Aristolochic acid I aggravates oxidative stress-mediated apoptosis by inhibiting APE1/Nrf2/HO-1 signaling.

Nephrotoxicity induced by aristolochic acid I (AAI) is related to redox stress and apoptosis. Apurinic/apyrimidine endonuclease 1 (APE1) has antioxidant and anti-apoptotic effects. This study investigated the potential role of APE1 in AAI-induced nephrotoxicity. Renal injury was successfully induced in C57BL/6J mice by intraperitoneal injection of AAI every other day for 28 days. Expressions of APE1, nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase 1 (HO-1) in renal tissues of the model mice was inhibited, accompanied by oxidative damage and apoptosis. Similar results were obtained in vitro in human proximal tubular (HK-2) cells damaged by AAI. In the presence of a low concentration of the APE1 inhibitor E3330, expression of Nrf2 and HO-1 proteins in HK-2 cells was decreased and AAI-induced apoptosis was aggravated. Overexpression of APE1 in HK-2 cells promoted the expression of Nrf2 and HO-1, and alleviated apoptosis and renal injury induced by AAI. The collective findings demonstrate that AAI can inhibit the induction of oxidative stress and apoptosis by the APE1/Nrf2/HO-1 axis, leading to AAI renal injury. Targeting APE1 may be an effective therapeutic strategy to treat AA nephrotoxicity.

Renal angiotensin I-converting enzyme-deficient mice are protected against aristolochic acid nephropathy.

The renal renin-angiotensin system (RAS) is involved in the development of chronic kidney disease. Here, we investigated whether mice with reduced renal angiotensin I-converting enzyme (ACE-/-) are protected against aristolochic acid nephropathy (AAN). To further elucidate potential molecular mechanisms, we assessed the renal abundances of several major RAS components. AAN was induced using aristolochic acid I (AAI). Glomerular filtration rate (GFR) was determined using inulin clearance and renal protein abundances of renin, angiotensinogen, angiotensin I-converting enzyme (ACE) 2, and Mas receptor (Mas) were determined in ACE-/- and C57BL/6J control mice by Western blot analyses. Renal ACE activity was determined using a colorimetric assay and renal angiotensin (Ang) (1-7) concentration was determined by ELISA. GFR was similar in vehicle-treated mice of both strains. AAI decreased GFR in controls but not in ACE-/- mice. Furthermore, AAI decreased renal ACE activity in controls but not in ACE-/- mice. Vehicle-treated ACE-/- mice had significantly higher renal ACE2 and Mas protein abundances than controls. AAI decreased renal ACE2 protein abundance in both strains. Furthermore, AAI increased renal Mas protein abundance, although the latter effect did not reach statistical significance in the ACE-/- mice. Renal Ang(1-7) concentration was similar in vehicle-treated mice of both strains. AAI increased renal Ang(1-7) concentration in the ACE-/- mice but not in the controls. Mice with reduced renal ACE are protected against AAN. Our data suggest that in the face of renal ACE deficiency, AAI may activate the ACE2/Ang(1-7)/Mas axis, which in turn may deploy its reno-protective effects.

Aristolochic acid I induces proximal tubule injury through ROS/HMGB1/mt DNA mediated activation of TLRs.

Aristolochic acids (AAs) are extracted from certain plants as folk remedies for centuries until their nephrotoxicity and carcinogenicity were recognized. Aristolochic acid I (AAI) is one of the main pathogenic compounds, and it has nephrotoxic, carcinogenic and mutagenic effects. Previous studies have shown that AAI acts mainly on proximal renal tubular epithelial cells; however, the mechanisms of AAI-induced proximal tubule cell damage are still not fully characterized. We exposed human kidney proximal tubule cells (PTCs; HK2 cell line) to AAI in vitro at different time/dose conditions and assessed cell proliferation, reactive oxygen species (ROS) generation, nitric oxide (NO) production, m-RNA/ protein expressions and mitochondrial dysfunction. AAI exposure decreased proliferation and increased apoptosis, ROS generation / NO production in PTCs significantly at 24 h. Gene/ protein expression studies demonstrated activation of innate immunity (TLRs 2, 3, 4 and 9, HMGB1), inflammatory (IL6, TNFA, IL1B, IL18, TGFB and NLRP3) and kidney injury (LCN2) markers. AAI also induced epithelial-mesenchymal transition (EMT) and mitochondrial dysfunction in HK2 cells. TLR9 knock-down and ROS inhibition were able to ameliorate the toxic effect of AAI. In conclusion, AAI treatment caused injury to PTCs through ROS-HMGB1/mitochondrial DNA (mt DNA)-mediated activation of TLRs and inflammatory response.

