Yonago Acta medica 2006;49:29–37

Effects of Endothelin-1 and Angiotensin-II on Extracellular
Matrix Metabolism in a Rat Mesangial Cell Line, CRL-2573
Satoru Yamamoto, Masahiko Koda, Masaru Ueki, Chishio Munemura, Satoko
Maeta and Yoshikazu Murawaki
Division of Medicine and Clinical Science, Department of Multidisciplinary Internal Medicine,
School of Medicine, Tottori University Faculty of Medicine, Yonago 683-8504 Japan

The proﬁbrogenic role of endothelin-1 (ET-1) and angiotensin-II (AT-II) in renal ﬁbrosis
remains disputed. We therefore studied the effect of ET-1 and AT-II on rat mesangial
cells (CRL-2573), the major source of extracellular matrix proteins in the kidney. ET-1
stimulated DNA synthesis assessed by BrdU uptake in microtiter tetrazolium assay in adose dependent manner, but AT-II did not. Proliferation of the mesangial cells by ET-1
was suppressed by 15% with BQ123, an endothelin receptor A antagonist, which suggested a signiﬁcant role of the endothelin receptor A on growth stimulation. In the mesangial cells, ET-1 increased the mRNA expressions of procollagen α1 (I), transforming
growth factor-β1, connective tissue growth factor, tissue inhibitor of metalloproteinase-1
and matrix metalloproteinase-13 in dose dependent manners, whereas AT-II had exerted
no effect on the expression of these mRNAs. These data suggested that ET-1 might beinvolved in renal ﬁbrosis through the mesangial cell proliferation and/or the increase inﬁbrogenic cytokines.
Key words: angiotensin-II; endothelin-1; extracellular matrix metabolism; mesangial cell

have potent vascular smooth muscle-contracting
activity (Yanagisawa et al., 1988), and promote
cell proliferation and protein synthesis. Indeed,
overexpression of ET-1 in transgenic mice causes
renal interstitial fibrosis (Hocher et al., 1997),
glomerulosclerosis (Herman et al., 1998; Hocher
et al., 1997), renal cyst formation (Hocher et al.,1997, 2003), and arteriolosclerosis (Boffa et al.,2001, 2003). By contrast, ET-1 antagonists attenuate progressive glomerulosclerosis in vivo (Boffaet al., 1999, 2001; Yao et al., 2001). Similarly,
AT-II plays an important role in the developmentof renal ﬁbrosis (Sun et al., 2000; Mezzano et al.,Renal fibrosis and nephrosclerosis are pathological states common to nephropathy of various types, such as glomerulonephritis, diabetic
nephropathy and hypertensive nephropathy. In
various diseases, accumulation of extracellularmatrix, that is, progression of renal ﬁbrosis causes
further deterioration of renal function (Sun et al.,
2000; Boffa et al., 2003). It has become increasingly clear that various cytokines and vasoactive
factors are involved in extracellular matrix accumulation (Eddy, 2000; Yao et al., 2001; Mishra
et al., 2003). Among the vasoactive factors,
endothelin-1 (ET-1) and angiotensin-II (AT-II)

Abbreviations: AT-II, angiotensin-II; BrdU, bromodeoxyuridine; CTGF, connective tissue growth factor; DMEM,
Dulbecco’s modiﬁed Eagle medium; ECE, endothelin-converting enzyme; ECM, extracellular matrix; ELISA, enzymelinked immunosorbent assay; ET-1, endothelin-1; ETAR, endothelin A receptor; ETBR, endothelin B receptor; FBS,
fetal bovine serum; MMP-13, matrix metalloproteinase-13; MTT, microtiter tetrazolium; OD, optical density; PBS,
phosphate buffered saline; TGF-β1, transforming growth factor-beta 1; TIMP-1, tissue inhibitor of metalloproteinases-1

29

S. Yamamoto et al.

Island, NY), containing 200 U/mL penicillin G
sodium (Gibco-BRL), 200 µg/mL streptomycin
sulfate (Gibco-BRL), 0.25 µg/mL amphotericin B(Gibco-BRL) and 10% fetal bovine serum (FBS;
JRH Bioscience, Lenexa, KS). Incubation was
performed in a humidiﬁed atmosphere of air/CO2(95:5) in an incubator maintained at 37˚C. The
cells were subcultured in DMEM with 10% FBS,
penicillin G sodium (200 U/mL), streptomycin
sulfate (200 µg/mL) and amphotericin B (0.25 µg/mL), and the medium was changed every 24 h.

