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Protective effects of cyclic helix B peptide on aristolochic acid induced acute kidney injury
Biomedicine & Pharmacotherapy 94 (2017) 1167–1175
Available online at
ScienceDirect www.sciencedirect.com
Original article
Protective effects of cyclic helix B peptide on aristolochic acid induced acute kidney injury Yigang Zenga,b,1, Long Zhenga,b,1, Zhangru Yanga,b,1, Cheng Yanga,b , Yi Zhanga,b , Jiawei Lia,b , Weitao Zhanga,b , Mingnan Zhanga,b , Mushuang Hua,b , Shuo Wanga,b , Sidikejiang Niyazia,b , Ming Xua,b , Ruiming Ronga,b , Tongyu Zhua,b,c,* a
Department of Urology, Zhongshan Hospital, Fudan University, 180 Fenglin Road, Shanghai, 200032, China Shanghai Key Laboratory of Organ Transplantation, Shanghai, China c Shanghai Public Health Clinical Center, Fudan University, Shanghai, China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 April 2017 Received in revised form 25 July 2017 Accepted 25 July 2017
Background: Aristolochic acid (AA) injuries remain a serious condition associated with acute renal dysfunction. Herein, the effect and mechanism of a novel tissue protective peptide, cyclic helical Bpeptide (CHBP) derived from erythropoietin, were investigated in a mice model. Methods: Mice were randomly divided into four groups, receiving the following treatments (1: saline; 2: AA 10 mg/kg; 3: AA 10 mg/kg +CHBP 4nmol/kg; 4: AA 10 mg/kg +CHBP 8nmol/kg). Results: Blood urea nitrogen and serum creatinine was increased by AA but decreased by CHBP in a dosedependent fashion. CHBP also significantly improved renal tubular injury and inflammatory infiltration, which was gradually increased by AA. Apoptotic cells, infiltrating inflammatory cells, and active caspase3+ cells were greatly reduced by CHBP. In addition, CHBP inhibited caspase-3, 9 and improved bcl-2, bcl-xl protein expression in vivo. Conclusion: Taken together, we demonstrated, for the first time, that CHBP effectively improved renal function and tissue damage caused by AA, which maybe through reducing caspase-3 activation, apoptosis, and inflammation. © 2017 Published by Elsevier Masson SAS.
Keywords: Cyclic Helix B peptide Aristolochic acid Acute kidney injury
1. Introduction The traditional Chinese medicine aristolochic acid (AA), despite being banned in many countries, is still sold on the Internet [1]. Aristolochic acid nephropathy (AAN) remains a clinical problem [2]. Short-term high-dose AA can directly damage proximal renal tubular epithelial cells, activate the renal tissue cysteinyl aspartate-specific protease (caspase) pathway, and induce a renal tissue inflammatory reaction, leading to degeneration, necrosis, and apoptosis [3].The typical characteristics of AAN are described as severe renal tubular damage and rapid progression to end-stage renal disease [4].It is reported that the 2-year renal survival is only 17%, which is worse than other tubule interstitial nephropathies (74%) [5].Clinically, no effective treatment can reverse the progression of AA induced renal injury[5].
* Corresponding author at: Department of Urology, Zhongshan Hospital, Fudan University, 180 Fenglin Road, Shanghai, 200032, China. E-mail address: [email protected] (T. Zhu). 1 These authors contributed equally to this article. http://dx.doi.org/10.1016/j.biopha.2017.07.131 0753-3322/© 2017 Published by Elsevier Masson SAS.
Erythropoietin (EPO) is a hematopoietic hormone produced mainly by adult kidneys and has been routinely used clinically to treat anemia, for nearly 20 years [6]. In addition to promoting erythropoiesis, EPO also exhibits a strong tissue protective effect against kidney damage caused by ischemia-reperfusion injury [7]. EPO can inhibit renal tubular epithelial cell apoptosis, downregulate renal tissue after ischemia-reperfusion injury, and reduce acute renal injury [7]. However, to have a kidney protective effect, high doses of EPO are required, which can lead to erythropoiesis and its resulting side effects, including a hypercoagulable state, thrombosis, and hypertension [8]. Dose limitations significantly disrupt the balance between the benefits and risks of EPO applications [9]. With the development of biochemistry and biotechnology, therapeutic peptides have become popular and more effective [10]. Recently, the new linear peptide helix B surface peptide (HBSP) from EPO shows satisfactory renal protection by inhibiting inflammation and apoptosis [10].However, the 2-min plasma half-life of HBSP limits its use in vivo[10]. Thus, we have recently used a new cyclization strategy to synthesize thioether cyclic helical B-peptide (CHBP), which significantly enhances metabolic
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2. Materials and methods
were blind-labeled and reviewed by two renal pathologists. Renal damage was graded based on the percentage of damaged tubules in the sample: 0 = normal kidney (no damage); 1 = minimal damage (<25% damage); 2 = mild damage (25%–50% damage); 3 = moderate damage (50%–75% damage); and 4 = severe damage (>75% damage), similar with previous descriptions [10]. Renal tubular epithelial degeneration, renal tubular lumen expansion, tubular necrosis, inflammatory cell infiltration, renal interstitial edema, and renal interstitial fibrosis were used as an evaluation indicator of renal tubular and renal interstitial injury. Mean values of these estimates were used in the analyses [13]. Masson trichrome staining was also performed according to the standard protocol to assess collagen deposition [3].
