1521-0103/351/2/373–382$25.00 http://dx.doi.org/10.1124/jpet.114.215228THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 351:373–382, November 2014Copyright ª 2014 by The American Society for Pharmacology and Experimental Therapeutics

Suramin Inhibits the Development and Progression ofPeritoneal Fibrosis s

Chongxiang Xiong, Na Liu, Lu Fang, Shougang Zhuang, and Haidong YanDepartment of Nephrology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China (C.X., N.L., L.F., S.Z.,H.Y.); and Department of Medicine, Rhode Island Hospital and Brown University School of Medicine, Providence, Rhode Island(S.Z.)

Received April 19, 2014; accepted August 27, 2014

ABSTRACTPeritoneal fibrosis is one of the most serious complications inpatients with peritoneal dialysis (PD) and is associated with theloss of peritoneal membrane ultrafiltration function. In this study,we investigated whether suramin, an inhibitor that blocks multiplegrowth factors by binding to their receptors, would preventdevelopment of peritoneal fibrosis in a rat model. Rats were givena daily intraperitoneal injection of chlorhexidine gluconate (CG) for3 weeks to induce peritoneal fibrosis. Administration of suraminat 5, 10, and 20 mg/kg dose-dependently attenuated peritonealmembrane thickening and expression of collagen I, fibronectin,and a-smooth muscle actin. Increased expression of transforminggrowth factor-b1 (TGF-b1) and phosphorylation of Smad3 was

detected in fibrotic peritoneum and inhibited by suramin treatment.Suramin was also effective in blocking CG-induced phosphor-ylation of inhibitor of kB (IkB) and nuclear factor (NF)-kBp65,expression of several inflammatory cytokines, and infiltration ofmacrophages in the peritoneum. Moreover, suramin suppressedangiogenesis and expression of vascular endothelial growth factor,a molecule associated with angiogenesis in the injured peritoneum.Therefore, our results indicate that suramin treatment can effectivelyalleviate the development of peritoneal fibrosis by suppression ofTGF-b1 signaling, inflammation, and angiogenesis, and suggestthat suramin may have therapeutic potential for prevention ofperitoneal fibrosis in PD patients.

IntroductionPeritoneal dialysis (PD) is one of the renal replacement ther-

apies used in patients with end-stage renal disease (Chaudharyet al., 2011). During the process of PD, the peritoneal membraneis continuously exposed to a variety of insults, including bio-incompatible solutions, peritonitis, uremia, and chronic inflam-mation (Blake et al., 2013). Long-term exposure to these insultsoften causes peritoneal injury and subsequently leads to peri-toneal fibrosis (Kaneko et al., 2007). As peritoneal fibrosis isclosely associated with the decline of peritoneal function andultimately leads to ultrafiltration and solute transport failure(Nakamura and Niwa, 2004), it is important to develop in-terventions for prevention and treatment of peritoneal fibrosis.Peritoneal fibrosis is characterized by reduction or loss of

mesothelial cells, changes in the structure and number of bloodvessels, and enlargement of the submesothelial compact zonebecause of interstitial fibrosis (Tomino, 2012; de Lima et al.,

2013). Activation of fibroblasts with expression of a-smoothmuscle actin (a-SMA) is the cellular basis of interstitial fibro-sis, which is accompanied by excessive deposition of extracel-lular matrix components, such as collagen I and fibronectin.Although the pathophysiologic role of fibroblasts in the peri-toneum remains poorly understood, multiple factors andmechanisms, such as inflammation (Tomino, 2012; de Limaet al., 2013), neoangiogenesis (Sekiguchi et al., 2012), andactivation of transforming growth factor-b1 (TGF-b1) signaling(Chegini, 2008; Liu et al., 2012), have been shown to contributeto this process. TGF-b1 exerts its biologic actions throughactivation of two downstream signaling molecules, Smad2/3.Previous studies have indicated an increase in the expression ofTGF-b1 and phosphorylation of Smad2/3 in the peritoneum ina rat model of chlorhexidine digluconate–induced peritonealfibrosis (Lee et al., 2014). It is also evident that TGF-b1 causesperitoneal injury through a Smad-dependent signaling path-way (Patel et al., 2010). These data suggest that TGF-b sig-naling plays an important role in the development of PD.Inflammation is also critically connected with the progression

of peritoneal fibrosis (Lai and Leung, 2010). After peritonealinjury, a variety of cytokines/chemokines, such as interleukin-1(IL-1), IL-6, tumor necrosis factor-a (TNF-a), andmonocyte che-motactic protein-1 (MCP-1), are produced, and multiple leu-kocytes, in particular macrophages, are recruited to the injured

This study was supported by The National Nature Science Foundation ofChina [Grants 81170638 (to H.Y.), 81270778 and 81470920 (to S.Z.), 81200492and 81470991 (to N.L.)]; the Shanghai Scientific Committee of China [Grant13PJ1406900 (to N.L.)] and by Key Discipline Construction Project of PudongHealth Bureau of Shanghai [Grant PWZx2014-06 (to S.Z.)].

