Journal of Ethnopharmacology 156 (2014) 115–124

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Research Paper

Dahuang Fuzi Decoction ameliorates tubular epithelial apoptosis and renal damage via inhibiting TGF-β1-JNK signaling pathway activation in vivo Yue Tu a,b, Wei Sun a,n,1, Yi-Gang Wan c,1, Kun Gao d, Hong Liu b, Bing-Yin Yu b, Hao Hu b, Yan-Ru Huang b a

Department of Nephrology, The Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing, China Department of Graduate School, Nanjing University of Chinese Medicine, Nanjng, China Department of Traditional Chinese Medicine, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing, China d Department of Molecular Signaling, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Chuo, Yamanashi, Japan b c

art ic l e i nf o

a b s t r a c t

Article history: Received 2 June 2014 Received in revised form 3 August 2014 Accepted 24 August 2014 Available online 3 September 2014

Ethnopharmacological relevance: Dahuang Fuzi Decoction (DFD) is a traditional well-prescribed formula for the treatment of chronic kidney disease (CKD) in China. This study was carried out to examine the effects of DFD in adenine-induced tubular epithelial apoptosis and renal damage, in comparison with allopurinol (AP), then to clarify the therapeutic mechanisms in vivo. Materials and methods: A rat model of renal damage was created by adenine. Rats in Normal and Vehicle groups received distilled water, while rats in DFD and AP groups received DFD and AP, respectively. Proteinuria; urinary N-acetyl-β-D-glucosaminidase (NAG) levels; the blood biochemical parameters; renal histopathology damage; transferase-mediated dUTP nick-end labeling (TUNEL)-staining; the key molecular protein expressions in mitochondrial and transforming growth factor (TGF)-β1-c-JunNH2terminal kinase (JNK) pathways were examined, respectively. Results: Adenine administration induced severe renal damages, as indicated by the mass proteinuria, the heavy urinary NAG, and the marked histopathological injury in tubules and interstitium. This was associated with the activation of TGF-β1-JNK signaling pathway and tubular epithelial apoptosis. DFD treatment, however, significantly prevented proteinuria and urinary NAG elevation, and attenuated tubular epithelial apoptosis. It suppressed the protein expressions of Bax and cleaved caspase-3, whereas it enhanced the protein expression of Bcl-2. Furthermore, it also suppressed the protein levels of TGF-β1 as well as phosphorylated-JNK (p-JNK). Conclusion: DFD alleviated adenine-induced tubular epithelial apoptosis and renal damage in vivo, presumably through the suppression of TGF-β1-JNK pathway activation. & 2014 Elsevier Ireland Ltd. All rights reserved.

Chemical compounds studied in this article: rhein (PubChem CID: 10168) aloe-emodin (PubChem CID: 10207) emodin (PubChem CID: 3220) chrysophanol (PubChem CID: 10208) emodin-3-methyl ether (PubChem CID: 10639) (-) – asarinin (PubChem CID: 11869417) Keywords: Adenine-induced renal failure Dahuang Fuzi Decoction Tubular epithelial apoptosis Transforming growth factor-β1 c-JunNH2-terminal kinase

1. Introduction

Abbreviations: DFD, Dahuang Fuzi Decoction; CKD, chronic kidney disease; AP, Allopurinol; NAG, urinary N-acetyl-β-D-glucosaminidase; TUNEL, transferasemediated dUTP nick-end labeling; TGF, transforming growth factor; JNK, c-JunNH2terminal kinase; p-JNK, phosphorylated-JNK; CRF, chronic renal failure; MAPKs, mitogen-activated protein kinases; Bcl-2, B-cell lymphoma/leukemia-2; Bax, Bcl-2associated X protein; TCM, traditional Chinese medicine; α-SMA, α-smooth muscle actin; SUA, serum uric acid; BW, body weight; Scr, Serum creatinine; BUN, serum blood urea nitrogen; PAS, periodic acid-Schiff; PBS, phosphate buffered solution; BCA, bicinchoninic acid; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PVDF, polyvinylidene fluoride; GAPDH, glyceraldehyde-3phosphate dehydrogenase; TBST, Tris buffered saline tween; HRP, horseradish peroxidase; KW, kidney weight; ECM, extracellular matrix; UAN, uric acid nephropathy n Corresponding author. Tel.: þ 86 25 8661 7141-71416; fax: þ86 25 8661 8942. E-mail address: [email protected] (W. Sun). 1 These corresponding authors contributed equally to this work. http://dx.doi.org/10.1016/j.jep.2014.08.035 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.

The latest national survey of prevalence shows that chronic kidney disease (CKD) has become an important public health problem in China (Zhang et al., 2012). Previous studies have revealed that renal fibrosis is a common pathway for the progression of CKD to end-stage renal disease (Meguid El Nahas and Bello, 2005), and tubular epithelial apoptosis is a critical detrimental event that leads to tubular and interstitial injuries in association with irreversible renal fibrosis in human and animal models (Goumenos et al., 2002; Okamura et al., 2011). In rats, it has been shown that adenine feeding results in marked tubular and interstitial injuries and metabolic abnormalities, characterized by tubular dilation and renal dysfunction, which resembles chronic renal failure (CRF) in humans. Furthermore, the kidney injury was associated with the markers of apoptosis and the

