S. Yamamoto et al.

expression (Table 2). ET-1 at a concentration of10 –6 M significantly increased ET-1 mRNA expression 1.77 fold and ECE mRNA expression 1.38
fold, suggesting that it may mediate biological action through an autocrine loop (Fig. 6).

Discussion
In this study, we investigated the expressionof ETAR and ETBR in rat mesangial cells(CRL-2573). ET-1 has been reported to promote
the contraction, hypertrophy and proliferation ofcells, and the synthesis of the extracellular matrix(Herman et al., 1998; Duan et al., 1999; Chen etal., 2001). The addition of ET-1 to CRL-2573
cells increased DNA synthesis and cell numbers,
but the rates of increase were not very high. ET-1
stimulates mesangial cell proliferation in an autocrine fashion as well as through the paracrine
loop, where our data indicates that ET-1 increased
the expression of ET-1 and ECE. The proliferating effects of ET-1 in mesangial cells are primarily via ETAR (Benigni, 2000; Hocher et al., 2003),
although there are data suggesting that ETBR mediates human mesangial cell proliferation (Hermanet al., 1998; Benigni, 2000). In the present study,
although antagonist administration apparently did
not change DNA synthesis, the administration of
the ETAR antagonist suppressed cell proliferation, strongly suggesting that ETAR mediates cellproliferation. More interestingly, the addition of
ET-1 also increased the expression of mRNA for
ET-1 and ECE, suggesting that ET-1 functions in
an autocrine and paracrine manner. This indicates that much higher levels of locally produced
ET-1 than those in circulating plasma promote tissue ﬁbrosis.
A major role of ET-1 in mediating renal ﬁbrosis has recently been pointed out. Antagonismof ET-1 receptors delayed the evolution of renal
failure and increased survival rate in rats withrenal mass reduction (Hocher et al., 2003; Boffaet al., 2001; Goddard et al., 2004). Moreover, theuse of an endothelin receptor antagonist improved

Fig. 5. Effects of ET-1 or AT-II on CRL-2573 matrixrelated gene expression. The expressions of mRNA forprocollagen α1 (I) (a), TGF-β1 (b), CTGF (c), TIMP-1 (d)and MMP-13 (e) were determined by real-time quantitative PCR. Data were normalized to β2-microglobulin
mRNA levels. Error bars represent mean ± SD of 4 independent experiments.

34

Effects of ET-1 and AT-II on mesangial cells

Table 2. Effects of ET-1 and endothelin receptor antagonist on CRL-2573 matrix-related gene expression
mRNA
Procollagen α1 (I)ET-1 (10 –6 M)

Control
1.01 ± 0.23

2.16 ± 0.17
P < 0.01

TGF-β10.52 ± 0.25

CTGF

0.19 ± 0.08

TIMP-10.35 ± 0.11

MMP-130.51 ± 0.16

P < 0.05
P < 0.05
P < 0.05
P < 0.05

ET-1 (10 –6 M)
BQ123 (10 –6 M)
BQ788 (10 –6 M)
P < 0.05

1.02 ± 0.22
0.36 ± 0.04

P < 0.05

P < 0.05

0.91 ± 0.17
P < 0.01

0.75 ± 0.09

1.53 ± 0.09
0.95 ± 0.15

1.18 ± 0.24

P < 0.05

0.68 ± 0.27

0.19 ± 0.08

0.29 ± 0.19

1.57 ± 0.12

1.22 ± 0.23

0.70 ± 0.16

0.71 ± 0.12
P < 0.05

Data are mean ± SD of 4 independent experiments.
Each mRNA was normalized to β2-microglobulin mRNA.

renal structural damage and reduced extracellularmatrix formation (Benigni, 2000; Boffa et al.,
2001; Hocher et al., 2003; Chade et al., 2003). In
the present study, ET-1 increased procollagen α1(I) gene expression in a dose-dependent manner.
In addition, ET-1 dose-dependently increased theexpression of TGF-β1, as well as CTGF. TGF-β1
promotes fibroblast proliferation and increases
extracellular matrix production (Gomez-Garre etal., 1996; Boffa et al., 2003; Sorokin and Kohan,2003). TGF-β1 is thought to play a central role in
the progression of renal ﬁbrosis. CTGF is locateddownstream from TGF-β1 and acts proﬁbrogenically. As clearly indicated in these ﬁndings, ET-1
increases the production of the collagen-synthesizing system. According to one study, ET-1
increases the expression of the fibronectin andprocollagen α1 (I) and (IV) genes in mesangialcells (Gomez-Garre et al., 1996). ET-1 also increased the collagen-degrading system inhibitor
TIMP-1, suggesting that collagen degradation
may be inhibited, thereby promoting ﬁbrosis, although increased MMP-13 mRNA gene expression was observed.
In rat models of mesangial proliferative
glomerular nephritis, ETAR blocker reduced
mesangial cell proliferation, and bosentan(ETAR/ETBR blocker) improved renal function
and reduced mesangial cell ET-1 mRNA levels.
There is abundant evidence in vivo that ET-1 antagonists ameliorate glomerular injury in animalmodels with diabetic nephropathy (Nakamuraet al., 1995; Benigni, 2000). Bosentan reduced
albuminuria, increased glomerular filtration

Fig. 6. Effects of ET-1 on ET-1 and ECE transcript levels of CRL-2573. The expressions of mRNA for ET-1
and ECE were determined by real-time PCR. The data
were normalized by β2 -microglobulin mRNA. Error
bars represent mean ± SD of four independent experiments.

