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J Am Soc Nephrol 13:2058-2067, 2002
© 2002 American Society of Nephrology

A Mutant Form of the Wilms’ Tumor Suppressor Gene WT1 Observed in Denys-Drash Syndrome Interferes with Glomerular Capillary Development

Thomas A. Natoli*,{dagger}, Jing Liu*,{dagger}, Vera Eremina{ddagger}, Karen Hodgens§, Cong Li||, Yuki Hamano{dagger},¶, Peter Mundel#, Raghu Kalluri, Jeffrey H. Miner||, Susan E. Quaggin{ddagger},** and Jordan A. Kreidberg*,{dagger}

*Department of Medicine and §Laboratory for Skeletal Disorders and Rehabilitation, Department of Orthopedic Surgery, Children’s Hospital, Boston, Massachusetts; {dagger}Department of Pediatrics, Harvard Medical School, Boston, Massachusetts; {ddagger}Department of Maternal and Fetal Health, The Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada; ||Renal Division, Washington University School of Medicine, St. Louis, Missouri; Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts; #Departments of Medicine and Cell Biology, Albert Einstein College of Medicine, Bronx, New York; and **Division of Nephrology, St. Michael’s Hospital, Toronto, Ontario, Canada.

Correspondence to Dr. Jordan A. Kreidberg, Division of Nephrology, Department of Medicine, Hunnewell 3, Children’s Hospital, 300 Longwood Ave., Boston, MA 02115. Phone: 617-247-5194; Fax: 617-232-4315; E-mail: Jordan.Kreidberg{at}tch.harvard.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. The Wilms’ tumor suppressor gene WT1 encodes a zinc finger protein that is required for urogenital development. In the kidney, WT1 is most highly expressed in glomerular epithelial cells or podocytes, which are an essential component of the filtering system. Human subjects heterozygous for point mutations in the WT1 gene develop renal failure because of the formation of scar tissue within glomeruli. The relationship between WT1 expression in podocytes during development and glomerular scarring is not well understood. In this study, transgenic mice that expressed a mutant form of WT1 in podocytes were derived. The capillaries within transgenic glomeruli were dilated, indicating that WT1 might regulate the expression of growth factors that affect capillary development. Platelet endothelial cell adhesion molecule-1 expression was greatly reduced on glomerular endothelial cells of transgenic kidneys. These results suggest that WT1 controls the expression of growth factors that regulate glomerular capillary development and that abnormal capillary development might lead to glomerular disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In nephrons of vertebrate kidneys, blood is filtered within the glomerulus, which is a structure that develops at the proximal end of each nephron during kidney development. Within the glomerulus, cells known as podocytes extend a fine meshwork of foot processes, forming scaffolding around a plexus of six to eight capillary loops (Figure 1). Situated between the podocyte foot processes and the endothelial lining of the capillaries is the glomerular basement membrane (GBM), which is an epithelial basement membrane whose presence prevents the exit of large macromolecules from the circulation. The maintenance of this glomerular structure is essential for survival. Improper development of the glomeruli, as occurs in several human syndromes, or subsequent damage from disease processes results in the loss of protein from the circulation, leading to a disease state known as the nephrotic syndrome and ultimately to chronic renal failure, necessitating dialysis or kidney transplantation.



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  		Figure 1. (A) Schematic diagram of glomerular foot processes. Two foot processes are shown. Included in this schematic diagram are the interaction of {alpha}3{beta}1 integrin with the glomerular basement membrane (GBM) and the homophilic interaction of nephrin at the slit diaphragm. Podocin and CD2-associated protein (CD2-AP) also interact with nephrin. A fenestrated endothelial cell is indicated on the opposite side of the GBM. Filtration in the kidney occurs when filtrate passes from the capillary, across the GBM, across the slit diaphragm, and into Bowman’s space surrounding the podocytes. From Bowman’s space, the filtrate proceeds into the tubule of the nephron. (B) Scanning electron microscopic image, demonstrating how the podocyte foot processes form scaffolding around a loop of capillary within the glomerulus. Provided by Wilhelm Kriz, University of Heidelberg. Magnification, x75,000.

