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Mol Cell Biol. Nov 2004; 24(22): 9899–9910.
PMCID: PMC525476

The Wt1+/R394W Mouse Displays Glomerulosclerosis and Early-Onset Renal Failure Characteristic of Human Denys-Drash Syndrome

Abstract

Renal failure is a frequent and costly complication of many chronic diseases, including diabetes and hypertension. One common feature of renal failure is glomerulosclerosis, the pathobiology of which is unclear. To help elucidate this, we generated a mouse strain carrying the missense mutation Wt1 R394W, which predisposes humans to glomerulosclerosis and early-onset renal failure (Denys-Drash syndrome [DDS]). Kidney development was normal in Wt1+/R394W heterozygotes. However, by 4 months of age 100% of male heterozygotes displayed proteinuria and glomerulosclerosis characteristic of DDS patients. This phenotype was observed in an MF1 background but not in a mixed B6/129 background, suggestive of the action of a strain-specific modifying gene(s). WT1 encodes a nuclear transcription factor, and the R394W mutation is known to impair this function. Therefore, to investigate the mechanism of Wt1 R394W-induced renal failure, the expression of genes whose deletion leads to glomerulosclerosis (NPHS1, NPHS2, and CD2AP) was quantitated. In mutant kidneys, NPHS1 and NPHS2 were only moderately downregulated (25 to 30%) at birth but not at 2 or 4 months. Expression of CD2AP was not changed at birth but was significantly upregulated at 2 and 4 months. Podocalyxin was downregulated by 20% in newborn kidneys but not in kidneys at later ages. Two other genes implicated in glomerulosclerosis, TGFB1 and IGF1, were upregulated at 2 months and at 2 and 4 months, respectively. It is not clear whether the significant alterations in gene expression are a cause or a consequence of the disease process. However, the data do suggest that Wt1 R394W-induced glomerulosclerosis may be independent of downregulation of the genes for NPHS1, NPHS2, CD2AP, and podocalyxin and may involve other genes yet to be implicated in renal failure. The Wt1R394W mouse recapitulates the pathology and disease progression observed in patients carrying the same mutation, and the mutation is completely penetrant in male animals. Thus, it will be a powerful and biologically relevant model for investigating the pathobiology of the earliest events in glomerulosclerosis.

Glomerulosclerosis, whether primary or secondary to other disease processes, is a key and common feature of progressive renal failure, which is a major cause of morbidity and mortality in the United States. Although several genetic and environmental insults are known to cause primary glomerulosclerosis, the cellular mechanism by which they initiate this process is still largely unknown. A knowledge of these mechanisms would greatly aid in identifying strategies to prevent or slow the development of glomerulosclerosis, regardless of its etiology.

Glomeruli are complex and specialized structures responsible for blood filtration in the kidney and are targets of injury in a number of human diseases. The major functional features of the glomerulus are capillary loops lined with fenestrated endothelial cells, supporting mesangial cells, the glomerular basement membrane (GBM), and podocytes. The “octopus-like” (13) podocytes have long, actin-rich foot processes that interdigitate and interface with the extracapillary side of the GBM. Individual foot processes are separated by slits that are bridged by a porous diaphragm. The importance of the integrity of the podocyte foot processes and the slit diaphragm is underscored by the development of glomerulosclerosis and end-stage renal failure in individuals carrying mutations in genes encoding proteins thought to be important for the integrity of the podocyte cytoskeleton or the slit diaphragm (e.g., α-actinin-4, nephrin, podocin, and CD2-associated protein) (2, 21, 22, 23). In addition to these podocyte structural proteins, another protein whose mutation is known to predispose to early-onset glomerulosclerosis is that encoded by WT1.

The Wilms' tumor (WT) suppressor gene, WT1, encodes a zinc finger nuclear transcriptional factor that is normally expressed during development in induced renal mesenchyme and later in the crescent- and S-shaped bodies of the developing glomerulus (33). It is also expressed in the embryo in the gonadal ridge, mesothelium, spleen, brain, and spinal cord. In adults, WT1 is expressed in podocytes, in the Sertoli cells of the testes, in granulosa cells of the ovary, and in the uterus and oviduct (1, 24). WT1 was originally identified as playing an etiologic role in the development of WT and was subsequently identified as being mutated in patients with Denys-Drash syndrome (DDS), which consists of a triad of phenotypes: WT predisposition, male pseudohermaphrodism, and early-onset (<3 year of age) renal failure characterized by diffuse renal mesangial sclerosis (3, 5, 7, 10, 11, 18, 31, 32). Studies of patients with WT1 germ line mutations have revealed a strong phenotype-genotype correlation such that individuals with deletion or truncating mutations display genitourinary anomalies and WT predisposition, whereas individuals with zinc finger missense mutations display these phenotypes and the additional phenotype of early-onset renal failure characterized by renal mesangial sclerosis (17). The most common WT1 mutation found in DDS patients is a C-to-T transition that results in the substitution of tryptophan for arginine at amino acid 394 (R394W), and this mutation has been shown to alter the ability of WT1 to regulate downstream target genes (16, 29). To investigate the mechanism by which this WT1 missense mutation results in glomerulosclerosis and early-onset renal failure in humans, we generated a mouse strain that carries the analogous nucleotide and subsequent amino acid substitution (C1180T and R394W). As we report here, this Wt1R394W strain faithfully mimics the pathology and natural disease progression observed in DDS patients, and it provides a powerful and biologically relevant model for investigating the pathobiology of the earliest events in glomerulosclerosis.

