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Regular Articles |
From INSERM U.423*
and INSERM
U383,
Hôpital Necker-Enfants Malades,
Université René Descartes, Paris, France and the Department
of Pathology,
Howard Hughes Medical
Institute, Ann Arbor, Michigan
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Abstract |
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 Top Abstract IntroductionPatients and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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The WT1 gene comprises 10 exons and encodes a zinc finger (ZF) protein. Exons 1 to 6 encode a proline/glutamine rich region, and exons 7 to 10 encode the four ZF of the DNA-binding domain.9,10 Different functional domains involved in either repression or activation of transcription,11,12 a region involved in homodimerisation of the protein,13 and an RNA binding domain14 have been characterized. Two alternative splicing regions, one corresponding to the 17 amino acids encoded by exon 5 and the other corresponding to amino acids KTS encoded by the 3' end of exon 9, lead to the synthesis of four isoforms with definite and stable proportions15 and distinct subnuclear localization revealing different functions. WT1 plays a role in posttranscriptional processing of RNA as well as in transcription.14,16,17
Formation of the metanephros, the third and permanent renal organ in mammals, starts on the 5th week of gestation in human. It results from interactive events between the ureteric bud, an epithelial tube off the Wolffian duct, and the metanephric blastema, a loosely organized mesenchyme located at the caudal extremity of the nephrogenic cord.18,19 The mesenchymal blastema induces the growth and regular dichotomous branching of the ureteric bud leading to the formation of the collecting system. In contact with the ampullar tip of ureteral bud branches, mesenchymal cells condensate and transform into epithelial cells. Condensates undergo a series of transformation and elongation, resulting in the formation of mature nephrons. Vascularization seems to be provided by a double process of angiogenesis and vasculogenesis. Successive generations of nephrons are formed from the 8th week to the 36th week of gestation. Concomitant with the nephrogenesis, there are profound and definite changes in the expression of cellular and extra-cellular matrix proteins, growth factors and their receptors, proto-oncogenes, and transcription factors, with a tight spatial and temporal regulation.
WT1 is one of the transcription factors involved in nephrogenesis.20-22 In the fetal kidney, it is expressed in the metanephric blastema, condensing mesenchyme, renal vesicle, and developing and mature podocytes. The other main sites are the genital ridges, gonads and mesothelium. Knock-out of the gene in the mouse resulted in the absence of both kidneys and gonads.22 The expression of WT1 in the podocyte persists into adult life. In DDS, most WT1 mutations are de novo missense changes in exons 8 or 9 affecting zinc fingers 2 or 3 with a hotspot (R394W) in exon 9.4-7 However, rare DDS patients carry a WT1 mutation leading to putative truncated proteins lacking part of or the entire ZF domain.5-7 Changes in the zinc-finger structure are thought to behave in a dominant negative fashion in patients with DDS.23 In vitro, they abolish the binding of the WT1 ZF domain to its normal DNA targets.4,24 The gene PAX2, which encodes a transcription factor expressed in early kidney development, is one of the WT1 targets.25,26 It is down-regulated in precursor cells of the visceral glomerular epithelium, simultaneously with a marked increase in WT1 expression.27 Repression of PAX2 by WT1 has been demonstrated in vitro by the binding of WT1 to PAX2 regulatory sequences and by cotransfection assays using CAT reporter constructs under the control of PAX2 regulatory sequences showing WT1-dependent transcriptional repression.27 Transactivation of the WT1 promotor by PAX2 has also been described.28 PAX2 plays an important role in kidney development. Up-regulation of its expression in transgenic mice generates severe kidney abnormalities,29 whereas Pax2 null mutant mice lack kidneys, ureters, and genital tracts.30