Fetal exposure of Aristolochic Acid I undermines ovarian reserve by disturbing primordial folliculogenesis.

The primordial follicle pool established in early life determines the ovarian reserve in the female reproductive lifespan. Premature exhaustion of primordial follicles contributes to primary ovarian insufficiency (POI), that is dependent by the initial size of the primordial follicle pool and by the rate of its activation and depletion. AAI, a powerful nephrotoxin with carcinogenic potential, is present in the Aristolochiaceae species, which can release AAI into soil as a persistent pollutant. In order to assess the potential risk of Aristolochic Acid I (AAI) exposure on mammalian oogenesis, we uncovered its adverse effect on primordial folliculogenesis in the neonatal mouse ovary and its effect on female fertility in adulthood. Pregnant mice were orally administrated with doses of AAI without hepatic or renal toxicity during late-gestation. Ovaries from offspring of administered female displayed gross aberrations during primordial folliculogenesis. Also, unenclosed oocytes in germ-cell cysts showed increased DNA damage. Furthermore, several key factors, including NANOS3, SOX9, KLF4, that govern early gonad's differentiation were abnormally expressed in the exposed ovary, while the follicle formation was partially restored by knockdown of Nanos3 or sox9. In adulthood, these aberrations evolved into a significant reduction in offspring number and impaired ovarian reserve. Together, our results show that AAI influences primordial folliculogenesis and, importantly, affected female fertility. This study shows that administration of drugs herbs or consumption of vegetables that contain AAs during pregnancy may adversely influence the fertility of offspring.

Protective Effects of Mitochondrial Uncoupling Protein 2 against Aristolochic Acid I-Induced Toxicity in HK-2 Cells.

Aristolochic acid I (AA I) is one of the most abundant and toxic aristolochic acids that is reported to cause Aristolochic acid nephropathy (AAN). This paper was designed to assess whether mitochondrial Uncoupling Protein 2 (UCP2), which plays an antioxidative and antiapoptotic role, could protect human renal proximal tubular epithelial (HK-2) cells from toxicity induced by AA I. In this study, HK-2 cells were treated with different concentrations of AA I with or without UCP2 inhibitor (genipin). To upregulate the expression of UCP2 in HK-2 cells, UCP2-DNA transfection was performed. The cell viability was evaluated by colorimetric method using MTT. A series of related biological events such as Reactive Oxygen Species (ROS), Glutathione peroxidase (GSH-Px), and Malondialdehyde (MDA) were evaluated. The results showed that the cytotoxicity of AA I with genipin group was much higher than that of AA I alone. Genipin dramatically boosted oxidative stress and exacerbated AA I-induced apoptosis. Furthermore, the increased expression of UCP2 can reduce the toxicity of AA I on HK-2 cells and upregulation of UCP2 expression can reduce AA I-induced oxidative stress and apoptosis. In conclusion, UCP2 might be a potential target for alleviating AA I-induced nephrotoxicity.

[Quality control of substance benchmarks for Danggui Sini Decoction].