2.1. Aristolochic acid induced renal injury model
2.4. In situ end-labeling apoptotic cells
Male BALB/c mice (18–20 g) were obtained from Shanghai Slac Lab Animal, Co., Ltd. (Shanghai, China), and bred in a Specific pathogen Free (SPF)-grade experimental animal room. All animal experiments were performed according to the guidelines of the Care and Use of Laboratory Animals of the Laboratory Animal Ethical Commission of Fudan University with good animal surgical research practices, and were approved by the Animal Ethical Committee of Zhongshan Hospital, Fudan University. A total of 64 BALB/c mice were randomly divided into four groups (n = 16 each) according to the different treatments: (1) control group: intraperitoneal injection of equal doses of saline; (2) model group: intraperitoneal injection of AA sodium (Sigma, San Francisco, California, USA, 10 mg/kg in 1 mg/ml saline); (3) Intervention 1 group: intraperitoneal injection of AA sodium (Sigma10 mg/kg at the onset of 4 nmol/kg CHBP); (4) Intervention 2 group: intraperitoneal injection of AA sodium (Sigma10 mg/kg at the onset of 8 nmol/kg CHBP). The cardiac blood and renal tissue samples were harvested after ketamine anesthesia (Heng Rui Medicine Co., Ltd., Jiangsu, China), and then animals were ethically sacrificed (6 random mice per group, at 1, 3, and 7 days after injection, respectively).
Apoptotic cells in renal tissues were detected with TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling) staining using a pop Tag peroxidase kit (In Situ Cell Death Detection Kit-POD; Roche Diagnostics, Mannheim, Germany). After TUNEL labeling, nuclei were stained with hematoxylin and the TUNEL positive cells were observed at 200 magnification over 20 fields in the tubular, interstitial, and tubular lumen areas [11].
stability and renal protection [11]. In our previous ischemiareperfusion model, CHBP showed a strong protective effect on ischemia and apoptosis in a renal ischemia-reperfusion injury [11]; 8 nmol/kg of CHBP is the optimal dose [11]. In the pig model, CHBP administered with a preservative solution and autologous blood perfusate provides significant protection against renal injury, by increasing renal blood flow and oxygenation, and reducing apoptosis, inflammation, and tissue damage [12]. Based on these results, we hypothesized that CHBP may attenuate AA-induced renal injury. In this paper, the protective effect and mechanism of CHBP were studied in mouse models.
2.2. Renal function Whole blood was centrifuged at 1600g for 25 min at 4 C to obtain serum. A fully automatic biochemical analyzer was used for testing serum creatinine (SCr) and blood urea nitrogen (BUN) (BioAssay Systems,Hayward, CA,USA). 2.3. Histologic assessment
2.5. Immunostaining of myeloperoxidase (MPO) and active caspase-3 Immunohistochemical staining of MPO (Abcam, Cambridge, UK) and active caspase 3 (Abcam) was performed on frozen kidney sections using a DAKO ChemMate EnVision Detection Kit (DAKO, Carpinteria, CA, USA), as previously described [10]. Then, 10 non-overlapping fields of 200 were randomly selected from each slice. The average absorbance values for each field were calculated and semi-quantitative analysis was performed [10]. 2.6. Western blot analysis Membrane proteins were separated on SDS (sodium dodecyl sulfate, sodium salt)–polyacrylamide gels and transferred to polyvinylidene difluoride(PVDF) membranes. The membranes were blocked with 5% milk and incubated overnight with antiPI3 K p85, anti-AKt, anti-p-Akt Ser473, anti-cleaved caspase-3, anti- cleaved caspase- 9,anti-bcl-2, anti-bcl-xl, and anti-GAPDH antibodies, (1:1000; all from Cell Signaling Technology Inc., Beverly, MA, USA). The results were analyzed as previously described [11].
Hematoxylin and eosin (H&E) staining and Masson staining were performed to assess histologic injury. The tissue sections
Fig. 1. CHBP improved AA-induced acute renal dysfunction. Scr: serum creatinine; BUN: blood urea nitrogen; T1: 1 day after treatment; T2: 3 days after treatment; T3: 7 days after treatment;* P < 0.05 compared with control group; # P < 0.05 compared with model group; & P < 0.05 compared with Intervention 1 group.
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2.7. Statistical analysis
3. Results
SPSS software version 20.0 (SPSS 20.0 software, SPSS Inc., Armonk, NY, USA) was used for statistical analysis of the data by one-way analysis of variance, followed by a Bonferroni correction. Data are presented as the mean standard deviation. A p-value of less than 0.05 was considered to be statistically significant.
3.1. Identification of acute renal injury model
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An AA-induced acute renal injury model was successfully established by AA intraperitoneal injection (10 mg/kg). One day after treatment, creatinine and urea nitrogen levels of the model group increased significantly compared with the control group (p < 0.05) and peaked on day 7 (Fig. 1). Histological findings were confirmed on day 3 after the treatment, which showed renal tubular injury and inflammatory infiltration; on day 7, severe renal tubular damage appeared. The renal tubular damage grade of the model group was significantly increased compared to the control
Fig. 2. CHBP attenuated renal AA- induced renal tissue injury, inflammation. H&E: Hematoxylin and eosin staining; T1: 1 day after treatment; T2: 3 days after treatment; T3: 7 days after treatment;* P < 0.05 compared with control group; # P < 0.05 compared with model group; & P < 0.05 compare with Intervention 1 group;.
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group on day 7 after treatment, according to the renal tubule pathologic injury score (p < 0.05; Fig. 2). 3.2. CHBP can improve AA-induced acute renal dysfunction Compared with the model group, SCr and BUN levels significantly improved after CHBP, in a dose dependent manner (p < 0.05, days 1, 3, and 7; Fig. 1). 3.3. CHBP attenuates AA-induced renal tissue injury, inflammation, and collagen deposition To evaluate the renoprotective effects of CHBP in the mouse acute renal injury model, H&E and Masson staining of the kidneys was performed in each group. The tubular structure was found to have a healthy morphology and structure in the control group,
while severe renal tubular injury, interstitial dilatation, and inflammatory cell infiltration were observed in the model group. In contrast, CHBP treatment improved renal tubule interstitial disease (p < 0.05) compared with the model group, in a dosedependent manner (p < 0.05).Semi-quantitative analysis using a histological scoring system revealed that the kidney tissue in the CHBP-treated group was well protected, showing mild interstitial edema and cellular infiltration (Fig. 2). Masson staining was used to demonstrate extracellular matrix (ECM) deposition within the tubule interstitium. As shown in Fig. 3, collagen deposition was obviously elevated in the model group (p < 0.05) compared with the control group, but markedly reduced by CHBP treatment (p < 0.05), in a dose-dependent manner (p < 0.05).
