Schisandra chinensis Oil Attenuates Aristolochic Acid I-Induced Nephrotoxicity in vivo and in vitro / 中国结合医学杂志

OBJECTIVE@#To investigate the protective effects of Schisandra chinensis oil (SCEO) against aristolochic acid I (AA I)-induced nephrotoxicity in vivo and in vitro and elucidate the underlying mechanism.@*METHODS@#C57BL/6 mice were randomly divided into 5 groups according to a random number table, including control group, AA I group, and AA I +SCEO (0.25, 0.5 and 1 g/kg) groups (n=5 per group). Pretreatment with SCEO was done for 2 days by oral administration, while the control and AA I groups were treated with sodium carboxymethyl cellulose. Mice of all groups except for the control group were injected intraperitoneally with AA I (5 mg/kg) from day 3 until day 7. Histopathological examination and apoptosis of kidney tissue were observed by hematoxylin and eosin and TdT-mediated dUTP nick-end labeling (TUNEL) staining, respectively. The levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and serum creatinine (SCr), as well as renal malondialdehyde (MDA), glutathione, r-glutamyl cysteingl+glycine (GSH), and superoxide dismutase (SOD) were analyzed using enzyme-linked immunosorbent assay (ELISA). Expressions of hepatic cytochrome P450 1A1 (CYP1A1), CYP1A2, and nad(p)hquinonedehydrogenase1 (NQO1) were analyzed using ELISA, quantitative real-time polymerase chain reaction (qPCR) and Western blot, respectively. In vitro, SCEO (40 µ g/mL) was added 12 h before treatment with AA I (40 µ mol/mL for 48 h) in human renal proximal tubule cell line (HK-2), then apoptosis and reactive oxygen species (ROS) were analyzed by flow cytometry.@*RESULTS@#SCEO 0.5 and 1 g/kg ameliorated histopathological changes and TUNEL+ staining in the kidney tissues of mice with AA I-induced nephrotoxicity, and reduced serum levels of ALT, AST, BUN and SCr (P<0.01 or P<0.05). SCEO 0.5 and 1 g/kg alleviated the ROS generation in kidney, containing MDA, GSH and SOD (P<0.01 or P<0.05). SCEO 1 g/kg increased the expressions of CYP1A1 and CYP1A2 and decreased NQO1 level in the liver tissues (P<0.01 or P<0.05). Besides, in vitro studies also demonstrated that SCEO 40 µ g/mL inhibited apoptosis and ROS generation (P<0.05 or P<0.01).@*CONCLUSIONS@#SCEO can alleviate AA I-induced kidney damage both in vivo and in vitro. The protective mechanism may be closely related to the regulation of metabolic enzymes, thereby inhibiting apoptosis and ROS production.

Quantification of aristolochic acids and their DNA adducts in mice kidney and liver by HPLC-MS/MS / 中国中药杂志

This study aims to establish a quantitative method of 4 aristolochic acids-DNA adducts in mice kidney and liver based on high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS) for monitoring the content changes of aristolochic acids-DNA adducts. A Shiseido Capcellpak AQ C_(18) column(3 mm×100 mm, 3 μm) was used, with a mixture of 0.2% acetic acid-5 mmol·L~(-1) ammonium acetate as the aqueous phase and methanol as the organic phase for gradient elution. The multiple reaction monitoring(MRM) scanning method under positive mode by electrospray ionization(ESI) was performed for the detection of the aristolochic acids-DNA adducts which formed by combining aristolochic acid Ⅰ/Ⅱ with deoxyadenosine, deoxyguanosine, and deoxycytidine, respectively. Balb/c mice were given Guanmutong extract by gavage, and the relative content of aristolochic acids-DNA adducts in liver and kidney samples were analyzed within 60 days. It was found that the concentration of 4 aristolochic acids-DNA adducts in the kidney was significantly higher than that in the liver, and there were about 15.87 adducts in per 1×10~6 normal deoxynucleosides, which was 4.5-7.5 times than that of the liver. What's more, some adducts can still be detected on the 30 th day after administration. The concentration of the adducts in the liver was highest on the first day after administration, and a second peak appeared during the 7 th to 14 th days. The results indicated that aristolochic acids-DNA adducts are difficult to eliminate in vivo, and it is of great significance to study the mechanism of liver and kidney injury of aristolochic acid.