 
Studies of gene-targeted mice and human subjects with inherited kidney disease have defined two essential structural features of glomeruli that are necessary for maintenance of the structure of podocytes and the GBM (Figure 1). The first is the interaction of the laminin receptor {alpha}3{beta}1 integrin with the GBM (1). Mutation of either the {alpha}3 integrin gene or components of the GBM leads to an inability to form or maintain the foot process structure or an intact GBM. The second, equally important structure is the slit diaphragm, which is a protein matrix situated between the lateral aspects of each pair of adjacent foot processes. Nephrin, the product of the gene mutated in the Finnish type of congenital nephrotic syndrome (2), was identified as a component of the slit diaphragm (3–5), and podocin, the product of the NPHS2 gene (which is mutated in steroid-resistant nephrotic syndrome) (6), is a membrane protein associated with the slit diaphragm and CD2-associated protein (CD2-AP) (7–9).

The Wilms’ tumor-1 (WT1) gene encodes a protein, WT1, with a proline-rich amino-terminal domain and four carboxy-terminal zinc fingers, which is expressed throughout urogenital development and continues to be highly expressed in podocytes (10–12). A null mutation of WT1 obtained by gene targeting demonstrated that this gene is essential for the earliest phases of kidney and gonad development (13), but partial loss-of-function mutations identified in human subjects lead to glomerulosclerosis (14–16), a scarring process that occurs after loss of the normal podocyte structure. These mutations include those observed in Denys-Drash syndrome (DDS), in which mutations lead to an inability of the zinc fingers to bind DNA (16), and Frasier syndrome, in which a mutation at a splice donor site eliminates production of two of the four major splice forms of WT1 mRNA (14,15). Podocalyxin, a membrane-bound sialoprotein, was recently identified as a potential target of WT1 in podocytes (17). Other possible targets for regulation by WT1 during podocyte differentiation are unknown. The early steps leading to glomerulosclerosis in human subjects are not well understood, but the expression of WT1 throughout kidney development suggests that congenital malformations not apparent at birth might lead to eventual scarring. WT1 has been suggested to regulate the expression of several growth factors, and increased expression of transforming growth factor-{beta} and platelet-derived growth factor (PDGF) has been observed in kidneys from human subjects with DDS (18). Therefore, murine models of glomerular differentiation might facilitate elucidation of this complex process linking development and disease in the kidney.

The recent identification of a promoter region upstream from the nephrin gene as a sequence able to direct podocyte-specific gene expression in transgenic mice has expanded our ability to study genetic events that affect podocyte structure and function (19). Nephrin begins to be expressed in nascent podocytes in capillary loop-stage glomeruli (2), and this promoter-regulatory element confers faithful expression of adjacent transgenes (19,20). In this study, we used the nephrin promoter to investigate the effects of expressing a mutant form of the WT1 gene (originally identified in DDS) in podocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies
Monoclonal anti-WT1 (for immunofluorescence assays and Western blots) (SC-7385; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-podocalyxin (obtained from Marilyn Farquhar, University of California, San Diego), rabbit anti-{beta}-actin (Sigma Chemical Co., St. Louis, MO), and rabbit anti-vascular endothelial growth factor (VEGF) (SC-407; Santa Cruz) antibodies were used. Rat monoclonal antibody to laminin {alpha}1 (8B3, a gift from Dale Abrahamson, University of Kansas) and rabbit polyclonal antiserum to mouse laminin {beta}2 (a gift from Takako Sasaki and Rupert Timpl, Max Planck Institute for Biochemistry, Martinsried, Germany) have been described (21–24). Rabbit polyclonal antiserum to laminin {alpha}5 (25) and antibodies to nephrin (26), CD2-AP (27), synaptopodin (28), and {alpha}3 integrin (29) have also been described.

Transgenic Mice
Transgenic mice were produced by using standard methods of pronuclear injection. All protocols were approved by institutional animal care and use committees.

Transgenic Construct
The following PCR primers were used to produce a murine WT1 cDNA containing the R362X truncation mutation: 5'-gctgctcgtctcggatccagaaccgtccgcatccgag-3' and 5'-gctctagattcactcgcagtccttgaagtc-3'. The PCR product was digested with BamHI and XbaI. The final construct used to derive transgenic mice contained the mouse nephrin upstream fragment (20), the truncated WT1 cDNA, and a splice donor site, intron, splice acceptor site, and poly(A) sites from the {beta}-actin gene. The construct used to express the truncated WT1 protein in immortalized podocytes contained an IRES-green fluorescence protein cassette downstream of the WT1 cDNA; the promoter was a CAGG promoter obtained from Andras Nagy (Samuel Lunenfeld Research Institute, Toronto, Ontario, Canada).

