The aminoglycoside geneticin permits translational readthrough of the CTNS W138X nonsense mutation in fibroblasts from patients with nephropathic cystinosis.

BACKGROUND: Cystinosis is an ultrarare disorder caused by mutations of the cystinosin (CTNS) gene, encoding a cystine-selective efflux channel in the lysosomes of all cells of the body. Oral therapy with cysteamine reduces intralysosomal cystine accumulation and slows organ deterioration but cannot reverse renal Fanconi syndrome nor prevent the eventual need for renal transplantation. A definitive therapeutic remains elusive. About 15% of cystinosis patients worldwide carry one or more nonsense mutations that halt translation of the CTNS protein. Aminoglycosides such as geneticin (G418) can bind to the mammalian ribosome, relax translational fidelity, and permit readthrough of premature termination codons to produce full-length protein. METHODS: To ascertain whether aminoglycosides permit readthrough of the most common CTNS nonsense mutation, W138X, we studied the effect of G418 on patient fibroblasts. RESULTS: G418 treatment induced translational readthrough of CTNSW138X constructs transfected into HEK293 cells and expression of full-length endogenous CTNS protein in homozygous W138X fibroblasts. CONCLUSIONS: Reduction in intracellular cystine indicates that the CTNS protein produced is functional as a cystine transporter. Interestingly, similar effects were seen even in W138X compound heterozygotes. These studies establish proof-of-principle for the potential of aminoglycosides to treat cystinosis and possibly other monogenic diseases caused by nonsense mutations.

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.

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.

T-cell factor/ß-catenin activity is suppressed in two different models of autosomal dominant polycystic kidney disease.

During murine kidney development, canonical WNT signaling is highly active in tubules until about embryonic days E16-E18. At this time, ß-catenin transcriptional activity is progressively restricted to the nephrogenic zone. The cilial protein genes PKD1 and PKD2 are known to be mutated in autosomal dominant polycystic kidney disease (ADPKD), and previous studies proposed that these mutations could lead to a failure to suppress canonical WNT signaling activity. Several in vitro studies have found a link between cilial signaling and ß-catenin regulation, suggesting that aberrant activity might contribute to the cystic phenotype. To study this, we crossed T-cell factor (TCF)/ß-catenin-lacZ reporter mice with mice having Pkd1 or Pkd2 mutations and found that there was no ß-galactosidase staining in cells lining the renal cysts. Thus, suppression of canonical WNT activity, defined by the TCF/ß-catenin-lacZ reporter, is normal in these two different models of polycystic kidney disease. Hence, excessive ß-catenin transcriptional activity may not contribute to cystogenesis in these models of ADPKD.

WNT/ß-Catenin Signaling Is Required for Integration of CD24+ Renal Progenitor Cells into Glycerol-Damaged Adult Renal Tubules.

During development, nephron progenitor cells (NPC) are induced to differentiate by WNT9b signals from the ureteric bud. Although nephrogenesis ends in the perinatal period, acute kidney injury (AKI) elicits repopulation of damaged nephrons. Interestingly, embryonic NPC infused into adult mice with AKI are incorporated into regenerating tubules. Since WNT/ß-catenin signaling is crucial for primary nephrogenesis, we reasoned that it might also be needed for the endogenous repair mechanism and for integration of exogenous NPC. When we examined glycerol-induced AKI in adult mice bearing a ß-catenin/TCF reporter transgene, endogenous tubular cells reexpressed the NPC marker, CD24, and showed widespread ß-catenin/TCF signaling. We isolated CD24+ cells from E15 kidneys of mice with the canonical WNT signaling reporter. 40% of cells responded to WNT3a in vitro and when infused into glycerol-injured adult, the cells exhibited ß-catenin/TCF reporter activity when integrated into damaged tubules. When embryonic CD24+ cells were treated with a ß-catenin/TCF pathway inhibitor (IWR-1) prior to infusion into glycerol-injured mice, tubular integration of cells was sharply reduced. Thus, the endogenous canonical ß-catenin/TCF pathway is reactivated during recovery from AKI and is required for integration of exogenous embryonic renal progenitor cells into damaged tubules. These events appear to recapitulate the WNT-dependent inductive process which drives primary nephrogenesis.

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 CTNS(Red) 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.

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.

Canonical WNT signaling during kidney development.

