Aristolochic Acid Causes Albuminuria by Promoting Mitochondrial DNA Damage and Dysfunction in Podocyte

Aristolochic acid nephropathy, initially found in patients intaking of slimming herbs containing aristolochic acid (AA), was previously considered as a progressive renal interstitial fibrosis and urothelial malignancy. However, the presence of albuminuria in some patients with AAN suggests that AA may also damage the glomerular filtration barrier. In this study, mice AAN model was generated by daily administration of aristolochic acid I sodium salt intraperitoneally at a dose of 6 mg/kg body weight for 3 days. All of the mice developed heavy albuminuria at day 3 and 7 after receiving AA. In the mice received AA, morphologic change of glomeruli was minor under light microscopy but podocyte foot-process effacement was evident under electron microscopy. In mitochondria isolated from kidney, prominent mitochondrial DNA (mtDNA) damage was accompanied with marked decrease of mtDNA copy number and mitochondrial protein expression level. Similar to those in vivo results, AA treatment impaired the filtration barrier function of cultured podocytes. AA promoted mtDNA damage, decreased mtDNA copy number and mitochondrial protein expression in cultured podocytes. In addition, AA treatment also decreased ATP content, oxygen consumption rate and mitochondrial membrane potential as well as increased cellular reactive oxygen species in cultured podocytes. This study highlighted that AA could induce podocyte damage and albuminuria, which may be mediated by promoting mtDNA damage and mitochondrial dysfunction in podocytes.     

Aristolochic acid-induced accumulation of methylglyoxal and Nε-(carboxymethyl)lysine: an important and novel pathway in the pathogenic mechanism for aristolochic acid nephropathy

Aristolochic acid, found in the Aristolochia species, causes aristolochic acid nephropathy (AAN) and can develop into renal failure. Methylglyoxal (MGO) is a highly cytotoxic compound generated from the metabolic process of glucose or fatty acids. It binds to proteins and forms N(ε)-(carboxymethyl)lysine (CML), which contributes to aging and diabetes mellitus complications. However, no relevant literature explores the relationship of MGO and CML with AAN. By injecting AA (10mg/kg BW) into C3H/He mice for 5 consecutive days, we successfully developed an AAN model and observed tubular atrophy with decreased renal function. Creatinine clearance also decreased from 10.32 ± 0.79 ml/min/kg to 2.19 ± 0.29 ml/min/kg (p<0.01). The concentration of MGO in kidney homogenates increased 12 × compared to the control group (from 18.23 ± 8.05 μg/mg of protein to 231.16 ± 17.57 μg/mg of protein, p<0.01), and CML was observed in the renal tubules of the mice by immunohistochemistry. Furthermore, compared to the control group, GSH levels decreased by 0.32 × (from 2.46 ± 0.41 μM/μg of protein to 0.78 ± 0.15 μM/μg of protein, p<0.01), whereas intra-renal antioxidant capacity decreased by 0.54×(from 6.82 ± 0.97 U to 3.71 ± 0.25 U; unit is equivalent to μM Trolox/mg of protein, p<0.01). In this study, we found that serious kidney damage induced by AA is related to an increase and accumulation of MGO and CML.     

Metabolic activation of carcinogenic aristolochic acid, a risk factor for Balkan endemic nephropathy

Aristolochic acid (AA), a naturally occurring nephrotoxin and carcinogen, is associated with tumor development in patients suffering from Chinese herbs nephropathy (now termed aristolochic acid nephropathy, AAN) and may also be a cause for the development of a similar type of nephropathy, the Balkan endemic nephropathy (BEN). Major DNA adducts [7-(deoxyadenosin-N6-yl)-aristolactam and 7-(deoxyguanosin-N2-yl)aristolactam] formed from AA after reductive metabolic activation were found in renal tissues of patients with both diseases. Understanding which human enzymes are involved in AA activation and/or detoxication is important in the assessment of an individual's susceptibility to this plant carcinogen. This paper reviews major hepatic and renal enzymes responsible for AA-DNA adduct formation in humans. Phase I biotransformation enzymes play a crucial role in the metabolic activation of AA to species forming DNA adducts, while a role of phase II enzymes in this process is questionable. Most of the activation of AA in human hepatic microsomes is mediated by cytochrome P450 (CYP) 1A2 and, to a lower extent, by CYP1A1; NADPH:CYP reductase plays a minor role. In human renal microsomes NADPH:CYP reductase is more effective in AA activation. Prostaglandin H synthase (cyclooxygenase, COX) is another enzyme activating AA in human renal microsomes. Among the cytosolic reductases, NAD(P)H:quinone oxidoreductase (NQO1) is the most efficient in the activation of AA in human liver and kidney. Studies with purified enzymes confirmed the importance of CYPs, NADPH:CYP reductase, COX and NQO1 in the AA activation. The orientation of AA in the active sites of human CYP1A1, -1A2 and NQO1 was predicted from molecular modeling and explains the strong reductive potential of these enzymes for AA detected experimentally. We hypothesized that inter-individual variations in expressions and activities of enzymes activating AA may be one of the causes responsible for the different susceptibilities to this carcinogen reflected in the development of AA-induced nephropathies and associated urothelial cancer.     