MATERIALS AND METHODS

Generation of a mouse strain carrying the Wt1 R394W point mutation.

The R394W mutation was introduced into mouse embryonic stem (ES) cells by homologous recombination by a cre-loxP strategy (Fig. (Fig.1A).1A). With a genomic Wt1 clone isolated from a 129/SvEv genomic library, a loxP-PGKneobpA-loxP cassette was inserted in reverse orientation 3′ of exon 9 and an MC1tkpA herpes simplex virus thymidine kinase expression cassette (26, 41) was introduced 3′ of exon 10 of Wt1. The C1180T mutation was introduced via site-directed mutagenesis, and the entire construct was sequenced to verify the presence of the C-to-T transition and to ensure that no other alteration had been introduced into the construct.

FIG. 1.
Generation of a mouse strain carrying the Wt1R394W allele. (A) Targeting construct. (B) Southern blot demonstrating the presence of the Wt1R394W neo-tagged allele in ES cells. (C) Genomic sequence from a Wt1+/R394W mouse showing heterozygosity ...

AB1 (129/SvEv) ES cells were electroporated with the Wt1R394W targeting construct and selected for resistance to G418 and sensitivity to 1-(2-deoxy-2-fluoro-β-d-arabinofuranosyl)-s-iodouracil (26, 27). Clones were genotyped by Southern blotting of EcoRV-restricted clone DNA hybridized to a genomic probe 5′ of exon 7 (5′ probe); introduction of the neo cassette by homologous recombination resulted in a 7.14-kb fragment compared to the normal 5.34-kb fragment (Fig. (Fig.1B).1B). Following sequence verification, a targeted ES cell clone was injected into C57BL/6 (B6) blastocysts. Male chimeras were identified by coat color and subsequently crossed with wild-type females. Tail biopsies from agouti-pigmented F1 animals were genotyped by PCRs with a common forward primer (1.47 [5′ CTG CAG GTG GCT GTA CAG AA 3′]) paired with a mutant allele-specific primer (1.46 [5′ GTC TTC AGA TGG TCG GTC CA 3′]) (Fig. (Fig.1D).1D). Wt1+/R394W-neo offspring were crossed with ZP3-cre (Jackson Laboratory) females. Offspring were assessed for the excision of the neo cassette, and several Wt1+/R394W heterozygotes were identified. Genomic sequence from a heterozygote is shown in Fig. Fig.1C1C.

After obtaining germ line transmission of the Wt1 R394W mutation in a mixed B6/129 background, we maintained it in a mixed background. We also began mating heterozygotes with MF1 animals, an outbred line. This was motivated by a report that, when in an MF1 background, homozygotes for the Wt1 null allele (Wt1−/−) were viable throughout embryogenesis, in contrast to the embryonic day 12.5 lethality observed in Wt1−/− B6/129 animals (15, 24). All work with mice complied with all relevant federal guidelines and institutional policies.

Tissue collection and nucleic acid isolation.

Tissues from euthanized animals were snap-frozen in liquid nitrogen for nucleic acid analysis or immediately put into fixative appropriate for histologic analyses as described below. For TaqMan and SybrGreen assays, total RNA was extracted from kidney cortex by use of a RNAqueous-4PCR kit (Ambion) in accordance with the manufacturer's instructions.

Genotyping.

Once the Wt1R394W strain was established, animals were genotyped by amplification of tail snip DNA with PCR primers 1.75 (5′ TGC CTA CCC AAT GCT CAT TG 3′) and 1.55 (5′ GAA ACT GTT TGT AAC GAG AG 3′), corresponding to genomic sequences that flank the residual loxP present in animals carrying the C1180T mutation. The PCR product from the wild-type allele is 102 bp smaller than that from the mutant allele owing to the loxP and flanking sequences remaining in the mutant allele (Fig. (Fig.1D).1D). Animals were also genotyped with the common forward primer, 1.47, and the mutant allele-specific reverse primer, 1.46 (Fig. (Fig.1D1D).

TaqMan quantification of gene expression.

