Expression of connective tissue growth factor following Sodium Ferulate in rats with unilateral ureteral obstruction☆

Xie Xi-sheng1, Zuo Chuan1, Mi Xu-hua1, Ma Ai-jing1, Wang Dong-wen2, Fu Ping1

1Department of Nephrology, West China Hospital of Sichuan University, Chengdu 610041, Sichuan Province, China; 2College of Public Health, Si-chuan University, Chengdu 610041, Sichuan Province, China

Xie Xi-sheng☆, Studying for doctor-ate, Chief physician, Department of Nephrology, West China Hospital of Sichuan University, Chengdu 610041, Sichuan Province, China
xishengx@yahoo. com.cn

Xie Xi-sheng and Zuo Chuan Contrib-uted equally to this article

Correspondence to: Fu Ping, Department of Nephrology, West China Hospital of Sichuan University, Chengdu 610041, Sichuan Province, China
fupinghx@163.com

Received: 2007-11-12 Accepted: 2008-01-14 (07-50-10-5656/H)

Xie XS, Zuo C, Mi XH, Ma AJ, Wang DW, Fu P. Expression of connective tissue growth factor fol-lowing Sodium Ferulate in rats with unilateral ureteral obstruc-tion.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(28): 5562-5566 (China)
[www.zglckf.com/ zglckf/ejournal/ upfiles/08-28/ 28k-5562(ps).pdf]

Abstract
BACKGROUND: Connective tissue growth factor (CTGF) is a kind of factor that can mediate downstream action of transforming growth factor-β1 (TGF-β1). The upregulation of connective tissue growth factor expression plays an important role in pathological changes of renal interstitial fibrosis.
OBJECTIVE: To explore the effect of Sodium Ferulate on the expression of CTGF mRNA and protein in rats with unilateral ureteral obstruction (UUO) and pathological changes of renal interstitial fibrosis, and to compare with Losartan.
DESIGN: Randomized and controlled animal trial.
SETTING: Department of Nephrology, West China Hospital of Sichuan University, and College of Public Health, Sichuan University.
MATERIALS: Twenty-four healthy adult male SD rats were selected from the Experimental Animal Center of Sichuan University. Sodium Ferulate was provided by Sichuan Hengda Pharmacy Co, Ltd (No. 050302); rabbit anti-rat CTGF by Santa Cruz; Western blotting by BioRAD, USA; DNA Engine OpticonTM real-time fluorescent quantitation PCR device by MJ Research, USA.
METHODS: The experiment was performed at Research Laboratory of Clinical Medicine (grade BSL-1), College of Public Health, Sichuan University from May to December 2006. Twenty-four healthy rats were randomly divided into 4 groups (n=6): UUO model group, Sodium Ferulate group, Losartan group, and sham-operation group. According to the previous protocol, UUO models were established in UUO model group, Sodium Ferulate group, and Losartan group, and the other rats were subjected to sham operation. From the first day after UUO, Sodium Ferulate group was intragastrically (i.g.) administrated with 150 mg/kg/d Sodium Ferulate; Losartan group was administrated ig. with 20 mg/kg/d. Losartan; UUO and sham operation groups were administrated i.g. with matching normal saline. All rats were executed 14 days after UUO to harvest partial renal tissues. All experimental procedure was accorded with animal ethical standards.
MAIN OUTCOME MEASURES: The mRNA and protein expressions of CTGF were quantified by real-time PCR and Western blot. The pathological changes of renal interstitial tissues were observed by hematoxylin/eosin (HE) and Masson staining.
RESULTS: Twenty-four rats were included in final analysis. Fourteen days after UUO, CTGF mRNA and protein expressions in UUO model group were significantly increased compared with sham operation group, but the expressions in Sodium Ferulate group were significantly lower than model group (P < 0.05). Compared with Losartan treated group, there was no significant difference (P > 0.05). HE and Masson staining showed inflammatory cell infiltration and tubular and interstitial changes as well as collagen deposition in renal interstitial tissues on day 14 after UUO. Sodium Ferulate obviously improved the renal pathological changes in UUO rats (P < 0.05), and the effect was similar to Losartan (P > 0.05).
CONCLUSION: Sodium Ferulate inhibits UUO-induced renal interstitial fibrosis. This action, similar to the effect of Losartan, might be due to downregulation of CTGF expression.