C.X. and N.L. are co-first authors.dx.doi.org/10.1124/jpet.114.215228.s This article has supplemental material available at jpet.aspetjournals.org.

ABBREVIATIONS: CG, chlorhexidine gluconate; DAB, 3,39-diaminobenzidine (3,39,4,49-tetraamino-diphenyl); ECM, extracellular matrix; ELISA,enzyme-linked immunosorbent assay; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IkB, inhibitor of kB; IL, interleukin; MCP-1, monocytechemotactic protein-1; NF-kB, nuclear factor-kB; PD, peritoneal dialysis; a-SMA, a-smooth muscle actin; TGF-b1, transforming growth factor-b1;TNF-a, tumor necrotic factor-a; VEGF, vascular endothelial growth factor.

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area. In addition, nuclear factor-kB (NF-kB) has been consideredan important proinflammatory transcriptional factor (Muriel,2009). Under normal circumstances, IkBs, inhibitory proteins,constrain movement of NF-kB dimers into the nucleus. Duringthe inflammatory process, different stimuli induce ubiquitinationof phosphorylated IkBs and their subsequent degradation by theproteasome (Herrmann et al., 2004). IkB degradation allowsNF-kB to translocate into the nucleus (Karin, 2009), where itleads to the transcription ofmany genes involved in inflammatoryprocess (Hoesel andSchmid, 2013). Inflammatory targets regulatedby NF-kB include chemokines, cytokines, and adhesion molecules(Sun et al., 2004; Huang et al., 2013).Long-termPD often causes progressive angiogenesis, an event

associated with peritoneal fibrosis and ultrafiltration failure ofthe peritoneal membrane (Stavenuiter et al., 2011). As vascularendothelial growth factor (VEGF) is a potent proangiogenic cy-tokine (Fusshoeller, 2008;Saxena, 2008), several studieshaveex-amined the possible involvement of VEGF in peritoneal fibrosis.For example, expression of VEGF in mesothelial cells that haveundergone epithelial-to-mesenchymal transition is increasedin PD patients (Aroeira et al., 2005); a large number of VEGF-positive cells were observed in the thickening submesothelialarea (Yoshio et al., 2004); and treatment with a neutralizing anti-VEGF monoclonal antibody was able to prevent structural andfunctional microvascular alterations induced by hyperglycemia(De Vriese et al., 2001). As such, inhibition of VEGF expressionmay be an interesting therapeutic approach for prevention ofperitoneal fibrosis.Suramin is a polysulfonated naphthylurea thatwas originally

used in the prevention and treatment of early stages of humantrypanosomiasis; it also has antitumor activity (Liu et al., 2011).

Suramin exerts its pharmacologic effect through the interactionof several cytokines and growth factors with their receptors.Mechanistic studies demonstrate that suramin inhibits apopto-sis, suppresses expression of proinflammatory cytokines, in-activates myofibroblasts, and blocks multiple cell signalingpathways (Liu and Zhuang, 2011). Recent animal studies fromour laboratory and by other investigators have shown thatadministration of suramin can attenuate muscle, liver, andrenal fibrosis (Chan et al., 2003; Li et al., 2009; Liu et al., 2011).We have shown that suramin treatment can suppress theexpression of fibronectin and a-SMA and type I collagen andreduce the deposition of extracellular matrix (ECM).Currently, it is unknown whether suramin has an antifi-

brotic effect in the peritoneum. In this study, we investigatedthe effect of suramin on the development of peritoneal fibrosisinduced by chlorhexidine gluconate (CG) and the mechanismsinvolved in rats.

Materials and MethodsAntibodies and Chemicals. Antibodies to p-IkB, IkB, p-NF-kB,

NF-kB, p-Smad3, and Smad3 were purchased from Cell SignalingTechnologies (Danvers, MA). Antibodies to fibronection, collagen I (A2),and Smad7 were purchased from Santa Cruz Biotechnology, Inc. (Dallas,TX). Antibodies to GAPDH (glyceraldehyde 3-phosphate dehydrogenase),VEGF, and CD68 were purchased from BD Pharmingen (San Diego,CA). TNF-a, IL-1b, MCP-1, IL-6, TGF-b1 enzyme-linked immunosorbentassay (ELISA) kits were from R&D Systems (Minneapolis, MN).Suramin, a-SMA, CG, and all other chemicals were from Sigma-Aldrich(St. Louis, MO).

