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J Am Soc Nephrol 15: 3044-3051, 2004
© 2004 American Society of Nephrology
doi: 10.1097/01.ASN.0000146687.99058.25
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BASIC SCIENCE

The Major Podocyte Protein Nephrin Is Transcriptionally Activated by the Wilms’ Tumor Suppressor WT1

Nicole Wagner*, Kay-Dietrich Wagner*, Yiming Xing{dagger}, Holger Scholz{ddagger} and Andreas Schedl*

*INSERM U636, Centre de Biochimie, Faculté des Sciences, Nice, France; {dagger}Department of Development & Cell Biology, UCI, Irvine California; and {ddagger}Institut für Physiologie, Charité, Universitätsmedizin Berlin, Berlin, Germany

Correspondence to Dr. Andreas Schedl, INSERM U636, Centre de Biochimie, Parc Valrose, 06108 Nice, France. Phone: +33-04-92-07-6401; Fax: +33-04-92-07-6475; E-mail: schedl{at}unice.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
NPHS1 encodes the structural protein nephrin, which has a crucial role in the filtration barrier of the glomerular podocyte. Mutations or deregulation of NPHS1 are associated with a variety of renal diseases, including the Finnish type congenital nephrotic syndrome. This study analyzed a potential regulation of nephrin by the Wilms’ tumor protein, Wt1. Using an inducible U2OS osteosarcoma cell line, it is shown that upon Wt1 induction, endogenous nephrin mRNA becomes highly upregulated. Co-transfection studies demonstrate that Wt1 can activate the nephrin promoter >10-fold. DNase footprinting and mutation analysis identify a Wt1 responsive element in the nephrin promoter, which is required for the binding of Wt1 protein. Mutations or deletion of this Wt1 responsive element completely abolished transactivation of the nephrin promoter by Wt1. Moreover, transgenic analysis demonstrates the requirement of the identified binding site to direct podocyte-specific expression of a reporter gene in transgenic mice, thus confirming the importance of this site for the regulation of nephrin in vivo. Finally, it is shown that nephrin expression is lowest in kidneys of mice that lack specifically the Wt1(–KTS) splice variant, but in comparison with wild-type littermates, it is also reduced in animals with disruption of the Wt1(+KTS) splice variant. Taken together, these data identify nephrin as a direct transcriptional target for Wt1 and underline the importance of Wt1 as a key regulator in podocyte function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NPHS1 (encoding nephrin) has been identified by its mutational inactivation in patients who have congenital nephrotic syndrome of the Finnish type (1). The nephrin protein has been detected in glomerular podocytes, in different regions of the brain, and in {beta}-cells of the pancreas. In the kidney, it is predominantly localized to the glomerular slit diaphragm, which forms the filtration barrier of the kidney (1,2). Consistently, nephrin knockout mice show podocyte effacement, absence of the slit diaphragm, and massive proteinuria and die within 1 d after birth as a result of nephrotic syndrome (2). Recently, it was shown that nephrin is redistributed in the glomerular podocytes of patients with minimal-change nephropathy, membranoproliferative glomerulonephritis, and other renal disorders, suggesting a more general role for this protein in glomerular disease (3).

The extracellular segments of nephrin have been demonstrated to interact with Neph1 and other members of the nephrin gene family (4). The cytoplasmic segment interacts with the actin cytoskeleton via CD2AP (5,6) and directly with podocin (7), making nephrin a unique molecule for maintaining the structure of glomerular podocytes and the slit diaphragm. Furthermore, it was found that nephrin functions as a signaling molecule that can activate mitogen-activated protein kinase cascades (8). Despite its central role in podocyte function, the regulation of nephrin on the molecular level is unclear.

The Wilms’ tumor gene Wt1 encodes a zinc finger protein that has been identified on the basis of its involvement in nephroblastoma, an embryonic kidney tumor. Alternative splicing in the zinc finger region of Wt1 leads to the inclusion or omission of the three amino acids KTS, which influences the biochemical properties of the resulting protein. Isoforms that lack the KTS sequence (Wt1–KTS) are potent transcriptional activators and bind preferentially to DNA, whereas Wt1+KTS proteins may also have a role in RNA binding. Despite these biochemical differences, some of the functions of Wt1 seem to be overlapping (9). Knockout, transgenic, and siRNA analyses have demonstrated the importance of Wt1 at several stages of kidney development (10–12). However, expression of Wt1 continues in podocytes of adult kidneys, suggesting that this gene is also required during glomerular function. Consistent with this observation, we recently found that the two major podocyte proteins nephrin and podocalyxin were lowered in the kidneys of mice with reduced expression of Wt1 (13), suggesting co-regulation of nephrin and Wt1.