Fig. 3. CHBP attenuated renal AA- induced renal collagen deposition. T1: 1 day after treatment; T2: 3 days after treatment; T3: 7 days after treatment;* P < 0.05 compared with control group; # P < 0.05 compared with model group; & P < 0.05 compared with Intervention 1 group;.
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Fig. 4. CHBP improved AA-induced renal damage by inhibiting Inflammation. MPO: Myeloperoxidase; T1: 1 day after treatment; T2: 3 days after treatment; T3: 7 days after treatment;* P < 0.05 compared with control group; # P < 0.05 compared with model group; & P < 0.05 compared with Intervention 1 group.
3.4. CHBP improves AA-induced renal damage by inhibiting inflammation Inflammation was assessed by immunostaining of MPO+ cells, a marker primarily seen in neutrophil granulocytes. Control samples had limited MPO + cells. However, MPO + cells were significantly increased by AA, especially in peritubular and expanded interstitial areas. CHBP reduced AA-induced MPO + cells, mainly in the interstitial region, in a dose-dependent manner (p < 0.05). Some MPO + cells demonstrated cohesive nuclei, morphological features of apoptosis, or tubular lumens (Fig. 4).
3.5. CHBP inhibited AA-induced cell apoptosis in renal tissue Apoptotic cells were primarily located in the tubular and interstitial regions by TUNEL staining. The total number of apoptotic cells counted in tubular, interstitial, and tubular lumen areas was greatly increased in the model group compared with the control group (p < 0.05). CHBP reduced AA-induced apoptotic cells in kidney tissue (p < 0.05), and had the most important impact in the tubular region in a dose-dependent manner (p < 0.05; Fig. 5). 3.6. CHBP inhibited AA-induced active Caspase-3+ cells in renal tissue The distribution of active caspase-3+ cells detected by immunostaining, recognizing the 17 kD subunit, was mainly in the tubular and interstitial areas, fewer in glomerular areas. Almost
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Fig. 5. CHBP Inhibited AA-induced Cell Apoptosis in renal tissue. TUNEL: Terminal Deoxynucleotidyl Transferase mediated dUTP-biotin nick end labeling; T1: 1 day after treatment; T2: 3 days after treatment; T3: 7 days after treatment;* P < 0.05 compared with control group; # P < 0.05 compared with model group; & P < 0.05 compared with Intervention 1 group.
all positive cells showed condensed nuclei, the morphologic feature of apoptosis. Active caspase-3+ cells were increased in the model group compared with the control group (p < 0.05) but decreased by CHBP (p < 0.05) in a dose-dependent manner (p < 0.05; Fig. 6). 3.7. CHBP inhibits caspase-3, 9 and improve bcl-2, bcl-xl protein expression in vivo The expression of caspase-3, 9, bcl-2, and bcl-xl protein subunits in mouse kidneys was detected by western blot. The numerically highest expression of cleaved caspase-3, 9 was shown in the model group, but significantly reversed by CHBP (p < 0.05) in a dose-dependent manner (p < 0.05). The numerically lowest expression of bcl-2, bcl-xl was shown in the model group, but
significantly increased by CHBP (p < 0.05) in a dose-dependent manner (p < 0.05; Fig. 7) 4. Discussion AA is a major secondary metabolite of the aristolochic species and is known to be a human nephrotoxin [14]. AAN caused by Chinese medicine containing AA, has been reported at home and abroad; its clinical and pathological characteristics demonstrate an in-depth understanding. A large number of evidence-based studies have shown that AAN mainly involves renal tubules and interstitium, but not involve glomeruli [15].A single or short-term high-dose of AA, acting on the renal tubular epithelial cells in vivo, leads to significant cell necrosis and apoptosis, and can even lead to acute ANN [1]. A one-time dose of 10 mg/kg AA can lead to acute kidney injury in BALB/c mice, as confirmed in this study.
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Fig. 6. CHBP Inhibited AA-induced Active Caspase-3+ Cells in renal tissue.caspase: cysteinyl aspartate-specific protease; T1: 1 day after treatment; T2: 3 days after treatment; T3: 7 days after treatment;* P < 0.05 compared with control group; # P < 0.05 compared with model group; & P < 0.05 compared with Intervention 1 group.
AA can cause necrosis and apoptosis of proximal renal tubular epithelial cells of various genera (human, porcine, mouse, rabbit) [16]. In vivo, AA is able to rapidly stimulate extracellular calcium to enter the cytoplasm. Then, a rapid release of calcium of the intracellular calcium-endoplasmic reticulum and mitochondria into the cytoplasm [17].This leads to caspase activation, followed by damage to renal tubular epithelial cells, and eventual apoptosis [7]. In in vitro experiments, AA stimulated human renal tubular epithelial cells (HK-2) for 12, 24, and 48 h. And then the number of cells were reduced with a dose and time-dependent manner [18]. Intracellular ATP is decreased, followed by mitochondrial membrane depolarization and release of cytochrome c, leading to the activation of caspase-3 and ultimately resulting in cell damage and cell cycle arrest in the G2/M phase [18]. AA activates the caspase cascade, and ultimately causes apoptosis. We confirmed that the signal transduction pathway
of AA-induced renal tubular epithelial cell apoptosis is a caspase-3 dependent pathway [1]. As a terminal effect of apoptosis, caspase3 is a common key protease for substrate cleavage downstream of each pathway, its degradation of substrate activity is strongest, and it is called the executive of apoptosis [15].Caspase activation is associated with apoptosis. Inhibition of caspase activity can improve renal injury and reduce renal tubular epithelial cell apoptosis [15]. EPO is an endogenous protein that plays a protective role in a wide range of organs [7]. In ischemic-induced acute renal failure, the administration of EPO can reduce apoptosis of renal tubular cells and promote the recovery of renal function by inhibiting the activity of caspase-3, 9 [7]. The anti-apoptotic effect of EPO inhibits the activity of caspase-3 by phosphorylation of Akt and upregulates the expression of anti-apoptotic factors [18]. However, due to the very high dose requirements required to achieve tissue
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Fig. 7. CHBP inhibited caspase-3, 9 and improved bcl-2, bcl-xl protein expression in vivo. PI3 K: Phosphatidylinositol 3-kinase; AKt: protein kinase B; p-AKt: Phosphorylation AKt; caspase: cysteinyl aspartate-specific protease; bcl: B-cell lymphoma; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; T1: 1 day after treatment; T2: 3 days after treatment; T3: 7 days after treatment;* P < 0.05 compared with control group; # P < 0.05 compared with model group; & P < 0.05 compared with Intervention 1 group;.