Creation of the Rat Model with Peritoneal Fibrosis andSuramin Treatment. Thirty-six 6- to 8-week-old male Sprague-Dawley

Fig. 1. Suramin attenuates CG-induced peritoneal fibrosisin rats. Photomicrographs illustrating Masson trichromestaining of peritoneum in the rat model of GC-inducedperitoneal fibrosis administered with/without 20 mg/kgsuramin (A); thickening of the compact zone (B); andthe score of fibrosis (C) of peritoneal tissue. Data arerepresented as the mean 6 S.E.M. (n = 6). Means withdifferent lowercase letters are significantly different fromone another (P , 0.05).

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rats (1706 10 g) were purchased from Shanghai Super–B&K LaboratoryAnimal Corp. Ltd. (Shanghai, China). Animals were housed in stainlesssteel cages in a ventilated animal room at the Experimental AnimalCenter of Tongji University. Room temperature was maintained at20 6 2°C, relative humidity at 60 6 10%, and a 12-hour light/darkcycle. Distilled water and sterilized food for rats were available adlibitum. Animals were acclimated to this environment for 7 days prior toexperiments. Rats were randomly assigned to six groups with six ratsper group: the sham group, sham treated with suramin (20 mg/kg)group, the peritoneal fibrosis group, and rats with peritoneal fibrosistreated with different doses of suramin (5, 10, 20 mg/kg, respectively).The peritoneal fibrosis in rats was generated by daily intraperitonealinjection of 0.1% gluconate chlorhexidine (1 ml/100 g) as described pre-viously with a slight modification (Bozkurt et al., 2008). Normal saline(0.9%) was administered as a control. In the treatment groups, suraminwas intraperitoneally injected when the first dose of gluconate chlorhex-idine was administered and then given weekly at 5, 10, 20 mg/kg. At theend of 3 weeks, all rats were sacrificed and the parietal peritoneum awayfrom the injection points was harvested for further analysis. All experi-ments were conducted in accordance with the animal experimentationguideline of Tongji University School of Medicine.

Morphologic Studies of Peritoneum. Tissue sections wereprepared at 4-mm thickness by a routine procedure followed by fixationin 4%paraformaldehyde, dehydration and clearance in conventional-seriesethanols and xylene, and embedding in paraffin. Sections were stainedwith Masson’s trichrome or H&E for general histology, especially changesin the thickness of peritoneum. The thickness of the submesothelial tissuewas evaluated (in micrometers), and the average of ten independentmeasurements was calculated for each section (400� magnification).

Immunoblot Analysis. Immunoblot analysis of peritoneumtissue samples were conducted as described previously (Pang et al.,

2009). The densitometry analysis of immunoblot results was con-ducted by using NIH ImageJ software (NIH, Bethesda, MD).

Immunohistochemical Staining. The tissue sectionswere treatedfor 30 minutes in methanol containing 30% hydrogen peroxide (H2O2)after being routinely deparaffinized and hydrated. After washing withphosphate-buffered saline three times, microwave antigen retrievalwas conducted in citric acid buffer (pH 6.0) microwave, blocked with 5%bovine serum albumin blocking buffer at 37°C for 20 minutes, andwashed with phosphate-buffered saline three times. Sections were thenincubated with primary antibodies at 4°C overnight and homologoussecondary antibodies at room temperature.

Following DAB [3,39-diaminobenzidine (3,39,4,49-tetraamino-diphenyl)]coloration at room temperature, counterstaining in H&E, and mountingwith Neutral balsam, morphologic analyses were performed by usinglight microscopy. The antibody dilutions are: collagen-I antibody(1:250), anti–a-SMA (1:200), fibronectin antibody (1:200), CD68antibody (1:200), CD31 (1:200), and VEGF antibody (1:200). Forquantitative assessment, 10 areas were selected and the positivestaining area of collagen I and fibronectin was determined by ImagePro-Plus Software (NIH) and the average ratio to each microscopicfield (400�) was calculated and graphed. The numbers of a-SMA–expressing cells, CD68-positive macrophages, CD31-positive ves-sels, and VEGF-positive cells and were calculated in 10 fields at400� magnification.

ELISA Detection. ELISA detection of TGF-b1, MCP-1, IL-1b,TNF-a, IL-6 protein was performed in accordance with the manu-facturer’s instructions.