The aim of our study was to analyze the glomerular expression of WT1 and PAX2 in patients presenting with DDS. Normal fetal and postnatal kidneys, and kidneys from patients presenting various types of nephropathies were used as controls. We also analyzed the expression of these two transcription factors in patients affected with IDMS. In the present study, we show that podocyte distribution of WT1 was abnormal in the majority of patients affected with DDS, a finding consistent with the decreased DNA-binding capacity of the abnormal WT1 protein. Persistent expression of PAX2 was observed in the podocyte, a finding likely to result from the loss of WT1 dependent transcriptional repression. Interestingly, the same anomalies were also found in most patients with IDMS suggesting that a defect in WT1 could also be operative in at least some IDMS, a hypothesis recently confirmed in some patients by our molecular studies.7 Primary involvement of PAX2 is an alternative hypothesis.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Renal tissue from 10 patients with DDS and 13 patients with IDMS
was obtained at biopsy performed for diagnostic purposes (15 patients)
at nephrectomy because of Wilms' tumor or before renal transplantation
(6 and 9 patients, respectively). Clinical and molecular data
concerning these patients are presented in Table 1
. Clinical data from 14 of these
patients have been previously reported3,8
as well
as results of molecular studies of DDS patients (patients (pts)
3, 4, and 5),4
DDS patients (pts 1, 2, and 6),
and IDMS patients (pts 22, 23, and 24).7
Clinical
and molecular findings in three patients with Wilms' tumor and
abnormal genitalia but with no glomerular symptoms are also presented
in Table 1
.7
Analysis of the expression of PAX2
and WT1 was performed on normal renal tissue adjacent to the tumor. In
this retrospective study, molecular analysis of the WT1 gene
was done only in patients observed in the most recent period.
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Antibodies
WT(C-19)(Santa Cruz Biotechnology, Santa Cruz, CA) is an affinity-purified rabbit polyclonal antibody raised against a peptide corresponding to amino acid residues 327–345 mapping at the carboxy terminus of the human Wilms' tumor protein. The first batch of antibody (6063) gave abnormal staining of vascular smooth muscle cell nuclei in addition to the expected normal pattern, a reaction previously observed by Charles et al31 with similar antibody. Faint but diffuse cytoplasmic staining of tubular cell cytoplasm was also observed. For these reasons, two other batches (D124 and H 277) were tested. Both gave the same specific labeling on normal kidneys without any staining of smooth muscle cells; tubular cell cytoplasm was strictly negative. Staining was stronger with H277. Consequently, only results obtained with this last antibody are presented in this paper. The PAX2 antibodies were generated against a bacterial fusion protein containing amino acids 188–385 of the PAX2 coding region.26 This region does not include the conserved paired domain. Studies in transfected cells demonstrate minimal cross-reactivity with the related proteins, PAX5 and PAX8.32 Within the PAX2 sequence used for the antigen, the mouse and human genes are 97.5% identical. These antibodies recognize PAX2 proteins from a diverse array of species, including mouse, human, zebrafish, and chick. PC10 (Pharmingen, San Diego, CA) is a purified monoclonal antibody raised again the proliferating cell nuclear antigen (PCNA). Goat anti-rabbit immunoglobulins were purchased from Biosource International (Camarillo, CA). Affinity-purified fluorescein isothiocyanate-conjugated swine anti-goat IgG(H+L) was purchased from Caltag Laboratories (Burlingame, CA). Biotinylated horse anti-mouse immunoglobulins, avidin-biotin blocking kit, VECTASTAIN Elite ABC kit, diaminobenzidine, and substrates were from Vector Laboratory (Burlingame, CA).
Methods
Both immunofluorescence and immunoperoxidase methods were used on Dubosq-Brazil or formalin-fixed, paraffin-embedded tissues from 10 DDS patients (four biopsy and six nephrectomy specimens), 14 IDMS patients (11 biopsy and three nephrectomy specimens), three patients with Wilms' tumor, and 28 controls. In four patients (pts 6, 8, 14, and 21), a second specimen obtained later on in the course of the disease was also studied. In six patients (pts 2, 3, 5, 14, 15, and 21), immunolabeling was performed in parallel on frozen tissues.