Here we investigated whether Wt1 might act as a direct activator of nephrin. Using a combination of in vitro and in vivo approaches, we show that Wt1 can bind and activate the nephrin promoter and that this binding is essential for podocyte-specific expression in vivo. Consistently, nephrin expression is reduced in mouse models that lack specific isoforms of Wt1.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mouse Strains, Generation, and Analysis of Transgenic Mice
A description of the generation of mutant mice, which lacked specifically either the –KTS or the +KTS splice variant of Wt1, and a genotyping protocol of the embryos are given by Hammes et al. (9). Transgenic animals were generated by pronuclear microinjection into fertilized mouse oocytes according to standard procedures (11). Superovulation was performed using the F1 generation of CBAxC57B6 crosses (Charles River, L’Arbresle, France). Fosters were killed at embryonic day 17.5 (E17.5; the day of microinjection was taken as E0.5), and embryos were dissected for PCR and lacZ analysis. Transgenic embryos were identified using the primers Neph-LacZ forward 5'-AAGACTGCGACAGTCACAGACA-3' and Neph-LacZ backward 5'-GCTGCAAGGCGATTAAGTTGG-3'. LacZ staining was performed on isolated embryonic kidneys following a protocol described in Hogan et al. (14). After staining, kidneys were postfixed for 3 h in 4% paraformaldehyde; washed two times in PBS; and embedded in a mixture of 13% BSA, 0.5% gelatin, and 2.5% glutaraldehyde (Sigma, St. Quentin Fallavier, France) in PBS. Vibratome sections were cut to 100 µm on a Leica VT1000S. Sections were transferred onto gelatin-coated glass slides and viewed under a DMLB microscope (Leica) connected to a digital camera (Spot RT Slider, Diagnostic Instruments) using the Spot Software.

Cell Culture
U2OS osteosarcoma cells (ATCC HTB-96) were obtained from the American Type Culture Collection. U2OS cells (clone UB27) with tetracycline-repressible expression of Wt1(–KTS) were the gift of Dr. C. Englert (15). The cells were grown as described elsewhere (16).

Cloning
The nephrin promoter (accession no. AY183460) was a gift of Dr. K. Tryggvason (17). This 6.2-kb promoter fragment was subcloned into the KpnI and HindIII restriction sites of the luciferase reporter plasmid pGl2basic (Promega). Deletion and mutations of the WT1 binding site were performed with the aid of the Quik Change II site-directed mutation kit (Stratagene) according to the manufacturer’s instruction using the following primers: {Delta}WTB, 5'-ACAGAAAATGAGAAGGGAGGAGGGGGGAGATG-3' (forward); Mut1, 5'-AATGAGAAGGGGGTAGGAATTAATTAGGAGGAGGAGGGGGGAGATG-3' (forward); Mut2, 5'-GAAGGGGGTAGGAATGGATTATAAGGAGGAGGGGGGAGATGG-3' (forward); Mut3, 5'-GAAGGGGGTAGGAATGGAGGATTATAAGGAGGGGGGAGATGGAATTAAAG-3' (forward); and Mut4, 5'-GGGGTAGGAATGGAGGAGGATTATAAGGGGGGAGATGGAATTAAAGAC-3' (forward). Reverse primers were in the corresponding antisense orientation. All plasmids were sequenced (ABI Prism310 instrument) using the BigDye Terminator v1.1 Cycle sequencing kit (Applied Biosystems) according to the manufacturer’s instructions.

Cell Transfections and Reporter Gene Assays
A total of 0.3 µg of the reporter constructs together with 1.6 µg either of Wt1(–KTS) or Wt1(+KTS) expression vectors (Wt1 cDNA in pCB6+ plasmid) and 0.1 µg of a cytomegalovirus-driven {beta}-galactosidase plasmid were transiently co-transfected into U2OS osteosarcoma cells using the Fugene reagent (Roche). Appropriate control experiments were performed, in which we transfected identical amounts (1.6 µg) of the empty pCB6+ expression plasmid. Luciferase and {beta}-galactosidase activities were measured as described previously (18,19). Luciferase activities are given as relative light units normalized to {beta}-galactosidase activity. Results shown are averages of 10 transfection experiments. P < 0.05 was considered significant (ANOVA with Bonferroni test as post hoc test).