Evaluation of renal oxygenation in rats with acute aristolochic acid nephropathy using blood oxygenation level-dependent magnetic resonance imaging / 南方医科大学学报

OBJECTIVE@#To evaluate the changes in renal oxygenation in rats with acute aristolochic acid nephropathy using blood oxygenation level-dependent (BOLD) magnetic resonance imaging (MRI) at 7.0T.@*METHODS@#Wistar rats were randomly divided into AAN group (=18) and control group (=6) for intraperitoneal injections of AAI at 40 mg/kg and PEG400, respectively, on a daily basis for 6 consecutive days. All the control rats and 6 rats from AAN group underwent BOLD MRI scan before and at 2, 4, and 6 days after the initial injection for measuring renal cortical and medullary R2 values. At each of the 4 time points, 3 rats in AAN group were sacrificed for histological evaluation; the control rats were examined at 6 days after the initial injection.@*RESULTS@#The cortical and medullary R2 values of the rats in AAN group on days 4 and 6 were significantly higher than those in the control group ( < 0.05). In AAN group, the cortical R2 values showed no obvious changes on day 2 as compared with the baseline values, but increased significantly on day 4 ( < 0.05) and day 6 ( < 0.01); the medullary R2 values increased progressively and were significantly higher than the baseline values on day 4 ( < 0.01) and day 6 ( < 0.01). In the control group, no significant changes were detected in either cortical or medullary R2 values throughout the experiment.@*CONCLUSIONS@#BOLD MRI allows non-invasive measurement of renal oxygenation levels in rats with AAN. The increase of renal cortical and medullary R2 values, and particularly the latter, indicates a lowered renal oxygenation level, which provides potentially useful information for clinical decisions.

Carcinogens that induce the A:T > T:A nucleotide substitutions in the genome / 医学前沿

Recently, Ng et al. reported that the A:T > T:A substitutions, proposed to be a signature of aristolochic acid (AA) exposure, were detected in 76/98 (78%) of patients with hepatocellular carcinoma (HCC) from the Taiwan Province of China, and 47% to 1.7% of HCCs from the Chinese mainland and other countries harbored the nucleotide changes. However, other carcinogens, e.g., tobacco carcinogens 4-aminobiphenyl and 1,3-butadiene, air toxic vinyl chloride and its reactive metabolites chloroethylene oxide, melphalan and chlorambucil, also cause this signature in the genome. Since tobacco smoke is a worldwide public health threat and vinyl chloride distributes globally and is an air pollutant in Taiwan Province, the estimation of the patients' exposure history is the key to determine the "culprit" of the A:T > T:A mutations. Apparently, without estimation of the patients' exposure history, the conclusion of Ng et al. is unpersuasive and misleading.

Characterization and quantitation of aristolochic acid analogs in different parts of Aristolochiae Fructus, using UHPLC-Q/TOF-MS and UHPLC-QqQ-MS / 中国天然药物

Aristolochiae Fructus, a Chinese herbal medicine derived from the fruit of Aristolochia contorta Bge., contains nephrotoxic aristolochic acid analogues (AAAs). According to ancient medical texts, various medicinal parts of the fruit of A. contorta were ever used. In order to reveal which part could be safely and effectively used, it is necessary to analyze the chemical profiles of different medicinal parts. Herein we compared the chemical compositions and determined aristolochic acid I (AA-I) and aristolochic acid II (AA-II) in the four parts viz. outer pericarp, inner pericarp, septum, and seed. Ultra-high performance liquid chromatography equipped with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) was applied for chemical profiling. Ultra-high performance liquid coupled with triple quadrupole mass spectrometry (UHPLC-QqQ-MS) was employed to quantify AA-I and AA-II in different parts. It was found that the chemical compositions of the four parts varied both qualitatively and quantitatively. A total of 10 AAAs, including 5 aristolochic acids and 5 aristolactams, together with 3 alkaloids, were unambiguously or tentatively identified by UHPLC-QTOF-MS. The quantitatively analytical results obtained by UHPLC-QqQ-MS showed that AA-I and AA-II exclusively accumulate in the seeds of A. contorta. These findings provide supporting data for the rational selection of medicinal parts.

