Aristolochic Acid Nephropathy Research Articles

Delineation of renal protein profiles in aristolochic acid I-induced nephrotoxicity in mice by label-free quantitative proteomics

Introduction: Aristolochic acid nephropathy (AAN) is a kidney injury syndrome caused by aristolochic acids exposure. Our study used label-free quantitative proteomics to delineate renal protein profiles and identify key proteins after exposure to different doses of aristolochic acid I (AAI).Methods: Male C57BL/6 mice received AAI (1.25 mg/kg/d, 2.5 mg/kg/d, or 5 mg/kg/d) or vehicle for 5 days.Results and discussion: The results showed that AAI induced dose-dependent nephrotoxicity. Differences in renal protein profiles between the control and AAI groups increased with AAI dose. Comparing the control with the low-, medium-, and high-dose AAI groups, we found 58, 210, and 271 differentially expressed proteins, respectively. Furthermore, protein-protein interaction network analysis identified acyl-CoA synthetase medium-chain family member 3 (Acsm3), cytochrome P450 family 2 subfamily E member 1 (Cyp2e1), microsomal glutathione S-transferase 1 (Mgst1), and fetuin B (Fetub) as the key proteins. Proteomics revealed that AAI decreased Acsm3 and Cyp2e1 while increasing Mgst1 and Fetub expression in mice kidneys, which was further confirmed by Western blotting. Collectively, in AAI-induced nephrotoxicity, renal protein profiles were dysregulated and exacerbated with increasing AAI dose. Acsm3, Cyp2e1, Mgst1, and Fetub may be the potential therapeutic targets for AAN.

Integrative informatics analysis identifies that ginsenoside Re improves renal fibrosis through regulation of autophagy.

We previously demonstrated that ginsenoside Re (G-Re) has protective effects on acute kidney injury. However, the underlying mechanism is still unclear. In this study, we conducted a meta-analysis and pathway enrichment analysis of all published transcriptome data to identify differentially expressed genes (DEGs) and pathways of G-Re treatment. We then performed in vitro studies to measure the identified autophagy and fibrosis markers in HK2 cells. In vivo studies were conducted using ureteric obstruction (UUO) and aristolochic acid nephropathy (AAN) models to evaluate the effects of G-Re on autophagy and kidney fibrosis. Our informatics analysis identified autophagy-related pathways enriched for G-Re treatment. Treatment with G-Re in HK2 cells reduced autophagy and mRNA levels of profibrosis markers with TGF-β stimulation. In addition, induction of autophagy with PP242 neutralized the anti-fibrotic effects of G-Re. In murine models with UUO and AAN, treatment with G-Re significantly improved renal function and reduced the upregulation of autophagy and profibrotic markers. A combination of informatics analysis and biological experiments confirmed that ginsenoside Re could improve renal fibrosis and kidney function through the regulation of autophagy. These findings provide important insights into the mechanisms of G-Re's protective effects in kidney injuries.

LRG1 Contributes to the Pathogenesis of Multiple Kidney Diseases: A Comprehensive Review

<b><i>Background:</i></b> The increasing prevalence of kidney diseases has become a significant public health issue, with a global prevalence exceeding 10%. In order to accurately identify biochemical changes and treatment outcomes associated with kidney diseases, novel methods targeting specific genes have been discovered. Among these genes, leucine-rich α-2 glycoprotein 1 (LRG1) has been identified to function as a multifunctional pathogenic signaling molecule in multiple diseases, including kidney diseases. This study aims to provide a comprehensive overview of the current evidence regarding the roles of LRG1 in different types of kidney diseases. <b><i>Summary:</i></b> Based on a comprehensive review, it was found that LRG1 was upregulated in the urine, serum, or renal tissues of patients or experimental animal models with multiple kidney diseases, such as diabetic nephropathy, kidney injury, IgA nephropathy, chronic kidney diseases, clear cell renal cell carcinoma, end-stage renal disease, canine leishmaniosis-induced kidney disease, kidney fibrosis, and aristolochic acid nephropathy. Mechanistically, the role of LRG1 in kidney diseases is believed to be detrimental, potentially through its regulation of various genes and signaling cascades, i.e., fibronectin 1, GPR56, vascular endothelial growth factor (VEGF), VEGFR-2, death receptor 5, GDF15, HIF-1α, SPP1, activin receptor-like kinase 1-Smad1/5/8, NLRP3-IL-1b, and transforming growth factor β pathway. <b><i>Key Messages:</i></b> Further research is needed to fully comprehend the molecular mechanisms by which LRG1 contributes to the pathogenesis and pathophysiology of kidney diseases. It is anticipated that targeted treatments focusing on LRG1 will be utilized in clinical trials and implemented in clinical practice in the future.

