2007 Fiscal Year Final Research Report Summary
Studies of mechanisms for secretion and synthesis of renal kallikrein, a salt-sensitive factor, on immortalized cell lines and its geneticpolymorlphism
| Project/Area Number |
17590842
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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 | Kitasato University |
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Principal Investigator |
FUJITA Tomoe Kitasato University, School of Medicine, Assistant Professor (20296510)
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| Co-Investigator(Kenkyū-buntansha) |
MASATAKA Majima Kitasato University, School of Medicine, Professor (70181641)
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| Project Period (FY) |
2005 – 2007
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| Keywords | kallikrein / Immortalized gene / connecting tubule / calcium |
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| Research Abstract |
1. An experiment of establishment of connecting tubular cell lines from the transgenic rats expressing the SV40 temperature-sensitive T-antigen gene, an immortalized gene.
Distal convoluted tubules (DCT), connecting tubules (CNT), and initial collecting tubules (ICT) were each isolated from normal rat kidney by a microdissection technique. Cellular amounts of mRNA expression of marker genes such as Na^<+->CL cotransporter (NCC), kallikrein, Na^+/Ca^<2+> exchanger 1 (NCX1), and 11β-hydroxysteroid dehydrogenase 2 (11βHSD2) were examined in primary cultured cells from each part of the tubules. Highly expressed NCC was observed in the DCT and the latter three genes were all highly expressed in the CNT and ICT which were comparable to the previous report In the next study CNT and ICT were isolated from the above transgenic rats harboring an immortalized gene. As for the CNT, cells derived from the CNT were almost replaced by fibroblasts during passages. ICT cells which were obtained by a sam
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e procedure contained less fibroblasts, but expressed a few kallikrein mRNA. Therefore, an additional experiment is needed to obtain CNT and ICT cells from the above transgenic rats. Before the additional experiment, minimum dilution or flow cytometory with lectins will be tried to obtain kallikrein containing cells, connecting tubular cells and principal cells in the CNT and ICT, respectively.
2. Ca^<2+> imaging on primary cultured CNT cells.
CNT cells 4 to 5 days after primary culture isolated from Wistar rats were used for the experiment. Cells were loaded with Fura-2 AM, an indicator of the cytosolic free Ca^<2+> concentration. Intracellular changes in Ca^<2+> concentration were measured by digital Ca^<2+> imaging system with excitation wavelengths of 340/380 nm and an emission wavelength of 510 nm. In order to examine involvement of intracellular Ca^<2+> in stimulatory secretion of renal kallikrein, effects of secretagogues of renal kallikrein such as high K^+ solution and low Na^+ solution and agonists which induce phosphatidyl-inositol response such as vasopressin and bradykinin on intracellular Ca^<2+> have been studied. However, there is a problem with respect to tubular attachment to collagen type I-coat glasses, i. e. only a few cells can form small islets after more than 10 CNT are cultured on the glass chamber slide and such cells are easily detached from the glasses.
3. Prospects
Renal kallikrein secretion is restricted in the kidney where CNT cells and principal cells distributed in the CNT and ICT, respectively, are major resources of renal kallikrein. One of the roles of renal kallikrein is sodium handling at the late distal tubules and its secretion is reduced in several hypertensive models in rats. Thus, examination of mechanisms for renal kallikrein secretion with respect to Ca^<2+> imaging is expected to find etiologic factor of salt-sensitive hypertension Less
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Research Products
(6 results)
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[Journal Article] Functional analysis of polymorphisms in the organic anion transporter, SLC22A6 (OAT1)2005
Author(s)
Fujita, T, Brown, C, Carlson, EJ, Taylor, T, Cruz, M, Johns, SJ, Stryke, D, Kawamoto, M, Fujita, K, Castro, R, Chen, CW, Lin, ET, Brett, CM, Burchard, EG, Ferrin, TE, Huang, CC, Leabman, MK, Giacomini, KM
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Journal Title
Pharmacogenet Genomics 15
Pages: 201-209
Description
「研究成果報告書概要(欧文)」より
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