2006 Fiscal Year Final Research Report Summary
Gene expression and molecular mechanism of Annexins on urolithiasis
| Project/Area Number |
16591627
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Urology
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| Research Institution | Kanazawa Medical University |
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Principal Investigator |
MIYAZAWA Katsuhito Kanazawa Medical University, Urogenital Surgery, Associate professor, 医学部, 助教授 (60219772)
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| Co-Investigator(Kenkyū-buntansha) |
SUZUKI Koji Kanazawa Medical University, Urogenital Surgery, Professor, 医学部, 教授 (70064615)
MORIYAMA Manabu Kanazawa Medical University, Urogenital Surgery, Assistant professor, 医学部, 講師 (50278131)
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| Project Period (FY) |
2004 – 2006
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| Keywords | urolithiasis / calcium oxalate / calcium binding molecule / annexins / PAR |
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| Research Abstract |
Purpose : Kidney stone formation is a complex process, and numerous molecules participate in this cascade. Attachment of newly formed crystals to renal epithelial cells appears to be a critical step in the development of this cascade. Annexins are one of the calcium binding molecules and up-regulated by calcium oxalate crystals. Annexin V is routinely used as a probe to demonstrate phosphatidylserine exposed on the surface of apoptotic cells. To obtain more detailed insight into annexins on urolithiasis and renal epithelial injury, we examined an incidence of apoptotic cells and expression of apoptosis related genes in kidneys of stone-forming rat.
Materials and Methods : Male Wistar rats were administrated ethylene glycol (EG) in drinking water and force fed withl alpha-OH-D_3. Apoptosis was detected as a ladder of fragmented DNA in agarose gels of electrophoresed genomic DNA. Apoptotic cells were localized by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labe
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ling (TUNEL) method. Expression of apoptosis-related genes was analyzed by both reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry.
Results : While no labeling was detected in the control or the first day of administration by the TUNEL method, labeling began to be detected in the renal tubular epithelium of the outer medulla at day 3, and the number of labeled cells increased progressively during the observation period. A ladder of DNA fragments was demonstrated in the kidneys of rats administrated after 2 weeks. Immunohistochemical studies revealed the expressions of Fas ligand (Fas L), bax and interleukin-1 beta converting enzyme (ICE) in the renal tubular epithelium of the descending limb of loop of Henle and distal convoluted tubules. mRNA of ICE, c-myc, p53 and Fas L genes were also up-regulated in the kidneys of stone forming-rat model.
Conclusion : In the hyperoxaluric, subsequent stone-forming rat kidney caused by the administration of EG and 1 alpha-OH-D_3, apoptosis was detected in the renal epithelial cells, especially in those of the descending limb of the loop of Henle and distal convoluted tubules. RT-PCR and immunohistochemical analyses revealed up-regulations of Fas-L, ICE, Bax, c-myc and p53. These findings indicate that renal epithelial injury in the stone-forming rat model following EG and 1 alpha-OH-D_3 feeding may induce the expression of those apoptosis-related genes, and different pathways in the stone-forming rat model may induce apoptosis. Less
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