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From the Nephro-Urology Unit, Institute of Child Health, University College London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-smooth muscle actin, a structural protein up-regulated
during TGF-ß1-induced phenotypic switching between human renal
dysplastic epithelial and mesenchymal lineages in
vitro, was also observed in these aberrant epithelia.
These findings implicate increased TGF-ß1 signaling in the
early biological changes generated by fetal urinary tract
obstruction.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Experimental disruption of urine flow during renal development causes renal malformations in several animal species.6-8 Sheep constitute a particularly good experimental model because the urinary tract can be manipulated relatively early in gestation in vivo, hence mimicking potential events in utero in humans. The ovine metanephros appears at 27 to 30 days of gestation, urine flows at an estimated 5 to 6 ml/hour by 75 days,9 and full term is 145 days. Interruption of urine flow by either ureteral or urinary bladder outlet obstruction generates a spectrum of anatomical abnormalities, which are dependent on the timing of the surgery, ranging from growth failure and dysplasia to hydronephrosis and subcapsular cysts.6,10,11 Moreover, our previous studies have demonstrated both morphological and molecular changes after a relatively short period of fetal urine flow impairment: complete unilateral ureteric obstruction was performed at 90 days and, after 10 days, we observed disruption of nephrogenesis with diminished formation of nephrons and cystic dilatation of maturing glomeruli.12 These anatomical changes were accompanied by dysregulation of cell proliferation and apoptosis, and aberrant expression of molecules critical for normal nephrogenesis such as the transcription factor PAX2, molecular aberrations similar to those observed in human renal malformations.13-15
Transforming growth factor-ß1 (TGF-ß1) is a multifunctional
cytokine, which is widely expressed during normal development and
up-regulated in diverse adult pathological conditions, including kidney
diseases.16
TGF-ß1 signaling is transduced via cell
surface type I and type II receptors (TGF-ßR1 and TGF-ßR2):
TGF-ßR2 binds the ligand and forms a complex with TGF-ßR1 that
activates intracellular signaling cascades.17
Exogenous
TGF-ß1 blocks branching morphogenesis in metanephric cell and organ
culture,18,19
hence reproducing in vitro one of
the classic features of human dysplastic kidneys.4
We
therefore recently studied the expression of components of the TGF-ß1
axis in human dysplastic kidneys, a subset of which were associated
with impaired urine flow, and demonstrated up-regulation of the
cytokine and persistent expression of its receptors.20
In
addition, exogenous TGF-ß1 caused dysplastic epithelial cells in
culture to lose epithelial characteristics (eg, ZO1) and gain
mesenchymal markers such as -smooth muscle actin (SMA). Based on
these findings, we postulated that up-regulated TGF-ß1 signaling,
potentially induced by urinary tract obstruction, could have a number
of effects in human dysplastic kidneys including inhibitory effects on
PAX2-mediated cyst growth and induction of epithelial to mesenchymal
transformation with consequent loss of potential
nephrons.20
We therefore hypothesized that surgical ureteric obstruction of mid-gestation sheep would lead to up-regulation of components of the TGF-ß1 axis in the ipsilateral kidney. Our results confirm that a relatively short period of obstruction is sufficient to up-regulate TGF-ß1 expression during nephrogenesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Surgery and Collection of Samples
Sheep samples were obtained from a tissue bank established from previous operations.12 In brief, pregnant Mule cross-breed sheep (R. White, Oxfordshire, UK) were fasted for 1 day, at 90 days of gestation, and anesthesia was induced with sodium pentothal, then maintained with halothane/oxygen. The abdomen was incised in the midline, the uterus entered with diathermy and the fetal hindquarters delivered. Diathermy was used to make a lumbotomy incision through skin and muscle and the fetal ureter was identified and ligated. The muscle layers, uterus, and skin were closed with vicryl or silk sutures, anesthesia was stopped, and animals were allowed to recover. Gentamicin (80 mg) and penicillin (600 mg) were injected into the uterine cavity at operation, and intramuscular streptomycin (1 g) was administered for 5 days postoperatively. Obstructed kidneys from six animals were harvested 10 days after surgery at 100 days of gestation (obstructed group). Contralateral kidneys were not examined in this project, because no significant difference in weight or gross morphology was observed in our previous study of short-term obstruction,12 although longer term unilateral fetal obstruction or abnormal kidney development is associated with contralateral overgrowth and changes in gene expression.10,21 Control, or sham-operated, kidneys (n = 5) were obtained from the twin animals to the operated group, which underwent the same operative anesthesia but did not have ureteral ligation. Half of each freshly-removed fetal sheep kidney was fixed in 2% paraformaldehyde and embedded in paraffin wax and the symmetrical other half was snap-frozen in liquid nitrogen and stored at -70°C.