Allele-specific TaqMan probe-primer sets (purchased from Applied Biosystems) were used to quantitate expression from the wild-type and mutant alleles. PCR primers were as follows: sense, 5′ CCA GTG TAA AAC TTG TCA GCG AAA 3′; antisense, 5′ ATG AGT CCT GGT GTG GGT CT 3′. The allele-discriminating probes were 5′ TTT TCC CGG TCC 3′ (wild type) and 5′ TTT TCC TGG TCC GAC 3′ (R394W mutant). Assays were performed as previously described (43).

Quantitative reverse transcription-PCR.

To quantitate the expression of genes other than Wt1 that have been implicated in the development of glomerulosclerosis, real-time SybrGreen assays were performed with RNA isolated from the kidney cortexes of 2-month-old Wt1+/R394W MF1-N2 (n = 4) and Wt1+/+ MF1-N2 (n = 4) animals and also from 4-month-old mutant Wt1+/R394W MF1-N2 (n = 5) and Wt1+/+ MF1-N2 (n = 5) animals. Gene expression was quantitated relative to the expression of the gene for glyceraldehyde-3-phosphate dehydrogenase. The primers used were IGF1R (forward, 5′ GAA GAA CGC CGA CCT CTG TTA C 3′; reverse, 5′ GGA CAC CGC ATC CAA GAT G 3′), IGF1 (forward, 5′ AAG CAG CCC GCT CTA TCC 3′; reverse, 5′ TTC TGA GTC TTG GGC ATG TCA 3′), TGFB1 (forward, 5′ AAA CGG AAG CGC CAT CGA A 3′; reverse, 5′ TGG CGA GCC TTA GTT TGG A 3′), CD2AP (forward, 5′ TGG TGG AAG GGT GAA CTG AAC 3′; reverse, 5′ TTG CAG GAG GAG GTG GTT TC 3′), nephrin (forward, 5′ CGA GGC ACT TCG TGA AAC 3′; reverse, 5′ GCA CTT GCT CTC CCA GGA CT 3′), podocalyxin (forward, 5′ GTG TTC ATC TGT GCC CAT TCC 3′; reverse, 5′ AGC TAG CCG AGG CCA TGT TA 3′), podocin (forward, 5′ TTT GCC TTT GCC ATT TGA CA 3′; reverse, 5′ ATG CTC CCT TGT GCT CTG TTG 3′).

Urine analysis.

Urine was collected during handling of mice over plastic wrap. Urinary protein was analyzed by electrophoresis of 3 μl of urine on 10% denaturing sodium dodecyl sulfate-polyacrylamide gels, followed by Coomassie brilliant blue staining.

Histologic analysis.

For routine histologic analysis, kidney samples were obtained immediately following euthanasia, fixed in 4% paraformaldehyde, and embedded in paraffin. Sections 6 μm thick were cut and mounted on glass slides after deparaffinization and staining with hematoxylin and eosin or periodic acid-Schiff (PAS).

For electron microscopy, small wedge samples were cut from kidneys and fixed in 2% glutaraldehyde, dehydrated in graded ethanol, and embedded in epoxy resin. Thin sections were stained with uranyl acetate.

Immunohistochemical analysis.

Immunohistochemical analysis was carried out with a Vectastain ABC (avidin-biotin-peroxidase) kit (Vector Laboratories, Burlingame, Calif.) as recommended by the manufacturer. Deparaffinized sections were incubated with 10% goat serum in phosphate-buffered saline (PBS) for 30 min at room temperature and then incubated for 1 h at room temperature with either anti-Wt1 polyclonal antibody (Santa Cruz) (1:100), which detects both mutant and wild-type proteins, or anti-nephrin polyclonal antibody. After being washed three times with PBS, the sections were incubated with biotinylated secondary antibody (Santa Cruz) for 45 min at room temperature. After incubation with avidin-biotin-peroxidase complex for 45 min, the sections were washed with PBS. The color was developed with 3,3′-diaminobenzidine substrate. The sections were dehydrated and mounted with Permount (Fisher) and examined with a Leica DMR epifluorescence microscope, and images were captured by a Hamamatsu C5810 charge-coupled device camera.

RESULTS

Generation of the Wt1+/R394W mouse strain.

We introduced the most common DDS missense mutation, R394W, into the mouse germ line. As shown by Southern blot analysis (Fig. (Fig.1B),1B), recombination of the targeting construct into the ES cell genome was successful. Sequence analysis of genomic DNA from a Wt1+/R394W heterozygote recovered after cre-mediated excision of the neo cassette confirmed the presence of the R394W mutation; in Fig. Fig.1C,1C, both the wild-type C and mutant T nucleotides at position 1180 (relative to the AUG start codon) can be seen. This mutation is the same nucleotide change observed in DDS patients, and it results in the same amino acid change (arginine to tryptophan) at amino acid 394. Animals carrying the missense mutation were genotyped by verifying the presence of the 102-bp loxP sequence and flanking sequence that remains following cre-mediated excision of the neo cassette (Fig. (Fig.1D,1D, top) and by mutant allele-specific PCR (Fig. (Fig.1D,1D, bottom).