Genotyping and Reverse Transcription-PCR
Mice and embryos were genotyped by using the following primers: 5'-ccagcttgaatgcatgac-3' and 5'-gccaaaatgatgagacagcac-3'. The same primers were used for reverse transcription (RT)-PCR and, after observation of the ethidium bromide-stained bands, the DNA fragments were blot-transferred to Hybond-N+ membranes (Amersham, Arlington Heights, IL) and hybridized with a probe containing the entire murine WT1 cDNA.

Histologic Analyses
Histologic preparations were prepared from paraffin-embedded sections, and 5-µm sections were stained with hematoxylin and eosin, under standard conditions. All immunostaining was performed with frozen sections. For indirect immunofluorescence assays, 7-µm frozen sections were fixed with 2% paraformaldehyde in phosphate-buffered saline (PBS) and blocked with 10% normal goat serum. Antibodies diluted in PBS with 1% bovine serum albumin were applied for 1 h. Sections were rinsed with PBS and incubated with Cy3- or FITC-conjugated secondary antibodies (Chemicon, Temecula, CA). Sections were then rinsed, mounted in 0.1x PBS/90% glycerol with 1 mg/ml p-phenylenediamine, and viewed with a fluorescence microscope. Images were captured with a Spot 2 cooled color digital camera (Diagnostic Instruments, Sterling Heights, MI). All images are representative of at least five from each of two wild-type or three transgenic kidneys.

Electron Microscopy
Tissues were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, postfixed with 1% osmium tetroxide, dehydrated in graded alcohols, infiltrated, and embedded in LR white resin. Thin sections (80 nm) were cut with a diamond knife, collected on carbon-reinforced nickel grids, and stained with uranyl acetate and lead citrate. Sections were examined with a Philips 300 microscope (FEI Corp., Hillsboro, OR).

In Vitro Podocyte Cultures
Immortalized podocytes were cultured as described (30). Cells were transfected by using calcium phosphate transfection techniques. Because the transgene used to transfect immortalized podocytes contained an IRES-green fluorescence protein cassette but no antibiotic selection marker, two rounds of fluorescence-activated cell sorting (performed 1 and 3 wk after transfection) were used to enrich populations of transfected cells. Cells were then plated in 96-well plates at limiting dilution, and individual clonal populations were isolated and screened by Western blotting, to determine which were expressing high levels of the truncated WT1 protein.

Western Blotting
Western blotting was performed according to standard protocols, as previously described (31).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Production of Transgenic Mice
Two constructs were used to derive transgenic mice, both of which contained a deletion of the two zinc fingers at the carboxy-terminal end of the WT1 protein (Figure 2). This mutation duplicated a truncation mutation observed in DDS (32); the truncated protein is thought to function in a dominant-negative manner (33–35). Two alternative splice events and several alternative translational start sites determine the structure of the WT1 protein (36,37). The first alternative splice inserts exon 5, which contains 17 amino acids, between the proline-rich amino-terminal domain and the four zinc finger domains. The second splice inserts three amino acids, i.e., lysine-threonine-serine (KTS), at the end of the third zinc finger. DDS can result from truncation mutations that entirely eliminate the third and fourth zinc fingers, suggesting that the presence or absence of the KTS sequence is not an important factor in determining this phenotype (32). This is in contrast to the related Frasier syndrome, which results from an inability to express +KTS isoforms (14,15). However, there is no information regarding whether the WT1 peptide responsible for the DDS and Frasier phenotypes includes exon 5. Therefore, we constructed transgenes that contained or omitted exon 5 (Figure 2).



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  		Figure 2. Transgenic constructs. WT1 is shown as a protein with four zinc fingers and a proline-rich amino-terminal domain. The alternatively spliced exon 5 and the KTS sequence are indicated. Two transgenic constructs were used, which were truncated to remove the third and fourth zinc fingers. One construct included exon 5. The {beta}-actin intron and poly(A) sequence at the 3'-ends of the constructs are not indicated. DDS, Denys-Drash syndrome; Pr., promoter.