In Situ Hybridization
For TGF-ß1 in situ hybridization we used a human TGF-ß1 cDNA plasmid, as described.20 Other groups have previously confirmed the use of human probes to detect sheep TGF-ß1 mRNA.22 Plasmids were linearized with restriction enzymes and sense and anti-sense uridine triphosphate-digoxigenin-labeled riboprobes were prepared using an RNA polymerase kit (Digoxigenin RNA labeling kit; Boehringer Mannheim, Lewes, UK). In situ hybridization was performed on 7-µm paraffin sections of formation-fixed tissue, as described.20 After dewaxing, sections were digested for 10 minutes with proteinase K (20 µg/ml) at 37°C and then postfixed in 4% paraformaldehyde. Sections were then washed in 2x standard saline citrate, prehybridized, and then hybridized with digoxigenin-labeled sense or anti-sense TGF-ß1 probes at 60°C overnight in 100 µl of hybridization buffer containing 50% v/v formamide, 5x standard saline citrate, 1x Denhardt’s reagent, heat-denatured salmon sperm DNA (0.1 mg/ml), and 10% w/v dextran sulfate. Sections were finally washed at 60°C in 2x standard saline citrate with 25% formamide, followed by 1x standard saline citrate and 0.1% sodium dodecyl sulfate. Hybridized probe was detected by incubation with anti-digoxigenin antibody conjugated to alkaline phosphatase and color development with NBT/BCIP substrate (Promega, Southampton, UK). Slides were washed and mounted with Citifluor (Chemical Labs, University of Kent, UK).
Immunohistochemistry
For TGF-ß1 immunohistochemistry we used a rabbit polyclonal
antibody (sc146; Santa Cruz Biotechnology, Santa Cruz, CA) raised
against a peptide corresponding to amino acid residues 328 to 353
within the carboxy-terminal region of the human TGF-ß1. This antibody
shows no cross-reactivity with TGF-ß2 or TGF-ß3 and, despite being
directed against the human protein, has confirmed specific staining
patterns in sheep.22
For TGF-ß receptor
immunohistochemistry we used specific anti-TGF-ßR1 (sc402) and
TGF-ßR2 (sc220) antibodies raised against the carboxy terminus of the
human proteins. We have previously validated these antibodies in human
studies20
but found in this study that, although both gave
specific results on Western blotting, only the anti-TGF-ßR1 antibody
worked for immunohistochemistry on sheep tissue sections. A mouse
monoclonal antibody was used for -SMA (A5691). Immunohistochemistry
was performed as described.20
Four-µm sections were
dewaxed through Histo-Clear (National Diagnostics, Atlanta, GA) and
rehydrated through 100 to 30% alcohols. After washing in tap water and
phosphate-buffered saline (PBS) (pH 7.4) for 5 minutes, they were
treated with trypsin (1 mg/ml) for 10 to 15 minutes at 37°C.
Endogenous peroxidase was quenched with 3% hydrogen peroxide for 30
minutes at room temperature. Sections were washed with blocking buffer
(PBS, 10% goat serum, 0.1% Tween-20) and then incubated with the
primary antibody (TGF-ß1 antibody at 1:150 to 1:300 dilution,
TGF-ßR1 and TGF-ßR2 antibody at 1:500 to 1:1000, -SMA antibody
at 1:150 to 1:300) in blocking buffer overnight at 4°C. After
thorough washing in PBS/0.1% Tween-20, primary antibodies were
detected by the sensitive DAKO EnVision+ System (DAKO, Ely, UK).
Controls were omission of primary antibody or preincubation with a
10-fold excess of the appropriate peptide for 2 hours at room
temperature. Sections were counterstained with hematoxylin, mounted in
dextropropoxyphene (BDH, Poole, UK), examined on a Zeiss Axioplan
microscope (Carl Zeiss, Oberkochen, Germany), and photographed under
oil immersion with a x63 magnification lens.