Characterization of the Wt1R394W strain revealed that all homozygous mutants were embryonically lethal. Heterozygous mutant mice exhibited none of the developmental anomalies, such as male pseudohermaphrodism or gonadal dysgenesis, associated with DDS, and as has been observed for mice heterozygous for a Wt1 null allele, there is no evidence of a predisposition to WT in the Wt1R394W heterozygotes. However, as described in more detail below, Wt1R394W heterozygotes developed proteinuria and mesangial sclerosis in a strain- and genetic background-dependent manner.

Expression of the R394W mutant allele in kidneys from Wt1+/R394W animals.

With allele-specific TaqMan probes, expression of the wild-type and mutant alleles could independently be determined. Kidney RNAs from five wild-type and five heterozygous animals were assessed. As shown in Fig. Fig.1E,1E, both alleles are expressed in the kidneys of R394W heterozygotes, although the level of the mutant transcript is reduced relative to that of the wild-type transcript. Expression of the wild-type transcript in Wt1+/R394W kidneys was 50% of that in Wt1+/+ kidneys. Total Wt1 expression in R394W heterozygotes was 80 to 85% of that in wild-type animals.

Kidney development and Wt1-encoded protein expression in Wt1+/R394W mice.

There was no discernible difference in kidney development in Wt1+/R394W animals compared to that in wild-type littermates; kidneys from newborn wild-type and Wt1+/R394W mutant mice were indistinguishable. Normal mature glomeruli (Fig. (Fig.2A,2A, top, arrowheads) were observed in both wild-type and heterozygous newborns. Normal comma-shaped and S-shaped bodies of developing glomeruli in the nephrogenic zone of the renal cortex were also observed in Wt1+/R394W newborns (Fig. (Fig.2A,2A, middle, arrows). At 3 weeks of age, wild-type and heterozygous kidneys were still indistinguishable by light microscopy (Fig. (Fig.2A,2A, bottom).

FIG. 2.
(A) Normal kidney development in Wt1+/R394W mice. Both regions of fully differentiated kidney (top panels) and of nephrogenic zones of the kidney (middle panels) are shown for newborn animals. Arrowheads indicate normal mature glomeruli; arrows ...

Expression of Wt1-encoded protein was also similar in the kidneys of wild-type and heterozygous animals from birth to 4 months of age (Fig. (Fig.2B).2B). As expected, Wt1-encoded protein staining was specific to the glomerular podocytes (Fig. (Fig.2B,2B, arrows). All of the sections shown are from animals with an MF1 background. As described in detail below, Wt1+/R394W-MF1 animals developed proteinuria and glomerulosclerosis. The 2-month-old mutant kidney shown in Fig. Fig.2B2B was obtained from a Wt1+/R394W mutant animal that had not yet developed proteinuria. The 4-month mutant kidney was from a Wt1+/R394W mutant animal that did display proteinuria and glomerular sclerosis; however, for comparison with the wild-type kidney, shown in Fig. Fig.2B2B is a Wt1+/R394W mutant glomerulus selected for its relatively normal histology.

Early-onset renal mesangial sclerosis and proteinuria observed in R393W heterozygotes in an MF1 background.

As noted above, we began crossing the R394W allele onto an MF1 background. Unexpectedly, several of the N2 generation animals (heterozygotes backcrossed to the MF1 strain for two generations and designated Wt1+/R394W MF1-N2) died at 4 to 5 months of age. At necropsy, the kidneys were noted to have an aberrant histology. This prompted us to sacrifice several 4-month-old Wt1+/R394W MF1-N2 animals and control littermates to assess kidney pathology more rigorously.

Shown in Fig. Fig.33 are kidney sections from sacrificed animals. In contrast to the normal glomerulus and tubules in wild-type littermates (panels A and C, respectively), Wt1+/R394W MF1-N2 animals displayed severely sclerotic glomeruli (Fig. (Fig.3B,3B, arrow). In the more medullary regions of the kidneys (Fig. (Fig.3D),3D), dilated tubules (arrowheads) and protein casts (asterisks) were observed. PAS-stained sections revealed that, in contrast to the normal glomerular architecture observed in wild-type control kidneys (Fig. (Fig.3E,3E, arrowheads), mutant kidneys contained a few glomeruli with segmental sclerosis (in Fig. Fig.3F,3F, the arrowhead indicates the normal region of glomerulus and the arrow indicates the sclerotic portion). However, mutant kidneys predominantly contained glomeruli with global mesangial sclerosis (Fig. 3G and H, arrows), which were distributed throughout the kidney, like the global diffuse mesangial sclerosis observed in DDS patients.

