2001 Fiscal Year Final Research Report Summary
Functional analysis of thiazide-sensitive Na-Cl cotransporter gene
| Project/Area Number |
12671020
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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 |
Kidney internal medicine
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| Research Institution | Tohoku University |
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Principal Investigator |
TAKEUCHI Kazuhisa Tohoku Univ. Graduate Sch. Med., Associate Prof., 大学院・医学系研究科, 助教授 (40260426)
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| Co-Investigator(Kenkyū-buntansha) |
ITO Sadayoshi Tohoku Univ. Graduate Sch. Med., Prof., 大学院・医学系研究科, 教授 (40271613)
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| Project Period (FY) |
2000 – 2001
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| Keywords | Na-Cl transporter / Gitelman syndrome / Gene transcription / Transgenic rat / steroid hormone / HFH-3 / distal convuluted tubule |
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| Research Abstract |
Molecular mechanism underlying the renal localization of thiazide-sensitive Na-Cl cotransporter (TSC) gene expression was studied. Transcription function of 5'-flanking region of rat (r)TSC gene (5'FL/rTSC) was shown to be active in HEK293 cells which express TSC mRNA. Compared to the transcription activity of 5'FL/rTSC in HEK293 cells, it was little in both HepG2 and A10 cells lacking TSC mRNA expression, suggesting the potent tissue-specific transcription activity of the 5'FL/rTSC. To further examine the tissue-specific transcription of 5'FL/rTSC, transgenic rats harboring the 5'FL/rTSC fused upstream of the LacZ gene were generated. Immunohistochemical analysis clearly showed that LacZ gene expression was co-localized to distal convoluted tubules (DCT) with TSC, indicating that the 5'FL/rTSC regulates the renal tubule-specific expression of TSC. By deletion analysis and homology search, we identified a putative cis-acting element (HFH-3/rTSC, -400/-387 position) for binding of a transcription factor HFH-3 (hepatocyte nuclear factor-3/folk head homologue-3) which is also localized to DCT. Transcription activity of the truncated HFH-3/rTSC was stimulated by overexpression of HFH-3. Electrophoretic mobility-shift assay showed a direct binding of in vitro synthesized HFH-3 to the HFH-3/rTSC. In conclusion, the transcription regulatory region of TSC gene confers the DCT-specific gene expression in the kidney. A DCT-specific transcription factor HFH-3 may be involved in the renal tubule-specific transcription of TSC gene.
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