1 Division of Nephrology and
2 Division of Endocrinology, BMP-7, a member of
the bone morphogenic protein subfamily (BMPs) of the transforming
growth factor-
transforming growth factor- BONE MORPHOGENIC PROTEINS (BMPs) are members of a
unique subfamily of the transforming growth factor- Studies in mice revealed that the BMP-7 gene is highly expressed in the
kidney. BMP-7 mRNA is abundantly expressed during mouse renal
organogenesis. It localizes to the ureteric bud tips, in the comma- and
S-shaped bodies, and in premature glomeruli (10, 18). Recently, two
independent groups reported that BMP-7-deficient mice lack condensation
of the renal mesenchyme around the ureteric bud tips and
mesenchymal-to-epithelial differentiation with subsequent absence of nephrons (7, 17). This identified BMP-7 as an early inducer
of nephrogenesis. More recently, using organ culture of mouse
metanephric rudiments, Vukicevic et al. (38) demonstrated inhibition of
mesenchymal condensation and epithelial differentiation using BMP-7
antisense oligonucleotides or anti-BMP-7 antibodies. These experiments
suggest an important role for BMP-7 in fetal renal epithelial cell
differentiation. Tubular epithelial cells have been shown to undergo a
dedifferentiation process in renal ischemia. Growth factors
that function as inducers of cell differentiation or growth during
fetal organogenesis have been shown to afford a variable degree of
protection in models of acute renal ischemia (8, 9). Several
preliminary in vivo studies using animal models of acute and chronic
renal failure demonstrated that the administration of BMP-7 improves
renal function and preserves, to a variable degree, renal epithelial
cell morphology (6, 14, 35, 36).
To better understand the potential role of BMP-7 in the adult kidney,
we cloned a rat BMP-7 cDNA sequence and used it as a probe to localize
BMP-7 gene expression by in situ hybridization in normal rat kidney and
in rat kidneys with acute renal failure induced by
ischemia/reperfusion. Furthermore, we explored the role of
BMP-7 in the differentiation of metanephric mesenchymal cells into
epithelial cells.
Cloning of rat BMP-7 cDNA.
Poly(A)+ RNA from both fetal and
adult rat kidney RNA was used to prepare cDNA using Superscript (Moloney murine leukemia virus) reverse polymerase and
oligo(dT) priming. Two 30-cycle rounds of PCR amplification of the cDNA using nested primers with sequences conserved amongst human, mouse and
dog were performed within a rat sequence corresponding to bp
424-1399 of the human sequence. The sense primer sequence
corresponding to bp 424-444 was
5'-AGGGCTTCTCCTACCCCTACA-3'. The external and internal
antisense primers were 5'-CGGACCACCATGTTTCTGTATTTC-3' and
5'-GGAGCTGTCATCGAAGTAGAGGAC-3', respectively. The PCR
product was subcloned into pBluescript using TA cloning.
Sequencing and comparison with previously published human and mouse
sequences revealed 89% and 95% nucleotide identity, respectively,
within the uniquely determined 937-bp cloned region.
Renal ischemia model. Male
Sprague-Dawley rats weighing 225-250 g were used. Rats were fed ad
libitum and housed in an animal facility at 21°C with a 12:12-h
light-dark cycle for ~1 wk. Anesthesia was induced by injection of
pentobarbital sodium at 100 mg/kg body wt ip. Kidneys were accessed via
a mid-abdominal incision. The left renal artery was identified, freed
by blunt dissection, and occluded using the loop-clamp technique. Core
body temperature was maintained at 37°C by placing the animal on a
homeothermic table, and the abdominal viscera were covered with
saline-soaked gauze. After a clamping period of 60 min, the loop was
removed, and the kidney was carefully observed to ensure total
reperfusion as judged visually. If reperfusion was incomplete, then the
experiment was terminated and the animal was killed. Kidneys were
removed under anesthesia 3, 6, and 16 h after total reperfusion, cut in 3-5 mm coronal sections, frozen in liquid nitrogen,
and stored at RNA isolation and analysis by RNase protection
assay. Total RNA was isolated from kidney tissue by the
modified method of Chomczynski and Sacchi (4) using RNAzol. An RNase
protection assay was used to analyze BMP-7 mRNA expression. The DNA
template was constructed by subcloning a 300-bp
EcoR I fragment of the rat BMP-7 cDNA
(bp 668-968) into pGEM-3Zf(+). To generate the single-stranded 32P-labeled RNA
antisense probe, the plasmid was linearized with Xho I and transcribed with T7 RNA
polymerase using the T7/Sp6 transcription kit and 50 µCi
of [32P]UTP for each
transcription reaction under conditions described by the manufacturer.