FIG.3.
Glomerulosclerosis in Wt1+/+ and Wt1+/R394W animals at 4 months. Panels: A, representative glomerulus from a wild-type animal; B, representative glomerulus from a mutant animal; C, representative tubules from a wild-type animal; ...

Assessment of renal lesions in Wt1+/R394W MF1-N2 mice by electron microscopy.

Renal pathology characteristic of glomerulosclerosis in humans was also observed by electron microscopy in Wt1+/R394W MF1-N2 mice. Assessment of coded slides revealed that kidneys from wild-type animals (Fig. 4A and C) displayed glomeruli with a normal, uniform GBM (arrows) and discrete podocyte foot processes (arrowheads). In contrast, the glomeruli of Wt1+/R394W MF1-N2 animals (Fig. 4B and D) displayed severe thickening of the GBM (arrows) and effacement of podocyte foot processes (arrowheads). In short, the kidney pathology of the Wt1+/R394W MF1-N2 animals was identical to the pathology observed in DDS patients. With few exceptions, animals displaying a more severe renal pathology were also the animals exhibiting a higher degree of proteinuria by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis.

FIG. 4.
Electron micrographs of kidneys from 4-month-old Wt1+/+ and Wt1+/R394W mice. Arrows, normal GBM in wild-type kidney and thickened GBM in mutant kidney. Arrowheads, normal podocyte foot processes in wild-type and effaced processes ...

Background specificity of phenotype.

The development of renal disease in the Wt1 R394W mutant strain occurred in a mutation- and background-dependent manner (Fig. (Fig.55 and Table Table1).1). At 4 months of age, 100% (seven of seven) of the Wt1+/R394W MF1-N2 males and 43% (three of seven) of the Wt1+/R394W MF1-N2 females displayed proteinuria and glomerulosclerosis (Fig. (Fig.5,5, top). In contrast, no proteinuria was detected in animals carrying the R394W mutation in a B6/129 background or in wild-type littermates in either background (Fig. (Fig.5,5, bottom).

FIG. 5.
Genetic background-dependent development of proteinuria in Wt1+/R394W mice at 4 months of age. Arrows indicate animals with proteinuria.
TABLE 1.
Glomerulosclerosis and proteinuria at 4 months in R394W (MF1 background) but not in R394W (B6/129 background) mice

Temporal progression of the glomerulosclerosis phenotype.

To investigate the temporal progression of glomerulosclerosis in Wt1+/R394W MF1-N2 animals, we began systematically to assess animals at specific ages. As shown in Table Table22 and Fig. Fig.2A,2A, no aberrant kidney histology was observed in either newborn (0 of 6) or 3-week-old (0 of 11) Wt1+/R394W MF1-N2 animals. By 2 months of age, 33% (two of six) of the Wt1+/R394W MF1-N2 males had developed glomerulosclerosis. No aberrant pathology was observed in any of the four Wt1+/R394W MF1-N2 females at 2 months. By 4 months of age, 100% of the male and 43% of the female Wt1+/R394W MF1-N2 animals displayed proteinuria and glomerulosclerosis. In contrast, none of the wild-type littermates were affected at 4 months (Fig. (Fig.55 and Table Table1).1). By 10 to 12 months of age, 12 (71%) of 17 Wt1+/R394W MF1-N2 females displayed proteinuria.

TABLE 2.
Temporal progression of glomerulosclerosis in Wt1+/R394W-MF1 heterozygotes

These data demonstrate that the Wt1 R394W mutation predisposes to glomerulosclerosis and kidney failure. The mutation appears to be completely penetrant in males. Given the temporal progression in males and the fact that 43 and 71% of females displayed glomerulosclerosis by 4 months or 10 to 12 months, respectively, we expect that penetrance will be complete in females also, but at a later age.

Expression of genes implicated in glomerulosclerosis in Wt1+/R394W MF1-N2 and wild-type control mice.

The expression of several podocyte genes previously implicated in glomerulosclerosis was assessed in Wt1+/R394W MF1-N2 mutant mouse kidneys. As shown in Table Table3,3, there was a modest (20 to 30%) but statistically significant decrease in the expression of nephrin and podocalyxin in newborn mutant kidneys, but no change was noted at either 2 or 4 months. In mutant kidneys expression of podocin was decreased 25% at birth but was increased 50 and 85% at 2 and 4 months, respectively. A statistically significant upregulation of CD2AP expression (a 41 to 65% increase) and IGF1 expression (a 50 to 65% increase) was also observed at 2 and 4 months in mutant kidneys. TGFB1 expression was elevated by 70% in mutant kidneys at 2 months and by ~50% at 4 months. No significant difference between mutant and wild-type kidneys in the expression of IGF1R was observed at any age. It should be noted that the expression values are relative within a gene or time point; expression levels cannot be compared across genes or across ages.

