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Am J Physiol Renal Physiol (January 23, 2007). doi:10.1152/ajprenal.00423.2006
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Submitted on October 26, 2006
Accepted on January 8, 2007

Recovery and maintenance of nephrin expression in cultured podocytes and identification of HGF as a repressor of nephrin

Yosuke Takano1, Kozue Yamauchi1, Nobuhiko Hiramatsu1, Ayumi Kasai1, Kunihiro Hayakawa1, Makiko Yokouchi1, Jian Yao1, and Masanori Kitamura1*

1 Molecular Signaling, University of Yamanashi, Chuo, Yamanashi, Japan

* To whom correspondence should be addressed. E-mail: masanori{at}yamanashi.ac.jp.

Cultured podocytes easily lose expression of nephrin. In this report, we developed optimum media for recovery and maintenance of nephrin gene expression in murine podocytes. Using reporter podocytes, we found that activity of the nephrin gene promoter was enhanced by D-MEM/F12 or {alpha}-MEM when compared with RPMI-1640. In any of these basal media, addition of 1,25-dihydroxyvitamin D3, all-trans-retinoic acid or dexamethasone significantly increased activity of the nephrin promoter. The effects of the supplemental components were synergistic, and the maximum activation was achieved by D-MEM/F12 supplemented with three agents. This culture medium was designated as vitamin D3, retinoic acid and dexamethasone - supplemented D-MEM/F12 (VRADD). In reporter podocytes that express nephrin, VRADD induced activation of the nephrin gene promoter up to 60 fold. Even in podocytes that have lost nephrin expression during multiple passages, expression of nephrin mRNA was dramatically recovered by VRADD. However, VRADD caused damage of podocytes in prolonged cultures, which was avoided in the absence of dexamethasone (designated as VRAD). VRAD maintained expression of nephrin for extended periods, which was associated with the differentiated phenotype of podocytes. Using the VRAD-primed podocytes, we revealed that expression of nephrin mRNA as well as nephrin promoter activity was suppressed by a putative dedifferentiation factor of podocytes, hepatocyte growth factor.




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