DNase I Footprint Assay
Two consecutive nephrin promoter fragments, spanning 693 and 582 bp, respectively, were generated by PCR using one nonlabeled and one 32P-labeled primer (distal fragment: FP1 forward, 5'-TCCTGCAGGAGATAAGCAGG-3', FP1 reverse, 5'-AGGATGGAACGCAGAGC-3'; proximal fragment: FP2 forward, 5'-CTAGCTCTGCGTTCCATCCT-3', FP2 reverse, 5'-CACCAGCAGCTTGTTTGTTGC-3'). The PCR fragments were gel-purified, and probes (50,000 cpm) were incubated with 50 µg of GST, GST-WT1(–KTS), and GST-WT1(+KTS), respectively, in binding buffer (100 mM KCl, 5 mM MgCl2, 10 mM Tris [pH 7.5], 1 mM CaCl2, 2 mM dithiothreitol, 50 µg/ml BSA, and 2 µg/ml herring sperm DNA) for 30 min at room temperature. Samples were treated with RQ1-DNase I (Promega) in a total reaction volume of 100 µl, and digestion was terminated after 3 min by adding 90 µl of prewarmed (37°C) stop solution (200 mM NaCl, 30 mM EDTA, 1% SDS, and 100 µg/ml yeast RNA). DNA fragments were phenol/chloroform-extracted, ethanol-precipitated, and separated by electrophoresis on a 6% polyacrylamide sequencing gel in 1x TBE buffer. The sequence of the protected region was determined by alignment with a sequencing reaction using a Thermo Sequenase radiolabeled terminator cycle sequencing kit (Amersham) with the primer, which was labeled in the respective footprinting reaction.

Electrophoretic Mobility Shift Assays
Electrophoretic mobility shift assays using recombinant Wt1 protein are described in detail elsewhere (16). The end-labeled 22-bp double-stranded oligonucleotide (5'-GGTAGGAATGGAGGAGGAGGAG-3') contained the Wt1 responsive element from the mouse nephrin promoter. Control experiments were performed with mutated oligonucleotides). An unlabeled 21-bp DNA sequence including the previously identified Wt1(–KTS) binding site from the vitamin D receptor gene promoter (5'-TGAACTTAGTGGGCGTGGTTG-3') (14) was used at 10- to 250-fold molar excess amounts in competition experiments.

Reverse Transcription–PCR
Wt1 expression was induced in UB27 cells by omission of tetracycline from the culture medium for 24 h. Reverse transcription–PCR was performed as described in detail elsewhere (16) using the following primers for amplification: human {beta}-actin, 5'-TTCTACAATGAGCTGCGTGTG-3' (forward), 5'-CGTCACACTTCATGATGGAGT-3' (reverse); human nephrin, 5'-ACGACGCTCAGGGCTTCTCT-3' (forward), 5'-TCTAGCAGGGTCCCCTTCCA-3' (reverse); mouse Wt1, 5'-ATCAGATGAACCTAGGAG-3' (forward), 5'-CTGGGTATGCACACATGA-3' (reverse).

For real-time PCR analysis of the kidneys from splice-specific knockout mice, the LightCycler instrument (Roche) was used according to the following protocol: Denaturation at 95°C for 15 s, annealing at 55°C for 10 s, and elongation at 72°C for 20 s (40 cycles). Serial dilutions of cDNA were used to generate a standard curve. Primers and probes, which were designed by TIB Molbiol (Berlin, Germany), had the following sequences: nephrin, 5'-AGGGTCGGAGGAGGATCGAA-3' (forward), 5'-GGGAAGCTGGGGACTGAAGT-3' (reverse), 5'-GTCCCCAGTCCACTGACTCTCTCCTC-p-3' (LC-Red640), 5'-CTAACCGTGGAGCTTCTTGTGTCCC-X-3' (FL), glyceraldehyde-3-phosphate dehydrogenase, 5'-ATTCAACGGCACAGTCAAGG-3' (forward), 5'-TGGATGCAGGGATGATGTTC-3' (reverse), 5'-TGGAAAGCTGTGGCGTGATGGC-p-3' (LC-Red640), 5'-CCAGAAGACTGTGGATGGCCCCT-X-3' (FL).