A comprehensive quality control method was established to provide references for quality control and evaluation of substance benchmarks of Danggui Sini Decoction(DSD). The HPLC separation was performed on a Kromasil 100 C-8 column(4.6 mm×250 mm, 5 µm) with acetonitrile(A)-0.05% phosphoric acid in water(B) as mobile phase in a gradient elution mode at the flow rate of 1 mL·min~(-1). The column temperature was 25 ℃ and the detection wavelength was set at 275 nm. Under these conditions, the content of seven components, including paeoniflorin, liquiritin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhetate, ligustilide, and asarinin was simultaneously determined. Under the same chromatographic conditions, the HPLC fingerprint method for analysis of 15 batches of DSD was established. The content determination of aristolochic acid I, using the same test solution as the content determination item, was performed on an ACQUITY UPLC BEH C_(18) column(2.1 mm×50 mm, 1.7 µm) with methanol(A)-water(including 0.1% formic acid and 5 mmol·L~(-1) ammonium formate)(B) as the mobile phase in a gradient elution mode at the flow rate of 0.4 mL·min~(-1) and the column temperature of 40 ℃ by LC-MS/MS. The MS conditions included electrospray ionization(ESI) as an ion source, positive ion ionization, selective reaction monitoring(SRM), the parent ion of 359.3, and the daughter ion of 297.8. The results of the methodological investigation all met the requirements of content determination/fingerprint determination. As a result, the content ranges of paeoniflorin, liquiritin, cinnamic acid, cinnamaldehyde, ammonium glycyrrhetate, ligustilide, and asarinin were 5.419 8-11.267 3, 1.023-3.669 8, 0.145 6-0.444 1, 0.099 1-0.321 9, 3.159 1-7.731 9, 0.146 4-0.471 7, and 0.237 3-0.401 0 mg·g~(-1), respectively. Twenty-two common peaks were selected and 10 of them were identified by the comparison with the reference substances. The fingerprint similarity of 15 batches of DSD was in the range of 0.91-0.996 and the content of aristolochic acid I in DSD was 300.03-638.13 ng·g~(-1). The method established in this study is reliable and easy to operate and has great practical value, which can be used for overall quality control of substance benchmarks for DSD.

Aristolochic acid I promoted clonal expansion but did not induce hepatocellular carcinoma in adult rats.

Aristolochic acid I (AAI) is a well-known nephrotoxic carcinogen, which is currently reported to be also associated with hepatocellular carcinoma (HCC). Whether AAI is a direct hepatocarcinogen remains controversial. In this study we investigated the association between AAI exposure and HCC in adult rats using a sensitive rat liver bioassay with several cofactors. Formation of glutathione S-transferase placental form-positive (GST-P+) foci was used as the marker for preneoplastic lesions/clonal expansion. We first conducted a medium-term (8 weeks) study to investigate whether AAI had any tumor-initiating or -promoting activity. Then a long-term (52 weeks) study was conducted to determine whether AAI can directly induce HCC. We showed that oral administration of single dose of AAI (20, 50, or 100 mg/kg) in combination with partial hepatectomy (PH) to stimulate liver proliferation did not induce typical GST-P+ foci in liver. In the 8-week study, only high dose of AAI (10 mg · kg-1 · d-1, 5 days a week for 6 weeks) in combination with PH significantly increased the number and area of GST-P+ foci initiated by diethylnitrosamine (DEN) in liver. Similarly, only high dose of AAI (10 mg· kg-1· d-1, 5 days a week for 52 weeks) in combination with PH significantly increased the number and area of hepatic GST-P+ foci in the 52-week study. No any nodules or HCC were observed in liver of any AAI-treated groups. In contrast, long-term administration of AAI (0.1, 1, 10 mg· kg-1· d-1) time- and dose-dependently caused death due to the occurrence of cancers in the forestomach, intestine, and/or kidney. Besides, AAI-DNA adducts accumulated in the forestomach, kidney, and liver in a time- and dose-dependent manner. Taken together, AAI promotes clonal expansion only in the high-dose group but did not induce any nodules or HCC in liver of adult rats till their deaths caused by cancers developed in the forestomach, intestine, and/or kidney. Findings from our animal studies will pave the way for further large-scale epidemiological investigation of the associations between AA and HCC.

Cytotoxicity and genotoxicity of the carcinogen aristolochic acid I (AA-I) in human bladder RT4 cells.

