Rosuvastatin Attenuates Inflammation, Apoptosis and

Original Report: Laboratory Investigation American

Journal of

Nephrology

Received: November 1, 2012 Accepted: November 21, 2012 Published online: December 21, 2012

Am J Nephrol 2013;37:7–15 DOI: 10.1159/000345990

Rosuvastatin Attenuates Inflammation, Apoptosis and Fibrosis in a Rat Model of Cyclosporine-Induced Nephropathy Hyun Kyung Nam a Seong Joo Lee a Moo Hyun Kim b Jee Hyun Rho c Young Ki Son d Su Mi Lee d Seong Eun Kim d Ki Hyun Kim d Won Suk An d a

Department of Internal Medicine, Busan Medical Center, b Regional Clinical Trial Center, Dong-A University Hospital, and c Department of Anatomy and Cell Biology and Mitochondria Hub Regulation Center, and d Department of Internal Medicine, Dong-A University College of Medicine, Busan, Republic of Korea

Key Words Apoptosis ⴢ Cyclosporine ⴢ Fibrosis ⴢ Inflammation ⴢ Rosuvastatin

Abstract Background/Aim: Cyclosporine (CsA)-induced kidney injury is characterized by renal dysfunction with inflammatory cell infiltrations, apoptosis and fibrosis. Pleiotropic effects of statins may exert anti-inflammatory, antiapoptotic and antifibrotic actions beyond lipid control. The aim of this study is to investigate whether rosuvastatin (RUS) has anti-inflammatory, antiapoptotic and antifibrotic effects on chronic CsA-induced nephropathy in a rat model. Methods: Male Sprague-Dawley rats fed a low-sodium diet were divided into three treatment groups: control (0.9% saline injection), CsA (15 mg/kg/day by subcutaneous injection), CsA + RUS (10 mg/kg/day by gastric gavage). Renal function, CsA level and lipid levels were measured at the end of 4 weeks. The expression of ED-1, transforming growth factor-␤1 (TGF-␤1) and ␣-smooth muscle actin (␣-SMA) for inflammation and fibrosis were examined by Western blot analysis. The expression levels of apoptosis-associated factors were examined by Western blot analysis. Apoptosis was evaluated using the

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terminal deoxynucleotidyl transferase-mediated biotin nick end-labeling (TUNEL) method. Results: Kidney function was decreased in CsA-treated rats compared with controls, which was attenuated by RUS. RUS did not affect the lipid level or the blood CsA level. TUNEL staining showed that RUS inhibited CsA-induced tubular apoptosis. RUS decreased CsA-induced increased expression of Bax/Bcl-2 ratio. The expressions of ED-1, ␣-SMA, TGF-␤1, Smad2/3, Smad4 and p-JNK were increased in CsA-treated rats, which were attenuated by RUS. Tubular atrophy and interstitial fibrosis in CsA-treated rats were attenuated by RUS supplementation. Conclusion: RUS supplementation attenuates proinflammatory and apoptosis-related factors and inhibits the fibrotic pathways including the smad-dependent and smad-independent pathways in a rat model of CsA-induced nephropathy. Copyright © 2012 S. Karger AG, Basel

Introduction

Cyclosporin A (CsA), a cyclic decapeptide obtained from extracts of the soil fungus, Tolypocladium inflatum Gams, is the most effective and widely used first-line immunosuppressant in solid organ transplantation and auWon Suk An Department of Internal Medicine, Dong-A University 3Ga-1, Dongdaesin-Dong Seo-Gu, Busan 602-715 (Republic of Korea) E-Mail answ @ dau.ac.kr