As a control gene, a 90-bp Pst
I/Rsa I fragment of human ribosomal
RNA (36B4) in pGEM-3Zf(+) was cut with
Rsa I and transcribed with T7 RNA
polymerase. The labeled RNA (1 × 106 cpm) was
hybridized at 50°C to 20 µg of total RNA prepared from rat kidney
tissue. After hybridization, the samples were digested with RNase A (50 mg/ml) and RNase T1 (2 mg/ml) followed by the addition of proteinase K
to inactivate the remaining RNase. After multiple extractions in
phenol-chloroform, the samples were precipitated in ethanol,
redissolved in loading buffer containing 90% formamide, and separated
on a 6% denaturing polyacrylamide-urea gel. Gels were exposed to Kodak
X-Omat film with intensifying screens at In situ hybridization. The technique
was performed as described earlier (28). In brief, 2- to 3-µm
paraffin sections were mounted on charged glass slides and stored in
airtight boxes at Culture of metanephric mesenchymal
cells. Metanephric mesenchymal cell cultures were
prepared in Dr. Mazen Arar's laboratory as described by Herzlinger
with some modification (11). Briefly, 13-day gestation embryonic kidney
rudiments were isolated, and the ureteric bud was freed surgically. The
remaining mesenchyme was incubated in 0.2% collagenase for 5 min and
then transferred to DMEM media with 10% fetal calf serum. Metanephric
mesenchymal cells were then dissociated mechanically by gentle
aspiration through a pipette and plated in DMEM containing 10% serum.
Cells were grown at 37°C in 5%
CO2. Immunohistochemical analysis
using the avidin-biotin complex technique was performed as described previously (2).
Localization of BMP-7 mRNA expression in the adult rat
kidney. A low-power overview in dark-field microscopy
of a kidney section after in situ hybridization with BMP-7 antisense
RNA shows the renal expression of BMP-7 as a layer of bright silver
grains (Fig. 1A).
Strong BMP-7 expression can be seen in a stripe pattern throughout the
medulla with the highest signal expressed in the outer medulla (om).
The cortical tubulointerstitium (c) exhibits weak and scattered BMP-7
hybridization, which is specific compared with the nonspecific signal
using sense hybridization (Fig. 1B).
High-power bright-field microscopy of the outer medulla reveals the
BMP-7 signal over the tubules as black silver grains (Fig.
1C). This expression is highly
specific compared with the sense hybridization signal (Fig.
1D). Also, the tubules of the inner
medulla showed specific BMP-7 expression (Fig.
1E), although this signal was much
weaker compared with the outer medulla.
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
superfamily of secreted growth factors, is abundantly
expressed in the fetal kidney. The precise role of this protein in
renal physiology or pathology is unknown. A cDNA that encodes rat BMP-7
was cloned and used as a probe to localize BMP-7 mRNA expression by in
situ hybridization in the adult rat kidney. The highest expression of
BMP-7 mRNA could be seen in tubules of the outer medulla. In glomeruli,
a few cells, mainly located at the periphery of the glomerular tuft, showed specific and strong signals. Also, high BMP-7 mRNA expression could be localized to the adventitia of renal arteries, as well as to
the epithelial cell layer of the renal pelvis and the ureter. Preliminary evidence suggests that BMP-7 enhances recovery when infused
into rats with ischemia-induced acute renal failure. We examined BMP-7 mRNA expression in kidneys with acute renal failure induced by unilateral renal artery clamping. BMP-7 mRNA abundance as
analyzed by solution hybridization was reduced in ischemic kidneys
after 6 and 16 h of reperfusion compared with the contralateral kidney.