Aristolochic acid (AA-I) induces upper urothelial tract cancer (UUTC) and bladder cancer (BC) in humans. AA-I forms the 7-(2'-deoxyadenosin-N6-yl)aristolactam I (dA-AL-I) adduct, which induces multiple A:T-to-T:A transversion mutations in TP53 of AA-I exposed UTUC patients. This mutation is rarely reported in TP53 of other transitional cell carcinomas and thus recognized as an AA-I mutational signature. A:T-to-T:A transversion mutations were recently detected in bladder tumors of patients in Asia with known AA-I-exposure, implying that AA-I contributes to BC. Mechanistic studies on AA-I genotoxicity have not been reported in human bladder. In this study, we examined AA-I DNA adduct formation and mechanisms of toxicity in the human RT4 bladder cell line. The biological potencies of AA-I were compared to 4-aminobiphenyl, a recognized human bladder carcinogen, and several structurally related carcinogenic heterocyclic aromatic amines (HAA), which are present in urine of smokers and omnivores. AA-I (0.05-10 µM) induced a concentration- and time-dependent cytotoxicity. AA-I (100 nM) DNA adduct formation occurred at over a thousand higher levels than the principal DNA adducts formed with 4-ABP or HAAs (1 µM). dA-AL-I adduct formation was detected down to a 1 nM concentration. Studies with selective chemical inhibitors provided evidence that NQO1 is the major enzyme involved in AA-I bio-activation in RT4 cells, whereas CYP1A1, another enzyme implicated in AA-I toxicity, had a lesser role in bio-activation or detoxification of AA-I. AA-I DNA damage also induced genotoxic stress leading to p53-dependent apoptosis. These biochemical data support the human mutation data and a role for AA-I in BC.

Enteric Oxalate Secretion Mediated by Slc26a6 Defends against Hyperoxalemia in Murine Models of Chronic Kidney Disease.

BACKGROUND: A state of oxalate homeostasis is maintained in patients with healthy kidney function. However, as GFR declines, plasma oxalate (Pox) concentrations start to rise. Several groups of researchers have described augmentation of oxalate secretion in the colon in models of CKD, but the oxalate transporters remain unidentified. The oxalate transporter Slc26a6 is a candidate for contributing to the extrarenal clearance of oxalate via the gut in CKD. METHODS: Feeding a diet high in soluble oxalate or weekly injections of aristolochic acid induced CKD in age- and sex-matched wild-type and Slc26a6-/- mice. qPCR, immunohistochemistry, and western blot analysis assessed intestinal Slc26a6 expression. An oxalate oxidase assay measured fecal and Pox concentrations. RESULTS: Fecal oxalate excretion was enhanced in wild-type mice with CKD. This increase was abrogated in Slc26a6-/- mice associated with a significant elevation in plasma oxalate concentration. Slc26a6 mRNA and protein expression were greatly increased in the intestine of mice with CKD. Raising Pox without inducing kidney injury did not alter intestinal Slc26a6 expression, suggesting that changes associated with CKD regulate transporter expression rather than elevations in Pox. CONCLUSIONS: Slc26a6-mediated enteric oxalate secretion is critical in decreasing the body burden of oxalate in murine CKD models. Future studies are needed to address whether similar mechanisms contribute to intestinal oxalate elimination in humans to enhance extrarenal oxalate clearance.

Co-Exposure to Aristolochic Acids I and II Increases DNA Adduct Formation Responsible for Aristolochic Acid I-Mediated Carcinogenicity in Rats.

