Aliskiren ameliorates chlorhexidine digluconate-induced peritoneal fibrosis in rats

Authors


Bang-Gee Hsu, MD, PhD, No 707, Section 3, Chung-Yang road, Hualien, Taiwan. Tel.: +886 3 8561825; fax: +886 3 8577161; e-mail: gee.lily@msa.hinet.net

Abstract

Eur J Clin Invest 2010; 40 (4): 301–309

Abstract

Background  Peritoneal fibrosis (PF) is a recognized complication of long-term peritoneal dialysis (PD) and can lead to ultrafiltration failure. The present study was designed to investigate the protective effects of aliskiren on chlorhexidine digluconate-induced PF in rats.

Materials and methods  The PF was induced in Sprague–Dawley rats by daily administration of 0·5 mL 0·1% chlorhexidine digluconate in normal saline via PD tube for 1 week. Rats received daily intravenous injections of low-dose aliskiren (1 mg kg−1) or high-dose aliskiren (10 mg kg−1) for 1 week. After 7 days, conventional 4·25% Dianeal (30 mL) was administered via a PD catheter with a dwell time of 4 h and assessed of peritoneal function. At the end of dialysis, rats were sacrificed and the liver peritoneum was harvested for microscopically and immunohistochemistry.

Results  There was no significant difference in mean arterial pressure and heart rate between groups. After 4 h of PD, the D4/P4 urea level was reduced, the D4/D0 glucose level, serum and dialysate transforming growth factor-β1 (TGF-β1) level was increased, the liver peritoneum was markedly thicker, and the expression of TGF-β1, alpha-smooth muscle actin (α-SMA), fibronectin, collagen, and vascular endothelial growth factor (VEGF) were elevated in the PS group compared with the vehicle group. Aliskiren decreased the serum and dialysate TGF-β1 level, decreased the thickness of the liver peritoneum, and decreased the expression of TGF-β1, α-SMA, fibronectin, collagen, and VEGF-positive cells in liver peritoneum. Moreover, high-dose aliskiren had better protective effects against PF than low dose in rats.

Conclusions  Aliskiren protected against chlorhexidine digluconate-induced PF in rats by decreasing TGF-β1 production.

Introduction

In long-term peritoneal dialysis (PD), morphological and functional changes of the peritoneal membrane are common. Sub-mesothelial fibrosis, angiogenesis, vasculopathy and collagen deposition typically occur in the peritoneal membrane [1–3]. Peritoneal sclerosis is one of the most serious complications of PD and is associated with a high morbidity and mortality and is one of the most serious complications of long-term PD [4–6]. The renin–angiotensin system may have fibrotic effects on the peritoneum [7]. Local angiotensin II generating systems are also noted in human peritoneal mesothelial cells [8]. Injurious stimuli induce reactivation of the local peritoneal angiotensin II, which initiates the production of transforming growth factor-β1 (TGF-β1) and contributes to extracellular matrix accumulation and induces peritoneal fibrosis (PF) [7,9,10].

Aliskiren, a direct inhibitor of renin, has been shown in experimental and clinical studies to be effective in lowering blood pressure and to hold considerable potential for organ protection beyond blood pressure reduction [11,12]. Aliskiren is involved in the initial and rate-limiting step in the renin–angiotensin system cascade that decreases angiotensin II production [12] and may be beneficial to decrease TGF-β1 production in PF. In this study, we evaluated the effects of aliskiren on chlorhexidine digluconate-induced PF in rats.

Materials and methods

Preparation of animals and peritoneal tube insertion

Thirty-two male Sprague–Dawley rats weighing 280–300 g were purchased from the National Animal Center (Taipei, Taiwan) and housed in the university Animal Center in a controlled environment at 22 ± 1 °C with a 12 h light/dark cycle. Food and water were given ad libitum. The experimental protocol was approved by the Animal Care and Use Committee of Tzu Chi University. Rats were anaesthetized with ether inhalation for about 30 min. During the period of anaesthesia, fur over the abdominal wall and right inguinal area were closely shaved. A polyethylene catheter (PE-240; PD catheter), length about 30 cm, was inserted about 4 cm into the peritoneal cavity through a midline incision below the xiphoid process. The catheter was then tunnelled subcutaneously to the right inguinal area. A polyethylene catheter (PE-50) was inserted into the right femoral artery to collect blood samples and was connected to a pressure transducer (Gould Instruments, Cleveland, OH, USA) to record arterial pressure (AP) and heart rate (HR) on a polygraph recorder (Power Lab; AD Instruments, Mountain View, CA, USA). Another PE-50 catheter was inserted into the femoral vein for intravenous administration of drugs. All procedures were performed under sterile conditions. Sterilized stainless steel covers were used to cover the PD catheters, femoral artery PE catheters and femoral vein PE catheters to prevent the rats from biting and dislocating the catheters. After the operation, animals were placed in a conscious rat metabolic cage (Mike Biological Technologies, Hualien, Taiwan). Rats awakened soon after the operation and peritoneal sclerosis was induced 24 h later with the rats in a conscious state [13–15].