Genetic loci that affect aristolochic acid-induced nephrotoxicity in the mouse

Aristolochic acids (AA) are plant-derived nephrotoxins and carcinogens found in traditional medicines and herbal remedies. AA causes aristolochic acid nephropathy (AAN) and is a suspected environmental agent in Balkan endemic nephropathy (BEN) and its associated upper urothelial cancer. Approximately 5-10% of individuals exposed to AA develop renal insufficiency and/or cancer; thus a genetic predisposition to AA sensitivity has been proposed. The mouse is an established animal model of AAN, and inbred murine strains vary in AA sensitivity, confirming the genetic predisposition. We mapped quantitative trait loci (QTL) correlated with proximal tubule dysfunction after exposure to AA in an F2 population of mice, derived from breeding an AA-resistant strain (C57BL/6J) and an AA-sensitive strain (DBA/2J). A single main QTL was identified on chromosome 4 (Aanq1); three other interacting QTLs, (Aanq2-4) also were detected. The Aanq1 region was also detected in untreated mice, raising the possibility that preexisting differences in proximal tubule function may affect the severity of AA-elicited toxicity. This study lays the groundwork for identifying the genetic pathways contributing to AA sensitivity in the mouse and will further our understanding of human susceptibility to AA found widely in traditional medicines.     

Macrophage-derived,LRG1-enriched extracellular vesicles exacerbate aristolochic acid nephropathy in a TGFβR1-dependent manner

Cell Biology and Toxicology - Aristolochic acid nephropathy (AAN) is a progressive kidney disease caused by some herbal medicines, but treatment remains ineffective. We previously found that...     

A fluorescence-based analysis of aristolochic acid-derived DNA adducts

Aristolochic acids (AAs), major components of plant extracts from Aristolochia species, form (after metabolic activation) pro-mutagenic DNA adducts in renal tissue. The DNA adducts can be used as biomarkers for studies of AA toxicity. Identification of these adducts is a complicated and time-consuming procedure. We present here a fast, nonisotopic, fluorescence-based assay for the detection of AA-DNA adducts in multiple samples. This approach allows analysis of AA adducts in synthetic DNA with known nucleotide composition and analysis of DNA adducts formed from chemically diverse AAs in vitro. The method can be applied to compare AA-DNA adduct formation in cells and tissues.     

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.     

DNA adducts of aristolochic acid II: total synthesis and site-specific mutagenesis studies in mammalian cells

Aristolochic acids I and II (AA-I, AA-II) are found in all Aristolochia species. Ingestion of these acids either in the form of herbal remedies or as contaminated wheat flour causes a dose-dependent chronic kidney failure characterized by renal tubulointerstitial fibrosis. In ∼50% of these cases, the condition is accompanied by an upper urinary tract malignancy. The disease is now termed aristolochic acid nephropathy (AAN). AA-I is largely responsible for the nephrotoxicity while both AA-I and AA-II are genotoxic. DNA adducts derived from AA-I and AA-II have been isolated from renal tissues of patients suffering from AAN. We describe the total synthesis, de novo, of the dA and dG adducts derived from AA-II, their incorporation site-specifically into DNA oligomers and the splicing of these modified oligomers into a plasmid construct followed by transfection into mouse embryonic fibroblasts. Analysis of the plasmid progeny revealed that both adducts blocked replication but were still partly processed by DNA polymerase(s). Although the majority of coding events involved insertion of correct nucleotides, substantial misincorporation of bases also was noted. The dA adduct is significantly more mutagenic than the dG adduct; both adducts give rise, almost exclusively, to misincorporation of dA, which leads to AL-II-dA→T and AL-II-dG→T transversions.     

