Search

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.sub.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.sub.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. retinoic acid; vitamin D; dexamethasone; hepatocyte growth factor doi: 10.1152/ajprenal.00423.2006.

Macrophage-mesangial cell interaction plays a crucial role in the pathogenesis of glomerulonephritis. Activated macrophages trigger mesangial cells to express an array of inflammation-associated genes via activation of NF-kappaB and AP-1. However, this inflammatory response is often transient and subsides spontaneously. We found that mesangial cells activated by bystander macrophages showed blunted responses of NF-kappaB to subsequent macrophage exposure. It was associated with sustained levels of IkappaBbeta, but not IkappaBalpha. The tolerance observed was reversible and reproduced by conditioned media from activated macrophages (macrophage-conditioned medium (MphiCM)). In vivo priming of mesangial cells by activated glomerular macrophages also caused the tolerance of mesangial cells. The macrophage-derived tolerance inducers were heat-labile, and multiple molecules were involved. Among inflammatory cytokines produced by macrophages, TNF-alpha and IL-1beta were able to induce mesangial cell tolerance dose-dependently. The mesangial cell tolerance was also observed in activation of the MAPK-AP-1 pathway; i.e., phosphorylation of ERK, JNK, and p38 MAPK by macrophages was blunted when the cells were pre-exposed to MphiCM. Induction of c-fos and c-jun was also abrogated in mesangial cells pre-exposed to MphiCM, and the suppression was attenuated by blockade of MAPK activation during the first exposure to MphiCM. These data elucidated that mesangial cells, once exposed to macrophages, become insensitive to subsequent activation by macrophages and proinflammatory stimuli. This self defense of glomerular cells may play a role in the resolution of macrophage-mediated, acute glomerulonephritis.