Immunofluorescence
Indirect immunofluorescence was performed as follows: 3-µm thick sections were mounted on 3-aminopropyl triethoxysilane pretreated slides. Deparaffinized sections were treated with microwave heating (2 x 5 minutes in 0.8 mol/L urea, pH 6.4). Ten percent normal swine serum in PBST (0.01 mol/L phosphate-buffered saline (PBS) containing 0.05% Tween-20) was used as blocking solution. WT1 and PAX2 antibodies were diluted to 1:100 and 1:500, respectively. After incubation with primary antibodies for 1 hour at room temperature, sections were rinsed three times in PBS and incubated for 30 minutes with goat anti-rabbit immunoglobulins diluted to 1:40 in PBS. After washing in PBS, they were incubated for 40 minutes with fluorescein isothiocyanate-conjugated swine anti-goat IgG(H+L) diluted to 1:40 in PBS. Then slides were washed in PBS. A mounting media containing p-phenylene-diamine was used to delay fluorescence quenching. Labeling was examined with a Leitz Orthoplan microscope equipped for light, fluorescence, and phase contrast microscopy. Phase contrast microscopy allows the visualization of cell structure and the precise identification of cell nuclei.
Immunoperoxidase
Frozen Tissue
Renal samples were snap-frozen in liquid nitrogen using OCT compound (Miles Laboratories Inc, Naperville, IL). Four-µm thick cryostat sections were air dried and fixed in acetone for 10 minutes. Immunoperoxidase staining was carried out using the Vectastain Elite ABC kit (Vector). After washing in fresh buffer (0.01 mol/L PBS, pH 7.4), endogenous biotin was blocked by the biotin blocking agent according to the instructions of the manufacturer. Then sections were incubated for 1 hour at room temperature in a moist chamber with the appropriate dilution of primary antibodies in PBST:WT1, 1/1000 and PAX2, 1/500. They were washed in PBS and incubated with biotinylated secondary antibody for 30 minutes. For quenching the endogenous peroxidase, sections were treated with 3% hydrogen peroxide in methanol for 5 minutes and then washed in PBS for 20 minutes. They were then incubated for 30 minutes with Vectastain Elite ABC reagent. After washing, final staining of the sections by diaminobenzidine or very intense purple, chromogenes was monitored under the microscope. Sections incubated with anti-PCNA antibodies were counterstained with hematoxylin for 5 minutes. No counterstaining was performed on other sections.
Fixed Tissue
Three-µm thick deparaffinized sections were pretreated by microwave heating in a urea solution (2 x 5 minutes in 0.8 mol/L urea, pH 6.4). WT1, PAX2, and PCNA antibodies were used at a dilution of 1:1000, 1:800, and 1:1000, respectively. Staining procedures were as previously described.
Controls were performed in the same conditions, but the primary antibody was omitted. No labeling was observed in control sections.
In Situ Hybridization
The murine Pax2 probe is a BamHI-EcoRI fragment that corresponds to the 3' half of the PAX2 coding sequence and does not contain the conserved paired domain. On Northern blots, this fragment recognizes two PAX2 mRNAs of 4.2 and 4.5 kb in size but does not detect the 3.0-kb PAX8 mRNA.25 By in situ hybridization, the PAX2 probe does not detect PAX8 expression in the thyroid nor PAX5 expression in the spleen. PAX2 probe was subcloned into Bluescript vector (Stratagene, LaJolla, CA). The template was linearized with restriction endonuclease EcoRI (sense probe) or BamHI (antisense probe). Sense and antisense RNA probes were transcribed from the linearized templates with T7 (sense) and T3 (antisense) RNA polymerases (Boehringer Mannheim, Mannheim, Germany) using digoxigenin UTP (Boehringer-Mannheim) according to the manufacturer's instructions. They were DNaseI-treated, purified, and stored at -80°.