INTRODUCTION

Tubular interstitial fibrosis is the common pathway to the end-stage renal diseases. Transforming growth factor-β1 (TGF-β1) is a well-recognized profibrotic factor. It is reported that connective-tissue growth factor (CTGF) is the downstream action factor of TGF-β1. In addition, abnormal expression of CTGF plays an important role in the occurrence and progression of renal interstitial fibrosis[1-4].
Ferulaic acid, one of the derivates of cassic acid, is always found in angelica, ligustici, and other plants. Sodium Ferulate is synthesized sodium salt of ferulaic acid, and has been demonstrated as a nonpeptide endothelin receptor antagonist [5]. Our previous studies suggest Sodium Ferulate can improve serum creatinine and urine protein levels in 5/6 nephrectomized rats, inhibit fibroblast production and interstitial cells proliferation in remnant kidney and delay glomerular sclerosis and renal interstitial fibrosis[6]. In addition, Sodium Ferulate can suppress glomerular and interstitial fibrosis progression by inhibiting the expressions of cellular adhesion molecules, CD44 and CD54, in 5/6 nephrectomized rats[7]. Some studies have demonstrated Sodium Ferulate acts as a protective role for kidney of diabetic rats possibly by inhibiting the expression of TGF-β1 and type Ⅳ collagen[8]. In this study, we explored the effect of Sodium Ferulate on CTGF and renal fibrosis in rats with unilateral ureteral obstruction (UUO) to provide experimental evidence for Sodium Ferulate in clinical application.

MATERIALS AND METHODS

Materials
Animals: The experiment was performed at Research Laboratory of Clinical Medicine (grade BSL-1), College of Public Health, Sichuan University from May to December 2006. Twenty-four clean-grade healthy rats weighing 180-200 g were purchased from the Experimental Animal Center of Sichuan University (No. 011). All experimental procedure was accorded with animal ethical standards.
Agents: Sodium Ferulate (150 mg per pill, Sichuan
Hengda Pharmacy Co, Ltd, No. 050302), rabbit anti-rat CTGF (Santa Cruz), HRP-labelled goat anti-rabbit IgG (Beijing Zhongshan), RNA extract kit (Qiagen), reverse transcription agent (TakaRa ExScript TM RT Reagent Kit), and Perfect Real Time (TakaRa: Premix EX TaqTM).
Equipment: Included Western blotting (BioRAD, USA), and DNA Engine OpticonTM real-time fluorescent quantitation PCR device (MJ Research, USA).
Primers and Taqman probe sequence: Taqman probe 5'-FAM was fluorescence reporter, and 3'- TAMRA was fluo-rescence quencher. Randomized primers were purchased from Shanghai Shenergy Biocolor Bioscience & Technology Com-pany, and CTGFPCR primers and probes were synthesized by Shanghai Sangon Biological Engineering Technology & Ser-vices Co., Ltd. CTGFF: 5'-GCC TGT TCC AAG ACC TGT-3'; CTGFR: 5'-GGA TGC ACT TTT TGC CCT TCT TA-3'; CTGFTM: 5'-CTC CAC CCG GGT TAC CAA TGA C-3'; amplified fragment was 151 bp. GAPDH was used as internal reference. GAPDHF: 5'-TGG GTG TGA ACC AGG AGA A-3; GAPDHTM: 5'-ACT GCA CCA CCA ACT GCT TAG C-3'; GAPDHR: 5'-GGC ATG GAC TGT GGT CAT GA-3'; ampli-fied fragment was 143 bp.

