1 expression in rat with cyclosporine A-induced
chronic nephrotoxicity
Shi SH et al / Acta Pharmacol Sin 2004 Jan; 25 (1): 98-103
1 expression in rat with cyclosporine A-induced
chronic nephrotoxicity
Shao-hua SHI1, Shu-sen ZHENG, Chang-ku JIA, You-fa ZHU2, Hai-yang XIE3
Department of Hepatobiliary Surgery, the First Affiliated Hospital, Medical College, Zhejiang University; 2Department of Pathology, Medical College, Zhejiang University; 3Institute of Surgery, Zhejiang University, Hangzhou 310003, China
1 Correspondence to Dr Shao-hua SHI. Phn 86-571-8723-6570.
Received 2002-10-16 Accepted 2003-05-12
KEY WORDS tea; cyclosporine A; transforming growth
factor-; kidney; polymerase chain reaction;
immunohistochemistry; Western blotting
ABSTRACT
AIM: To investigate the inhibitory effect of tea polyphenols (TP)
on the transforming growth factor-1
(TGF-1) expression in rat model
of cyclosporine A (CsA)-induced chronic nephrotoxicity. METHODS: The
rat model of CsA-induced chronic nephrotoxicity was used, 4 groups of rats were
respectively treated with vehicle (0.1 mL ·kg-1·d-1
sc), TP (80 mg·kg-1·d-1 ig), CsA (15 mg·kg-1·d-1sc)
and TP plus CsA (CsA 15 mg·kg-1·d-1 sc+TP 80
mg·kg-1·d-1 ig). At the end of day 28 of treatment,
serum and urine are analyzed for creatinine clearance, kidney tissue for pathologic
analysis. The TGF-1 mRNA and its
protein expression were detected by RT-PCR, immunohistochemistry, and Western
blot. RESULTS: CsA-treated rats had increased renal expression
of TGF-1 mRNA and its protein,
compared with the vehicle- or TP- treated controls. The renal function and interstitial
fibrosis were ameliorated and renal expression of TGF-1
mRNA and its protein was decreased in animals treated with CsA plus TP, compared
with aminals treated with CsA alone (P<0.05). CONCLUSION:
TP significantly inhibits renal expression of TGF-1
in rat model of cyclosporine-induced chronic nephrotoxicity, suggesting that
the decreased renal expression of TGF-1
exerted by TP is one of mechanisms to protect renal function and tissue structure.
INTRODUCTION
Cyclosporine A (CsA) is the main immunosuppressive agent in the organ transplantation,
and its clinical impact is much enormous. Short-term (ex:1-year) kidney, heart,
liver, lung, and pancreas allograft survival rates have been greatly improved
since the clinical use of CsA. One-year hepatic allograft survival rates now
approach or surpass 80 %, when compared with the survival rates of 30 %-50 %
before clinical CsA use. However, the long-term viability of both renal and
nonrenal allograft has not shown similar improvement. At present five-year liver
allograft survival rates are 60 % or less[1-3]. Clinical retrospective
analysis has indicated that calcineurin inhibitor-based immunosuppressant such
as CsA and FK506 treatment is associated with a variety of side effects, especially
their chronic nephrotoxicity that can shorten the long-term survival rates of
allografts and patients[1,2]. The mechanisms leading to chronic nephrotoxicity
of calcineurin inhibitor are not clear. The following factors are associated
with chronic nephrotoxicity: transforming growth factor-1
(TGF-1), angiotensin II, nitric
oxide (NO), reactive oxygen species (ROS), thromboxane A2 (TXA2),
and leukotriene (LT). TGF-1 plays
an important role in the progressive interstitial fibrosis in the chronic CsA
nephrotoxicity[2]. CsA increases free radical formation in the process
of CsA nephrotoxicity. Vitamin E significantly suppresses CsA-induced lipid
peroxidation and improves glomerular filtration rate and reduces renal fibrosis[4].
The treatment of adenovirus to express superoxide dismutases significantly blocks
formation of ROS and minimizes pathological alterations and inhibition of renal
function caused by CsA[5]. Tea polyphenals (TP), as potent antioxidant,
can neutralize free oxygen radicals such as O2_, OH·
in vitro and in vivo[6]. TP effectively attenuates
the acute nephrotoxicity induced by CsA, which is contributed to the antioxidant
properties of TP[7]. The purpose of this study was to determine whether
TP could reduce the chronic CsA nephro-toxicity, and the expression of TGF-1
in tubular and interstitial cells in the rat model of CsA-induced chronic nephrotoxicity.
