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Possible role of mtDNA depletion and respiratory chain defects in aristolochic acid I-induced acute nephrotoxicity
Toxicology and Applied Pharmacology 266 (2013) 198–203
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Possible role of mtDNA depletion and respiratory chain defects in aristolochic acid I-induced acute nephrotoxicity Zhenzhou Jiang, Qingli Bao 1, Lixin Sun ⁎, Xin Huang, Tao Wang, Shuang Zhang, Han Li, Luyong Zhang ⁎⁎ Jiangsu Center for Drug Screening, China Pharmaceutical University, Nanjing 210009, China
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Article history: Received 23 March 2012 Revised 7 July 2012 Accepted 9 July 2012 Available online 20 July 2012 Keywords: Aristolochic acid I Nephrotoxin Respiratory chain complexes Mitochondrial DNA Respiratory control ratio
a b s t r a c t This report describes an investigation of the pathological mechanism of acute renal failure caused by toxic tubular necrosis after treatment with aristolochic acid I (AAI) in Sprague–Dawley (SD) rats. The rats were gavaged with AAI at 0, 5, 20, or 80 mg/kg/day for 7 days. The pathologic examination of the kidneys showed severe acute tubular degenerative changes primarily affecting the proximal tubules. Supporting these results, we detected significantly increased concentrations of blood urea nitrogen (BUN) and creatinine (Cr) in the rats treated with AAI, indicating damage to the kidneys. Ultrastructural examination showed that proximal tubular mitochondria were extremely enlarged and dysmorphic with loss and disorientation of their cristae. Mitochondrial function analysis revealed that the two indicators for mitochondrial energy metabolism, the respiratory control ratio (RCR) and ATP content, were reduced in a dose-dependent manner after AAI treatment. The RCR in the presence of substrates for complex I was reduced more significantly than in the presence of substrates for complex II. In additional experiments, the activity of respiratory complex I, which is partly encoded by mitochondrial DNA (mtDNA), was more significantly impaired than that of respiratory complex II, which is completely encoded by nuclear DNA (nDNA). A real-time PCR assay revealed a marked reduction of mtDNA in the kidneys treated with AAI. Taken together, these results suggested that mtDNA depletion and respiratory chain defects play critical roles in the pathogenesis of kidney injury induced by AAI, and that the same processes might contribute to aristolochic acid-induced nephrotoxicity in humans. © 2012 Published by Elsevier Inc.
Introduction Aristolochic acids (AAs) are present in extracts from plants of Aristolochia species (e.g., Aristolochia clematitis, Aristolochia fangchi, and Aristolochia manshuriensis) and are derivatives of 3, 4-methylenedioxy10‐nitro-1-phenanthrenecarboxylic acid. The main forms of the AAs are AAI and AAII. The extracts of Aristolochia species are used in traditional medicine in therapies for arthritis, rheumatism, gout, and festering wounds. However, AAs have been associated with the development of a severe human nephropathy, designated as aristolochic acid nephropathy (AAN) (Gillerot et al., 2001; Schmeiser et al., 2009; Vanhaelen et al., 1994). Herbal products that contain AAs were withdrawn from the market after animal studies showed that they caused tumor induction and Abbreviations: AAI, aristolochic acid I; BUN, blood urea nitrogen; Cr, creatinine; RCR, respiratory control ratio; mtDNA, mitochondrial DNA; nDNA, nuclear DNA; OK cell, opossum kidney proximal tubule cell; LLC-PK1 cell, Lilly Laboratories Culture-pig kidney cell; ERK, extracellular signal‐regulated kinase; ADP, adenosine diphosphate; ATP, adenosine triphosphate; OXPHOS, oxidative phosphorylation system; ND-1, NADH dehydrogenase subunit 1. ⁎ Corresponding author. ⁎⁎ Corresponding author. Fax: +86 25 83271142. E-mail addresses: [email protected] (Z. Jiang), [email protected] (Q. Bao), [email protected] (L. Sun), [email protected] (X. Huang), [email protected] (T. Wang), [email protected] (S. Zhang), [email protected] (H. Li), [email protected] (L. Zhang). 1 Co-first author. 0041-008X/$ – see front matter © 2012 Published by Elsevier Inc. doi:10.1016/j.taap.2012.07.008
renal toxicity (Krumbiegel et al., 1987). Both clinical and in vitro findings suggest that the proximal tubules are the target of aristolochic acid injury (Depierreux et al., 1994). Damage to the proximal tubule epithelium in patients was demonstrated by a progressive decrease in urinary neutral endopeptidase levels and the loss of the brush border of the tubules (Nortier et al., 1997). Experimentally, the toxicity of aristolochic acid to the proximal tubule was confirmed by an investigation of its effects on a well-established opossum kidney (OK) proximal tubule cell line. Exposure of the OK cell monolayers to aristolochic acid altered receptormediated endocytosis (Lebeau et al., 2001). Additional studies have shown that the formation of AA-related DNA adducts is toxic to the tubular epithelium and carcinogenic to the urethral epithelium (Arlt et al., 2000; Nortier et al., 2000). Cysteinyl leukotriene synthesis was shown to be involved in the AAI-induced renal proximal tubular epithelial cell injury in LLC-PK1 cells via an ERK activation pathway (Yang et al., 2011). However, the pathogenesis underlying aristolochic acid-induced toxicity in renal proximal tubular epithelial cells has not been completely elucidated and is still being debated. The kidney is a highly aerobic organ. A high density of mitochondria is required for its transport activity and the reabsorption of >99% of the daily glomerular filtrate, most of which are along the proximal tubule. The density of mitochondria varies along the proximal tubule and is higher in the convoluted segment, making this segment more vulnerable to mitochondrial dysfunction from insults such as genetic