The plant extract aristolochic acid (AA), containing aristolochic acids I (AAI) and II (AAII) as major components, causes aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN), unique renal diseases associated with upper urothelial cancer. Recently (Chemical Research in Toxicology 33(11), 2804-2818, 2020), we showed that the in vivo metabolism of AAI and AAII in Wistar rats is influenced by their co-exposure (i.e., AAI/AAII mixture). Using the same rat model, we investigated how exposure to the AAI/AAII mixture can influence AAI and AAII DNA adduct formation (i.e., AA-mediated genotoxicity). Using 32P-postlabelling, we found that AA-DNA adduct formation was increased in the livers and kidneys of rats treated with AAI/AAII mixture compared to rats treated with AAI or AAII alone. Measuring the activity of enzymes involved in AA metabolism, we showed that enhanced AA-DNA adduct formation might be caused partially by both decreased AAI detoxification as a result of hepatic CYP2C11 inhibition during treatment with AAI/AAII mixture and by hepatic or renal NQO1 induction, the key enzyme predominantly activating AA to DNA adducts. Moreover, our results indicate that AAII might act as an inhibitor of AAI detoxification in vivo. Consequently, higher amounts of AAI might remain in liver and kidney tissues, which can be reductively activated, resulting in enhanced AAI DNA adduct formation. Collectively, these results indicate that AAII present in the plant extract AA enhances the genotoxic properties of AAI (i.e., AAI DNA adduct formation). As patients suffering from AAN and BEN are always exposed to the plant extract (i.e., AAI/AAII mixture), our findings are crucial to better understanding host factors critical for AAN- and BEN-associated urothelial malignancy.

Aristolochic acid I: an investigation into the role of food crops contamination, as a potential natural exposure pathway.

Aristolochic acid I (AAI) is a potent nephrotoxic and carcinogenic compound produced by plants of the Aristolochiaceae family and thoroughly investigated as a main culprit in the etiology of Balkan endemic nephropathy (BEN). So far, the AAI exposure was demonstrated to occur through the consumption of Aristolochia clematitis plants as traditional remedies, and through the contamination of the surrounding environment in endemic areas: soil, food and water contamination. Our study investigated for the first time the level of AAI contamination in 141 soil and vegetable samples from two cultivated gardens in non-endemic areas, A. clematitis being present in only one of the gardens. We developed and validated a simple and sensitive ultra-high-performance liquid chromatography-ion trap mass spectrometry method for qualitative and quantitative AAI analysis. The results confirmed the presence of AAI at nanogram levels in soil and vegetable samples collected from the non-endemic garden, where A. clematitis grows. These findings provide additional evidence that the presence of A. clematitis can cause food crops and soil contamination and unveil the pathway through which AAI could move from A. clematitis to other plant species via a common matrix: the soil. Another issue regarding the presence of AAI, in a non-endemic BEN area from Romania, could underlie a more widespread environmental exposure to AAI and explain certain BEN-like cases in areas where BEN has not been initially described.

Defining in vivo dose-response curves for kidney DNA adduct formation of aristolochic acid I in rat, mouse and human by an in vitro and physiologically based kinetic modeling approach.

Aristolochic acid I (AAI) is a well-known genotoxic kidney carcinogen. Metabolic conversion of AAI into the DNA-reactive aristolactam-nitrenium ion is involved in the mode of action of tumor formation. This study aims to predict in vivo AAI-DNA adduct formation in the kidney of rat, mouse and human by translating the in vitro concentration-response curves for AAI-DNA adduct formation to the in vivo situation using physiologically based kinetic (PBK) modeling-based reverse dosimetry. DNA adduct formation in kidney proximal tubular LLC-PK1 cells exposed to AAI was quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry. Subsequently, the in vitro concentration-response curves were converted to predicted in vivo dose-response curves in rat, mouse and human kidney using PBK models. Results obtained revealed a dose-dependent increase in AAI-DNA adduct formation in the rat, mouse and human kidney and the predicted DNA adduct levels were generally within an order of magnitude compared with values reported in the literature. It is concluded that the combined in vitro PBK modeling approach provides a novel way to define in vivo dose-response curves for kidney DNA adduct formation in rat, mouse and human and contributes to the reduction, refinement and replacement of animal testing.

Molecularly imprinted ratiometric fluorescent probe for visual and fluorescent determination of aristolochic acid I based on a Schiff-base fluorescent compound.