MATERIALS AND METHODS
Experimental design Forty adult male
Sprague-Dawley rats weighing (200-230 g) were obtained from
the Center of Experimental Animals, Zhejiang University.
They were housed in individual cages and received a
low-salt diet (0.049 % sodium, measured by flame
photometer in the Zhejiang Povincial Center of Disease
Control and Prevention). After one week on the low-salt
diet, weight-matched pairs of rats were randomly
divided into 4 groups. Group 1: rats (n=10) were
administered vehicle olive oil 1.5
mL·kg-1·d-1 sc (subcutaneous
injection). Group 2: rats (n=10) were gavaged with TP
80 mg·kg-1·d-1. Group 3: rats
(n=10) were received CsA 15
mg·kg-1·d-1 sc. Group 4: rats
(n=10) were treated with CsA 15
mg·kg-1·d-1 sc plus TP 80
mg·kg-1·d-1 ig. TP was gifted by YANG Xian-Qiang (Institute of Tea
Scence, Zhejiang University). CsA was purchased from
Sandoz Company. Body weight was recorded daily. Ten rats of each group were treated for 28 d. At the
end of treatment period, 24-h urine samples were
collected in metabolic cages. After opening the abdomen
through a middle incision under the anesthesia with
ketamine, 2 mL blood was taken from inferior vein cava
and serum was harvested immediately by centrifugation at
4 ºC and stored at -70
ºC until use. The aorta was cannulated retrogradely below the renal arteries with a
9-gauge needle. After the left renal aorta was occluded
by ligation and the right renal veins were opened by a
small incision for outflow, the kidneys were perfused
with 20 mL of cold and heparinized saline (contained
heparin 500 IU). The kidneys were removed. The cortex was carefully dissected from the medulla. The
TGF-1 mRNA and its protein expression was
assayed by RT-PCR, immunohistochemistry, and Western blot.
Functional studies Creatinine of urinary and serum, were measured by Biochemical Autoanalyzer (model 7170, HITACHI). The creatinine clearance (Ccr) was calculated using standard formulae. CsA blood levels after 4-h administration were determined in whole blood by monoclonal radioimmunoassay.
Morphology Tissue samples were fixed in 10 % buffered formalin and embedded in paraffin. Sections at 4 µm were stained with hematoxylin and eosin, and Masson trichrome. Findings ascribed to tubular injury included cellular vacuolization, tubular collapse, tubular distention, and hyalinization of afferent arterioles. For tubular injury, the following semiquantitative score was utilized[8]: 0=no tubular injury, 0.5=<5% of tubules injured, 1=5 % to 20 %, 1.5=21 % to 35 %, 2=36 % to 50 %, 2.5=51 % to 65 %, and 3=>65 %. The findings of interstitial fibrosis consisted of matrix-rich expansion of the interstitium with distortion, collapse of the tubules, and thickening of the tubular basement membranes. Interstitial fibrosis was estimated by counting the percentage of injured areas per field and was scored semiquantitatively using the following[8]: 0=normal interstitium, 0.5=<5 % of areas injured, 1=5 % to 15 %, 1.5=16 % to 25 %, 2=26 % to 35 %, 2.5=36 % to 45 %, and 3=>45 %.