Analysis of aristolochic acids, aristololactams and their analogues using liquid chromatography tandem mass spectrometry / 中国天然药物

More than 80 aristolochic acids (AAs) and aristololactams (ALs) have been found in plants of the Aristolochiaceae family, but relatively few have been fully studied. The present study aimed at developing and validating a liquid chromatography tandem mass spectrometry (LC/MS(n)) for the analysis of these compounds. We characterized the fragmentation behaviors of 31 AAs, ALs, and their analogues via high performance liquid chromatography coupled with electrospray ionization mass spectrometry. We summarized their fragmentation rules and used these rules to identify the constituents contained in Aristolochia contorta, Ar. debilis, Ar. manshurensis, Ar. fangchi, Ar. cinnabarina, and Ar. mollissima. The AAs and ALs showed very different MS behaviors. In MS(1) of AAs, the characteristic pseudomolecular ions were [M + NH4](+), [M + H](+), and [M + H - H2O](+). However, only [M + H](+) was found in the MS(1) of ALs, which was simpler than that of AAs. Distinct MS(n)fragmentation patterns were found for AAs and ALs, showing the same skeleton among the different substituent groups. The distribution of the 31 constituents in the 6 species of Aristolochia genus was reported for the first time. 25 Analogues of AAs and ALs were detected in this genus. A hierarchical schemes and a calculating formula of the molecular formula of these nitrophenanthrene carboxylic acids and their lactams were proposed. In conclusion, this method could be applied to identification of similar unknown constituents in other plants.

Research on biological detoxification of Chinese medicine containing aristolochic acid A by ten microorganisms / 中国中药杂志

This paper was aim to screen microorganisms with attenualed efficiency for Chinese medicine containing aristolochic acid A by liquid-state fermentation. Twelve Chinese medicine were detected by UPLC and aristolochic acid A was only founded in four species of Aristolochia, those were Caulis Aristolochiae Manshuriensis, Aristolochiae Radix, Aistolochia Contorta Bunge and Herba Aristolochiae Mollissima,but not in the others. With the four Chinese medicine containing aristolochic acid A as raw material, ten microorganisms were tested, and the content of aristolochic acid A was detected by UPLC. The results showed that one microorganism can decrease content of aristolochic acid A in all those four Chinese medicine.