Muscle RING-finger protein-1 (MuRF1) as a connector of muscle energy metabolism and protein synthesis

During pathophysiological muscle wasting, a family of ubiquitin ligases, including muscle RING-finger protein-1 (MuRF1), has been proposed to trigger muscle protein degradation via ubiquitination. Here, we characterized skeletal muscles from wild-type (WT) and MuRF1 knockout (KO) mice under amino acid (AA) deprivation as a model for physiological protein degradation, where skeletal muscles altruistically waste themselves to provide AAs to other organs. When WT and MuRF1 KO mice were fed a diet lacking AA, MuRF1 KO mice were less susceptible to muscle wasting, for both myocardium and skeletal muscles. Under AA depletion, WT mice had reduced muscle protein synthesis, while MuRF1 KO mice maintained nonphysiologically elevated levels of skeletal muscle protein de novo synthesis. Consistent with a role of MuRF1 for muscle protein turnover during starvation, the concentrations of essential AAs, especially branched-chain AAs, in the blood plasma significantly decreased in MuRF1 KO mice under AA deprivation. To clarify the molecular roles of MuRF1 for muscle metabolism during wasting, we searched for MuRF1-associated proteins using pull-down assays and mass spectrometry. Muscle-type creatine kinase (M-CK), an essential enzyme for energy metabolism, was identified among the interacting proteins. Coexpression studies revealed that M-CK interacts with the central regions of MuRF1 including its B-box domain and that MuRF1 ubiquitinates M-CK, which triggers the degradation of M-CK via proteasomes. Consistent with MuRF1's role of adjusting CK activities in skeletal muscles by regulating its turnover in vivo, we found that CK levels were significantly higher in the MuRF1 KO mice than in WT mice. Glucocorticoid modulatory element binding protein-1 and 3-hydroxyisobutyrate dehydrogenase, previously identified as potential MuRF1-interacting proteins, were also ubiquitinated MuRF1-dependently. Taken together, these data suggest that, in a multifaceted manner, MuRF1 participates in the regulation of AA metabolism, including the control of free AAs and their supply to other organs under catabolic conditions, and in the regulation of ATP synthesis under metabolic-stress conditions where MuRF1 expression is induced.     

A novel and specific method for the determination of aristolochic acid-derived DNA adducts in exfoliated urothelial cells by using ultra performance liquid chromatography-triple quadrupole mass spectrometry

Aristolochic acid nephropathy (AAN) is associated with the prolonged exposure to nephrotoxic and carcinogenic aristolochic acids (AAs). DNA adducts induced by AAs have been proven to be critical biomarkers for AAN. Therefore, accurate and specific quantification of AA-DNA adducts is important. In this study, a specific method using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed and applied for the determination of 7-(deoxyadenosin-N(6)-yl)aristolactam I (dA-AAI) in exfoliated urothelial cells of AA-dosed rats. After the isolation from urine samples, DNA in urothelial cells were subjected to enzymatic digestion and solid-phase extraction on a C(18) Sep-Pak cartridge for the enrichment of DNA adducts. The sample extracts were analyzed by reverse-phase UPLC-MS/MS with electrospray ionization in positive ion mode. The quantification of the AA-DNA adduct was performed by using multiple reaction monitoring with reserpine as internal standard. The method provided good accuracy and precision with a detection limit of 1 ng/ml, which allowed the detection of trace of dA-AAI in exfoliated urothelial cells. After one-month oral dose of AAI at 10 mg/kg/day, 2.1±0.3 dA-AAI per 10(9) normal dA was detected in exfoliated urothelial cells of rats. Compared to the traditional methods such as (32)P-postlabelling and HPLC with fluorescence detection, the developed UPLC-MS/MS method is more specific and rapid with a retention time of 4 min. The outcome of this study may have clinical significance for diagnosing and monitoring AA-associated disease because detection of DNA adducts in exfoliated urothelial cells is non-invasive and convenient.     

