RhoGTPase activation is a key step in renal epithelial mesenchymal transdifferentiation.

ESRD is characterized by an interstitial infiltrate of inflammatory cells in association with tubular atrophy, epithelial mesenchymal transdifferentiation (EMT), and interstitial fibrosis. Human proximal tubular epithelial cells (HK2 cells) undergo EMT in response to activated PBMC conditioned medium (aPBMC-CM), showing acquisition of a fibroblastoid morphology, increased fibronectin-EDA (EDA) expression, loss of junctional E-cadherin localization, and cytokeratin 19 (K19) expression. The signaling pathway(s) that regulates EMT in response to aPBMC-CM is not well understood. This study shows that aPBMC-CM induces a rapid activation of RhoA, Rac1, and Cdc42 activity in HK2 cells from 15 min to 48 h. Moreover, infection with adenovirus expressing constitutively active RhoA, Rac1, and Cdc42 significantly increased the expression of EDA and downregulated expression of E-cadherin and K19. Dominant negative RhoA expression suppressed aPBMC-CM-induced upregulation of EDA but did not restore the expression of E-cadherin and K19. Constitutively active RhoA activated the Rho kinase and its downstream effectors, whereas constitutively active Rac1 and Cdc42 activated the P21-activated protein kinase in epithelial cells. In further experiments, HK2 cells were treated with toxin B, exoenzyme C3, Y-27632, and HA1077. These strategies, inhibiting the Rho/Rho kinase pathway, as well as the Rac1/Cdc42/P21-activated protein kinase pathway, blocked transdifferentiation of HK2 cells in response to aPBMC-CM. To conclude, these results indicate that aPBMC-CM activates RhoA, Rac1, and Cdc42 and their downstream effectors, leading to HK2 cells undergoing transdifferentiation. Therefore, activation of small RhoGTPases is a key step in the mechanism of EMT and likely to be a contributor to tubulointerstitial fibrosis.

Oncostatin M, a cytokine released by activated mononuclear cells, induces epithelial cell-myofibroblast transdifferentiation via Jak/Stat pathway activation.

Interactions between inflammatory infiltrates and resident tubular epithelial cells may play important roles in the development of tubulointerstitial fibrosis, by promoting epithelial cell-myofibroblast transdifferentiation (EMT). Human proximal tubular epithelial cells transdifferentiated to myofibroblasts after treatment with activated PBMC conditioned medium. mRNA and protein levels for alpha-smooth muscle actin, collagen I, and fibronectin EDA(+) (markers for the myofibroblastic phenotype) were increased, whereas those for E-cadherin and cytokeratin 19 (markers for the epithelial phenotype) were decreased. cDNA microarray analysis was used to identify other changes in gene expression that might point to novel molecular mechanisms driving EMT. Of 1176 array genes, 61 demonstrated at least a twofold change at at least two consecutive time points, of the five time points examined (0.5, 4, 8, 16, and 48 h). Of these genes, 59% were upregulated and 41% were downregulated. The array indicated upregulation of expression of the oncostatin M (OSM)-specific receptor beta subunit from 4 to 48 h after exposure of kidney epithelial cells to activated PBMC conditioned medium, which contained high levels of OSM. In additional experiments, it was demonstrated that OSM induced EMT. OSM activated the Jak/Stat signaling pathway in epithelial cells, and a specific inhibitor of Jak2 blocked both its phosphorylation after exposure to OSM and the induction of alpha-actin and loss of cytokeratin 19 expression. Therefore, OSM is a novel inducer of EMT and is likely to be one of several cytokines produced by inflammatory infiltrates that contribute to this and subsequent tubulointerstitial fibrosis.