2001; Boffa et al., 2003). Rats chronically infusedwith AT-II develop tubulointestitial injuries withtubular atrophy and dilation, cast formation andintestinal ﬁbrosis (Hocher et al., 1997; Mezzano etal., 2001). It has been shown that ET-1 and AT-II
are involved in the progression of various pathological states, such as acute renal failure, chronic
renal failure and chronic heart failure (Benigni
et al., 2000; Muller et al., 2002). However, muchof their association with renal matrix metabolism
remains unclear. In this study, we examined theeffects of ET-1 and AT-II on cell proliferation andmatrix metabolism in mesangial cells, which are
the main cells producing extracellular matrix inthe kidney (Schnaper et al., 1996; Yao et al., 2001;
Sorokin et al., 2003).

Measurement of DNA synthesis and cell
multiplication
Cell multiplication was measured by MTT assay.
DNA synthesis was measured as the incorporation of BrdU into DNA. Cells were plated in
96-well plates at a density of 2 × 103 cells/well,
4 × 103 cells/well, 8 × 103 cells/well or 16 × 103
cells/well in DMEM with 15% FBS. After 24 h,
the cells were washed with phosphate bufferedsaline (PBS; Gibco-BRL) (pH 7.2) and placed in
DMEM with 0.125% FBS for 6 h. This medium
was removed and the cells were placed in fresh
DMEM with 0.125% FBS and BrdU (2 × 10 –5 L/well). After 6 h of incubation with BrdU at 37˚C,
the BrdU incorporated into DNA was measured
by enzyme-linked immunosorbent assay (ELISA).
After 2 h of incubation with the cell counting
kit-8 solution (1 ×10 –5 L/well), the absorbance of
formosan, which is produced by WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodiumsalt], the main component of cell counting kit-8,
was measured at 450 nm using microplate reader.
Subsequent experiments were performed at 4 ×
103 cells/well. The cells were plated in 96-welldishes at a density of 4 × 103 cells/well in DMEM
with 15% FBS for 24 h. After the cells were
washed with PBS, they were placed in fresh
DMEM with 0.125% FBS containing ET-1 or ATII at various concentrations (10 –6 M, 10 –8 M or
10 –10 M). After 6 h of incubation with BrdU (2× 10 –5 L/well) or cell counting kit-8 solution (1 ×Materials and Methods
Materials
CRL-2573, a rat mesangial cell line was purchased from ATCC (Manassas, VA). BQ123, anantagonist for endothelin A receptor (ETAR),
and BQ788, for endothelin B receptor (ETBR),
as well as ET-1, were purchased from Alexis
Biochemicals (San Diego, CA). A bromodeoxyuridine (BrdU) colorimetric cell proliferation
enzyme-linked immunosorbent assay kit waspurchased from (Roche Diagnostics, Mannheim,
Germany). A cell count kit-8 was purchased from
Dojindo Laboratories (Kumamoto, Japan). The
primers for real-time polymerase chain reaction(PCR) were synthesized at Nihon Gene ResearchLaboratories (Sendai, Japan), and Superscript II
RNaseH(–) reverse transcriptase was purchasedfrom Invitrogen (Carlsbad, CA). Random hexamers and oligo (dT) primers were purchased from
Promega (Tokyo, Japan).
Cell culture
A cell pellet was suspended in Dulbecco’s modiﬁed Eagle medium (DMEM; Gibco-BRL; Grand30

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

Master SYBR Green I kit (Roche). The appropriate size of each PCR product was veriﬁed on an
ethidium bromide-stained 3% agarose gel. Conditions for real-time PCR were as follows: 10 min
denaturing at 95˚C, 10 s annealing at 55˚C, and3 s to 6 s ampliﬁcation at 72˚C. Forty-ﬁve cycles
were performed and then followed by meltingcurve analysis to verify the correctness of theamplification. Amplification efficiency was determined for all primer pairs. The efﬁciency of
the PCR was determined by analyzing a dilution
series of control cDNA. Using the analysis modeof the Light Cycler software package, the slope oflog concentrations of the control cDNA dilutions
was calculated. Efﬁciency was calculated as follows: Eff = 10 –1/slope.
T he measu re d ef f iciencies were use dfor the calculation of the mRNA values. β2 Microglobulin, a stable housekeeping gene in thiscell system, was analyzed in parallel with every
PCR. The resulting β2 -microglobulin values
were used as the standard for the presentation of
the mRNA data of the different transcriptions.
Analysis of the data was performed according
to the manufacturer’s instructions, using Light
Cycler software version 3.5.3. Oligonucleotide
primers were designed according to the publishedsequences (Table 1).