Exploring the Correlation between Pi and Shen from the Excretion of AA-I and Expressions of Or- ganic Anion Transporting Polypeptide 2al and 2 b1 in Pi Deficiency Model Rats / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To explore the correlation between Pi and Shen by observing the relationship between the metabolism of aristolochic acid (AA) and mRNA and protein expression levels of organic anion transporting polypeptide (oatp) superfamily member 2a1 and 2 b1 (oatp2al and oatp2bl) in renal, small intestinal, and large intestinal tissues of Pi deficiency syndrome (PDS) model rats.</p><p><b>METHODS</b>Totally 46 Sprague-Dawley (SD) rats were randomly divided into four groups, i.e., the blank group (n = 12), the PDS group (n = 22), the AA-I group (n = 6), and the PDS AA-I group (n = 6). PDS model was established by subcutaneously injecting Reserpine at the daily dose of 5 mg/kg for 16 successive days. Carotid intubation was performed in 6 rats selected from the blank group and the PDS group. Pharmacokinetics of AA-I were detected at 5, 15, 30, 45, and 60 min after gastrogavage of AA-I. AA-I concentrations in renal, small intestinal, and large intestinal tissues of 10 rats selected from the PDS group were determined. Normal saline was administered to 6 rats selected from the PDS group and the blank group by gastrogavage. Renal, small intestinal, and large intestinal tissues were collected in the AA-I group and the PDS AA-I group at 60 min after gastrogavage of AA-I. mRNA and protein expression levels of oatp2a1 and oatp2b1 in each tissue were detected using real-time polymerase chain reaction (RT- PCR) and Western blot.</p><p><b>RESULTS</b>Compared with the blank group, plasma concentrations of in vivo AA-I were obviously higher in the PDS group at 15, 30, 45, and 60 min after gastrogavage of AA-I with statistical difference (P < 0.05). Plasma concentrations of AA-I were obviously decreased at 60 min after gastrogavage of AA-I; AA-I concentrations in renal and large intestinal tissues were elevated; AA-I concentrations in small intestinal tissues were obviously reduced in the PDS group. There was no statistical difference in mRNA expression levels of oatp2a1 and oatp2b1 in the aforesaid three tissues of rats between the blank group and the PDS group. Compared with the blank group, mRNA expression levels of oatp2a1 and oatp2b1 decreased in small intestinal tissues of the AA-I group, and the mRNA expression level of oatp2a1 in large intestinal tissues significantly decreased (P < 0.05, P < 0.01). Compared with the PDS group, mRNA expression levels of oatp2a1 and oatp2b1 increased in renal tissues of the PDS AA-I group (P < 0.05); mRNA expression levels of oatp2b1 increased in large intestinal tissues of the PDS AA-I group (P < 0.05).</p><p><b>CONCLUSIONS</b>The difference in AA-I metabolism might be associated with changed expression levels of oatp2a1 and oatp2b1 in renal, small intestinal, and large intestinal tissues under Pi deficiency induced loss of transportation. Shen and Dachang played important roles in substance metabolism under Pi deficiency state, which proved Pi-Shen correlated in Chinese medical theories.</p>

Advance in studies on toxicity of aristolochic acid and analysis on risk factors / 中国中药杂志

The renal toxicity and mutagenicity of aristolochic acid (AA) as well as its carcinogenicity on upper urinary tract transitional epithelial cells have been widely known. Since 2003, drug regulatory departments have successively cancelled the quality standards for AA-containing medicines such as Aristolochiae Radix, Aristolochiae Manshuriensis Caulis and Aristolchiae Fangchi Radix, and adopted measures for strengthening regulation and revising package insert or quality standards for other AA-containing medicines, including Aristolochia Cinnabarina Radix, Aristolochiae Fructus, Aristolochiae Mollissimae Herba, in order to control its safety risk. In recent years, domestic and foreign studies on AA have mainly involved action mechanism and clinical performance of AA toxicity, early-stage diagnosis and treatment method. In this paper, authors gave a brief summary and evaluation on risk factors for using AA-containing medicines, and offered measures and suggestions for preventing and controlling AA toxicity.

Aristolochic acid nephropathy: an issue worth more attention / 中国中西医结合杂志

Aristolochic acid nephropathy (AAN), a progressive renal interstitial fibrosis frequently resulting in end stage renal disease, still remains a common chronic interstitial nephropathy in China. Therefore, great attention should be paid to AAN. This review summarized recent research progress of AAN in terms of in vivo aristolochic acid metabolism, epithelial mesenchymal transition, proteomics, immunity-inflammation, and autophagy, which will help to further understand the pathogenesis of AAN, and to search effective intervention targets.