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activation of caspase-3 (Ikeda et al., 2010; Shuvy et al., 2011). Transforming growth factor (TGF)-β has been recognized as an important mediator in the genesis of CKD. It has been demonstrated that TGF-β activates apoptosis in the cultured tubule epithelial cell lines (Bhaskaran et al., 2003). Moreover, overexpression of TGF-β1 leads to mesangial expansion, interstitial fibrosis and proteinuria in transgenic mice (Kopp et al., 1996). Additionally, the TGF-β signaling pathway can activate the mitogen-activated protein kinases (MAPKs) and induce apoptosis (Schuster and Krieglstein, 2002). Many studies have shown that the c-JunNH2-terminal kinase (JNK) signaling pathway contributes to tubular cell apoptosis and fibrosis processes in different models of diseases (Alcorn et al., 2009; Ma et al., 2007). TGFβ1 induces JNK activation in a bimodal manner, and it is reported that JNK1 mediates TGF-β1-induced renal fibrosis (Liu et al., 2012). B-cell lymphoma/leukemia-2 (Bcl-2) and Bcl-2-associated X protein (Bax), members of the Bcl-2 family, play key roles in initiating mitochondrial dysfunction. Bcl-2 is associated with the nuclear membrane, endoplasmic reticulum and outer mitochondrial membrane, and has been verified to regulate cell survival and prevent apoptosis (Harris and Thompson, 2000). In contrast, Bax acts as a dominant-negative inhibitor of Bcl-2, promoting apoptosis (Pastorino et al., 1998). Mitochondrial dysfunction induces downstream effector caspases, such as caspase-3, followed by proteolytic cleavage of a wide range of substrates responsible for the morphological and biochemical changes that are the hallmarks of apoptosis (Kroemer and Reed, 2000). JNK activation, the upstream of Bax conformational change and the translocation of Bax and Bak from the cytosol to the mitochondria, results in an apoptotic execution phase involving the activation of caspase 3 (cleaved caspase-3) (Yu et al., 2003). Therefore, we believe that intercepting TGF-β1-JNK signaling pathway may have the useful applications for treating adenine-induced tubular epithelial apoptosis and renal damage in model rats. The use of traditional Chinese medicine (TCM) has a unique treatment effect on CKD in China for many years. Dahuang Fuzi Decoction (DFD), from the Synopsis of Golden Chamber (Jin Kui Yao Lue) in Han Dynasty, is a classical prescription consisting of 3 medicinal plants: Radix et Rhizoma Rhei (Rheum L., Da huang, Rhubarb), Radix Aconiti Lateralis Praeparata (Aconitum carmichaeli Debx., Fu zi, Prepared common monk shood branched root), and Radix et Rhizoma Asari (Asarum sieboldii Miq., Xi xin, Manchurian wildginger). Li et al. reported a comparative pharmacokinetics study of DFD, showing that rhein, aloe-emodin and emodin are 3 anthraquinones present in rat plasma after oral administration (Li et al., 2013). In clinic in China, DFD is frequently used to treat the patients with gastropathy, gynaopathy, severe acute pancreatitis and CKD, in particular, renal dysfunction and the quality of life for CKD patients can be significantly ameliorated with DFD treatment (Liang et al., 2008; Liu et al., 2010; Lu et al., 2010; Zhao, 2013). In the basic pharmacological research area, the bioactive components of DFD such as rhein and emodin could improve fibrosis and apoptosis in vivo. It has been reported preliminarily, rhein not only suppresses the protein expressions of TGF-β1, α-smooth muscle actin (SMA) and fibronectin but also down-regulates the protein expression of caspase-3 in the obstructed kidneys in unilateral nephrectomy and adriamycininduced glomerulosclerosis models (He et al., 2011; Ji et al., 2005). In addition, emodin inhibits the activity of NF-κB signaling pathway and the protein overexpressions of TGF-β1 and fibronectin in diabetic nephropathy model, and that, it prevents the increased Bax/Bcl-2 ratio and apoptotic injury in cyclosporine-induced nephropathy rats (Son et al., 2013; Yang et al., 2013). In spite of these, the deepgoing therapeutic mechanisms in vivo by which DFD alleviates tubular epithelial apoptosis and renal damage remain poorly understood. Hence, in this report, our goal was to research the precise mechanisms in vivo of DFD in order to deepen our understanding

of its therapeutic effects in clinic, by using the approaches of observing kidney lesions and tubular epithelial apoptosis in rats intervening with adenine and DFD or allopurinol (AP) treatment, which has been proved to improve serum uric acid (SUA) level and renal dysfunction in CKD patients (Sezer et al., 2014), as well as evaluating the protein expressions of Bcl-2, Bax and cleaved caspase-3 in mitochondrial pathway, and detecting the changes in TGF-β1-JNK signaling pathway activation. 2. Materials and methods 2.1. Preparation of DFD The granules of DFD were composed of Radix et Rhizoma Rhei (9 g, voucher specimen no. 1201080), Radix Aconiti Lateralis Praeparata (12 g, voucher specimen no. 1210003) and Radix et Rhizoma Asari (3 g, voucher specimen no. 1208107), which were purchased from Tianjiang Pharmacology Co. Ltd (Jiangyin, China). All of the herb granules, with a total weight of 24 g, were dissolved in 24 mL distilled water to a concentration of 1 g/mL for experimental use. 2.2. Chromatographic analysis of DFD DFD was dissolved in methonal, 8% hydrochloric acid and chloroform, as well as filtered through a 0.45 μm filter (Microgen, Laguna Hills, CA, USA) before high performance liquid chromatography (HPLC) analyses. Agilent 1260 serious system (Agilent Technologies, Santa Clara, CA, USA) including pump, degrasser, autosampler, diode array detector and temperature controller was used for HPLC analyses. HPLC analyses were performed using Agilent ZORBAX SB-C18 (4.6  250 mm, particle size 5 μm) with methanol (as Solvent A) – acetic acid – sodium acetate buffer solution (pH 4.5; as Solvent B) as mobile phase at a flow rate of 1.0 mL/min at the column temperature of 35 1C. A gradient elution was applied from 60% of Solvent A and 40% of Solvent B starting from 0 to 8 min, 60–75% of Solvent A and 40–25% of Solvent B starting from 8 to 10 min, 75% of Solvent A and 25% of Solvent B starting from 10 to 35 min, 75–60% of Solvent A and 25–40% of Solvent B starting from 35 to 38 min, 60% of Solvent A and 40% of Solvent B starting from 38 to 40 min. Pure standards of rhein (voucher specimen no. 110757-200206), aloe-emodin (voucher specimen no. 110795-201007), emodin (voucher specimen no. 110756200110) and chrysophanol (voucher specimen no. 110796-201319) purchased from National Institutes for Food and Drug Control, as well as emodin-3-methyl ether (voucher specimen no. 130103) and (-) – asarinin (voucher specimen no. 130510) purchased from Chengdu Purechen-Shandard Co. Ltd, were used as external standards in the HPLC analyses. DFD mainly contained the bioactive compositions as follows, aloe-emodin (1.56%), rhein (0.12%), emodin (0.11%), chrysophanol (1.98%), emodin-3-methyl ether (1.96%) and (-) – asarinin (0.02%) for quality control (Fig. 1). 2.3. Reagent, drug, and animal Adenine (voucher specimen no. 0183) was obtained from Amresco (Solon, OH, USA) and fresh adenine was prepared every day. The 2% adenine was prepared from dissolving 1 g adenine in 50 mL flour solution. AP (voucher specimen no. 100502), obtained from Sine Wanxing Pharmacology Co. Ltd. (Shanghai, China), was produced by dissolving 100 mg AP in 50 mL distilled water to a concentration of 2 mg/mL. Twenty-seven Sprague-Dawley (SD) male rats, weighing approximately 200 g each, were purchased from the Animal Center of the Nanjing Military District General Hospital (Nanjing, China). The experiments were performed in accordance with protocols approved by the Animal Ethics Committee of Nanjing University

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Fig. 1. Results of HPLC analyses of DFD (below) and six compounds including rhein, aloe-emodin, (-) – asarinin, emodin, chrysophanol and emodin-3-methyl ether (above) in 287 nm. Column: Agilent ZORBAX SB-C18 (4.6  250 mm2, 5 μm); mobile phase: methanol (A) – acetic acid – sodium acetate buffer solution (pH 4.5; B) (A: 60% and B: 40% in 0– 8 min, A: 60–75% and B: 40–25% in 8–10 min, A: 75% and B: 25% in 10–35 min, A: 75–60% and B: 25–40% in 35–38 min, A: 60% and B: 40% in 38–40 min); flow rate: 1.0 mL/min; detection wavelength: 287 nm.

