No Incidence of Liver Cancer Was Observed in A Retrospective Study of Patients with Aristolochic Acid Nephropathy.

OBJECTIVE: To assess the risk of aristolochic acid (AA)-associated cancer in patients with AA nephropathy (AAN). METHODS: A retrospective study was conducted on patients diagnosed with AAN at Peking University First Hospital from January 1997 to December 2014. Long-term surveillance and follow-up data were analyzed to investigate the influence of different factors on the prevalence of cancer. The primary endpoint was the incidence of liver cancer, and the secondary endpoint was the incidence of urinary cancer during 1 year after taking AA-containing medication to 2014. RESULTS: A total of 337 patients diagnosed with AAN were included in this study. From the initiation of taking AA to the termination of follow-up, 39 patients were diagnosed with cancer. No cases of liver cancer were observed throughout the entire follow-up period, with urinary cancer being the predominant type (34/39, 87.17%). Logistic regression analysis showed that age, follow-up period, and diabetes were potential risk factors, however, the dosage of the drug was not significantly associated with urinary cancer. CONCLUSIONS: No cases of liver cancer were observed at the end of follow-up. However, a high prevalence of urinary cancer was observed in AAN patients. Establishing a direct causality between AA and HCC is challenging.

No Incidence of Liver Cancer Was Observed in A Retrospective Study of Patients with Aristolochic Acid Nephropathy / 中国结合医学杂志

OBJECTIVE@#To assess the risk of aristolochic acid (AA)-associated cancer in patients with AA nephropathy (AAN).@*METHODS@#A retrospective study was conducted on patients diagnosed with AAN at Peking University First Hospital from January 1997 to December 2014. Long-term surveillance and follow-up data were analyzed to investigate the influence of different factors on the prevalence of cancer. The primary endpoint was the incidence of liver cancer, and the secondary endpoint was the incidence of urinary cancer during 1 year after taking AA-containing medication to 2014.@*RESULTS@#A total of 337 patients diagnosed with AAN were included in this study. From the initiation of taking AA to the termination of follow-up, 39 patients were diagnosed with cancer. No cases of liver cancer were observed throughout the entire follow-up period, with urinary cancer being the predominant type (34/39, 87.17%). Logistic regression analysis showed that age, follow-up period, and diabetes were potential risk factors, however, the dosage of the drug was not significantly associated with urinary cancer.@*CONCLUSIONS@#No cases of liver cancer were observed at the end of follow-up. However, a high prevalence of urinary cancer was observed in AAN patients. Establishing a direct causality between AA and HCC is challenging.

Aristolochic acid-containing Chinese herbal medicine and upper urinary tract urothelial carcinoma in Taiwan: a narrative review.

PURPOSE: The high incidence of upper urinary tract urothelial carcinoma (UTUC) in Taiwan is largely due to exposure to aristolochic acid (AA), a principal component of Aristolochia-based herbal medicines. Here we systematically review the molecular epidemiology, clinical presentation and biomarkers associated with AA-induced UTUC. METHODS: This is a narrative review. Medline, Embase, and Web of Science were searched from inception to December 31, 2021. Studies evaluating the association, detection, and clinical characteristics of AA and UTUC were included. RESULTS: A nationwide database revealed 39% of the Taiwanese population had been exposed to AA-containing herbs between 1997 and 2003. Epidemiological reports revealed AA posed a significantly higher hazard for renal failure and UTUC in herbalists and the general population who ingested AA-containing herbs. The presence of aristolactam-DNA adducts and a distinctive signature mutation, A:T to T:A transversions, located predominantly on the non-transcribed DNA strand, with a strong preference for deoxyadenosine in a consensus sequence (CAG), was observed in many UTUC patients. Clinically, AA-related UTUC patients were characterized by a younger age, female gender, impaired renal function and recurrence of contralateral UTUC. To date, there are no preventive measures, except prophylactic nephrectomy, for subjects at risk of AA nephropathy or AA-related UTUC. CONCLUSION: AA exposure via Aristolochia-based herbal medicines is a problem throughout Taiwan, resulting in a high incidence of UTUC. Aristolactam-DNA adducts and a distinctive signature mutation, A:T to T:A transversions, can be used as biomarkers to identify AA-related UTUC. AA-related UTUC is associated with a high recurrence rate of contralateral UTUC.