Immunohistochemistry
Tissue was fixed overnight at 4°C in 3% paraformaldehyde in PBS. Thereafter, tissue was washed 3 x 20 min in PBS and snap-frozen in prechilled isopentane. Snap-frozen tissue samples were embedded in Tissue-Tek OCT compound (Sakura Finetek). Ten-micrometer tissue sections were cut on a cryostat and transferred onto gelatin-coated glass slides. The tissue was permeabilized with 0.1% Triton X-100 in PBS and blocked by incubation for 1 h in 10% normal donkey serum (in PBS, 0.1% Triton X-100, and 3% BSA). An indirect immunofluorescent double-labeling technique was used to mark Wt1-, nephrin-, and synaptopodin-expressing cells (20). Staining was performed with the following primary antibodies each diluted 1:50 in PBS, 0.1% Triton X-100, 3% normal donkey serum, and 3% BSA: Polyclonal rabbit anti-Wt1 antibody (C-19; Santa Cruz Biotechnology), polyclonal goat anti-nephrin antibody (N-20; Santa Cruz Biotechnology), or polyclonal rabbit anti-nephrin antibody (gift from L. Holzman). Monoclonal mouse antisynaptopodin antibody (Progen) was used undiluted. The slides were viewed under an epifluorescence microscope (DMLB, Leica) connected to a digital camera (Spot RT Slider, Diagnostic Instruments) using the Spot Software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To investigate whether Wt1 can activate the endogenous nephrin gene, we made use of an osteosarcoma cell line, which expresses Wt1(–KTS) under control of a tetracycline-repressible promoter (U2OS, clone UB27) (15). The Wt1(–KTS) splice variant lacks the tripeptide insertion (lysine, threonine, serine, and KTS) between zinc fingers 3 and 4 of the molecule and functions as a transcriptional regulator (21). As expected, removal of tetracycline from the culture medium induced the expression of Wt1 (Figure 1A). This induction of Wt1 was associated with a clear increase in the amount of endogenous mRNA for nephrin. For analyzing whether nephrin is activated directly by Wt1, the published mouse nephrin promoter (17) was cloned in front of a luciferase reporter plasmid and co-transfected with plasmids coding for the major Wt1 isoforms into U2OS osteosarcoma cells (21). Co-transfection with Wt1(–KTS) plasmid stimulated the basal activity of the nephrin promoter >10-fold (Figure 1B). For comparison, the Wt1(+KTS) isoform, which has a lower DNA binding affinity than the Wt1(–KTS) protein, stimulated the nephrin promoter activity to a lesser extent.



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  		Figure 1. (A) Wt1, nephrin, and {beta}-actin mRNA detected by reverse transcription–PCR (RT-PCR) in an osteosarcoma-derived cell line (clone UB27) with tetracycline-repressible expression of the mouse Wt1(–KTS) variant. Nephrin mRNA is upregulated upon induction of Wt1 by removal of tetracycline from the culture medium. (B) Activation of a luciferase reporter harboring a 6.2-kb sequence of the mouse nephrin promoter by transient co-transfection of expression constructs for the Wt1(–KTS) and Wt1(+KTS) variants. Shown are relative luciferase activities normalized to {beta}-galactosidase in each sample. Values are means ± SEM of 10 experiments. *Statistical significance (P < 0.05). pGL2basic is the empty reporter vector. (C) DNase I footprint assay to reveal binding of Wt1 protein to the nephrin promoter. Two different promoter segments (FP1, –1.2 to 0.6 kb; FP2, –0.6 kb to 4 bp), spanning 582 and 693 bp, respectively, were analyzed. The 22-bp sequence, which was protected from DNase I digest by the Wt1(–KTS) protein, is indicated. (D) Electrophoretic mobility shift assay (EMSA) demonstrating binding of the Wt1(–KTS) and, to a lesser extent, the Wt1(+KTS) protein to the 22-bp protected element (WTB) in the nephrin promoter. Binding could be competed with a 10- to 250-fold molar excess of an unlabeled Wt1(–KTS) binding site from the mouse vitamin D receptor promoter (18).