Medical School (Permit Number: SCXK (SU) 2008.0010). All rats were housed at 22 73 1C and 50710% humidity using a 12 h light/ dark cycle and were fed a standard rat chow and given tap water ad libitum in the Experimental Animal Center of The Affiliated Hospital of Nanjing University Medical School. The rats were allowed 1 week to acclimatize before the experiment. 2.4. Experimental design According to the previous studies (Deng et al., 1998; Wang et al., 2004), we administered 2% adenine at a dose of 150 mg/kg for 2 weeks to generate rats with renal failure. The experimental procedure is shown in Fig. 2. Twenty-seven rats were divided into four groups according to the random number table: ① 5 rats in Normal group (distilled water), ② 6 rats in Vehicle group (adenine þdistilled water), ③ 8 rats in DFD group (adenine þDFD), and ④ 8 rats in AP group (adenine þ AP). In clinic, 24 g/d DFD is used to treat a 60 kg patient. According to the animal standard conversion formula, the effective amount in rats is equivalent to 2.5 g/kg/d. Similarly, the effective amount of AP in rats is 0.03 g/kg/d. Following the administration of adenine for 2 weeks, DFD and AP were given to the rats in DFD and AP groups, respectively, by daily, morning gastric gavage for 3 weeks, while the rats in Vehicle and Normal groups were treated with 2 mL distilled water. Every 3 days, in the afternoon of the treatment period, the rats in Vehicle, DFD and AP groups were given 2% adenine at a total dose of 150 mg/kg to avoid a quick recovery of renal function. At the end of 5 weeks, all rats were anesthetized by intraperitoneal injection of ketamine and diazepam at the rate of 1 to 1 and at the dose of 1 mL/kg body weight (BW), and sacrificed via cardiac puncture. Blood samples and the kidneys were collected for the detection of various indicators.

appearance, and were removed and weighed before the rats were sacrificed by cardiac puncture. 2.6. Serum and urinary parameters Blood was drawn from the eye ground venous plexus at week 0 and 2. At week 5, blood was drawn from the heart while the rats were under anesthesia with ketamine and diazepam. Urine samples were collected from the rats, which were housed individually in metabolic cages. Urinary samples were obtained at week 0, 2 and 5 before and after the intervention of adenine and drugs. Serum creatinine (Scr), blood urea nitrogen (BUN), SUA, urinary protein as well as urinary N-acetyl-β-D-glucosaminidase (NAG) were measured using an automatic biochemical analyzer in the Department of Laboratory Medicine of Nanjing Drum Tower Hospital. 2.7. Histopathology Renal cortices were separated and trimmed mid-longitudinally, and 5 μm-paraffin embedded sections were prepared. These sections were stained with a periodic acid-Schiff (PAS) or a Masson reagent. Using light microscopy, changes in the glomeruli, tubules and interstitium were observed. The sections were analyzed in a double-blinded manner, and the degree of tubular damage was scored from grade 0 to 4 grades according to the percentage of tubular dilation and fibrosis as follows: grade 0, well-preserved renal architecture; grade 1, less than 25% of the field involved; grade 2, 25 to 50% of the field involved; grade 3, 50% to 75% of the field involved; and grade 4, 75% to 100% of the field involved (Wang et al., 2001). The number of tubular epithelial cells and the fibrosis area were calculated with Image-Pro Plus 5.0 software (Media Cybernetics, Silver Spring, MD). 2.8. Apoptosis assessment using TUNEL

2.5. General status and renal macroscopic morphology The spirit, diet, drinking water and activities of rats in each group were observed everyday and they were weighed every weekend. The kidneys were photographed to document their

Paraffin-embedded kidney tissue was cut into 5 μm-thick sections and processed for transferase-mediated dUTP nick-end labeling (TUNEL) staining with an in situ cell apoptosis detection kit (Boster, China). The nuclei labeled with a dark-brown color

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Fig. 2. Experimental procedure and sample collection.

were considered apoptotic cells when visualized at a magnification of 200  using a light microscope. The percentage of apoptotic cells in the tubules of control and treated rats are presented as the percentage of positive apoptotic tubular cells in 5 nonoverlapping random fields.

2.9. Semiquantitative Western blot analysis The protein expressions of Bcl-2, Bax, cleaved caspase-3, TGF-β1, JNK, and p-JNK in the kidney were evaluated by Western blot. The kidney samples, stored in  80 1C before use, were 100 mg. After washing with phosphate buffered solution (PBS) and centrifugation, the total protein was extracted by total protein lysate, which contains protease inhibitors and phosphatase inhibitors. After extracting the total protein, the protein concentrations were measured with a bicinchoninic acid (BCA) protein concentration assay kit (Keygentec, Nanjing, China) and the total protein was mixed with 5  electrophoresis sample buffer (Beyotime, Haimen, China). After equal amounts of protein in each sample were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 10% acrylamide gels, the separated proteins were transferred from the gel to a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, USA). The strips of membrane were incubated with the following antibodies: rabbit anti-rat Bcl-2 monoclonal antibody (1:500 dilution), rabbit antirat Bax monoclonal antibody (1:500 dilution), rabbit anti-rat cleaved caspase-3 monoclonal antibody (1:1000 dilution), rabbit anti-rat TGFβ1 monoclonal antibody (1:500 dilution), rabbit anti-rat JNK monoclonal antibody (1:500 dilution), rabbit anti-rat p-JNK monoclonal antibody (1:500 dilution), which were all purchased from Abcam Ltd. (HKSP, New Territories, HK), and mouse anti-rat glyceraldehyde-3phosphate dehydrogenase (GAPDH) monoclonal antibody (1:10000 dilution, Bioworld Technology Inc., MN, USA) after being blocked with 10% nonfat dry milk (Yili, Inner Mongolia, China). The membranes were then washed with Tris buffered saline tween (TBST) and incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulins (1:800–2000 dilution) and anti-mouse immunoglobulins (1:10000 dilution) (Bioworld Technology Inc., MN, USA). The membranes were coated using HRP-labeled chemiluminescent substrates (Millipore, Bedford, USA), exposed and fixed in the dark box. This procedure was carried out 3 times. Semiquantitative analyses of the protein contents were performed using the Quantity One 4.1.1. The results were compared with the densitometric signal of GAPDH, respectively, and the ratios were expressed as the relative protein contents.

2.10. Statistical analysis Differences among groups were analyzed by one-way analysis of variance (ANOVA), LSD method was used for multiple comparison. Qualitative data were analyzed using Fisher's exact test as indicated. The P-value reported was two-sided and value of less than 0.05 was considered statistically significant. All analyses were performed using the SPSS software (Version 13.0, SPSS Inc., USA).

3. Results 3.1. DFD improves rat's general status and renal macroscopic morphology The rats with adenine-induced renal damage were listless, lost their appetite and had low activity, which were all improved in DFD and AP groups. The water intake of all rats was measured every week and was approximately 30–50 mL per day. Fig. 3 shows renal macroscopic morphology in 4 groups. The macroscopic morphology of the kidneys was intact, ruddy and smooth in Normal group. In Vehicle group, adenine induced significant enlargement of the kidneys with a granular appearance. Compared to Vehicle group, the size and macroscopic morphology of the kidneys were improved in DFD and AP groups. The treatment with DFD has more of an effect on the renal appearance than that with AP. In comparison with Normal group, the BW of Vehicle, DFD and AP groups decreased significantly from week 3 (Fig. 4). Moreover, in week 4 and 5, the BW of AP group was significantly reduced compared with Vehicle and DFD groups (Fig. 4). Compared to Normal group, the kidney weight (KW) over the BW increased notably in Vehicle, DFD and AP groups (Fig. 5). However, there is no difference among Vehicle, DFD, and AP groups. In sum, these results showed that DFD and AP could significantly improve the general status and renal morphological appearance of the rats with adenine-induced renal damage. Furthermore, DFD has the obvious beneficial effect on BW in the rats with adenine administration. 3.2. DFD ameliorates adenine-induced proteinuria, urinary NAG excretion, and renal dysfunction Rats in Normal group had normal proteinuria, urinary NAG, and renal function throughout the 5-week period. In contrast, the mass proteinuria and impaired renal function in Vehicle, DFD and AP

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Fig. 3. Macroscopic morphology of the kidneys in 4 groups. (A) Normal group; (B) Vehicle group; (C) DFD group; (D) AP group. Abbreviations: DFD, Dahuang Fuzi Decoction; AP, Allopurinol.