In situ hybridization in ischemic kidneys showed a marked decrease of
BMP-7 mRNA in the outer medulla and in glomeruli. Utilizing rat
metanephric mesenchymal cells in culture, we also demonstrate that
BMP-7 induces epithelial cell differentiation. Taken together, these
data suggest that BMP-7 is important in both stimulating and
maintaining a healthy differentiated epithelial cell phenotype.
superfamily; cytokines; growth
factors; differentiation; metanephric mesenchyme
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(TGF-
)
superfamily of secreted growth factors (22). The relationship between
members of the family is based on amino acid sequence homology of a
highly conserved seven-cysteine domain in the
COOH-terminal region of the proteins. In addition, there appears to be
a functional similarity, since all members of the TGF-
superfamily
have been implicated in organ development (19, 22, 30, 41). BMPs were
isolated from bone matrix and demonstrated to induce ectopic bone
formation in various tissues in vivo (27, 33, 34, 40). Expression in
the kidney or a potential role in renal biology has been shown so far
for BMP-2, -3, -4, -5, -7, and -9 (3, 10, 13, 15, 16, 20, 21, 25, 31,
32, 37).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
70°C. For in situ hybridization, one section
was fixed in freshly prepared, buffered 4% paraformaldehyde overnight
and processed further in a paraffin-embedding automat using a standard
protocol, supplied by the manufacturer.
70°C.
70°C with desiccant up to 4 wk without a
change in results. Riboprobes were generated as described for the RNase
protection assay. Some of the experiments were repeated using a
pGEM3(Zf+) plasmid containing a 469-bp cDNA insert of murine BMP-7 (bp
673-1141) to generate antisense transcripts for in situ
hybridization, with similar results (17). In vitro transcription was
carried out using a commercial kit and 200 µCi
[35S]UTP for each
transcription reaction under conditions described by the manufacturer.
The probes were run on a 6% denaturing
polyacrylamide-urea gel and visualized by autoradiography to ensure
full-length transcripts. For in situ hybridization,
[35S]mRNAs were
diluted to 2 × 104 to 5 × 105 cpm/ml in
hybridization buffer containing 2 mM EDTA (pH 7.5), 20 mM Tris (pH
7.5), 0.6 M NaCl, 2× Denhardt's solution, 20% dextran sulfate,
0.1 mg/ml tRNA, and 0.2 M dithiothreitol. After deparaffinization, kidney sections were digested with 20 mg/ml proteinase K in PBS for 10 min. Sections were postfixed for 5 min in 4%
paraformaldehyde and acetylated using 0.25% acetic
anhydride in 0.1 M triethanolamine for 10 min. A volume of 25-50
µl of hybridization mixture was placed on each section and covered
with a siliconized glass coverslip. Hybridization was performed in
moist chambers at 50°C for 16 h. Coverslips were removed by washing
in 4× saline sodium citrate (SSC) and 2× SSC for 10 min
each at 37°C. Slides were then washed in 0.5× SSC for 15 min
at 37°C followed by a washing step in 0.5× SSC for 30 min at
60°C. After rinsing the slides in NTE buffer (10 mM Tris, 10 mM
NaCl, and 1 mM EDTA) for 15 min at 37°C, RNase A was
added at a concentration of 20 mg/ml and incubated for 30 min at
37°C. Slides were washed in NTE for 15 min, 0.5× SSC for 30 min at 37°C and in 0.5× SSC for 15 min and 0.1× SSC for
15 min at room temperature and dehydrated. Sections were dried for 1 h
at room temperature, and emulsion autoradiography was performed using
Kodak NTB2 emulsion. The coated slides were exposed at 4°C for
10-14 days. Slides were developed in Kodak D19, fixed in Kodak Unifix, and counterstained with hematoxylin and eosin.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
A and
B: dark-field microscopy of kidney
medulla at low-power magnification (×40, green filter).
