Autosomal dominant polycystic kidney disease (ADPKD) and other forms of PKD are associated with dysregulated cell cycle and proliferation. Although no effective therapy for the treatment of PKD is currently available, possible mechanism-based approaches are beginning to emerge. A therapeutic intervention targeting aberrant cilia-cell cycle connection using CDK-inhibitor R-roscovitine showed effective arrest of PKD in jck and cpk models that are not orthologous to human ADPKD. To evaluate whether CDK inhibition approach will translate into efficacy in an orthologous model of ADPKD, we tested R-roscovitine and its derivative S-CR8 in a model with a conditionally inactivated Pkd1 gene (Pkd1 cKO). Similar to ADPKD, Pkd1 cKO mice developed renal and hepatic cysts. Treatment of Pkd1 cKO mice with R-roscovitine and its more potent and selective analog S-CR8 significantly reduced renal and hepatic cystogenesis and attenuated kidney function decline. Mechanism of action studies demonstrated effective blockade of cell cycle and proliferation and reduction of apoptosis. Together, these data validate CDK inhibition as a novel and effective approach for the treatment of ADPKD.
Development of novel therapies for polycystic kidney disease (PKD) requires assays that adequately reflect disease biology and are adaptable to high-throughput screening. Here we describe an embryonic cystic kidney organ culture model and demonstrate that a new mutant allele of the Pkd1 gene (Pkd1(tm1Bdgz)) modulates cystogenesis in this model. Cyst formation induced by cAMP is influenced by the dosage of the mutant allele: Pkd1(tm1Bdgz) -/- cultures develop a larger cystic area compared with +/+ counterparts, while Pkd1(tm1Bdgz) +/- cultures show an intermediate phenotype. A similar relationship between the degree of cystogenesis and mutant gene dosage is seen in cystic kidney organ cultures derived from mice with a mutated Nek8 gene (Nek8(jck)). Both Pkd1- and Nek8- cultures display altered cell-cell junctions, with reduced E-cadherin expression and altered desmosomal protein expression, similar to ADPKD epithelia. Additionally, characteristic ciliary abnormalities are identified in cystic kidney cultures, with elevated ciliary polycystin 1 expression in Nek8 homozygous cultures and elevated ciliary Nek8 protein expression in Pkd1 homozygotes. These data suggest that the Nek8 and Pkd1 genes function in a common pathway to regulate cystogenesis. Moreover, compound Pkd1 and Nek8 heterozygous adult mice develop a more aggressive cystic disease than animals with a mutation in either gene alone. Finally, we validate the kidney organ culture cystogenesis assay as a therapeutic testing platform using the CDK inhibitor roscovitine. Therefore, embryonic kidney organ culture represents a relevant model for studying molecular cystogenesis and a rapid tool for the screening for therapies that block cystic growth.
The WT1 tumor suppressor gene is a zinc finger-containing transcription factor which is required for development of the kidney and gonads. A mammal-specific alternative splicing event within this gene results in the presence or absence of a 17-amino-acid sequence within the WT1 protein. To determine the function of this sequence in vivo, gene targeting was utilized to specifically eliminate the exon encoding this sequence in mice. Mice lacking WT1 exon 5 develop normally. Adult mice lacking this exon are viable and fertile, and females are capable of lactation.The WT1 gene was originally identified as a gene that is deleted or rearranged in many cases of Wilms' tumor (reviewed in reference 23). It encodes a zinc finger-containing nuclear protein with DNA-and RNA-binding activity. Two alternative splicing events within this gene result in the production of four distinct transcripts, and alternative translation start sites and RNA editing result in the production of at least 24 distinct proteins (8). One alternative splicing event results in the presence or absence of a 3-amino-acid sequence, KTS, in the third zinc finger domain of the protein. Proteins lacking this sequence (hereafter referred to as ϪKTS) demonstrate a diffuse nuclear localization and DNA-binding and transcriptional regulatory activity (23). Isoforms that contain this insertion (hereafter referred to as ϩKTS) show a punctate nuclear localization (colocalizing with spliceosomes) and impaired DNA-binding and transcriptional regulatory activities (23). The other alternative splicing event results in the presence or absence of a 17-amino-acid sequence between the transcriptional activation and DNA-binding domains. This sequence has been suggested to contain a transcriptional repression domain and is involved in an interaction with the transcriptional regulatory protein Par4 (11,22). The in vivo function of proteins containing this 17-amino-acid insertion is unknown.
