Cultured rat glomerular epithelial cells show gene expression and production of transforming growth factor-beta: expression is enhanced by thrombin.
BACKGROUND
Glomerular crescents play an important role in progressive glomerular injury. The lesions consist of epithelial cells, macrophages, and deposits of fibrin and extracellular matrix. Transforming growth factor beta (TGF-beta) contributes to the modulation of cell growth and extracellular matrix synthesis. Thrombin is involved in fibrin formation in crescents. The purpose of this study was to examine whether glomerular epithelial cells (GEC) could produce TGF-beta, and if so, to clarify the role of TGF-beta in GEC proliferation. We also investigated whether thrombin could modulate the production of TGF-beta and extracellular matrix by GEC.


METHODS
Bioassay using the TGF-beta-dependent mink pulmonary epithelial cell line (CCL-64), immunoblot analysis, and reverse transcriptase polymerase chain reaction (RT-PCR) were used to demonstrate TGF-beta production by rat GEC. TGF-beta gene expression was examined by RT-PCR in GEC incubated with thrombin, and type IV collagen and fibronectin were quantified by enzyme immunoassay in culture supernatants of GEC incubated with thrombin or TGF-beta.


RESULTS
TGF-beta activity was demonstrated in GEC supernatants by bioassay. Immunoblot analysis of concentrated culture supernatants using anti-TGF-beta antibody revealed a 12.5-kDa protein, which was compatible with TGF-beta. Concentrated GEC supernatants inhibited GEC proliferation as well as porcine TGF-beta. RT-PCR demonstrated TGF-beta gene expression in GEC. Thrombin (0.5-5.0 U/ml) enhanced TGF-beta mRNA expression in a dose-dependent manner. Thrombin (5.0 U/ml) and porcine TGF-beta (5.0 ng/ml) stimulated the production of type IV collagen and fibronectin by GEC.


CONCLUSIONS
Rat GEC produce TGF-beta in vitro. Thrombin may participate in the progression of glomerulosclerosis in crescentic glomerulonephritis through the stimulation of TGF-beta production by GEC.
Tsunoda
S
S
Second Department of Internal Medicine, Hirosaki University School of Medicine, Zaifu-cho 5, Hirosaki 036-8216, Japan.
Yamabe
H
H
Osawa
H
H
Kaizuka
M
M
Shirato
K
K
Okumura
K
K
D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D024141:Collagen Type IV / Q000096:biosynthesis; D004847:Epithelial Cells / Q000166:cytology / Q000502:physiology; D005353:Fibronectins / Q000096:biosynthesis; D015870:Gene Expression / Q000187:drug effects; D007678:Kidney Glomerulus / Q000166:cytology / Q000502:physiology*; D051381:Rats; D013552:Swine; D013917:Thrombin / Q000494:pharmacology*; D016212:Transforming Growth Factor beta / Q000096:biosynthesis* / Q000235:genetics*
D024141:Collagen Type IV; D005353:Fibronectins; D016212:Transforming Growth Factor beta; D013917:Thrombin