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*
Department of Anatomy, Catholic University Medical College, Seoul,
Korea
Division of Nephrology, Hypertension and Transplantation, University of
Florida, Gainesville, Florida.
Correspondence to Dr. Kirsten M. Madsen, Division of Nephrology, Box 100224, University of Florida, Gainesville, FL 32610-0224. Phone: 352-846-1665; Fax: 352-392-3581; E-mail: madsekm{at}medicine.ufl.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The loop of Henle is formed as an outgrowth from the proximal and distal tubule anlage of the nephron, close to the vascular pole of the glomerulus. Concomitant with the growth and development of the renal papilla, the loops of Henle grow and descend from the glomerulus toward the tip of the papilla (3,4,5). Although numerous mitoses have been reported in the anlage of the loop of Henle (4), the site or sites of cell proliferation during the growth and development of the loop have not been established.
Two types of nephrons, juxtamedullary and superficial, can be distinguished in the mature kidney (6,7). Juxtamedullary nephrons have long loops of Henle that descend into the inner medulla and often reach the tip of the papilla. They have a long descending thin limb and an ascending thin limb that continues into the thick ascending limb. The abrupt transition from the squamous epithelium of the ascending thin limb to the cuboidal epithelium of the thick ascending limb constitutes the border between the inner and outer medulla. In contrast, superficial nephrons have short loops of Henle that terminate in the outer medulla close to the border between the outer and inner medulla, and they do not have an ascending thin limb. The transition between the descending thin limb and the thick ascending limb epithelium is located in the descending limb 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 zone. Thick ascending limbs are present throughout the renal papilla, which therefore has the structural composition characteristic of the mature inner stripe of the outer medulla (3,4,5,8). Thus, all loops of Henle have the structural configuration of the short-looped nephrons of the adult kidney, and there are no ascending thin limbs. However, during the first 2 wk of life, the cuboidal epithelium of the thick ascending limb in the renal papilla is gradually transformed into the ascending thin limb by a process that starts just before the bend of the loop and proceeds toward the outer medulla (4,8).
A recent study from our laboratory demonstrated that the transformation of the ascending limb epithelium occurs by deletion of thick ascending limb cells by apoptosis, followed by a structural transformation of remaining thick ascending 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 epithelium. Neither is it known whether the thick ascending limb epithelium is the only source of ascending thin limb cells or whether cell proliferation occurs in the adjacent descending thin limb, followed by migration of cells into the ascending limb. Furthermore, our previous study does not rule out the presence of a putative as-yet-undetected stem cell that might also give rise to ascending thin limb cells.
The purpose of the present study was to identify the site(s) of cell proliferation in the loop of Henle and to establish whether there is evidence of cell proliferation in the epithelium undergoing transformation, in the newly formed ascending thin limb epithelium, or in the adjacent descending thin limb at the transition between the two epithelia.
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Materials and Methods |
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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 through the heart. The animals initially were perfused
briefly with phosphate-buffered saline (PBS), osmolality 298 mOsm/kg
H2O (pH 7.4), to rinse away all blood. This was followed by
perfusion with a periodate-lysine-paraformaldehyde solution for 5 min. After
perfusion, the kidneys were removed and cut into 1- to 2-mm-thick slices that
were fixed additionally by immersion in the periodate-lysine-paraformaldehyde
solution overnight at 4°C. Sections of tissue were cut transversely
through the entire kidney on a Vibratome Pelco 101, Sectioning series 1000
(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
The thick ascending limb of the loop of Henle was identified by use of a
rabbit polyclonal antibody against the human serotonin receptor,
5-HT1A (kindly provided by Dr. John Raymond, Medical University of
South Carolina, Charleston, SC). The antibody labels the basolateral plasma
membrane of all distal tubule cells, including those of the thick ascending
limb, distal convoluted tubule, and connecting tubule
(11). The proximal tubule and
descending thin limb of the loop of Henle were identified by use of an
affinity-purified rabbit polyclonal antibody raised against a synthetic
peptide corresponding to the terminal 22 amino acids of rat aquaporin-1
(AQP1). The antibody recognizes AQP1 in the rat kidney and has been
characterized in detail in previous studies
(12). For the detection of
BrdU, a mouse monoclonal antibody against BrdU (Boehringer Mannheim) was
used.