TABLE 3.
Real-time PCR quantitation of expression of genes implicated in glomerulosclerosis in the kidneys of mutant and wild-type newborn and 2- and 4-month-old animals

Nephrin protein expression in glomeruli from Wt1+/R394W MF1-N2 mice.

We also assessed wild-type and mutant kidneys for nephrin protein expression by immunohistochemical analysis. In this analysis, both normal- and abnormal-appearing glomeruli from mutant animals were examined. At both 2 and 4 months of age, wild-type levels of nephrin protein were observed in mutant animals that had not yet developed proteinuria or glomerulosclerosis (Fig. (Fig.6A,6A, compare +/+ panels and +/R394W top panels). In mutant animals that did exhibit proteinuria and glomerulosclerosis, nephrin expression was only marginally reduced in normal-appearing glomeruli (Fig. (Fig.6A,6A, +/R394W, middle) but more obviously reduced in the sclerotic glomeruli (Fig. (Fig.6A,6A, +/R394W, bottom).

FIG. 6.
(A) Immunohistochemical analysis of nephrin protein expression in Wt1+/R394W mice and control littermates at 2 months (2M) and 4 months (4M) of age (MF1 background). From mutant animals (+/R394W) are shown glomeruli (GM) from presymptomatic ...

Cell proliferation in the kidneys from Wt1+/R394W MF1-N2 mice.

Mesangial cell proliferation has been noted in some models of glomerulosclerosis (38). Therefore, we assessed cell proliferation in mutant and wild-type kidneys by immunohistochemical analysis with the proliferation nuclear marker PCNA. As shown in Fig. Fig.6B,6B, many proliferation-positive cells were found in tubules in both Wt1+/R394W MF1-N2 and wild-type control mice at 3 weeks of age, but very few proliferating cells were observed in the glomeruli at this stage in either the mutant or wild-type animals (top panels). However, by 2 and 4 months of age, a low frequency of proliferating cells in glomeruli was observed (middle and bottom panels). Importantly, there was no difference in mitotic activity between Wt1+/R394W MF1-N2 and wild-type control mice at any of these ages.

Expression of Wt1 in different genetic backgrounds.

To investigate whether the expression of Wt1 differed between mouse strains, we assessed the expression of the wild-type Wt1 allele in animals heterozygous for the Wt1 R394W allele in either an MF1-N2 (n = 5) or a B6/129 background (n = 5) with the wild-type-specific probe for the TaqMan assays. Animals were 2 months of age and did not display proteinuria or glomerulosclerosis. There was a significant (P = 0.0002) difference between the two genetic backgrounds, with Wt1 expression in MF1-N2 animals (14.1 ± 4.5) being only ~50% of that in B6/129 animals (37.4 ± 5.1).

DISCUSSION

Although glomerulosclerosis in many forms (focal, segmental, diffuse, etc.) is a common feature in progressive renal failure, little is known about its pathobiology. The study of patients genetically predisposed to end-stage renal failure has resulted in the identification of several genes (e.g., NPHS1, NPHS2, and ACTN4) that encode structural proteins (nephrin, podocin, and α-actinin-4) critical for the architecture of the glomerulus, specifically for the integrity of the podocyte foot processes and/or the slit diaphragm (2, 21, 22). These data, along with data from mutant mouse strains (e.g., nphs1 and nphs2 cd2ap) (35, 37, 40), have greatly increased our understanding of the composition of the slit diaphragm and its tethering to the podocyte. However, most of the time glomerulosclerosis, primary or secondary, occurs independently of mutations in these genes. Although dysregulation or functional disruption of these structural proteins may occur secondarily to other causes of glomerulosclerosis, it is not clear that this is the case. Thus, the continued investigation of the mechanisms by which various genetic and nongenetic insults result in glomerular pathology will likely provide further insight into the pathobiology of glomerulosclerosis and ultimately identify points at which intervention measures may slow the disease process.

The Wt1+/R394W mutant mouse provides an excellent and biologically relevant animal model for understanding the natural progression of glomerulosclerosis. DDS patients heterozygous for WT1 mutations most commonly carry the R394W mutation, and in an MF1 background, mice heterozygous for this mutation develop proteinuria and severe glomerulosclerosis within the first few months of life. In male animals, the penetrance of the mutation is 100% by 4 months of age, and even at 2 months of age a third of Wt1+/R394W males displayed glomerulosclerosis. In female animals the progression to a detectable disease state is slower (0% at 2 months, 43% at 4 months, and 71% at 10 to 12 months), but given what is seen in males, it is likely that all heterozygous females will ultimately develop proteinuria and glomerulosclerosis. Interestingly, the earlier progression to proteinuria and glomerulosclerosis in male animals is similar to the situation in human populations, in which males generally develop symptoms of renal failure at an earlier age and progress to endpoint renal failure faster than females (14).