 
Published binding sites for Wt1 have been demonstrated to be GC rich or contain TCC (opposite strand GGA) motifs [for review, see (22)]. Sequence analysis of the nephrin promoter revealed several potential elements in a 1.2-kb fragment upstream of the predicted transcription start site. To identify potential Wt1 binding site(s) in this fragment, we performed DNase I footprint analysis of two segments covering 582 and 693 bp, respectively (designated as FP1 and FP2). A 22-bp element in the FP1 fragment was protected by the Wt1(–KTS) protein but not by the Wt1(+KTS) product (Figure 1C). No additional footprints were obtained. To confirm further specific binding of Wt1 to the identified sequence, we tested the protected element for its ability to bind Wt1(–KTS) and Wt1(+KTS) protein in electrophoretic mobility shift assays. A strong retardation band was detected when the oligonucleotide from the nephrin promoter was incubated with Wt1(–KTS) protein (Figure 1D). Binding could be competed with increasing amounts of unlabeled oligonucleotide, which was identical to a Wt1 consensus element from the vitamin D receptor promoter (18). Wt1(+KTS) protein also bound the identified element but apparently with a much lower affinity (Figure 1D).

To test whether the identified element is required for the activation of the nephrin promoter by Wt1, we decided to mutate this sequence in our reporter plasmid and test its ability to respond to Wt1 in co-transfection assays. Remarkably, a 22-bp deletion of the Wt1 binding element (pNeph{Delta}WTB) completely abolished activation of the 6.2-kb promoter construct by Wt1(–KTS) (Figure 2A). Because the identified 22-bp sequence contained four degenerate repeats of a 5'-GGAGG-3' binding site, we mutated each of the GG doublets (Figure 2A). Any mutation alone abolished activation of the nephrin promoter construct by Wt1(–KTS), suggesting a cooperative effect of the repeats for transactivation by Wt1. Accordingly, electrophoretic mobility shift assay analysis revealed that the binding affinity for Wt1(–KTS) protein was clearly reduced by each single mutation in the 22-bp fragment (Figure 2B).



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  		Figure 2. (A) Activity of the wild-type and mutated nephrin promoter in U2OS cells in response to transient co-transfection of the Wt1(–KTS) isoform. Shown are relative luciferase activities. Values are means ± SEM of 10 experiments. *Statistical significance (P < 0.05). (B) EMSA demonstrating binding of Wt1 to the 22-bp element (WTB) in the nephrin promoter. Binding of Wt1(–KTS) could be competed with a 10- to 250-fold molar excess of an unlabeled Wt1 consensus sequence from the mouse vitamin D receptor promoter. Introduction of mutations identical to those in the promoter constructs clearly reduced binding of Wt1(–KTS). (C) Schematic illustration of the nephrin promoter constructs used for transfection experiments. The localization and sequence of the identified Wt1-binding element (WTB) is indicated. In construct pNeph{Delta}WTB, the Wt1-binding element has been deleted selectively.

 
The molecular analysis described above clearly identified a Wt1 responsive element in the U2OS osteosarcoma cell line. However, there remained the small possibility that this binding site was present only in the heterologous system. Because of difficulties of transforming currently available podocyte cell lines, confirmation of our data using co-transfection assays in podocytes was impossible. Hence, to confirm the importance of the identified Wt1 binding site in vivo, we decided to use a transgenic approach. The 6.2-kb nephrin promoter has been shown previously to direct expression of a lacZ reporter gene (nephA) in glomeruli of transgenic mice (17). To test the importance of the above-identified Wt1 binding site, we introduced the same 22-bp deletion as in the pNeph{Delta}WTB plasmid into the nephA lacZ construct and injected both the wild-type and the mutant constructs into fertilized mouse oocytes. Foster mothers were killed at E17.5, and embryos were analyzed by PCR for the integration of the transgene. For identifying promoter activity, the kidneys of all embryos were isolated and stained for {beta}-galactosidase activity. As expected, five of six transient transgenic embryos showed blue staining within the podocytes (Figure 3A). In contrast, none of four transgenic animals that were generated with the 22-bp deletion construct showed lacZ-positive cells in the kidney, indicating that this promoter could not direct podocyte-specific expression of the reporter gene. Taken together, these data clearly demonstrate that the identified binding site is also relevant for tissue-specific expression in vivo.