A: hybridization signal with bone
morphogenetic protein-7 (BMP-7) antisense probe is seen as a layer of
intense bright silver grains in a stripe pattern throughout the medulla
particularly in the outer part (om). Only weak scattered BMP-7
expression is seen in the cortical tubulointerstitium (c). Moderate
BMP-7 signal can be seen also in the inner medulla (im).
B: hybridization with sense probe
shows a weak nonspecific background.
C-F: bright-field microscopy of
outer medulla at high-power magnification (×400).
C: specific hybridization signal with
BMP-7 antisense probe is seen as black grains over epithelial cells in
tubules of outer medulla. D:
hybridization with sense probe shows only weak nonspecific
hybridization. E: hybridization with
BMP-7 antisense probe demonstrates widespread signal involving almost
all tubules in inner medulla. This signal is much less intense but
still specific compared with sense hybridization
(F).
A strong and specific hybridization signal for BMP-7 mRNA expression
could be seen in glomeruli in a peripheral pattern suggesting expression in glomerular visceral epithelial cells. Also, some parietal
epithelial cells of the Bowman's capsule show a positive signal (Fig.
2A)
compared with nonspecific signal using control sense probe (Fig.
2B). Also, there is a diffuse but specific signal in the cortical tubulointerstitium surrounding the glomerulus.
|
Strong BMP-7 mRNA expression was seen surrounding renal arteries (Fig. 2C and inset). At higher power, this signal clearly could be localized to the adventitia of the arteries (Fig. 2C), whereas the smooth muscle layer and the endothelium of the artery showed only nonspecific background signal (Fig. 2D). The epithelial cell layer of the renal pelvis and the ureter was found to be another site of high BMP-7 mRNA expression (Fig. 2, E and F). The smooth muscle layer of the pelvis and ureter again were negative.
BMP-7 mRNA abundance is diminished in ischemic
kidneys. Whole kidney BMP-7 mRNA was analyzed
quantitatively using an RNase protection assay (Fig.
3). Kidneys subjected to
ischemia/reperfusion injury following clamping the renal artery
for 60 min showed no change in mRNA abundance after 3 h of reperfusion.
However, whole kidney BMP-7 mRNA abundance was decreased after 6 h and
even more so after 16 h of reperfusion.
|
BMP-7 mRNA expression is decreased in the outer
medulla and glomeruli. The in situ hybridization for
BMP-7 mRNA expression in the ischemic kidneys after 16 h of reperfusion
showed a diminished signal over the medulla (dark-field microscopy)
(Fig.
4B). The overall expression of BMP-7 mRNA was nearly undetectable particularly in the outer medulla (high-power bright-field) (Fig.
4C). Tubules in the outer medulla
showed signs of injury with loss of brush-border membranes, detachment
of tubular cells from their basement membrane, and loss of cells due to
necrosis. Some cells were still viable and attached to
the basement membrane; however, no or only weak expression of BMP-7
mRNA could be detected (Fig. 4D).
|
Expression of BMP-7 was also diminished in glomeruli of kidneys
subjected to ischemia and 16 h of reperfusion. The peripheral glomerular pattern seen in the control kidneys remained detectable, but
to a much lesser degree (Fig.
5B).
Also, the expression in the cortical tubulointerstitium surrounding the
glomeruli was weaker after ischemia/reperfusion compared with
the control kidneys (Fig. 5A).
|
The expression of BMP-7 mRNA in the adventitia of renal arteries and in the epithelial cell layer of the renal pelvis and the ureter was not affected by renal ischemia/reperfusion (data not shown).
BMP-7 induces differentiation of metanephric
mesenchymal cells into epithelial cells. Metanephric
mesenchymal cells maintained in culture retain their phenotypic
characteristics including fibroblastic morphology and expression of
-smooth muscle actin and vimentin. Upon treatment of confluent cells
with 250 ng/ml BMP-7, foci of condensed rounded polyhedral cells
develop in the cultures. These condensations of cells appear as early
as two days after treatment with BMP-7. The positive staining for
cytokeratin is consistent with epithelial cell phenotype (Fig.