Widespread use of unconventional targeting signals in mitochondrial ribosome proteins

Mitochondrial ribosomes are complex molecular machines indispensable for respiration. Their assembly involves the import of several dozens of mitochondrial ribosomal proteins (MRPs), encoded in the nuclear genome, into the mitochondrial matrix. Proteomic and structural data as well as computational predictions indicate that up to 25% of yeast MRPs do not have a conventional N‐terminal mitochondrial targeting signal (MTS). We experimentally characterized a set of 15 yeast MRPs in vivo and found that five use internal MTSs. Further analysis of a conserved model MRP, Mrp17/bS6m, revealed the identity of the internal targeting signal. Similar to conventional MTS‐containing proteins, the internal sequence mediates binding to TOM complexes. The entire sequence of Mrp17 contains positive charges mediating translocation. The fact that these sequence properties could not be reliably predicted by standard methods shows that mitochondrial protein targeting is more versatile than expected. We hypothesize that structural constraints imposed by ribosome assembly interfaces may have disfavored N‐terminal presequences and driven the evolution of internal targeting signals in MRPs.     

Acute nephrotoxicity of aristolochic acid in vitro: metabolomics study for intracellular metabolic time-course changes

Time-course metabolic changes of aristolochic acid nephrotoxicity (AAN) was investigated using acute AAN HK-2 model. And the AAN-related biomarkers were selected. In the results, 11 potential identified biomarkers were selected and validated using multivariate method combined with time-course analysis. Several metabolic pathways, including vitamin metabolism, lipids acalytion, trytophan metabolism and protein degradation were found to be associated with AAN pathology. This research will provide a valuable reference for the discovery of more potential biomarkers of AAN progression in clinic.     

Krebs durch Chemikalien

Aristolochia as human carcinogen Aristolochic acid (AA), the active principle of the old drug Aristolochia ssp. has recently been classified as human carcinogen. The elucidation of the molecular mechanism of carcinogenesis of AA in animals led to the establishment of two specific biomarkers which were used to prove a causative role of AA in human cancers. These are AA‐DNA adducts, biomarkers of exposure to AA and AT→TA transversion mutations induced by these DNA adducts, biomarkers of effect. By detecting both biomarkers in individuals who ingested Aristolochia herbs during a weight‐reduction regimen in Belgium and in farmers in the Balkans where Aristolochia ssp are growing as weeds in cereal fields AA was identified as the cause for urothelial cancer and a disease of the kidney (AAN).     

Diabetes-induced changes in the renal cortical proteome assessed with two-dimensional gel electrophoresis and mass spectrometry

To understand the spectrum of proteins affected by diabetes and to characterize molecular functions and biological processes they control, we analyzed the renal cortical proteome of db/db mice using 2-DE combined with MALDI-TOF, MALDI-TOF/TOF, and LC-MS/MS. This approach yielded 278 high confidence identifications whose expression levels were significantly increased or decreased >two-fold by diabetes, of which 170 mapped to gene identifiers representing 147 nonredundant proteins. Gene Ontology classification demonstrated that 80% of these proteins modulated physiological functions, 55% involved metabolism, approximately 25% involved carboxylic and organic acid metabolism, 14% involved biosynthesis or catabolism, and 12% involved fatty acid metabolism. Predominant molecular functions were catalytic (61%), oxidoreductase (20%), and transferase (17%) activities, and nucleotide and ATP binding (11-15%). Twenty eight percent of the proteins identified as significantly altered by diabetes were mitochondrial proteins. The top-ranked network described by Ingenuity Pathway Analysis indicated PPARalpha was the most common node of interaction for the numerous enzymes whose expression levels were influenced by diabetes. These differentially regulated proteins create a foundation for a systems biology exploration of molecular mechanisms underlying the pathophysiology of diabetic nephropathy.     

