A novel model to study renal myofibroblast formation in vitro
1Abteilung Nephrologie und Rheumatologie, Georg-August-Universität, Göttingen, Germany. cgrupp@gwdg.de
Background:
In chronic renal disease, the decrease of excretory renal function closely correlates with the extent of interstitial fibrosis. A common feature of interstitial fibrosis is the occurrence of myofibroblasts, which are regarded as the predominant cells in matrix synthesis. We studied the transformation of renal fibroblasts into myofibroblasts in vitro as a model to elucidate the mechanisms underlying this process.
Methods:
Primary cultures of freshly isolated rat inner medullary fibroblasts were established as reported previously. mRNA expression of myofibroblast markers and interstitial collagens was examined by Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR). Phenotypic expression was investigated by immunolabeling. Endogenous transforming growth factor-beta (TGF-beta) production was inhibited by addition of a neutralizing antibody or by TGF-beta 1 antisense oligodeoxynucleotides (ODNs).
Results:
Initially and during the first 36 hours, primary culture cells expressed neither alpha-smooth muscle actin nor desmin mRNA. From day 2 of primary culture, we observed a strong increase in these mRNAs, as evaluated by RT-PCR, followed by the phenotypic expression of these myofibroblast markers. Collagen type I mRNA was first detectable from day 4 of primary culture and showed a strong increase in its expression level during the following days, with phenotypic expression predominantly in myofibroblasts. The transformation rate of fibroblasts to myofibroblasts largely decreased in cocultures with collecting duct cells. This effect could be reversed by reducing the seeding density. We examined in this system the effect of TGF-beta 1 to define further its putative fibrogenic activity. However, neither the addition of exogenous TGF-beta 1 nor the inhibition of endogenous TGF-beta production showed any significant effect on fibroblast transformation, suggesting that this cytokine exerts its effects at other levels or requires a cofactor.
Conclusions:
We present a novel model to examine the de novo expression of myofibroblast markers and collagen type I in freshly isolated fibroblasts under defined conditions in primary culture. This model could provide a strategy for the molecular characterization of myofibroblast formation and the phenomena associated with this process.