Peritoneal fibrosis

Peritoneal fibrosis was induced by daily 0·5 mL 0·1% chlorhexidine digluconate (Sigma-Aldrich, St Louis, MO, USA) in normal saline solution via a PD catheter and later 1 mL of normal saline was administrated after chlorhexidine digluconate injection via PD catheter.

Experimental design

Rats were randomly divided into four groups. The vehicle group (= 8) received 0·5 mL normal saline and later 1 mL of normal saline daily via a PD catheter and 0·5 mL daily intravenously via the femoral vein for 1 week. The PF group (= 8) received 0·5 mL 0·1% chlorhexidine digluconate and later 1 mL of normal saline daily and intravenously 0·5 mL normal saline via the femoral vein daily for 1 week. The low-dose aliskiren group (= 8) received 0·5 mL 0·1% chlorhexidine digluconate and later 1 mL of normal saline daily and intravenously 1 mg kg−1 aliskiren (Novartis Pharmaceuticals, Cambridge, MA, USA) in 0·5 mL normal saline via the femoral vein daily for 1 week. The high-dose aliskiren group (= 8) received 0·5 mL 0·1% chlorhexidine digluconate and later 1 mL of normal saline daily and intravenously 10 mg kg−1 aliskiren [16] in 0·5 mL normal saline via the femoral vein daily for 1 week.

Peritoneal dialysis

After the end of PF (7 days), the rats were given 30 mL of conventional 4·25% glucose-containing peritoneal fluid (Dianeal; Baxter Healthcare SA, Singapore Branch, Singapore) via PD catheter to the peritoneal cavity, with a dwell time of 4 h. After 4 h of PD, animals were sacrificed for pathological examination.

Blood and dialysate fluid sample analysis

Blood and dialysate fluid samples (0·5 mL) were collected for measurement of glucose at 0 and 4 h after initiation of PD. Within 1 h of collection, these samples were centrifuged at 3000 g for 10 min prior to subsequent biochemical analysis. The serum was decanted and separated into two parts; one part was stored at 4 °C within 1 h after collection for biochemical analysis. Serum and dialysate fluid levels of glucose, and urea were measured with an autoanalyzer (COBAS Integra C111; Roche Diagnostics, Basel, Switzerland) to obtain various biochemical data [13–15]. The other part of the serum collected at 4 h after PD was stored at −80 °C for later measurement of TGF-β1 concentrations.

Serum and dialysate TGF-β1 measurement by ELISA

At 4 h of PD, TGF-β1 concentrations in the blood and dialysate samples were measured separately by antibody enzyme-linked immunosorbent assay (ELISA) with commercial antibody pairs, recombinant standards and a biotin–streptavidin–peroxidase detection system (Assay Designs, Ann Arbor, MI, USA) as described previously [13,14]. All reagents, samples and working standards were brought to room temperature and prepared according to the manufacturer’s directions. Reactions were quantified by optical density using an automated ELISA reader (Sunrise; Tecan Co., Grödingen, Austria) at 450/540 nm wavelength.

Peritoneal solutes transport analysis

Peritoneal solute transport was calculated from the dialysate concentration relative to its concentration in the initial infused dialysis solution (D4/D0 glucose) for glucose, and the dialysate-to-plasma concentration ratio (D4/P4 urea) at 4 h for urea.

Histological assessment

The liver peritoneum were fixed overnight in 4% buffered formaldehyde, processed by standard methods, and stained with haematoxylin and eosin (H&E). Liver peritoneal surfaces were evaluated by morphometry and immunohistochemistry (IHC). Thickening of the liver peritoneum was defined from the liver surface to the peritoneal cavity. Quantification of the liver peritoneum was performed on paraffin-embedded tissue sections and was counted digitally using a 200× objective lens via a computer imaging analysis system (Image-Pro Plus 4·5; Media Cybernetics, Bethesda, MD, USA) as described previously [17,18]. Briefly, the thickness of the liver peritoneum was measured at 10 points in each rat.

