Recovery and maintenance of nephrin expression in cultured podocytes and identification of HGF as a repressor of nephrin.
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 DMEM/F12 or alpha-MEM compared with RPMI-1640. In any of these basal media, addition of 1,25-dihydroxyvitamin D(3), 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 DMEM/F12 supplemented with three agents. This culture medium was designated as vitamin D(3), retinoic acid and dexamethasone-supplemented DMEM/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.
Takano
Yosuke
Y
Departments of Molecular Signaling, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Yamanashi, Japan .
Yamauchi
Kozue
K
Hiramatsu
Nobuhiko
N
Kasai
Ayumi
A
Hayakawa
Kunihiro
K
Yokouchi
Makiko
M
Yao
Jian
J
Kitamura
Masanori
M
D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002117:Calcitriol / Q000494:pharmacology; D002454:Cell Differentiation; D002460:Cell Line; D003470:Culture Media / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology; D004338:Drug Combinations; D015870:Gene Expression / Q000187:drug effects; D005938:Glucocorticoids / Q000494:pharmacology; D017228:Hepatocyte Growth Factor / Q000494:pharmacology*; D006801:Humans; D008565:Membrane Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D050199:Podocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000187:drug effects; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D013997:Time Factors; D014212:Tretinoin / Q000494:pharmacology
D015415:Biomarkers; D003470:Culture Media; D004338:Drug Combinations; D005938:Glucocorticoids; D008565:Membrane Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; C113267:nephrin; D014212:Tretinoin; D017228:Hepatocyte Growth Factor; D003907:Dexamethasone; D002117:Calcitriol