Molecular profiles and urinary biomarkers of upper tract urothelial carcinomas associated with aristolochic acid exposure.

Recurrent upper tract urothelial carcinomas (UTUCs) arise in the context of nephropathy linked to exposure to the herbal carcinogen aristolochic acid (AA). Here we delineated the molecular programs underlying UTUC tumorigenesis in patients from endemic aristolochic acid nephropathy (AAN) regions in Southern Europe. We applied an integrative multiomics analysis of UTUCs, corresponding unaffected tissues and of patient urines. Quantitative microRNA (miRNA) and messenger ribonucleic acid (mRNA) expression profiling, immunohistochemical analysis by tissue microarrays and exome and transcriptome sequencing were performed in UTUC and nontumor tissues. Urinary miRNAs of cases undergoing surgery were profiled before and after tumor resection. Ribonucleic acid (RNA) and protein levels were analyzed using appropriate statistical tests and trend assessment. Dedicated bioinformatic tools were used for analysis of pathways, mutational signatures and result visualization. The results delineate UTUC-specific miRNA:mRNA networks comprising 89 miRNAs associated with 1,862 target mRNAs, involving deregulation of cell cycle, deoxyribonucleic acid (DNA) damage response, DNA repair, bladder cancer, oncogenes, tumor suppressors, chromatin structure regulators and developmental signaling pathways. Key UTUC-specific transcripts were confirmed at the protein level. Exome and transcriptome sequencing of UTUCs revealed AA-specific mutational signature SBS22, with 68% to 76% AA-specific, deleterious mutations propagated at the transcript level, a possible basis for neoantigen formation and immunotherapy targeting. We next identified a signature of UTUC-specific miRNAs consistently more abundant in the patients' urine prior to tumor resection, thereby defining biomarkers of tumor presence. The complex gene regulation programs of AAN-associated UTUC tumors involve regulatory miRNAs prospectively applicable to noninvasive urine-based screening of AAN patients for cancer presence and recurrence.

Probing the Hidden Role of Mitochondrial DNA Damage and Dysfunction in the Etiology of Aristolochic Acid Nephropathy.

Aristolochic acid nephropathy (AAN) is a unique type of progressive renal interstitial fibrotic disease caused by prolonged exposure to aristolochic acids (AAs) through AA-containing herbal medicines or AA-tainted food. Despite decades of research and affecting millions of people around the world, the pathophysiology of AAN remains incompletely understood. In this study, we tested the potential causative role of mitochondrial dysfunction in AAN development. Our findings revealed AA exposure induces an exposure concentration and duration dependent lowering of adenosine triphosphate in both cultured human kidney and liver cells, highlighting an AA exposure effect on mitochondrial energy production in the kidney and liver, which both are highly metabolically active and energy-demanding organs. Analysis with liquid chromatography-tandem mass spectrometry coupled with stable isotope dilution method detected high levels of mutagenic 8-oxo-2'-deoxyguanosine and 7-(deoxyadenosine-N6-yl)-aristolactam adduct on mitochondrial DNA isolated from AA-treated cells, unmasking a potentially important causative, but previously unknown role of mitochondrial DNA mutation in the pathophysiology of AAN development.

Aristolochic acid nephropathy: A scientometric analysis of literature published from 1971 to 2019.