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  		Figure 3. LacZ analysis of transgenic mice. (A) Design of transgenic constructs. Nephrin:LacZ contains a 6.2-kb nephrin promoter fragment cloned in front of a lacZ reporter construct (17). {Delta}WTB:LacZ represents the identical construct but carrying a 22-bp deletion of the identified Wt1 binding site. (B) Vibratome sections of kidneys isolated at embryonic day 17.5 (E17.5) and stained for {beta}-galactosidase activity. Nephrin:LacZ transgenic kidneys showed strong staining within the podocyte layer of glomeruli (a and arrows in c). In contrast, kidneys from {Delta}WTB:LacZ transgenic animals (b and d) lacked {beta}-galactosidase activity, indicating the absence of transgene expression. Scale bars = 200 µm.

 
The published expression pattern of Wt1 and nephrin strongly suggest that both are expressed in the same cell type (2,23–25). To explore whether Wt1 and nephrin share overlapping expression patterns, we performed double-immunofluorescent labeling for both proteins in the kidney. Indeed, Wt1 was detected in the nuclei of glomerular podocytes, whereas nephrin was localized in podocyte cell membranes and to some extent in the cytoplasm (Figure 4). To investigate further the role of alternatively spliced Wt1 products for nephrin expression in vivo, we made use of our recently established mouse lines lacking either the Wt1(–KTS) or the Wt1(+KTS) splice variant (9). Using quantitative reverse transcription–PCR, nephrin expression at E16.5 was lowest in kidneys of animals that lacked Wt1(–KTS) (P < 0.001; Figure 5). However, also Wt1(+KTS)-deficient mice had lower nephrin mRNA levels than wild-type littermates. Theoretically, the observed reduction of nephrin mRNA expression could represent a delay of kidney differentiation in the Wt1 mutants rather than a reduction of nephrin on a cellular level. To distinguish between this two possibilities, we analyzed nephrin protein expression and tissue distribution at E18.5 using immunohistochemistry on kidney sections. As already seen for mRNA expression, nephrin protein levels were lowest in the Wt1(–KTS)-deficient mice but in comparison with wild-type littermates were also reduced in animals with disruption of the Wt1(+KTS) splice variant. Synaptopodin as a marker for early glomerular development was used in double-immunofluorescence experiments together with nephrin to exclude a developmental delay in the splice-specific knockout animals. Although the number of glomeruli was reduced in Wt1(–KTS–/–) mice (9), those glomeruli that were present showed similar levels of synaptopodin expression when compared with Wt1(+KTS–/–) mice or wild-type littermates (Figure 6).



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  		Figure 4. Double-immunofluorescent labeling of nephrin (red) and Wt1 (green) in adult mouse kidney. Note the overlapping expression in glomerular podocytes. Counterstaining with DAPI (blue) was used to visualize nuclei (c). Scale bars = 100 µm.

 


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  		Figure 5. Quantitative RT-PCR for nephrin in kidneys of mice lacking specifically either the Wt1(–KTS) or the Wt1(+KTS) splice variant at E16.5. Wild-type littermates (wt) served as controls. Nephrin expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. Values are means ± SEM of five animals each. *Statistical significance (P < 0.001).

 