Six-µm thick deparaffinized sections were pretreated by microwave heating for 5, 1, and 5 minutes in a urea solution (0.8 mol/L urea, pH 6.4) and then washed in PBST (PBS containing 5% bovine serum albumin and 0.05% Tween 20) for 30 minutes. Prehybridization was carried out for 1 hour at 48°C in 100 µl of hybridization solution containing 50% deionized formamide, 5x SSC, 5x Denhardt's solution, 250 µg/ml of fish sperm DNA, 250 µg/ml yeast tRNA, 4 mmol/L EDTA, and 10% dextran sulfate. Hybridization was performed at 48°C for 16 hours in 50 µl of the hybridization buffer (50% deionized formamide, 5x SSC, 5x Denhardt's solution, 250 µg/ml yeast tRNA, 4 mmol/L EDTA) containing 10 ng/ml of the denatured digoxigenin-labeled Pax2 probe (antisense or sense). After hybridization, the slides were washed twice with 2x SSC at 42°C, followed by 0.2x SSC and 0.1x SSC at room temperature. After blocking with 0.5% blocking reagent in the buffer (0.1 mol/L Tris-HCl, 150 mmol/L NaCl, pH 7.9) for 30 minutes at room temperature, sections were incubated overnight at 4°C with alkaline phosphatase-conjugated sheep anti-digoxigenin IgG (Boehringer Mannheim) 1:800 in the blocking solution. The color was then developed with the chromogenic agents nitro blue tetrazolium, 5-bromo-4-chloro-3-indolyl phosphate (Boehringer Mannheim), and 2 mmol/L levamisole in the dark at room temperature. After the color was fully developed, all of the sections were washed at the same time with the buffer (100 mmol/L Tris-HCl, 1 mmol/L EDTA, pH 8) to allow comparison of the hybridization signal and then was mounted in an aqueous medium.
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Normal Human Fetal and Postnatal Kidneys
In the kidneys (10, 19, 24, and 28 gestational weeks) from the 4 fetuses, undifferentiated mesenchyme was seen in the outer cortex. Progression of nephron differentiation from condensation of mesenchymal cells around the ampullar tip of the ureteral branches (future collecting ducts) to vesicle, comma-shaped body, S-shaped body, and later stages of differentiation was observed from the outer to the deep cortex.
Denys-Drash Syndrome (10 Patients)
Diagnosis of diffuse mesangial sclerosis had been based on
evaluation of renal tissue obtained by biopsy (5 pts) or nephrectomy
performed because of Wilms' tumor (5 pts), in patients under 1 year of
age (7 pts) or between 2 and 5 years (3 pts). Various degrees of
severity of glomerular lesions were observed. In the early stages, the
glomerular lesions were characterized by a fibrillar increase in
mesangial matrix with no mesangial cell proliferation and the capillary
walls were lined by hypertrophied podocytes. In the fully developed
lesion, there was a combination of thickening of the glomerular
basement membranes and massive enlargement of mesangial areas leading
to reduction in the patency of the capillary lumens (Figure 1a)
. The expanded mesangial matrix had a
spongy appearance with mesangial cells embedded in a delicate, fine
periodic acid-Schiff and silver positive network. In advanced
stages, the contracted solidified glomeruli were still surrounded by a
layer of hypertrophied and vacuolized podocytes. The deepest glomeruli
were usually the least severely affected. Tubulointerstitial lesions
were associated and included focal tubular dilatations involving
primarily proximal tubules. Dilatations were prominent in patients with
fully developed or advanced glomerular lesions.
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Diagnosis of diffuse mesangial sclerosis had been made on renal
biopsy specimens obtained between the ages of 1 month and 5 years in 13
patients presenting with nephrotic syndrome and on nephrectomy specimen
obtained at the time of transplantation in one. Glomerular and tubular
lesions were similar to those described in DDS (Figure 1b)
.
Wilms' Tumor and Abnormal Male Genital Development (3 Patients with WT1 Mutations)
Kidney tissue excluding the Wilms' tumor was normal. No glomerular changes were detected.
WT1 and PAX2 Expression and Immunohistology
Similar results were obtained whatever the technique used (immunofluorescence or immunohistochemistry, frozen or paraffin-embedded tissues). No modification in immunolabeling was observed on renal specimens from the same patient obtained at different times in the course of the disease.
Normal Human Fetal and Postnatal Kidneys
In normal fetal kidneys (10, 19, 24 and 28 gestational weeks), WT1
was faintly expressed in nuclei of mesenchymal blastema. The nuclear
expression increased in condensates, renal vesicles, and prepodocytes
of comma-shaped bodies, and it persisted in podocytes of mature
glomeruli. No cytoplasmic labeling was observed in these structures.