BACKGROUND: Aristolochic acid nephropathy (AAN) is a type of drug-induced nephropathy that may result in acute kidney injury and is associated with a potentially progressive course of kidney fibrosis and upper tract urothelial carcinoma. Aristolochic acids (AAs) are a group of toxins commonly present in plants of the genera Aristolochia and Asarum, which are found worldwide. AAN still occurs in Asian and Balkan regions. The progressive lesions and mutational events initiated by AAs are irreversible, and no effective therapeutic regimen for AAN has been established. Furthermore, more people are at risk of this disease due to casual exposure to AAs. This study performed a scientometric analysis of global research literature focusing on AAN. METHODS: The Web of Science database was searched to identify all publications pertaining to "aristolochic acid nephropathy" or "Balkan endemic nephropathy" using these terms as key words to search the literature from 1971 to 2019. The collected data included the document type, author, journal, publication year, citation reports, and country of publication, and were analyzed using the VOSviewer software. RESULTS: A total of 1251 records were initially obtained. Publication types, including "meeting abstract," "letter," "editorial material," and "proceedings paper" were excluded, which left 1083 publications comprising 923 articles and 160 reviews. English was the predominant language of the publications. China had the most number of articles published with 217 (20.0%), followed by the USA with 186 articles (17.2%), and Germany with 138 articles (12.7%). Kidney International, Food and Chemical Toxicology, and Toxins were the 3 most active journals in publishing articles related to AAN. The total number of citations received by all publications was 39,970, with an average of 36.91 citations per article (range: 0-1769). The literature mainly focused on apoptosis, oxidative stress, and inflammation in AAN. CONCLUSION: This study indicated that AAN is a significant topic in nephrology research, as shown by the large number of publications. The literature has mainly focused on the mechanisms of AA-induced nephropathy.

Proteome-wide analysis of protein alterations in response to aristolochic acids in rat kidney and liver tissues.

Aristolochic acids (AAs), nephrotoxic components of herbs, have been previously demonstrated to cause DNA damage by forming DNA-AA adducts. However, the changes of tissue proteome profiles revealing AA toxicity need to be further studied. We conducted a proteomic study on the kidney and liver tissues of AA treated rats by a shotgun proteomics approach coupled with LC-MS/MS technology. A total of 1543 and 1641 proteins were identified and quantified in the kidneys and liver. Due to AA dosage, 10 and 4 proteins significantly changed in kidneys and the liver after multiple testing correction. Pathway enrichment analysis results were variant in kidneys and the liver. The enrichment analysis of metabolic pathways showed that gene expression and protein biosynthesis disorders were the common causes of AA toxicity to organs. Biological processes that positively responded to AAs in the liver probably have a detoxification function. SEC14-like protein 2 and synaptic vesicle membrane protein VAT-1 homolog were the mostly downregulated proteins in the liver and kidneys respectively.

Protective effect of panaxydol against repeated administration of aristolochic acid on renal function and lipid peroxidation products via activating Keap1-Nrf2/ARE pathway in rat kidney.

Panaxydol (PX), a polyacetylenic compound isolated from the roots of Panax notoginseng, is found to possess various biological functions. However, its protective effects against aristolochic acid (AA)-induced renal injury have not been elucidated yet. The present study was undertaken to elucidate the renoprotective effect of PX on Wistar male rats via activating Keap1-Nrf2/ARE pathway. Experimental animals were randomized into four groups, such as control group, I/R group, AA (5 mg/kg/d; ip for 10 days), and AA-induced rats treated with PX (10 and 20 mg/kg/d; po for 20 days). At the end of the experimental period, the rats were killed, and the biochemical parameters denoting renal functions were evaluated; histological analysis displaying the renal tissue architecture, real-time quantitative reverse-transcription polymerase chain reaction, and immunohistochemistry (IHC) analysis of Keap1-Nrf2/ARE genes were elucidated. The results demonstrated that the rats administered with AA displayed a significant increase in the blood urea nitrogen level with an increased urine creatinine and protein excretion. Also, the serum levels of urea, uric acid, and albumin levels were increased. Furthermore, the histological evaluation denoted the cellular degeneration with increased tissue lipid peroxidation levels. In contrast, rats administered with PX significantly prevented the tissue degeneration with improved antioxidant levels. Conversely, PX treatment increased the messenger RNA expression of Nrf2, NQO1, HO-1 with an attenuated expression of 4HNE and NOX-4 levels in IHC analysis. Thus, the results of the present study suggest that PX could suppress AA-induced renal failure by suppressing oxidative stress through the activation of Keap1-Nrf2 signaling pathway.