Methods
Grouping and processing
Twenty-four rats were randomly divided into 4 groups (n=6): UUO model group, Sodium Ferulate group, Losartan group, and sham-operation group. According to the previous proto-col[9], UUO models were established in UUO model group, Sodium Ferulate group, and Losartan group, and the other rats were subjected to sham operation. From the first day after UUO, Sodium Ferulate group was intragastrically (i.g.) ad-ministrated with 150 mg/kg per day Sodium Ferulate; Losar-tan group was administrated i.g. with 20 mg/kg per day. Losartan; UUO and sham operation groups were administrated ig. with matching normal saline. All rats were fed separately and drank freely at (23±2) ℃ and relative humidity of (55±2)% for 14 days. Then the rats were executed to harvest renal tissues. Partial renal tissues were preserved in 4% para-formaldehyde solution for HE staining, Masson staining and immunohistochemical determination, and partial tissues were frozen by liquid nitrogen and preserved at -70℃ to extract mRNA and protein.

CTGF expression by immunohistochemical staining
Renal tissues of each rat were embedded by paraffin, sliced into 3 μm, baked for 2 hours, deparaffinaged, and put into 1.5% H2O2-methanol solution at 37 ℃ for 30 minutes. The tissues were incubated with rabbit anti-rat CTGF polyclonal antibody (1∶100) at 4 ℃ overnight. Tissues incubated with rabbit serum (5%) served as blank controls. After 14-16 hours, HRP-labelled goat anti-rabbit IgG was added, and incubated at 37 ℃ for 30 minutes. The slices were mounted using neutral gum after DAB coloration, hematoxylin staining, and di-methyl benzene transparency. Yellow-brown tissues were CTGF-positive. Data were analyzed using Image Pro plus color pathological image analysis software. Ratio of positive staining area and tubulointerstitial total area (except for lumina) in 20 nonoverlapping 200 fold visual fields of each section was calculated and the mean value was obtained.
CTGF mRNA levels by real-time fluorescent quantitation PCR
The concentration of tissue RNA harvested by Trizol method was detected using ultraviolet spectrophotometer, and 2 μg total RNA was extracted for reverse transcription. DNA Engine OpticonTM real-time fluorescent quantitation PCR device was used for PCR amplification in 30 μL reaction system. Ampli-fication was 94 ℃ for 2 minutes, 40 cycles; 94 ℃ for 30 seconds, 60 ℃ for 30 seconds (fluorescence signal monitor-ing); preserved at 16 ℃. △CT value of each group was compared. △CT value=target gene Ct value-reference gene Ct value; Ct value was cycle threshold, representing amplifi-cation cycles of cDNA. The more the original copy number of target gene, the less the cycle number of amplification, and accordingly, the smaller △CT value was.

CTGF protein levels by Western blotting
Tissue total protein was split and extracted according to Mo-lecular Clone: a Laboratory Manual. Protein concentration was quantified by BCA method. Protein was subjected to electrophoresis in 100 g/L SDS-polyacrylamide gel, and transformed to PVDF membrane. After sealed with 5% skimmed milk at 4 ℃ overnight, the protein was added rabbit anti-rat CTGF polyclonal antibogy (1∶500) at 37 ℃ for 2 hours. Membrane was washed for three times with each for 10 minutes. Then HRP-labelled goat anti-rabbit IgG (1: 20 000) was added at 37 ℃ for 1 hour followed by ECL radioauto-graphy. Absorbance value of radioautography results was analyzed using Quantity one software, and corrected using GAPDH.

Renal tubular interstitial injury extent by HE staining
HE staining was performed by routine procedure. Pathological changes were scaled by previous protocol [10].

Renal interstitium relative area by Masson staining
Masson staining was performed by routine procedure. Renal in-terstitium relative area was calculated by previous protocol [11].

Statistical analysis
Data were analyzed using SPSS 13.0 software. Measurement data were represented by Mean±SD. Mean value in one group before and after intervention was compared using paired t-test, and in two or more groups was compared using one-way analysis of variance (ANOVA).

RESULTS

Quantitative analysis of animals
Twenty-four rats were all included in final analysis with no loss.