TGF-1 mRNA expression by RT-PCR
The primers were designed by computer assistance
according to the gene bank. TGF-1: forward,
5´-ACTGATA-CGCCTGAGTGGCTGT-3´; reverse,
5´-CTCTGTGGA-GCTGAAGCAGTAG-3´, the size of
amplified fragment is 303 bp. Internal control GAPDH:
forward, TGGCA-CAGTCAAGGCTGAGA; reverse, CTTCTGAGTG-GCAGTGATGG; the size of amplified
fragment is 387 bp. Total RNA from each sample was
isolated using TRIzol solution (Gibco Inc, USA). Total
RNA was quantified with the ratio of absorption values
of RNA samples at 260 nm and 280 nm. For each sample, 4 µg of total RNA was reverse transcribed into
first strand of cDNA in 20-µL reaction system at
37 ºC for 50 min. Then polymerase chain reaction was per
formed from the synthesized cDNA in a 50 µL solution
containing 3 µL of cDNA, 1 µL of 25 mmol primers
(up-stream and down-stream) of TGF-1, 0.5
µL of 25 mmol primers (up-stream and down-stream)
of GAPDH, 10 mmol dNTP 1 µL, 25 mmol
MgCl2 4 µL, 10×buffer 5 µL, 0.3 µL of
Taq DNA polymerase (Promega). Amplification was performed in a thermal
cycler (MJ Research Co, USA) under the following conditions: 26 cycles of denaturation at
94 ºC for 50 s, annealing at
59 ºC for 45 s, extension at
70 ºC for 40 s, followed by a final extension for 5 min. PCR product
10 µL was electrophoresed on a 1.5 % agarose gel, and
stained with EB. The TGF-1mRNA level in each
sample was semi-quantified by comparing the
intensities of TGF-1mRNA band with those of the
internal control GAPDH band.
Immunohistochemistry Formalin-fixed tissues
were paraffin-embedded and cut into 4-µm sections.
The sections were deparaffinized in xylene and
rehydrated in graded ethanol to PBS. After blocking
endogenous peroxidase activity with 3 %
H2O2/methanol (1/32 vol/vol) for 10 min. Tissue sections was boiling for the
antigen epitope retrieval in 0.1 mol/L citrate buffer (pH
6.0) for 10 min, the tissue sections were then
interacted overnight at 4 ºC with the 1:200 diluted primary
rabbit anti-rat TGF-1 antibodies (Santa Cruz
Biotechnology), controls were incubated with the
normal rabbit serum and the PBS instead of primary
antibodies. After ample washings with PBS, the slides
were incubated for 30 min with EnVision+horseradish
peroxidase-labeled secondary antibody goat anti-rabbit
IgG (Dako Company) at room temperature, again washed extensively in PBS. The slides were then
developed in diaminobenzidine for 2-5min and rinsed with
water. The slides were then counterstained with haematoxylin. A minimum of 20 randomly selected
areas per sample were observed at ×400 magnification.
The intensity of staining for the glomerular, tubulointerstitial, and vascular compartments was
evaluated by an observer blinded to the treatment groups
using the following semiquantitative scale: 0=diffuse,
very weak, or absent staining, 1=staining involving less
than 25 %, 2=25 %-50 %, 3= 50 %-75 %, 4=70 %-100
%. Photographs were obtained at identical exposure and
development time intervals.
Western blot analysis Protein extracts were
prepared by homogenizing the 500 mg renal cortex or 500
mg medulla respectively in cold 1.5 mL RIPA buffer
containing 1 % Nonidet P-40 (Amaresco), 0.5 %
sodiumdeoxycholate (Sigma), 0.1 % sodium dodecyl sulfate (Amaresco), 15 µg phenylmethylsulfonyl
fluoride (Sigma), and 45 U aprotinin (Sigma) with a glass
tissue homogenizer. The tissue homogenates were
centrifuged at at 10000×g 4
ºC for 10 min. The supernatants were collected and centrifuged at at
10000×g,4 ºC for 10 min again. Protein concentration in the
supernatant was determined by Brandford method. 2×Loading
buffer (Tris HCl 42 mmol/L, 10 % glycerol, 2.3 %
sodium dodecyl sulfate, 5 % 2-mercaptoethanol, and
0.002 % bromophenol blue) was then added to each supernatant with equal volume containing equal amounts
of protein, which was subsequently boiled for 3 min
and then electrophoresed on a SDS-polyacrylaminde gel.
Proteins were transferred to nitrocellulse and incubated
sequentially with rabbit anti-rat TGF-1 (Santa
Cruz Biotechnology), and then with
peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology)
in the second reaction. Detection was performed with
enhanced chemiluminescence reagent (Santa Cruz Biotechnology).
Statistical analysis Results are presented as mean±SD. Analysis of variance (ANOVA), Kruska-Wallis test, and Tukey method were used by SAS8.0 statistical software. The level of statistical significance was chosen as P<0.05.
RESULTS
Physiologic studies Animals treated with CsA and CsA plus TP failed to gain body weight when compared with those receiving vehicle and TP at d 28 (P<0.05). The glomerular filtration rate (Ccr) was lower in the CsA-treated rats compared with (CsA+TP)-treated group at d 28 (P<0.05, Tab 1). There was no statistical difference in whole blood CsA level between CsA-treated animals and (CsA+TP)-treated animals (Tab 1).