protection, it causes adverse side effects, limiting the use of EPO in anti-cell apoptosis [17].We synthesized the small molecule polypeptide HBSP by analyzing the three-dimensional structure of EPO, according to the most critical 11 amino acid residue sequence on the hydrophilic interface of helix B [13]. HBSP is different from EPO, which has no erythropoietic activity and only binds to tissue protective receptor, and fully simulates the mechanism of tissue protection of endogenous EPO [10].The short half-life of HBSP restricts its clinical transformation [10].For this purpose, we invented the conformation constraints of the cyclic peptide CHBP [11]. CHBP has stability and biological activity [11]. We found that CHBP significantly reduced apoptosis and necrosis of renal tubular epithelial cells after acute renal injury, relieved its associated inflammatory response, and induced autophagy of renal tubular epithelial cells, demonstrating promising renal protection [11]. It was confirmed that the protective effect of 8 nmol/kg CHBP on ischemia-reperfusion injury was the most effective [11]. Based on this, we speculate that CHBP can protect AA from acute renal injury by inhibiting apoptosis. In order to reveal the protective effect of CHBP on renal injury induced by AA, we first evaluated renal function. On day 7, BUN and SCr levels were significantly increased in the model group. Compared with the model group, BUN and SCr levels were
significantly decreased in the intervention group after CHBP treatment, in a dose-dependent manner. Pathologically, CHBP significantly improved AA-induced renal tubular injury, interstitial expansion, and inflammatory cell infiltration. CHBP can reduce the destruction of renal tubular structure, retain more functional kidneys, and shows a good renal protective effect on histology. Immunostaining of active caspase-3 showed that the activity of caspase-3 + cells in the model group exists mainly in the tubule interstitial region and renal tubular lumen, and was significantly increased compared with the control group. CHBP significantly reduced the amount of active caspase-3 + cells in a dose-dependent manner. Further studies showed that CHBP significantly downregulated the expression of caspase-3, 9 protein and upregulated the expression of bcl-2, bcl-xl protein after AA injury, and inhibited apoptosis of renal tubular epithelial cells induced by AA.Caspase-9 is the key starting enzyme upstream in the caspase cascade. After activation, full-length caspase-9 was cleaved into a 37/39 kDa fragment to catalyze the downstream caspase, including caspase3, which ultimately resulted in the execution of apoptotic cell death [19]. Bcl-xL is an anti-apoptotic protein that is similar to bcl2 in the bcl-2 family of apoptotic proteins [7]. Bcl-2, bcl-xL, and other anti-apoptotic proteins can directly bind to mitochondria to prevent the release of cytochrome C in the mitochondria, or
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binding to caspase-9 to prevent the caspase-9 activation pathway, so that blocked caspase-3 activation inhibited cell apoptosis, and has a good renal protective effect [18]. Although CHBP had no significant effect on the expression of PI3K-p85 and Akt protein in our study, it had a significant effect on the phosphorylation of AKt Ser473. It has been reported that activated Akt can promote caspase-9 phosphorylation; and the latest studies have found that activated Akt can inhibit the proapoptotic effect by regulating caspase-9 shear variation [15,20]. In addition, CHBP minimizes MPO + cells in the interstitial region [12]. Neutrophil infiltrates in the interstitial region in acute AA injury. Local strong synthesis of proinflammatory cytokines can produce defensive physiologic activities that increase tissue damage and dysfunction by producing oxygen free radicals and raising more inflammatory cells. There is no doubt that AAinduced nephrotoxicity is associated with neutrophil infiltration. CHBP may attenuate renal damage by reducing neutrophil infiltration, thereby improving AA-induced tissue damage and renal dysfunction. 5. Conclusion In summary, CHBP can inhibit apoptosis, reduce the inflammatory response, reduce mouse AA induced kidney injury, and improve kidney function. This study provides a new methodology for the treatment of acute AAN; the specific mechanism of CHBP protection in AA-induced renal injury requires further exploration. Conflict of interest Authors declare no conflict of interest. Acknowledgements This study was supported by National Natural Science Foundation of China (grants numbers 81270833, 81570674 to TZ) References [1] W. Chan, N.M. Pavlovic, W. Li, C.K. Chan, J. Liu, K. Deng, Y. Wang, B. Milosavljevic, E.N. Kostic, Quantitation of aristolochic acids in corn, wheat grain, and soil samples collected in Serbia: identifying a novel exposure pathway in the etiology of balkan endemic nephropathy, J. Agric. Food Chem. 64 (2016) 5928–5934. [2] T. Fleischer, Y.C. Su, S.J. Lin, How do government regulations influence the ability to practice Chinese herbal medicine in western countries, J. Ethnopharmacol. 196 (2017) 104–109. [3] X. Liu, J. Wu, J. Wang, J. Fan, X. Feng, X. Yu, X. Yang, Possible role of mitochondrial injury in Caulis Aristolochia manshuriensis-induced chronic aristolochic acid nephropathy, Drug Chem. Toxicol. 40 (2017) 115–124.