Immunohistochemistry

For IHC of liver peritoneums, serial 4-μm sections were deparaffinized, rehydrated and incubated with different mouse monoclonal antibodies at 4 °C overnight, according to the manufacturer’s directions. Antigen retrieval was used for TGF-β1, alpha-smooth muscle actin (α-SMA), fibronectin, type-I collagen (collagen) and vascular endothelial growth factor (VEGF). Dilutions were 1 in 100 for TGF-β1 (Abcam, Cambridge, MA, USA), fibronectin, collagen (Rockland, PA, USA), VEGF and α-SMA (Bio SB, Santa Barbara, CA, USA). After incubation, tissue sections were covered with biotinylated goat anti-mouse polyvalent secondary antibody and incubated at room temperature for 10 min. Sections were then washed and the slides were incubated in peroxidase conjugated streptavidin–biotin complex (Dako, Copenhagen, Denmark) for 10 min. The areas where there were positive cells for TGF-β1, α-SMA, fibronectin, collagen and VEGF were evaluated from a 0·01 mm2 region (200× magnification field; 0·1 mm width × 0·1 mm length) of four fields, and was counted digitally via a computer imaging analysis system (Image-Pro Plus 4·5; Media Cybernetics). Results are reported as the percentage of positive cells per mm2 of peritoneal tissue [13,15,17,19]. All scoring was performed in a blinded manner on coded slides.

Statistical analysis

Data are expressed as mean ± SD. Statistical comparisons between groups were made by repeated measures two-way anova followed by a post hoc test (Bonferroni’s method). Histological scores were analysed by the Kruskal–Wallis test followed by the Mann–Whitney U-test. A P-value less than 0·05 was considered statistically significant.

Results

Mean arterial pressure, heart rate and peritoneal function

There was no significant difference in mean arterial pressure (MAP) and HR between groups (Fig. 1a,b). The D4/D0 glucose level was significantly higher, and D4/P4 urea level was significantly lower in the PF group than the vehicle group (*< 0·05; Fig. 2a,b). The D4/P4 urea level was significantly higher in the low-dose aliskiren group than the PF group ($< 0·05; Fig. 2b). The D4/D0 glucose level was significantly lower, and D4/P4 urea level was significantly higher in the high-dose aliskiren group than the PF group (#< 0·05; Fig. 2a,b). Moreover, The D4/D0 glucose level was significantly lower in the high-dose aliskiren group than the low-dose aliskiren group (+< 0·05; Fig. 2a).

Figure 1.

 Change in mean artery pressure (MAP) (a), and heart rate (HR) (b) during chlorhexidine digluconate-induced peritoneal fibrosis in rats. There was no significant difference in MAP and HR between groups.

Figure 2.

 The D4/D0 glucose level (a), and D4/P4 urea level (b) after chlorhexidine digluconate-induced peritoneal fibrosis in rats. *< 0·05 for the PF group compared with the vehicle group. #< 0·05 for the high-dose aliskiren group compared with the PF group. $< 0·05 for the low-dose aliskiren group compared with the PF group. +< 0·05 for the high-dose aliskiren group compared with the low-dose aliskiren group.

Serum and dialysate TGF-β1 level

PS greatly elevated the serum and dialysate TGF-β1 level compared with the vehicle group (Fig. 3a,b). The serum and dialysate TGF-β1 level was significantly lower for the low-dose aliskiren group compared to the PF group ($< 0·05; Fig. 3a,b) or high-dose aliskiren group compared to the PF group (#< 0·05; Fig. 3a,b). Moreover, compared with the low-dose aliskiren group, the serum and dialysate TGF-β1 level was lower in the high-dose aliskiren group (+< 0·05; Fig. 3a,b).

Figure 3.

 Serum (a) and dialysate (b) transforming growth factor-beta 1 (TGF-β1) level after chlorhexidine digluconate-induced peritoneal fibrosis in rats. *< 0·05 for the PF group compared with the vehicle group. #< 0·05 for the high-dose aliskiren group compared with the PF group. $< 0·05 for the low-dose aliskiren group compared with the PF group. +< 0·05 for the high-dose aliskiren group compared with the low-dose aliskiren group.

Histopathology of liver peritoneum

Daily chlorhexidine digluconate increased the thickness of the liver peritoneum (Fig. 4b). The PF group had thicker liver peritoneum than the vehicle group (*< 0·05; Fig. 4e). The thickness of the liver peritoneum was significant lower for the low-dose aliskiren group compared to the PF group ($< 0·05; Fig. 4e) or high-dose aliskiren group compared to the PF group (#< 0·05; Fig. 4e). Moreover, compared with the low-dose aliskiren group, the thickness of liver peritoneum was lower in the high-dose aliskiren group (+< 0·05; Fig. 4e).