Aristolochic acid induces acute kidney injury through ferroptosis.

Aristolochic acid (AA)-induced acute kidney injury (AKI) presents with progressive decline in renal function and rapid progression to end-stage renal disease. Among the multiple mechanisms identified in AKI, ferroptosis has been shown to be involved in various forms of AKI. But few studies have elucidated the role of ferroptosis in AA-induced AKI. In this study, we investigated the role of ferroptosis in AA-induced acute renal tubular injury in vivo and in vitro. Mice with acute aristolochic acid nephropathy showed increased malondialdehyde levels, aggravated lipid peroxidation, decreased superoxide dismutase activity, and glutathione depletion. The expression of glutathione peroxidase 4 was decreased and the expression of acyl-CoA synthetase long-chain family member 4 was increased. Inhibition of ferroptosis by ferrostatin-1 significantly improved the renal function, reduced histopathological lesions, partially alleviated lipid peroxidation, and restored the antioxidant capacity. In vitro studies also revealed that AA significantly reduced cell viability, induced reactive oxygen species production, increased intracellular iron level and decreased ferroptosis-related protein expression. Inhibition of ferroptosis significantly increased cell viability and attenuated AA-induced renal tubular epithelial cell injury. It is suggested that ferroptosis plays an important role in AA-induced acute tubular injury. And inhibition of ferroptosis may exert renoprotective effects possibly by preventing lipid peroxidation, restoring the antioxidant activity or regulating iron metabolism.

Ionizable copolymer functionalized magnetic nanocomposite as an adsorbent for boosting the extraction selectivity of aristolochic acids.

Aristolochic acid nephropathy (AAN) has drawn increasing public attention. Organic anion transporters (OATs) are considered to be responsible for mediating nephrotoxicity of aristolochic acids (AAs), as AAs are typical OAT1 substrates that exhibit anionic properties and contain one hydrophobic domain. Inspired by the OAT1 three-dimensional structure or substrate/protein interactions involved in transport, we designed a magnetic polymeric hybrid, mimicking the effect of basic and aromatic residues of OAT1, for efficient enriching aristolochic acid I (AA I) and aristolochic acid II (AA II) in Traditional Chinese patent medicines (TCPM). N, N-dimethylaminopropyl acrylamide (DMAPAm) was used as a cationic monomer and copolymerized with divinylbenzene (DVB) onto the surface of monodisperse magnetic nanoparticles (denoted as MNs@SiO2T-DvbDam). The magnetic polymer hybrid demonstrated high selectivity and capacity for AAs, which was mainly attributed to (1) electrostatic interactions from the cationic or basic moiety of DMAPAm and (2) the hydrophobic and π-π stacking interactions from the aromatic ring of DVB. Additionally, the surface of the hybrid exhibited amphiphilic property according to the ionization of DMAPAm, thus improving the compatibility of the adsorbent with the aqueous sample matrix. This strategy was proven to be robust in the analysis of real drug samples, which was characterized by a good linearity, high recovery and satisfactory reusability. This work confirmed that the proposed tool could be a promising candidate for enhancing the extraction selectivity of AAs in Traditional Chinese medicines (TCM).

PSTPIP2 ameliorates aristolochic acid nephropathy by suppressing interleukin-19-mediated neutrophil extracellular trap formation.