Statistical Analysis. All the experiments were conductedat least three times. Data depicted in graphs represent the means 6S.E.M. for each group. Intergroup comparisons were made usingone-way analysis of variance. Multiple means were compared using

Fig. 2. Suramin inhibits a-SMA expression in the ratmodel of CG-induced peritoneal fibrosis. Immunostain-ing of a-SMA in the submesothelial compact zone (A).Peritoneal lysates were prepared and subject to immu-noblot analysis with antibodies to a-SMA or GAPDH (B).Representative immunoblots from three or more experi-ments are shown. (C) Expression levels of the proteinswere quantified by densitometry and normalized withGAPDH. Data are represented as the mean 6 S.E.M.Means with different lowercase letters are significantlydifferent from one another (P , 0.05).

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Tukey’s test. The differences between two groups were determined byStudent’s t test. Statistical significant difference between mean valueswas marked in each graph. P , 0.05 is considered significant.

ResultsSuramin Prevents Chlorhexidine Gluconate–Induced

Peritoneal Fibrosis in Rats. We recently showed thatadministration of suramin alleviates progression of renal fi-brosis in a mouse model of unilateral ureteral obstruction (Liuet al., 2011). However, the pharmacological effect of suramin inperitoneal fibrosis remains unclear. The pathogenesis of peri-toneal fibrosis is characterized by overproduction and de-position of extracellular matrix, which leads to fibrotic lesionsand peritoneal scarring. Thus, we first examined the effect ofsuramin on the expression of peritoneal collagen fibrils byMasson’s trichrome staining. Rat peritoneal fibrosis wasestablished by daily injection of CG for 3 weeks. Suramintreatment started immediately after injection of CG at con-centrations of 5, 10, 20 mg/kg, which was then administeredweekly until animals were sacrificed. Peritoneum adminis-tered with CG displayed severe morphologic lesions charac-terized by the thickening of submesothelial compact zone,peritoneal mesothelial cell loss, and peritoneal interstitialexpansion with collagen accumulation and deposition. Treat-ment with suramin reduced the thickness of peritoneum ina dose-dependent manner with maximum effect at 20 mg/kg

(Fig. 1, A and B; Supplemental Figs. 1 and 2). The quanti-tative analysis of thickness of peritoneum indicated an 8-foldincrease in chlorhexidine gluconate–injured rats compared withsham-operated animals. Suramin treatment dose dependentlyattenuated this fibrotic response: at 20 mg/kg, suramin wassufficient to prevent it (Fig. 1C). These data suggest thatsuramin is a potent agent in preventing the development ofperitoneal fibrosis.Suramin Inhibits Expression of a-SMA in the Rat

Model of Chlorhexidine Gluconate–Induced PeritonealFibrosis. To investigate the ability of suramin in suppress-ing peritoneal myofibroblast activation, we examined theeffect of suramin on the expression of a-SMA, a hallmarkof myofibroblasts in the peritoneum. Western blot and im-munohistochemistry analysis of peritoneum showed themarked upregulation of a-SMA expression in the rat modelof peritoneal fibrosis, and administration of suramin signif-icantly reduced this response (Fig. 2). Inhibition of a-SMAexpression by suramin also occurred in a dose-dependentmanner, with the observed effect at 5 mg/kg, a greater inhibitionat 10 mg/kg, and the maximum effect at 20 mg/kg (Fig. 2, Aand B; Supplemental Fig. 1). Suramin administration sup-pressed peritoneal expression of a-SMA by approximately75% in chlorhexidine gluconate–treated animals comparedwith those treated with chlorhexidine gluconate alone (Fig.2C). This suggests that suramin has a potent capability toinhibit activation of peritoneal fibroblasts.

Fig. 3. Suramin blocks fibronection expression in therat model of CG-induced peritoneal fibrosis. Immuno-staining of fibronectin in the submesothelial compactzone (A). Peritoneal tissue lysates were subjected toimmunoblot analysis with specific antibodies againstfibronectin (B). Expression levels of fibronectin werequantified by densitometry and normalized withGAPDH (C). Data are represented as the mean 6 S.E.M.(n = 6). Means with different lowercase letters aresignificantly different from one another (P , 0.05).