Effect of fermented cordyceps powder and prednisone on the Notch2/Hes-1 signaling activation in the kidney tubules of rats with acute aristolochic acid nephropathy / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To investigate the effect of both fermented Cordyceps powder (CS) and prednisone on the Notch2/hes-1 signaling activation in the kidney tubules of rats with acute aristolochic acid nephropathy (AAAN).</p><p><b>METHODS</b>Totally 50 SD rats were randomly divided into 4 groups, i.e., the normal group, the model group, the CS group, the prednisone group, and the CS plus prednisone group, 10 in each group. The AAAN rat model was induced by intragastric administration of pure aristolochic acid A at the daily dose of 100 mg/kg for 3 days. Rats in the CS group were administered with CS at the daily dose of 5.0 g/kg by gastrogavage, while those in the prednisone group were administered with prednisone at the daily dose of 0.5 mg/kg. Rats in the CS plus prednisone group were treated by CS and prednisone. All treatment lasted for 3 successive weeks. Kidney functions [urea nitrogen (BUN) and serum creatinine (SCr)] were detected. The pathological changes of kidneys were observed by Hematoxylin-Eosin staining. The apoptosis of the renal tubular epithelial cells was detected by TUNEL. The protein expressions of Notch2 and Hes-1 in the renal tissue were detected by immunohistochemical assay and Western blot.</p><p><b>RESULTS</b>Results of HE staining showed the structure in the nephridial tissue was regular in rats of the normal group. The renal tubular necrosis occurred in the rats of the model group. The pathological changes of kidneys were obviously improved in the CS group, the prednisone group, and the CS plus prednisone group. Compared with the normal group, levels of BUN and SCr, semi-quantitative score of the tubular interstitial tissue, ratio of apoptotic cells, and expressions of Notch2 and Hes-1 proteins significantly increased in the model group (P < 0.01). Compared with the model group, the aforesaid indices significantly decreased in the 3 treatment groups (P < 0.01). All indices decreased most obviously in the CS plus prednisone group (P < 0.05, P < 0. 01).</p><p><b>CONCLUSIONS</b>Notch2/hes-1 signaling activation might be associated with apoptosis of renal tubular epithelial cells. Both CS and prednisone could play a nephroprotective role for AAAN. But CS plus prednisone could achieve the best effect. Inhabiting the Notch2/hes-1 signaling activation could be its nephroprotective mechanism.</p>

Development of gastric precancerous lesion animal model / 中国中药杂志

<p><b>OBJECTIVE</b>To establish a model of gastric precancerous lesion by using Aristolochic manshuriensis which contains aristolochic acids.</p><p><b>METHOD</b>The SD rats were randomly divided into four groups: control and three different doses of ethanol extractive of A. manshuriensis (EEA) (corresponding to aristolochic acid I 2.5, 5.0, 10.0 mg x kg(-1)), respectively. EEA was intragastrically given to rats every other day. At the end of the 10th, 15th, 20th week, part of the rats in each group was sacrificed and the stomachs were weighed. The gastric tumor was assessed by the weight and the relative stomach weight to the body weight. The stomachs were fixed in 4% neutral formalin, and the paraffin imbedding tissues were sliced and HE stained. Histomorphology was observed under the light microscope to determine gastric hyperplasia, mucosa precancerosis (atypical hyperplasia) and gastric cancer formation.</p><p><b>RESULT</b>The rats treated with different doses of EEA for 10 weeks induced mucosa papillary, epithelioma hyperplasia. Histological observation showed mucosa precancerosis lesions characterized as atypical hyperplasia at the dose levels corresponding to aristolochic acid I 5.0 and 10.0 mg x kg(-1) treated for 10 weeks. The incidence rate of gastric precancerosis in those two groups was 100% at the 15th week. Malignant tumors were observed in most of the animals in 10.0 mg x kg(-1) group. The animals in 5.0 mg x kg(-1) group were well tolerant compared to 10.0 mg x kg(-1) group during the course of experiment, so the dose of aristolochic acid I 5.0 mg x kg(-1) and 10-15 weeks treatment were considered to be optimum to establish the model of gastric precancerosis.</p><p><b>CONCLUSION</b>A rat model of gastric precancerosis can be induced within a short duration by giving an oral administration of the ethanol extract of A. manshuriensis which contains aristolochic acids.</p>