Preembedding Immunolabeling for 5-HT1A Receptor
Fifty-µm Vibratome sections were processed for immunohistochemistry by
use of an indirect preembedding immunoperoxidase method. All sections were
washed with 50 mM NH4Cl in PBS, three times for 15 min. Before
incubation with the primary antibody, the sections were pretreated with a
graded series of ethanol for antigen retrieval and incubated for 2 h with PBS
containing 1% bovine serum albumin (BSA), 0.05% saponin, and 0.2% gelatin
(solution A). The tissue sections then were incubated overnight at 4°C
with the antibody against the 5-HT1A receptor, diluted 1:50 in 1%
BSA-PBS (solution B). After several washes with solution A, the tissue
sections were incubated for 2 h in peroxidase-conjugated goat anti-rabbit IgG,
Fab fragment (Jackson ImmunoResearch Laboratories, West Grove, PA), diluted
1:50 in solution B. The tissues then were rinsed, first in solution A and
subsequently in 0.05 M tris(hydroxymethyl)aminomethane (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 washing with 0.05 M Tris buffer,
the sections were dehydrated in a graded series of ethanol and embedded in
Poly/Bed 812 Resin (Polysciences Inc., Warrington, PA). From all animals,
50-µm-thick Vibratome sections through the entire kidney were mounted in
Epon 812 between polyethylene vinyl sheets.
Postembedding Immunolabeling for AQP1 and BrdU
From flat-embedded 50-µm-thick Vibratome sections of kidney processed
for immunohistochemical identification of the thick ascending limb of the loop
of Henle, sections from the cortex, outer medulla, and outer and inner parts
of the papilla were excised and glued onto empty blocks of Epon 812. Two
consecutive 1-µm-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 with graded ethanol and rinsed in tap water. To increase
accessibility of the DNA, the sections were treated with 20 µg/ml
proteinase K (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 being rinsed in tap water for 10 min, the tissue
sections were incubated for 30 min with methanolic H2O2,
rinsed in tap water for 10 min, and treated with 0.5% triton X-100 in PBS for
15 min. The sections then were rinsed in PBS three times for 10 min before
being treated with 1% BSA for 1 h. One of the consecutive sections was
incubated with the mouse anti-BrdU antibody (1:60), whereas the other section
was incubated with a mixture of rabbit anti-AQP1 (1:500) and mouse anti-BrdU
(1:60) antibodies overnight 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). For detection of peroxidase, Vector SG (Vector
Laboratories, Burlingame, CA) was used as the chromogen to produce a gray-blue
color, which is easily distinguished from the brown staining produced by
3,3'-diaminobenzidine in the preembedding procedure used for detection
of 5-HT1A. The sections were washed with distilled water,
dehydrated with graded ethanol and xylene, mounted in Canada balsam, and
examined by light microscopy.
Quantitation of BrdU-Positive Cells
Thick ascending limb cells, identified by basolateral 5-HT1A
receptor immunolabeling, and proximal tubule and descending thin limb cells,
identified by AQP1 immunolabeling, were counted on 1-µm-thick semithin
sections form the cortex, outer medulla, and proximal and distal papilla from
four animals derived from two different litters at each of the following ages:
1, 3, 5, 7, and 14 d. On the same sections, 5-HT1A—positive
and AQP1-positive cells with BrdU-labeled nuclei were counted and expressed as
percentages of the total number of 5-HT1A- and AQP1-positive cells,
respectively, counted in the different tubule segments for each animal. The
values are presented as mean ± SD from four animals.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Identification of the Descending Limb of Henle's Loop
Descending limb cells were identified by immunolabeling with antibodies to
AQP1. In developing nephrons, AQP1 immunoreactivity was confined to the plasma
membrane of proximal tubules and the descending thin limb of the loop of Henle
(Figures
5,6,7,8).
At birth, the transition from the pars recta of the proximal tubule to the
descending thin limb was subtle, with a gradual change in tubule diameter, a
gradual decrease in cell height, and a loss of microvilli. From 3 to 7 d of
age, there was an increase in the number of loops of Henle that all exhibited
a gradual transition from the cuboidal epithelium of the proximal tubule to
the squamous epithelium of the immature descending thin limb
(Figure 6). At 14 d of age,
there was an abrupt transition from the cuboidal epithelium of the proximal
tubule to the squamous epithelium of the descending thin limb, thus defining
the border between the outer and inner stripe of the outer medulla.
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Detection of Cell Proliferation
Cell proliferation in the loop of Henle was determined by incorporation of
BrdU into DNA of dividing cells followed by immunohistochemical detection by
use of a double- and triple-labeling procedure for the detection of BrdU,
5-HT1A, and AQP1 immunoreactivity on the same section. In all
animals, cells with BrdU-positive nuclei, identified by their gray-blue color,
were present throughout the renal cortex and outer medulla (Figures
1,
2,
3,
5, and
6). During the first week of
life, numerous BrdU-labeled cells were located in the nephrogenic zone in the
outer cortex (Figures 1A and
5). They were most prevalent in
the undifferentiated mesenchyme but also were observed frequently in the
ampullae and the ureteric bud as well as in developing glomeruli and tubules.