10 –5 L/well) at 37˚C, the BrdU uptake or formosan was measured. To assess the effect of ETAR
or ETBR on DNA synthesis and cell proliferation,
the cells were incubated with ETAR antagonist
BQ123 (10 –6 M) or ETBR antagonist BQ788 (10 –6
M).
Expression of matrix related genes
Cell culture with ET-1 and AT-II
The cells were plated on 25 cm2 dishes at a density of 4 × 103 cells/dish in DMEM containing 10%
FBS. After 48 h of culture, the cells were washedwith PBS and placed in DMEM with 0.125% FBS
for 12 h. Cells were incubated for 12 h in fresh
DMEM with 0.125% FBS containing ET-1 or ATII at various concentrations (0, 10 –8 and 10 –6 M),
and the total RNA was extracted. To assess theeffect of ETAR or ETBR on expression of procollagen α1 (I), transforming growth factor-beta1(TGF-β1), connective tissue growth factor (CTGF),
tissue inhibitor of metalloproteinases-1 (TIMP-1),
matrix metalloproteinase-13 (MMP-13), ET-1 andendothelin-converting enzyme (ECE) mRNA,the cells were incubated using BQ123 (10 –6 M) or
BQ788 (10 –6 M) with ET-1 (10 –6 M).
RNA isolation and reverse transcription
Total RNA of the conﬂuent CRL-2573 in 25 cm2
dishes was extracted using the acid-phenol guanidium method. Total RNA (1 × 10 –5 L) was reverse transcripted in a ﬁnal volume of 2 × 10 –5 L
containing 1× RT buffer (5 × 10 –4 M each dNTP,
3 × 10 –3 M MgCl2, 7.5 × 10 –2 M KCl, 5 × 10 –2
M Tris-HCl, pH 8.3), 10 units of Superscript II
RNaseH(–) reverse transcriptase, 50 mg/ L random hexamers 1 × 10 –6 L, 1 × 10 –7 M oligo (dT)primer 5 × 10 –7 L.

Statistical analysis
Statistical analysis was performed using the Statview Version 5.0 (SAS Institute, Cary, NC). Signiﬁcance was calculated using the unpaired t-test.
Statistical signiﬁcance was regarded as P < 0.05.

Results

Real-time PCR with light cycler system
We used real-time PCR to quantify mRNAs ofprocollagen α1 (I), TGF-β1 , CTGF, TIMP-1,
MMP-13, ET-1 and ECE mRNA. Real-time
PCR was performed using a Light Cycler System(Roche, Tokyo) and a Light Cycler-FastStart DNA

Effects of ET-1 and AT-II on DNA synthesis and multiplication of CRL-2573 cells
In the cells, BrdU incorporation into DNA increased with cell density: 1.04 ± 0.09 OD at 450
nm at 2 × 103 cells/well, 1.51 ± 0.16 OD at 4 × 103
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S. Yamamoto et al.

Table 1. Primer nucleotide-sequence

β 2-Microglobulin

Forward primer 5'-CCGATGTATATGCTTGCAGAGTTAA-3'
Reverse primer 5'-CAGATGATTCAGAGCTCCATAGA-3'

Procollagen α 1( I )Forward primer 5'-TCCGGCTCCTGCTCCTCTTA-3'
Reverse primer 5'-GTATGCAGCTGACTTCAGGG-3'

Transforming growth factor-beta 1 (TGF-β 1)

Forward primer 5'-TTGCCCTCTACAACCAACAG-3'
Reverse primer 5'-GGCTTGCGACCCACGTAGTA-3'

Connective tissue growth factor (CTGF)

Forward primer 5'-TCCCTGCGACCCACACAA-3'
Reverse primer 5'-TGCAACTGCTTTGGAAGGAC-3'

Tissue inhibitor of metalloproteinases-1 (TIMP-1) Forward primer 5'-TGCTCTTGTTGCTATCATTGATAGCTT-3'
Reverse primer 5'-CGCTGGTATAAGGTGGTCTCGAT-3'
Matrix metalloproteinase-13 (MMP-13)

Forward primer 5'-GGAAGACCCTCTTCTTCTCA-3'
Reverse primer 5'-TCATAGACAGCATCTACTTTGTC-3'