6).
|
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DISCUSSION |
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Cloning of the BMP-7 gene and screening for its expression in mice revealed the kidney as the organ with highest BMP-7 mRNA content (26). Also, BMP-7 mRNA has been shown to be expressed throughout renal development including the earliest stages of metanephric maturation (10, 18, 39). These reports suggest a potential role for BMP-7 in the adult kidney. Our data characterize the distribution of BMP-7 mRNA in the adult rat kidney. Strong expression is found in tubular epithelial cells of the outer medulla and in glomerular epithelial cells. Thus the pattern of BMP-7 expression in the adult kidney is similar to the expression pattern during various stages of fetal kidney development, demonstrating BMP-7 expression in differentiating epithelial cells and structures (10, 18, 39). Moreover, our results show that the abundance of BMP-7 mRNA in the intact kidney during ischemia is significantly diminished compared with the contralateral kidney. This decrease in BMP-7 mRNA abundance is prominent at two major sites, e.g., the outer medullary tubules and glomerular epithelial cells. The decrease in BMP-7 mRNA abundance in the outer medulla might be due at least in part to cell detachment and necrosis following ischemia. However, despite the marked loss of renal function that occurs in the ischemia/reperfusion model, the number of epithelial cells that undergo necrosis and detachment cannot account for this decrease in mRNA abundance. Equal amounts of mRNA were loaded as confirmed by the inclusion of 36B4, a ribosomal RNA, as a housekeeping gene. In addition, tubular epithelial cells in the outer medulla of the ischemic kidney that appear normal and remain attached to their basement membrane also have diminished BMP-7 mRNA expression by in situ hybridization. These results suggest that renal ischemia leads to a decrease in BMP-7 mRNA abundance specifically in these two structures. Two key studies point to an important role for BMP-7 in inducing and/or maintaining epithelial cell differentiation. First, there is severe renal dysorganogenesis in the BMP-7-deficient mouse with an absence of nephron formation (7, 17). Second, recombinant BMP-7 protein is capable of inducing epithelial differentiation with subsequent nephron formation in metanephric organ culture. Moreover, the induction of epithelial differentiation of the metanephric mesenchyme in organ culture by spinal cord tissue can be blocked completely by BMP-7 antisense oligonucleotides or anti-BMP-7 antibodies (38). Our data utilizing metanephric mesenchymal cells in culture confirm these observations. The addition of BMP-7 to the cells results in differentiation of a subpopulation of these cells into epithelial cells that express cytokeratin. Also, the data in BMP-7-deficient mice suggest that BMP-7, perhaps in concert with other factors, is necessary for renal organogenesis (17). Potential mechanisms of action of BMP-7 in addition to differentiation include effects on proliferation and/or prevention of apoptosis prior to mesenchymal epithelial differentiation. In the adult kidney, epithelial cells already possess a fully differentiated and highly specialized phenotype. Renal injury results in morphological and functional changes in epithelial cells (23, 29). Tubular epithelial cells are particularly susceptible and appear to dedifferentiate during renal ischemia.
The diminished expression of BMP-7 in ischemic kidneys is consistent
with a recent report (1) demonstrating a marked decrease in the
expression of BMP-7 mRNA and protein in the medulla of ischemic kidneys
and with several reports showing a beneficial effect of BMP-7 on renal
structure and function. The studies reported a beneficial effect of
recombinant BMP-7 infused in rat models of acute and chronic renal
injury. Two preliminary studies published as abstracts reported an
increase in glomerular filtration rate and renal blood flow and a
decrease in serum creatinine and blood urea nitrogen in rats that
received BMP-7 injections (14, 36). Another study demonstrated that
BMP-7 infusion preserves the integrity of vascular smooth muscle cells
and maintains actin expression in -smooth muscle cells (35). In this
study, BMP-7 decreased endothelial expression of the intercellular
adhesion molecule ICAM, evidence for suppression of the inflammatory
response associated with ischemia/reperfusion injury. In the
five-sixths nephrectomy model, there is an increase in BMP-7 mRNA
expression in response to the injection of recombinant BMP-7 associated
with partial preservation of renal function and structure (6). Taken
together, the results of these studies and the high constitutive
expression of BMP-7 in the adult kidney and its decreased expression in
renal ischemia shown in this study suggest that BMP-7 may play
a role in adult kidney physiology as well pathophysiology. It is
unclear whether the decrease in BMP-7 in ischemic renal epithelial
cells contributes in some way to the reported injury of these cells. However, it is most likely that BMP-7 functions to maintain a fully
differentiated, healthy epithelial cell phenotype in the adult kidney.
