tris buffer for 5 min, after which H2O2 was added to Animals and Bromo-2'-Deoxy-Uridine Treatment a final concentration of 0.01% and the incubation was continued for Sprague-Dawley rats were used in all experiments. Kidneys were 10 min. After washing with 0.05 M Tris buffer, the sections were obtained from 1-, 3-, 5-, 7-, and 14-d-old pups. For each age group, dehydrated in a graded series of ethanol and embedded in Poly/Bed animals from two separate litters were used. All animals were admin- 812 Resin (Polysciences Inc., Warrington, PA). From all animals, istered 5-bromo-2'-deoxy-uridine (BrdU; Boehringer Mannheim, 50-␮m-thick Vibratome sections through the entire kidney were GmbH, Mannheim, Germany), 50 ␮g/g body wt, as a single subcu- mounted in Epon 812 between polyethylene vinyl sheets. taneous injection 18 h before preservation of kidneys for immunohis- tochemistry. BrdU is a thymidine analog that is incorporated into DNA during the S phase of the cell cycle and subsequently can be Postembedding Immunolabeling for AQP1 and BrdU ␮ detected on tissue sections by use of specific antibodies against BrdU From flat-embedded 50- m-thick Vibratome sections of kidney (9,10). processed for immunohistochemical identification of the thick ascend- ing limb of the loop of Henle, sections from the cortex, outer medulla, Tissue Preservation and outer and inner parts of the papilla were excised and glued onto empty blocks of Epon 812.