Characterization of the Collagen Phenotype of Rabbit Proximal Tubule Cells in Culture

Abstract

Studies were performed to characterize the collagen phenotype of cultured rabbit proximal tubule (RPT) epithelial cells grown on plastic and on the reconstituted basement membrane preparation, Matrigel. When grown on a plastic substratum, RPT cells display a cobblestone appearance characteristic of glomerular epithelial cells. While initially forming an interlocking network of cells after subculture on Matrigel, this pattern of culture morphology rapidly develops into one characterized by isolated, organized groups of cells. Notwithstanding the effects of Matrigel on culture morphology, total cellular proliferation was reduced only 25% when RPT cells were grown on this substrate.Greater than 90% of the collagen synthesized by RPT cells grown on plastic was secreted into the culture medium. Qualitative analysis by SDS-PAGE revealed components exhibiting electrophoretic mobilities corresponding to the chains present in type IV and type I collagens. Quantitative analysis by CM-Trisacryl chromatography established that ∼ 2/3 of the total collagen synthesized by RPT cells grown on plastic was type IV and ∼ 1/3 type I. Quantitative analysis of the collagens produced by RPT cells grown on Matrigel again indicated the synthesis of only type IV and type I molecules but in a slightly more equal ratio of both collagen types and in the ratio of secreted to cell-associated molecules. However, the total amount of collagen synthesized by RPT cells grown on Matrigel was reduced to ∼ 1% of the level synthesized by the cells grown on plastic. On plastic, ∼ 3/4 of the type I collagen produced was recovered as the type I homotrimer, but on Matrigel type I homotrimers represented only ∼ 55% of the total type I collagen synthesized. On Matrigel, the majority of the type IV collagen was recovered as heterotrimers containing α1(IV) and α2(IV) chains. In contrast, RTP cells grown on plastic predominantly produced type IV homotrimers containing only the α1(IV) chain. These data represent the initial report describing the collagens produced by non-immortalized cultured proximal tubule cells. The finding that a significant amount of the total collagen synthesized was type IV (basement membrane) collagen, regardless of culture substrate, suggests that the RPT cells have maintained a significant degree of differentiation in culture, and thus establishes RPT cells as an appropriate model for investigating ECM changes in proximal tubule cells that occur in kidney disease. Finally, the observation that culture of RPT cells on a reconstituted basement membrane preparation results in a significant reduction in total collagen production and alterations in the molecular forms of type IV and type I molecules synthesized indicates that integrity of the tubular basement membrane may represent an important component in preventing the development of tubulointerstitial fibrosis.