The renoprotective effect of diosgenin on aristolochic acid I-induced renal injury in rats: impact on apoptosis, mitochondrial dynamics and autophagy.

Aristolochic acid I (AA-I) remains a leading cause of aristolochic acid nephropathy (AAN), however few prevention and treatment strategies exist. In this work, the nephroprotective effect of diosgenin, a steroidal saponin distributed abundantly in several plants, on AA-I-induced renal injury and its underlying mechanism were investigated. Sprague-Dawley rats were intragastrically administered with 30 mg kg-1 d-1 diosgenin two hours before exposure to 10 mg kg-1 d-1 AA-I for consecutive four weeks, and the histological change, the renal and liver function, apoptosis, autophagy and the involved pathways were investigated. The results showed that diosgenin relieved AA-I-induced renal histological damage, including mild edematous disorder of renal tubular arrangement and widening of renal tubular lumen. No obvious changes in the hepatic tissue structure were observed in all treatment groups. Moreover, diosgenin up-regulated the expression of Bcl-2 and down-regulated Bax, and subsequently inhibited AIF expression and the cleaved form of Caspase-3, thereby alleviating apoptosis triggered by AA-I. Diosgenin also mitigated AA-I-induced renal mitochondrial dynamics disorder by increasing the expression of mitochondrial dynamics-related proteins including DRP1 and MFN2. Diosgenin inhibited AA-I-evoked autophagy via ULK1-mediated inhibition of the mTOR pathway. Overall, these results suggest that diosgenin has a protective effect against AA-I-induced renal damage and it may be a potential agent for preventing AA-I-induced AAN.

Determination of Aristolochic Acids in Vegetables: Nephrotoxic and Carcinogenic Environmental Pollutants Contaminating a Broad Swath of the Food Supply and Driving Incidence of Balkan Endemic Nephropathy.

Balkan endemic nephropathy (BEN) is a slowly progressive interstitial fibrotic disease affecting numerous people living along the Danube River in the Balkan Peninsula, of which aristolochic acids (AAs) produced naturally in Aristolochia plants are key etiological agents. However, the exposure biology of the disease remains poorly understood. Initially, the high incidence of BEN in the Balkan Peninsula was thought to occur through ingestion of bread prepared from flour made with wheat grains comingled with the seeds of Aristolochia clematitis L., an AA-containing weed that grows abundantly in the wheat fields of the affected areas. In this study, by a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, we show for the first time that vegetables, in particular root vegetables of endemic areas, are extensively contaminated with AAs taken up through root absorption from the AA-tainted soil. Furthermore, we found a pH dependence of the n-octanol/water partition coefficient (Kow) of AAs, which resulted in a dramatically higher hydrophobicity-driven plant uptake efficiency of AAs into food crops in endemic areas, characterized by higher acidity levels, compared to non-endemic areas. We believe the results of this study have significantly unraveled the mystery surrounding the uneven distribution of BEN incidence.

Clinicopathologic characteristics and prognosis of upper tract urothelial carcinoma complicated with aristolochic acid nephropathy after radical nephroureterectomy.

BACKGROUND: The purpose of this study was to identify the clinicopathologic characteristics and prognosis of upper tract urothelial carcinoma (UTUC) patients complicated with aristolochic acid nephropathy(AAN) after radical nephroureterectomy (RNU). METHODS: The clinical data of 42 UTUC patients with AAN (AAN group) and 238 UTUC patients without AAN (Non-AAN group) were retrospectively reviewed. All patients received a RNU with excision of bladder cuff. Demographic and clinical data, including preoperative indexes, intraoperative indexes and surgical outcomes were compared. RESULTS: There were no significant differences in age, tumor location, surgery approach, tumor pathologic grade, stage, the mean operative time and estimated blood loss between the two groups (all p > 0.05). There were more female patients in the AAN group (p < 0.001), and 57.1% were high grade tumors. The AAN group showed a higher complications rate (p = 0.003). The median follow-up time was 43.2 months. The AAN group showed a worse estimated 5-year overall survival rate (35.1% vs. 63.0%, p = 0.014), however, no significant difference was found between the two groups with regard to disease specific survival (63.5% vs. 81.5%, p = 0.091). Multivariate binary logistic regression analysis showed that AAN was an independent factor related with overall and disease specific survival. 38.9% of all patients experienced any types of recurrence, and the estimated 5-year recurrence-free survival rate was lower in the AAN group (37.1% vs. 63.7%, p = 0.001). In the comparison of subgroups stratified by recurrence type, the AAN group had a higher intravesical (p = 0.030) and contralateral recurrence rate (p = 0.040). CONCLUSION: UTUC with AAN occurred more frequently in female patients who were more likely to develop high-grade tumors. However, these patients showed a worse overall survival and a lower recurrence-free survival rate than the other patients. AA-related UTUC might be associate with an increased risk of intravesical and contralateral recurrence after RUN.