Table 1 Kinetics of 24 h urinary protein excretion and urinary NAG excretion in 4 groups. Group (n)

Normal (5) Vehicle (6) DFD (8) AP (8)

Proteinuria (mg/24 h)

Urinary NAG (U/L)

Week 0

Week 2

Week 5

10.29 71.73 11.55 74.45 10.6771.66 10.61 72.60

10.81 7 1.28 38.79 7 6.60nn 38.56 7 9.63nn 37.377 13.89nn

10.60 7 1.74 30.38 7 3.83nn 19.39 7 5.76n## 24.50 7 8.24nn#

3.38 7 0.97 6.677 0.81nn 4.497 1.50## 5.25 7 1.05nn#

Abbreviations: NAG, N-acetyl-β-D-glucosaminidase; DFD, Dahuang Fuzi Decoction; AP, Allopurinol. The data are expressed as the mean 7S.E. Fig. 4. Changes in BW before and after the treatment in 4 groups. Abbreviations: BW, body weight; DFD, Dahuang Fuzi Decoction; AP, Allopurinol. n¼ 5–8 rats per group. The data are expressed as the mean 7 S.E. nnPo 0.01 vs. Normal group, # P o 0.05, ##Po 0.01 vs. Vehicle group, φ Po 0.05 vs. DFD group. Symbols are: (○) Normal group; (■) Vehicle group; (△) DFD group; (◇) AP group.

Fig. 5. KW over BW when the rats were sacrificed in 4 groups. Compared to Normal group, the ratio of KW to BW was increased notably in Vehicle, DFD and AP groups. There is no difference among Vehicle, DFD, and AP groups. The data are expressed as the means 7 S.E. n¼ 5–8 rats per group. nnPo 0.01 vs. Normal group. Abbreviations: BW, body weight; KW, kidney weight; DFD, Dahuang Fuzi Decoction; AP, Allopurinol.

groups were detected in adenine-administrated rats at week 2 (Tables 1 and 2). Proteinuria was declined significantly with DFD and AP treatment during the next 3 weeks (Table 1). Adenine induced the urinary NAG excretion elevation. Compared to Vehicle group, the urinary NAG excretion was reduced in DFD and AP groups (Table 1). The BUN, Scr and SUA levels were significantly lowered as a result of treatment with DFD and AP for 3 weeks, compared with Vehicle group (Table 2). In comparison with AP group, DFD was more effective in improving Scr (Table 2). In brief, DFD and AP decreased the proteinuria, urinary NAG, BUN, Scr and SUA in rats with adenine-induced renal damage. Moreover, DFD was more effective than AP in improving renal dysfunction. 3.3. DFD improves adenine-induced tubules and interstitium injury Histopathological changes in the tubules and interstitium in renal cortex in all groups were observed by light microscopy after staining with periodic acid-Schiff (PAS) and Masson. Compared to Normal

nn

#

Po 0.01 vs. Normal group. Po 0.05, ##Po 0.01 vs. Vehicle group at the same time point.

group (Fig. 6A and E), Fig. 6B and F shows significant morphological abnormality in renal cortex in Vehicle group, characterized by the loss of tubular epithelial cells, the accumulation of extracellular matrix (ECM), and interstitial fibrosis. This damage to renal cortex was ameliorated in DFD group (Fig. 6C and G) and AP group (Fig. 6D and H). Compared to Normal group, the tubule damage score was significantly increased in Vehicle group (Fig. 6I). With the treatment of DFD and AP, the tubular damage score was significantly lower, in comparison with that in Vehicle group (Fig. 6I). The tubular damage score was lower in DFD group, compared with AP group (Fig. 6I). Furthermore, compared to Normal group, the average number of tubular epithelial cells per tubule in Vehicle group was reduced significantly. The average number of tubular epithelial cells per tubule was higher in DFD and AP groups (Fig. 6J). Compared to AP group, DFD significantly increased the average number of tubular epithelial cells per tubule (Fig. 6J). As shown in Fig. 6K, almost no fibrosis area was observed in Normal group. After intervention with adenine, the fibrosis area in renal cortex increased; however, it became smaller after DFD and AP treatment. In addition, DFD showed the more effectiveness than AP in decreasing the fibrosis area (Fig. 6K) In short, histopathological injury including the tubular damage score, the number of tubular epithelial cells/tubule and the fibrosis area were attenuated in model rats with DFD and AP treatment. Furthermore, the therapeutic effect of DFD was more outstanding than that of AP in progressing tubules and interstitium lesion. 3.4. DFD attenuates renal tubular apoptosis Measured by the TUNEL assay, normal rats showed occasional TUNEL-positive cells (Fig. 7A). However, the number in tubules increased after the administration of adenine to normal rats (Fig. 7B). With the treatment of DFD and AP, the number of condensed pyknotic nuclei with apoptotic bodies was reduced, and the antiapoptotic effect of DFD was better than that of AP (Fig. 7C and D). Quantitation of TUNEL-stained apoptotic cells in the kidney in Vehicle group demonstrated a significant increased number of apoptotic cells, mainly in the tubules, in comparison with Normal group.

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Table 2 Blood biochemical parameters in 4 groups. Parameters

BUN (mmol/L)

Scr (μmol/L)

SUA (μmol/L)

Week

Group (n) Normal (5)

Vehicle (6)

0 2 5 0 2 5

3.98 7 0.53 4.58 7 0.43 4.727 0.37 19.80 7 1.30 20.80 7 1.92 21.80 7 2.86

3.98 70.43 22.7073.37nn 20.477 2.96nn 19.00 73.22 118.33711.89nn 110.83 78.61nn

0 2 5

75.60 7 6.73 84.40 7 6.66 92.80 7 8.53

70.17 74.07 199.3378.26nn 194.83 736.66nn

DFD (8) 3.90 7 0.53 21.94 7 3.15nn 12.83 7 3.84nn## 19.38 7 3.46 114.137 8.68nn 71.38 7 14.93nn## 69.63 7 6.55 203.007 3.85nn 138.63 7 10.60n##

AP (8)

φ

3.95 7 0.50 21.20 7 3.09nn 15.767 3.56nn# 19.137 2.95 115.38 7 10.21nn 90.50 7 18.24nn# 71.137 5.11 206.887 10.58nn 155.63 7 32.47nn#

Abbreviations: BUN, blood urea nitrogen; Scr, serum creatinine; SUA, serum uric acid, DFD, Dahuang Fuzi Decoction; AP, Allopurinol. The data are expressed as the mean7S.E. nn