Histological changes CsA-treated rats had characteristic morphologic changes similar to the chronic human lesion, mostly evident at 15 mg·kg-1·d-1 for 28 d. Tubular injury consisted of cellular and intercellular vacuolization, tubular collapse, and tubular dilation. Striped interstistial fibrosis was present with thickening of the tubular basement membranes and Bowman's caspsule. Both tubular injury and tubulointerstitial fibrosis were improved in (CsA+TP)-treated group when compared with CsA-treated group at d 28 (P<0.05, Tab1).
Tab 1. Changes in cyclosporine A (CsA) whole blood level, serum creatinine clearance, and semiquantitative scores of tubular injury and tubulointerstitial fibrosis in the experimental groups at d 28. bP<0.05 vs (CsA+TP)-treated group.
VH: vehicle olive oil 1.5 mL·kg-1·d-1 sc; TP: tea polyphenols 80 mg·kg-1·d-1 ig; CsA: cyclosporine A 15 mg·kg-1·d-1 sc; CsA+TP: CsA 15 mg·kg-1·d-1 sc+TP 80 mg·kg-1·d-1 ig.
Expression of TGF-1 mRNA by
RT-PCR TGF-1 mRNA was markedly
increased by d 28 (P< 0.05) in the CsA-treated rats when compared
with control vehicle and TP-treated rats. But expression of TGF-1
mRNA in both cortex and medulla in (CsA+TP) group was lower than that in CsA-treated
group by d 28 (P<0.05, Fig 1).
Fig 1. TGF-1 mRNA expression
in the kidney. A) The expression of TGF-1
mRNA relative to GAPDH detected by RT-PCR in vehicle, tea polyphenals (TP),
cyclosporine A (CsA), and (CsA+TP)-treated rats. B) Quantitation of mRNA expression
of TGF-1. C and M are representative
of cortex and medulla, respectively. C1-C4 indicates tissues taken from cortex
of rats treated by CsA, CsA+TP, TP, and vehicle respectively. M1-M4 indicates
tissues taken from medulla of rats treated by CsA, CsA+TP, TP, and vehicle,
respectively. The expression of mRNA was significantly increased in both cortex
and medulla in the CsA group by d 28. bP<0.05 vs
CsA+TP group.
Expression of TGF-1 by
immunohistochemistry Expression of TGF-1
by immunohistochemistry was mainly present at tubular cells, interstitial cells,
and Bowman's caspsule. The total amount of TGF-1
expression was reduced in the kidneys of (CsA+TP)-treated rats as compared to
CsA-treated rats at d 28 (P<0.05, Fig 2). In the control groups the
expression of TGF-1 protein was
very weak.
Fig 2. Expression of transforming growth factor-1
(TGF-1) by immunohistochemistry
assay. Photomicrograph (A) showed that cyclosporine A (CsA) 15 mg×kg-1×
d-1 sc induced great expression of TGF-1.
(B) In rats treated with CsA 15 mg×kg-1× d-1
sc+TP 80 mg×kg-1× d-1 ig showed mild to moderate
staining (×600). (C) Pathological semiquantitative scores for TGF-1
expression. bP<0.05 vs the CsA-treated group.
Expression of TGF-1 by
Western blot analysis The total amount of TGF-1
protein expression in both cortex and medulla was inhibited in CsA plus TP-treated
group by 28 d (P<0.05), when compared with the CsA-treated group.
In the control group the expression of TGF-1
protein was very weak (Fig 3).
Fig 3. TGF-1 expression in
the kidney by Western blot. A) TGF-1
expression in cortex and medulla of rat kidneys. B) Quantitative scoring of
TGF-1 expression. C and M are representative
of cortex and medulla, respectively. C1-C4 indicates tissues taken from cortex
of rats treated by CsA, CsA+TP, TP, and vehicle, respectively. M1-M4 indicates
tissues taken from medulla of rats treated by CsA, CsA+TP, TP, and vehicle,
respectively. bP<0.05 vs the CsA-treated group.