Analysis of urinary aristolactams by on-line solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry

Aristolochic acids (AAs), nephrotoxicants and known human carcinogens, are a mixture of structurally related derivatives of nitrophenanthrene carboxylic acids with the major components being aristolochic acid I and aristolochic acid II. People may ingest small amounts of AAs from its natural presence in medicinal plants and herbs of the family Aristolochiaceae, including the genera Aristolochia and Asarum, which have been used worldwide in folk medicine for centuries. In order to assess AA intake, an on-line solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry (on-line SPE-LC/MS/MS) method was developed to analyze their most abundant corresponding metabolites, aristolactams (ALs), in urine to serve as biomarkers. The limits of quantitation were 0.006 ng for aristolactam I (AL-I), and 0.024 ng for aristolactam II (AL-II) on column. Recovery varied from 98.0% to 99.5%, and matrix effects were within 75.3-75.4%. This method was applied to analyze ALs in the urine samples collected on days 1, 2, 4, and 7 from mice treated with 30 mg/kg or 50mg/kg AAs. Their half lives were estimated to be 3.55 h and 4.00 for AL-I, and 4.04 and 4.83 h for AL-II, depending on AAs doses. These results demonstrated that the first simple on-line SPE-LC/MS/MS method was successfully developed to analyze urinary ALs with excellent sensitivity and specificity to serve as biomarkers to assess current AA intake from AAs-containing Chinese herbs.     

Atherogenicity of amino acids in the lipid-laden macrophage model system in vitro and in atherosclerotic mice: a key role for triglyceride metabolism

Atherosclerosis-related research has focused mainly on the effects of lipids on macrophage foam cell formation and atherogenesis, whereas the role of amino acids (AAs) was understudied. The current study aimed to identify anti- or pro-atherogenic AA in the macrophage model system and to elucidate the underlying metabolic and molecular mechanisms. J774A.1 cultured macrophages were treated with increasing concentrations of each 1 of the 20 AAs. Macrophage atherogenicity was assessed in terms of cellular toxicity, generation of reactive oxygen species (ROS) and cellular cholesterol or triglyceride content. At nontoxic concentrations (up to 1 mM), modest effects on ROS generation or cholesterol content were noted, but six specific AAs significantly affected macrophage triglyceride content. Glycine, cysteine, alanine and leucine significantly decreased macrophage triglyceride content (by 24%–38%), through attenuated uptake of triglyceride-rich very low-density lipoprotein (VLDL) by macrophages. In contrast, glutamate and glutamine caused a marked triglyceride accumulation in macrophages (by 107% and 129%, respectively), via a diacylglycerol acyltransferase-1 (DGAT1)-dependent increase in triglyceride biosynthesis rate with a concurrent maturation of the sterol regulatory element-binding protein-1 (SREBP1). Supplementation of apolipoprotein E-deficient (apoE^−/−) mice with glycine for 40 days significantly decreased the triglyceride levels in serum and in peritoneal macrophages (MPMs) isolated from the mice (by 19%). In contrast, glutamine supplementation significantly increased MPM ROS generation and the accumulation of cholesterol and that of triglycerides (by 48%), via enhanced uptake of LDL and VLDL. Altogether, the present findings reveal some novel roles for specific AA in macrophage atherogenicity, mainly through modulation of cellular triglyceride metabolism.     

Glutamate dehydrogenase requirement for apoptosis induced by aristolochic acid in renal tubular epithelial cells

Ingestion of aristolochic acids (AA) contained in herbal remedies results in a renal disease and, frequently, urothelial malignancy. The genotoxicity of AA in renal cells, including mutagenic DNA adduct formation, is well-documented. However, the mechanisms of AA-induced tubular atrophy and renal fibrosis are largely unknown. Epithelial cell death is a critical characteristic of these pathological conditions. To elucidate the mechanisms of AA-induced cytotoxicity, we explored AA-interacting proteins in tubular epithelial cells (TEC). We found that AA interacts with a mitochondrial enzyme glutamate dehydrogenase (GDH) and moderately inhibits its activity. We report that AA induces cell death in GDH-knockdown TEC preferentially via non-apoptotic means, whereas in GDH-positive cells, death was executed by both the non-apoptotic and apoptotic mechanisms. Apoptosis is an energy-reliant process and demands higher adenosine 5′-triphosphate (ATP) consumption than does the non-apoptotic cell death. We found that, after AAI treatment, the ATP depletion is more pronounced in GDH-knockdown cells. When we reduced ATP in TEC cells by inhibition of glycolysis and mitochondrial respiration, cell death mode switched from apoptosis and necrosis to necrosis only. In addition, in cells incubated at low glucose and no glutamine conditions, oxaloacetate and pyruvate reduced AAI-induced apoptosis our data suggest that AAI-GDH interactions in TEC are critical for the induction of apoptosis by direct inhibition of GDH activity. AA binding may also induce changes in GDH conformation and promote interactions with other molecules or impair signaling by GDH metabolic products, leading to apoptosis.     