Throughout kidney development, most of the BrdU-labeled tubule cells were
located in medullary rays in the cortex
(Figure 2) and in the outer
medulla (Figures 3 and
6). BrdU-labeled cells were
much less common in the renal papilla at any of the ages studied, and they
were rarely observed in the terminal part of the papilla (Figures
4 and
8), where they seemed to
represent mainly interstitial cells and collecting duct cells. In the 20
animals studied, a total of only two BrdU-positive cells were detected in the
5-HT1A-positive epithelium of the thick ascending limb in the inner
part of the renal papilla. There were no BrdU-positive nuclei in the
5-HT1A-positive cells undergoing transformation in the ascending
limb of Henle's loop or in the already transformed epithelium that still
exhibited weak 5-HT1A immunostaining. BrdU-labeled nuclei were
rarely observed in the AQP1-positive descending thin limb in the renal papilla
(Figures 7 and
8).
Quantitation of BrdU-Positive Nuclei in Ascending Limb of Henle's
Loop
The number of BrdU-positive nuclei, expressed as percentages of the total
number of 5-HT1A-positive cells in the ascending limb of the loop
of Henle, are listed in Table
1. Among the 5-HT1A-positive tubules, including the
thick ascending limb, distal convoluted tubule, and connecting tubule, the
largest number of BrdU-positive nuclei were observed in the cortical thick
ascending limb in the medullary ray of the renal cortex at all ages studied
(approximately 15% in 1-, 3-, and 5-d-old pups; 13% in 7-d-old pups; and 5% in
14-d-old pups). In contrast, there were few BrdU-labeled cells in
5-HT1A-positive tubules in the cortical labyrinth (2 to 3% of the
total number of 5-HT1A-positive cells in the distal convoluted
tubule and connecting tubule). Numerous cells with BrdU-labeled nuclei were
observed in the 5-HT1A-positive thick ascending limbs in the outer
medulla of all animals (9 to 11% in 1- and 3-d-old pups and 6 to 7% in 5-, 7-,
and 14-d-old pups). In sharp contrast, BrdU-labeled cells were rare (<3%)
in 5-HT1A-positive cells in the renal papilla at all ages, and they
were absent in the lower half of the papilla after 3 d of age. BrdU-labeled
nuclei were not observed in 5-HT1A-positive cells undergoing
apoptosis or in the 5-HT1A-positive, newly formed ascending thin
limb epithelium.
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Quantitation of BrdU-Positive Nuclei in Descending Limb of Henle's
Loop
The number of BrdU-positive nuclei, expressed as percentage of the total
number of AQP1-positive cells in the descending limb of the loop of Henle, are
listed in Table 2. Among the
AQP1-positive tubules, including the proximal tubule and descending thin limb,
the largest number of BrdU-labeled nuclei was observed in the pars recta of
the proximal tubule in the medullary ray (15 to 17% in 1- and 3-d-old pups and
7 to 8% in 5-, 7-, and 14-d-old pups) and outer medulla (13 to 20%) at all
ages studied. In contrast, there were few BrdU-labeled cells in AQP1-positive
proximal convoluted tubules in the cortical labyrinth (2 to 3%). BrdU-labeled
cells were rare (<4%) in AQP1-positive immature descending thin limbs in
both the outer medulla and the renal papilla at all ages, and they were absent
from the lower half of the renal papilla after 3 d of age.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In a previous study of the developing rat kidney, we demonstrated that the ascending thin limb is derived from the epithelium of the primitive thick ascending limb by a process that involves apoptotic deletion of thick ascending limb cells in the inner medulla, followed by transformation of the remaining thick ascending limb cells into a thin squamous epithelium (8). However, these observations did not rule out the possibility that ascending thin limb cells might derive from proliferating cells in the adjacent descending thin limb or by proliferation of already transformed thick ascending limb cells or as-yet-unidentified stem cells in the segment undergoing transformation. The present study was designed to test these possibilities by identifying sites of cell proliferation in the loop of Henle and in particular by determining whether proliferating cells could be detected in or adjacent to the segment undergoing transformation. The results, which are summarized in Figure 9, demonstrate that cell proliferation in the loop of Henle occurs mainly in the outer medulla and in medullary rays in the cortex. In the renal papilla, <4% of the cells in the loop of Henle were undergoing proliferation, and, in the terminal part of the renal papilla, cell proliferation was not observed in the loop of Henle after 3 d of age. Cell proliferation was not observed in the transforming part of the primitive thick ascending limb that contained apoptotic cells or in the newly formed ascending thin limb epithelium, which was easily identified because 5-HT1A immunoreactivity was retained for a short period of time after the transformation occurred. These observations indicate that ascending thin limb cells are not derived from unidentified stem cells located in the immature thick ascending limb epithelium undergoing transformation. Moreover, the absence of detectable BrdU-labeled cells in the already transformed