Western Blotting
Kidney samples were homogenized in RIPA buffer (PBS, 1% Nonidet
P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate) plus
protease inhibitors (30 µg/ml aprotinin, 100 mmol/L sodium
orthovanadate, 100 mmol/L phenylmethyl sulfonyl fluoride) and
supernatants were collected by centrifugation at 13,000 rpm for 15
minutes. Protein concentration was determined using the bicinchoninic
acid protein assay (Pierce, Rockford, IL). Samples were boiled for 5
minutes, then 50 µg of total protein was loaded per well and
electrophoresed through an 8 or 12% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis gel (8% for TGF-ß1
analysis or 12% for the other proteins). Equality of loading was
determined by staining representative gels with Coomassie blue (data
not shown). After electrophoresis, proteins were transferred to
nitrocellulose membranes (Amersham Life Science Ltd., Little Chalfont,
UK) by electroblotting (Bio-Rad, Hemel Hempstead, UK) and left
overnight at 4°C in blocking solution [5% (w/v) fat-free milk
powder, 0.3% (v/v) Tween-20 in PBS]. They were then incubated with
primary antibodies, as above, including anti-TGF-ß1, TGF-ßR1, and
TGF-ßR2 at 1:200 to 500 dilution, or -SMA (A2547) at 1:1000
dilution, at 4°C for 1 hour. After washing in blocking solution,
blots were incubated for 30 minutes with appropriate horseradish
peroxidase-conjugated second antibodies diluted 1:1000 to 1500 in
blocking solution. Blots were washed three times with blocking solution
and once with PBS. The blot was then developed using the ECL detection
kit (Amersham Life Science Ltd.), and intensity of staining was
determined by optical densitometry after scanning on a Hewlett-Packard
ScanJet 5100C (Hewlett-Packard Ltd., Bracknell, UK). Results were
standardized so that protein levels in the sham-operated kidneys were
nominally designated as 100% and statistical assessment of staining
intensity was performed using a Student’s t-test. Rainbow
markers were used to determine protein size. In some experiments, after
visualizing the immobilized proteins the antibody complex was stripped
by rocking the blot in stripping buffer (Chemistrip; Chemicon, Harrow,
UK) for 1 hour at room temperature. The blot was then reprobed with
other antibodies, as above. This procedure allowed direct comparison of
staining intensity on the same samples with different antibodies.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, containing ureteric bud branch tips,
mesenchymal condensates and early nephron precursors, such as S-shaped
bodies, as previously described.12
In contrast, in organs
obstructed from 90 to 100 days of gestation, the superficial cortex was
grossly abnormal with dilated ureteric bud branches, sparse mesenchymal
condensates, and numerous cysts (Figure 1B)
. Normal S-shaped bodies
were not detected and cystic dilatation of developing glomeruli was
frequently observed (Figure 1B)
. Obstruction of urine flow also
perturbed development of the medulla, with marked dilatation of
collecting ducts (Figure 1; C, D, and E
).
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. Weak signals were also observed in some
glomeruli in the deeper cortex, particularly over endothelial cells and
podocytes but not in the parietal epithelium of Bowman’s capsule (data
not shown). Using the same antisense probe, prominent TGF-ß1 signal
was detected in the superficial cortex of obstructed kidneys at 100
days (Figure 1B)
: main sites of expression were dilated ureteric bud
branches, dilated tubules, and both the tufts and parietal epithelia of
cystic glomeruli. Normal medullary collecting ducts in sham-operated
organs expressed low levels of TGF-ß1 transcripts (Figure 1C)
,
whereas strong signals were detected in dilated medullary collecting
ducts in obstructed kidneys (Figure 1D)
. Significant signals were not
detected in any of these sites in control hybridizations with a
TGF-ß1 sense probe (Figure 1E
and data not shown).
Histochemistry demonstrated faint immunoreactivity for TGF-ß1 in
ureteric bud branch tips in the sham-operated nephrogenic cortex
(Figure 2A)
, whereas strong
immunostaining was observed in epithelia of dilated ureteric buds and
developing tubules from obstructed organs (Figure 2B)
. Deeper in the
cortex of sham-operated kidneys, TGF-ß1 immunostaining was weakly
positive in normal glomeruli and maturing distal tubules (Figure 2C)
.