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Suramin Blocks Expression of Fibronectin and Colla-gen I in the Rat Model of Chlorhexidine Gluconate–Induced Peritoneal Fibrosis. Tissue fibrosis is characterizedby accumulation of ECM components, such as fibronectinand collagen I, two major ECM proteins (Liu et al., 2011;Subeq et al., 2011). As such, we examined the effect of suraminon their expression in rats with peritoneal fibrosis. Immuno-histochemistry staining and Western blot analysis of perito-neum indicated increased expression of fibronectin (Fig. 3) andcollagen I (Fig. 4) in this model. Suramin treatment also dosedependently reduced their expression with the significantinhibition at 5 mg/kg, and to the basal level when 20 mg/kgwas used (Supplemental Fig. 1). Quantitative analysis ofWestern blotting results indicates that treatment with20 mg/kg suramin reduced the expression of fibronectin andcollagen I by approximately 80 and 70%, respectively, inchlorhexidine gluconate–treated rats (Figs. 3C and 4C).Collectively, these results indicate that suramin treatmentsuppresses expression of ECM proteins.Suramin Inhibits TGF-b1 Expression and Smad3

Phosphorylation in the Rat Model of ChlorhexidineGluconate–Induced Peritoneal Fibrosis. TGF-b signal-ing pathway is associated with most fibrotic diseases, and anincrease in its markers has been considered as a major mech-anism of peritoneal fibrosis (Liu, 2006; Wynn, 2008). To in-vestigate the effect of suramin on the TGF-b signaling in the ratmodel of peritoneal fibrosis, we first examined the expression

level of TGF-b1 in the peritoneum by ELISA. Figure 5A showsthat chlorhexidine gluconate injury induced a marked increasein the expression of TGF-b1, and administration of suraminreduced its expression by 50%. We next examined the effect ofsuramin on the phosphorylation of Smad3 and expression ofSmad7, two key mediators in TGF-b signaling (Bhogal et al.,2005; Kaminska et al., 2005). Chlorhexidine gluconate injuryresulted in increased phosphorylation of Smad3. Treatmentwith suramin reduced Smad3 phosphorylation (Fig. 5, B andD),and increased expression of Smad7 (Fig. 5, C andD). Expressionof total Smad3 was not affected by chlorhexidine gluconateand suramin (Fig. 5B). As decreased Smad3 phosphorylationand increased expression of Smad7 have been shown to beassociated with inhibition of peritoneal fibrosis progression inan experimental rat model, we suggest that inhibition of TGF-bsignalingmay be largely associatedwith the antifibrotic effect ofsuramin.Suramin Inhibits NF-kB Signaling in the Rat Model

of Chlorhexidine Gluconate–Induced Peritoneal Fibro-sis. Activation of NF-kB signaling pathway is a key regula-tor of angiogenesis and inflammation in peritoneal fibrosis(Kitamura et al., 2012). IkB is the inhibitor of NF-kB (Manand Zhang, 2011). To examine the effect of suramin on theNF-kB pathway in peritoneal fibrosis, experiments wereconducted to analyze total IkB and phosphorylated IkB levelsby immunoblot analysis. Chlorhexidine gluconate injuryinduced IkB phosphorylation in the peritoneum, which was

Fig. 4. Suramin blocks collagen I expression in the ratmodel of CG-induced peritoneal fibrosis. Immunostainingof collagen I in the submesothelial compact zone (A).Peritoneal tissue lysates were subjected to immunoblotanalysis with specific antibodies against collagen I (B).Expression levels of collagen I were quantified bydensitometry and normalized with GAPDH (C). Dataare represented as the mean 6 S.E.M. (n = 6). Meanswith different lowercase letters are significantly differentfrom one another (P , 0.05).

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inhibited in the presence of suramin (Fig. 6). The phosphor-ylated IkB was not detected in the peritoneum of sham andsham-treated animals with suramin.Next, we examined the effect of suramin on the expression of

phosphorylatedNF-kBp65. The phosphorylatedNF-kBp65wasbarely detected in the peritoneum of sham and sham-treatedsuramin rats but its expression level was significantly up-regulated in the rats with peritoneal fibrosis. Administrationof suramin significantly inhibited NF-kBp65 phosphorylationwithout affecting expression of its total levels (Fig. 7, A and B).These data suggest that suramin is effective in inhibiting acti-vation of NF-kB signaling pathway in the peritoneum.Suramin Suppresses Expression of Multiple Inflam-

matory Cytokines in Chlorhexidine Gluconate–InducedPeritoneal Fibrosis in Rats. Proinflammatory cytokinesand adhesionmolecules play an important role in the developmentand progression of peritoneal fibrosis. To determine the effect ofsuramin on inflammatory cytokines, we measured their expres-sion levels in rats with peritoneal fibrosis, treated or untreatedwith suramin, by ELISA (Fig. 8). The expression levels ofproinflammatory cytokines and chemokines, including IL-6, -a,MCP-1, and IL-1b, were significantly increased in rats withperitoneal fibrosis. Suramin treatment decreased their expres-sion levels (Fig. 8, A–D).Suramin Inhibits Infiltration of Macrophages in