Derivatives of the ureteric bud were WT1 negative. (Figure 1c)
. PAX2
was absent in the uninduced blastema. In contrast, strong nuclear
expression was observed as soon as the cells condensed in contact with
the ampullae. The expression persisted in the comma-shaped body and
disappeared in the visceral epithelium of the future glomeruli of the
S-shaped body (Figure 1d)
. Progressive effacement of PAX2 labeling in
the proximal and then the distal tubule was observed in the following
stage of nephron differentiation, whereas parietal epithelial cells of
the mature glomeruli remained positively labeled with PAX2 antibodies.
PAX2 was expressed in the ureteral bud ramifications; the expression
was maximal in the ampullar cells.
In postnatal kidneys (2 to 42 years), WT1 antibody uniformly stained
the nuclei of the podocytes and the parietal epithelium lining the
Bowman's capsule. The cytoplasm was consistently negative (Figure 1, e and g)
. No tubular structure was labeled. PAX2 was strongly expressed
in the parietal epithelium of Bowman's capsules and in the collecting
tubules (Figure 1, f and i)
with a nuclear localization. Nuclei from
distal tubules were also focally labeled. No PAX2 staining was detected
in podocytes, vessels, or interstitial cells.
Denys-Drash Syndrome (10 Patients)
Marked decrease in podocyte nuclear staining with anti-WT1
antibodies was observed in eight cases with focal disappearance of the
labeling in two of them (Figure 2, a and b)
. Compared with staining in controls, irregular podocyte staining was
a striking feature with the presence within the same glomerulus of
positive, negative, and faintly stained podocyte nuclei. Three patients
with abnormal distribution of WT1 also showed abnormal nuclear
expression of PAX2 in the podocytes (Figure 2c)
. In the others, no
change in the expression of PAX2 was detected. In two patients, the
expression of WT1 and PAX2 was normal. Normal expression of PAX2 was
observed in Bowman's capsule, distal, and collecting duct cells. No
neoexpression of PAX2 was detected in dilated proximal tubules (Figure 2k)
.
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In 11 patients, abnormal distribution of the WT1 protein was
observed, consisting of decrease or absence of nuclear labeling of the
podocytes (Figure 2, d and e)
. In seven of these patients, nuclear
labeling of the podocytes with anti-PAX2 antibodies was observed
(Figure 2f)
. In two patients, the glomerular distribution of WT1 and
PAX2 was normal. Findings in other renal cells were the same as in DDS
patients.
Wilms' Tumor and Abnormal Male Genital Development (3 Patients with WT1 Mutations)
Focal decrease or disappearance of WT1 nuclear staining was
observed in the adjacent kidney of the tumor of one patient (Figure 2, g and h)
. In the two other patients, the expression of WT1 and PAX2 was
normal in nontumoral tissue. No glomerular symptoms were
detected.
Controls (22 Patients)
Renal biopsy or nephrectomy specimens from patients presenting various types of nephropathies were studied. No change in WT1 or PAX2 expression was observed in any of these cases.
PAX2 Expression and in Situ Hybridization
Normal Human Fetal and Postnatal Kidneys
In fetal kidney, no PAX-2 expression was seen in uninduced
blastema cells. PAX-2 transcripts were detected in the condensing
mesenchyme around the ampullar extremities of the developing collecting
ducts and in early epithelial structures (Figure 3, a and c)
. Expression decreased at
later developmental stages and was absent from future podocytes. Pax-2
transcripts were also seen in collecting duct cells.
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Diffuse Mesangial Sclerosis
In two patients with positive labeling of podocyte nuclei with
anti-PAX-2 antibodies, abnormal expression of PAX2 mRNA was seen in the
cytoplasm of podocytes (Figure 3d)
. PAX-2 expression was normal in
epithelial cells of Bowman's capsules, distal, and collecting tubules.
Controls
Normal expression of PAX-2 was observed in control kidneys. PAX-2 transcripts were not detected in the podocytes.
Correlations between Mutations of WT1 and Podocyte Expression of WT1 and PAX2
WT1 mutations were identified in six DDS and three IDMS patients.