Aristolochic acid nephropathy (AAN) is a progressive kidney disease caused by herbal medicines. Proline-serine-threonine phosphatase-interacting protein 2 (PSTPIP2) and neutrophil extracellular traps (NETs) play important roles in kidney injury and immune defense, respectively, but the mechanism underlying AAN regulation by PSTPIP2 and NETs remains unclear. We found that renal tubular epithelial cell (RTEC) apoptosis, neutrophil infiltration, inflammatory factor, and NET production were increased in a mouse model of AAN, while PSTPIP2 expression was low. Conditional knock-in of Pstpip2 in mouse kidneys inhibited cell apoptosis, reduced neutrophil infiltration, suppressed the production of inflammatory factors and NETs, and ameliorated renal dysfunction. Conversely, downregulation of Pstpip2 expression promoted kidney injury. In vivo, the use of Ly6G-neutralizing antibody to remove neutrophils and peptidyl arginine deiminase 4 (PAD4) inhibitors to prevent NET formation reduced apoptosis, alleviating kidney injury. In vitro, damaged RTECs released interleukin-19 (IL-19) via the PSTPIP2/nuclear factor (NF)-κB pathway and induced NET formation via the IL-20Rβ receptor. Concurrently, NETs promoted apoptosis of damaged RTECs. PSTPIP2 affected NET formation by regulating IL-19 expression via inhibition of NF-κB pathway activation in RTECs, inhibiting RTEC apoptosis, and reducing kidney damage. Our findings indicated that neutrophils and NETs play a key role in AAN and therapeutic targeting of PSTPIP2/NF-κB/IL-19/IL-20Rβ might extend novel strategies to minimize Aristolochic acid I-mediated acute kidney injury and apoptosis.

Histone deacetylase-mediated silencing of PSTPIP2 expression contributes to aristolochic acid nephropathy-induced PANoptosis.

Aristolochic acid nephropathy (AAN) is a progressive kidney disease caused by using herbal medicines. Currently, no therapies are available to treat or prevent aristolochic acid nephropathy. Histone deacetylase (HDAC) plays a crucial role in the development and progression of renal disease. We tested whether HDAC inhibitors could prevent aristolochic acid nephropathy and determined the underlying mechanism. HDACs expression in the aristolochic acid nephropathy model was examined. The activation of PANoptosis of mouse kidney and renal tubular epithelial cell were assessed after exposure to HDAC1 and HDAC2 blockade. Kidney-specific knock-in of proline-serine-threonine-phosphatase-interacting protein 2 (PSTPIP2) mice were used to investigate whether PSTPIP2 affected the production of PANoptosome. Aristolochic acid upregulated the expression of HDAC1 and HDAC2 in the kidneys. Notably, the HDAC1 and HDAC2 specific inhibitor, romidepsin (FK228, depsipeptide), suppressed aristolochic acid-induced kidney injury, epithelial cell pyroptosis, apoptosis and necroptosis (PANoptosis). Moreover, romidepsin upregulated PSTPIP2 in renal tubular epithelial cells, which was enhanced by aristolochic acid treatment. Conditional knock-in of PSTPIP2 in the kidney protected against aristolochic acid nephropathy. In contrast, the knockdown of PSTPIP2 expression in PSTPIP2-knock-in mice restored kidney damage and PANoptosis. PSTPIP2 function was determined in vitro using PSTPIP2 knockdown or overexpression in mouse renal tubular epithelial cells (mTECs). Additionally, PSTPIP2 was found to regulate caspase 8 in aristolochic acid nephropathy. HDAC-mediated silencing of PSTPIP2 may contribute to aristolochic acid nephropathy. Hence, HDAC1 and HDAC2 specific inhibitors or PSTPIP2 could be valuable therapeutic agents for preventing aristolochic acid nephropathy.

Spatially resolved multi-omics unravels region-specific responses, microenvironment remodeling and metabolic reprogramming in aristolochic acid nephropathy

<p>Aristolochic acid nephropathy (AAN), primarily caused by overexposure to aristolochic acid I (AAI), is characterized by acute renal injury, interstitial nephritis, and metabolic dysfunction. Previous studies have revealed the cell-specific responses to AAI and its role in inducing metabolic dysfunction. However, the lack of structural information in these datasets hinders our full understanding of the spatially specific pathological mechanisms of AAN. To address this limitation, we propose an integration of spatial transcriptomics with spatial metabolomics methods to establish a spatial multi-omics analysis, which allows for deciphering region-specific responses, microenvironmental remodeling, and metabolic reprogramming <i>in situ</i> in AAN. The pathological differences between renal regions indicate that AAI-induced renal injury exhibits spatial heterogeneity. With prolonged AAI treatment, we observed an increased proportion and co-localization of the injured proximal tubule (PT-inj) and immune cells in the cortex region, accompanied by intercellular crosstalk involving the MHC-I and CCL pathways. In addition, we identified a divergent cellular response along nephron segments, with up-regulation of multiple renal stress markers and pathways after AAI treatment. Regional heterogeneity of metabolic activities was also observed, with PT-inj cells exhibiting dysregulation of carbohydrate, lipid, and amino acid metabolic pathways, as well as increased purine and pyrimidine metabolism after AAI treatment. These findings provide a more comprehensive understanding of the cellular and molecular mechanisms of AAN in a spatial context, and suggest potential intervention pathways to alleviate the global burden of AAN.</p>

Detection and Removal of Aristolochic Acid in Natural Plants, Pharmaceuticals, and Environmental and Biological Samples: A Review.

