An Integrated View of Aristolochic Acid Nephropathy: Update of the 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) as part of traditional phytotherapies (formerly known as "Chinese herbs nephropathy"), or by the environmental contaminants in food (Balkan endemic nephropathy). It is frequently associated with urothelial malignancies. Although products containing AA have been banned in most of countries, AAN cases remain regularly reported all over the world. Moreover, AAN incidence is probably highly underestimated given the presence of AA in traditional herbal remedies worldwide and the weak awareness of the disease. During these two past decades, animal models for AAN have been developed to investigate underlying molecular and cellular mechanisms involved in AAN pathogenesis. Indeed, a more-in-depth understanding of these processes is essential to develop therapeutic strategies aimed to reduce the global and underestimated burden of this disease. In this regard, our purpose was to build a broad overview of what is currently known about AAN. To achieve this goal, we aimed to summarize the latest data available about underlying pathophysiological mechanisms leading to AAN development with a particular emphasis on the imbalance between vasoactive factors as well as a focus on the vascular events often not considered in AAN.

Familial versus environmental factors in Balkan endemic nephropathy in Mehedinti county, Romania, by means of albuminuria and tubular biomarkers: preliminary study.

INTRODUCTION AND AIMS: Balkan endemic nephropathy (BEN), a regional tubulointerstitial kidney disease encountered in South-Eastern Europe, with still undefined etiology and inexorable evolution towards end stage renal disease, raises the question of the relative contribution of family and environmental factors in its etiology. In order to evaluate the intervention of these factors, markers of tubular injury have been assessed, this lesion being considered an early renal involvement in BEN. METHODS: The paper studies relatives of BEN patients currently included in dialysis programmes (for involvement of the family factor) and their neighbors (for involvement of environmental factors) and analyzes them with regard to tubular injury by means of tubular biomarkers (N-acetyl-beta-d-glucosaminidase-NAG and alpha-1-microglobulin), and albuminuria. At the same time, glomerular filtration rate (GFR) (CKD-EPI) was measured. It is considered that, in order to acquire the disease, one should have lived for 20 years in the BEN area. The relatives have been classified according to this criterion. RESULTS: More evident tubular injury was found in the neighbors of BEN patients living for more than 20 years in the endemic area, which argues in favor of environmental factors. Higher levels of urinary alpha-1-microglobulin and albumin in relatives of BEN patients who had been living for more than 20 years in the area than in relatives with a residence under 20 years, plead for the same hypothesis. GFR was lower in persons who had been living for more than 20 years in the BEN area (neighbors and relatives). CONCLUSIONS: Environmental factors could be more important in BEN than family factors.

[Endemic nephropathy in Croatia]. / Endemska nefropatija u Hrvatskoj.

Endemic nephropathy (EN) is a chronic tubulointerstitial aristolochic acid nephropathy (AAN) affecting residents of the certain villages in the valleys of the major tributaries of the Danube river in the south-east Europe including Croatia. Patients with EN have a significantly higher incidence of transitional cell carcinoma of the ureter than the general population. A-T transversion of the p53 gene is now considered to be a mutational "signature" of aristolochic acid, which is a cause of endemic nephropathy. Currently used diagnostic criteria for EN are outdated, uneven (three types of criteria) and are not in agreement with proposed new guidelines for kidney diseases. Therefore, based on current knowledge and expertise of a group of scientists and experts from all countries with EN as well as world where AAN has been reported, new diagnostic criteria and the new classification of the population of endemic villages were created at a symposium on EN. EN presents a major public health problem and current knowledge about this disease as well as new diagnostic criteria should help us in its early detection and treatment and maybe in a near future its eradication.

Update on endemic nephropathies.

PURPOSE OF REVIEW: A large number of patients worldwide suffer from chronic kidney disease (CKD) of unknown cause. Endemic nephropathies possibly contribute to this. The purpose of this review is to give a brief review of endemic nephropathies and to summarize what is known about their cause. RECENT FINDINGS: The cause of Balkan endemic nephropathy was eventually resolved, after 50 years of research. The cause was exposure to aristolochic acid from food. A new type of endemic nephropathy has recently been identified in Central America; Mesoamerican nephropathy. This kidney disease mainly affects agricultural workers in hot climates. Renal biopsy studies suggest that repeated dehydration and kidney ischemia is involved in the pathogenesis. SUMMARY: Endemic nephropathies may comprise an important cause of CKD. Epidemiological studies are needed to describe the occurrence and distribution of the diseases. However, biopsy studies, in combination with careful clinical evaluation of the patients, are necessary to find out the cause of endemic nephropathies and thereby help in their prevention.