Chlorhexidine Gluconate–Induced Peritoneal Fibrosisin Rats. One of the characteristic pathologic changes inperitoneal fibrosis is the infiltration of macrophages into thethickened submesothelial compact zone (Nishino et al., 2012).Reduced macrophage infiltration would have a potential forpreventing peritoneal fibrosis. To elucidate suramin on thisprocess, we conducted immunohistochemistry staining usingan antibody against CD68, a marker of active macrophages.

Figure 9A demonstrated that macrophage infiltration was in-creased in the peritoneum of rats after chlorhexidine gluconateinjection. Comparedwith the sham animals, suramin treatment

Fig. 5. Suramin inhibits expression of TGF-b1and Smad3 phosphorylation in the rat model ofCG-induced peritoneal fibrosis. Protein from peri-toneal tissues from sham-operated or CG-treatedrats with/without suramin administration wassubjected to the determination of TGF-b1 levelsby the ELISA (A). Peritoneal lysates were subjectto immunoblot analysis with antibodies to phos-phorylated Smad3, Smad3, or GAPDH (B) as wellas Smad7 or GAPDH (C). Expression levels ofp-Smad3 were quantified by densitometry andnormalized with total Smad3 (D). Data arerepresented as the mean 6 S.E.M. (n = 6). Meanswith different lowercase letters are significantlydifferent from one another (P , 0.05).

Fig. 6. Suramin inhibits I-kB phosphorylation in the rat model ofCG-induced peritoneal fibrosis. Peritoneal tissue lysates were subjectedto immunoblot analysis with specific antibodies against phospho-IkB(p-IkB), IkB, or GAPDH (A). Expression levels of p-IkB were quantified bydensitometry and normalized with IkB (B). Data are represented as themean 6 S.E.M. (n = 6). Means with different lowercase letters aresignificantly different from one another (P , 0.05).

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significantly reduced the number of macrophages in the in-jured peritoneum (Fig. 9A). The quantitative analysis indi-cated that the number of CD68-postive macrophages wassignificantly increased in the ratwith peritoneal fibrosis (Fig. 9B),

and suramin treatment significantly reduced macrophage in-filtration in the peritoneum. Therefore, suramin is also able toinhibit infiltration of macrophage cells into the submesotheliallayer.Suramin Decreases VEGF Expression in Chlorhex-

idine Gluconate–Induced Peritoneal Fibrosis in Rats.Long-term PD is frequently accompanied by neo-angiogenesiswith vasculopathy (Ro et al., 2007). Previous studies haveshown that the expression level of VEGF is elevated in theperitoneal membrane of PD patients, leading to angiogenesisof the peritoneum (Fusshoeller, 2008; Saxena, 2008). We thusalso examined expression of VEGF and CD31 (a marker ofvascular endothelial cells) by immunohistochemistry stain-ing. Angiogenesis was evaluated by counting the number ofCD31-positive blood vessels in the peritoneal tissue injured bychlorhexidine gluconate. A marked increase in VEGF-positivecells and CD31-positive blood vessels was observed, whichwas inhibited by suramin administration (Fig. 10, A and B).These data suggest that suramin may also prevent peritonealfibrosis through suppression of neo-angiogenesis.

DiscussionIn this study, we report that suramin can prevent the

progression of peritoneal fibrosis induced by chlorhexidinegluconate. Suramin significantly inhibited thickening of thesubmesothelial compact zone, expression of a-SMA, anddeposition of ECM proteins (i.e., type I collagen and fibro-nectin) in the peritoneum of rats with peritoneal fibrosis.Suramin treatment also reduced macrophage infiltration andexpression of VEGF. Furthermore, suramin administrationinhibited expression of multiple cytokines and chemokines inthe injured peritoneum. These data are consistent with theinhibitory effect of suramin on renal fibrosis and suggest that

Fig. 7. Suramin inhibits NF-kB phosphorylation in the rat model ofCG-induced peritoneal fibrosis. Peritoneal tissue lysates were subjected toimmunoblot analysis with specific antibodies against phospho-NF-kB p65,NF-kB p65 and GAPDH (A). Expression levels of phospho-NF-kBp65 werequantified by densitometry and normalized with NF-kBp65 (B). Dataare represented as the mean 6 S.E.M. (n = 6). Means with different lower-case letters are significantly different from one another (P , 0.05).