Aristolochic acids (AAs) are a toxic substance present in certain natural plants. Direct human exposure to these plants containing AAs leads to a severe and irreversible condition known as aristolochic acid nephropathy (AAN). Additionally, AAs accumulation in the food chain through environmental mediators can trigger Balkan endemic nephropathy (BEN), an environmental variant of AAN. This paper presents a concise overview of the oncogenic pathways associated with AAs and explores the various routes of environmental exposure to AAs. The detection and removal of AAs in natural plants, drugs, and environmental and biological samples were classified and summarized, and the advantages and disadvantages of the various methods were analyzed. It is hoped that this review can provide effective insights into the detection and removal of AAs in the future.

TNF- α from the Proximal Nephron Exacerbates Aristolochic Acid Nephropathy.

Aristolochic acid nephropathy (AAN) presents with tubular epithelial cell (TEC) damage and tubulointerstitial inflammation. Although tumor necrosis factor-α (TNF) regulates cell apoptosis and inflammatory responses, the effects of tubular TNF in the progression of AAN require elucidation. Floxed TNF mice on the 129/SvEv background were crossed with PEPCK-Cre mice to generate PEPCK-Cre+TNFflox/flox (TNF PTKO) mice or bred with Ksp-Cre mice to generate KSP-Cre+TNFflox/flox (TNF DNKO) mice. TNF PTKO, TNF DNKO, and WT controls (Cre negative littermates) were subjected to acute and chronic AAN. Deletion of TNF in the proximal but not distal nephron attenuated kidney injury, renal inflammation, and tubulointerstitial fibrosis after acute or chronic aristolochic acid exposure. The TNF PTKO mice did not have altered numbers of infiltrating myeloid cells in AAN kidneys. Nevertheless, kidneys from aristolochic acid-treated TNF PTKO mice had reduced levels of proteins involved in regulated cell death, higher proportions of TECs in the G0/G1 phase, and reduced TEC proportions in the G2/M phase. Pifithrin-α, which restores the cell cycle, abrogated differences between the wild-type and PTKO cohorts in G2/M phase arrest of TECs and kidney fibrosis after aristolochic acid exposure. TNF from the proximal but not the distal nephron propagates kidney injury and fibrogenesis in aristolochic acid nephropathy in part by inducing G2/M cell cycle arrest of TECs.

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

To assess the risk of aristolochic acid (AA)-associated cancer in patients with AA nephropathy (AAN). 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. 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. 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.

Simultaneous toxicokinetic studies of aristolochic acid I and II and aristolactam I and II using a newly-developed microdialysis liquid chromatography-tandem mass spectrometry.

Aristolochic acids (AAs) are naturally occurring genotoxic carcinogens linked to Balkan endemic nephropathy and aristolochic acid nephropathy. Aristolochic acid I and II (AA-I and AA-II) are the most abundant AAs, and AA-I has been reported to be more genotoxic and nephrotoxic than AA-II. This study aimed to explore metabolic differences underlying the differential toxicity. We developed a novel microdialysis sampling coupled with solid-phase extraction liquid chromatography-tandem mass spectrometry (MD-SPE-LC-MS/MS) to simultaneously study the toxicokinetics (TK) of AA-I and AA-II and their corresponding aristolactams (AL-I and AL-II) in the blood of Sprague Dawley rats co-treated with AA-1 and AA-II. Near real-time monitoring of these analytes in the blood of treated rats revealed that AA-I was absorbed, distributed, and eliminated more rapidly than AA-II. Moreover, the metabolism efficiency of AA-I to AL-I was higher compared to AA-II to AL-II. Only 0.58% of AA-I and 0.084% of AA-II was reduced to AL-I and AL-II, respectively. The findings are consistent with previous studies and support the contention that differences in the in vivo metabolism of AA-I and AA-II may be critical factors for their differential toxicities.