Prominent TGF-ß1 immunoreactivity was observed, however, in
obstructed kidneys, particularly in the epithelia of glomerular cysts
and dilated tubules (Figure 2D)
. Occasional cells around these
distorted epithelia were also positive. TGF-ß1 was also detected in
vessels in both sham-operated and obstructed kidneys (Figure 2C
and
data not shown). In the medulla, faint TGF-ß1 immunoreactivity was
detected in collecting ducts in control kidneys whereas, in parallel
with our in situ hybridization findings, more intense
TGF-ß1 immunostaining was recorded in large, dilated collecting ducts
in obstructed kidneys (data not shown). Significant TGF-ß1
immunostaining was not detected in sham-operated or obstructed kidney
sections when the primary antibody was either omitted or preabsorbed
with TGF-ß1 peptide (Figure 2, E and F
, respectively).
|
. Low
levels of receptor protein were also detected in normal
loosely-arranged uninduced mesenchyme (Figure 3A)
. In the obstructed
kidneys, prominent TGF-ßR1 immunostaining was detected in both
normal-caliber and dilated tubules, and in glomerular cysts (Figure 3B)
. Weak receptor immunoreactivity was also noted in a subset of
surrounding cells (Figure 3B)
. Large collecting ducts were positive for
TGF-ßR1 in the medulla of sham-operated animals (Figure 3C)
and this
receptor was strongly expressed in abnormal dilated collecting ducts in
obstructed kidneys (Figure 3D)
.
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-SMA staining was not detected in the sham-operated
nephrogenic cortex (Figure 4A)
. In
contrast, there was marked up-regulation of this mesenchymal marker in
the superficial cortex of obstructed kidneys, both within dilated
epithelia and in cells around distorted glomeruli and tubules (Figure 4B)
. Deeper in the normal cortex, -SMA staining was observed in the
mesangium of normal maturing glomeruli (Figure 4C)
, but there was
stronger staining in obstructed kidneys, particularly within the tufts
in cystic glomeruli (Figure 4D)
. In the medulla, very little -SMA
immunoreactivity was detected in normal collecting ducts (Figure 4E)
,
whereas there was prominent staining in the epithelia of collecting
ducts in obstructed kidneys (Figure 4F)
. Strong -SMA staining was
also observed in vessels in both normal and abnormal kidneys (Figure 4
;
C, E, and F).
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-SMA
antibodies was confirmed by Western blotting (Figure 5)
, because these antibodies were
originally intended for human studies. Major bands, of the expected
size,17,20
were detected in both sham-operated and
obstructed kidneys as follows: 25 kd corresponding to TGF-ß1 dimers,
55 kd for TGF-ßR1, 70 kd for TGF-ßR2, and 43 kd for -SMA (Figure 5)
. Compared to sham-operated kidneys, where protein levels were
designated as 100%, there were increases of 281% in TGF-ß1
(P = 0.02), 379% in TGF-ßR1
(P = 0.02), 291% in TGF-ßR2
(P = 0.02), and 54% in -SMA
(P = 0.01).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We recorded up-regulation of TGF-ß1 mRNA and protein in the malformed kidneys attached to obstructed ureters, particularly in aberrant epithelia. These changes were observed after only 10 days of obstruction, suggesting that TGF-ß1 may be important in the early biological changes generated by impairment of urinary flow. A potential explanation for these findings could be that the increased hydrostatic pressure generated by impaired urinary flow causes direct stretch of the epithelia with resultant epithelial up-regulation of TGF-ß1, as reported for other renal cells subjected to cyclical stretch in culture.23 This is the first time that up-regulation of TGF-ß1 has been reported after such short-term fetal obstruction, although the results are consistent with our observations of up-regulated TGF-ß1 in human dysplastic kidneys,20 some of which were attached to obstructed lower urinary tracts. Up-regulation of TGF-ß1 has also been described by Medjebeur and colleagues22 in longer term urinary tract obstruction in fetal sheep: they reported that bladder obstruction, generated by combined ligation of the urethra and urachus between 60 and 80 days of gestation, caused increased TGF-ß1 mRNA and protein expression when assessed at 120 days of gestation, after nephrogenesis was complete.22 Increased TGF-ß1 protein expression was observed in both tubular epithelia and the renal interstitium in this model22 and it would have been intriguing to examine either protein distribution at earlier time points or mRNA expression by in situ hybridization to determine whether the cytokine was produced within the epithelia, as we observed. Other reports relevant to the current study describe increased TGF-ß1 in rats with congenital hydronephrosis,24 and angiotensin II-mediated up-regulation of TGF-ß1 with perturbed renal growth after neonatal ureteric ligation in the rat.8 TGF-ß1 has also been implicated in the progressive fibrosis induced by obstruction of the adult kidney.25
TGF-ß1 signaling is transduced via TGF-ßR1 and TGF-ßR2, but the distribution of these receptors has not previously been reported in fetal urinary tract obstruction. In this study, we detected up-regulation of both receptors on Western blot, and TGF-ßR1 protein immunolocalized to abnormally dilated tubule and glomerular epithelia in the cortex, sites in which we detected its ligand. Moreover, there was persistent expression of TGF-ßR1 in abnormally dilated medullary collecting ducts. These expression sites would be consistent with an autocrine TGF-ß1 signaling system within epithelia, although we were unable to confirm co-localization with TGF-ßR2, which is necessary for initial TGF-ß1 binding, because the anti-human TGF-ßR2 antibody did not work on sheep sections in this study. Up-regulation of TGF-ßR1 has also been reported after postnatal ureteric obstruction in the rat26 but was not observed in ureteric tumor-associated obstruction in humans.27
What are the likely biological effects of elevated TGF-ß1 signaling
within developing renal epithelia? We postulate that there would be at
least two distinct effects: firstly, TGF-ß1 could perturb epithelial
cell turnover by restricting proliferation and promoting apoptosis, as
described in other epithelia.20,28,29
In this context, it
is of note that apoptosis was detected in cyst walls and glomerular
tuft epithelia in our previous experiments using this short-term
obstruction model (see Figures 5C and D
in Attar et al12
),
sites where we observed TGF-ß1 expression in the present study.