In five DDS and one IDMS patients, localization of WT1 was abnormal and
was associated with podocyte expression of PAX2 in four patients (Table 2)
. In contrast, three patients with WT1
missense mutations lying in exon 9 had normal WT1 and PAX2 expression.
Two of these mutations are the most frequently found in DDS and affect
amino acids involved in DNA binding, arginine 394 in one patient and
aspartic acid 396 in the other.
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Normal Postnatal, DDS, and IDMS Kidneys
In normal mature kidneys, PCNA positive cells were not found in
glomeruli, whereas very occasional tubular or interstitial cells were
stained by anti-PCNA antibodies. In all patients with DDS or IDMS,
marked increase in tubular (from dilated or normal tubules) and
interstitial cell labeling was observed (Figure 2j)
. In contrast, no
podocyte labeling was detected in one third of patients. In the others,
PCNA-positive podocytes were focally present: they were rare and
observed in 6 to 12% of glomeruli (Figure 2i)
in most cases, but in
two patients, one with normal, the second with abnormal PAX2
expression, they were present in 20 and 50% of glomeruli,
respectively.
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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In normal fetal kidneys, we clearly showed expression of WT1 in nuclei of uninduced blastema, confirming the initial findings of Grubb et al.36 The expression eventually increased in condensing blastema, renal vesicle, comma, and S-shaped bodies and peaked in podocytes, whereas it was down-regulated in tubular structures. In normal mature kidneys, a strong, regular labeling of podocyte nuclei was observed without cytoplasmic staining.
In 8 of the 10 DDS patients studied, the distribution of WT1 protein was abnormal. Podocytes were irregularly stained with disappearance or focal decrease of the nuclear signal. These changes were not observed in the control group. Most mutations in DDS patients are missense exon 8 or 9 mutations affecting zinc fingers 2 or 3.4-7 We have no information on the molecular defect in four patients of our series. In the six others, a mutation was identified. One patient has a frameshift mutation leading to a protein truncated in zinc finger 3. Five patients carry the most commonly reported DDS missense mutations resulting in the replacement of arginine 366 in zinc finger 2 (one patient) or arginine 394 in zinc finger 3 (4 patients). These highly conserved arginine residues are critical for contact with guanine in the DNA target and consequently for protein-DNA binding. The consequences of DDS mutations on the nuclear targeting and DNA binding capacity of the WT1 protein have been analyzed in cellular models. Missense mutations such as 394Arg, altering amino acids that directly interact with the DNA target4,24 but also mutations leading to the removal of the last zinc fingers,24 abolish WT1 binding to its DNA targets. More recently, targeting signals required for nuclear localization of the WT1 protein have been identified, one within ZF 1 and another within ZF 2 and 3.38 In this reported study, the WT1 product of 3T3 cells transfected with DDS WT1 mutant (CMV-PM) was unable to concentrate in the nucleus. Our immunohistochemical findings in podocytes of DDS patients are the in vivo morphological counterpart of in vitro laboratory data. However, two DDS patients (pts 4 and 10), one of them carrying the classic R394W missense mutation, had normal WT1 nuclear localization. WT1 self-associates in vitro and in vivo through the amino-terminal part of the protein and dimerisation of the mutated protein with wild-type WT1 may prevent DNA binding of the complex.13,16,39 The low nuclear labeling observed in most podocytes is in agreement with this dominant negative effect of the mutated protein.