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  		Figure 6. Double-immunofluorescent labeling for nephrin (green) and synaptopodin (red) in kidney sections from mice with specific lack either of the Wt1(–KTS) (b, e, h, and k) or the Wt1(+KTS) (c, f, i, and l) splice variant and from wild-type littermates (a, d, g, and h) at E18.5. Counterstaining with DAPI (blue) was used to visualize nuclei (j, k, and l). Scale bars = 100 µm. Note that the differences in nephrin protein expression match those of nephrin mRNA in the splice-specific Wt1 knockouts, whereas synaptopodin protein expression is only slightly reduced in the splice-specific knockouts.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The nephrin expression pattern during kidney development is strikingly overlapping with that of Wt1 and can first be found in the presumptive podocyte layer of the S-shaped body, a time point when also Wt1 expression increases (2,25). This observation prompted us to analyze whether Wt1 acts as a direct transcription factor on the nephrin promoter. Here we have shown that Wt1 stimulated nephrin expression in cultured cells and that specific ablation of either the Wt1(–KTS) or the Wt1(+KTS) proteins resulted in reduced expression levels of nephrin in vivo. It is interesting that the promoter of the nephrin gene was stimulated not only by the Wt1(–KTS) variant but also by the Wt1(+KTS) protein, albeit to a lesser extent. This may seem surprising considering that the Wt1(+KTS) protein is believed to play a role mainly in posttranscriptional mechanisms, such as mRNA splicing (26,27). However, our previous observations both in vitro and in vivo support the idea of some functional redundancy between +KTS and –KTS isoforms (9,18). The analysis of splice-specific mutants performed in this study showed a reduction of nephrin in glomeruli of both strains. This seems to be at least in part a direct rather than an indirect effect, as the early glomerular marker synaptopodin remained unaffected, suggesting that initial differentiation of podocytes occurs in both models. Because overall levels of Wt1 expression are largely unchanged in Wt1 splice-specific mutants and because compound heterozygotes (Wt1–KTS–/Wt1+KTS–) survive normally (9), we may speculate that both Wt1 isoforms are required for the activation of nephrin in vivo. This may either involve two independent/successive differentiation steps or alternatively require the interaction of both proteins. In support of the latter hypothesis, Wt1 can heterodimerize at least in vitro (28), and mutant proteins have been shown to act in a dominant negative manner in a mouse model for Denys-Drash syndrome (29).

The Wt1 responsive element in the nephrin promoter identified here consists of several repeats of a predicted Wt1 core-binding site (16,30). Presumably, these repeats are functioning in a cooperative manner, as mutations of each one of the elements resulted in a strong reduction in the binding affinity for Wt1(–KTS) in vitro. Our transgenic analysis suggested an absolute requirement of the identified element for podocyte-specific expression not only in vitro but also in vivo. Two other groups have used fragments of the nephrin promoter to drive transgene expression in the kidney. The position of the Wt1 responsive element identified here is in agreement with the promoter fragment identified by Moeller et al. (31). In contrast, promoter studies by Beltcheva et al. (17) suggested a region farther upstream to be required for podocyte-specific expression, suggesting that an additional element(s) is required together with the Wt1 binding site to allow kidney-specific activation of the nephrin promoter. Hence, Wt1 may require a cooperative interaction with other as-yet-unknown factors to stimulate nephrin expression. Thus, our data support but not directly prove that Wt1 is regulating nephrin expression in vivo solely through the identified binding site.

From patient analyses as well as knockout studies, it is clear that nephrin serves a crucial role in glomerular filtration. Given the importance of the regulatory element for nephrin expression identified here, we can speculate that mutations or polymorphisms in this sequence may contribute to glomerular diseases in human patients. Furthermore, that expression of nephrin and podocalyxin (30), both key structural proteins of the podocyte, are regulated by Wt1 may suggest that Wt1 acts as a central regulatory molecule to protect the structural and functional integrity of glomerular podocytes. It is interesting that the respective Wt1 binding elements in the nephrin and podocalyxin promoter differ in sequence. This suggests that the podocyte-specific regulation of these genes is not the result of a duplication of regulatory elements but rather that regulatory sequences have evolved independently. In either case, it will be interesting to study other potential targets, such as podocin, Neph1, and CD2AP, which may also depend on the expression of Wt1. A detailed knowledge of the relevant downstream effectors of Wt1 would permit us to evaluate the regulatory pathways that have evolved to establish podocyte-specific morphology and function.


   Acknowledgments
 
This study was supported in part by a grant from the Deutsche Forschungsgemeinschaft (DFG; Scho 634/4-1) and the Asssociation pour le Recherche contre le Cancer (#5198). K.-D.W. and N.W. are recipients of a fellowship from EMBO (K.D.W.), from the DFG, and from the Charité (Rahel-Hirsch Program; N.W.).

The expert technical assistance of M. Magliano, A. Richter, and I. Grätsch is gratefully acknowledged. The nephrin promoter was a gift of K. Tryggvason. We thank D. Haber and C. Englert for the gift of the Wt1 expression constructs and the U2OS osteosarcoma cells with inducible Wt1 expression and Larry Holzman for the antinephrin antibody.


   Footnotes
 
N.W. and K.-D.W. contributed equally to this work.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication May 6, 2004. Accepted for publication September 6, 2004.




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