toimmune disease [1]. Despite its clinical benefits, the clinical use of CsA is limited by its nephrotoxic potential. CsA-induced nephropathy is characterized by progressive renal insufficiency, afferent arteriopathy, tubulointerstitial inflammation, and striped fibrosis [2]. Long-standing hypoxic injury to the kidney has been regarded as the main etiology of CsA-induced injury [3], and mediators such as angiotensin II and transforming growth factor-␤1 (TGF-␤1) are involved in the pathogenesis of chronic CsA nephropathy [4, 5]. CsA-induced cell death caused by apoptosis of renal tubular cells results in renal tubular atrophy and the loss of tubular mass observed in chronic CsA nephropathy [6, 7]. Therefore, it is necessary to control inflammation, apoptosis and fibrosis for delaying the progression of chronic CsA nephropathy. Statins, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, are drugs proven for the management of dyslipidemia to prevent primary and secondary cardiovascular disease. The benefits of statins are usually derived from reducing atherogenic lipoprotein levels. In addition to modulating lipid levels, clinical and experimental studies have shown that statins have pleiotropic effects including anti-inflammatory, antiapoptotic and antifibrotic effects [8–11]. The renoprotective effects of statins have also been reported in animal models [8, 9]. Rosuvastatin (RUS), a fully synthetic 3-hydroxy-3methylglutaryl coenzyme A reductase inhibitor, reduced inflammation in nitric oxide-deficient rats and had a renoprotective effect in hypertensive rat models [12, 13]. However, the benefits of RUS in a rat model of chronic CsA-induced nephropathy have not been reported. Furthermore, there is no evidence that RUS simultaneously attenuates inflammation, apoptosis and fibrosis in vivo which are prominent in CsA-induced nephropathy. Thus, the aim of this study is to investigate the anti-inflammatory, antiapoptotic and antifibrotic effects of RUS in a well-described rat model of chronic CsA-induced nephropathy.

Materials and Methods Animals and Experimental Design Male Sprague-Dawley rats initially weighing 180–200 g were housed in cages with a temperature- and light-controlled environment. They were allowed free access to a low-salt diet (0.05% sodium; Teklad Premier, Madson, Wisc., USA) and tap water. CsA (Chong Kun Dang, Seoul, Korea) was diluted with saline to a final concentration of 15 mg/ml.

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Am J Nephrol 2013;37:7–15

The experimental protocol was approved by the Institutional Animal Care and Use Committee of Dong-A University Medical School. Rats were randomized into three groups and treated daily for 4 weeks as shown below. All of the controls and treated rats survived during the experiments. Group 1 (G1; n = 5): rats received saline (1 ml/kg/day by subcutaneous injection). Group 2 (G2; n = 5): rats received CsA (15 mg/kg/day by subcutaneous injection). Group 3 (G3; n = 5): rats received CsA and RUS (10 mg/kg/day by gastric gavage). The dose and route of administration of CsA (15 mg/kg/day) was chosen based on a previous study [14]. The rats assigned to the RUS-treated group were given 10 mg/kg/day of RUS by gastric gavage for 4 weeks using a Gastight microsyringe (Gastight, Hamilton, Reno, Nev., USA). The dose of RUS was decided by previous evidence preventing progression of renal injury in deoxycorticosterone acetate (DOCA)-salt hypertensive rats [12]. After starting the treatment, rats were pair-fed and daily body weight was monitored. On the day of sacrifice, blood samples were collected from the inferior vena cava. We evaluated each test with prior knowledge of each group to avoid mixing the samples. Blood urea nitrogen and serum creatinine concentrations were determined spectrophotometrically using a 917 Hitach autoanalyzer. Wholeblood CsA level was measured by a monoclonal radioimmunoassay (Incstar, Stillwater, Minn., USA). Total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were measured using kits purchased from specified manufacturers. Histopathologic Evaluation On the day of sacrifice, kidneys were retrieved, washed with heparinized saline, fixed in periodate-lysine-paraformaldehyde solution and embedded in wax. After dewaxing, 4-␮m sections were processed and stained with periodic acid-Schiff (PAS). Kidney lesions were looked for included vacuolization of tubular cells, tubular atrophy and dilatation, inflammatory cellular infiltrate and interstitial fibrosis. Western Blotting Kidney tissue was homogenized in lysis buffer (pH 7.6, containing 300 mM NaCl, 50 mM Tris-Cl, 0.5% Triton X-100, protease-inhibitor cocktail) and incubated at 4 ° C for 30 min. The lysates were centrifuged at 14,000 rpm for 20 min at 4 ° C. Protein concentrations of lysates were determined with Bradford protein assay reagent (Bio-Rad, Hercules, Calif., USA) and 40 ␮g of proteins were loaded onto 7.5–15% SDS/PAGE. The gels were transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, Piscataway, N.J., USA) and reacted with each antibody. Antibodies against TGF-␤, ED-1, iNOS, E-cadherin, rabbit polyclonal anti-human caspase-3, and caspase-7 were obtained from Santa Cruz Biotechnology (Santa Cruz, Calif., USA). Mouse monoclonal anti-human caspase-8, monoclonal anti-Bax antibodies, anti-Bcl-2 antibodies, extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), smad-2/3, smad-4 and smad-7 antibodies were purchased from Cell Signaling Technology (Beverly, Mass., USA). Antibody against ␣-smooth muscle actin (␣-SMA) was purchased from Abcam (Cambridge, Mass., USA). Monoclonal anti-␤-actin was obtained from Sigma (St. Louis, Mo., USA).