Po 0.01 vs. Normal group. Po 0.05, ##Po 0.01 vs. Vehicle group. φ P o0.05 vs. AP group. #

Fig. 6. Histopathological changes in the tubules and interstitium in 4 groups. Photomicrographs of periodic acid-Schiff (PAS) staining (A-D) and Masson staining (E–H) (original magnification, 200  ). The glomerular capillary loops were open and the tubular epithelial cells were arranged in order in Normal group (A and E). With adenineinduced renal failure, loss of tubular epithelial cells and the accumulation of extracellular matrix (ECM) were detected (B and F). Damage to the renal tissues was ameliorated in DFD group (C and G) and AP group (D and H). I: The tubular damage score (grade). J: The number of tubular epithelial cells/tubule. K: Fibrosis area. Abbreviations: DFD, Dahuang Fuzi Decoction; AP, Allopurinol. The data are expressed as the means 7 S.E. n¼ 5–8 rats per group. nn P o0.01 vs. Normal group; #P o0.05, ##Po 0.01 vs. Vehicle group; φφPo 0.01 vs. AP group.

The percentage of TUNEL-positive cells was significantly declined in DFD and AP groups. DFD decreased the number of apoptotic cells more successfully than AP (Fig. 7E). We also found that DFD can ameliorate adenine-induced mitochondrial apoptosis. The results of one representative Western blot

are shown in Fig. 8. Compared to Normal group, Bax and cleavedcaspase-3 protein expression levels were significantly increased in Vehicle group. The increased protein expression of Bcl-2 in Vehicle group was significantly lower than that in Normal group. The protein expressions of Bax and cleaved-caspase-3 were significantly reduced

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Fig. 7. Immunohistochemical detection of TUNEL-positive cells in renal tissue in 4 groups, including A–D (original magnification, 200  ). TUNEL staining was performed in kidney sections from Normal group (A), Vehicle group (B), DFD group (C) and AP group (D). A representative area from each section is shown. An increase in the number of apoptotic cells (dark-brown color), mainly localized in the tubules, was observed in renal sections from Vehicle group, compared with Normal group. With DFD and AP treatment, the number of condensed pyknotic nuclei with apoptotic bodies was reduced, and the anti-apoptotic effect of DFD is better than that of AP. (E): Bar graph showing the percentage of apoptotic cells. Abbreviations: DFD, Dahuang Fuzi Decoction; AP, Allopurinol. The data are expressed as the means 7 S.E. n ¼5–8 rats per group. nnPo 0.01 vs. Normal group; #Po 0.05, ##P o 0.01 vs. Vehicle group; φφPo 0.01 vs. AP group.

Fig. 8. Protein expressions of Bcl-2, Bax, and cleaved caspase-3 in the kidney in 4 groups. (A) Normal group; (B) Vehicle group; (C) DFD group; (D) AP group. The Bcl-2, Bax, and cleaved caspase-3 proteins were extracted and subjected to semiquantitative Western blot analysis with an anti-Bcl-2 antibody, anti-Bax antibody, or anti-cleaved caspase-3 antibody. Equal loading of the protein per lane was verified by reprobing the blot with an anti-GAPDH antibody. The blot shown is representative of the Bcl-2, Bax, cleaved caspase-3 and GAPDH levels, from left to right, respectively, from four independent experiments (upper panel). The data in the lower panel are shown as the ratios relative to GAPDH. Abbreviations: DFD, Dahuang Fuzi Decoction; AP, Allopurinol. The data are expressed as the means 7 S.E. n¼5–8 rats per group. * Po 0.05 vs. Normal group; #P o0.05, ##P o0.01 vs. Vehicle group; φPo 0.05, φφPo 0.01 vs. AP group.

in DFD group, in comparison with Vehicle group. In addition, the protein expression of Bcl-2 increased significantly in DFD and AP groups. In a nutshell, DFD and AP could significantly reduce the number of apoptotic cells, mainly in tubules in model rats, through down-regulating the protein expressions of Bax and cleaved caspase-3, as well as up-regulating the expression of Bcl-2, as the key molecules in mitochondrial pathway.

3.5. DFD inhibits TGF-β1-JNK pathway Given the vital role of TGF-β1-JNK pathway in apoptosis (Ma et al., 2007; Yu et al., 2003), we examined the influence of DFD on this pathway. Western blot analysis showed that the expressions of TGF-β1 and p-JNK at the protein level were significantly elevated in Vehicle group (Fig. 9). In DFD group, however, they were significantly suppressed. In other words, the protein expressions of TGF-β1 and

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Fig. 9. Protein expressions of TGF-β1, JNK and p-JNK in the kidneys of 4 groups. (A) Normal group; (B) Vehicle group; (C) DFD group; (D) AP group. The TGF-β1, JNK and p-JNK proteins were extracted and subjected to semiquantitative Western blot analysis with an anti-TGF-β1 antibody, anti-JNK antibody or anti-p-JNK antibody. Equal loading of the protein per lane was verified by reprobing the blot with the anti-GAPDH antibody. The data blot shown is representative of TGF-β1, JNK, p-JNK and GAPDH levels, from left to right, respectively, from four independent experiments (upper panel). The data in lower panel are shown as the ratios relative to GAPDH. Abbreviations: DFD, Dahuang Fuzi Decoction; AP, Allopurinol. The data are expressed as the means7 S.E. n¼ 5–8 rats per group. nPo 0.05, nnPo 0.01 vs. Normal group; #Po 0.05, ##Po 0.01 vs. Vehicle group; φPo 0.05 vs. AP group.

p-JNK initiated by adenine could be significantly reduced by DFD and AP.

4. Discussion In this study, we provided the first evidence that DFD can ameliorate tubular epithelial apoptosis. The mechanisms involved are schematically illustrated in Fig. 10. We demonstrated that adenine-induced renal damage was associated with the activation of TGF-β1-JNK signaling pathway, and the induction of mitochondria apoptosis in tubular epithelial cells. Treatment of rats with DFD significantly improved tubular epithelial cell injury via blocking the activity of TGF-β1-JNK signaling pathway. According to Synopsis of Golden Chamber (Jin Kui Yao Lue), DFD, composed of Radix et Rhizoma Rhei, Radix Aconiti Lateralis Praeparata and Radix et Rhizoma Asari, is a typical prescription for the method of warm and purgation, with the effect of warming yang-deficiency and eliminating turbidity or stasis. In recent years, DFD has been reported to dramatically reverse the increased levels of BUN and Scr in CKD patients (Liu et al., 2010). Despite of the remarkable clinical effects, little is known about its therapeutic mechanisms. In the present study, using a rat model of renal damage induced by adenine, we explored the potential mechanisms of DFD in vivo. Models for CRF consist of partial nephrectomy and the administration of drugs, which are usually irreversible (Oite, 2011). Koeda et al. found that oral administration of adenine in rats leads to the kidney injury (Koeda et al., 1998), characterized by massive cystic dilatation and crystal deposition in the tubules. In addition to this, Shuvy et al. further demonstrated that high adenine phosphate diet cessation in rats could significantly reduce renal injury, which was coupled with the regression of apoptotic features, such as histological apoptotic features and apoptosis related pathways (Shuvy et al., 2011). Accordingly, we developed a renal damaged rat model, which was given adenine at a suitable dose of 150 mg/kg for 2 weeks, and the same dose every 3 days in the afternoon for the next 3 weeks. Using this strategy, renal dysfunction can be maintained at a stable level, and when rats with adenine-induced renal damage were

