Characterization of the collagen phenotype of rabbit proximal tubule cellsin culture

Studies were performed to characterize the collagen phenotype of cultured r abbit proximal tubule (RPT) epithelial cells grown on plastic and on the re constituted basement membrane preparation, Matrigel. When grown on a plasti c substratum, RPT cells display a cobblestone appearance characteristic of glomerular epithelial cells. While initially forming an interlocking networ k of cells after subculture on Matrigel, this pattern of culture morphology rapidly develops into one characterized by isolated, organized groups of c ells. Notwithstanding the effects of Matrigel on culture morphology, total cellular proliferation was reduced only 25% when RPT cells were grown on th is substrate. Greater than 90% of the collagen synthesized by RPT cells grown on plastic was secreted into the culture medium. Qualitative analysis by SOS-PAGE reve aled components exhibiting electrophoretic mobilities corresponding to the chains present in type IV and type I collagens. Quantitative analysis by CM -Trisacryl chromatography established that similar to 2/3 of the total coll agen synthesized by RPT cells grown on plastic was type IV and similar to 1 /3 type I. Quantitative analysis of the collagens produced by RPT cells gro wn on Matrigel again indicated the synthesis of only type IV and type T mol ecules 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 simil ar to 1% of the level synthesized by the cells grown on plastic. On plastic , similar to 3/4 of the type I collagen produced was recovered as the type I homotrimer, but on Matrigel type I homotrimers represented only similar t o 55% of the total type I collagen synthesized. On Matrigel, the majority o f the type IV collagen was recovered as heterotrimers containing alpha 1(IV ) and alpha 2(IV) chains. In contrast, RTP cells grown on plastic predomina ntly produced type IV homotrimers containing only the alpha 1(IV) chain. Th ese data represent the initial report describing the collagens produced by nonimmortalized cultured proximal tubule cells. The finding that a signific ant amount of the total collagen synthesized was type IV (basement membrane ) collagen, regardless of culture substrate, suggests that the RPT cells ha ve 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 observ ation that culture of RPT cells on a reconstituted basement membrane prepar ation results in a significant reduction in total collagen production and a lterations in the molecular farms of type IV and type I molecules synthesiz ed indicates that integrity of the tubular basement membrane may represent an important component in preventing the development of tubulointerstitial fibrosis.