Secondly, TGF-ß1 may divert potential functional renal epithelia
toward a mesenchymal lineage, as reported in vitro in mature
rat kidney tubular epithelial cells.30
Evidence supporting
this second mechanism comes from our current observation that -SMA
was up-regulated in malformed epithelia in obstructed kidneys. We
suggest that this is unlikely to be a nonspecific stress response in
view of our previous findings that excess TGF-ß1 directly causes
increased -SMA in human renal epithelial cells in
vitro.20
Hence, the -SMA-positive epithelia
observed in obstructed kidneys may represent a first step of
transitional cells that are undergoing TGF-ß1-induced phenotypic
transformation toward a mesenchyme-like lineage.
TGF-ß1 is not the only molecule that is known to be dysregulated in
urinary tract maldevelopment, however, and other potential conflicting
or modifying factors may also affect the final renal phenotype. The
transcription factor PAX2, for example, is up-regulated in both human
cystic epithelia14
and in this sheep obstruction
model.12
PAX2 has been implicated in the survival and
expansion of metanephric precursor cells during development and cyst
growth in transgenic mice.31
One would therefore predict
that excess PAX2 alone should promote cyst growth, which is consistent
with our earlier observation of increased proliferating cell nuclear
antigen expression in dilated epithelia in the obstructed sheep kidneys
(see Figures 3 and 4
in Attar et al12
). In this location,
therefore, it is likely that the biological results of the
up-regulation of TGF-ß1 reported in our present study would be to
limit the rate of cyst growth by antagonizing the effects of PAX2. One
potential mechanism for this would be a TGF-ß1-mediated reduction in
the stability of PAX2 mRNA, as demonstrated in cultured renal
epithelial cells.32
Moreover, we have demonstrated that
expression of PAX2 protein is reduced after TGF-ß1 treatment of human
dysplastic epithelial cells.20
These complex potential
interactions comprise part of the working model, linking up-regulation
of TGF-ß1 and Pax2 to fetal urinary tract obstruction in the
pathogenesis of kidney malformations, recently proposed by our group
(see Figure 10 in Yang et al20
). Other potential modifying
factors described in obstructed kidneys include angiotensin II, the
survival factor BCL2, and epidermal growth factor.8
In summary, in the present study we observed up-regulation of the
multifunction cytokine TGF-ß1 and its receptors TGF-ßR1 and
TGF-ßR2, co-localization of the cytokine with TGF-ßR1 in aberrant
epithelia and elevated levels of -SMA in a sheep model of short-term
mid-gestation unilateral ureteric obstruction. These observations raise
the possibility that therapeutic interventions to reduce the activity
of the TGF-ß1 signaling axis (eg, using substances such as
decorin33
) may ameliorate the severity of human renal
malformations. Some of the effects of excess TGF-ß1 may, however, be
beneficial by preventing or restricting the actions of other factors,
such as PAX2. Further clarification of the biological effects of
TGF-ß1 blockade will therefore be required in animal models such as
this one before such therapies could be contemplated in humans.
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Acknowledgements |
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Footnotes |
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Supported by project grants from Action Research (S/P/3178), the National Kidney Research Fund (R11/2/1996 and R18/1/2000), and the Kidney Research Aid Fund.
Accepted for publication April 18, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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