Effect of Sodium Ferulate on CTGF expression
Immunohistochemical determination
Few CTGF expressions were observed in the sham operation group. CTGF expression was significantly increased in renal tissues 14 days after UUO, but the expressions in Sodium Ferulate group and Losartan group were significantly decreased (P < 0.05). There was no significant difference between Sodium Ferulate group and Losartan group (P > 0.05, Figure 1, Table 1).

CTGF mRNA expression
CTGF mRNA expression in UUO model group was signifi-cantly up-regulated compared with sham operation group 14 days after UUO (△CT was significantly decreased), while CTGF mRNA expression in Sodium Ferulate group was sig-nificantly decreased compared with UUO model group (P < 0.05). There was no significant difference between Sodium Ferulate group and Sodium Ferulate group (P > 0.05). This indicated that Sodium Ferulate greatly inhibited CTGF mRNA expression.
Western hybridization of CTGF
CTGF protein expression was significantly increased 14 days after UUO compared with sham operation group (P < 0.05), and significantly decreased in Sodium Ferulate group, but still markedly higher than sham operation group (P < 0.05, Figure 2).

Effect of Sodium Ferulate on UUO-induced morphological changes
HE staining results
Compared with sham operation group, renal interstitium in model group was widened 14 days after UUO, with much inflammatory cell infiltration, renal tubule expansion, granu-locyte aggregation in renal tubular lumina; in addition, renal tubular epithelial cell atrophy, necrosis and shedding was found. All above-mentioned changes were lessened in Sodium Ferulate group and Losartan group (Figure 3, Table 1).

Masson staining results
Compared with sham operation group, renal interstitial colla-gen deposition was significantly increased in UUO model group. After Sodium Ferulate and Losartan treatment, collagen deposition was significantly decreased compared with UUO model group (P < 0.05). There was no significant difference between Sodium Ferulate group and Losartan group (P > 0.05, Figure 4, Table 1).

DISCUSSION

Many studies show that CTGF overexpression can induce glomerular sclerosis and renal interstitial fibrosis. With high success rate and repeatability, unilateral ureteral obstruction (UUO) acts as a typical animal model to observe renal tubular and interstitial fibrosis, and accordingly widely used in studies for renal interstitial fibrosis[12]. In our study, HE staining and Masson staining show obvious renal fibrosis characteristics such as inflammatory cell infiltration, renal tubular and inter-stitial changes, and interstitial collage deposition, indicating UUO induces notable pathological changes of renal interstitial fibrosis. In addition, both CTGF mRNA and protein expres-sions are significantly up-regulated, indicating CTGF plays an important role in renal interstitial fibrosis, which is accorded with previous reports[13-15]. Sodium Ferulate is a kind of non-peptide endothelin receptor antagonists. Some studies have demonstrated that Sodium Ferulate can protect kidney from chronic pathological lesion, and the mechanism is correlated with endothelin suppressing[16]. There are few studies about Sodium Ferulate therapy for CTGF. Losartan as angio-tensin-converting plays a renoprotective role [17-18]. It has been proved that Losartan can relieve UUO-induced renal lesion[19]. Thus, we selected Losartan as positive control.
HE and Masson staining results show that after Sodium Ferulate treatment for UUO rats, renal fibrosis is significantly improved (P < 0.05), but not significantly different from Losartan (P > 0.05). CTGF mRNA levels are markedly ele-vated 14 days after UUO compared with sham operation group (△CT value is significantly decreased), while CTGF mRNA levels after Sodium Ferulate and Losartan treatment are markedly down-regulated compared with UUO group (P < 0.05), but there is no significant difference between Sodium Ferulate and Losartan groups (P > 0.05). Immunohistochemical and Western hybridization results show that Sodium Ferulate greatly inhibits CTGF protein expression (P < 0.05), which is similar to Losartan (P > 0.05). The findings of the study sug-gest Sodium Ferulate can effectively improve UUO-induced renal fibrosis in rats, and significantly inhibit CTGF mRNA and protein expressions. The mechanism of action may be correlated with the inhibitory effect of Sodium Ferulate on CTGF expression.

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