Transitional cell carcinoma associated with aristolochic acid nephropathy: most common cancer in chronic hemodialysis patients in China / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The research of cancer in patients on hemodialysis (HD) in China has not been reported. The aim of this study was to investigate the clinical and histological features and outcomes of cancer in Chinese HD patients.</p><p><b>METHODS</b>The study subjects were 49 cancer patients (1.4%) out of 3448 end stage renal disease (ESRD) patients maintained on HD at China-Japan Friendship Hospital from October 1997 to July 2011.</p><p><b>RESULTS</b>Urinary tract cancer (74%) was the most common followed by gastrointestinal tract cancer (12%), breast cancer (6%), lung cancer (4%), thyroid cancer (2%), and hematologic cancer (2%). Thirty-three patients (67%) had urinary tract transitional cell carcinoma (TCC) and 29 of them had aristolochic acid nephropathy (AAN) as underlying disease. Death occurred in eight patients out of 49, and the survival rate of HD patients with cancer was similar to those without cancer (P = 0.120).</p><p><b>CONCLUSION</b>The urinary tract TCC is the most common cancer in HD patients with AAN in one of the centers of northern China.</p>

A new aristolochic acid derivative from Asarum himalaicum / 药学学报

To study the chemical constituents of Asarum himalaicum, fifteen compounds were isolated from a 70% ethanol extract by using a combination of various chromatographic techniques including column chromatography over silica gel, Sephadex LH-20, and semi-preparative HPLC. By spectroscopic techniques including 1H NMR, 13C NMR, and HR-ESI-MS, these compounds were identified as 4-demethoxyaristolochic acid BII (1), aristolochic acid I (2), aristolochic acid Ia (3), 7-hydroxyaristolochic acid I (4), aristolochic acid IV (5), aristolic acid II (6), debilic acid (7), aristololactam I (8), 9-hydroxyaristololactam I (9), 7-methoxyaristololactam IV (10), (2S)-narigenin-5, 7-di-O-beta-D-pyranosylglucoside (11), 4-hydroxybenzoic acid (12), 3, 4-dihydroxybenzoic acid (13), 4-hydroxycinnamic acid (14), and beta-sitosterol (15). All of these compounds (1-15) were obtained from A. himalaicum for the first time. Among them, 1 was identified as a new compound, and compounds 3-6, 9, 12-14 were isolated from Asarum genus for the first time. Since the kidney toxicity of aristolochic acids and aristololactams has been reported, the result of this investigation suggests that it should be cautioned to use A. himalaicum as a medicine.

Clinical characteristics and long-term follow-up analysis of three cases with newborn aristolochic acid nephropathy / 中华儿科杂志

<p><b>OBJECTIVE</b>To summarize the clinical characteristics and prognosis of newborn aristolochic acid nephropathy induced by akebia.</p><p><b>METHOD</b>Retrospective analysis of clinical manifestations, therapy and prognosis was made upon data of 3 newborn infants with renal function lesion induced by akebia.</p><p><b>RESULT</b>Three infants who were fed with Chinese herbal medicines containing akebia trifoliate suffered from acute renal failure, renal glomerular and tubular injury, with symptoms of vomiting, diarrhea, and oliguria. Laboratory tests manifested hyperpotassemia, hyponatremia, elevation of serum creatinine and urea nitrogen, and metabolic acidosis. Renal glomerular lesion was mild, presented with proteinuria and increased serum β(2) microglobin. Renal dysfunction was manifested with alkaline urine, glucosuria, positiveness of urine glucose, ketone and aminoaciduria, and increased urine β(2) microglobin excretion. After symptomatic treatment for 3 to 4 weeks, the renal function of these infants recovered. Proteinuria, aminoaciduria and glucosuria turned negative within 5 to 8 months, 3 months to 1 year, and 9 months to 3 years, respectively. Urine pH decreased to 7.0 after 5.0 - 5.5 years. All cases took citric acid mixtures for 5.5 to 6 years. A 12-years follow-up demonstrated that serum creatinine of 3 cases were within normal range during the first 11 years of life, however recent follow-up showed increased serum creatinine of case 1 and case 2, except for serum creatinine of case 3 remained normal. The estimated glomerular filtration rate (eGFR) of all the 3 cases decreased. Among which, eGFR of case 1 and case 2 were lower than 90 [ml/(min·1.73 m(2))], and decreased 1.1 [ml/(min·1.73 m(2))] and 0.6 [ml/(min·1.73 m(2))] per year during recent six years, respectively. No obvious decrease of eGFR was observed in case 3. Blood gas analysis and urine routine were normal, yet blood and urine β(2) microglobin excretion were still high. Urinary N-acetyl-β-D-glucosaminidase increased again after having returned to normal.</p><p><b>CONCLUSION</b>Newborn aristolochic acid nephropathy induced by akebia might induce acute renal failure and renal tubules injury. Renal function could recover after symptomatic treatment in short-term. Nevertheless, glomerular filtration rate presents a slow descending tendency and renal tubules lesion lasted for many years, which requires a long-term follow-up.</p>