One of the WT1 targets is PAX2, the expression of which is down-regulated by WT1 during normal kidney development.27 In the metanephros, PAX2 RNA and protein are detected in the ureteric bud and its derivatives, and after birth, the expression persists in collecting ducts.26,33,40-41 PAX2 is absent from the uninduced blastema and transiently expressed in the developing nephron. It progressively disappears from the presumptive precursors of the podocytes at the S-shaped body stage and is no longer expressed in the following stages of nephron development.26,27,33 In parallel with the disappearance of PAX2, podocytes stop proliferating and definitively lose the ability to divide.42 Using immunohistochemical and in situ hybridization techniques, we observed this pattern of expression in normal fetal and postnatal kidneys and in control kidneys showing various types of nephropathies. In contrast, the podocytes of three DDS patients showed PAX2 nuclear immunolabeling. These dramatic changes in PAX2 expression, which were observed in DDS patients with poor or absent WT1 nuclear staining, are likely to result from the loss of WT1 dependent transcriptional repression. No abnormal PAX2 expression was detected in patients with normal WT1 podocyte distribution. Deregulation of PAX2 expression may be implicated in the mechanisms leading from WT1 mutation to podocyte alteration and the development of DMS as PAX2 dominant gain-of-function mutation in transgenic mice leads to congenital nephrotic syndrome with severe tubular microcystic dilatations, a frequent morphological finding in DDS.3,29 Other target genes of WT1, such as PDGFA,43,44 IGF2,45 TGF-ß1,46 or nov, encoding a putative IGFBP,47 may also be involved in the pathological pathway leading from WT1 mutation to podocyte dysfunction and glomerular sclerosis.
Abnormal WT1 and PAX2 expression was also detected in nephrotic infants
with IDMS. As glomerular lesions are identical, this
clinicopathological entity may be difficult (if not impossible) to
differentiate from DDS, especially in females, in the absence of other
symptoms of the triad. Actually, most 46XX DDS females do not develop
genital abnormalities.7
In addition, development
of Wilms' tumor may be prevented by bilateral nephrectomy performed at
the time of transplantation, which should occur early in life because
of rapid progression to end-stage renal failure. However, the
occurrence of IDMS in normal 46XY males, the frequent familial
incidence of the disease and the occurrence of IDMS in children born to
consanguinous parents, strongly suggest that it is a separate entity
with a possible autosomal recessive inheritance (Table 1)
.8,48-57
Abnormal podocyte distribution of WT1
was observed in 9 of the 14 IDMS patients studied and was associated
with PAX2 nuclear expression in seven patients. This anomaly, similar
to that found in DDS patients suggested that WT1 could also be
implicated in the occurrence of IDMS, making IDMS a clinical variant of
DDS. This hypothesis was confirmed by the finding of WT1 exon 8 or 9
mutation in three patients, one male and two females. Two of these
mutations (H377Y and D396N) have been previously described in DDS
patients.5
An alternative hypothesis is that IDMS
is a multigenic disease caused by mutations of WT1 or one of
the genes involved in the regulatory pathway of WT1. The
finding of abnormal distribution of WT1 and PAX2 proteins in one
patient in whom the WT1 mutation has been excluded by sequencing of the
5' part of exon 1 and of all of the exons 2 to 10 supports this
hypothesis. One candidate gene is PAX2, abnormally expressed
in the podocyte, especially because its deregulation in transgenic mice
leads to early and severe nephrotic syndrome with renal changes
compatible with those observed in DMS29
and because some reported familial cases of IDMS are associated with
ocular defects, another target tissue of PAX2
dysfunction.8,48,50,52,55
No change in the distribution of WT1 or PAX2 was detected in the nontumoral renal tissue from two patients with Wilms' tumor, genital anomalies in the male, a constitutional WT1 missense mutation in exon 3 in the female, and stop codon in exon 7 leading to the loss of the four zinc fingers in the male. These patients had no glomerular symptoms. However, in one male patient with the same phenotype and a stop codon in exon 4, WT1 nuclear labeling of podocytes was focally absent. At this time, this 9-year-old patient has not developed glomerular symptoms.
It is noteworthy that the up-regulation of PAX2 observed in podocytes, which is known to stimulate proliferation, was associated with podocyte hypertrophy but not with patent podocyte hyperplasia. However, using PCNA as a marker of proliferation, we observed PCNA positive podocytes in two thirds of patients; they were abundant in two and rare in most cases. No glomerular expression of PCNA was found in normal controls. Interestingly, marked increase in PCNA expression was observed in tubular and interstitial cells. This finding may be correlated with the early occurrence of severe tubulointerstitial lesions with tubular dilatation in diffuse mesangial sclerosis. Recently, it has been shown that in human dysplastic kidney, PAX2 is expressed in cystic and hyperproliferative epithelia, which are thought to be malformed branches of the ureteral bud, suggesting that persistent expression of PAX2 is involved in cyst formation.33 In diffuse mesangial sclerosis, tubular dilatations develop primarily from proximal tubules, and we did not detect any abnormal PAX2 expression in these dilated tubules, suggesting that the mechanism of tubular cell proliferation and dilatation is not PAX2 dependent in this condition. Contrarily to the findings of Winyard et al,33 we observed persistent expression of PAX2 in nondilated collecting tubules of patients as well as of normal mature kidneys.