Nam /Lee /Kim /Rho /Son /Lee /Kim /Kim / An

Table 1. Basic parameters

Number of rats Final body weight, g Creatinine, mg/dl Blood urea nitrogen, mg/dl Total cholesterol, mg/dl Triglycerides, mg/dl HDL-C, mg/dl LDL-C, mg/dl CsA concentration, ng/ml

G1

G2

G3

5 352.5816.8 0.2180.06 1581 110811 15587 110811 68811 NM

5 282.5825.7* 1.1280.22* 93825* 105814 158813 110811 6182 2,816.98257.9

5 312.586.4* 0.4880.12*, # 52821*, # 10389 151812 110811 6588 2,918.08133.8

NM = Not measured. * p < 0.05 compared to G1, # p < 0.05 compared to G2.

TUNEL Staining Apoptosis was assessed using terminal deoxynucleotidyl transferase-mediated biotin nick end-labeling (TUNEL) assay. Assay specimen undergoing apoptosis was identified by the ApopTag in situ apoptosis detection kit (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) according to the manufacturer’s protocol. Immunofluorescent Staining For immunofluorescent staining, the tissue sections were fixed in cold 100% methanol for 2–3 min at room temperature and then washed three times with phosphate-buffered saline (PBS). Next, the tissues were blocked with 0.1% bovine serum albumin in PBS for 1 h, after which they were incubated with terminal deoxynucleotidyl transferase and ED-1 antibody for 1 h at 37 ° C. The tissues were then incubated with FITC-conjugated secondary antibody for 1 h after serial washing with PBS. Counterstaining with propidium iodide (5 ␮g/ml) was conducted to verify the location and integrity of the nuclei. FITC-conjugated goat anti-rabbit IgG antibodies were purchased from Vector (Burlingame, Calif., USA) and propidium iodide was purchased from Sigma. Fluorescent images were observed and analyzed using a Zeiss LSM 510 laserscanning confocal microscope.

Statistical Analysis Results were expressed as mean 8 SD. Ten tests per group were conducted for parametric analysis with enough power in this animal model. ANOVA and Tukey posttests for multiple groups were used in multiple comparisons using SPSS software version 19.0 (Statistical Package for Social Science version 19.0, SPSS Inc., an IBM Company, Chicago, Ill., USA). p ! 0.05 was considered significant.

