Renal fibroblasts are thought to play a major role in the development of renal fibrosis (RF). The mechanisms leading to this renal alteration remain poorly understood. In order to identify new molecular factors and pathways potentially associated with RF, we performed differential proteomic analyses with two established fibroblast cell lines with RF phenotype. Differential two dimensional gel electrophoresis combined with mass spectrometry analysis revealed the alteration of more than 30 proteins in fibrotic kidney interstitial fibroblasts (TK188) compared to normal kidney interstitial fibroblast (TK173). Among these proteins, markers of the endoplasmic reticulum (ER) stress- and the unfolded protein response (UPR) pathway (GRP78, GRP94, ERP57, ERP72, and CALR) and the oxidative stress pathway proteins (PRDX1, PRDX2, PRDX6, HSP70, HYOU1) were highly up-regulated in fibrotic cells. Activation of these stress pathways through long time exposition of TK173, to high NaCl or glucose concentrations resulted in TK188 like phenotype. Parallel to an increase in reactive oxygen species the stressed cells showed a significant alteration in expression of fibrosis markers, ER-stress and oxidative stress proteins. Similar effects of osmotic stress could be also observed on renal proximal tubule cells. Our data suggest an important role of the ER-stress proteins in fibrosis and highlights the pro-fibrotic effect of osmotic stress through activation of oxidative stress and ER-stress pathways.