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mitochondrial cytopathy (Martin-Hernandez et al., 2005; Niaudet and Rotig, 1997) or toxic xenobiotics (Izzedine et al., 2005). Interestingly, AAI causes mitochondrial swelling, leakage of Ca 2+, membrane depolarisation, and release of cytochrome c in isolated kidney mitochondria. The swelling effect was monitored at A540 in mitochondria energized by 5 mM succinate (Qi et al., 2007). As the main constituent of AA, AAI has been found to be responsible for the nephrotoxicity of AA (Shibutani et al., 2007). Therefore, we hypothesised that the toxicity of AAI to the proximal tubule involves mitochondrial dysfunction. In this study, we investigated the effect of AAI on respiratory chain function. The mechanisms underlying AAI-induced mitochondrial dysfunction were further examined. The results provided the first evidence that mtDNA depletion and respiratory chain defects might play important roles in AAI-induced acute nephrotoxicity. Methods Materials. AAI (>98%), purchased from Zhengzhou Haoxinren Medicine Research and Consulting Co., Ltd. (lot no.: 20090217, Henan, China), was dissolved in 0.5% CMCNa prior to use. Animals and experimental treatments. Sprague–Dawley (SD) rats (weighing from 180 to 200 g) were used in this study. They were kept at a constant ambient temperature and under a steady light/ dark cycle with free access to food and water. The rats were randomly assigned to the following 4 groups: a control group, a low dose group (5 mg/kg/day), a middle dose group (20 mg/kg/day) and a high dose group (80 mg/kg/day). The rats were gavaged daily with AAI or control solution for 7 days and monitored for the appearance of diarrhoea, body weight loss, and other signs of distress. The rats were euthanized 24 h after the final treatment. The left kidneys were stored at − 80 °C for subsequent analysis. The right kidney was divided into two sections, one of which was fixed with formalin for histologic examination and the other with glutaraldehyde (5%) for subsequent electron microscopy. BUN and Cr were measured by microplate-based colorimetric assays. All procedures were approved by the local review board for animal studies. Histological examination. The tissue samples were preserved in 10% neutral buffered formalin, embedded in paraffin wax and cut to a thickness of 4 μm. These tissue sections were stained with hematoxylin and eosin (HE), and two pathologists who were blind to the treatment conditions examined each section using light microscopy. The degree of renal toxicity was graded on a scale of 0 to 2.5, where scores of 0 indicated no histological toxicity, 0.5 to 1.0 indicated mild histological toxicity, 1.0 to 2.0 indicated moderate histological toxicity and 2.5 indicated severe histological toxicity. Ultrastructural examination. The portions of the kidneys designated for electron microscopic analysis were cut into 1-mm fragments, fixed in 5% glutaraldehyde, post-fixed in 1% OsO4, dehydrated in ethyl alcohol (30–96%) and 100% acetone, and subsequently embedded in epoxy resin. Ultrathin sections were cut with a Reichert OmU2 ultramicrotome and examined using a JEM-1200 EX transmission electron microscope. The evaluator was blind to the treatment status of the rats. Isolation of mitochondria. All steps for the isolation of mitochondria were performed at 4 °C. The kidney mitochondria were isolated essentially as previously described (Gerschenson et al., 2000). Briefly, a 500 mg portion of the kidney was minced and then homogenised in 5 ml of homogenisation buffer (210 mM mannitol, 70 mM sucrose, 1 mM EDTA, 20 mM HEPES [pH 7.4], 2 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride). The homogenate was centrifuged twice at 1000 × g for 5 min to remove the cellular debris and nuclei. The mitochondria were collected at 20,000 × g for 20 min, gently resuspended in 50–100 μl homogenisation buffer, frozen in liquid
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nitrogen, and stored at −70 °C. The mitochondrial protein concentrations were measured using the BCA protein determination method using bovine serum albumin (BSA) as a standard. Measurement of oxygen consumption. The mitochondria (1 mg protein/ml) were incubated in 130 mM sucrose, 50 mM KCl, 5 mM MgCl2, 0.1 mM EGTA, 5 mM KH2PO4, and 5 mM HEPES; pH 7.4 at 25 °C with a Clark-type oxygen electrode (Hansatech Instruments, Oxygraph) under continuous stirring (Custodio et al., 1994). The mitochondrial oxygen respiration rates were measured in the presence of glutamate (1.0 mM) and malate (1.0 mM) that enter the electron transport chain selectively at complex I or succinate (2.0 mM) plus NADH dehydrogenase inhibitor (2 μM rotenone) that enters at complex II. The adenosine diphosphate (ADP)-stimulated respiration (state 3) was measured in the presence of 150 μM ADP, and the ADP-independent respiration (state 4) was monitored after all the ADP was consumed. The respiratory control ratio (RCR), defined as the ratio of oxygen consumption during state 3 to state 4 (a measure of the coupling of adenosine triphosphate synthesis to respiration), was calculated to evaluate the degree of mitochondrial damage. Measurement of ATP levels. The kidneys were homogenised in deionised boiling water (Yang et al., 2002) using a homogeniser and then centrifuged at 4000 rpm for 10 min. The clear supernatants were collected for the analyses. The protein concentrations were measured using the BCA method. The ATP levels were determined using the creatine kinase method (ATP Content Detection Kit; Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer's instructions. Respiratory chain complexes. After the isolation of mitochondria, the specific activities of the oxidative phosphorylation system (OXPHOS) enzyme complexes I and II were quantified on a Hewlett Packard diode array spectrophotometer as previously described (Trounce et al., 1996). Complex I (NADH:ubiquinone oxidoreductase) activity was determined by measuring the reduction of the ubiquinone analogue decylubiquinone. Complex II (succinate:ubiquinone oxidoreductase) specific activity was measured by monitoring the reduction of the dye 2, 6-dichlorophenolindophenol. mtDNA studies. DNA from the kidneys was extracted using the phenol: chloroform precipitation method, and the total amount of DNA recovered was determined by spectrophotometry. To quantify the mtDNA copy number, well-conserved single-copy genes were selected. Pyruvate kinase was used as a marker for nDNA and NADH dehydrogenase subunit 1 (ND-1) for mtDNA. The primers used to amplify a 267-bp region of the ND-1 gene were as follows: fwd, 5′-GGCTACATACAATTACGCAAAG-3′; and rev. 5′-TAGAATGGAGTAGACCGAAAGG-3′. To amplify a 347-bp fragment of the pyruvate kinase gene, the primer sequences consisted of the following: fwd, 5′-ACTGGCCGGTGTCATAGTGA-3′; and rev, 5′-TGTTGACCAGCCGTATGGATA-3′. The real-time PCR reactions were performed according to the manufacturer's instructions (SYBR® Premix Ex Taq™ II, TaKaRa), and thermal cycling was performed in an iCycler® (Bio-Rad). The mtDNA content was normalised to the nucleic DNA content. Statistical analysis. The Student's t-test was employed to analyse the differences between the sets of data. Statistical analyses were performed using GraphPad Prism (GraphPad, San Diego, CA). A value of pb 0.05 was considered statistically significant. Results AAI-induced acute nephrotoxicity in rats To characterise the nephrotoxicity of AAI, the kidneys were subjected to pathologic examination. A dramatic loss of the proximal