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Available online at
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Original article
Protective effects of cyclic helix B peptide on aristolochic acid induced acute kidney injury Yigang Zenga,b,1, Long Zhenga,b,1, Zhangru Yanga,b,1, Cheng Yanga,b , Yi Zhanga,b , Jiawei Lia,b , Weitao Zhanga,b , Mingnan Zhanga,b , Mushuang Hua,b , Shuo Wanga,b , Sidikejiang Niyazia,b , Ming Xua,b , Ruiming Ronga,b , Tongyu Zhua,b,c,* a
Department of Urology, Zhongshan Hospital, Fudan University, 180 Fenglin Road, Shanghai, 200032, China Shanghai Key Laboratory of Organ Transplantation, Shanghai, China c Shanghai Public Health Clinical Center, Fudan University, Shanghai, China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 April 2017 Received in revised form 25 July 2017 Accepted 25 July 2017
Background: Aristolochic acid (AA) injuries remain a serious condition associated with acute renal dysfunction. Herein, the effect and mechanism of a novel tissue protective peptide, cyclic helical Bpeptide (CHBP) derived from erythropoietin, were investigated in a mice model. Methods: Mice were randomly divided into four groups, receiving the following treatments (1: saline; 2: AA 10 mg/kg; 3: AA 10 mg/kg +CHBP 4nmol/kg; 4: AA 10 mg/kg +CHBP 8nmol/kg). Results: Blood urea nitrogen and serum creatinine was increased by AA but decreased by CHBP in a dosedependent fashion. CHBP also significantly improved renal tubular injury and inflammatory infiltration, which was gradually increased by AA. Apoptotic cells, infiltrating inflammatory cells, and active caspase3+ cells were greatly reduced by CHBP. In addition, CHBP inhibited caspase-3, 9 and improved bcl-2, bcl-xl protein expression in vivo. Conclusion: Taken together, we demonstrated, for the first time, that CHBP effectively improved renal function and tissue damage caused by AA, which maybe through reducing caspase-3 activation, apoptosis, and inflammation. © 2017 Published by Elsevier Masson SAS.
Keywords: Cyclic Helix B peptide Aristolochic acid Acute kidney injury
1. Introduction The traditional Chinese medicine aristolochic acid (AA), despite being banned in many countries, is still sold on the Internet [1]. Aristolochic acid nephropathy (AAN) remains a clinical problem [2]. Short-term high-dose AA can directly damage proximal renal tubular epithelial cells, activate the renal tissue cysteinyl aspartate-specific protease (caspase) pathway, and induce a renal tissue inflammatory reaction, leading to degeneration, necrosis, and apoptosis [3].The typical characteristics of AAN are described as severe renal tubular damage and rapid progression to end-stage renal disease [4].It is reported that the 2-year renal survival is only 17%, which is worse than other tubule interstitial nephropathies (74%) [5].Clinically, no effective treatment can reverse the progression of AA induced renal injury[5].
* Corresponding author at: Department of Urology, Zhongshan Hospital, Fudan University, 180 Fenglin Road, Shanghai, 200032, China. E-mail address: [email protected] (T. Zhu). 1 These authors contributed equally to this article. http://dx.doi.org/10.1016/j.biopha.2017.07.131 0753-3322/© 2017 Published by Elsevier Masson SAS.
Erythropoietin (EPO) is a hematopoietic hormone produced mainly by adult kidneys and has been routinely used clinically to treat anemia, for nearly 20 years [6]. In addition to promoting erythropoiesis, EPO also exhibits a strong tissue protective effect against kidney damage caused by ischemia-reperfusion injury [7]. EPO can inhibit renal tubular epithelial cell apoptosis, downregulate renal tissue after ischemia-reperfusion injury, and reduce acute renal injury [7]. However, to have a kidney protective effect, high doses of EPO are required, which can lead to erythropoiesis and its resulting side effects, including a hypercoagulable state, thrombosis, and hypertension [8]. Dose limitations significantly disrupt the balance between the benefits and risks of EPO applications [9]. With the development of biochemistry and biotechnology, therapeutic peptides have become popular and more effective [10]. Recently, the new linear peptide helix B surface peptide (HBSP) from EPO shows satisfactory renal protection by inhibiting inflammation and apoptosis [10].However, the 2-min plasma half-life of HBSP limits its use in vivo[10]. Thus, we have recently used a new cyclization strategy to synthesize thioether cyclic helical B-peptide (CHBP), which significantly enhances metabolic
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2. Materials and methods
were blind-labeled and reviewed by two renal pathologists. Renal damage was graded based on the percentage of damaged tubules in the sample: 0 = normal kidney (no damage); 1 = minimal damage (<25% damage); 2 = mild damage (25%–50% damage); 3 = moderate damage (50%–75% damage); and 4 = severe damage (>75% damage), similar with previous descriptions [10]. Renal tubular epithelial degeneration, renal tubular lumen expansion, tubular necrosis, inflammatory cell infiltration, renal interstitial edema, and renal interstitial fibrosis were used as an evaluation indicator of renal tubular and renal interstitial injury. Mean values of these estimates were used in the analyses [13]. Masson trichrome staining was also performed according to the standard protocol to assess collagen deposition [3].