Fig. 10. Hypothetical scheme showing the proposed mechanisms involved in the anti-fibrotic and anti-apoptotic effects of Dahuang Fuzi Decoction in adenineinduced rat renal damage.

sacrificed, they all had renal failure with tubular and interstitial injury, which indicated that our modeling method was successful. It is widely acknowledged that uric acid nephropathy (UAN) in human beings is due to excessive production of UA, induced by disorders of purine metabolism or decreasing renal excretion of UA, which lead to hyperuricemia, renal injury via the deposition of urate microcrystals in the kidneys, and eventually to CRF (Shimizu and Hori, 2009). Similarly in rats, adenine, being a purine base, may be the source of the UA, as a product of xanthine oxidase, and can induce hyperuricemia, following kidney damage, which result from disequilibrium between the synthetic and elimination rates of UA (Sanders et al., 1997; So, 2007). AP, a structural analog of hypoxanthine and a xanthine oxidase inhibitor, could decrease serum levels of UA and reverse damaged kidney function and structure in rats fed adenine (Diwan et al., 2013).

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Using the newly established rat model, we investigated the effects of DFD and AP on renal damage. We firstly observed the general status and detected the serum and urinary parameters. Our results showed that DFD and AP significantly improved the general status and the morphological appearance in renal cortex, reduced proteinuria and urinary NAG, and decreased the levels of BUN, Scr as well as SUA in adenine-induced renal damaged rats. Furthermore, DFD could ameliorate the BW loss in the rats with adenine administration, while AP has no beneficial effect on it. Ikeda et al. found that renal failure, produced by feeding rats an adenine-containing diet for 4 weeks, is associated with the elevated serum inorganic phosphorus (Pi), Scr and BUN levels (Ikeda et al., 2010). However, we did not find a significant alternation in the Pi (data not shown), the reasons of which might be the different administration methods, adenine doses, and time periods for establishing the animal models. It has been demonstrated that, cell apoptosis, such as renal tubular epithelial apoptosis, is a critical cause of renal fibrosis, which eventually results in CKD (Okamura et al., 2011). In adenine administrated rats, tubular epithelial apoptosis and the activation of caspase-3 have been previously observed (Shuvy et al., 2011). In this investigation, we found the deterioration of renal histopathology in tubules and interstitium, as well as an increase in tubular epithelial apoptosis in adenine-induced renal damaged rats, which are consistent with the above-mentioned research works, and that, renal histopathological injury including the tubular damage score, the number of tubular epithelial cells/tubule and the fibrosis area, as well as the marked features of tubular epithelial apoptosis were attenuated after 3 weeks of DFD and AP therapies, compared with the model rats in Vehicle group. Our results thus indicated that DFD and AP have anti-fibrosis and anti-apoptosis effects in vivo, of note, these effects of DFD were more powerful than AP. Apoptosis is a highly regulated intracellular process that can be initiated through several signaling mechanisms, including two main intracellular signaling pathways, the extrinsic pathway (the death receptors) and the intrinsic pathway (the mitochondrian) (Green and Kroemer, 2004; Riedl and Salvesen, 2007). Bcl-2 family members are the arbiters of the mitochondrial apoptotic pathway, including anti-apoptotic members (such as Bcl-2) and proapoptotic members (such as Bax), which can regulate the activation of caspases that cleave a set of cellular proteins, such as caspase-3 (Mohamed et al., 2010; Tsujimoto, 1998). It has been reported that the overexpression of Bcl-2 can prevent apoptosis in renal proximal tubules (Lin et al., 1999). A previous study found that adenine-induced CRF model rats had tubular epithelial apoptosis, up-regulated Fas expression, and down-regulated Bcl2 expression in renal tissues (Wang et al., 2004). Moreover, Ryan et al. documented that, in aged skeletal muscles during repetitive in situ electrically stimulated isometric contractions, AP could reduce caspase-3 activity and Bax accumulation in mitochondria, but it did not affect other indicators of mitochondria-associated apoptotic signaling (Ryan et al., 2011). Interestingly, compared to AP, we found in this study that DFD dramatically reduced the number of apoptotic cells, mainly in tubules, via mitochondrial pathway in adenine-induced renal damaged rats. Not only so, DFD could down-regulate the protein expressions of Bax and cleaved caspase-3, as well as up-regulate the protein expression of Bcl-2 in the kidney in vivo. We unhappily have no idea whether DFD attenuates tubular epithelial apoptosis through the extrinsic death receptor pathway, which should be investigated in further studies. TGF-β is a multifunctional growth factor that has a principal role in regulating cell growth, development, differentiation and apoptosis in a variety of cell types, including epithelial cell types (Massagué, 1990). TGF-β1, one of the TGF-β isoforms, is considered to be a vital factor in promoting apoptosis in human renal proximal tubular epithelial cells (Xu et al., 2012). It has been demonstrated that JNK signaling plays a pathogenic role in tubular apoptosis and renal

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fibrosis (Alcorn et al., 2009; Ma et al., 2007). Nevertheless, under some circumstances, JNK plays a protective role and supports cell survival. JNK has diverse functions because it operates in a manner that depends on cell type, stimulus, and context, etc. (Davis, 2000). Liu et al. reported that JNK1 is critical for TGF-β1-induced renal fibrosis (Liu et al., 2012). It has been reported that rhubarb, a major herbal medicine of DFD, could attenuate TGF-β1-mediated migration of hepatic stellate cells by interfering the phosphorylation of JNK (Lin et al., 2009). Furthermore, JNK can activate some non-transcription factors, such as Bcl-2 (Yamamoto et al., 1999). For this reason, by means of modulating the expression of JNK, it is possible to block apoptosis and fibrosis signaling pathway reaction. In our report, JNK plays a pathogenic role in tubular epithelial apoptosis, namely, activating the mitochondrial pathway in rats with adenine-induced renal damage. In addition, DFD and AP markedly reduced the protein expressions of TGF-β1 and p-JNK, and that, had an anti-apoptotic effect via the Bcl-2/Bax-caspase-3 mitochondrial pathway, compared with the model rats in Vehicle group. These results impel us to believe that anti-apoptotic actions of DFD in vivo are analogous to the JNK signaling inhibitor. It happens that there is a similar case in the unilateral ureteral obstruction rats. Ma et al. (2007) reported that, CC-401, a specific inhibitor of JNK in vivo could not only reduce tubular apoptosis in the obstructed kidney, but also prevent the progression of renal fibrosis. However, it is a pity that we found the administration of adenine combined with the injection of this JNK signaling inhibitor resulted in the mass mortality in adenine-induced renal damaged rats. Thus, we considered that, although the JNK signaling inhibitor is a key modulator in the progression of renal fibrosis, the complete block of JNK signaling pathway in adenineinduced renal damaged rats could lead to the activation of other vital signaling pathways. By comparison, DFD similar to AP is safer and more helpful than the JNK signaling inhibitor in ameliorating tubular epithelial apoptosis and renal fibrosis in vivo. Here, we unavoidably think of the causal relationship between TGF-β1 and JNK, as two crucial signaling molecules in TGF-β1-JNK signaling pathway. The previous observation was reported that, in the invasion of stromal myofibroblasts, TGF-β1 stimulates JNK activity causing JNK to phosphorylate the JNK substrate Jun, and pharmacological inhibition of JNK alleviates TGF-β stimulated invasion (De Wever et al., 2004). Our data showed that the expression of JNK protein was increased accompanied by TGF-β1 protein over-expression in the kidney, and DFD decreased the expression of TGF-β1 protein together with the suppression of protein expression for JNK in adenine-induced renal damaged rats. Unfortunately, we could not affirm whether DFD in vivo directly affect TGF-β1 or JNK in TGF-β1-JNK pathway based upon this model. In future, more detailed analyses in vitro of TGF-β1-JNK pathway are needed to address this hypothesis. Given that in vivo experiments only demonstrated a small part of this complicated process, we cautiously conclude that, in rats with adenine-induced renal damage, TGF-β1-JNK pathway is activated, which in turn activates the Bcl-2/Bax-caspase-3 mitochondrial pathway and ultimately induces tubular epithelial apoptosis. In addition, DFD is a natural therapeutic option for alleviating adenine-induced renal damage and tubular epithelial apoptosis by inhibiting the activation of TGF-β1-JNK signaling pathway.