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Fig. 1. AAI-induced acute nephrotoxicity in rats. (A) Randomly selected images of the histopathology of kidneys from rats treated with AAI at 0 (a), 5 (b), 20 (c), or 80 mg/kg/day (d) for 7 days. Loss of the proximal tubule brush borders is observed in each group treated with AAI (arrow). Congestion in peritubular capillaries is observed in (c) and (d). Protein casts, cellular debris desquamation of tubular epithelial cells are observed in (d). All images are shown at identical magnification. (B) The pathologic scores of rats treated with AAI. The grading system used for classifying changes in renal pathology is described under the Methods section. (C) Serum markers of renal injury in rats treated with AAI. The blood collected from rats treated with AAI for 7 days was used for analysis of blood urea nitrogen and serum creatinine. All results are shown as means ± SEM for n= 5 animals for each group. Symbols indicate the statistical significance relative to vehicle control within each group: *p b 0.05, **p b 0.01.
tubule brush borders was confirmed by histopathology in the animals in each of the groups treated with AAI. In the AAI (5 mg/kg/day) group, loss of the brush borders, degeneration, and necrosis of the tubular epithelial cells were observed in 3/6 rats. In the AAI (20 mg/kg/day) group, degeneration and necrosis of the tubular epithelial cells were observed in 4/6 rats, and congestion was observed in some peritubular capillaries. In the AAI (80 mg/kg/day) group, loss of the brush borders, degeneration, and necrosis of the tubular epithelial cells were observed
in all rats. Protein casts, cellular debris, desquamation of tubular epithelial cells, and congestion were observed in several peritubular capillaries (Fig. 1A). The kidneys from the control rats showed no necrosis. High overall pathologic scores were recorded for the kidneys collected from the rats treated with AAI (Fig. 1B). Supporting these results, we detected significant increases of BUN and Cr in rats treated with the high dose of AAI, indicating damage to the kidney (Fig. 1C). Thus, AAI administration is associated with strong nephrotoxic effects in rats.
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AAI-induced ultrastructural mitochondrial alterations in kidney The ultrastructural examination showed that the proximal tubular mitochondria were extremely enlarged and dysmorphic, with loss and disorientation of their cristae. The ultrastructure of the kidneys from the AAI-exposed rats was characterised by morphologically aberrant mitochondria. In the AAI (5 mg/kg/day) group, mitochondrial swelling and small vacuoles were observed in all rats. In the AAI (20 mg/kg/ day) group, the cristae were markedly distorted, the mitochondrial structure was degraded, and the quantity of mitochondria was slightly decreased in each rat. In the AAI (80 mg/kg/day) group, the mitochondrial morphology was completely disrupted and the decrease in mitochondrial quantity was apparent in all rats (Fig. 2).
Effects of AAI on respiratory chain function in the kidney To study the effects of AAI treatment on the mitochondrial function in rat kidneys, we first evaluated the effects of AAI on the ATP content and RCR, two indicators of mitochondrial energy metabolism in these tissues. Our results indicated that the ATP contents were reduced in a dose-dependent manner after AAI treatment (Fig. 3A). In Fig. 3B, the RCR in mitochondria is shown in the presence of succinate or glutamate/malate. In the presence of substrates for complex I
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(glutamate/malate), AAI (5–80 mg/kg/day) dose-dependently and markedly reduced the RCR. With substrate for complex II (succinate), the lowest concentration of AAI treatment (5 mg/kg/day) failed to affect the RCR, and a minor decrease in RCR was detected in the AAI 20 and 80 mg/kg/day group compared with that in the presence of glutamate/ malate. Substrate specificity suggests that the inhibition of complex I was more significant than respiratory complex II. So in the next experiments, the activities of complexes I and II were examined. As presented in Figs. 3C and D, there were slight decreases in complex II-specific activities in the kidneys of AAI-exposed rats as compared to controls. However, the activities of complex I in the kidneys of rats exposed to 5, 20, and 80 mg/kg/day of AAI decreased to approximately 66.23% (P=0.16), 25.33% (Pb 0.05), and 11.6% (Pb 0.05), respectively, of the values for the unexposed controls. These results indicated that the activity of respiratory complex I, which is partly encoded by mtDNA, was reduced more significantly than that of respiratory complex II, which is completely encoded by nDNA.
Effects of AAI on mtDNA content The mtDNA content in kidneys, normalised to nDNA, was reduced after AAI treatment. The relative mtDNA ratio in the rats exposed to 5, 20, and 80 mg/kg/day of AAI decreased to approximately 74.16%
Fig. 2. Transmission electron micrographs of AAI-induced ultrastructural mitochondrial alterations in kidney. (A) Typical mitochondria in the control group. (B) Swollen mitochondria and small vacuoles (circled) are indicated in the AAI group (5 mg/kg/day). (C) The cristae (arrow) are distorted and the mitochondrial structure is degraded in the AAI group (20 mg/kg/day). (D) The mitochondrial morphology is completely disrupted in the AAI group (80 mg/kg/day). The image is shown at 30,000× magnification.