2.1. Aristolochic acid induced renal injury model
2.4. In situ end-labeling apoptotic cells
Male BALB/c mice (18–20 g) were obtained from Shanghai Slac Lab Animal, Co., Ltd. (Shanghai, China), and bred in a Specific pathogen Free (SPF)-grade experimental animal room. All animal experiments were performed according to the guidelines of the Care and Use of Laboratory Animals of the Laboratory Animal Ethical Commission of Fudan University with good animal surgical research practices, and were approved by the Animal Ethical Committee of Zhongshan Hospital, Fudan University. A total of 64 BALB/c mice were randomly divided into four groups (n = 16 each) according to the different treatments: (1) control group: intraperitoneal injection of equal doses of saline; (2) model group: intraperitoneal injection of AA sodium (Sigma, San Francisco, California, USA, 10 mg/kg in 1 mg/ml saline); (3) Intervention 1 group: intraperitoneal injection of AA sodium (Sigma10 mg/kg at the onset of 4 nmol/kg CHBP); (4) Intervention 2 group: intraperitoneal injection of AA sodium (Sigma10 mg/kg at the onset of 8 nmol/kg CHBP). The cardiac blood and renal tissue samples were harvested after ketamine anesthesia (Heng Rui Medicine Co., Ltd., Jiangsu, China), and then animals were ethically sacrificed (6 random mice per group, at 1, 3, and 7 days after injection, respectively).
Apoptotic cells in renal tissues were detected with TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling) staining using a pop Tag peroxidase kit (In Situ Cell Death Detection Kit-POD; Roche Diagnostics, Mannheim, Germany). After TUNEL labeling, nuclei were stained with hematoxylin and the TUNEL positive cells were observed at 200 magnification over 20 fields in the tubular, interstitial, and tubular lumen areas [11].
stability and renal protection [11]. In our previous ischemiareperfusion model, CHBP showed a strong protective effect on ischemia and apoptosis in a renal ischemia-reperfusion injury [11]; 8 nmol/kg of CHBP is the optimal dose [11]. In the pig model, CHBP administered with a preservative solution and autologous blood perfusate provides significant protection against renal injury, by increasing renal blood flow and oxygenation, and reducing apoptosis, inflammation, and tissue damage [12]. Based on these results, we hypothesized that CHBP may attenuate AA-induced renal injury. In this paper, the protective effect and mechanism of CHBP were studied in mouse models.
2.2. Renal function Whole blood was centrifuged at 1600g for 25 min at 4 C to obtain serum. A fully automatic biochemical analyzer was used for testing serum creatinine (SCr) and blood urea nitrogen (BUN) (BioAssay Systems,Hayward, CA,USA). 2.3. Histologic assessment
2.5. Immunostaining of myeloperoxidase (MPO) and active caspase-3 Immunohistochemical staining of MPO (Abcam, Cambridge, UK) and active caspase 3 (Abcam) was performed on frozen kidney sections using a DAKO ChemMate EnVision Detection Kit (DAKO, Carpinteria, CA, USA), as previously described [10]. Then, 10 non-overlapping fields of 200 were randomly selected from each slice. The average absorbance values for each field were calculated and semi-quantitative analysis was performed [10]. 2.6. Western blot analysis Membrane proteins were separated on SDS (sodium dodecyl sulfate, sodium salt)–polyacrylamide gels and transferred to polyvinylidene difluoride(PVDF) membranes. The membranes were blocked with 5% milk and incubated overnight with antiPI3 K p85, anti-AKt, anti-p-Akt Ser473, anti-cleaved caspase-3, anti- cleaved caspase- 9,anti-bcl-2, anti-bcl-xl, and anti-GAPDH antibodies, (1:1000; all from Cell Signaling Technology Inc., Beverly, MA, USA). The results were analyzed as previously described [11].
Hematoxylin and eosin (H&E) staining and Masson staining were performed to assess histologic injury. The tissue sections
Fig. 1. CHBP improved AA-induced acute renal dysfunction. Scr: serum creatinine; BUN: blood urea nitrogen; T1: 1 day after treatment; T2: 3 days after treatment; T3: 7 days after treatment;* P < 0.05 compared with control group; # P < 0.05 compared with model group; & P < 0.05 compared with Intervention 1 group.
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2.7. Statistical analysis
3. Results
SPSS software version 20.0 (SPSS 20.0 software, SPSS Inc., Armonk, NY, USA) was used for statistical analysis of the data by one-way analysis of variance, followed by a Bonferroni correction. Data are presented as the mean standard deviation. A p-value of less than 0.05 was considered to be statistically significant.
3.1. Identification of acute renal injury model
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An AA-induced acute renal injury model was successfully established by AA intraperitoneal injection (10 mg/kg). One day after treatment, creatinine and urea nitrogen levels of the model group increased significantly compared with the control group (p < 0.05) and peaked on day 7 (Fig. 1). Histological findings were confirmed on day 3 after the treatment, which showed renal tubular injury and inflammatory infiltration; on day 7, severe renal tubular damage appeared. The renal tubular damage grade of the model group was significantly increased compared to the control
Fig. 2. CHBP attenuated renal AA- induced renal tissue injury, inflammation. H&E: Hematoxylin and eosin staining; T1: 1 day after treatment; T2: 3 days after treatment; T3: 7 days after treatment;* P < 0.05 compared with control group; # P < 0.05 compared with model group; & P < 0.05 compare with Intervention 1 group;.
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group on day 7 after treatment, according to the renal tubule pathologic injury score (p < 0.05; Fig. 2). 3.2. CHBP can improve AA-induced acute renal dysfunction Compared with the model group, SCr and BUN levels significantly improved after CHBP, in a dose dependent manner (p < 0.05, days 1, 3, and 7; Fig. 1). 3.3. CHBP attenuates AA-induced renal tissue injury, inflammation, and collagen deposition To evaluate the renoprotective effects of CHBP in the mouse acute renal injury model, H&E and Masson staining of the kidneys was performed in each group. The tubular structure was found to have a healthy morphology and structure in the control group,
while severe renal tubular injury, interstitial dilatation, and inflammatory cell infiltration were observed in the model group. In contrast, CHBP treatment improved renal tubule interstitial disease (p < 0.05) compared with the model group, in a dosedependent manner (p < 0.05).Semi-quantitative analysis using a histological scoring system revealed that the kidney tissue in the CHBP-treated group was well protected, showing mild interstitial edema and cellular infiltration (Fig. 2). Masson staining was used to demonstrate extracellular matrix (ECM) deposition within the tubule interstitium. As shown in Fig. 3, collagen deposition was obviously elevated in the model group (p < 0.05) compared with the control group, but markedly reduced by CHBP treatment (p < 0.05), in a dose-dependent manner (p < 0.05).