A molecularly imprinted ratiometric fluorescent probe (MIRF probe) was synthesized for the determination of aristolochic acid I (AAI) based on the Schiff-base fluorescent compound N,N'-bis(o-carboxybenzylidene)-p-4,4'-diaminobiphenyl (BDDB). The BDDB was immobilized in the silica nanoparticle (BDDB@SiO2) as an internal standard material. The blue-emitting BDDB@SiO2 and the yellow-emitting carbon quantum dots (y-CDs) were wrapped in the molecularly imprinted polymer (MIP) to provide a reliable reference signal at 440 nm and a fluorescent response signal at 530 nm at the excitation wavelength of 365 nm, respectively. In the preparation of the MIP of the MIRF probe, 4-vinylbenzoic acid as the functional monomer and AAI as the template molecule were used. An imprinting factor of 2.25 was obtained. Under the optimum conditions, the fluorescent response signal at 530 nm was quenched gradually by AAI in the range 1.0 to 120.0 µmol/L, while the reference signal at 440 nm remained unchanged. The limit of detection was 0.45 µmol/L, and the fluorescent color of the MIRF probe changed gradually from yellow to green to blue, which illustrated that the developed probe had a specific AAI recognition ability, a good anti-interference ability, and a sensitively visual determination ability. The probe was successfully applied to the AAI determination in traditional Chinese medicine (TCM) Asarum. The results showed that it had satisfactory recoveries (95.5-107.3%) and low relative standard deviations (2.0%). Furthermore, this method has a potential for the onsite naked eye determination of AAI in TCM samples.Graphical abstract.

Untargeted LC-MS-based metabonomics revealed that aristolochic acid I induces testicular toxicity by inhibiting amino acids metabolism, glucose metabolism, ß-oxidation of fatty acids and the TCA cycle in male mice.

As the main toxic component of aristolochic acid, aristolochic acid I (AAI) is primarily found in Aristolochiaceae plants such as Aristolochia, Aristolochia fangchi and Caulis aristolochiae manshuriensis. AAI has been proven to be carcinogenic, mutagenic and nephrotoxic. Although the role of AAI in testicular toxicity has been reported, its mechanism of action is unknown. Using metabonomics and molecular biology techniques, we tried to identify the differential endogenous metabolites of AAI that may affect the changes in testicular function in mice, map the network of metabolic pathways, and systematically reveal the molecular mechanism of AAI-induced testicular toxicity. We found that AAI inhibited amino acid metabolism in mouse testicular cells, impeded the uptake and oxidative decomposition of fatty acids, prevented normal glucose uptake by testicular cells, which inhibited glycolysis and gluconeogenesis, affected the mitochondrial tricarboxylic acid (TCA) cycle, which impaired the ATP energy supply, decreased the number of spermatogenic cells and sperm in the testes, induced changes in the mitochondrial state of spermatogonial cells, and ultimately led to physiological and pathological changes in the testes. AAI also regulated the testicular physiological activity by regulating the androgen receptor and hormone levels. This study used metabonomics and other methods to elucidate the mechanism of AAI-induced testicular toxicity from a new angle.

Isolation of aristolochic acid I from herbal plant using molecular imprinted polymer composited ionic liquid-based zeolitic imidazolate framework-67.

Aristolochic acid I is a toxic compound found in the genus of Aristolochia plants, which are commonly used as herbal cough treatment medicines. To remove the aristolochic acid I in extract efficiently and selectively, a molecularly imprinted polymer composed of ethylimidazole ionic liquid-based zeolitic imidazolate framework-67 was synthesized and used as the adsorbent. Under the conditions optimized by the software design expert, the sorbent showed highest adsorption amount of 34.25 mg/g in methanol/water (95:5, v/v) at 39°C for 138 min. The sorbent was then applied to solid phase extraction to isolate aristolochic acid I from the extract of the herbal plant Fibraurea Recisa Pierre. 0.043 mg/g of aristolochic acid I was obtained after the loading, washing, and elution processes. The limit of detection of 2.41 × 10-5  mg/mL and good recoveries provided evidence for the accuracy of this method.

The impact of p53 on aristolochic acid I-induced nephrotoxicity and DNA damage in vivo and in vitro.