Figure 4.

 Haematoxylin and eosin staining of liver peritoneum. Histologic sections from the vehicle group (a), peritoneal fibrosis (PF) group (b), low-dose aliskiren group (c) and high-dose aliskiren group (d) (magnification × 200). Thickness of liver peritoneum after chlorhexidine digluconate-induced PF in rats (e). *< 0·05 for the PF group compared with the vehicle group. #< 0·05 for the high-dose aliskiren group compared with the PF group. $< 0·05 for the low-dose aliskiren group compared with the PF group. +< 0·05 for the high-dose aliskiren group compared with the low-dose aliskiren group. Bar = 20 μm.

Immunohistochemistry of liver peritoneum

Compared with vehicle group, rats in the PF group had more cells that were TGF-β1-positive, α-SMA-positive, fibronectin-positive, collagen-positive and VEGF-positive (Fig. 5b,f,j,n,r). The TGF-β1-positive, α-SMA-positive, fibronectin-positive, collagen-positive and VEGF-positive cells were significantly lower for the low-dose aliskiren group compared to the PF group ($< 0·05; Fig. 6a–e) or high-dose aliskiren group compared to the PF group (#< 0·05; Fig. 6a–e). Moreover, compared with the low-dose aliskiren group, the TGF-β1-positive, α-SMA-positive, fibronectin-positive, collagen-positive and VEGF-positive cells were lower in the high–dose aliskiren group (+< 0·05; Fig. 6a–e).

Figure 5.

 Immunohistochemical (IHC) staining for transforming growth factor-beta 1 (TGF-β1; a, b, c, d), alpha-smooth muscle actin (α-SMA; e, f, g, h), fibronectin (i, j, k, l), collagen (m, n, o, p) and vascular endothelial growth factor (VEGF; q, r, s, t) in the liver peritoneum. Histologic sections from the vehicle group (a, e, i, m, q), PF group (b, f, j, n, r), low-dose aliskiren group (c, g, k, o, s) and high-dose aliskiren group (d, h, l, p, t) (magnification × 200) after chlorhexidine digluconate-induced peritoneal fibrosis in rats.

Figure 6.

 Percentage of TGF-β1-positive cell score (a), α-SMA-positive cell score (b), fibronectin-positive cell score (c), collagen-positive cell score (d) and VEGF-positive cell score (e) after chlorhexidine digluconate-induced peritoneal fibrosis in rats. *< 0·05 for the PF group compared with the vehicle group. #< 0·05 for the high-dose aliskiren group compared with the PF group. $< 0·05 for the low-dose aliskiren group compared with the PF group. +< 0·05 for the high-dose aliskiren group compared with the low-dose aliskiren group.

Discussion

This study found that administration of aliskiren decreased chlorhexidine digluconate-induced thickness of the liver peritoneum by decreasing the serum and dialysate TGF-β1 level, and also significantly decreased the expression of TGF-β1, α-SMA, fibronectin, collagen and VEGF in liver peritoneum in rats. Moreover, high-dose aliskiren had better protective effects against PF than low dose in rats.

The PF is characterized by activation of peritoneal resident cells, accumulation and deposition of excess matrix proteins within the interstitium, and neoangiogenesis and vasculopathy of the peritoneal microvasculature [1,20]. A common animal model of PF employs rats that are given an intraperitoneal injection of 0·1% chlorhexidine gluconate with 15% ethanol [6,21]. In this rat model, there is an increased expression of TGF-β1, α-SMA, collagen and VEGF in the peritoneum [17,19,22,23]. Our model, using chlorhexidine digluconate-induced PF, also noted an increased expression of TGF-β1, α-SMA, fibronectin, collagen and VEGF in the liver peritoneum and an increase in the thickness of the liver peritoneum.

TGF-β1 is a profibrotic cytokine that has been shown to regulate PF [10,24]. The overexpression of TGF-β1 further increases α-SMA, VEGF and promotes PF through extensive collagen deposition and neoangiogenesis [25]. A recent study found that aliskiren suppressed the renal gene expression of TGF-β1 in vivo and inhibited TGF-β1-mediated pathways toward renal fibrosis [26]. Our study also noted that aliskiren decreased serum TGF-β1 levels after chlorhexidine digluconate-induced PF in rats.