Overview of aristolochic acid nephropathy: an update.

Aristolochic acid nephropathy (AAN) is a rapidly progressive renal interstitial fibrosis caused by medical or environmental exposure to aristolochic acid (AA). Since the outbreak of AAN in Belgium was reported nearly 30 years ago, the safety of herbal remedies has drawn considerable attention, and AAN has become a global public health problem. Breakthroughs have been made to better understand the disease, including the toxicity of AAs, the possible mechanisms of AAN, the disease patterns, and the pathological features; however, some critical problems remain unresolved. Because of the insidious onset of the disease, the incidence of AAN and the prevalence of exposure to AAs are unknown and might be largely underestimated. During the past decades, AA-containing herbs have been strictly administrated in many regions and the occurrence of AAN has declined sharply, yet cases of AAN are still sporadically reported. Despite the progress in the understanding of the disease's pathogenesis, there is no effective treatment for delaying or reversing the renal deterioration caused by AAN. Therefore, the risk of exposure to AAs should be taken seriously by public health workers and clinicians. In this review, we updated the latest data on AAN, summarized the advances throughout these years, and put forward some challenges for future research.

#6818 UNVEIL THE IMMUNE CELL LANDSCAPE IN AGEING KIDNEYS WITH AKI AND CKD

Abstract Background and Aims Immunosenescence, or aging process of the immune system, leads to either defective immune response or increased systemic inflammation. Previous studies have shown that kidney immune system may coordinate the process of kidney inflammation, resolution, or progressive fibrosis in kidney diseases. However, the exact role of individual immune cell type has not been systematically characterized, especially in the context of ageing kidneys. Method Firstly, we induced acute and chronic aristolochic acid nephropathy (AAN) in 15-month-old mice. At different stages of AAN (Steady state, acute inflammation, recovery from acute injury, and chronic fibrosis), mice were then sacrificed, and kidneys were harvested for further processing. We flow-sorted kidney CD45+ immune cells (live cells) and applied to 10 x Genomics platform for single cell isolation and cDNA library construction. Illumina NovaSeq 6000 system was used for RNA sequencing. Finally, we performed downstream bioinformatics analysis through CellRanger and Seurat pipelines. Results Utilizing AAN mouse model, we successfully conducted flow sorted kidney immune cells single-cell RNA sequencing (scRNAseq) from 15 M/O kidneys with AKI and CKD. We clearly identified 25 distinctive immune cell types, representing distinctive lymphoid and myeloid populations (Figure 1a). We showed dynamic change of immune cell compositions upon different phases of AAN (i.e., CCR2+ monocytes and proliferative macrophages increased during acute inflammation, activated T cells expanded during recovery phase, CD206+CCL8+ macrophages enriched within the fibrotic kidneys) (Figure 1b). By analysis of differentially expressed genes (DEG), we identified 30 top-ranked genes, which uniquely enriched in specific cell subset (Figure 1c). Conclusion We have successfully unveiled the immune cell landscape in ageing kidneys with AKI and CKD. Our preliminary data has demonstrated that distinct immune cell subsets at different phases of injury may have unique roles. Future work will focus on the cell-cell interactions and immune cell activation/differentiation.

Molecular Characterization of Microrna Interference and Aristolochic Acid Intoxication Found in Upper Tract Urothelial Carcinoma in Patients with Balkan Endemic Nephropathy: A Systematic Review of the Current Literature.

The term "aristolochic acid nephropathy" (AAN) is used to include any form of toxic interstitial nephropathy that is caused either by ingestion of plants containing aristolochic acids (AA) or by the environmental contaminants in food such as in Balkan endemic nephropathy (BEN). Aristolochic acid (AA) intoxication is strongly associated with the development of upper tract urothelial carcinoma (UTUC); however, the underlying molecular mechanism remains to be defined. MicroRNAs (miRNA) regulate several biological processes, including cell proliferation, differentiation, and metabolism, acting as oncogenes or tumor suppressors. A unique miRNA expression profile suggested that miRNAs could function as regulators in UTUC developmental processes. This review aimed to summarize data available in the literature about underlying molecular mechanisms leading to the expression of miRNAs in AA-UTUC patients with BEN. Strong correlation in AA-UTUC has a distinctive gene alteration pattern, AL-DNA adducts, and a unique tumor protein (TP53) mutational spectrum AAG to TAG (A: T→T: A) transversion in codon 139 (Lys → Stop) of exon 5 activates the p53 tumor suppressor protein. Further, p53 protein is responsible not only for the expression of miRNAs but also acts as a target molecule for miRNAs and plays a crucial function in the AA-UTUC pathogenicity through activation of cyclin-dependent kinase (CyclinD1) and cyclin protein kinase 6(CDK6) to support cell cycle arrest. This study, proposed a molecular mechanism that represented a possible unique relationship between AA intoxication, miRNAs expression, and the progression of UTUC in patients with BEN.