Fig. 8. Suramin suppresses the expression ofMCP-1, TNF-a, IL-1b, and IL-6 in the rat modelof CG-induced peritoneal fibrosis. Peritoneallysates were subjected to the ELISA as describedunder Materials and Methods. The expressionlevels of MCP-1 (A), IL-1b (B), TNF-a (C), andIL-6 (D) are indicated over control (sham withvehicle). Data are represented as themean6S.E.M.(n = 6). Means with different lowercase letters aresignificantly different from one another (P , 0.05).

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suramin is also a potent agent for attenuation of peritonealfibrosis.As in renal fibrogenesis, multiple signaling pathways are

activated and involved in the development of peritoneal fi-brosis. Of these pathways, TGF-b signaling has been shown toplay a predominant role in this process. To understand themechanism by which suramin inhibits peritoneal fibrosis, weexamined the effect of suramin on the expression of TGF-b1and activation of Smad3, a key mediator in TGF-b signaling.Our results clearly showed that administration of suraminblocked TGF-b1 expression as well as Smad3 phosphorylation.In addition, we also observed that suramin treatment in-creased expression of Smad7. These data, together with ourprevious observations that suramin is able to inhibit expres-sion of ECM proteins and Smad3 phosphorylation in renalfibroblasts exposed to TGF-b1 suggest that suramin mayattenuate peritoneal fibrosis through suppression of the TGF-bsignaling pathway at different levels. However, this may not bethe sole mechanism, as our previous studies also indicated that

suramin inhibits phosphorylation of epidermal growth factorreceptor and platelet-derived growth factor receptors, twogrowth factor receptors associatedwith tissue fibrosis in variousorgans, including peritoneum. Further studies are needed toinvestigate the effect of suramin on the activation of these tworeceptors in peritoneal fibrosis.It is well documented that inflammation contributes to

peritoneal fibrosis. Inflammatory response is characterized byexpression of multiple cytokines/ chemokines and infiltration ofproinflammatory cells, in particularmacrophages. In this study,we demonstrated that suramin treatment inhibits expressionof proinflammatory cytokines/chemokines, such as IL-1, IL-6,TNF-a, andMCP-1, and infiltration of macrophages. As such, itis likely that another mechanism of suramin inhibition ofperitoneal fibrosis is through suppression of inflammation inthe injured peritoneum. In support of this speculation, we foundthat suramin inhibits phosphorylation of NF-kBp65, a keytranscriptional factor for the expression of multiple proinflam-matory chemokines and cytokines and phosphorylation of

Fig. 9. Effect of suramin on the leukocyte infiltration in the peritoneum inthe rat model of CG-induced peritoneal fibrosis. Immunostaining ofleukocytes with CD68 in the submesothelial compact zone (A). Quantita-tive analysis was conducted in three random fields of each sample, and 10fields (1 � 100) were analyzed for each condition. Data are represented asthe mean 6 S.E.M. (n = 6). Means with different lowercase letters aresignificantly different from one another (P , 0.05) (B).

Fig. 10. Effect of suramin on the peritoneal expression of VEGF in therat model of CG-induced peritoneal fibrosis. The protein expression ofVEGF was detected by immunohistochemistry staining (A). Quantita-tive analysis of peritoneal VEGF expression in three random fields ofeach sample, and 10 fields (1 � 100) were analyzed for each condition(B). Data are represented as the mean 6 S.E.M. (n = 6). Means withdifferent lowercase letters are significantly different from one another(P , 0.05).

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p-IkBa. Because IkBa is an inhibitor of NF-kB signaling and itsphosphorylation is required for activation of NF-kB activation,inhibition of IkBa phosphorylation would contribute to in-activation of NF-kB signaling and subsequent inhibition oftranscription of inflammatory genes.Angiogenesis and vasculopathy in the peritoneum play an

important role in the regulation of water and solute transportin the peritoneum (Williams et al., 2002). However, increasedvascularity and/or peritoneal blood flow resulted in progressiveperitoneal fibrosis (Davies et al., 2011). This manifests itselfas submesothelial thickening and is associated with increasedvascularization that leads to ultrafiltration dysfunction(Sekiguchi et al., 2012). Abundance of in vitro and in vivostudies (Margetts et al., 2001; Ghosh and Karin, 2002) haveshown that vascular endothelial growth factor induced byTGF-b1 can upregulate the expression of VEGF receptor 1and interaction of VEGF with VEGF receptor 1 leads toangiogenesis and ultimately ultrafiltration failure. On thisbasis, we speculated that reduction of VEGF expression wouldalleviate angiogenesis and improve ultrafiltration of perito-neal membrane. In the present study, we examined the effectof suramin on the expression of VEGF and found that suraminwas effective in suppressing its expression in rat peritoneumafter chlorhexidine gluconate injection. In addition, we alsoobserved that suramin treatment inhibited expression of CD31, amarker of vascular endothelial cells (Fig. 11). Therefore, suraminmay inhibit neo-angiogenesis and protect the peritoneumfrom fibrosis by suppressing production of angiogenic growthfactors.PD has been widely accepted as a treatment of end-stage

