TGF-βbgr;1 stimulates the release of pre-formed bFGF from renal proximal tubular cells

TGF-βbgr;1 stimulates the release of pre-formed bFGF from renal proximal tubular cells.BackgroundIt is now clear that the progression of renal disease is closely correlated to the degree of renal interstitial fibrosis. We have previously demonstrated that the renal proximal tubular epithelial cell may contribute to the fibrotic response by the generation of profibrotic cytokines. Transforming growth factor-βbgr;1 (TGF-βbgr;1) and basic fibroblast growth factor (bFGF) are two of a group of profibrotic cytokines that have been associated with the development of renal interstitial fibrosis. In this study, we have examined the influence of TGF-βbgr;1 on the generation of bFGF by renal tubular epithelial cells.MethodsHK2 cells were grown to confluence and were serum deprived and stimulated with recombinant TGF-βbgr;1 under serum-free conditions. Subsequently, supernatant, cell-associated, intracellular, and matrix-associated bFGF concentrations were determined by enzyme-linked immunosorbent assay (ELISA). bFGF mRNA expression was examined by reverse transcription-polymerase chain reaction (RT-PCR).ResultsThe exposure of confluent serum-deprived HK2 cells to TGF-βbgr;1 led to a significant increase in bFGF concentration in the cell culture supernatant. Twenty-four hours following the addition of 10 ng/ml TGF-βbgr;1, this represented a twofold increase in bFGF concentration (control, 102 pg/ml, N = 24, vs. 202 pg/ml, N = 19, P = 0.0001). Despite the increase in bFGF concentration in the supernatant, there was no change in the expression of bFGF mRNA following the addition of TGF-βbgr;1. The addition of 10 ng/ml of TGF-βbgr;1 led to a 30% decrease in the total cell-associated bFGF concentration (control, 8.51 ng/ml, N = 16, TGF-βbgr;1, 6.01 ng/ml, N = 13, P = 0.0042). This decrease in intracellular bFGF was associated with a 15% reduction in anti-bFGF antibody binding to fixed permeabilized cells, following the addition of 10 ng/ml of recombinant TGF-βbgr;1 (N = 9, P = 0.0007), suggesting that the mechanism of stimulation of bFGF by TGF-βbgr;1 involved the release of preformed bFGF from within the cells. In addition, following the addition of TGF-βbgr;1, there was a significant dose-dependent decrease in the amount of bFGF sequestered in the extracellular matrix. At a dose of 10 ng/ml TGF-βbgr;, this represented a greater than sevenfold decrease (N = 9, P = 0.0007) in matrix-bound bFGF, although this represented less than 3% of the total bFGF released into the supernatant.ConclusionThe data presented suggest that the main mechanism by which TGF-βbgr;1 stimulates bFGF generation by proximal tubular epithelial cells is by stimulation of the secretion of preformed cytokine from within the cells