The kidney pathology observed in Wt1+/R394W heterozygous animals also mimics that seen in DDS patients. By light and electron microscopy, key features of DDS nephropathy are observed: diffuse global mesangial sclerosis, shrunken glomerular tufts, effacement of the podocyte foot processes, thickening of the GBM, deposition of extracellular matrix, dilated tubules, and protein casts. Thus, the insight gained by studying the pathobiology and natural course of glomerulosclerosis in the Wt1 R394W strain will be highly relevant to understanding the human disease process.

Because Wt1 is a nuclear transcriptional factor specifically expressed in the podocytes, mutation of Wt1 likely results in the dysregulation of other podocyte-expressing genes, which in turn ultimately results in glomerulosclerosis. As noted above, loss of one of several proteins (nephrin, podocin, and CD2AP) thought to be critical for the structure and interaction of the podocyte foot processes with the GBM and the slit diaphragm results in a glomerular pathology similar to that caused by heterozygosity for the Wt1 R394W allele. Therefore the biologic effect of the Wt1 mutation could plausibly be due to downregulation of one or more of these known podocyte genes.

Our real-time PCR quantitation of gene expression in the kidneys of mutant animals, however, argues against this to some extent. In newborn Wt1+/R394W kidneys, which were histologically indistinguishable from newborn wild-type kidneys, we observed no downregulation of CD2AP. We did detect a statistically significant 20 to 30% reduction in nephrin and podocin gene expression in kidneys from newborn Wt1+/R394W animals, but this reduction would not be expected to result in a renal phenotype since mice heterozygous for nephrin or podocin null alleles do not display glomerular sclerosis (34, 35, 37). At 2 and 4 months, no decreased expression of podocin, nephrin, or CD2AP RNA was observed in kidneys from mutant animals compared to that in those of wild-type littermates. In fact, podocin was upregulated to a statistically significant degree (P = 0.003) in 4-month-old mutant kidneys and also in 2-month-old animals (P = 0.058), and CD2AP was significantly upregulated in both 2- and 4-month-old affected animals. These data for podocin and CD2AP are in striking contrast to the known role that ablation of these genes plays in the etiology of some forms of glomerulosclerosis (2, 23, 37, 40). For another podocyte gene whose protein product, podocalyxin, also plays a role in podocyte-slit diaphragm interaction (42) and whose expression is reported to be upregulated by WT1 in an in vitro cell system (30), we observed a 20% reduction in expression in newborn mutant kidneys but normal expression in mutant kidneys at 2 and 4 months. As with nephrin and podocin, the modest decrease in podocalyxin expression in Wt1+/R394W newborns is unlikely to be the mechanism by which the Wt1 mutation results in the severe and early-onset glomerular sclerosis observed in these animals since mice heterozygous for podocalyxin mutations exhibit no renal phenotype (9).

These gene expression data, then, suggest that the role of Wt1 mutation in glomerulosclerosis in the Wt1R394W strain is apparently not mediated through downregulation of nephrin, podocin, CD2AP, or podocalyxin, although the modest decrease in nephrin, podocin, and podocalyxin observed in newborn mutant kidneys suggests that analysis of the prenatal expression of the genes for these proteins may be of interest in the future. Similar to the significant increase in CD2AP expression we observed in Wt1+/R394W mutant mouse kidneys at 2 and 4 months of age, upregulation of CD2AP has also been reported in kidneys from podocin−/− mice (23). These data suggest that the perturbation of podocyte function due to ablation (e.g., podocin, nephrin, or CD2AP) or mutation (WT1) of any one of several podocyte genes subsequently initiates a series of adaptive (and maladaptive) processes that can include a general dysregulation of other podocyte-expressed genes.

In contrast to the normal levels of nephrin gene expression we observed in Wt1+/R394W mice at 2 and 4 months (ages at which glomerulosclerosis is observed), nephrin gene expression has been reported to be greatly reduced in phenotypically abnormal Wt1−/− mice rescued with Wt1-expressing YAC transgenes (12). To investigate further the role of nephrin dysregulation in our Wt1+/R394W animals, we compared nephrin protein expression in kidneys from both presymptomatic and symptomatic animals at 2 and 4 months with that in kidneys from wild-type littermates. We observed that at both 2 and 4 months, nephrin protein expression was normal in the glomeruli of presymptomatic Wt1+/R394W animals. In animals with proteinuria and glomerulosclerosis, nephrin expression was marginally normal in histologically normal glomeruli but dramatically reduced in sclerotic glomeruli. This is similar to the loss of nephrin protein observed only in abnormal glomeruli of animals heterozygous for a Wt1 null allele (28). These data, the absence of sclerosis in Nphs1+/− animals, the only modestly reduced Nphs1 expression in newborn Wt1+/R394W mouse kidneys, the normal levels of Nphs1 RNA in Wt1+/R394W mouse kidneys at 2 and 4 months, and the concordance of nephrin protein expression with the degree of glomerular pathology in Wt1 mutant mouse kidneys suggest, in toto, that dysregulation of Nphs1 gene expression does not mediate Wt1R394W-induced glomerulosclerosis. These data further suggest that in the Wt1R394W model, reduced nephrin protein is secondary to the disease process.