Author contributions Y.T., W.S., and YG.W. provided conception and design of research; Y.T., K.G., H.L., BY.Y., H.H. and YR.H. performed experiments; Y.T., K.G., W.S., and YG.W. analyzed data and interpreted results of experiments; Y.T. prepared figures and drafted manuscript; Y.T., W.S., and YG.W. edited and revised manuscript; Y.T., W.S. and YG.W. approved final version of manuscript.

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Acknowledgments This work was supported by: 1. 2013 Program for Excellent Doctoral Dissertation of Nanjing University of Chinese Medicine; 2. The National Natural Science Foundation of China (No. 81373607); 3. A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). We thank Dr. Yan Chen (Key Laboratory of Multi-delivery System of Chinese Materia Medica of State Administration of TCM, Key Laboratory of New Drug Delivery System of Chinese Meteria Medica, Jiangsu Provincial Institution of TCM,Nanjing, China) for HPLC analyses of DFD. We also thank Dr. Xun-Yang Luo and Dr. Le Zhang (Department of Laboratory Medicine, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medicine School, Nanjing, China) as well as Dr. Xue-jiao Yin (Department of Chinese Medicine, Huanghe Hospital, Zhengzhou, China) for their helpful technical assistance or in setting up the experiments. References Alcorn, J.F., van der Velden, J., Brown, A.L., McElhinney, B., Irvin, C.G., Janssen-Heininger, Y.M., 2009. c-Jun N-terminal kinase 1 is required for the development of pulmonary fibrosis. American Journal of Respiratory Cell and Molecular Biology 40, 422–432. Bhaskaran, M., Reddy, K., Radhakrishanan, N., Franki, N., Ding, G., Singhal, P.C., 2003. Angiotensin II induces apoptosis in renal proximal tubular cells. American Journal of Physiology – Renal Physiology 284, 955–965. Deng, H.Z., Jin, W.J., Hou, L.B., Liao, X.L., 1998. Methodology of pharmacodynamic study on chronic renal failure. China Journal of Chinese Material Medicine 23, 243–246. Davis, R.J., 2000. Signal transduction by the JNK group of MAP kinases. Cell 103, 239–252. De Wever, O., Westbroek, W., Verloes, A., Bloemen, N., Bracke, M., Gespach, C., Bruyneel, E., Mareel, M., 2004. Critical role of N-cadherin in myofibroblast invasion and migration in vitro stimulated by colon-cancer-cell-derived TGF-beta or wounding. Journal of Cell Science 117, 4691–4703. Diwan, V., Mistry, A., Gobe, G., Brown, L., 2013. Adenine-induced chronic kidney and cardiovascular damage in rats. Journal of Pharmacological and Toxicological Methods 68, 197–207. Goumenos, D.S., Tsamandas, A.C., El Nahas, A.M., Thomas, G., Tsakas, S., Sotsiou, F., Bonikos, D.S., Vlachojannis, J.G., 2002. Apoptosis and myofibroblast expression in human glomerular disease: a possible link with transforming growth factorbeta-1. Nephron 92, 287–296. Green, D.R., Kroemer, G., 2004. The pathophysiology of mitochondrial cell death. Science 305, 626–629. Harris, M.H., Thompson, C.B., 2000. The role of the Bcl-2 family in the regulation of outer mitochondrial membrane permeability. Cell Death and Differentiation 7, 1182–1191. He, D., Lee, L., Yang, J., Wang, X., 2011. Preventive effects and mechanisms of rhein on renal interstitial fibrosis in obstructive nephropathy. Biological and Pharmaceutical Bulletin 34, 1219–1226. Ikeda, R., Imai, Y., Maruyama, W., Mizoguchi, K., 2010. Systemic disorders of calcium dynamics in rats with adenine-induced renal failure: implication for chronic kidney disease-related complications. Nephrology 15, 54–62. Ji, Z.Q., Huang, C.W., Liang, C.J., Sun, W.W., Chen, B., Tang, P.R., 2005. Effects of rhein on activity of caspase-3 in kidney and cell apoptosis on the progression of renal injury in glomerulosclerosis. Chinese Medical Journal 85, 1836–1841. Kopp, J.B., Factor, V.M., Mozes, M., Nagy, P., Sanderson, N., Böttinger, E.P., Klotman, P.E., Thorgeirsson, S.S., 1996. Transgenic mice with increased plasma levels of TGF-beta 1 develop progressive renal disease. Laboratory Investigation 74, 991–1003. Koeda, T., Wakaki, K., Koizumi, F., Yokozawa, T., Oura, H., 1998. Early changes of proximal tubules in the kidney of adenine-ingesting rats, with special reference to biochemical and electron microscopic studies. Nippon Jinzo Gakkai Shi 30, 239–246. Kroemer, G., Reed, J.C., 2000. Mitochondrial control of cell death. Nature Medicine 6, 513–519. Lin, H.H., Yang, T.P., Jiang, S.T., Yang, H.Y., Tang, M.J., 1999. Bcl-2 overexpression prevents apoptosis-induced Madin-Darby canine kidney simple epithelial cyst formation. Kidney International 55, 168–178. Liang, X.X., Zhang, B.G., Liu, Q.F., 2008. Pharmacodynamic research and clinical application of Dahuang Fuzi Tang. Chinese Traditional Patent Medicine 30, 1670–1673. Lin, Y.L., Wu, C.F., Huang, Y.T., 2009. Effects of rhubarb on migration of rat hepatic stellate cells. Journal of Gastroenterology and Hepatology 24, 453–461. Liu, G.F., Zhou, Q., Tong, X.L., 2010. The clinical application and pharmacological research progress of Dahuang fuzi Decoction. Chinese Archives of Traditional Chinese Medicine 28, 1848–1851. Liu, Q., Zhang, Y., Mao, H., Chen, W., Luo, N., Zhou, Q., Chen, W., Yu, X., 2012. A crosstalk between the Smad and JNK signaling in the TGF-β-induced epithelial-mesenchymal transition in rat peritoneal mesothelial cells. PLoS One 7, e32009.

