2001 Fiscal Year Final Research Report Summary
Establishment of Renal Stone Formation Model in Cell Culture and Isolation Study of Genetic Calculogenesis Factors
| Project/Area Number |
10671467
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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 |
Urology
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| Research Institution | Hamamatsu University School of Medicine |
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Principal Investigator |
NAITO Yasuhisa Hamamatsu University School of Medicine, 1st Department of Pathology, Associate Professor, 医学部, 助教授 (10107815)
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| Co-Investigator(Kenkyū-buntansha) |
SUGIMURA Haruhiko Hamamatsu University School of Medicine, 1st Department of Pathology, Professor, 医学部, 教授 (00196742)
OHTAWARA Yoshihisa Hamamatsu University School of Medicine, Urological Department, Assistant, 医学部, 助手 (80124717)
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| Project Period (FY) |
1998 – 2000
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| Keywords | Renal stone / Cell culture / MDCK cell line / Ca2+ flux / Fura-2 / Calcium oxalate / Gene of calculogenesis |
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| Research Abstract |
Cell culture models of renal stone formation were established using the MDCK established cell line derived from canine kidney distal epithelial cells. These microliths corresponding to their location in human renal tubules first detected microscopically after 5 days of culture. Renal microliths were composed of calcium phosphate with a Ca : P ratio of 2.7 : 1 as demonstrated by X-ray and microinfrated spectroscopy and were morphologically closely similar in appearance to those observed in human specimens by scanning electron microscopy.
The measurement of extracellular Ca2+ concentrations in cell culture system is a critical importance, because renal stone formation may be tightly regulated by [Ca2+]i. We measured [Ca2+]i of two phase culture medium with added various concentrations of Ca^<2+> by double chamber culture system with Ca^<2+> waves, using fura-2 fluorescence image processing. In conclusion, it was shown that values of Ca^<2+> were higher in lower chamber corresponding to ba
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sal layer of cells than in upper one corresponding to the region of the tubular lumen. These data suggest that MDCK cells might actively transport [Ca2+]i from apical region to basal area in cell culture system and that the deposits form due to active concentration of calcium and phosphate by the MDCK cells under the micro-environments where media are supersaturated and heteronuleation may occur.
Establishment of cell culture model of calcium oxalate stone formation was studied by adding sodium oxalate, ascorbic acid, ethylene glycol, and sodium, glycolate into the cell culture medium. However, formed tiny crystals were composed of calcium phosphate in all experiments. Further examinations including metabolical enzymes for calcium oxalate formation in cell culture are needed
We also isolated two types of colony, one with renal microliths and the other with none, for comparison of mutants that do and do not generate renal stones might be useful to obtain further information on the mechanisms of calculogenesis. Different DNAs between two types of colony were studied by the differential display method. Now, some different DNAs between them are obtained. It would be of great interest to ascertain the complicated genetic factors associated with pathogenesis of renal stone formation including the predispositions to lithogenesis by using our culture models. Less
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Research Products
(4 results)