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Fig. 3. Effects of AAI on respiratory chain function in the kidney. The respiratory control ratio (A) and ATP content (B) of kidneys from SD rats treated with AAI for 7 days. Respiratory complex I (C) is reduced more significantly than respiratory complex II (D) in the kidneys of rats treated with AAI. All results are shown as the means ± SEM for n = 5 animals for each group. Symbols indicate the statistical significance relative to vehicle control within each group, *p b 0.05, **p b 0.01.
(P = 0.12), 59.69% (P b 0.05), and 29.20% (P b 0.01), respectively, compared to the unexposed control (Fig. 4). Discussion In the present study, we found that AAI induced renal toxicity in rats, and the mechanism of toxicity was related to mtDNA depletion and respiratory chain defects. Our conclusion is based on the following observations: first, the pathologic examination revealed loss of brush borders in only 3/6 rats in the low dose group. However, the ultrastructural examination showed enlarged and dysmorphic proximal
Fig. 4. MtDNA content in the kidneys measured using real-time PCR. The mtDNA content was normalised to nuclear DNA. The graphs depict data from three independent experiments. All results are shown as the means ± SEM for n= 5 animals for each group. Symbols indicate the statistical significance relative to the vehicle control within each group, *p b 0.05, **p b 0.01.
tubular mitochondria with loss and disorientation of their cristae in all rats of this group (Fig. 2). This suggests that changes in renal mitochondria may occur prior to damage to and loss of the brush-border plasma membrane and provides evidence that renal mitochondria are the most important intracellular target. Second, respiratory complex I, which is partly encoded by mtDNA, was reduced more significantly than respiratory complex II, which is completely encoded by nDNA, in the kidneys of rats treated with AAI (Figs. 3C and D). In agreement with this, the RCR in the presence of substrates for complex I was reduced more significantly than in the presence of substrates for complex II. Third, the real-time PCR assay revealed depletion of mtDNA in the kidneys of rats treated with AAI (Fig. 4). To our knowledge, mtDNA depletion and respiratory chain defects associated with AAI in the kidney have not previously been reported, and this mechanism could explain the renal toxicity of this chemical. The mitochondria represent a potential drug target because of their unique structure and function. Mitochondria have a unique milieu, with an alkaline and negatively charged interior (pH ≈ 8) and a series of specific channels and carrier proteins. These proteins facilitate the selective accumulation of xenobiotics in the matrix and/or the inner mitochondrial membrane through efficient trapping mechanisms (Murphy and Smith, 2000; Smith et al., 2003). Moreover, mitochondria are the only organelles outside the nucleus that contain DNA, and they are capable of synthesising some proteins. Unlike nDNA, mtDNA is not protected by histone proteins. Furthermore, mtDNA repair processes are generally less efficient than those for nDNA. As a result, mtDNA is more likely to be damaged than nDNA (Scatena et al. 2007). We demonstrated that AAI affected mitochondrial bioenergetic function by measuring the effects of this chemical on RCR. The RCR indicates how tightly the oxidative phosphorylation process is coupled,
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making this ratio a useful indicator of dysfunction. A lower RCR indicates a respiratory dysfunction, suppression of ATP synthesis, and the generally poor condition of the mitochondria. Consistent with the reduced RCR, the ATP content was decreased in a dose-dependent manner after AAI treatment. A previous study in HK-2 cells also showed decreased ATP content after AA treatment (Qi et al., 2007). Together, these results suggest that AAI adversely affects energy metabolism. The mtDNA encodes 13 structural proteins or enzymes, 2 ribosomal RNAs, and 22 transfer RNAs. The other structural proteins of the mitochondria and enzymes involved in mtDNA replication and repair are encoded by nDNA. ATP is produced in the mitochondria by the oxidative phosphorylation machinery, which involves 5 respiratory complexes. Complex II (SDH) of the respiratory chain is entirely encoded by nDNA. The other 4 complexes are encoded partially by mtDNA and partially by nDNA. Complex I (NADH dehydrogenase) of the respiratory chain is composed of 11 subunits (Hall et al., 2008). Seven of these subunits (ND1, ND2, ND3, ND4, ND4L, ND5, and ND6) are encoded entirely by mtDNA and are responsible for the catalytic and proton-pumping activities of the enzyme (Anderson et al., 1981). We compared the activities of complexes I and II and found that AAI reduced complex I activities in a dose-dependent manner without significantly affecting complex II activities. In agreement with this, the RCR in the presence of substrates for complex I was reduced more significantly than in the presence of substrates for complex II. These results demonstrated the selective depletion of mtDNA-encoded proteins and enzymatic function with no identifiable loss of nDNA-encoded mitochondrial proteins. To further define the effects of AAI, the mtDNA levels were measured. Real-time PCR revealed that the mtDNA content in the kidneys of the AAI-treated animals was significantly less than that of the untreated animals. This result is consistent with the activities of respiratory chain complexes. These findings show that AAI induces nephrotoxicity by impairing mtDNA replication, thus suppressing oxidative phosphorylation. This observation may explain the particular sensitivity of the convoluted proximal tubule to AAI toxicity because this segment exhibits one of the highest densities of mitochondria in the kidney (Hall et al., 2009). Mitochondria DNA may be more susceptible than nDNA to AAI-induced damage due to the absence of efficient DNA repair mechanisms in mitochondria and the lack of protective histones in mtDNA (Gredilla, 2010). In summary, our data suggest that mtDNA depletion and respiratory chain defects occur in the kidney after acute AAI exposure. Our results provide new insight into the involvement of mtDNA depletion and respiratory chain defects as a possible mechanism for AAI-induced acute nephrotoxicity. Conflict of interest The authors declare that there are no conflict of interest. Acknowledgments This work was supported by the Specific Fund for Public Interest Research of Traditional Chinese Medicine, Ministry of Finance of China (no. 200707008), and the 2011 Program for Excellent Scientific and Technological Innovation Team of Jiangsu Higher Education and partially supported by the 111 Project (111-2-07).