Exposure to aristolochic acid (AA) is associated with human nephropathy and urothelial cancer. The tumour suppressor TP53 is a critical gene in carcinogenesis and frequently mutated in AA-induced urothelial tumours. We investigated the impact of p53 on AAI-induced nephrotoxicity and DNA damage in vivo by treating Trp53(+/+), Trp53(+/-) and Trp53(-/-) mice with 3.5 mg/kg body weight (bw) AAI daily for 2 or 6 days. Renal histopathology showed a gradient of intensity in proximal tubular injury from Trp53(+/+) to Trp53(-/-) mice, especially after 6 days. The observed renal injury was supported by nuclear magnetic resonance (NMR)-based metabonomic measurements, where a consistent Trp53 genotype-dependent trend was observed for urinary metabolites that indicate aminoaciduria (i.e. alanine), lactic aciduria (i.e. lactate) and glycosuria (i.e. glucose). However, Trp53 genotype had no impact on AAI-DNA adduct levels, as measured by 32P-postlabelling, in either target (kidney and bladder) or non-target (liver) tissues, indicating that the underlying mechanisms of p53-related AAI-induced nephrotoxicity cannot be explained by differences in AAI genotoxicity. Performing gas chromatography-mass spectrometry (GC-MS) on kidney tissues showed metabolic pathways affected by AAI treatment, but again Trp53 status did not clearly impact on such metabolic profiles. We also cultured primary mouse embryonic fibroblasts (MEFs) derived from Trp53(+/+), Trp53(+/-) and Trp53(-/-) mice and exposed them to AAI in vitro (50 µM for up to 48 h). We found that Trp53 genotype impacted on the expression of NAD(P)H:quinone oxidoreductase (Nqo1), a key enzyme involved in AAI bioactivation. Nqo1 induction was highest in Trp53(+/+) MEFs and lowest in Trp53(-/-) MEFs; and it correlated with AAI-DNA adduct formation, with lowest adduct levels being observed in AAI-exposed Trp53(-/-) MEFs. Overall, our results clearly demonstrate that p53 status impacts on AAI-induced renal injury, but the underlying mechanism(s) involved remain to be further explored. Despite the impact of p53 on AAI bioactivation and DNA damage in vitro, such effects were not observed in vivo.

The Impact of p53 on Aristolochic Acid I-Induced Gene Expression In Vivo.

Exposure to aristolochic acid (AA) is linked to kidney disease and urothelial cancer in humans. The major carcinogenic component of the AA plant extract is aristolochic acid I (AAI). The tumour suppressor p53 is frequently mutated in AA-induced tumours. We previously showed that p53 protects from AAI-induced renal proximal tubular injury, but the underlying mechanism(s) involved remain to be further explored. In the present study, we investigated the impact of p53 on AAI-induced gene expression by treating Trp53(+/+), Trp53(+/-), and Trp53(-/-) mice with 3.5 mg/kg body weight (bw) AAI daily for six days. The Clariom™ S Assay microarray was used to elucidate gene expression profiles in mouse kidneys after AAI treatment. Analyses in Qlucore Omics Explorer showed that gene expression in AAI-exposed kidneys is treatment-dependent. However, gene expression profiles did not segregate in a clear-cut manner according to Trp53 genotype, hence further investigations were performed by pathway analysis with MetaCore™. Several pathways were significantly altered to varying degrees for AAI-exposed kidneys. Apoptotic pathways were modulated in Trp53(+/+) kidneys; whereas oncogenic and pro-survival pathways were significantly altered for Trp53(+/-) and Trp53(-/-) kidneys, respectively. Alterations of biological processes by AAI in mouse kidneys could explain the mechanisms by which p53 protects from or p53 loss drives AAI-induced renal injury in vivo.

The potential role of aquaporin 1 on aristolochic acid I induced epithelial mesenchymal transition on HK-2 cells.