Results

Basic Parameters The results obtained at 4 weeks are summarized in table 1. Body weights were similar among the groups at the beginning of the study. Compared with the control Rosuvastatin and Cyclosporine-Induced Nephropathy

group (G1), the CsA-treated group (G2) and the CsA plus RUS-treated group (G3) showed significantly reduced body weight. At 4 weeks, G2 showed increased serum creatinine (1.12 8 0.22 vs. 0.21 8 0.06 mg/dl) and blood urea nitrogen (93 8 25 vs. 15 8 1 mg/dl) as compared with G1. G3 had significantly lower serum creatinine levels compared with G2 (p ! 0.05). The levels of TC, TG, HDL-C and LDL-C did not show any differences among the three groups. CsA blood levels were 12,000 ng/ml in G2 and G3 and were not different between G2 and G3 at the end of study. ED-1 and iNOS Data Compared with G1, G2 significantly upregulated ED-1 protein expression. RUS supplementation attenuated increased ED-1 expression (fig. 1a). The expression of inducible nitric oxide synthase (iNOS) was increased in G2 compared to G1, which was not reversed by RUS (fig. 1b). Confocal microscopy also showed that CsA induced ED-1 in rat kidney and RUS prevented expression of ED-1 (fig. 1c). Apoptosis Data We found that caspase-3, caspase-7, and caspase-8 were activated in the CsA-treated group (G2) and that RUS prevented the activation of these effector caspases. CsA increased the ratio of Bax and Bcl-2, which was attenuated by RUS in G2 (fig. 2a). Confocal microscopy showed that CsA increased TUNEL-positive cells and that RUS cotreatment attenuated these effects (fig. 2b).

␣-SMA and E-Cadherin Data The data are illustrated in figure 3. CsA-treated rats (G2) showed significant upregulation of ␣-smooth musAm J Nephrol 2013;37:7–15

9

G2

G3

G1

iNOS

␤-Actin

␤-Actin

iNOS (AU)

ED-1 (AU)

600 500 400 300 200

#

100 0

a

G2

G1

ED-1

400 350 300 250 200 150 100 50 0

* *

b

G3

PI

G3

ED-1

*

700

G2

Color version available online

G1

G2

G1

Merge

G3

Merge (×2)

G1

G2

Fig. 1. a Expression of ED-1 was significantly increased in CsA-treated rats (G2) compared to the controls (G1), which was ameliorated by RUS treatment (G3). b Expression of iNOS was significantly increased in CsA-treated rats, which was not attenuated by RUS cotreatment. c Increased expression of ED-1 was shown in CsA-treated rats, which was reversed by RUS treatment in confocal microscopy images. * p ! 0.05 compared to G1, # p ! 0.05 compared to G2.

G3

cle actin (SMA), a molecular marker of fibroblasts. Expression of E-cadherin was downregulated in G2. RUS supplementation inhibited the induction of ␣-SMA and increased E-cadherin expression compared to G2. TGF-␤1, Smads, ERK1/2, and JNK Data TGF-␤1, an important profibrotic molecule, was significantly increased in CsA-treated rats (G2), and TGF␤1 was markedly attenuated by RUS supplementation. Total Smad-2/3 and Smad-4 levels were significantly in10


50 μm

c

20 μm

creased in G2, and RUS supplementation significantly attenuated the upregulation of Smad-2/3 and Smad-4 (fig. 4a). Inhibitory Smad-7 abundance was partially decreased in G2 and restored with RUS in G3. Phosphorylated JNK expression was significantly increased in G2, and this effect was reversed by RUS supplementation (fig. 4b). G2 showed a slight increase of phosphorylated ERK1/2 compared to G1, which was not prevented with RUS.