Trace analysis of aristolochic acid A / 中国中药杂志

A HPLC method for limit detection of aristolochic acid A in the Chinese herbs containing aristolochic acid or suspected-containing aristolochic acid and their preparations was established. The samples were analyzed on an Alltima C18 column eluted with methanol-water-acetic acid (68:32:1.5) as the mobile phase. Flow rate was at 1.0 mL x min(-1) and the detection wavelength was at 390 nm. The calibration curve was linear over the range from 0.016 to 0.51 g (r = 0.9993) and LOD was 4 ng. The average recovery was 101.2% with RSD of 2.01%. The procedures of sample preparation were systematically investigated. The contents of aristolochic acid A in Radix et Rhizoma Asari bought from market or drugstore were fluctuated from 3.1 to 26.6 microg x g(-1) and 3 of 11 samples accorded with the quality requirement of current Chinese Pharmacopoeia. Among 15 batches samples of Chinese medicaments, only one sample was found to contain aristolochic acid A. The present investigation shows that the method is sensitive and repeatable and it could be used for the limit detection of aristolochic acid A in the Chinese herbal medicines containing trace amount of aristolochic acid A or suspected-containing aristolochic acid A and their preparations.

Effect of bone morphogenetic protein-7 on aristolochic acid induced renal tubular epithelial cells transdifferentiation / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe the effect of bone morphogenetic protein-7 (BMP-7) on aristolchic acid induced renal tubular epithelial cell trans-differentiation to look for new therapeutic approach for aristolchic acid nephropathy (AAN).</p><p><b>METHODS</b>In vitro cultured human proximal renal tubular epithelial cell line HK-2 cells were treated with different concentrations of BMP-7 (75 ng/mL, 150 ng/mL and 300 ng/mL) after trans-differentiation of the cells was induced by AA (10 microg/mL). Levels of alpha-SMA mRNA and protein expressions were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blotting respectively.</p><p><b>RESULTS</b>BMP-7 reversed the AA inducing alpha-SMA expressions in HK-2 cells in a dose-dependent manner.</p><p><b>CONCLUSION</b>BMP-7 can inhibit the trans-differentiation of human renal tubular epithelial cell induced by AA, thereby might be a new potential drug for AAN prevention and treatment.</p>

Effects of Hirsutella sinensis on TGF-beta1 and Snail expressions and transdifferentiation of tubular epithelial-myofibroblast in renal tissue of rats with chronic aristolochic acid nephropathy / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To investigate the antagonizing effect of Hirsutella sinensis (HS) on renal tubular epithelial-myofibroblast transdifferentiation (TEMT) and its possible pathogenic mechanism in rats with chronic aristolochic acid nephropathy (CAAN).</p><p><b>METHODS</b>Eighteen male Sprague-Dawley rats were equally divided into 3 groups, the model (M) group, the intervention (I) group and the control (C) group. The 24 h urinary protein (UP) in rats was measured before intervention and at the end of the 1st, 4th, 8th, and 12th week, and creatinine clearance rate (CCr) was measured before intervention and at the end of the 12th week respectively. All rats were sacrificed at the end of the 12th week, their kidney was taken for examining the degree of fibrosis in renal interstitial with Masson's stain and determining mRNA and protein expressions of transforming growth factor-beta1 (TGF-beta1), Snail, alpha-smooth muscle actin (alpha-SMA) and cytokeratin in renal tissue by Real-time RT-PCR and immunohistochemistry staining, respectively.</p><p><b>RESULTS</b>Compared with the C group, CCr was significantly lower, while 24 h UP was higher; the relative area of interstitial fibrosis was significantly larger in the M group; besides, the mRNA and protein expressions of TGF-beta1, Snail and alpha-SMA were significantly up-regulated (P < 0.01 or P < 0.05), and those of cytokeratin were significantly down-regulated (P < 0.01) in renal tissue of the M group. While in the I group, all the above-mentioned abnormalities were restored to some extent (P < 0.05) and showed significant difference (all P < 0.05) as compared with those in the M group.</p><p><b>CONCLUSION</b>HS can downregulate TGF-beta1 and Snail expressions in renal tissue, antagonize TEMT and renal interstitial fibrosis, and improve renal function in CAAN rats.</p>