Biotransformation and Toxicities of Aristolochic Acids.

Environmental and iatrogenic exposures contribute significantly to human diseases, including cancer. The list of known human carcinogens has recently been extended by the addition of aristolochic acids (AAs). AAs occur primarily in Aristolochia herbs, which are used extensively in folk medicines, including Traditional Chinese Medicine. Ingestion of AAs results in chronic renal disease and cancer. Despite importation bans imposed by certain countries, herbal remedies containing AAs are readily available for purchase through the internet. With recent advancements in mass spectrometry, next generation sequencing, and the development of integrated organs-on-chips, our knowledge of cancers associated with AA exposure, and of the mechanisms involved in AA toxicities, has significantly improved. DNA adduction plays a central role in AA-induced cancers; however, significant gaps remain in our knowledge as to how cellular enzymes promote activation of AAs and how the reactive species selectively bind to DNA and kidney proteins. In this review, I describe pathways for AAs biotransformation, adduction, and mutagenesis, emphasizing novel methods and ideas contributing to our present understanding of AA toxicities in humans.

Toward clinical understanding of aristolochic acid upper-tract urothelial carcinoma.

A cluster of patients poisoned by herbal medicine in the 1990s revealed that aristolochic acid (AA) causes kidney failure and upper tract urothelial carcinoma (UTUC). Recent research demonstrated that this was not an isolated incident; on the contrary, AA exposure is widespread in East Asia. This editorial highlights research by Lu and colleagues that investigates clinical characteristics of AA and non-AA UTUCs from 90 patients in Beijing based on the AA mutational signature. The study also detected AA mutations in non-tumor tissue of AA exposed patients and showed that AA mutations can be detected in urine, which might form the basis for non-invasive tests for AA exposure.

Aristolochic acid mutational signature defines the low-risk subtype in upper tract urothelial carcinoma.

Rationale: Dietary exposure to aristolochic acids and similar compounds (collectively, AA) is a significant risk factor for nephropathy and subsequent upper tract urothelial carcinoma (UTUC). East Asian populations, who have a high prevalence of UTUC, have an unusual genome-wide AA-induced mutational pattern (COSMIC signature 22). Integrating mutational signature analysis with clinicopathological information may demonstrate great potential for risk ranking this UTUC subtype. Methods: We performed whole-genome sequencing (WGS) on 90 UTUC Chinese patients to extract mutational signatures. Genome sequencing data for urinary cell-free DNA from 26 UTUC patients were utilized to noninvasively identify the mutational signatures. Genome sequencing for primary tumors on 8 out of 26 patients was also performed. Metastasis-free survival (MFS) and cancer-specific survival (CSS) were measured using Kaplan-Meier methods. Results: Data analysis showed that a substantial proportion of patients harbored the AA mutational signature and were associated with AA-containing herbal drug intake, female gender, poor renal function, and multifocality. Field cancerization was found to partially contribute to multifocality. Nevertheless, AA Sig subtype UTUC patients exhibited favorable outcomes of CSS and MFS compared to the No-AA Sig subtype. Additionally, AA Sig subtype patients showed a higher tumor mutation burden, higher numbers of predicted neoantigens, and infiltrating lymphocytes, suggesting the potential for immunotherapy. We also confirmed the AA signature in AA-treated human renal tubular HK-2 cells. Notably, the AA subtype could be ascertained using a clinically applicable sequencing strategy (low coverage) in both primary tumors and urinary cell-free DNA as a basis for therapy selection. Conclusion: The AA mutational signature as a screening tool defines low-risk UTUC with therapeutic relevance. The AA mutational signature, as a molecular prognostic marker using either ureteroscopy and/or urinary cell-free DNA, is especially useful for diagnostic uncertainty when kidney-sparing treatment and/or immune checkpoint inhibitor therapy were considered.