DISCUSSION
We examined an experimental model of chronic CsA nephrotoxicity with pathologic
features similar to the human lesion characterized by tubular atrophy, progressive
striped interstitial fibrosis with loss of cellularity in areas of fibrosis,
afferent arteriole hyalinization
and glomerulosclerosis[2,8]. In our experiment CsA promoted the total
protein expression of renal tissue TGF-1
which was mainly expressed in tubular and interstitial cells. Antioxidant TP
inhibited the CsA-induced TGF-1
expression and interstitial fibrosis. Bioptic tissues taken from renal allografts
with CsA nephrotoxicity are detected by immunohistochemistry that TGF-1
expression is also mostly concentrated in tubular cells[9]. CsA in
a dose-dependent manner stimulates expression of TGF-1
protein and steady-state mRNA levels in both proximal tubular cells and tubulointerstitial
fibroblasts[10]. TGF-1
mimics and anti-TGF-1 antiboby
or soluble TGF- II receptor rogates
the chronic nephrotoxicity of CsA in vivo. TGF-1
promotes fibronectin and collagen synthesis[11,12] . These data suggest
that TGF-1 play an important role
in mediating CsA chronic nephrotoxicity.
The mechanisms by which antioxidant TP inhibits the TGF-1
expression in CsA-induced chronic nephrotoxicity may be the following: (1) TP
increases tissue bioactivity of superoxide dismutase and glutathione peroxidase
which can inactivate free oxygen radicals such as O2-.
Because O2- can react with NO by forming ONOO2-
to loose bioactivity of NO, TP may reduce nitric oxide (NO) loss and maitain
NO physical function[7,13]. In the experiment of chronic CsA nephrotoxi-city,
N-nitro-L-arginine-methyl ester (L-NAME) as the NO synthase
inhibitor, strikingly worsens the glomerular filtration rate and the CsA-induced
fibrosis, and up-regulates expression of TGF-1,
plasminogen activator inhibitor-1 (PAI-1); L-arginine, as the substrate
for NO synthase, has the opposite beneficial effect[14]. (2) TP may
inhibit the synthesis of TXA2 and LT which are mediated by CsA-induced
lipid peroxidation. CsA induces the glomerular synthesis of O2-,
H2O2, malondial-dehyde. Vitamin E minimizes the adverse
effects of CsA on kidney function and the glomerular synthesis of these compounds[15].
The treatment of adenovirus to express superoxide dismutase significantly blocks
formation of ROS and minimizes pathological alterations and inhibition of renal
function caused by CsA[5]. CsA can promote the expression of cycloxy-genase
I (COX I), which was completely inhibited by vitamin E in chronic CsA nephrotoxicity[16].
COX I can promote the synthesis of TXA2. F2-isoprostane
produced by a noncyclo-oxygenase mechanism involving lipid peroxidation, as
a potent preglomerular vasoconstrictor acting principally through the interaction
with TXA2 receptor, also may be involved in the CsA nephrotoxicity
by CsA lipid peroxidation[17]. The synthesis of leukotrienes (LT)
which are produced by the 5-lipoxygenase pathway is caused by the CsA-induced
lipid peroxidation. Peptidoleukotriene as one kind of LT can stimulate fibroblast
proliferation and extracellular matrix synthesis. LT receptor antagonist can
largely block the CsA-induced renal vasoconstriction and morphological impairment[18].
Treatment with TXA2 receptor antagonist GR32191 has a similar effect
as thromboxane inhibitors in CsA nephrotoxicity[19]. (3) The potent
vasoconstriction effects of TXA2 and LT contribute to the activation
of the renin-angiotensin system. Angiotensin II can cause the excessive expression
of TGF-1 and osteopontin which
are associated with renal interstitial fibrosis. Osteopontin is also a macrophage
chemoat-tract[20]. There are intrarenal angiotensin II deposits and
excessive expression of angiotensin II type I receptor in renal medulla[21].
Angiotensin II type 1 receptor antagonist losartan can markedly reduce the degree
of CsA-induced tubulointerstitial fibrosis, and the expression of TGF-1
and osteopontin[22] .
These data suggested that antioxidant TP significantly ameliorated CsA-induced
chronic nephrotoxicity and inhibited the expression of TGF-1
in CsA-induced chronic nephrotoxicity.
REFERENCES
II receptor in rats with
overload proteinuria. J Am Soc Nephrol 1999; 10: 537A (abstract).