Differential effects of MYH9 and APOL1 risk variants on FRMD3 Association with Diabetic ESRD in African Americans

Single nucleotide polymorphisms (SNPs) in MYH9 and APOL1 on chromosome 22 (c22) are powerfully associated with non-diabetic end-stage renal disease (ESRD) in African Americans (AAs). Many AAs diagnosed with type 2 diabetic nephropathy (T2DN) have non-diabetic kidney disease, potentially masking detection of DN genes. Therefore, genome-wide association analyses were performed using the Affymetrix SNP Array 6.0 in 966 AA with T2DN and 1,032 non-diabetic, non-nephropathy (NDNN) controls, with and without adjustment for c22 nephropathy risk variants. No associations were seen between FRMD3 SNPs and T2DN before adjusting for c22 variants. However, logistic regression analysis revealed seven FRMD3 SNPs significantly interacting with MYH9—a finding replicated in 640 additional AA T2DN cases and 683 NDNN controls. Contrasting all 1,592 T2DN cases with all 1,671 NDNN controls, FRMD3 SNPs appeared to interact with the MYH9 E1 haplotype (e.g., rs942280 interaction p-value = 9.3E⁻⁷ additive; odds ratio [OR] 0.67). FRMD3 alleles were associated with increased risk of T2DN only in subjects lacking two MYH9 E1 risk haplotypes (rs942280 OR = 1.28), not in MYH9 E1 risk allele homozygotes (rs942280 OR = 0.80; homogeneity p-value = 4.3E⁻⁴). Effects were weaker stratifying on APOL1. FRMD3 SNPS were associated with T2DN, not type 2 diabetes per se, comparing AAs with T2DN to those with diabetes lacking nephropathy. T2DN-associated FRMD3 SNPs were detectable in AAs only after accounting for MYH9, with differential effects for APOL1. These analyses reveal a role for FRMD3 in AA T2DN susceptibility and accounting for c22 nephropathy risk variants can assist in detecting DN susceptibility genes.     

Plasma profile and urine excretion of amino acids in children with celiac disease on gluten-free diet after oligofructose-enriched inulin intervention: results of a randomised placebo-controlled pilot study

Amino Acids - The circulating amino acid (AAs) concentrations are indicators of dietary protein intake and metabolic status. In celiac disease (CD), the AA imbalance is frequently observed....     

Aristolochic acid downregulates monocytic matrix metalloproteinase-9 by inhibiting nuclear factor-κB activation

Aristolochic acid (AA)-associated nephropathy was described as being characterized by a rapid progressive enhancement of interstitial renal fibrosis. Renal tissue fibrosis occurs because of an imbalance of extracellular matrix (ECM) accumulation and matrix metalloproteinase (MMP) activation. Much evidence indicates that inflammatory renal disease including monocyte and mesangial interactions is linked to the development and progression of renal remodeling. In this study, we found that AA showed concentration-dependent inhibition of tumor necrosis factor (TNF)-α-induced MMP-9 activation with an IC₅₀ value of 6.4 ± 0.5 μM in human monocytic THP-1 cells. A similar effect was also noted with different ratios of AAs (types I and II). However, AA had no inhibitory effect on the intact enzymatic activity of MMP-9 at a concentration of 20 μM. On the other hand, the level of tissue inhibitor of metalloproteinase (TIMP)-1 was not induced by AA, but it suppressed TNF-α-induced MMP-9 protein and messenger RNA expressions. AA also significantly inhibited TNF-α-induced IκBα degradation. Furthermore, an electrophoretic mobility shift assay and a reported gene study, respectively, revealed that AA inhibited TNF-α-induced NF-κB translocation and activation. In addition, compared to other NF-κB inhibitors, AA exerted significant inhibition of MMP-9 activation and monocyte chemotactic protein-1-directed invasion. From these results, we concluded that AA, a natural compound, inhibits TNF-α-induced MMP-9 in human monocytic cells possibly through the NF-κB signal pathway. These results also imply that AA may be involved in alteration of matrix homeostasis during renal fibrosis in vivo.