| Co-Investigator(Kenkyū-buntansha) |
NONOGUCHI Hiroshi Kumamoto University, Department of Nephrology, Associate professor, 医学部附属病院・腎臓内科, 助教授 (30218341)
TOMITA Kimio Kumamoto University, Department of Nephrology, Professor, 医学部附属病院・腎臓内科, 教授 (40114772)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 2001: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
In this study, we aimed to clone mammalian osmosensor by degenerative PCR technique using cDNA of Sho1 and Sln1, which are osmosensors of S. cerevisiae, and to establish mouse macula densa cell line to test the hypothesis that Tubulo-glomerular feedback (TGF) might sense osmolar change in interstitiium of juxta-glomerular apparatus via osmosensor. Degenerative PCR products are being sequenced. However, the candidate of mammalian osmosensor has not been founded yet.
To make mouse macula densa cell line, the cell suspension of the kidney cortex of mice transgenic for the SV40 large T-antigen were labeled with FITC-conjugated Dolichos biflorus lectin recognizing sugar residues in collecting duct cells and phycoerythrin-conjugated Helix pomatia lectin recognizing sugar residues on macula densa cells, collecting duct cells, and cells in S1 and S2 segments of proximal tubules. The cells were analyzed by flow cytometry. Cells that were phycoerythrin-positive and FITC-negative were collected aseptically into culture medium, and were cultured at 37°C in a humidified atmosphere of 95% air, 5% CO2 until confluent. The cells were then cloned by limiting dilution. Now we are screening for expression of macula densa cell markers by RT-PCR using positive selection (mRNAs of COX-2, bNOS, NKCC2, ROMK, and oxytocin receptors) and negative selection {Tamm-Horsfall protein (THP), vasopressin receptor type 2 (V2R), and glucose-6-phosphatase (Glu-6-Pase)} to identify macula densa cell line.
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