TRANSFORMATION OF RAT INNER MEDULLARY FIBROBLASTS TO MYOFIBROBLASTS IN-VITRO

Renal fibroblasts play a major role in the pathogenesis of renal inter stitial fibrosis. This process is associated at least in some forms of interstitial fibrosis with a differentiation of fibroblasts into myof ibroblasts, characterized by the de novo expression of alpha-smooth mu scle (alpha-sm) actin and/or desmin. Both the mechanisms underlying th is differentiation and their effects on cellular function are poorly u nderstood. In vitro studies are difficult since the phenotypes of fibr oblasts in culture have as yet not been well defined. We have, therefo re, examined the phenotype of inner medullary fibroblasts (IMF) during the transition from in vivo to in vitro in various cell fractions der ived from the inner medulla of healthy rats. IMF were positive for the lectin BSL-1 and negative for markers of endothelial cells. IMF first lost their prominent lipid droplets in vitro. Subsequently they devel oped cytoplasmic processes accompanied by a decrease in their reactivi ty for the lectin BSL-1 from strong to weak. From day 3 in primary cul ture, exclusively these weakly positive BSL-1 cells showed a de novo e xpression of alpha-sm actin (day 4 of primary culture, 75 +/- 4%; day 20, 94 +/- 2%) and desmin (day 4, 43 +/- 8%; day 20, 66 +/- 6%), class ifying them as myofibroblasts. This transformation depended on culture conditions. In a mixed coculture with inner medullary collecting duct (IMCD) cells the transformation of IMF was largely absent: a signific antly greater number of strong BSL-1 positive cells contained prominen t lipid droplets (39 +/- 4 vs. 19 +/- 40%, P < 0.05) an day 4 of prima ry culture, and the transition of strongly to weakly positive BSL-1 IM F was almost completely blocked. By reducing the seeding density of IM CD cells the effect of this condition on IMF transformation could be l argely abolished. This first detailed phenotypic characterization of r at fibroblasts during the transition from in vivo to in vitro demonstr ates that these cells-depending on culture conditions-differentiate to myofibroblasts within a few days of primary culture and that subcultu red IMF exhibit predominantly this phenotype. The presented model may serve as a useful toot for the in vitro study of myofibroblast formati on and the consequences of such a differentiation for the physiologica l functions of IMF.