G3 Caspase-3 Cleaved caspase-3 Caspase-7 Cleaved caspase-7

300

200 150 #

100 50

Cleaved caspase-7 (AU)

G2


G1


*

250

250

*

200 150

#

100

0

50 0

G1

G2

G3

G1

G2

G3

Caspase-8

Cleaved caspase-8 Bax Bcl-2 ␤-Actin

5

*

250 200 #

150 100

Bax/Bcl-2 (AU)


300

50 0

a

*

4 3 # 2 1 0

G1

TUNEL

G2

G3

PI

G1

Merge

G2

G3

Merge (×2)

G1

G2

Fig. 2. a Expression of caspase-3, proapoptotic Bax, and antiapoptotic Bcl-2 before (G1) and after (G2) CsA treatment. RUS attenuated CsA-induced production of caspase-3 cleaved products and upregulated Bax protein (G3). RUS also increased antiapoptotic protein Bcl-2. b TUNEL staining. Confocal microscopy images showed CsA-induced TUNEL-positive apoptotic cells and that RUS prevented apoptosis. * p ! 0.05 compared to G1, # p ! 0.05 compared to G2.

G3

Renal Pathology Figure 5 shows the morphological changes among the three groups. PAS-stained specimens from the CsAtreated group (G2) revealed vacuolization of tubular cells, tubular atrophy and interstitial fibrosis. The RUS cotreatment group (G3) had less tubulointerstitial changes than G2. Rosuvastatin and Cyclosporine-Induced Nephropathy

50 μm

b

20 μm

Discussion

The pathogenesis of CsA nephropathy is multifactorial and several factors including inflammation, apoptosis, fibrosis and transformation of renal proximal tubular cells by the epithelial mesenchymal transition (EMT) process are involved [3, 15, 16]. We observed increased Am J Nephrol 2013;37:7–15

11

G1

G2

␣-SMA (AU)

150

G3

*

100 # 50

␣-SMA 0 E-cadherin

G1

G2

G3

120 E-cadherin (AU)

␤-Actin

Fig. 3. Expression of ␣-SMA and E-cad-

herin. CsA treatment significantly increased the expression of ␣-SMA and decreased that of E-cadherin (G2) compared to the controls (G1). RUS treatment prevented these effects (G3). * p ! 0.05 compared to G1, # p ! 0.05 compared to G2.

inflammatory process by showing ED-1-positive macrophage infiltrations in the rat model with CsA nephropathy. We also noted increased expressions of apoptotic products such as cleaved caspases and the ratio of Bax and Bcl-2 in CsA-induced apoptosis. In addition, CsA induced ␣-SMA leading to EMT and elevated TGF-␤1, which is a well-known profibrogenic cytokine in present study. First of all, these main processes related to renal injury were simultaneously attenuated by RUS treatment. Therefore, our study presents the necessity of a clinical study with RUS for the prevention of CsA-induced nephropathy. Renal fibrosis, which is characterized by the accumulation of ECM proteins including collagen and fibronectin, results in a deterioration of kidney function [17]. TGF-␤1 has been implicated in the fibrosis via several signaling pathways. TGF-␤-activated kinase 1 stimulated by TGF-␤1 induces Smad-dependent and Smad-independent signaling pathways. The Smad-independent TGF-␤ signaling pathways including ERK1/2 and JNK play a role in the pathological processes of kidney disease [18]. Smad2 and Smad-3 mediate fibrosis by stimulating ECM production, inhibiting ECM degradation, and inducing tubular EMT in Smad-dependent TGF-␤ signaling pathways [19]. Phosphorylated Smad-2 and Smad-3 form a complex with common Smad-4 and then translocate to 12


#

100 80 60

*

40 20 0 G1

G2

G3

nuclei to bind and regulate target genes [20]. Smad-7 is an inhibitory Smad and negatively regulates Smad-2 and Smad-3 activation by its negative feedback mechanism. In this study, we demonstrated that Smad-dependent and Smad-independent signaling pathways are activated in a CsA nephropathy rat model. Furthermore, we showed that the activated Smad-dependent pathway was inhibited and that the Smad-independent JNK signaling pathway was more targeted by RUS treatment than the ERK1/2 signaling pathway. Apoptosis is an essential process in the development and tissue homeostasis of most multicellular organisms; deregulation of apoptosis has been implicated in the pathogenesis of CsA-induced nephropathy [21]. Caspases which are essential in most types of apoptosis are a 12-member family of specific cysteine proteases [22]. In this study, we demonstrated that not only caspase-3 and caspase-7, which are the effector caspases, but also the processing of the initiator caspase-8 were inhibited by RUS in CsA-induced nephropathy. The involvement of Bax and Bcl-2-related proteins in apoptosis is well documented; the Bcl-2 protein protects cells from a variety of stimuli that induce apoptosis, while the death-repressing activity of Bcl-2 may be counteracted by dimerization with Bax [23]. It has been proposed that the relative ratio of Bax and Bcl-2 determines cell survival following apopNam /Lee /Kim /Rho /Son /Lee /Kim /Kim / An