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Possible role of mtDNA depletion and respiratory chain defects in aristolochic acid I-induced acute nephrotoxicity Zhenzhou Jiang, Qingli Bao 1, Lixin Sun ⁎, Xin Huang, Tao Wang, Shuang Zhang, Han Li, Luyong Zhang ⁎⁎ Jiangsu Center for Drug Screening, China Pharmaceutical University, Nanjing 210009, China
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Article history: Received 23 March 2012 Revised 7 July 2012 Accepted 9 July 2012 Available online 20 July 2012 Keywords: Aristolochic acid I Nephrotoxin Respiratory chain complexes Mitochondrial DNA Respiratory control ratio
a b s t r a c t This report describes an investigation of the pathological mechanism of acute renal failure caused by toxic tubular necrosis after treatment with aristolochic acid I (AAI) in Sprague–Dawley (SD) rats. The rats were gavaged with AAI at 0, 5, 20, or 80 mg/kg/day for 7 days. The pathologic examination of the kidneys showed severe acute tubular degenerative changes primarily affecting the proximal tubules. Supporting these results, we detected significantly increased concentrations of blood urea nitrogen (BUN) and creatinine (Cr) in the rats treated with AAI, indicating damage to the kidneys. Ultrastructural examination showed that proximal tubular mitochondria were extremely enlarged and dysmorphic with loss and disorientation of their cristae. Mitochondrial function analysis revealed that the two indicators for mitochondrial energy metabolism, the respiratory control ratio (RCR) and ATP content, were reduced in a dose-dependent manner after AAI treatment. The RCR in the presence of substrates for complex I was reduced more significantly than in the presence of substrates for complex II. In additional experiments, the activity of respiratory complex I, which is partly encoded by mitochondrial DNA (mtDNA), was more significantly impaired than that of respiratory complex II, which is completely encoded by nuclear DNA (nDNA). A real-time PCR assay revealed a marked reduction of mtDNA in the kidneys treated with AAI. Taken together, these results suggested that mtDNA depletion and respiratory chain defects play critical roles in the pathogenesis of kidney injury induced by AAI, and that the same processes might contribute to aristolochic acid-induced nephrotoxicity in humans. © 2012 Published by Elsevier Inc.
Introduction Aristolochic acids (AAs) are present in extracts from plants of Aristolochia species (e.g., Aristolochia clematitis, Aristolochia fangchi, and Aristolochia manshuriensis) and are derivatives of 3, 4-methylenedioxy10‐nitro-1-phenanthrenecarboxylic acid. The main forms of the AAs are AAI and AAII. The extracts of Aristolochia species are used in traditional medicine in therapies for arthritis, rheumatism, gout, and festering wounds. However, AAs have been associated with the development of a severe human nephropathy, designated as aristolochic acid nephropathy (AAN) (Gillerot et al., 2001; Schmeiser et al., 2009; Vanhaelen et al., 1994). Herbal products that contain AAs were withdrawn from the market after animal studies showed that they caused tumor induction and Abbreviations: AAI, aristolochic acid I; BUN, blood urea nitrogen; Cr, creatinine; RCR, respiratory control ratio; mtDNA, mitochondrial DNA; nDNA, nuclear DNA; OK cell, opossum kidney proximal tubule cell; LLC-PK1 cell, Lilly Laboratories Culture-pig kidney cell; ERK, extracellular signal‐regulated kinase; ADP, adenosine diphosphate; ATP, adenosine triphosphate; OXPHOS, oxidative phosphorylation system; ND-1, NADH dehydrogenase subunit 1. ⁎ Corresponding author. ⁎⁎ Corresponding author. Fax: +86 25 83271142. E-mail addresses: [email protected] (Z. Jiang), [email protected] (Q. Bao), [email protected] (L. Sun), [email protected] (X. Huang), [email protected] (T. Wang), [email protected] (S. Zhang), [email protected] (H. Li), [email protected] (L. Zhang). 1 Co-first author. 0041-008X/$ – see front matter © 2012 Published by Elsevier Inc. doi:10.1016/j.taap.2012.07.008
renal toxicity (Krumbiegel et al., 1987). Both clinical and in vitro findings suggest that the proximal tubules are the target of aristolochic acid injury (Depierreux et al., 1994). Damage to the proximal tubule epithelium in patients was demonstrated by a progressive decrease in urinary neutral endopeptidase levels and the loss of the brush border of the tubules (Nortier et al., 1997). Experimentally, the toxicity of aristolochic acid to the proximal tubule was confirmed by an investigation of its effects on a well-established opossum kidney (OK) proximal tubule cell line. Exposure of the OK cell monolayers to aristolochic acid altered receptormediated endocytosis (Lebeau et al., 2001). Additional studies have shown that the formation of AA-related DNA adducts is toxic to the tubular epithelium and carcinogenic to the urethral epithelium (Arlt et al., 2000; Nortier et al., 2000). Cysteinyl leukotriene synthesis was shown to be involved in the AAI-induced renal proximal tubular epithelial cell injury in LLC-PK1 cells via an ERK activation pathway (Yang et al., 2011). However, the pathogenesis underlying aristolochic acid-induced toxicity in renal proximal tubular epithelial cells has not been completely elucidated and is still being debated. The kidney is a highly aerobic organ. A high density of mitochondria is required for its transport activity and the reabsorption of >99% of the daily glomerular filtrate, most of which are along the proximal tubule. The density of mitochondria varies along the proximal tubule and is higher in the convoluted segment, making this segment more vulnerable to mitochondrial dysfunction from insults such as genetic