In addition to the podocyte structural genes that play an etiological role in some forms of glomerulosclerosis, other more globally expressed genes such as TGFB1 and genes in the IGF1 axis have also been implicated in the development of glomerulosclerosis (4, 6, 8, 19, 25, 36, 39, 45). To assess whether dysregulation of TGFB1, IGF1, or IGFR1 could be a mechanism by which Wt1 mutation results in glomerular pathology, we also measured their expression in mutant and wild-type kidneys. No changes in the expression of these genes was observed in the newborn mutant kidney. Interestingly, TGFB1 was significantly upregulated in Wt1R394W kidneys at 2 months and IGF1 was significantly upregulated in mutant kidneys at both 2 and 4 months. These data are similar to the upregulation of TGFB1 that is observed in glomerulosclerosis due to a variety of etiologies (20, 44). However, as with the data for nephrin, podocin, and podocalyxin, our data for TGFB1 and IGF1 cannot discriminate between upregulation that is due to Wt1 mutation and is a key initiator of glomerular pathology versus that which is a consequence of glomerulosclerosis. A more extensive analysis of mutant kidneys at earlier ages and stratification of samples based on both genotype and degree of pathology are necessary to investigate this further.

Mesangial cell hyperproliferation has been implicated in the development of glomerulosclerosis (38). However, we observed no increase in proliferating cells in glomeruli from mutant animals, indicating that mesangial proliferation is also not the mechanism by which Wt1 mutation leads to glomerulosclerosis.

The phenotype displayed by carriers of the Wt1 R394W allele is dramatically modulated by the genetic background of the animals. In contrast to the 100% penetrance of the mutation by 4 months in Wt1+/R394W males (and 71% penetrance for both genders) in an MF1 background, in a B6/129 background 0% of Wt1+/R394W animals displayed proteinuria at 4 months of age. These data point to a role for a modifying gene that differs among mouse backgrounds and modulates susceptibility to Wt1 R394W-induced glomerulosclerosis. The mechanism by which this modifying gene acts is not known but may relate to the difference in the levels of wild-type Wt1 expression we observed between B6/129 and MF1 animals; the modifying gene(s) in the MF1 background may function to generally reduce Wt1 expression. Data from Wt1−/− animals that have been rescued by Wt1-containing YAC transgenes also suggest that the level of wild-type Wt1 expression is a critical factor in the development of Wt1-related phenotypes (12), and our data support this model.

Missense mutations in exons encoding zinc finger domains of WT1 are known to cause early-onset renal failure in humans. With this report, we have demonstrated that the most commonly occurring human mutation, R394W, when introduced into the mouse genome, results in the same early-onset proteinuria and glomerulosclerosis as observed in human DDS. The mechanism by which this mutation initiates the disease process is not known but is presumably dysregulation of downstream target genes. This aberrant regulation may result in the downregulation of genes critical for glomerular function, in particular, podocyte architecture and viability. We observed no dramatically decreased expression of nephrin, podocin, podocalyxin, or CD2AP in mutant kidneys, but other podocyte genes have yet to be assessed. Alternative to a mechanism whereby Wt1 mutation alters expression of glomerular proteins, which then triggers a cascade of events culminating in glomerulosclerosis, Wt1 mutation-induced dysregulated gene expression may result in an abnormal composition of the GBM, which in turn disrupts the normal attachment of the podocytes. Further gene expression studies with Wt1+/R394W mice will help to identify genes that play a role both primarily and secondarily in glomerulosclerosis and to clarify the mechanism of Wt1 R394W-induced renal failure. As this animal model recapitulates the renal pathology and disease progress observed in humans carrying the analogous mutation, its study will elucidate the mechanism of glomerulosclerosis in DDS patients. This, in turn, will likely provide important insight into the pathobiology of glomerulosclerosis in general.

Acknowledgments

This work was supported by NIH grants CA78257, CA34936, and HD30284. DNA sequencing, blastocyst injection, and veterinary resources were supported by NIH Cancer Center support (core) grant CA16672.

We thank Allan Bradley for ABI ES and SNL 76/7 STO cells and John Stewart for discussions and initial pathological analyses.

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