renal disease patients. Success of this modality is largely de-pendent on the structural and functional integrity of the peri-toneal membrane. However, the duration of PD can be limited byperitoneal alteration in both structure and function, includingperitoneal fibrosis and ultrafiltration failure (Saxena, 2008;Davies et al., 2011). In vitro and in vivo studies (Liu et al., 2014)have demonstrated that peritoneal fibrosis is characterized bydetachment of the mesothelial layer, dilatation of intercellularspaces, and deposition of extracellular matrix (Devuyst et al.,2010). Our current studies have also shown that suramin caninhibit submesothelial thickening, deposition of fibrils, andexpression of multiple ECM proteins, suggesting its usefulnessin attenuating peritoneal fibrosis and reducing ultrafiltrationfailure. Given the fact that there is no available therapy forhalting or preventing this complication thus far, it would beinteresting to conduct clinical trials in the future to investigatewhether suramin can be used as an additive component in thePD dialysate for preventing development of peritoneal fibrosisduring PD.However, there are two limitations for this study. First,

suramin administration only started immediately after in-jection of chlorhexidine gluconate, the inducer of perito-neal fibrosis. As such, the current results only suggest thatsuramin is an effective agent that prevents development ofperitoneal fibrosis, but they do not show whether it also hasa therapeutic effect in established peritoneal fibrosis. To gainmore information about its therapeutic effect, it is necessaryto administer suramin at different time points after injectionof chlorhexidine gluconate to study its effects on peritonealfibrosis. Second, we only examined the antifibrotic effect ofsuramin in one animal model of peritoneal fibrosis. Becauseperitoneal fibrosis is a complex process that is caused by

multiple pathogenic factors and is involved in different mech-anisms, it is possible that the antifibrotic effect of suraminmayvary with animal models of this disease. Therefore, additionalanimal studies with delayed administration of suramin indifferent models of peritoneal fibrosis are required prior toinitiation of a clinical trial to test its effectiveness in humanperitoneal fibrosis.In summary, we demonstrate that suramin is able to pre-

vent the development of peritoneal fibrosis. The antifibroticeffect of suramin is associated with inactivation of TGF-b1and NF-kB signaling, inhibition of macrophage infiltrationand cytokine/chemokine expression, as well as suppression ofangiogenesis. As peritoneal fibrosis is involved in the in-creased production of numerous cytokines/growth factors andsubsequent activation of their receptors and signaling path-ways, suramin, with its ability to inhibit the interaction ofmultiple cytokines/growth factors with their receptors, mayhave therapeutic potential in the treatment of peritoneal fibrosis.Thus, suramin may become a novel therapeutic approach toprevent the development of peritoneal fibrosis in PD patients.

Fig. 11. Effect of suramin on peritoneal angiogenesis in the rat model ofCG-induced peritoneal fibrosis. The protein expression of CD31 wasdetected by immunohistochemistry staining (A). Quantitative analysis ofperitoneal CD31 expression in three random fields of each sample, and 10fields (1� 100) were analyzed for each condition (B). Data are representedas the mean 6 S.E.M. (n = 6). Means with different lowercase letters aresignificantly different from one another (P , 0.05).

Suramin Inhibits Peritoneal Fibrosis 381

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Authorship Contributions

Participated in research design: Yan, Liu, Zhuang.Conducted experiments: Xiong, Liu.Contributed new reagents or analytic tools: Xiong, Yan, Liu,

Zhuang.Performed data analysis: Xiong, Fang, Liu.Wrote or contributed to the writing of the manuscript: Xiong, Fang,

Yan, Liu, Zhuang.

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Address correspondence to: Dr. Haidong Yan, Department of Nephrology,Tongji University School of Medicine, 150 Ji Mo Road, Pudong New District,Shanghai 200120, China. E-mail: yhdcmu@sina.com; or Dr. Shougang Zhuang,Brown University School of Medicine, Rhode Island Hospital, Middle House301, 593 Eddy Street, Providence, RI 02903. Email: szhuang@lifespan.org

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