 
We obtained four adult mice with the transgene containing exon 5. These mice appeared normal, with no compromise of renal function. Histologic analyses of kidneys from these mice revealed normal-appearing glomeruli (data not shown). In contrast, we were able to obtain only one adult mouse with the transgene without exon 5, suggesting that expression of this transgene might lead to kidney defects incompatible with postnatal survival. This finding led us to produce additional transgenic litters that were euthanized on embryonic day 18 (E18), to determine whether expression of the mutant form of WT1 in developing podocytes had interfered with the assembly of normal glomeruli. From these litters, transgenic embryos were obtained at the expected frequency. A total of eight E18 transgenic embryos were obtained, and all except one exhibited abnormal-appearing glomeruli. Because glomeruli comprise only a small fraction of the overall kidney mass, RT-PCR was required for detection of transgene expression. Blot-hybridization was used to confirm that the bands observed after RT-PCR resulted from transcripts produced from the WT1 transgenes (Figure 3C).



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  		Figure 3. WT1 expression. (A and B) Immunofluorescence staining for WT1 in wild-type (A) and transgenic (B) glomeruli. Staining was restricted to podocytes. Magnification, x60. (C) Southern blot of reverse transcription-PCR analysis of transgenic embryos. Lanes 1, 2, 3, 5, and 6, RNA samples from kidneys from embryonic day 18 embryos identified as transgenic; lanes 4 and 7, identically prepared, nontransgenic control samples. A full-length WT1 cDNA probe was used to probe the PCR products. The highest band (Gen) is the expected size of the unspliced transgene and indicates the presence of contaminating genomic DNA in the RNA preparation. The lower band (RNA) is the correct size for the expected splice product. The middle band is thought to represent the use of a cryptic splice site within the transgene.

 
Expression of WT1 in Transgenic Glomeruli
To evaluate whether the truncated WT1 peptide expressed from the transgene was acting in a dominant-negative manner, it was necessary to determine whether wild-type levels of WT1 were affected by transgene expression. Because glomeruli comprise a small portion of the kidney mass and because WT1 is also expressed in the nephrogenic zone of E18 kidneys, it was not possible to compare the levels of wild-type and truncated WT1 in Western blot analyses. Therefore, immunofluorescence staining was used to qualitatively assess whether expression of the transgene affected wild-type levels of WT1 in podocytes (Figure 3, A and B). An antibody to an amino-terminal domain was used for detection of both wild-type and truncated WT1. Staining with the amino-terminal domain-specific antibody revealed only slightly more intense staining in the transgenic kidneys, suggesting that the expression of the transgene did not greatly increase the overall amount of WT1 in podocytes.

Glomeruli in Transgenic Embryos
Examination of E18 transgenic embryos demonstrated the presence of abnormally developed glomeruli (Figure 4). At E18, the murine kidney is still developing nascent nephrons but mature glomeruli are also present. The most mature glomeruli in wild-type E18 kidneys have well developed capillary loops (each with the diameter of a single red blood cell) and mature foot processes extending from the podocytes, which form complete scaffolding around the capillary loops. In contrast, fewer capillary loops were present in transgenic glomeruli and each was abnormally wide, such that several red blood cells were observed to be adjacent to each other within a single capillary loop (Figure 5). These observations suggest that the process by which an original single capillary loop in a nascent glomerulus undergoes branching to form a capillary plexus involves WT1-dependent interactions between the capillaries and the podocytes.



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  		Figure 4. Histologic analysis of transgenic glomeruli. (A) Wild-type glomeruli. (B and C) Transgenic glomeruli. Kidney sections were stained with hematoxylin and eosin. In the transgenic glomeruli, larger-than-normal capillaries are indicated by the collections of red blood cells, a feature not present in the small capillaries of wild-type glomeruli.

 


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  		Figure 5. Electron micrographs of glomeruli. Wild-type (A and B) and transgenic (C and D) glomeruli are shown at low (x1300) (A and C) and medium (x18,700) (B and D) magnification. In each case, the most mature glomeruli observed are shown. The low-power view makes evident the large capillaries of the transgenic glomeruli. Several red blood cells can be observed within a capillary loop (CL), a finding not present in normal glomeruli. At higher power, foot processes (FP) are evident in both wild-type (B) and transgenic (D) glomeruli but are more irregularly formed in the transgenic sample. CAP, capillary.