epithelium indicates that the ascending thin limb is not a major site of cell proliferation. Finally, the absence of detectable cell proliferation in the AQP1-positive descending thin limb immediately adjacent to the epithelium undergoing transformation indicates that ascending thin limb cells are not derived from proliferating cells in the descending limb of Henle's loop. It is generally accepted that the cells of the descending limb of the loop of Henle are derived from the proximal tubule anlage of the S-shaped body, whereas the ascending limb is derived from the distal tubule anlage (3,4,5). Our results confirm that the boundary between the descending and ascending limb epithelium is located shortly before the bend of the loop in the terminal part of the descending limb, and there is no evidence that descending thin limb cells are being transformed into ascending thin limb cells. Taken together, these observations support the results of previous studies from our laboratory (8) and by other investigators (4,5) suggesting that the ascending thin limb is derived mainly by a transformation of the immature thick ascending limb epithelium in the renal papilla during the first 2 wk after birth.
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The results of the present study also revealed that cell proliferation in both the descending and ascending limb of the loop of Henle occurs mainly in the medullary ray of the renal cortex and in the outer stripe of the outer medulla. Thus, two distinct areas of cell proliferation exist in the developing kidney: the nephrogenic zone in the outer cortex under the renal capsule and the area around the corticomedullary junction, which appears to constitute a major growth zone for medullary structures, including the loop of Henle. In the developing kidney, the transition from the proximal tubule to the descending thin limb is initially subtle with a gradual decrease in cell height and loss of the brush border (4). Cell proliferation was detected in the part of the descending limb corresponding to the primitive or immature S3 segment of the proximal tubule. Of interest, in adult animals, proximal tubules and thick ascending limbs that are located in medullary rays in the cortex and in the outer stripe of the outer medulla are especially sensitive to ischemic as well as nephrotoxic injury (13). The recovery phase from acute renal failure in these conditions is characterized by significant cell proliferation in the pars recta of the proximal tubule as well as the thick ascending limb in this region (14,15,16). Thus, it appears that the capacity to undergo cell proliferation is preserved in these segments of the nephron throughout life. The low percentage of proliferating cells observed in the loop of Henle in the outer part of the renal papilla during the first three days after birth was very similar to that found in both proximal and distal convoluted tubules throughout postnatal development. After 3 d of age, cell proliferation was rare in the papillary portion of the loop of Henle. The rarity of proliferating cells in the renal papilla is in agreement with the results of previous studies reporting a very low mitotic index in the papilla of the developing rat kidney (17,18). Studies in the developing rat kidney by Coles et al. (17) reported that, at all ages examined, mitotic figures were mainly observed in the nephrogenic zone in the renal cortex. However, results were presented only from the nephrogenic zone and from the renal papilla, where mitotic figures were very rare. There was no information about cell proliferation in the inner cortex or outer medulla. In contrast, Kahane et al. (18) reported that in 10-d-old rats, the highest mitotic index was found in tubules in the renal medulla, which did not include the papilla. This indicates that cell proliferation was observed in the outer medulla, which is in agreement with the results of our study. In a study of cell proliferation in the developing human kidney, Nadasdy et al. (19) reported that the highest proliferation index was observed in immature tubules in the nephrogenic zone and in the distal nephron, including the thick ascending limb and distal convoluted tubule. However, no distinction was made between the different regions of the kidney.
In summary, the main site of cell proliferation in both the descending and ascending limb of the loop of Henle was found in the medullary ray in the inner cortex and in the outer stripe of the outer medulla. There was no evidence for the presence of stem cells or proliferating cells in the segments undergoing transformation. We conclude that the growth zone for the loop of Henle is located at the corticomedullary junction, and the ascending thin limb is derived mainly, if not exclusively, from cells of the primitive thick ascending limb.
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Acknowledgments |
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Footnotes |
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).
However, BrdU-positive nuclei are present in the medullary collecting duct
(MCD) and in the interstitium. Arrowhead indicates apoptotic body in TAL cell,
and asterisk indicates descending thin limb. Magnification, x660.
