Thrombin stimulates production of fibronectin by human proximal tubular epithelial cells via a transforming growth factor-beta-dependent mechanism.
BACKGROUND
Tubulointerstitial fibrosis contributes to the progression of many forms of glomerular disease and to end-stage renal failure. Inflammatory mediators generated during glomerular injury may induce tubulointerstitial lesions by stimulating tubular cells. Thrombin has multiple biological functions in addition to its role in haemostasis and has been detected in the urine of patients with glomerular diseases. The present study investigated whether thrombin can modulate the production of fibronectin (FN) in cultured human proximal tubular epithelial cells (PTEC).


METHODS
Cultured PTEC were incubated with or without thrombin to examine the effect of thrombin on FN production in PTEC. FN and transforming growth factor-beta (TGF-beta) levels were measured in culture supernatants by enzyme-linked immunosorbent assay (ELISA). Expression of FN mRNA was analysed by reverse transcriptase-polymerase chain reaction. Effects of thrombin on matrix metabolism were examined by enzyme immunoassay for the detection of secreted matrix metalloproteinase (MMP) and its inhibitors (TIMPs) as well as by zymography.


RESULTS
Thrombin stimulated FN secretion in PTEC. Thrombin also stimulated TGF-beta secretion in PTEC in a dose-dependent manner. Expression of FN mRNA by PTEC was augmented by thrombin. The stimulatory effect of thrombin on FN secretion was inhibited by neutralizing antibodies against TGF-beta but not by an irrelevant antibody. Thrombin-induced FN secretin was also inhibited by thrombin inhibitors, such as antithrombin III, hirudin and argatroban. Although thrombin stimulated TIMP-1 and -2 secretion by PTEC, the stimulatory effect of thrombin on MMP-2 was not statistically significant. Thrombin did not affect the expression of MMP-2 in zymography studies.


CONCLUSIONS
We found that thrombin stimulates FN production in PTEC without causing matrix degradation, an effect that may contribute to the formation of tubulointerstitial fibrosis associated with glomerular disease. The stimulatory effect of thrombin on FN production in PTEC is, at least in part, mediated by TGF-beta.
Shirato
Kenichi
K
Second Department of Internal Medicine, Hirosaki University School of Medicine, Hirosaki, Japan.
Osawa
Hiroshi
H
Kaizuka
Mitsuaki
M
Nakamura
Norio
N
Sugawara
Toshiyuki
T
Nakamura
Masayuki
M
Tamura
Michiko
M
Yamabe
Hideaki
H
Okumura
Ken
K
D000199:Actins / Q000096:biosynthesis / Q000235:genetics; D018929:Cell Culture Techniques; D004847:Epithelial Cells / Q000378:metabolism*; D005353:Fibronectins / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D013917:Thrombin / Q000502:physiology*; D016212:Transforming Growth Factor beta / Q000378:metabolism*
D000199:Actins; D005353:Fibronectins; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D013917:Thrombin