 
Electron microscopy was also used to evaluate whether podocyte foot processes were present in glomeruli of transgenic mice (Figure 5). Foot processes were present in transgenic mice, although they were more irregularly shaped than those observed in wild-type glomeruli. To evaluate whether this was attributable to deficient expression of proteins known to be required for the maintenance of normal podocyte and foot process morphologic features, the expression of these proteins was examined.

Integrin and Basement Membrane Expression in Transgenic Glomeruli
Aberrant glomerular development or glomerular disease has been observed in human subjects and mice with mutations in the {alpha}3 integrin gene or genes encoding components of the GBM (1,38). The expression of {alpha}3{beta}1 integrin and basement membrane components was therefore examined in WT1-transgenic kidneys (Figure 6). These examinations were performed with immunofluorescence microscopy; more quantitative techniques could not be used because glomeruli comprise such a small proportion of the kidney. During the maturation of glomeruli, there is a switch in the expression of laminin isoforms, from laminin-1 ({alpha}1{beta}1{gamma}1) to laminin-10/11 ({alpha}5{beta}2{gamma}1 or -2) (25,38,39). Expression of the {alpha}5 and {beta}2 peptides is required for full glomerular maturation ({alpha}5) and maintenance of glomerular integrity ({beta}2) (23,40). Immunodetection of the {alpha}1, {alpha}5, and {beta}2 laminin subunits with specific antibodies revealed that this switch in isoform expression occurred normally in transgenic glomeruli and was apparently not regulated by WT1. Moreover, staining with {alpha}1 or {beta}1 subunit-specific antibodies demonstrated the expected low levels of laminin-1 in nearly mature glomeruli. Staining of type IV collagen subunits and {alpha}3{beta}1 integrin did not reveal any differences between wild-type and transgenic glomeruli. Therefore, expression of the truncated WT1 transgene did not seem to affect the expression of basement membrane protein-encoding genes or {alpha}3{beta}1 integrin, and altered levels of these proteins are unlikely to account for the phenotype of WT1-transgenic mice.



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  		Figure 6. GBM and integrin expression. Components of the GBM, including {alpha}1, {alpha}5, and {beta}2 laminin (Lam) subunits, the {alpha}3 subunit of type IV collagen (Col), and {alpha}3{beta}1 integrin (Int), were examined, as indicated at the left. The {alpha}1, {alpha}4, and {alpha}5 subunits of type IV collagen were also examined and were found to be expressed at identical levels in wild-type and transgenic animals (not shown). (Left) Wild-type control littermates. (Right) Transgenic mice. Magnification, x60.

 
Podocyte Structural Proteins
An intact slit diaphragm between adjacent foot processes is also required for maintenance of the structural integrity of glomeruli, especially for assembling and maintaining individual foot processes (41). Expression of the known components of the slit diaphragm, including nephrin, podocin, and CD2-AP, was examined in wild-type and transgenic glomeruli. Little difference in the expression of slit diaphragm components in wild-type and transgenic glomeruli was observed (Figure 7).



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  		Figure 7. Podocyte structural protein expression. Nephrin, podocin, CD2-AP, synaptopodin, and podocalyxin expression was assessed, as indicated. (Left) Wild-type control littermates. (Right) Transgenic mice. Staining was similar in wild-type and transgenic kidneys. Magnification, x60.

 
Synaptopodin is a cytoskeleton-associated protein whose expression in the kidney is restricted to podocytes, primarily in foot processes (42). Synaptopodin seemed to be expressed at equal levels in wild-type and transgenic podocytes (Figure 7). Podocalyxin is a highly charged membrane protein that is expressed on the lateral surfaces of podocytes and might have a role in maintaining some distance between adjacent foot processes and podocyte cell bodies, thus also maintaining space for the glomerular filtrate until it empties into proximal tubules (43,44). Expression of podocalyxin was demonstrated to be regulated by WT1 in a cell culture differentiation system (17). Mice with a targeted mutation of the podocalyxin gene were unable to form podocyte foot processes, although wide glomerular capillaries were not present in those mice (43). As indicated by immunofluorescence staining, podocalyxin seemed to be present at equal levels in wild-type and transgenic mice.