In conclusion, changes in podocyte distribution of WT1 and PAX2 observed in DDS patients are consistent with the transcription role of WT1 and the WT1 down-regulation of PAX2. However, no correlation could be established between the presence of any particular mutation and the expression pattern of WT1 or PAX2. The same anomalies have been observed in males and females patients with IDMS suggesting that WT1 is likely to play a significant pathophysiological role not only in DDS but also in IDMS.
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Acknowledgements |
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Footnotes |
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Supported by the Institut National de la Santé et de la Recherche Médicale, the Association Claude Bernard, and the Association pour l'Utilisation du Rein Artificiel.
Accepted for publication October 4, 1998.
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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J. Bariety, C. Mandet, G. S. Hill, and P. Bruneval Parietal Podocytes in Normal Human Glomeruli J. Am. Soc. Nephrol., October 1, 2006; 17(10): 2770 - 2780. [Abstract] [Full Text] [PDF]
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 J.-K. Guo, A. Schedl, and D. S. Krause Bone Marrow Transplantation Can Attenuate the Progression of Mesangial Sclerosis Stem Cells, February 1, 2006; 24(2): 406 - 415. [Abstract] [Full Text] [PDF]
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J. S. Jaggi, S. V. Seshan, M. R. McDevitt, K. LaPerle, G. Sgouros, and D. A. Scheinberg Renal Tubulointerstitial Changes after Internal Irradiation with {alpha}-Particle-Emitting Actinium Daughters J. Am. Soc. Nephrol., September 1, 2005; 16(9): 2677 - 2689. [Abstract] [Full Text] [PDF]
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 N. Sabherwal, K. U. Schneider, R. J. Blaschke, A. Marchini, and G. Rappold Impairment of SHOX nuclear localization as a cause for Leri-Weill syndrome J. Cell Sci., June 15, 2004; 117(14): 3041 - 3048. [Abstract] [Full Text] [PDF]
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H. Schmid, A. Henger, C. D. Cohen, K. Frach, H.-J. Grone, D. Schlondorff, and M. Kretzler Gene Expression Profiles of Podocyte-Associated Molecules as Diagnostic Markers in Acquired Proteinuric Diseases J. Am. Soc. Nephrol., November 1, 2003; 14(11): 2958 - 2966. [Abstract] [Full Text] [PDF]
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 C. E. Patek, S. Fleming, C. G. Miles, C. O. Bellamy, M. Ladomery, L. Spraggon, J. Mullins, N. D. Hastie, and M. L. Hooper Murine Denys-Drash syndrome: evidence of podocyte de-differentiation and systemic mediation of glomerulosclerosis Hum. Mol. Genet., September 15, 2003; 12(18): 2379 - 2394. [Abstract] [Full Text] [PDF]
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J.-K. Guo, A. L. Menke, M.-C. Gubler, A. R. Clarke, D. Harrison, A. Hammes, N. D. Hastie, and A. Schedl WT1 is a key regulator of podocyte function: reduced expression levels cause crescentic glomerulonephritis and mesangial sclerosis Hum. Mol. Genet., March 1, 2002; 11(6): 651 - 659. [Abstract] [Full Text] [PDF]
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 V. Chauvet, F. Qian, N. Boute, Y. Cai, B. Phakdeekitacharoen, L. F. Onuchic, T. Attie-Bitach, L. Guicharnaud, O. Devuyst, G. G. Germino, et al. Expression of PKD1 and PKD2 Transcripts and Proteins in Human Embryo and during Normal Kidney Development Am. J. Pathol., March 1, 2002; 160(3): 973 - 983. [Abstract] [Full Text] [PDF]
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