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mitochondrial cytopathy (Martin-Hernandez et al., 2005; Niaudet and Rotig, 1997) or toxic xenobiotics (Izzedine et al., 2005). Interestingly, AAI causes mitochondrial swelling, leakage of Ca 2+, membrane depolarisation, and release of cytochrome c in isolated kidney mitochondria. The swelling effect was monitored at A540 in mitochondria energized by 5 mM succinate (Qi et al., 2007). As the main constituent of AA, AAI has been found to be responsible for the nephrotoxicity of AA (Shibutani et al., 2007). Therefore, we hypothesised that the toxicity of AAI to the proximal tubule involves mitochondrial dysfunction. In this study, we investigated the effect of AAI on respiratory chain function. The mechanisms underlying AAI-induced mitochondrial dysfunction were further examined. The results provided the first evidence that mtDNA depletion and respiratory chain defects might play important roles in AAI-induced acute nephrotoxicity. Methods Materials. AAI (>98%), purchased from Zhengzhou Haoxinren Medicine Research and Consulting Co., Ltd. (lot no.: 20090217, Henan, China), was dissolved in 0.5% CMCNa prior to use. Animals and experimental treatments. Sprague–Dawley (SD) rats (weighing from 180 to 200 g) were used in this study. They were kept at a constant ambient temperature and under a steady light/ dark cycle with free access to food and water. The rats were randomly assigned to the following 4 groups: a control group, a low dose group (5 mg/kg/day), a middle dose group (20 mg/kg/day) and a high dose group (80 mg/kg/day). The rats were gavaged daily with AAI or control solution for 7 days and monitored for the appearance of diarrhoea, body weight loss, and other signs of distress. The rats were euthanized 24 h after the final treatment. The left kidneys were stored at − 80 °C for subsequent analysis. The right kidney was divided into two sections, one of which was fixed with formalin for histologic examination and the other with glutaraldehyde (5%) for subsequent electron microscopy. BUN and Cr were measured by microplate-based colorimetric assays. All procedures were approved by the local review board for animal studies. Histological examination. The tissue samples were preserved in 10% neutral buffered formalin, embedded in paraffin wax and cut to a thickness of 4 μm. These tissue sections were stained with hematoxylin and eosin (HE), and two pathologists who were blind to the treatment conditions examined each section using light microscopy. The degree of renal toxicity was graded on a scale of 0 to 2.5, where scores of 0 indicated no histological toxicity, 0.5 to 1.0 indicated mild histological toxicity, 1.0 to 2.0 indicated moderate histological toxicity and 2.5 indicated severe histological toxicity. Ultrastructural examination. The portions of the kidneys designated for electron microscopic analysis were cut into 1-mm fragments, fixed in 5% glutaraldehyde, post-fixed in 1% OsO4, dehydrated in ethyl alcohol (30–96%) and 100% acetone, and subsequently embedded in epoxy resin. Ultrathin sections were cut with a Reichert OmU2 ultramicrotome and examined using a JEM-1200 EX transmission electron microscope. The evaluator was blind to the treatment status of the rats. Isolation of mitochondria. All steps for the isolation of mitochondria were performed at 4 °C. The kidney mitochondria were isolated essentially as previously described (Gerschenson et al., 2000). Briefly, a 500 mg portion of the kidney was minced and then homogenised in 5 ml of homogenisation buffer (210 mM mannitol, 70 mM sucrose, 1 mM EDTA, 20 mM HEPES [pH 7.4], 2 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride). The homogenate was centrifuged twice at 1000 × g for 5 min to remove the cellular debris and nuclei. The mitochondria were collected at 20,000 × g for 20 min, gently resuspended in 50–100 μl homogenisation buffer, frozen in liquid
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nitrogen, and stored at −70 °C. The mitochondrial protein concentrations were measured using the BCA protein determination method using bovine serum albumin (BSA) as a standard. Measurement of oxygen consumption. The mitochondria (1 mg protein/ml) were incubated in 130 mM sucrose, 50 mM KCl, 5 mM MgCl2, 0.1 mM EGTA, 5 mM KH2PO4, and 5 mM HEPES; pH 7.4 at 25 °C with a Clark-type oxygen electrode (Hansatech Instruments, Oxygraph) under continuous stirring (Custodio et al., 1994). The mitochondrial oxygen respiration rates were measured in the presence of glutamate (1.0 mM) and malate (1.0 mM) that enter the electron transport chain selectively at complex I or succinate (2.0 mM) plus NADH dehydrogenase inhibitor (2 μM rotenone) that enters at complex II. The adenosine diphosphate (ADP)-stimulated respiration (state 3) was measured in the presence of 150 μM ADP, and the ADP-independent respiration (state 4) was monitored after all the ADP was consumed. The respiratory control ratio (RCR), defined as the ratio of oxygen consumption during state 3 to state 4 (a measure of the coupling of adenosine triphosphate synthesis to respiration), was calculated to evaluate the degree of mitochondrial damage. Measurement of ATP levels. The kidneys were homogenised in deionised boiling water (Yang et al., 2002) using a homogeniser and then centrifuged at 4000 rpm for 10 min. The clear supernatants were collected for the analyses. The protein concentrations were measured using the BCA method. The ATP levels were determined using the creatine kinase method (ATP Content Detection Kit; Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer's instructions. Respiratory chain complexes. After the isolation of mitochondria, the specific activities of the oxidative phosphorylation system (OXPHOS) enzyme complexes I and II were quantified on a Hewlett Packard diode array spectrophotometer as previously described (Trounce et al., 1996). Complex I (NADH:ubiquinone oxidoreductase) activity was determined by measuring the reduction of the ubiquinone analogue decylubiquinone. Complex II (succinate:ubiquinone oxidoreductase) specific activity was measured by monitoring the reduction of the dye 2, 6-dichlorophenolindophenol. mtDNA studies. DNA from the kidneys was extracted using the phenol: chloroform precipitation method, and the total amount of DNA recovered was determined by spectrophotometry. To quantify the mtDNA copy number, well-conserved single-copy genes were selected. Pyruvate kinase was used as a marker for nDNA and NADH dehydrogenase subunit 1 (ND-1) for mtDNA. The primers used to amplify a 267-bp region of the ND-1 gene were as follows: fwd, 5′-GGCTACATACAATTACGCAAAG-3′; and rev. 5′-TAGAATGGAGTAGACCGAAAGG-3′. To amplify a 347-bp fragment of the pyruvate kinase gene, the primer sequences consisted of the following: fwd, 5′-ACTGGCCGGTGTCATAGTGA-3′; and rev, 5′-TGTTGACCAGCCGTATGGATA-3′. The real-time PCR reactions were performed according to the manufacturer's instructions (SYBR® Premix Ex Taq™ II, TaKaRa), and thermal cycling was performed in an iCycler® (Bio-Rad). The mtDNA content was normalised to the nucleic DNA content. Statistical analysis. The Student's t-test was employed to analyse the differences between the sets of data. Statistical analyses were performed using GraphPad Prism (GraphPad, San Diego, CA). A value of pb 0.05 was considered statistically significant. Results AAI-induced acute nephrotoxicity in rats To characterise the nephrotoxicity of AAI, the kidneys were subjected to pathologic examination. A dramatic loss of the proximal