Reduced Platelet Endothelial Cell Adhesion Molecule Expression in Glomerular Endothelial Cells
The aberrant capillary development in transgenic glomeruli suggested abnormal expression of angiogenic growth factors by podocytes during glomerular capillary development. Platelet endothelial cell adhesion molecule-1 (PECAM-1) expression on endothelial cells could be an indicator of an endothelial response to exogenous production of angiogenic and other growth factors, such as transforming growth factor-{beta} and VEGF (45,46). PECAM-1 expression on endothelial cells of transgenic glomeruli was greatly reduced or undetectable, compared with that observed in wild-type glomeruli (Figure 8). PECAM-1 on nonglomerular endothelium stained identically in wild-type and transgenic kidneys (data not shown). These results suggest that WT1 may indeed regulate the expression of growth factors that affect vascular development in glomeruli.



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  		Figure 8. Platelet endothelial cell adhesion molecule (PECAM) expression. (A) Wild-type glomerulus. (B) Transgenic glomerulus. PECAM staining (green) outlines the presence of capillary endothelial cells. Staining for {alpha}3{beta}1 integrin (red) is included to indicate the location of podocytes. Little or no PECAM is present on endothelial cells of transgenic glomeruli. Magnification, x60.

 
Podocyte Differentiation In Vitro
For more-quantitative assessment of the effect of expression of the dominant-negative WT1 in podocytes, an in vitro system for podocyte differentiation was used. Podocyte cell lines have been isolated from mice carrying a temperature-sensitive T antigen transgene (30). After being shifted to the nonpermissive temperature, these immortalized podocytes differentiate and begin to express podocyte-specific markers. Stable transfectants that expressed the dominant-negative WT1 transgene used to derive transgenic mice were obtained, and clonal populations were derived (Figure 9A). In contrast to published findings indicating that podocalyxin was a potential regulatory target of WT1 (17), podocalyxin expression was not decreased in podocyte cell lines expressing the dominant-negative WT1 construct, compared with wild-type cells (Figure 9B). Because the most striking aspect of the transgenic phenotype was the abnormal capillary development, the expression of angiogenic proteins known to be expressed by podocytes, including VEGF and ephrin-B2 (47), was examined. Ephrin-B2 could not be detected in these cell lines, and VEGF was present at levels similar to those detected in Western blots (Figure 9B). Further work is needed to determine whether other growth factors that affect capillary development might be regulated by WT1.



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  		Figure 9. Western blots of WT1 and proteins expressed by immortalized podocytes. (A) WT1 expression in wild-type and transfected podocytes. Lane WT, wild-type podocytes; only the wild-type band is present. This lane is overloaded, in comparison with the two transfected lanes, to demonstrate the position of the wild-type band and to indicate that no other band is present. Lanes DN, two transfected lines, showing the lower band from the truncation product and the upper wild-type band. The truncation product is more abundant than the wild-type band. (B) Expression of podocalyxin and growth factors. Podocytes were maintained at the nonpermissive temperature and allowed to differentiate for 2 wk before collection. Podocalyxin and vascular endothelial growth factor (VEGF) are shown. The {beta}-actin control demonstrates equivalent loading of all lanes. Podocalyxin and VEGF bands appear similar in all lanes.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results confirm that WT1 is critically important for glomerular differentiation. Capillaries of transgenic glomeruli are abnormally wide, and there is reduced PECAM expression on glomerular endothelial cells. These results indicate that WT1 regulates the expression of factors that affect endothelial cells and capillary structure.

The identification of WT1 mutations among individuals with DDS and Frasier syndrome established WT1 as a critical gene required to maintain the stability of glomeruli (14–16). DDS is caused by mutations similar to that in our transgene or by point mutations that affect the DNA-binding ability of WT1, and it is therefore thought to be attributable to dominant-negative effects of a mutant WT1 peptide (16,34). There is molecular evidence from in vitro systems to support this interpretation (33,34). However, it has been difficult to distinguish dominant-negative effects from decreases in the levels of wild-type WT1 protein in accounting for the phenotype. In this study, both chromosomal copies of WT1 were intact, and levels of wild-type WT1 protein appeared similar in wild-type and transgenic glomeruli. Therefore, the appearance of a mutant phenotype in WT1-transgenic mice strongly supports the interpretation that DDS is attributable to dominant-negative actions of mutant forms of WT1.

