WT1 regulates expression of DNA repair gene Neil3 during nephrogenesis.

Mammalian nephrons arise from a population of nephron progenitor cells (NPCs) expressing the master transcription factor Wilms tumor-1 (WT1), which is crucial for NPC proliferation, migration, and differentiation. In humans, biallelic loss of WT1 precludes nephrogenesis and leads to the formation of Wilms tumor precursor lesions. We hypothesize that WT1 normally primes the NPC for nephrogenesis by inducing expression of NPC-specific DNA repair genes that protect the genome. We analyzed transcript levels for a panel of DNA repair genes in embryonic day 17.5 (E17.5) versus adult mouse kidneys and noted seven genes that were increased >20-fold. We then isolated Cited1+ NPCs from E17.5 kidneys and found that only one gene, nei-like DNA glycosylase 3 (Neil3), was enriched. RNAscope in situ hybridization of E17.5 mouse kidneys showed increased Neil3 expression in the nephrogenic zone versus mature nephron structures. To determine whether Neil3 expression is WT1 dependent, we knocked down Wt1 in Cited1+ NPCs (60% knockdown efficiency) and noted a 58% reduction in Neil3 transcript levels. We showed that WT1 interacts with the Neil3 promoter and that activity of a Neil3 promoter-reporter vector was increased twofold in WT1+ versus WT1- cells. We propose that Neil3 is a WT1-dependent DNA repair gene expressed at high levels in Cited1+ NPCs, where it repairs mutational injury to the genome during nephrogenesis. NEIL3 is likely just one of many such lineage-specific repair mechanisms that respond to genomic injury during kidney development.NEW & NOTEWORTHY We studied the molecular events leading to Wilms tumors as a model for the repair of genomic injury. Specifically, we showed that WT1 activates DNA repair gene Neil3 in nephron progenitor cells. However, our observations offer a much broader principle, demonstrating that the embryonic kidney invests in lineage-specific expression of DNA repair enzymes. Thus, it is conceivable that failure of these mechanisms could lead to a variety of "sporadic" congenital renal malformations and human disease.

The novel aminoglycoside, ELX-02, permits CTNSW138X translational read-through and restores lysosomal cystine efflux in cystinosis.

BACKGROUND: Cystinosis is a rare disorder caused by recessive mutations of the CTNS gene. Current therapy decreases cystine accumulation, thus slowing organ deterioration without reversing renal Fanconi syndrome or preventing eventual need for a kidney transplant.15-20% of cystinosis patients harbour at least one nonsense mutation in CTNS, leading to premature end of translation of the transcript. Aminoglycosides have been shown to permit translational read-through but have high toxicity level, especially in the kidney and inner ear. ELX-02, a modified aminoglycoside, retains it read-through ability without the toxicity. METHODS AND FINDINGS: We ascertained the toxicity of ELX-02 in cells and in mice as well as the effect of ELX-02 on translational read-through of nonsense mutations in cystinotic mice and human cells. ELX-02 was not toxic in vitro or in vivo, and permitted read-through of nonsense mutations in cystinotic mice and human cells. CONCLUSIONS: ELX-02 has translational read-through activity and produces a functional CTNS protein, as evidenced by reduced cystine accumulation. This reduction is comparable to cysteamine treatment. ELX-02 accumulates in the kidney but neither cytotoxicity nor nephrotoxicity was observed.

Wilms tumor suppressor, WT1, suppresses epigenetic silencing of the ß-catenin gene.

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 ß-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 ß-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.

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.

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.

WT1 is a modifier of the Pax2 mutant phenotype: cooperation and interaction between WT1 and Pax2.

Metanephric kidney development requires an inductive interaction between the ureteric bud and progenitor mesenchyme, where the early expression of two genes, Wilms' tumour 1 (WT1) and paired box 2 (Pax2), establishes critical but unknown developmental pathways. Indeed, transgenic mice with deregulated overexpression of Pax2 exhibit structural kidney defects and impaired renal function, as do mice harboring targeted disruptions and/or spontaneous mutations of either the Pax2 or WT1 genes. WT1 and Pax2 are thought to regulate each other's expression during renal development. To better define the relationship between WT1 and Pax2, we generated mouse embryos containing heterozygous mutations in both genes. WT1(+/-)/Pax2(1Neu/+) kidneys were 50% smaller than wild-type kidneys. They were characterized by severe attenuation of the renal medulla, and reduced development of calyces and the renal pelvis. Renal cortex development in compound heterozygotes culminated in fewer nephrons than in WT1(+/-), Pax2(1Neu/+) or wild-type mice. Only minor variations in the mesenchymal expression pattern of Pax2 protein, and the mRNA expression levels of Pax2 and WT1, were noted in mutant kidneys. We show that WT1 and Pax2 proteins interact in vitro and in vivo, demonstrating that WT1 and Pax2 can form a molecular complex. Our data suggest that WT1 is a modifier of the Pax2 mutant phenotype.