Diana M. Iglesias

PAX2 activates WNT4 expression during mammalian kidney development

The transcription factor PAX2 is expressed during normal kidney development and is thought to influence outgrowth and branching of the ureteric bud. Mice with homozygous null Pax2 mutations have developmental defects of the midbrain-hindbrain region, optic nerve, and ear and are anephric. During nephrogenesis, PAX2 is also expressed by mesenchymal cells as they cluster and reorganize to form proximal elements of each nephron, but the function of PAX2 in these cells is unknown. In this study we hypothesized that PAX2 activates expression of WNT4, a secreted glycoprotein known to be critical for successful nephrogenesis. PAX2 protein was identified in distal portions of the “S-shaped” body, and the protein persists in the emerging proximal tubules of murine fetal kidney. PAX2 activated WNT4 promoter activity 5-fold in co-transfection assays with JTC12 cells derived from the proximal tubule. Inspection of the 5′-flanking sequence of the human WNT4 gene identified three novel PAX2 recognition motifs; each exhibited specific PAX2 protein binding in electromobility shift assays. Two motifs were contained within a completely duplicated 0.66-kb cassette. Transfection of JTC12 cells with a PAX2 expression vector was associated with a 7-fold increase in endogenous WNT4 mRNA. In contrast, Wnt4 mRNA was decreased by 60% in mesenchymal cell condensates of fetal kidney from mice with a heterozygous Pax2 mutation. We speculated that a key function of PAX2 is to activate WNT4 gene expression in metanephric mesenchymal cells as they differentiate to form elements of the renal tubules.

Stem cell microvesicles transfer cystinosin to human cystinotic cells and reduce cystine accumulation in vitro.

Cystinosis is a rare disease caused by homozygous mutations of the CTNS gene, encoding a cystine efflux channel in the lysosomal membrane. In Ctns knockout mice, the pathologic intralysosomal accumulation of cystine that drives progressive organ damage can be reversed by infusion of wildtype bone marrow-derived stem cells, but the mechanism involved is unclear since the exogeneous stem cells are rarely integrated into renal tubules. Here we show that human mesenchymal stem cells, from amniotic fluid or bone marrow, reduce pathologic cystine accumulation in co-cultured CTNS mutant fibroblasts or proximal tubular cells from cystinosis patients. This paracrine effect is associated with release into the culture medium of stem cell microvesicles (100–400 nm diameter) containing wildtype cystinosin protein and CTNS mRNA. Isolated stem cell microvesicles reduce target cell cystine accumulation in a dose-dependent, Annexin V-sensitive manner. Microvesicles from stem cells expressing CTNSRed transfer tagged CTNS protein to the lysosome/endosome compartment of cystinotic fibroblasts. Our observations suggest that exogenous stem cells may reprogram the biology of mutant tissues by direct microvesicle transfer of membrane-associated wildtype molecules.

A variant OSR1 allele which disturbs OSR1 mRNA expression in renal progenitor cells is associated with reduction of newborn kidney size and function

Human nephrons are formed during fetal life through an interaction between the branching ureteric bud and progenitor cells. The wide variation in final nephron number has been attributed to allelic variants of genes regulating ureteric bud arborization. Here, we hypothesize that dysfunctional variants of the Odd-Skipped Related 1 (OSR1) gene which compromise the renal progenitor cell pool might also limit newborn kidney size and function. We show that OSR1 is expressed in human mesenchymal stem cells, the blastemal component of Wilms tumors and CD24+/CD133+ progenitor cells isolated from the mature kidney. We identified an OSR1(rs12329305(T)) allele in 6% of normal Caucasians which alters an exon2 splice enhancer. This variant is predicted to reduce spliceosome-binding affinity and stability of the OSR1 mRNA. In cultured cells, the OSR1(rs12329305)(T) allele produced no identifiable transcript. Normal Caucasian newborns from Montreal with the OSR1(rs12329305)(T) allele had kidney volume 11.8% smaller (P= 0.006) and cord blood cystatin C levels 12.6% higher (P = 0.005) than those with wild-type genotype. Effects of the OSR1(rs12329305)(T) allele are additive with genes that alter ureteric bud branching. Kidney volume was reduced more in newborns bearing both RET(rs1800860)(A) and OSR1(rs12329305)(T) alleles (22%, P= 0.0008) and cystatin C was increased by 17% (P= 0.006) versus newborns with wild-type alleles. Although only two subjects had PAX2(rs11599825)(A) and OSR1(rs12329305)(T) alleles, kidney size was reduced by 27% and cystatin C was increased by 14% versus wild-types (P= NS).

In vivo validation of PAX2 as a target for renal cancer therapy

PAX genes are frequently overexpressed in human cancer tissue and appear to contribute to the tumor phenotype, suggesting that they may be potential targets for cancer therapy. In particular, aberrant PAX2 expression has been reported in a high proportion of primary tumors, including the majority of renal cell carcinomas (RCC). We recently demonstrated that PAX2 suppresses cisplatin-induced apoptosis in cultured RCC cells. We hypothesized that silencing of PAX2 expression might partially overcome the notorious resistance of renal cell carcinomas to chemotherapy in vivo. In this report, we show that a PAX2 shRNA successfully knocks down PAX2 mRNA and protein levels in an RCC cell line (ACHN). ACHN cells stably transfected with shRNAs targeted against the PAX2 homeodomain are 3-6-fold more susceptible to cisplatin-induced caspase-3 activation than control ACHN cells line. Furthermore, growth of subcutaneous ACHN/shPAX2 xenografts in nude mice is significantly more responsive to cisplatin therapy than control ACHN cell tumors. Our observations validate PAX2 as a potential therapeutic gene target in renal cancer and suggest that adjunctive PAX2 knockdown may enhance the efficacy of other chemotherapeutic agents.