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Fig. 1. AAI-induced acute nephrotoxicity in rats. (A) Randomly selected images of the histopathology of kidneys from rats treated with AAI at 0 (a), 5 (b), 20 (c), or 80 mg/kg/day (d) for 7 days. Loss of the proximal tubule brush borders is observed in each group treated with AAI (arrow). Congestion in peritubular capillaries is observed in (c) and (d). Protein casts, cellular debris desquamation of tubular epithelial cells are observed in (d). All images are shown at identical magnification. (B) The pathologic scores of rats treated with AAI. The grading system used for classifying changes in renal pathology is described under the Methods section. (C) Serum markers of renal injury in rats treated with AAI. The blood collected from rats treated with AAI for 7 days was used for analysis of blood urea nitrogen and serum creatinine. All results are shown as means ± SEM for n= 5 animals for each group. Symbols indicate the statistical significance relative to vehicle control within each group: *p b 0.05, **p b 0.01.
tubule brush borders was confirmed by histopathology in the animals in each of the groups treated with AAI. In the AAI (5 mg/kg/day) group, loss of the brush borders, degeneration, and necrosis of the tubular epithelial cells were observed in 3/6 rats. In the AAI (20 mg/kg/day) group, degeneration and necrosis of the tubular epithelial cells were observed in 4/6 rats, and congestion was observed in some peritubular capillaries. In the AAI (80 mg/kg/day) group, loss of the brush borders, degeneration, and necrosis of the tubular epithelial cells were observed
in all rats. Protein casts, cellular debris, desquamation of tubular epithelial cells, and congestion were observed in several peritubular capillaries (Fig. 1A). The kidneys from the control rats showed no necrosis. High overall pathologic scores were recorded for the kidneys collected from the rats treated with AAI (Fig. 1B). Supporting these results, we detected significant increases of BUN and Cr in rats treated with the high dose of AAI, indicating damage to the kidney (Fig. 1C). Thus, AAI administration is associated with strong nephrotoxic effects in rats.
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AAI-induced ultrastructural mitochondrial alterations in kidney The ultrastructural examination showed that the proximal tubular mitochondria were extremely enlarged and dysmorphic, with loss and disorientation of their cristae. The ultrastructure of the kidneys from the AAI-exposed rats was characterised by morphologically aberrant mitochondria. In the AAI (5 mg/kg/day) group, mitochondrial swelling and small vacuoles were observed in all rats. In the AAI (20 mg/kg/ day) group, the cristae were markedly distorted, the mitochondrial structure was degraded, and the quantity of mitochondria was slightly decreased in each rat. In the AAI (80 mg/kg/day) group, the mitochondrial morphology was completely disrupted and the decrease in mitochondrial quantity was apparent in all rats (Fig. 2).
Effects of AAI on respiratory chain function in the kidney To study the effects of AAI treatment on the mitochondrial function in rat kidneys, we first evaluated the effects of AAI on the ATP content and RCR, two indicators of mitochondrial energy metabolism in these tissues. Our results indicated that the ATP contents were reduced in a dose-dependent manner after AAI treatment (Fig. 3A). In Fig. 3B, the RCR in mitochondria is shown in the presence of succinate or glutamate/malate. In the presence of substrates for complex I
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(glutamate/malate), AAI (5–80 mg/kg/day) dose-dependently and markedly reduced the RCR. With substrate for complex II (succinate), the lowest concentration of AAI treatment (5 mg/kg/day) failed to affect the RCR, and a minor decrease in RCR was detected in the AAI 20 and 80 mg/kg/day group compared with that in the presence of glutamate/ malate. Substrate specificity suggests that the inhibition of complex I was more significant than respiratory complex II. So in the next experiments, the activities of complexes I and II were examined. As presented in Figs. 3C and D, there were slight decreases in complex II-specific activities in the kidneys of AAI-exposed rats as compared to controls. However, the activities of complex I in the kidneys of rats exposed to 5, 20, and 80 mg/kg/day of AAI decreased to approximately 66.23% (P=0.16), 25.33% (Pb 0.05), and 11.6% (Pb 0.05), respectively, of the values for the unexposed controls. These results indicated that the activity of respiratory complex I, which is partly encoded by mtDNA, was reduced more significantly than that of respiratory complex II, which is completely encoded by nDNA.
Effects of AAI on mtDNA content The mtDNA content in kidneys, normalised to nDNA, was reduced after AAI treatment. The relative mtDNA ratio in the rats exposed to 5, 20, and 80 mg/kg/day of AAI decreased to approximately 74.16%
Fig. 2. Transmission electron micrographs of AAI-induced ultrastructural mitochondrial alterations in kidney. (A) Typical mitochondria in the control group. (B) Swollen mitochondria and small vacuoles (circled) are indicated in the AAI group (5 mg/kg/day). (C) The cristae (arrow) are distorted and the mitochondrial structure is degraded in the AAI group (20 mg/kg/day). (D) The mitochondrial morphology is completely disrupted in the AAI group (80 mg/kg/day). The image is shown at 30,000× magnification.
