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| Status |
Public on Dec 09, 2011 |
| Title |
Global gene expression profiling in early-stage polycystic kidney disease in the Han:SPRD Cy rat identifies a role for RXR signaling |
| Organism |
Rattus norvegicus |
| Experiment type |
Expression profiling by array
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| Summary |
Han:SPRD Cy is a spontaneous rat model of polycystic kidney disease (PKD) caused by a missense mutation in Pkdr1. Cystogenesis in this model is not clearly understood. In the current study, we performed global gene expression profiling in early-stage PKD cyst development in Cy/Cy kidneys and normal (+/+) kidneys, at 3 and 7 days of postnatal age. Expression profiles were determined by microarray analysis, followed by validation with real-time RT-PCR. Genes were selected with over 1.5 fold expression changes compared with age-matched +/+ kidneys for canonical pathway analysis. We found 9 pathways in common between 3-day and 7-day Cy/Cy kidneys. Three significantly changed pathways were designated 'VDR/RXR Activation,' 'LPS/IL-1 Mediated Inhibition of RXR Function,' and 'LXR/RXR Activation'. These results suggest that RXR mediated signaling is significantly altered in developing kidneys with mutated Pkdr1. In gene ontology analysis, the functions of these RXR-related genes were found to be involved in regulating cell proliferation and organ morphogenesis. With real-time RT-PCR analysis, the up-regulation of Ptx2, Alox15b, OSP and PCNA, major markers of cell proliferation associated with the RXR pathway, were confirmed in 3- and 7-day Cy/Cy kidneys compared with 3-day +/+ kidneys. The increased RXR protein was observed both in nuclei and cytoplasm of cystic epithelial cells in early-stage Cy/Cy kidneys, and the RXR-positive cells were strongly positive for PCNA staining. Taken together, cell proliferation and organ morphogenesis signals transduced by RXR mediated pathways may have important roles for cystogenesis in early-stage PKD in this Pkdr1-mutated Cy rat.
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| Overall design |
Rats for microarray assay were anesthetized at 3 and 7 days of age with sodium pentobarbital (Schering-Plough Corporation, Kenilworth, NJ, USA), and both kidneys were removed rapidly, causing exsanguination. The left kidney was frozen in liquid nitrogen for RNA extraction. RNA was extracted from kidneys of 3- and 7-day-old rats using a monophasic solution of phenol/guanidine isothiocyanate and TRIzol® reagent (Invitrogen Co., Carlsbad, CA, USA) in accordance with their manual, and the samples were incubated with RNase-free DNase I (Ambion, TX, USA). The quality and concentration of each sample was confirmed by spectrophotometry (NanoDropTM ND-1000; Asahi glass Co. Ltd., Tokyo, Japan). Genotypes were determined using polymerase chain reaction-restriction fragment length polymorphism. 200 ng aliquots of total RNA obtained from kidneys of 3- or 7-day-old rats were labeled using an Agilent Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies, Inc., Santa Clara, CA, USA) according to the manufacturer’s instructions. The pooled renal RNAs of +/+, Cy/+ or Cy/Cy rats were labeled with the Cy5-fluorescence dye, and the pooled renal RNAs of +/+ rats adopted as the template control were labeled with the Cy3-fluorescence dye. After determination of labeling efficiency, 1 μg aliquots of Cy3-labeled RNA obtained from age-matched +/+ kidneys and Cy5-labeled RNA from +/+, Cy/+, or Cy/Cy kidneys were mixed and hybridized onto Agilent Rat Oligo Microarrays (product no. G4130A) according to the manufacturer’s hybridization protocol. After washing, the microarray slides were examined with an Agilent microarray scanner and software (scanner model G2565BA). Data analysis was performed with Agilent Feature Extraction software (version A.7.1.1) and Excel 2003 (Microsoft).
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| Contributor(s) |
Kugita M, Nishii K, Morita M, Yoshihara D, Sugiyama HK, Yamada K, Yamaguchi T, Wallace DP, Calvet JP, Kurahashi H, Nagao S |
| Citation(s) |
20926632 |
| Submission date |
Jul 22, 2010 |
| Last update date |
Dec 21, 2016 |
| Contact name |
Kugita Masanori |
| E-mail(s) |
m-kugi@fujita-hu.ac.jp
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| Organization name |
Fujita Health University
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| Department |
Center of Animal Models for Human Diseases
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| Street address |
kutsukake-cho Denngakugakubo 1-98
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| City |
Toyoake |
| State/province |
Aichi |
| ZIP/Postal code |
470-1192 |
| Country |
Japan |
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| Platforms (1) |
| GPL4135 |
Agilent-014879 Whole Rat Genome Microarray 4x44K G4131F (Feature Number version) |
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| Samples (6)
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| Relations |
| BioProject |
PRJNA131691 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE23079_RAW.tar |
83.5 Mb |
(http)(custom) |
TAR (of TXT) |
| Processed data included within Sample table |
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