Wilms Tumor Suppressor, WT1, Suppresses Epigenetic Silencing of the β-Catenin Gene

Background: Hereditary Wilms tumors are preceded by WT1(−) clones with an inhibitory chromatin histone pattern established by EZH2. Results: In amniotic mesenchymal stem cells, WT1 suppresses EZH2, derepresses β-catenin (CTNNB1), and enhances responsiveness to WNT9b. Conclusion: WT1 regulates transition from the epigenetically silenced chromatin state. Significance: Developmental blockade in nephrogenic rests may be mediated by loss of the WT1-EZH2-CTNNB1 axis. The mammalian kidney is derived from progenitor cells in intermediate mesoderm. During embryogenesis, progenitor cells expressing the Wilms tumor suppressor gene, WT1, are induced to differentiate in response to WNT signals from the ureteric bud. In hereditary Wilms tumors, clonal loss of WT1 precludes the β-catenin pathway response and leads to precancerous nephrogenic rests. We hypothesized that WT1 normally primes progenitor cells for differentiation by suppressing the enhancer of zeste2 gene (EZH2), involved in epigenetic silencing of differentiation genes. In human amniotic fluid-derived mesenchymal stem cells, we show that exogenous WT1B represses EZH2 transcription. This leads to a dramatic decrease in the repressive lysine 27 trimethylation mark on histone H3 that silences β-catenin gene expression. As a result, amniotic fluid mesenchymal stem cells acquire responsiveness to WNT9b and increase expression of genes that mark the onset of nephron differentiation. Our observations suggest that biallelic loss of WT1 sustains the inhibitory histone methylation state that characterizes Wilms tumors.

PAX3 is Expressed in the Stromal Compartment of the Developing Kidney and in Wilms Tumors with Myogenic Phenotype

Wilms tumor (WT) is the most frequent renal neoplasm of childhood; a myogenic component is observed in 5% to 10% of tumors. We demonstrate for the first time that myogenic WTs are associated with expression of PAX3, a transcription factor known to specify myoblast cell fate during muscle development. In a panel of 20 WTs, PAX3 was identified in 13 of 13 tumor samples with myogenic histopathology but was absent in 7 of 7 tumors lacking a myogenic component. Furthermore, we show that PAX3 is expressed in the metanephric mesenchyme and stromal compartment of developing mouse kidney. Modulation of endogenous PAX3 expression in human embryonic kidney (HEK293) cells influenced cell migration in in vitro assays. Mutations of WT1 were consistently associated with PAX3 expression in WTs, and modulation of WT1 expression in HEK293 cells was inversely correlated with the level of endogenous PAX3 protein. We demonstrate abundant PAX3 and absence of PAX2 expression in a novel cell line (WitP3) isolated from the stromal portion of a WT bearing a homozygous deletion of the WT1 gene. We hypothesize that PAX3 sets stromal cell fate in developing kidney but is normally suppressed by WT1 during the mesenchyme-to-epithelium transition leading to nephrogenesis. Loss of WT1 permits aberrant PAX3 expression in a subset of WTs with myogenic phenotype.

Priming the renal progenitor cell

The mammalian kidney arises from OSR1(+) progenitor cells in the intermediate mesoderm. However, these cells must acquire unique properties before they can respond to inductive signals that launch the differentiation program. Recent data indicate that the transcription factor, WT1, plays a master role in this transition. Interestingly, some of these embryonic nephron progenitor cells are retained in the adult organ where they may participate in tissue regeneration after acute kidney injury. A better understanding of the biology of these cells may one day allow progenitor cell-based therapeutic strategies to help regenerate damaged adult nephrons.

Wilms tumor arising in a child with X-linked nephrogenic diabetes insipidus

We report on a child with X-linked nephrogenic diabetes insipidus (NDI) who developed Wilms tumor (WT). Nephrogenic diabetes insipidus is caused by mutations of the arginine vasopressin receptor (AVPR2) or aquaporin-II (AQP2) genes. Wilms tumor is also genetically heterogeneous and is associated with mutations of WT1 (15–20%), WTX (20–30%) and other loci. The boy presented at 5 months with failure to thrive, polyuria, hypernatremia and abdominal mass. Analysis of leukocyte DNA showed a novel missense mutation (Q174H) of the AVPR2 gene, which was not present in his mother. In cells (WitS) isolated from the tumor, WTX mRNA expression and coding sequence were intact. However, we identified a 44-kb homozygous deletion of the WT1 gene spanning exons 4 to 10. The WT1 deletion was not present in leukocyte DNA from the patient or his mother. We also noted strong β-catenin (CTNNB1) expression in the tumor cells and identified a heterozygote missense Ser45Cys mutation of exon 3 of CTNNB1. However, the mutation was absent both in the constitutional DNA of the patient and his mother. The concurrence of WT and NDI has not been previously reported and may be unrelated. Nevertheless, this case nicely illustrates the sequence of events leading to sporadic Wilms tumor.