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Fig. 3. Effects of AAI on respiratory chain function in the kidney. The respiratory control ratio (A) and ATP content (B) of kidneys from SD rats treated with AAI for 7 days. Respiratory complex I (C) is reduced more significantly than respiratory complex II (D) in the kidneys of rats treated with AAI. All results are shown as the means ± SEM for n = 5 animals for each group. Symbols indicate the statistical significance relative to vehicle control within each group, *p b 0.05, **p b 0.01.
(P = 0.12), 59.69% (P b 0.05), and 29.20% (P b 0.01), respectively, compared to the unexposed control (Fig. 4). Discussion In the present study, we found that AAI induced renal toxicity in rats, and the mechanism of toxicity was related to mtDNA depletion and respiratory chain defects. Our conclusion is based on the following observations: first, the pathologic examination revealed loss of brush borders in only 3/6 rats in the low dose group. However, the ultrastructural examination showed enlarged and dysmorphic proximal
Fig. 4. MtDNA content in the kidneys measured using real-time PCR. The mtDNA content was normalised to nuclear DNA. The graphs depict data from three independent experiments. All results are shown as the means ± SEM for n= 5 animals for each group. Symbols indicate the statistical significance relative to the vehicle control within each group, *p b 0.05, **p b 0.01.
tubular mitochondria with loss and disorientation of their cristae in all rats of this group (Fig. 2). This suggests that changes in renal mitochondria may occur prior to damage to and loss of the brush-border plasma membrane and provides evidence that renal mitochondria are the most important intracellular target. Second, respiratory complex I, which is partly encoded by mtDNA, was reduced more significantly than respiratory complex II, which is completely encoded by nDNA, in the kidneys of rats treated with AAI (Figs. 3C and D). In agreement with this, the RCR in the presence of substrates for complex I was reduced more significantly than in the presence of substrates for complex II. Third, the real-time PCR assay revealed depletion of mtDNA in the kidneys of rats treated with AAI (Fig. 4). To our knowledge, mtDNA depletion and respiratory chain defects associated with AAI in the kidney have not previously been reported, and this mechanism could explain the renal toxicity of this chemical. The mitochondria represent a potential drug target because of their unique structure and function. Mitochondria have a unique milieu, with an alkaline and negatively charged interior (pH ≈ 8) and a series of specific channels and carrier proteins. These proteins facilitate the selective accumulation of xenobiotics in the matrix and/or the inner mitochondrial membrane through efficient trapping mechanisms (Murphy and Smith, 2000; Smith et al., 2003). Moreover, mitochondria are the only organelles outside the nucleus that contain DNA, and they are capable of synthesising some proteins. Unlike nDNA, mtDNA is not protected by histone proteins. Furthermore, mtDNA repair processes are generally less efficient than those for nDNA. As a result, mtDNA is more likely to be damaged than nDNA (Scatena et al. 2007). We demonstrated that AAI affected mitochondrial bioenergetic function by measuring the effects of this chemical on RCR. The RCR indicates how tightly the oxidative phosphorylation process is coupled,
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making this ratio a useful indicator of dysfunction. A lower RCR indicates a respiratory dysfunction, suppression of ATP synthesis, and the generally poor condition of the mitochondria. Consistent with the reduced RCR, the ATP content was decreased in a dose-dependent manner after AAI treatment. A previous study in HK-2 cells also showed decreased ATP content after AA treatment (Qi et al., 2007). Together, these results suggest that AAI adversely affects energy metabolism. The mtDNA encodes 13 structural proteins or enzymes, 2 ribosomal RNAs, and 22 transfer RNAs. The other structural proteins of the mitochondria and enzymes involved in mtDNA replication and repair are encoded by nDNA. ATP is produced in the mitochondria by the oxidative phosphorylation machinery, which involves 5 respiratory complexes. Complex II (SDH) of the respiratory chain is entirely encoded by nDNA. The other 4 complexes are encoded partially by mtDNA and partially by nDNA. Complex I (NADH dehydrogenase) of the respiratory chain is composed of 11 subunits (Hall et al., 2008). Seven of these subunits (ND1, ND2, ND3, ND4, ND4L, ND5, and ND6) are encoded entirely by mtDNA and are responsible for the catalytic and proton-pumping activities of the enzyme (Anderson et al., 1981). We compared the activities of complexes I and II and found that AAI reduced complex I activities in a dose-dependent manner without significantly affecting complex II activities. In agreement with this, the RCR in the presence of substrates for complex I was reduced more significantly than in the presence of substrates for complex II. These results demonstrated the selective depletion of mtDNA-encoded proteins and enzymatic function with no identifiable loss of nDNA-encoded mitochondrial proteins. To further define the effects of AAI, the mtDNA levels were measured. Real-time PCR revealed that the mtDNA content in the kidneys of the AAI-treated animals was significantly less than that of the untreated animals. This result is consistent with the activities of respiratory chain complexes. These findings show that AAI induces nephrotoxicity by impairing mtDNA replication, thus suppressing oxidative phosphorylation. This observation may explain the particular sensitivity of the convoluted proximal tubule to AAI toxicity because this segment exhibits one of the highest densities of mitochondria in the kidney (Hall et al., 2009). Mitochondria DNA may be more susceptible than nDNA to AAI-induced damage due to the absence of efficient DNA repair mechanisms in mitochondria and the lack of protective histones in mtDNA (Gredilla, 2010). In summary, our data suggest that mtDNA depletion and respiratory chain defects occur in the kidney after acute AAI exposure. Our results provide new insight into the involvement of mtDNA depletion and respiratory chain defects as a possible mechanism for AAI-induced acute nephrotoxicity. Conflict of interest The authors declare that there are no conflict of interest. Acknowledgments This work was supported by the Specific Fund for Public Interest Research of Traditional Chinese Medicine, Ministry of Finance of China (no. 200707008), and the 2011 Program for Excellent Scientific and Technological Innovation Team of Jiangsu Higher Education and partially supported by the 111 Project (111-2-07).
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