G2

G3

250

*

1,500

Smad-2/3 (AU)

G1

TGF-␤1 (AU)

2,000

1,000 500

100 50

0

0 G1

Smad-2/3

␤-Actin

400 350 300 250 200 150 100 50 0

*

#

a

G1

G1

G3

Smad-7 (AU)

Smad-4 (AU)

Smad-7

G2

G2

G2

G3 p-ERK1/2

G1

p-JNK (AU)

␤-Actin

b

G2

G3

G1

G2

G3

160 140 120 100 80 60 40 20 0

p-JNK

Fig. 4. a CsA treatment significantly increased the expression of TGF-␤1, Smad2/3 and Smad-4. Smad-7 was partially decreased (G2) compared to the control (G1). RUS treatment significantly prevented upregulated TGF-␤1, Smad-2/3 and Smad-4 (G3). b Expression of p-ERK1/2 and p-JNK. CsA treatment significantly increased the expression of p-JNK. RUS treatment prevented this effect. * p ! 0.05 compared to G1, # p ! 0.05 compared to G2.

G1 160 140 120 100 80 60 40 20 0

G3

p-ERK1/2 (AU)

Smad-4

#

150

#

TGF-␤1

*

200

400 350 300 250 200 150 100 50 0

G2

G3

* #

G1

G2

G3

totic stimuli [24]. In this study, the ratio of Bax and Bcl-2 was reversed by RUS, indicating that RUS prevents apoptosis in a CsA-treated rat model. NO dysregulation has been linked to CsA-related vasoconstriction, inflammatory reaction, and fibrosis [25].

In this study, enhancement of iNOS expression in the CsA-treated group is evident as described in a previous study [26]. However, RUS at the dose of 10 mg/kg/day did not suppress the CsA-induced overexpression of iNOS. Di Napoli et al. [27] found that 2 mg/kg/day RUS signifi-

Rosuvastatin and Cyclosporine-Induced Nephropathy


13


G1

G2

G3

cantly decreased the expression of iNOS compared to the control group, but the beneficial effect of RUS at the dose of 20 mg/kg/day was lost in the expression of iNOS in a reperfusion injury rat heart model. Therefore, an adequate dose of RUS should be found for acquiring benefits in the point of iNOS. In our study, treatment with RUS did not alter the serum lipid levels. Therefore, the renoprotective effects of RUS were less related with the lipid-lowering effects of RUS in a rat model. Further studies are required to determine whether lipid-independent pleiotropic actions would be equally effective in CsA-induced nephropathy in clinical settings. In conclusion, RUS supplementation attenuates CsAinduced nephropathy by mitigating inflammation, apoptosis, EMT and fibrotic processes including the smad-dependent and smad-independent pathways. These findings suggest that RUS supplementation may have an important role in preventing CsA-induced nephropathy.

Acknowledgements This study was supported by a grant from the Korea Healthcare technology R&D project, Ministry of Health & Welfare, Republic of Korea (A070001).

Fig. 5. PAS-stained sections were examined using a light microscope. Original magnification !100. CsA treatment for 4 weeks induced interstitial fibrosis, inflammatory cell infiltration and tubular atrophy (G2) compared to control (G1). RUS cotreatment decreased pathologic changes (G3). Each image represents 5–6 samples.

Disclosure Statement The authors have nothing to declare.

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