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Basic research in gene therapy for urolithiasis using osteopontin antisense

Research Project

Project/Area Number 11470340
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Urology
Research InstitutionNagoya City University

Principal Investigator

KOHORK Kenjiro  Nagoya City University, Medical School, Professor, 医学部, 教授 (30122047)

Co-Investigator(Kenkyū-buntansha) TOZAWA Keiichi  Nagoya City University, Medical School, Assistant Professor, 医学部, 講師 (40264733)
HAYASHI Yutaro  Nagoya City University, Medical School, Associate Professor, 医学部, 助教授 (40238134)
佐々木 昌一  名古屋市立大学, 医学部, 講師 (50225869)
Project Period (FY) 1999 – 2001
Project Status Completed (Fiscal Year 2001)
Budget Amount *help
¥14,600,000 (Direct Cost: ¥14,600,000)
Fiscal Year 2001: ¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2000: ¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 1999: ¥8,100,000 (Direct Cost: ¥8,100,000)
Keywordsurolithiasis / osteopontin / gene transfection / gene therapy / アンチセンス / 細胞接着 / リポフェクション
Research Abstract

Biochemical analyses of urinary stones have shown that 1 to 5 % of their weight consists of proteinaceous ingredients, and several studies have emphasized the importance of proteins in stone formation. We previously extracted the proteinaceous fraction from calcium oxalate and calcium phosphorus stones using EDTA and identified osteopontin (OPN) as one of the major constituents of urinary stones. In another study, we found a strong expression of OPN mRNA on distal tubular cells in the kidneys of stone-forming rats. OPN is clearly produced in the kidney, although its function and significance there remain unclear. As we speculated that OPN played a role in stone formation, we tried to block the production of OPN using transfection of antisense OPN to protect stone formation in the kidney. The Tetracycline-Regulated Expression System was used. Plasmid pTet-OPN as was constructed by cloning mouse OPN cDNA sequence in an inverted (antisense) orientation. Two clones of antisense transfectans were identified which showed marked reduction in OPN syntesis. Intact NRK-52E cells, NRK-52E cells transfected with empty vector, and tetracycline-treated antisense clones, which did not show inhibition of OPN expression, all under identical conditions associated with calcium oxalate (CaOx) crystals. In contrast, the expression of antisense OPN RNA prevents the association of CaOx crystals with NRK-52E cells. Moreover, experimental kidney stone rat model in vivo transfection were inhibited OPN expression in the renal outer medulla where were expressed antisense OPN RNA, and were not observed the formation of CaOx microlith.
We suggest that the regulation of OPN protect the stone formation, and that OPN regulations is one of the methods of gene therapy to urolithiasis.

Report

(4 results)
  • 2001 Annual Research Report   Final Research Report Summary
  • 2000 Annual Research Report
  • 1999 Annual Research Report
  • Research Products

    (21 results)

All Other

All Publications (21 results)

  • [Publications] Sakakura T et al.: "Calcium phosphate stones produced by Madin-Darby canine kidney (MDCK) cells inoculated in nude mice"Urol Res. 27. 200-205 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Yasui T et al.: "Quantification of osteopontin in the urine of healthy and stone-forming men"Urol Res. 27. 225-230 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Yasui T et al.: "Expression of bone matrix proteins in urolithiasis model rats"Urol Res. 27. 255-261 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Yasui T et al.: "The effect of takusha, a kampo medicine, on renal stone formation and osteopontin expression in a rat urolithiasis model"Urol Res. 27. 194-199 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Tawada T et al.: "Distribution of osteopontin and calprotectin as matrix protein in calcium-containing stone"Urol Res. 27. 238-242 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Takahiro Yasui, et al.: "Effects of allopurinol on renal stone formation and osteopontin expression in a rat urolithiasis model"Nephron. 87. 170-176 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Sakakura T., Fujita K., Yasui T., Sasaki S., Mabuchi Y., Iguchi M., Kohri K.: "Calcium phosphate stones produced by Madin-Darby canine kidney (MDCK) cells inoculated in nude mice"Urol Res.. 27. 200-205 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Yasui T., Fujita K., Hayashi Y., Ueda K., Kon S., Maeda M., Ueda T., Kohri K.: "Quantification of osteopontin in the urine of healthy and stone-forming men"Urol Res.. 27. 225-230 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Yasui T., Fujita K., Sasaki S., Sato M., Sugimoto M., Hirota S., Kitamura Y., Nomura S., Kohri K.: "Expression of bone matrix proteins in urolithiasis model rats"Urol Res.. 27. 255-261 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Yasui T., Fujita K., Sato M., Sugimoto M., Iguchi M., Nomura S., Kohri K.: "The effect of takusha, a kampo medicine on renal stone formation and osteopontin expression in a rat urolithiasis model"Urol Res.. 27. 194-199 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Tawada T., Fujita K., Sakakura T., Shibutani T., Nagata T., Iguchi M., Kohri K.: "Distribution of osteopontin and calprotectin as matrix protein in calcium-containing stone"Urol Res.. 27. 238-242 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Inada M., Yasui T., Nomura S., Sato M., Yamagiwa H., Kimura T., Yasui N., Ochi T., Endo N., Kitamura Y., Kishimoto T. Komori T.: "Maturatinal Disturbance of Chondrocytes in Cbfal-Deficient Mice"Developmental Dynamics. 214. 279-290 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Takahiro Yasui, et al.: "Effects of allopurmol on renal stone formation and osteopontin expression in a rat urolithiasis model"Nephron. 87(2). 170-176 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Takahiro Yasui, et al.: "Effects of citrate on renal stone formation and osteopontin expression in a rat urolithiasis model"Urological Research. 29(1). 50-56 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Takahiro Yasui, et al.: "Effects of eicosapentaenoic acid on urinary calcium excretion in calcium stone formers"European Urology. 39(5). 580-585 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Takahiro Yasui, et al.: "Calcium oxalate crystal attachment to cultured rat kidney epithelial cell, NRK-52E"Urologia Internationalis. 67(1). 73-76 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Takahiro Yasui, et al.: "Osteopontin regulates adhesion of calcium oxalate crystals to renal epithelial cells"Eurolithiasis-9^<th> European Symposium on Urolithiasis. 199-200 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Keiichi Tozawa, et al.: "Signal transduction mediated by focal adhesion kinase (FAK) and the expression of osteopontin in the renal tubular cells: Role in stone formation"Euxolitbiasis-9^<th> European Symposium on Urolithiasis. 215-216 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Masuda K. et.al: "N-gGycan sturctures of an osteopontin from human bone"Biochemical and Biophysical Research Communications. 268. 814-817 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Kohri K. et.al: "Clinical features of primary hyperparathoyroidism : preoperative localization and parathyroidectory"Biomed & Pharmacother. 54 Suppll. 69-71 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Yamate T. et.al: "Urol.Res."Aanalysis of osteopontin DNA in patients with urolithiasis. 28. 159-166 (2000)

    • Related Report
      2000 Annual Research Report

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Published: 1999-04-01   Modified: 2016-04-21  

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