35

S. Yamamoto et al.

5 Boffa JJ, Tharaux PL, Placier S, Ardaillou R,Dussaule JC, Chatziantoniou C. Angiotensin II activates collagen type I gene in the renal vasculatureof transgenic mice during inhibition of nitric oxidesynthesis. Circulation 1999;100:1901–1908.
6 Chade AR, Best PJ, Rodriguez-porcel M, Herrmann
J, Zhu X, Sawamura T, et al. Endothelin-1 receptor
blockade prevents renal injury in experimental hypercholesterolemia. Kidney Int 2003;64:962–969.
7 Chen HC, Guh JY, Chang JM, Lai YH. Differential
effects of FMLP-activated neutrophils from patientswith IgA nephropathy enhanced endothelin 1 production of glomerular mesangial cells. Nephron2001;89:274–279.
8 Duan SB, Liu FY, Luo JA, Peng YM. Assessment ofurinary endothelin-1 and nitric oxide levels and theirrelationship with clinical and pathologic types inprimary glomerulonephritic. Yonsei Med J 1999;40:425–429.
9 Eddy AA. Molecular basis of renal ﬁbrosis. Pediatr
Nephrol 2000;15:290–301.
10 Goddard J, Johnstone NR, Hand MF, Cumming AD,Rabelink TJ, Rankin AJ, et al. Endothelin-A receptor antagonism reduces blood pressure and increases
renal blood ﬂow in hypertensive patients with chronic renal failure. Circulation 2004;109:1186–1193.
11 Gomez-Garre D, Ruiz-Ortega M, Ortego M, Largo R,
Lopez-Armada MJ, Plaza JJ, et al. Effects and interactions of endothelin-1 and angiotensin II on matrix
protein expression and synthesis and mesangial cell
growth. Hypertension 1996;27:885–892.
12 Her ma n W H, Ema ncipator SN, R hoten R LP,
Simonson MS. Vascular and glomerular expressionof Endothelin-1 in normal human kidney. Am J
Physiol 1998;275:F8–F17.
13 Hocher B, Thone-Reineke C, Rohmeiss P, Schmager
F, Slowinski T, Burst V, et al. Endothelin-1 transgenic mice develop glomerulosclerosis, interstitialﬁbrosis, and renal cysts but not hypertension. J Clin
Invest 1997;99:1380–1389.
14 Hocher B, Kalk P, Slowinski T, Godes M, Mach A,
Herzfeld S, et al. ETA receptor blockade induces
tubular cell proliferation and cyst growth in rats
with polycystic kidney disease. J Am Soc Nephrol
2003;14:367–376.
15 Mezzano SA, Ruiz-Ortega M, Egido J. Angiotensin
II and renal fibrosis. Hypertension 2001;38(Pt 2):
635–638.
16 Mishra R, Leahy P, Simonson MS. Gene expression proﬁle of endothelin-1-induced growth in glomerular mesangial cells. Am J Physiol Cell Physiol
2003;285:C1109–C1115.
17 Muller D, Mullally A, Dechend R, Park JK, Fiebeler
A, Pilz B, et al. Endothelin-converting enzyme inhibition ameliorates angiotensin II-induced cardiac
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rate and ameliorated the mesangial matrix indiabetic rats (Benigni et al., 1998, 2000). Ourﬁndings also indicated that the increased expression of procollagen α1 (I) induced by ET-1 wasattenuated by both ETAR and ETBR blockers(BQ123 and BQ788). However, the ET-1 enhanced expression of TGF-β1 and TIMP-1 wasreduced by ETBR blocker. The causes of thesereceptor-related differences are not clear. Since
nephropathy is improved by the administrationof ETAR antagonists, ETAR has been considered responsible. As the data we obtained in thepresent study suggest, ETBR is also involved
in increased production of ECM. These results
may reflect our culture conditions for the cellline CRL-2573, necessitating further study. Touse ET-1 antagonists as therapeutic agents, it isnecessary to compare the effects of an ETAR antagonist and a combination of ETAR and ETBR
antagonists.
On the other hand, the addition of AT-II did
not change the cell growth or the gene expressionof procollagen α1 (I) or TGF-β1, although theexpression of AT-II type 1a receptor mRNA wasobserved in the cell line. Therefore, there may
be abnormalities in the receptors or the signaltransduction system, and this cell line would not
be suitable for the study of the angiotensin system.
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Received December 19, 2005; accepted January 4, 2006
Corresponding author: Satoru Yamamoto37