Li, H., Guo, H., Wu, L., Zhang, Y., Chen, J., Liu, X., Cai, H., Zhang, K., Cai, B., 2013. Comparative pharmacokinetics study of three anthraquinones in rat plasma after oral administration of Radix et Rhei Rhizoma extract and Dahuang Fuzi Tang by high performance liquid chromatography-mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis 76, 215–218. Lu, X.G., Zhan, L.B., Kang, X., Liu, L., Li, Y.Z., Yu, J., Fan, Z.W., Bai, L.Z., Ji, C.Y., Wang, X.Z., 2010. Clinical research of Dahuang Fuzi decoction in auxiliary treatment of severe acute pancreatitis: a multi-center observation in 206 patients. Chinese Critical Care Medicine 22, 723–728. Massagué, J., 1990. The transforming growth factor-beta family. Annual Review of Cell Biology 6, 597–641. Meguid El Nahas, A., Bello, A.K., 2005. Chronic kidney disease: the global challenge. Lancet 365, 331–340. Ma, F.Y., Flanc, R.S., Tesch, G.H., Han, Y., Atkins, R.C., Bennett, B.L., Friedman, G.C., Fan, J.H., Nikolic-Paterson, D.J., 2007. A pathogenic role for c-Jun aminoterminal kinase signaling in renal fibrosis and tubular cell apoptosis. Journal of the American Society of Nephrology 18, 472–484. Mohamed, S.A., Misfeld, M., Hanke, T., Charitos, E.I., Bullerdiek, J., Belge, G., Kuehnel, W., Sievers, H.H., 2010. Inhibition of caspase-3 differentially affects vascular smooth muscle cell apoptosis in the concave versus convex aortic sites in ascending aneurysms with a bicuspid aortic valve. Annals of Anatomy 192, 145–150. Oite, T., 2011. Exploring the mechanisms of renoprotection against progressive glomerulosclerosis. Proceedings of the Japan Academy – Series B: Physical & Biological Sciences 87, 81–90. Okamura, D.M., Pasichnyk, K., Lopez-Guisa, J.M., Collins, S., Hsu, D.K., Liu, F.T., Eddy, A.A., 2011. Galectin-3 preserves renal tubules and modulates extracellular matrix remodeling in progressive fibrosis. American Journal of Physiology – Renal Physiology 300, 245–253. Pastorino, J.G., Chen, S.T., Tafani, M., Snyder, J.W., Farber, J.L., 1998. The overexpression of Bax produces cell death upon induction of the mitochondrial permeability transition. Journal of Biological Chemistry 273, 7770–7775. Riedl, S.J., Salvesen, G.S., 2007. The apoptosome: signalling platform of cell death. Nature Reviews Molecular Cell Biology 8, 405–413. Ryan, M.J., Jackson, J.R., Hao, Y., Leonard, S.S., Always, S.E., 2011. Inhibition of xanthine oxidase reduces oxidative stress and improves skeletal muscle function in response to electrically stimulated isometric contractions in aged mice. Free Radical Biology and Medicine 51, 38–52. Sanders, S.A., Eisenthal, R., Harrison, R., 1997. NADH oxidase activity of human xanthine oxidoreductase – generation of superoxide anion. European Journal of Biochemistry 245, 541–548. Schuster, N., Krieglstein, K., 2002. Mechanisms of TGF-beta-mediated apoptosis. Cell and Tissue Research 307, 1–14. Sezer, S., Karakan, S., Atesagaoglu, B., Acar, F.N., 2014. Allopurinol reduces cardiovascular risks and improves renal function in pre-dialysis chronic kidney disease patients with hyperuricemia. Saudi Journal of Kidney Diseases and Transplantation 25, 316–320. So, A., 2007. Recent advances in the pathophysiology of hyperuricemia and gout. Revue Médicale Suisse 3, 722–724. Shimizu, T., Hori, H., 2009. The prevalence of nephrolithiasis in patients with primary gout: a cross-sectional study using helical computed tomography. Journal of Rheumatology 36, 1958–1962. Shuvy, M., Nyska, A., Beeri, R., Abedat, S., Gal-Moscovici, A., Rajamannan, N.M., Lotan, C., 2011. Histopathology and apoptosis in an animal model of reversible renal injury. Experimental and Toxicologic Pathology 63, 303–306. Son, Y.K., Lee, S.M., An, W.S., Kim, K.H., Rha, S.H., Rho, J.H., Kim, S.E., 2013. The role of protein kinase CK2 in cyclosporine-induced nephropathy in rats. Transplantation Proceedings 45, 756–762. Tsujimoto, Y., 1998. Role of Bcl-2 family proteins in apoptosis: apoptosomes or mitochondria? Genes to Cells 3, 697–707. Wang, W.S., Tzanidis, A., Divjak, M., Thomson, N.M., Stein-oakley, AN, 2001. Altered signaling and regulatory mechanisms of apoptosis in focal and segmental glomerulosclerosis. Journal of the American Society of Nephrology 12, 1422–1433. Wang, S.H., Chen, J., Fu, Q., 2004. Influence of shenshuai capsule on renal tissue apoptosis of rats with chronic renal failure. Journal of Chinese Medicinal Materials 27, 195–197. Xu, Y., Yang, S., Huang, J., Ruan, S., Zheng, Z., Lin, J., 2012. Tgf-β1 induces autophagy and promotes apoptosis in renal tubular epithelial cells. International Journal of Molecular Medicine 29, 781–790. Yamamoto, K., Ichijo, H., Korsmeyer, S.J., 1999. BCL-2 is phosphorylated and inactivated by an ASK1/Jun N-terminal protein kinase pathway normally activated at G(2)/M. Molecular and Cellular Biology 19, 8469–8478. Yu, W.P., Sanders, B.G., Kline, K., 2003. RRR-α-tocopheryl succinate-induced apoptosis of human breast cancer cells involves Bax translocation to mitochondria. Cancer Research 63, 2483–2491. Yang, J., Zeng, Z., Wu, T., Yang, Z., Liu, B., Lan, T., 2013. Emodin attenuates high glucose-induced TGF-β1 and fibronectin expression in mesangial cells through inhibition of NF-κB pathway. Experimental Cell Research 319, 3182–3189. Zhang, L.X., Wang, F., Wang, L., Wang, W., Liu, B.C., Liu, J., Chen, M.H., He, Q., Liao, Y.H., Yu, X.Q., Chen, N., Zhang, J.E., Hu, Z., Liu, F.Y., Hong, D.Q., Ma, L.J., Liu, H., Zhou, X.L., Chen, J.H., Pan, L., Chen, W., Wang, W.M., Li, X.M., Wang, H.Y., 2012. Prevalence of chronic kidney disease in China: a cross-sectional survey. Lancet 379, 815–822. Zhao, P., 2013. Study of the quality of life for patients with chronic renal failure treating by retentive enema using Dahuang Fuzi Decoction. Chinese Journal of Integrative Traditional and Western Nephrology 14, 535–536.