The canonical WNT signaling pathway plays a crucial role in patterning of the embryo during development, but little is known about the specific developmental events which are under WNT control. To understand more about how the WNT pathway orchestrates mammalian organogenesis, we studied the canonical beta-catenin-mediated WNT signaling pathway in kidneys of mice bearing a beta-catenin-responsive TCF/betaGal reporter transgene. In metanephric kidney, intense canonical WNT signaling was evident in epithelia of the branching ureteric bud and in nephrogenic mesenchyme during its transition into renal tubules. WNT signaling activity is rapidly downregulated in maturing nephrons and becomes undetectable in postnatal kidney. Sites of TCF/betaGal activity are in proximity to the known sites of renal WNT2b and WNT4 expression, and these WNTs stimulate TCF reporter activity in kidney cell lines derived from ureteric bud and metanephric mesenchyme lineages. When fetal kidney explants from HoxB7/GFP mice were exposed to the canonical WNT signaling pathway inhibitor, Dickkopf-1, arborization of the ureteric bud was significantly reduced. We conclude that restricted zones of intense canonical WNT signaling drive branching nephrogenesis in fetal kidney.

Pax2 gene dosage influences cystogenesis in autosomal dominant polycystic kidney disease.

Mutations in PKD1 cause dominant polycystic kidney disease (PKD), characterized by large fluid-filled kidney cysts in adult life, but the molecular mechanism of cystogenesis remains obscure. Ostrom et al. [Dev. Biol., 219, 250-258 (2000)] showed that reduced dosage of Pax2 caused increased apoptosis, and ameliorated cystogenesis in Cpk mutant mice with recessive PKD. Pax2 is expressed in condensing metanephrogenic mesenchyme and arborizing ureteric bud, and plays an important role in kidney development. Transient Pax2 expression during fetal kidney mesenchyme-to-epithelial transition, as well as in nascent tubules, is followed by marked down-regulation of Pax2 expression. Here, we show that in humans with PKD, as well as in Pkd1(del34/del34) mutant mice, Pax2 was expressed in cyst epithelial cells, and facilitated cyst growth in Pkd1(del34/del34) mutant mice. In Pkd1(del34/del34) mutant kidneys, the expression of Pax2 persisted in nascent collecting ducts. In contrast, homozygous Pkd1(del34/del34) fetal mice carrying mutant Pax2 exhibited ameliorated cyst growth, although reduced cystogenesis was not associated with increased apoptosis. Pax2 expression was attenuated in nascent collecting ducts and absent from remnant cysts of Pkd1(del34/del34)/Pax2(1Neu/+) mutant mice. To investigate whether the Pkd1 gene product, Polycystin-1, regulates Pax2, MDCK cells were engineered constitutively expressing wild-type Pkd1; Pax2 protein levels and promoter activity were both repressed in MDCK cells over-expressing Pkd1, but not in cells without transgenic Pkd1. These data suggest that polycystin-1-deficient tubular epithelia persistently express Pax2 in ADPKD, and that Pax2 or its pathway may be an appropriate target for the development of novel therapies for ADPKD.

Poliquistosis renal autosomica dominante: deteccion de una nueva mutacion en el gen PKD1 / Autosomal dominant polycystic kidney disease: detection of a novel mutation in the PKD1 gene

La poliquistosis renal autosómica (ADPKD) es una enfermedad hereditaria que presenta heterogeneidad genética. Al menos tres genes son responsables del desarrollo de la enfermedad: PKD1 en el cromosoma 16p 13.3, PKD2 en 4q21 y PKD3, aún no localizado. A partir de la descripción de la secuencia del gen PKD1, el interés general se volcó a la búsqueda de mutaciones causantes de la enfermedad. La mayoría de las mutaciones halladas es de diverso origen y localiza a lo largo del gen, no pudiendo hallarse correlación fenotípica alguna. En este trabajo se describe el hallazgo de una mutación en el exón 44 del gen PKD1 en una familia previamente caracterizada por análisis de ligamiento. La mutación consiste en una sustitución de una base C por una T en la posición 12220 originado un codón stop donde se produce la mutación. Esto llevaría a una terminación prematura en la traducción produciendo una proteína en la cual estaría ausente parte del extremo carboxílico.

Poliquistosis renal autosomica dominante: deteccion de una nueva mutacion en el gen PKD1 / Autosomal dominant polycystic kidney disease: detection of a novel mutation in the PKD1 gene

La poliquistosis renal autosómica (ADPKD) es una enfermedad hereditaria que presenta heterogeneidad genética. Al menos tres genes son responsables del desarrollo de la enfermedad: PKD1 en el cromosoma 16p 13.3, PKD2 en 4q21 y PKD3, aún no localizado. A partir de la descripción de la secuencia del gen PKD1, el interés general se volcó a la búsqueda de mutaciones causantes de la enfermedad. La mayoría de las mutaciones halladas es de diverso origen y localiza a lo largo del gen, no pudiendo hallarse correlación fenotípica alguna. En este trabajo se describe el hallazgo de una mutación en el exón 44 del gen PKD1 en una familia previamente caracterizada por análisis de ligamiento. La mutación consiste en una sustitución de una base C por una T en la posición 12220 originado un codón stop donde se produce la mutación. Esto llevaría a una terminación prematura en la traducción produciendo una proteína en la cual estaría ausente parte del extremo carboxílico. (AU)