Long-term oral administration of Asarum heterotropoides f. mandshuricum (Maxim.) Kitag. decoction and its aristolochic acid analogs do not cause renal toxicity in mice.

Asarum heterotropoides f. mandshuricum (Maxim.) Kitag. (AH) is widely used to treat influenza, COVID-19, allergic rhinitis, headache, toothache, rheumatoid arthritis, and peptic ulcer. However, its clinical use is controversial due to the concern of aristolochic acid nephropathy (AAN) caused by its component aristolochic acid analogs (AAs). The chronic toxicity of AH decoction and its main components AA IVa (AA-IVa) and aristolactam I (AL-I) was evaluated in mice. AAs contents in AH were quantitated by liquid chromatography-mass spectrometry. A parallel design was employed to examine the potential chronic toxicity of AH decoction at doses equivalent to 0.5, 1.6, and 5.0g/kg AH (approximately 10-100 times the clinical doses for humans) and its major AA components at doses equivalent to that in 5.0g/kg AH to mice after consecutive daily oral administration for 12 and 24 weeks, and at 32 weeks after withdrawal for 8 weeks. AH crude herb contained 2.18μg/g of AA-I, 48.49μg/g of AA-IVa, and 14.0μg/g of AL-I. AH decoction contained 5.45μg/g of AA-IVa and 2.71μg/g of AL-I. None of AA-II and AA-IIIa were detected in AH. After long-term administration of AH decoction and its major components AA-IVa and AL-I, mice showed no signs of illness or body weight changes. In addition, biochemical and pathohistological examinations showed that long-term administration of AH decoction and its major components AA-IVa and AL-I did not alter 1) serum levels of glutamic-pyruvic transaminase, glutamic oxalacetic transaminase, alkaline phosphatase, creatinine, and urea nitrogen, 2) renal tissue mRNA expression of kidney injury molecule 1 and neutrophil gelatinase-associated lipocalin, and 3) pathological morphology in the mouse liver, kidney, stomach, and bladder. AH has no obvious toxicity to mice and is relatively safe when it is used in the form of decoction. AA-IVa and AL-I, the two major AAs in AH, are not toxic to mice at the dose equivalent to that in the high dose of AH decoction. Considering the limited toxicological data on AH, we recommend that AH decoction medication should not overdose and the duration should not be too long.

Aristolocholic acid I promotes renal tubular epithelial fibrosis by upregulating matrix metalloproteinase-9 expression via activating the C3a/C3aR axis of macrophages

Aristolochic acid I (AAI) can cause nephrotoxicity and is characterized by interstitial fibrosis. The C3a/C3aR axis of macrophages and matrix metalloproteinase-9 (MMP-9) play important roles in fibrosis, but whether they are involved in AAI-induced renal interstitial fibrosis and are related remains to be elucidated. In this study, we investigated whether C3a/C3aR axis of macrophages promotes renal interstitial fibrosis by regulating MMP-9 in aristolochic acid nephropathy (AAN). Intraperitoneal injection of AAI for 28 days successfully induced AAN in C57bl/6 mice. The content of C3a in the kidney of AAN mice was increased, and there was a significant distribution of macrophages in the renal tubules. The same results were observed in the in vitro experiment. We also explored the role and mechanism of macrophages after AAI administration in the epithelial–mesenchymal transformation (EMT) of renal tubular epithelial cells (RTECs) and found that AAI could activate the C3a/C3aR axis of macrophages to upregulate p65 expression in macrophages. p65 upregulated MMP-9 expression in macrophages not only directly but also by promoting the secretion if interleukin-6 by macrophages and then activating STAT3 in RTECs. The upregulation of MMP-9 expression could promote the EMT of RTECs. Taken together, our study demonstrated that the AAI-activated the C3a/C3aR axis of macrophages, which induced MMP-9 production, was one of the causes of renal interstitial fibrosis. Therefore, targeting the C3a/C3aR axis of macrophages is an effective therapeutic strategy for the prevention and treatment of renal interstitial fibrosis in AAN.