Hammes et al. (48) recently published a study of WT1-mutant mice that were engineered to exclusively express either the +KTS or -KTS form of WT1; the -KTS-only mice represent a model of Frasier syndrome, in which a mutation in a splice site eliminates expression of the +KTS isoform. Both +KTS-only and -KTS-only mice demonstrated abnormal glomerular development, although those phenotypes were distinct from that observed for the WT1-transgenic mice, in that abnormally wide capillary loops were not observed. Taken together, these results suggest that the dominant-negative mutations that result in DDS act through a mechanism different from that responsible for Frasier syndrome, which remains poorly understood.

Previously published studies of the effects of WT1 mutations on glomerular development or sclerosis, in either human subjects or mice, were unable to determine whether alternatively spliced exon 5 plays a role in podocyte differentiation. Exon 5 of WT1 is observed in all mammalian forms of WT1 but not in forms in other vertebrates (49). An interaction between the peptide encoded within exon 5 and the apoptosis-related protein Par4 has been identified (50), but this interaction seems not to be involved in glomerular differentiation. The results presented here indicate that the isoforms of WT1 that do not contain exon 5 have a greater, if not exclusive, role in directing podocyte differentiation. These findings are in agreement with our recent results demonstrating that a targeted deletion of WT1 exon 5 has no adverse effect on kidney development (51). Together, these results lead us to hypothesize that the WT1 amino terminal domains with and without exon 5 interact with different protein complexes and only the complex without exon 5 is required for glomerular development.

Several structural proteins have been identified as being crucial for correct podocyte differentiation and foot process assembly. These proteins include {alpha}3{beta}1 integrin, podocalyxin, CD2-AP, nephrin, podocin, and {alpha}5 laminin (1,6,23,43,52,53). All of these proteins seemed to be expressed at similar levels in wild-type and transgenic kidneys, although small differences in expression would not be detected with immunofluorescence staining. Podocalyxin expression was not decreased in immortalized podocytes expressing the dominant negative transgene. Moreover, the phenotype of the transgenic glomeruli was distinct from that of podocalyxin-mutant mice, which fail to form foot processes but were not noted to have abnormal capillary loops (43). Therefore, these results are unable to support a role for WT1 as a regulator of podocalyxin expression, as recently suggested (17). This discrepancy might be attributable to the difference in the cell types studied or the fact that WT1 expression was increased in one case, whereas its function was inhibited in our studies.

Capillary branching is an essential feature of glomerular development (47). During early glomerular development, endothelial cells migrate into the glomerular cleft and form an initial capillary loop, which then undergoes several rounds of branching. During this branching process, podocytes migrate around the capillary loops and extend foot processes.

Podocytes are known to express several angiogenic growth factors, including PDGF-A, VEGF, ephrin-B2, and angiopoietin-1, and expression of these factors is thought to regulate capillary expansion (47). This has been demonstrated most dramatically in the case of PDGF; targeted mutation of PDGF-B or PDGF receptor {beta} results in a loss of mesangial cells, dramatically disrupting the glomerular vasculature (54,55). Future experiments should determine whether any of these growth factors are targets of WT1 in podocytes. It is possible that one or more of these factors, or perhaps unidentified factors, are regulated by WT1 and are responsible for stimulating the expression of PECAM on glomerular endothelial cells.

The function of PECAM-1 in endothelial cells is less well understood than its function in leukocytes, where it is known to have an important role in attachment to endothelial cells and migration into extravascular tissue during inflammation (56). Recent studies suggested a role for PECAM-1 in cell-cell attachment (57). This could be of critical importance in glomeruli, where proper endothelial structure and attachment of adjacent endothelial cells might be required to maintain normal filtration across the GBM.

Because WT1-transgenic mice seem not to survive beyond the newborn period, they are not directly informative regarding the pathogenesis of the glomerular disease that occurs in individuals with DDS. Nevertheless, it is interesting to speculate that the maintenance of normal glomeruli might require the continued expression of growth factors, such that, in their absence, capillaries are damaged and sclerosis ensues. This possibility deserves additional study, which will be possible when conditionally WT1-mutant mice become available. Studies demonstrating that VEGF administration might ameliorate experimental models of glomerulosclerosis suggest a role for angiogenic growth factors in postnatal glomeruli (58–60).


   Acknowledgments
 
This work was supported by grants from the NIDDK (DK50118) and the Emerald Foundaiton. Dr. Natoli was supported by an American Society for Nephrology/National/Kidney Foundation/SangStat fellowship.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication March 21, 2002. Accepted for publication April 29, 2002.




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