ABSTRACT: Precise regulation of transduction signaling pathways genes is crucial for correct differentiation of kidney cells and perturbation may lead to tumor onset. Wilms tumors (WT), or nephroblastoma, originates from the metanephric blastema cells, which were unable to complete the differentiation, resulting in a triphasic tumor composed by distinct cell types, blastemal, epithelial and stromal, where the former harbor molecular characteristics similar to cells of the earliest stages of kidney development. In this study, to identify key early events involved in the disruption of correct cell signaling during kidney differentiation that can drive WT, we developed a cDNA platform containing genes from signaling transduction pathways. By using bioinformatics tools, we defined high conserved regions between human and mouse orthologous genes and assessed the gene modulation in blastemal cells captured from WT and differentiated kidneys (DK) in human and from four temporal stages of kidney differentiation in mouse. We found 18 genes, whose transcriptional level of the earliest phases of blastemal cell differentiation was recapitulated in WT. PAX2, FZD10, SHPK, WNT5B, FZD2, CRABP2, FRZB, GRK7, TESK1, HDGF and IGF2 were up- and MAPK9, PIK3CA, FRAT2, ITPR3, CDH6, HIPK1 and TIMP3 were down-regulated in WT respectively. Hierarchical clustering based on the expression of this gene set grouped DK from both species, human and mouse, and discriminated them from fetal kidney and WT in human, and the earliest kidney stages in mouse, validating the model proposed by this study and reveling a transduction signaling signature of WT. High robustness of this data was detected since expression level was tested by quantitative RT-PCR in the initial and independent sample set, with 75 and 56% of agreement. Agreement of 62% was also observed in protein level assessing blastemal component of an independent group of 137 WT. Protein expression of CRABP2, IGF2, GRK7, TESK1, HDGF, WNT5B, FZD2 and TIMP3 was also characterized in human fetal kidneys revealing interesting aspects of kidney differentiation. This study identified key genes modulated during kidney differentiation which may play a determinant role for WT onset. As far as we know, FZD10, FRZB, HDGF, MAPK9, FRAT2, SHPK, WNT5B, GRK7, TESK1, HDGF, PIK3CA, ITPR3, HIPK1 and TIMP3 were not previously associated to WT. The strong connection between nephrogenesis and WT highlights the importance of a detailed characterization of modulated genes belonged to signal transduction. Identification of gene expression involved in kidney differentiation arresting might be imperative to point out early alterations that drive WT onset and consequently are potential candidate as molecular marker. Here, we characterized expression pattern of genes belonged to transduction signaling pathways in blastemal cells during kidney development stages in mouse and WT in human. For that, we established a model of inter- and intra-specific hybridization to assess gene expression modulation to identify key events that drive WT onset. Dye-swap was performed for each sample as control for dye bias and used as replicate. In total, 24 frozen favorable histology WT and differentiated kidney samples were laser microdisscted for blastemal cells. Three kidneys from CD1 lineage from Mus Musculus were isolated from embryos (E) and postnatal animals (P) at ages E15.5, E17.5, P1.5 and P7.5 for laser microdissection of the blastemal cells in all stages of kidney differentiation. NA were purified with PicoPure™ RNA Isolation kit (Arcturus Engineering #KT0204) and treated with RNase-Free DNase Set (Qiagen #79254; Qiagen-Germantown MD USA).
