In glomerulosclerosis matrix accumulation causes chronic, progressive renal disease that ultimately leads to the need for dialysis or transplantation. Matrix accumulates because of a shift in the net balance between synthesis and degradation. Although most previous studies have focused on the role of matrix synthesis in glomerulosclerosis recent reports increasingly support a role for decreased matrix breakdown as well. Decreased degradation may result from lower susceptibility of matrix proteins to proteases, decreased synthesis or activity of matrix proteases, or increased production of endogenous protease inhibitors. Despite advances in understanding matrix synthesis and in characterizing matrix proteases, the pathways that coordinate this balance are unknown. The present project seeks to address the mechanism(s) by which the net balance of matrix protein turnover is regulated. An in vitro model using fetal glomerular mesangial cells has been developed which mimics the type of matrix accumulation observed in glomerulosclerosis. In this model, steady-state expression of mRNA and synthesis of protein for type IV collagen and laminin increase as the cells are subjected to serial passage in culture. Concomitantly, the expression of mRNA and protein for extracellular matrix proteases decreases. Changes in the expression of protease inhibitors also occur and could further favor matrix accumulation. This comprehensive model of matrix turnover will be studied to determine how these changes are regulated.
Two specific aims are proposed. First, the changes in cell phenotype related to matrix turnover will be examined. Steady-state expression and stability of mRNAs for matrix proteins, proteases and protease inhibitors will be investigated. Protein accumulation and synthetic rates will be determined and compared with steady-state mRNA expression. TGF-beta's, growth hormone and interleukin-6, each of which has been associated with glomerulosclerosis, will be evaluated for effects on matrix turnover in the cultured mesangial cells.
The second aim of the project is to identify pathways by which matrix turnover is regulated in this model. Genes that are differentially expressed by early-(classical) or late-passage (matrix accumulating) cells will be identified by a technique of subtractive hybridization. After differential expression is confirmed by Northern analysis of mesangial cell RNA using identified cDNAs, the clones will be sequenced, and cultured cells of both early- and late-passage phenotype will be evaluated for expression of the genes or gene products. Finally, the effects on matrix turnover of sense or antisense cDNAs, or of antibodies to the gene products, will be examined in the mesangial cell model. Our long-range goal is to examine expression of identified genes in renal disease. The studies described in this proposal will elucidate mechanisms of regulation of matrix turnover in glomerular mesangial cells, and may suggest mechanisms by which cell phenotype is altered in vivo to promote glomerulosclerosis.
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