Effects of Diet on Aristolochic Acid-DNA Adduct Formation: Implications for Balkan Endemic Nephropathy Etiology.

Prolonged exposure to aristolochic acids (AAs) through AA-containing herbal medicine or AA-contaminated food is associated with the development of aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN), both public health risks to which the World Health Organization is calling for global action to remove exposure sources. The AA exposure-induced DNA damage is believed to be related to both the nephrotoxicity and carcinogenicity of AA observed in patients suffering from BEN. While the chemical toxicology of AA is well-studied, we investigated in this study the understated effect of different nutrients, food additives, or health supplements on DNA adduct formation by aristolochic acid I (AA-I). By culturing human embryonic kidney cells in an AAI-containing medium enriched with different nutrients, results showed that cells cultured in fatty acid-, acetic acid-, and amino acid-enriched media produced ALI-dA adducts at significantly higher frequencies than that cultured in the normal medium. ALI-dA adduct formation was most sensitive to amino acids, indicating that amino acid- or protein-rich diets might lead to a higher risk of mutation and even cancer. On the other hand, cells cultured in media supplemented with sodium bicarbonate, GSH, and NAC reduced ALI-dA adduct formation rates, which sheds light on their potential use as risk-mitigating strategies for people at risk of AA exposure. It is anticipated that the results of this study will help to better understand the effect of dietary habits on cancer and BEN development.

Aristolochic acid induces an inflammatory response with prostaglandin E2 production and apoptosis in NRK-52E proximal tubular cells

Aristolochic acid nephropathy (AAN) is a type of drug-induced nephropathy in which ingestion of aristolochic acid (AA) causes acute kidney injury, with progressive renal fibrosis and upper urothelial carcinoma. Although the pathological features of AAN have been reported to involve significant cell degeneration and loss in the proximal tubules, the details of the toxic mechanism in the acute phase of the disease remain unclear. This study investigates the cell death pathway and intracellular metabolic kinetics of AA exposure in rat NRK-52E proximal tubular cells. AA exposure induces dose- and time-dependent apoptotic cell death in NRK-52E cells. We examined the inflammatory response to further investigate the mechanism of AA-induced toxicity. AA exposure increased the gene expression of inflammatory cytokines IL-6 and TNF-α, suggesting that AA exposure induces inflammation. Furthermore, analysis of lipid mediators by LC-MS revealed increases in intra- and extra-cellular arachidonic acid and prostaglandin E2 (PGE2). To investigate the relationship between the AA-induced increase in PGE2 production and cell death, celecoxib, an inhibitor of cyclooxygenase-2 (COX-2), which is involved in the production of PGE2, was administered, and a marked inhibition of AA-induced cell death was observed. These results suggest that exposure to AA induces concentration- and time-dependent apoptosis in NRK-52E cells, which is attributed to inflammatory responses mediated by COX-2 and PGE2.

Bioaccumulation and DNA Adduct Formation of Aristolactam I: Unmasking a Toxicological Mechanism in the Pathophysiology of Aristolochic Acid Nephropathy.

Prolonged exposure to aristolochic acid (AA) through AA-containing herbal medicines or AA-tainted food is putting a large portion of the global population at risk of developing renal fibrosis and tumors of the upper urinary tract. In an effort to better understand the organotropic property of AA, we studied the cytotoxicity, absorption, oxidative-stress inducing potential, and DNA adduct formation capability of aristolactam I (ALI), one of the major urinary metabolites of aristolochic acid I (AAI) in human cells. Despite ALI having a slightly lower cytotoxicity than that of AAI, the analysis revealed, for the first time, that ALI is bioaccumulated 900 times more than that of AAI inside cultured kidney cells. Furthermore, ALI induced a significantly larger glutathione depletion than that of AAI in the exposed cells. Together with the formation of ALI-DNA adduct at a reasonably high abundance, results of this study unmasked a previously disregarded causative role of ALI in the organotropic tumor-targeting property of AA.