Endothelin-1 (ET-1)

Forward primer 5'-CAAGCTGGGAAAGAAGTGT-3'
Reverse primer 5'-ATGTGCTCGGTTGTGTATC-3'

Endothelin-converting enzyme (ECE)

Forward primer 5'-AGCTCTTCTTCCTGGGATT-3'
Reverse primer 5'-CCAGACTTCGCATTTGTG-3'

Angiotensin-II (AT-II) type 1a receptor

Forward primer 5'-TGTTCTGACCTTCTTGGATG-3'
Reverse primer 5'-AAACACACTACGGAGCTTCT-3'

Endothelin A receptor (ETAR)

Forward primer 5'-ACCAGTCGAAAAGCCTCA-3'
Reverse primer 5'-TCTGCACAGGGTTAGTTCA-3'

Endothelin B receptor (ETBR)

Forward primer 5'-AACTTCCGCTCGAGCAAT-3'
Reverse primer 5'-TCCCGAGGCTTCATTCAT-3'

Fig. 1. Reverse transcription-PCR. Expressions of mRNA for AT-II type 1a receptor,
ETAR and ETBR were found by 3% agarose gel electrophoresis.

cells/well, 1.81 ± 0.09 OD at 8 × 103 cells/welland 2.08 ± 0.06 OD at 16 × 103 cells/well. Subsequent experiments were performed at 4 × 103
cells/well. The expression of mRNA for the AT-

II type 1a receptor, for ETAR and for ETBR in
mesangial cells was conﬁrmed by electrophoresis
on 3% agarose gels (Fig. 1). The addition of ET-1
to CRL-2573 cells increased DNA synthesis by32

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

Fig. 2. Effects of ET-1 or AT-II on DNA synthesis of
CRL-2573. BrdU incorporation into DNA was measured in 4.0 × 103 cells/well over 6 h at various concentrations of ET-1 or AT-II. The mean value of absorbancefor controls was set at 100%. Data represent mean ± SD
(8 wells).

Fig. 3. Effects of ET-1 or AT-II on CRL-2573 multiplication. Cell number was measured in 4.0 × 103 cells/
well over 6 h at various concentrations of ET-1 or AT-II,
using a cell count kit-8. The mean value of absorbancefor was set as 100%. Data represent mean ± SD.

20% at 10 –10 M, 29% at 10 –8 M and 33% at 10 –6
M (Fig. 2) and the number of cells by 2% at 10 –10
M, 4% at 10 –8 M and 9% at 10 –6 M (Fig. 3) ina dose-dependent manner. AT-II did not affectDNA synthesis (–2% at 10 –10 M, –2% at 10 –8 M
and 10% at 10 – 6 M) or multiplication (–2% at
10 –10 M, 0% at 10 –8 M and –1% at 10 –6 M) of

CRL-2573 (Figs. 2 and 3). The ETAR antagonist,
BQ123 inhibited the ET-1 enhanced multiplication of CRL-2573 by 11%. Similarly, the ETBR
antagonist BQ788, inhibited the ET-1 enhanced
multiplication of CRL-2573 by 8% (Fig. 4).
Effects of ET-1 and AT-II on mRNA expression of CRL-2573 cells
ET-1 at concentration of 10 – 8 M and 10 – 6 M
increased the expression of mRNA for procollagen α1 (I) (1.27- to 1.57-fold), TGF-β1 (1.83to 1.90-fold), TIMP-1 (1.60- to 1.80-fold) and
MMP-13 (1.76- to 2.62-fold), compared with thatof controls (Figs. 5a to e). Meanwhile, AT-II
had no affect on these mRNA expressions (Figs.
5a to e).
Both BQ123 and BQ788 suppressed the ET-1
enhanced expression of mRNA for procollagen α1(I), TGF-β1 and MMP-13 by 54%, 73% and 74%
respectively. While, BQ123 completely blocked
CTGF mRNA expression enhanced by ET-1, but
BQ788 exerted no effect. BQ123 did not affectthe expression of mRNA for TIMP-1, but BQ788
tended to suppress ET-1 enhanced TIMP-1 mRNA

Fig. 4. Effects of endothelin receptor antagonists on cellnumber of CRL-2573. Cell number was measured in 4.0
× 103 cells/well over 6 h at 10 –6 M of ET-1. The cells
were treated with BQ123, ETAR antagonist, or BQ788,
ETBR antagonist, at a concentration of 10 –6 M in thepresence of 10 –6 M of ET-1.

33