Fig. 3. CHBP attenuated renal AA- induced renal collagen deposition. T1: 1 day after treatment; T2: 3 days after treatment; T3: 7 days after treatment;* P < 0.05 compared with control group; # P < 0.05 compared with model group; & P < 0.05 compared with Intervention 1 group;.
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Fig. 4. CHBP improved AA-induced renal damage by inhibiting Inflammation. MPO: Myeloperoxidase; T1: 1 day after treatment; T2: 3 days after treatment; T3: 7 days after treatment;* P < 0.05 compared with control group; # P < 0.05 compared with model group; & P < 0.05 compared with Intervention 1 group.
3.4. CHBP improves AA-induced renal damage by inhibiting inflammation Inflammation was assessed by immunostaining of MPO+ cells, a marker primarily seen in neutrophil granulocytes. Control samples had limited MPO + cells. However, MPO + cells were significantly increased by AA, especially in peritubular and expanded interstitial areas. CHBP reduced AA-induced MPO + cells, mainly in the interstitial region, in a dose-dependent manner (p < 0.05). Some MPO + cells demonstrated cohesive nuclei, morphological features of apoptosis, or tubular lumens (Fig. 4).
3.5. CHBP inhibited AA-induced cell apoptosis in renal tissue Apoptotic cells were primarily located in the tubular and interstitial regions by TUNEL staining. The total number of apoptotic cells counted in tubular, interstitial, and tubular lumen areas was greatly increased in the model group compared with the control group (p < 0.05). CHBP reduced AA-induced apoptotic cells in kidney tissue (p < 0.05), and had the most important impact in the tubular region in a dose-dependent manner (p < 0.05; Fig. 5). 3.6. CHBP inhibited AA-induced active Caspase-3+ cells in renal tissue The distribution of active caspase-3+ cells detected by immunostaining, recognizing the 17 kD subunit, was mainly in the tubular and interstitial areas, fewer in glomerular areas. Almost
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Fig. 5. CHBP Inhibited AA-induced Cell Apoptosis in renal tissue. TUNEL: Terminal Deoxynucleotidyl Transferase mediated dUTP-biotin nick end labeling; T1: 1 day after treatment; T2: 3 days after treatment; T3: 7 days after treatment;* P < 0.05 compared with control group; # P < 0.05 compared with model group; & P < 0.05 compared with Intervention 1 group.
all positive cells showed condensed nuclei, the morphologic feature of apoptosis. Active caspase-3+ cells were increased in the model group compared with the control group (p < 0.05) but decreased by CHBP (p < 0.05) in a dose-dependent manner (p < 0.05; Fig. 6). 3.7. CHBP inhibits caspase-3, 9 and improve bcl-2, bcl-xl protein expression in vivo The expression of caspase-3, 9, bcl-2, and bcl-xl protein subunits in mouse kidneys was detected by western blot. The numerically highest expression of cleaved caspase-3, 9 was shown in the model group, but significantly reversed by CHBP (p < 0.05) in a dose-dependent manner (p < 0.05). The numerically lowest expression of bcl-2, bcl-xl was shown in the model group, but
significantly increased by CHBP (p < 0.05) in a dose-dependent manner (p < 0.05; Fig. 7) 4. Discussion AA is a major secondary metabolite of the aristolochic species and is known to be a human nephrotoxin [14]. AAN caused by Chinese medicine containing AA, has been reported at home and abroad; its clinical and pathological characteristics demonstrate an in-depth understanding. A large number of evidence-based studies have shown that AAN mainly involves renal tubules and interstitium, but not involve glomeruli [15].A single or short-term high-dose of AA, acting on the renal tubular epithelial cells in vivo, leads to significant cell necrosis and apoptosis, and can even lead to acute ANN [1]. A one-time dose of 10 mg/kg AA can lead to acute kidney injury in BALB/c mice, as confirmed in this study.
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Fig. 6. CHBP Inhibited AA-induced Active Caspase-3+ Cells in renal tissue.caspase: cysteinyl aspartate-specific protease; T1: 1 day after treatment; T2: 3 days after treatment; T3: 7 days after treatment;* P < 0.05 compared with control group; # P < 0.05 compared with model group; & P < 0.05 compared with Intervention 1 group.
AA can cause necrosis and apoptosis of proximal renal tubular epithelial cells of various genera (human, porcine, mouse, rabbit) [16]. In vivo, AA is able to rapidly stimulate extracellular calcium to enter the cytoplasm. Then, a rapid release of calcium of the intracellular calcium-endoplasmic reticulum and mitochondria into the cytoplasm [17].This leads to caspase activation, followed by damage to renal tubular epithelial cells, and eventual apoptosis [7]. In in vitro experiments, AA stimulated human renal tubular epithelial cells (HK-2) for 12, 24, and 48 h. And then the number of cells were reduced with a dose and time-dependent manner [18]. Intracellular ATP is decreased, followed by mitochondrial membrane depolarization and release of cytochrome c, leading to the activation of caspase-3 and ultimately resulting in cell damage and cell cycle arrest in the G2/M phase [18]. AA activates the caspase cascade, and ultimately causes apoptosis. We confirmed that the signal transduction pathway
of AA-induced renal tubular epithelial cell apoptosis is a caspase-3 dependent pathway [1]. As a terminal effect of apoptosis, caspase3 is a common key protease for substrate cleavage downstream of each pathway, its degradation of substrate activity is strongest, and it is called the executive of apoptosis [15].Caspase activation is associated with apoptosis. Inhibition of caspase activity can improve renal injury and reduce renal tubular epithelial cell apoptosis [15]. EPO is an endogenous protein that plays a protective role in a wide range of organs [7]. In ischemic-induced acute renal failure, the administration of EPO can reduce apoptosis of renal tubular cells and promote the recovery of renal function by inhibiting the activity of caspase-3, 9 [7]. The anti-apoptotic effect of EPO inhibits the activity of caspase-3 by phosphorylation of Akt and upregulates the expression of anti-apoptotic factors [18]. However, due to the very high dose requirements required to achieve tissue
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Fig. 7. CHBP inhibited caspase-3, 9 and improved bcl-2, bcl-xl protein expression in vivo. PI3 K: Phosphatidylinositol 3-kinase; AKt: protein kinase B; p-AKt: Phosphorylation AKt; caspase: cysteinyl aspartate-specific protease; bcl: B-cell lymphoma; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; T1: 1 day after treatment; T2: 3 days after treatment; T3: 7 days after treatment;* P < 0.05 compared with control group; # P < 0.05 compared with model group; & P < 0.05 compared with Intervention 1 group;.
