TGF-beta isoforms in renal fibrogenesis

Transforming growth factor-beta1 (TGF-beta1) is generally considered to be the major or predominant isoform involved in fibrosis, with the roles of TGF-beta2 and -beta3 being less clear. Because anti-TGF-beta-specific isoform treatment is in development, it is important to know more precisely about isoform action. Here we compared the actions of each isoform on production and degradation of extracellular matrix proteins by cultured rat mesangial cells, renal fibroblasts, and tubular epithelial cells. We investigated endogenous production of each isoform, the effect of adding one isoform on the production of the other isoforms, and the response to addition of isoform combinations on matrix protein production. Isoform-specific antibodies were used to determine the relative contribution of these isoforms to matrix protein production.

Each cell type was treated with TGF-beta (0.01 to 10 ng/mL) alone or in different combinations. Living cell number was determined by 3-[4,5]dimethylthiazol-2,5-diphenyltetrazolium bromide (MTT) assay. Supernatant fibronectin and TGF-beta isoform concentration were measured by enzyme-linked immunosorbent assay (ELISA). Collagen and proteoglycan production were measured by [3H]-proline and [35S]-sulfate incorporation, respectively. Matrix protein and TGF-beta isoform gene expression were determined by Northern blot. Release of 3H from preformed radiolabeled matrix by fibroblasts was used as a measure of matrix degradation.

Each isoform increased matrix protein synthesis and reduced matrix degradation by renal cells similarly. Combination of TGF-beta isoforms showed additive effects. No antifibrotic effect was observed with TGF-beta3. TGF-beta1 increased -beta2 and -beta3 production in a small and inconsistent manner. In contrast, TGF-beta2 and -beta3 stimulated TGF-beta1 in all three cell types. Eighty percent of TGF-beta3's fibrogenic effect was mediated by TGF-beta1. A pan-specific antibody to TGF-beta most effectively blocked plasminogen activator inhibitor type 1 (PAI-1) synthesis by epithelial cells under oxidative stress.

All three TGF-beta isoforms have fibrogenic effects on renal cells. TGF-beta2 and TGF-beta3 effects may be partially mediated by TGF-beta1. These data suggest that blockade of all isoforms together may yield the best therapeutic effect in reducing renal fibrosis.