Drug-Related Carcinogenesis: Risk Factors and Approaches for Its Prevention.

The review summarizes the data on the role of metabolic and repair systems in the mechanisms of therapy-related carcinogenesis and the effect of their polymorphism on the cancer development risk. The carcinogenic activity of different types of drugs, from the anticancer agents to analgesics, antipyretics, immunomodulators, hormones, natural remedies, and non-cancer drugs, is described. Possible approaches for the prevention of drug-related cancer induction at the initiation and promotion stages are discussed.

Hirsutella sinensis inhibits NLRP3 inflammasome activation to block aristolochic acid-induced renal tubular epithelial cell transdifferentiation.

In recent years, kidney damage caused by ingestion of Chinese medicinal herbs containing Aristolochic acid (AA) has attracted extensive attention. However, whether the nephrotoxicity of AA is related to NLRP3 inflammasome has not been reported. Hirsutella sinensis (HS) has a certain therapeutic effect on aristolochic acid nephropathy (AAN) and is related to NLRP3 inflammasome. Therefore, this study explores whether HS plays a role in renal injury induced by AA through NLRP3 inflammasome pathway. AA-stimulated renal tubular epithelial cells showed that AA could promote the expression of NLRP3, ASC, and α-SMA, increase the secretion and expression of caspase-1, IL-1ß, and IL-18, and inhibit the expression of E-cadherin in a dose- and time-dependent manner. When NLRP3 was down-regulated, the expression of α-SMA and E-cadherin did not change significantly, but significantly blocked the regulation of α-SMA and E-cadherin expression by AA. When AA and HS were added to renal tubular epithelial cells at the same time, the effects of AA on the expression of NLRP3, ASC, caspase-1, IL-1ß, IL-18, and α-SMA gradually decreased to the level of control group with the increase of HS dosage. At the same time, HS can reduce the transdifferentiation of renal tubular epithelial cells by inhibiting the activation of NLRP3 inflammasome. These findings will provide important pharmacological references for the treatment of AAN and the clinical application of HS.

Protective Effects of Melatonin Against Aristolochic Acid-Induced Nephropathy in Mice.

Melatonin, a pineal hormone, is well known to regulate the sleep-wake cycle. Besides, the hormone has been shown to display pleiotropic effects arising from its powerful anti-oxidant and anti-inflammatory activities. Recent studies have reported that melatonin exerts protective effects in animal models of kidney disease. However, the potential effects of melatonin on aristolochic acid (AA)-induced nephropathy (AAN) have not yet been investigated. Here, we found that the administration of melatonin ameliorated AA-induced renal dysfunction, as evidenced by decreased plasma levels of blood urea nitrogen and creatinine and histopathological abnormalities such as tubular dilatation and cast formation. The upregulation of tubular injury markers after AA injection was reversed by melatonin. Melatonin also suppressed AA-induced oxidative stress, as evidenced by the downregulation of 4-hydroxynonenal and reduced level of malondialdehyde, and modulated expression of pro-oxidant and antioxidant enzymes. In addition, p53-dependent apoptosis of tubular epithelial cells, infiltration of macrophages and CD4+ T cells into damaged kidneys, and renal expression of cytokines and chemokines were inhibited by melatonin. Moreover, melatonin attenuated AA-induced tubulointerstitial fibrosis through suppression of the tumor growth factor-ß/Smad signaling pathway. These results suggest that melatonin might be a potential therapeutic agent for AAN.