Proteomische Analyse eines in-vitro-Modells für die interstitielle renale Fibrose: Der osmotische Stress als Fibrose-triggernder Faktor

Abstract

Renal fibrosis (RF) is the final stage of many renal diseases leading to organ function loss. RF is characterised by the deposition and accumulation of extracellular matrix components (ECM). Renal fibroblasts are playing a major role in this process. To identify mechanisms associated with renal interstitial fibrosis, we performed a differential proteomic analysis of two established immortalized cell lines, normal kidney fibroblasts (TK173) and fibrotic kidney fibroblasts (TK188). 2-D electrophoresis combined with mass spectrometry analysis revealed the alteration of 43 proteins in TK188 compared to TK173. Among these proteins were markers that have been described to be involved in fibrogenesis (e.g. FIN, ACTA2, VIN, VIM, DES and KRT), protein markers of the ER stress and the unfolded protein response (UPR) pathway (GRP78, GRP94, ERP57, ERP72, PDI and CALR) and proteins of the oxidative stress pathway (PRDX1, PRDX2, PRDX6, SOD, PARK7 and HYOU1). All these proteins were highly upregulated in TK188 cells. The results of selected proteins were confirmed by western blot and immunofluorescence staining. The upregulation of stress proteins in the fibrotic cell line suggests a correlation between the activation of stress pathways and the development of fibrosis. After long time exposition of TK173 to osmotic stress with high NaCl concentration, we observed a transformation of the stressed cell line (TK173-NaCl) in a TK188-like phenotype. The proteomic analysis shows an alteration in the expression of 27 proteins in TK173-NaCl compared to TK173. 22 proteins were upregulated. Among them were fibrosis markers, proteins of ER stress and oxidative stress. The results of selected proteins were confirmed by western blot and immunofluorescence staining. Based on proteomic analysis of in vitro cell model for renal fibrosis (TK173/TK188), we identified typical fibrosis markers and illustrated the role of different stress pathways in fibrosis. We could also show the profibrotic effect of osmotic stress on renal fibroblasts through activation of oxidative stress and ER stress pathways.