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.
The WT1 tumor-suppressor gene is expressed by many forms of acute myeloid leukemia. Inhibition of this expression can lead to the differentiation and reduced growth of leukemia cells and cell lines, suggesting that WT1 participates in regulating the proliferation of leukemic cells. However, the role of WT1 in normal hematopoiesis is not well understood. To investigate this question, we have used murine cells in which the WT1 gene has been inactivated by homologous recombination. We have found that cells lacking WT1 show deficits in hematopoietic stem cell function. Embryonic stem cells lacking WT1, although contributing efficiently to other organ systems, make only a minimal contribution to the hematopoietic system in chimeras, indicating that hematopoietic stem cells lacking WT1 compete poorly with healthy stem cells. In addition, fetal liver cells lacking WT1 have an approximately 75% reduction in erythroid blastforming unit (BFU-E), erythroid colonyforming unit (CFU-E), and colony-forming unit-granulocyte macrophage-erythroidmegakaryocyte (CFU-GEMM). However, transplantation of fetal liver hematopoietic cells lacking WT1 will repopulate the hematopoietic system of an irradiated adult recipient in the absence of competition. We conclude that the absence of WT1 in hematopoietic cells leads to functional defects in growth potential that may be of consequence to leukemic cells that have alterations in the expression of WT1. IntroductionWT1 was isolated as a tumor-suppressor gene on the basis of its deletion in a subset of patients with Wilms tumor, a pediatric cancer of the kidney. 1 In addition to its mutation in 5% to 10% of Wilms tumor patients, mutations of WT1 have been implicated in a variety of other cancers including mesotheliomas, desmoplastic round cell tumors, and leukemias. 1 WT1 encodes a protein of 52 to 54 kD, containing 4 zinc fingers of the Cys2 His2 class, that has been demonstrated to act as transcription factor. 1 Potential transcriptional targets for WT1 include a variety of growth factor and growth factor receptor genes. 1 An additional role for WT1 in RNA processing has also been proposed. 1 Many leukemias, including up to 80% of acute myeloid leukemias (AMLs), have been demonstrated to express WT1. [2][3][4][5][6][7][8][9][10] Mutations of WT1 have been found in approximately 5% to 10% of AMLs, similar to the level seen in Wilms tumors, and sporadically in a variety of other leukemias. [11][12][13][14][15][16][17][18] Manipulation of WT1 expression in leukemia cells results in alterations in differentiation and growth potential. [19][20][21][22][23][24][25][26][27][28] In the treatment of leukemia, expression of WT1 in the peripheral blood and bone marrow has been suggested to be an indicator of clinical relapse. Attempts have been made to correlate WT1 expression with progression of disease and prognosis in several types of hematologic malignancies. 3,[29][30][31][32][33][34][35][36][37] The clinical use of anti-WT1 cytotoxic T-lymphocytes (CTLs) for preferential destruction of WT1-expre...
The early development of the metanephric kidney is characterized by the induced differentiation of mesenchymal cells into a stem cell population that undergoes a mesenchymal to epithelial transformation in response to stimuli from the ureteric bud. The Wilms' tumor suppressor gene, Wt1, is required for mesenchymal cells to complete this developmental program. In the absence of WT1, a prospective metanephric mesenchyme appears, but becomes apoptotic, and outgrowth of the ureteric bud from the Wolffian duct does not occur. Therefore, the examination of Wt1 -/- embryos allows the determination of those markers of early metanephric differentiation that do not require the ureteric bud or WT1 for their expression. Here, we demonstrate that several markers, including Pax-2, Six-2, and GDNF, were present as RNAs in the metanephric mesenchyme of Wt1 -/- embryos. These findings demonstrate that the metanephric mesenchyme in mutant embryos has begun to differentiate towards the nephrogenic lineage, and that this early differentiation does not require either WT1 or the presence of the ureteric bud. To determine whether WT1 functions other than to induce expression of factors that stimulate ureteric bud outgrowth, Wt1 -/- metanephric mesenchymes were recombined with wild-type ureteric buds in organ culture, but this failed to rescue tubulogenesis. However, the Wolffian duct from Wt1 -/- embryos was a competent inducer of wild-type metanephric mesenchyme.
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