protection, it causes adverse side effects, limiting the use of EPO in anti-cell apoptosis [17].We synthesized the small molecule polypeptide HBSP by analyzing the three-dimensional structure of EPO, according to the most critical 11 amino acid residue sequence on the hydrophilic interface of helix B [13]. HBSP is different from EPO, which has no erythropoietic activity and only binds to tissue protective receptor, and fully simulates the mechanism of tissue protection of endogenous EPO [10].The short half-life of HBSP restricts its clinical transformation [10].For this purpose, we invented the conformation constraints of the cyclic peptide CHBP [11]. CHBP has stability and biological activity [11]. We found that CHBP significantly reduced apoptosis and necrosis of renal tubular epithelial cells after acute renal injury, relieved its associated inflammatory response, and induced autophagy of renal tubular epithelial cells, demonstrating promising renal protection [11]. It was confirmed that the protective effect of 8 nmol/kg CHBP on ischemia-reperfusion injury was the most effective [11]. Based on this, we speculate that CHBP can protect AA from acute renal injury by inhibiting apoptosis. In order to reveal the protective effect of CHBP on renal injury induced by AA, we first evaluated renal function. On day 7, BUN and SCr levels were significantly increased in the model group. Compared with the model group, BUN and SCr levels were
significantly decreased in the intervention group after CHBP treatment, in a dose-dependent manner. Pathologically, CHBP significantly improved AA-induced renal tubular injury, interstitial expansion, and inflammatory cell infiltration. CHBP can reduce the destruction of renal tubular structure, retain more functional kidneys, and shows a good renal protective effect on histology. Immunostaining of active caspase-3 showed that the activity of caspase-3 + cells in the model group exists mainly in the tubule interstitial region and renal tubular lumen, and was significantly increased compared with the control group. CHBP significantly reduced the amount of active caspase-3 + cells in a dose-dependent manner. Further studies showed that CHBP significantly downregulated the expression of caspase-3, 9 protein and upregulated the expression of bcl-2, bcl-xl protein after AA injury, and inhibited apoptosis of renal tubular epithelial cells induced by AA.Caspase-9 is the key starting enzyme upstream in the caspase cascade. After activation, full-length caspase-9 was cleaved into a 37/39 kDa fragment to catalyze the downstream caspase, including caspase3, which ultimately resulted in the execution of apoptotic cell death [19]. Bcl-xL is an anti-apoptotic protein that is similar to bcl2 in the bcl-2 family of apoptotic proteins [7]. Bcl-2, bcl-xL, and other anti-apoptotic proteins can directly bind to mitochondria to prevent the release of cytochrome C in the mitochondria, or
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binding to caspase-9 to prevent the caspase-9 activation pathway, so that blocked caspase-3 activation inhibited cell apoptosis, and has a good renal protective effect [18]. Although CHBP had no significant effect on the expression of PI3K-p85 and Akt protein in our study, it had a significant effect on the phosphorylation of AKt Ser473. It has been reported that activated Akt can promote caspase-9 phosphorylation; and the latest studies have found that activated Akt can inhibit the proapoptotic effect by regulating caspase-9 shear variation [15,20]. In addition, CHBP minimizes MPO + cells in the interstitial region [12]. Neutrophil infiltrates in the interstitial region in acute AA injury. Local strong synthesis of proinflammatory cytokines can produce defensive physiologic activities that increase tissue damage and dysfunction by producing oxygen free radicals and raising more inflammatory cells. There is no doubt that AAinduced nephrotoxicity is associated with neutrophil infiltration. CHBP may attenuate renal damage by reducing neutrophil infiltration, thereby improving AA-induced tissue damage and renal dysfunction. 5. Conclusion In summary, CHBP can inhibit apoptosis, reduce the inflammatory response, reduce mouse AA induced kidney injury, and improve kidney function. This study provides a new methodology for the treatment of acute AAN; the specific mechanism of CHBP protection in AA-induced renal injury requires further exploration. Conflict of interest Authors declare no conflict of interest. Acknowledgements This study was supported by National Natural Science Foundation of China (grants numbers 81270833, 81570674 to TZ) References [1] W. Chan, N.M. Pavlovic, W. Li, C.K. Chan, J. Liu, K. Deng, Y. Wang, B. Milosavljevic, E.N. Kostic, Quantitation of aristolochic acids in corn, wheat grain, and soil samples collected in Serbia: identifying a novel exposure pathway in the etiology of balkan endemic nephropathy, J. Agric. Food Chem. 64 (2016) 5928–5934. [2] T. Fleischer, Y.C. Su, S.J. Lin, How do government regulations influence the ability to practice Chinese herbal medicine in western countries, J. Ethnopharmacol. 196 (2017) 104–109. [3] X. Liu, J. Wu, J. Wang, J. Fan, X. Feng, X. Yu, X. Yang, Possible role of mitochondrial injury in Caulis Aristolochia manshuriensis-induced chronic aristolochic acid nephropathy, Drug Chem. Toxicol. 40 (2017) 115–124.
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