Angiotensin II has growth factor properties, is involved in the effects of TGF-β1 and contributes to inducing PF [7,9,10]. A renin–angiotensin system inhibitor could be a powerful tool in preventing the progression of PF. Angiotensin receptor blockers or angiotensin converting-enzyme inhibitors have been shown to ameliorate chlorhexidine gluconate-induced PF in rats [27]. Aliskiren inhibits angiotensin I generation from angiotensinogen by direct renin inhibitors and further decreases angiotensin II production [12]. Our study also noted that aliskiren ameliorated chlorhexidine digluconate-induced liver peritoneum thickness and significantly decreased the expression of TGF-β1, α-SMA, fibronectin, collagen and VEGF in liver peritoneums in rats. Administration of 10 mg kg−1 day−1 aliskiren was shown to effectively lower blood pressure in spontaneous hypertensive rats [16], although the doses needed were up to 10-times higher than those used in marmosets, which reflects the differences in the in vitro inhibitory activity of human renin inhibitors between species [12]. Our research used intravenous 10 mg kg−1 day−1 aliskiren as the high-dose group and 1 mg kg−1 day−1 as the low-dose group to study the effects on PF in rats. The D4/D0 glucose level was significantly lower, and D4/P4 urea level was significantly higher in the high-dose aliskiren group than the PF group. The D4/D0 glucose level was significantly lower in the high-dose aliskiren group than the low-dose aliskiren group. Moreover, a high dose of aliskiren further decreased serum TGF-β1 production, ameliorated chlorhexidine digluconate-induced liver peritoneum thickness and decreased the expression of TGF-β1, α-SMA, fibronectin, collagen and VEGF in liver peritoneum in rats. High-dose aliskiren had better protective effects against PF than the low-dose in rats and did not induce hypotension. Our results suggest the need for clinical randomized trials to investigate whether treatment with aliskiren can elicit significant protective effects for PD patients who suffer from PF.

Limitations of the present study include that it is an animal study of short duration. The clinical presentation of encapsulating peritoneal sclerosis (EPS) is a rare but serious complication of PD patients [4–6]. EPS is felt to be a separate entity that occurs in a minority of patients with underlying peritoneal membrane fibrosis – the aetiology is unclear but felt to be due to a possible ‘second hit’ that occurs to the peritoneal membrane that is exposed to chronic PD [5]. In this study, by stating that PF occurs after 1 week of chlorhexidine digluconate and that PF is the same as clinical presentation of EPS is incorrect. Another limitation is that the doses of aliskiren in rats is very higher than those used in human, which reflects the differences in the in vitro inhibitory activity of human renin inhibitors between species [12,28]. Our study noted there was no apparent change in MAP and HR after aliskiren treatment for 1 week in rats. The dose of aliskiren and preventive strategies for treatment of peritoneal sclerosis in the chronic PD patients remains uncertain. Moreover, recent study noted angiotensin receptor blocker (valsartan) or angiotensin converting-enzyme inhibitor (enalapril) have been shown to ameliorate chlorhexidine gluconate-induced PF in rats [27]. Angiotensin-converting enzyme inhibitor (perindoprilat) or angiotensin receptor blocker (candesartan) inhibited glucose-induced TGF-β1 production by cultured human peritoneal mesothelial cells and combination of both agents had a stronger effect than each single drug alone [29]. The effect of angiotensin converting-enzyme inhibitor/angiotensin receptor blocker may be different to that of renin inhibitor because of renin-independent angiotensin generation (e.g. by trypsin or kallikrein) [30]. Furthermore, a combination of aliskiren and angiotensin receptor blockers may be more effective inhibition of plasma renin activity than with monotherapy, greater reduction of blood pressure in some clinical studies [31]. Additional studies will be required to ascertain whether the combination of aliskiren and angiotensin converting-enzyme inhibitor/angiotensin receptor blocker may be more protective effects from PF.

In conclusion, aliskiren protected against chlorhexidine digluconate-induced PF by decreasing serum and dialysate TGF-β1 levels and significantly decreased the expression of TGF-β1, α-SMA, fibronectin, collagen and VEGF in liver peritoneum in rats.

Acknowledgements

This work was supported by grants from the National Science Council (NSC 94-2314-B-303-012 and NSC 96-2314-B-303-004-MY2) and Tzu Chi General Hospital (TCRD98-02).

Address

Department of Laboratory Medicine and Biotechnology (C.-Y. Ke); Department of Nursing (Y.-M. Subeq, R.-P. Lee); School of Medicine, Tzu Chi University (B-G. Hsu), Hualien, Taiwan; Department of Nursing, Tzu Chi College of Technology (C-C. Lee, C-J. Lee); Division of Nephrology, Buddhist Tzu Chi General Hospital, Hualien, Taiwan (B-G. Hsu).

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