CHARACTERIZATION OF A MESANGIAL CELL PHENOTYPE ASSOCIATED WITH EXTRACELLULAR MATRIX (ECM) ACCUMULATION. • 2197

Abstract

Progressive glomerular disease (glomerulosclerosis) is associated with altered turnover of ECM proteins, leading to accumulation of ECM components and collapse of functional structures. Although a variety of mediators have been identified as stimulating synthesis or inhibiting degradation of ECM, the cellular events involved in this process remain unknown. Previously, we described a cell model consistent with matrix accumulation, in which fetal glomerular mesangial cells subjected to an extended series of passages (P) in culture produce more mRNA for certain collagens and for laminin, but express less ECM proteases, after P12. At P14, TGFβ expression increases. To better understand the cellular events involved in matrix accumulation, we sought to determine whether the changes we have observed in mesangial ECM turnover are associated with assumption of a specific cellular phenotype. Human fetal mesangial cells were evaluated from P9 through P17 for matrix accumulation, cytokine production, cytoskeletal arrangement and protein tyrosine phosphorylation. Immunoblot analysis indicated that, consistent with our previous evaluation of mRNA, cells from P13 and later accumulate laminin and collagen IV, but not collagen I, in the cell layer. At all passages, the cells continue to express angiotensin II receptors. They secrete no TNFα at any P; low levels of secreted IL-1β are detectable but are unchanged from P9 through P17. In contrast, by indirect immunofluorescence, F-actin decreases with serial passage, whereas α-smooth muscle actin shows increasingly intense staining and appears to be organized into a more fibrillar pattern. Vinculin staining at the edge of the cell membrane is lost with increasing passage number. Finally, between P11 and P13, an increase occurs in tyrosine phosphorylation of four proteins with Mr≈ 40, 45, 80 and 140 kDa. Taken together, these results confirm that ECM protein accumulation occurs in serially-passaged glomerular mesangial cells and indicate that this occurrence is associated with a switch in cell phenotype defined by specific changes in the cytoskeleton and in cell protein phosphorylation.