The structural and functional improvements that follow BMP-7
administration and our data supporting epithelial cell differentiation
of metanephric mesenchymal cells confirm this contention.
Interestingly, BMP-7 has been shown to stimulate proliferation of
primary smooth muscle cell cultures, whereas in established smooth
muscle cell cultures, BMP-7 inhibits cell growth and maintains the
expression of markers that are characteristic of fully differentiated
smooth muscle cell phenotype (5).
BMP-7 mRNA was also expressed normally in two other anatomical sites that deserve comment. The biological relevance of the expression of BMP-7 mRNA in the adventitia of renal arteries remains to be determined. The abundance of BMP-7 mRNA in the adventitia of renal arteries and on the epithelial cell layer of the pelvis and the ureter was not affected by the ischemia. However, there is evidence that this family of proteins may be involved in the biology of vascular wall. BMP-2, another member of this family of proteins, is expressed in atherosclerotic plaques. In vivo BMP-2 inhibits smooth muscle cell proliferation in a rat carotid artery balloon injury model (24). Similar inhibitory effects of BMP-2 on cell proliferation are seen in cultured smooth muscle cells and in mesangial cells (unpublished data) (24).
BMP-7 expression was also high and specific in the epithelial cell layer of the renal pelvis and the ureter. This pattern of expression fits well with the original observation of Huggins (12) in 1931, who demonstrated that urinary tract epithelium induces new bone formation in adjacent tissue after surgical approximation. It is possible that BMP-7 may act to protect urogenital epithelium from the constant exposure to urinary constituents. Further studies are needed to evaluate whether a loss of BMP-7 is involved in pathological conditions of the ureter and the bladder, such as urinary tract obstruction, interstitial cystitis or carcinogenesis.
In summary, this study demonstrates the constitutive expression of BMP-7 mRNA in the adult kidney. Specific BMP-7 mRNA expression is localized to tubular and glomerular epithelial cells, the adventitia of renal arteries, and the epithelium of the renal pelvis and ureter. Renal ischemia/reperfusion injury decreases BMP-7 expression specifically in the epithelium of outer medullary tubules and glomerular epithelial cells. We hypothesize that BMP-7 functions to maintain epithelial cell differentiation in normal adult kidneys. A decrease of its expression in ischemia may be involved in pathobiological manifestations of ischemia/reperfusion injury.
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ACKNOWLEDGEMENTS |
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We thank Merna Gonzales and Sergio Garcia for technical help and Janet Ortiz for typing the manuscript.
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FOOTNOTES |
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This study was supported in part by the Department of Veterans Affairs Medical Research Service and National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-33665 and DK-43988 (to H. E. Abboud) and DK-19473 (to M. S. Olson) and by Grants-in-Aid from the American Heart Association-Texas Affiliate (to M. Arar). M. Simon was a recipient of a research fellowship of the Deutsche Forschungsgemeinschaft and is currently a recipient of a fellowship of the National Kidney Foundation. J. D. Hernandez was supported by a National Institutes of Health Minority Supplement Award.
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. §1734 solely to indicate this fact.
Address for reprint requests: H. E. Abboud, Division of Nephrology, Dept. of Medicine, Univ. of Texas Health Science Center, 7703 Floyd Curl Dr., San Antonio, TX 78284-7882 (E-mail: abboud{at}uthscsa.edu).
Received 21 July 1998; accepted in final form 8 October 1998.
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