Aristolochic acid I (AA-I), one of the main active components in Aristolochaia herbs, may induce aristolochic acid nephropathy (AAN). Renal interstitial fibrosis is one of the most typical features of AAN. To investigate the mechanism of Aristolochic acid I (AA-I) -induced renal epithelial-mesenchymal transition (EMT) and determine the role of aquaporin-1 (AQP1) in this process, we established an AA-I-induced EMT model in human proximal tubular epithelial cells (HK-2 cells). Morphological examination, MTT assay, and Western blot analysis were performed. Aquaporin 1 (AQP1) and several EMT-related proteins were detected, thereby suggesting the occurrence of AA-I-induced EMT. Two main pathways of transforming growth factor-ß (TGF-ß) signaling, namely, Smad-dependent and Smad-independent signaling pathways, were also detected. The results showed that the TGF-ß / Smad-independent signaling pathways (ß-catenin, Ras-Raf-Erk1/2 signaling pathways) were activated, and AQP1 expression was decreased during the AA-I induced EMT on HK-2 cells. With the presence of TGF-ß1 receptor inhibitor (LY364947) and Erk1/2 inhibitor (PD98059), AQP1 expression was altered by PD98059, suggested that AQP1 could be adjusted by Erk1/2 signaling. Moreover, the inhibitory effect of AA-I on AQP1 was stronger than that of TGF-ß1, suggested that AQP1 may be an important target on AAN clinical therapy.

Selective analysis of aristolochic acid I in herbal medicines by dummy molecularly imprinted solid-phase extraction and HPLC.

In this study, surface molecularly imprinted polymers were prepared as the selective sorbents for separation of aristolochic acid I in herbal medicine extracts by a facile approach. A less toxic dummy template, ofloxacin, was used to create specific molecule recognition sites for aristolochic acid I in the synthesized polymers. The polymers were characterized by Fourier-transfer infrared spectroscopy, scanning electron microscopy, thermogravimetric analysis, elemental analysis, and nitrogen adsorption-desorption test. The adsorption capacity was calculated using adsorption kinetics, selectivity, and recycling experiments. The obtained polymers exhibited high thermostability, fast equilibrium time, and excellent binding ability. Subsequently, the polymers applied as the solid-phase extraction absorbent was proposed and used for the enrichment and analysis of aristolochic acid I in herbal plants. The result showed that the aristolochic acid I was enriched up to 16 times after analysis by using high-performance liquid chromatography. The good linearity for aristolochic acid I was obtained in the range of 0.1-200 µg/mL (R2  = 0.9987). The recovery and precision values were obtained (64.94-77.73%, RSDs% ≤ 0.8%, n = 3) at three spiked concentration levels. This work provided a promising method for selective enrichment, extraction, and purification of aristolochic acid I from complex herbal plants.

Plasma-specific microRNA response induced by acute exposure to aristolochic acid I in rats.

Aristolochic acid I (AAI) derived from a natural herbal alkaloid is a nephrotoxicant. AAI-induced acute kidney injury (AKI), a devastating clinical disease associated with high mortality rates, is difficult for early diagnosis. To address this issue, we identified and validated early-detection biomarkers for AAI-induced acute kidney injury via profiling microRNA expression in rats. Global miRNA expression profile analysis found that 21 miRNAs were significantly dysregulated in kidney of rats treated by 40 mg/kg AAI on day 2, day 4, or day 6, among which 5 miRNAs were upregulated at all three time points. Quantitative RT-PCR confirmed that miR-21-3p on day 4 and day 6 was obviously upregulated in kidney of rats treated by 40 mg/kg AAI. Further examination found that miR-21-3p was increased in plasma early on day 2 in 10 mg/kg AAI-treated rats, but not in non-target organs. Importantly, the elevation of plasma miR-21-3p preceded the increase in blood urea nitrogen and creatinine, and the presence of renal tubular injury, characterized by differential increase before and after the presence of renal tubular lesions. Our findings thus show that miRNA expression is upregulated in kidney and plasma of AKI rat induced by AAI, and plasma miR-21-3p may be served as a new potential biomarker for early diagnosing AAI-induced acute kidney injury in rats, and possibly in humans.