| Project/Area Number |
11671027
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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 | Third Department of Internal Medicine, Gunma University |
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Principal Investigator |
TSUKADA Yoshito (2000) Third Department of Internal Medicine, Gunma University, lecturer, 医学部, 講師 (60311601)
成清 卓二 (1999) 群馬大学, 医学部, 教授 (50010369)
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| Co-Investigator(Kenkyū-buntansha) |
成清 卓二 群馬大学, 医学部, 教授 (50010369)
塚田 義人 群馬大学, 医学部, 助手 (60311601)
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| Project Period (FY) |
1999 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2000: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1999: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Keywords | Membranous nephropathy / Heymann nephritis / Renal tubular brush border / Megalin / Experimental nephritis |
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| Research Abstract |
Using monoclonal antibodies (MoAbs), we have previously demonstrated that the pathogenetic antigen of experimental membranous nephropathy (rat Heymann nephritis) is a 120kD fragment of membrane protein derived from renal proxymal brush border. Recently, we have confirmed that two kinds of amino acid sequence of the V8 protease-digested fragments of this 120 kD protein are identical with N-terminal sequence of megalin.
We had cDNAs of megalin (clone 83, 226, Proc Natl Aca Sci, 1994) encoding this 120 kD portion kindly sent from Dr. Akihiko Saito in the second department of internal medicine of Niigata university. Then we subcloned DNA fragments encoding part of 120 kD into pTrcHis vector (Invitrogen) and successfully produced two kinds of 6xHis tagged recombinant protein (50 kD, respectively). These proteins were purified and immunized into rabbits. Polyclonal antibodies were tested against cryostat sections of normal rat kidney by indirect immunofluorescence study. We confirmed that these antibodies react only with proxymal brush border, and not with glomerular epithelium, consistent with the result with MoAbs.
In the ongoing study, we will produce another protein of 50 kD.And we will examine whether these three kinds of recombinant protein can evoke active Heymann nephritis by immunization into rats, which will enable us to identify the minimal pathogenetic antigen. Moreover with mouse MoAbs and rabbit polyclonal antibodies, recognizing different epitopes each other, it is possible to quantitate a small amount of circulating megalin antigen.
These studies promise us more precise identification of pathogenetic antigen and pathogenesis of experimental membranous nephropathy.
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