| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2002: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2001: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Research Abstract |
Purpose :
Kidney stone formation is a complex process, and numerous genes participate in this cascade. The binding and internalization of calcium oxalate monohydrate (CON) crystals, the most common crystal in renal stones by renal epithelial cells may be a critical step leading to kidney stone formation. Exposure to CON crystals alters the expression of various genes, but previous studies on gene expression have generally been limited. To obtain more detailed insight into gene expression, we examined gene expression profiles in renal epithelial cells exposed to CON crystals using cDNA macroarray. Materials and Methods : NRK-52E cells were exposed to CON crystals for 60 and 120 minutes. Poly (A)^+ RNA was isolated and converted into ^<32>P-labeled first-strand cDNA, then the cDNA probe was hybridized to the membrane. Hybridization images were scanned and the signal intensities were quantified. Expression of mRNA of 1176 genes were analyzed with global sum normalization methods.
Results :
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xposure to CON crystals altered the expression of some of the genes reported previously. Furthermore, novel genes were also identified. The genes for 35 proteins were altered in the expression level by more than 2-fold : annexin V, p53-binding mouse double minute 2 homolog, osteopontin, fibronectin, steroidogenic acute regulatory protein, clusterin, coagulation factor II (thrombin) receptor, mitogen-activated protein kinase 3, cathepsin B, cathepsin L, plasminogen activator inhibitor 1, cytokeratin 8, vimentin, hydrophobic surfactant associated protein C, inhibitor of DNA binding 1, 7-dehydrocholesterol reductase, cytochrome P-45O 2C23, myeloid cell differentiation protein 1, high mobility group protein 2, 5-hydroxytryptamine receptor 5B receptor, cytochrome c expressed in somatic tissues, connective tissue growth factor, tissue inhibitor of metalloproteinase 1, V(D)J recombination activating protein 1, Tclone15, thymosin beta 10, junD proto-oncogene, inhibitor of DNA binding 2, inhibitor of DNA-binding protein 3, G1/S-specific cyclin D1, collagen, insulin-like growth factor binding protein 2, insulin-like growth factor-binding protein 6, profilin 1 and metallothionein 1. Twenty genes had altered expression after a 60-minute exposure to COM crystals. Thirteen genes were up-regulated and seven genes were down-regulated. Nineteen genes had altered expression after a 120-minute exposure to COM crystals. Nine genes were up-regulated and ten genes were down-regulated.
Conclusions :
We performed a large-scale analysis of gene expression in renal epithelial cells exposed to COM crystals, and identified the genes differentially regulated. cDNA macroarray is a useful tool for evaluating gene expression in urolithiasis research. Less
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