Organic anion transporter system mediates the transport of aristolochic acid in tubular epithelial cells / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate whether aristolochic acid can be transported into human kidney proximal tubular cell (HKC) and its potential mechanism.</p><p><b>METHODS</b>Intracellular aristolochic acid was measured by liquid chromatography-tandem mass spectrometry. The release of lactate dehydrogenase (LDH) induced by aristolochic acid in the presence of organic anion transporter inhibitor (probenecid) or organic cation transporter inhibitor (tetraethylammonium) was evaluated. The effects of probenecid on aristolochic acid induced connective tissue growth factor (CTGF) mRNA and protein expression were also examined by real time polymerase chain reaction and Western blot, respectively.</p><p><b>RESULTS</b>Aristolochic acid was detected in the suspension of the denatured HKC after incubation with aristolochic acid sodium salt. The release of LDH from HKC, which was induced by 60 mg/L aristolochic acid sodium salt, was significantly inhibited by 1 mmol/L probenecid (P < 0.01), but not by 1 mmol/L tetraethylammonium. The increased CTGF mRNA and protein expression in HKC stimulated by 40 mg/L aristolochic acid sodium salt was significantly down-regulated by 1 mmol/L probenecid (P < 0.05), with an inhibition rate of 16% and 21%, respectively.</p><p><b>CONCLUSION</b>Aristolochic acid can be transported into HKC by organic anion transport system, and then exerts its biological effects.</p>

Dynamical distribution of bone marrow mesenchymal stem cells in rat model of chronic aristolochic acid nephropathy / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To investigate the distributive path and proliferative rule of marrow mesenchymal stem cells (MSCs) in the rat transplanted via caudal vein from male rat to female rats model of chronic aristolochic acid nephropathy (CAAN).</p><p><b>METHODS</b>Cells taken from femoral bone marrow of male Wistar rats were made into single cell suspension, cultured, purified and identified as MSCs. MSCs were transplanted via caudal vein into 50 female Wistar CAAN model rats allocated in the test group, they were killed, 10 rats in a batch, at various time points (6 h, 48 h, 10 d, 30 d and 60 d after transplantation). Besides, 10 rats allocated in the control group were killed on the 30th day after received sham-transplantation. Kidney tissue of all rats was taken for detecting cells originated from the donors by fluorescence in situ hybridization test with FAM-labeled sex determining region of Y chromosome (SRY FISH) probe, and their number in SRY was counted using SRY PCR.</p><p><b>RESULTS</b>MSCs were mainly distributed in the glomerular capillaries at the time points of 6 h and 48 h, but the number of MSCs in glomerular capillaries decreased and those in renal mesenchyma increased at the time points from 10 d to 60 d gradually, then tended to a steady state, meanwhile it showed a stable increasing trend in renal tubule. Cell colony of MSCs could be found in mesenchyma with a slowed down increasing between 30 d to 60 d, but the increasing in tubule was still steady.</p><p><b>CONCLUSION</b>MSCs originated from the donor can enter the kidney of acceptor and distribute from blood capillary to renal mesenchyma and tubule, and they can long time inhabit there and make propagation.</p>