Consensus statement on screening, diagnosis, classification and treatment of endemic (Balkan) nephropathy.

Currently used diagnostic criteria in different endemic (Balkan) nephropathy (EN) centers involve different combinations of parameters, various cut-off values and many of them are not in agreement with proposed international guidelines. Leaders of EN centers began to address these problems at scientific meetings, and this paper is the outgrowth of those discussions. The main aim is to provide recommendations for clinical work on current knowledge and expertise. This document is developed for use by general physicians, nephrologists, urologist, public health experts and epidemiologist, and it is hoped that it will be adopted by responsible institutions in countries harboring EN. National medical providers should cover costs of screening and diagnostic procedures and treatment of EN patients with or without upper urothelial cancers.

The epidemiology, diagnosis, and management of aristolochic acid nephropathy: a narrative review.

It has been 20 years since the first description of a rapidly progressive renal disease that is associated with the consumption of Chinese herbs containing aristolochic acid (AA) and is now termed aristolochic acid nephropathy (AAN). Recent data have shown that AA is also the primary causative agent in Balkan endemic nephropathy and associated urothelial cancer. Aristolochic acid nephropathy is associated with a high long-term risk for renal failure and urothelial cancer, and the potential worldwide population exposure is enormous. This evidence-based review of the diagnostic approach to and management of AAN draws on the authors' experience with the largest and longest-studied combined cohort of patients with this condition. It is hoped that a better understanding of the importance of this underrecognized and severe condition will improve epidemiologic, preventive, and therapeutic strategies to reduce the global burden of this disease.

Unusual Urothelial Tumors and Refractory Uremia Due to Balkan Endemic Nephropathy: A Case Report.

Balkan endemic nephropathy (BEN) is a rare progressive chronic renal disease found in residents living along the Balkan peninsula. We present a 92-year-old female who complained initially of cardio-respiratory symptoms and was found to have an acute hypoxemic respiratory failure with hypervolemia. The patient underwent computed tomography imaging and was found to have bilateral pleural effusions and moderate left-sided renal atrophy with left-sided hydronephrosis. The patient underwent diuresis for fluid overload and was treated with broad-spectrum antibiotics for hospital-acquired pneumonia. Further urological work-up revealed masses in the posterior bladder wall and left ureteropelvic junction. A biopsy of the posterior bladder wall mass confirmed high-grade papillary urothelial carcinoma. A review of the epidemiological history revealed the patient lived in Kosovo/former Yugoslavia for several decades following birth. A review of old records revealed the patient had chronic kidney disease (CKD) that was not fully explained by other causes, such as hypertension or diabetes. Given the epidemiological history, accelerated CKD, and unusual locations of urothelial carcinoma, the patient was diagnosed with BEN. Despite medical management and hemodialysis, the patient's renal function and mental status continued to deteriorate, and the decision was made to proceed with palliative care measures.

Variation in presentation and presence of DNA adducts and p53 mutations in patients with endemic nephropathy--an environmental form of the aristolochic acid nephropathy.

BACKGROUND: Endemic nephropathy (EN) and associated urothelial cell cancers (UUC) are an environmental form of aristolochic acid nephropathy where the most probable rout of ingestion of aristolochic acid (AA) was made by bread contaminated with AA, leading to chronic dietary intoxication. Clinical courses of three members of the same family, similarly exposed to toxin, who exhibited different clinical courses of the disease are presented. METHODS: Questionnaires on AA exposure were taken. Tissue samples were obtained during therapeutic nephrouretectomies. Histopathology, immunohistochemical detection of p53, p53 mutation screening in tumor DNA and analysis on the presence of aristolactam (AL)-DNA adducts were performed. RESULTS: Case 1 had UUC with typical EN histopathological signs, whereas Case 2 had bilateral UUCs with typical EN histopathological signs. In contrast, the patient in Case 3 initially showed renal insufficiency, complicated afterwards by right UUC, and later on by left UUC with histopathological end-stage chronic changes but without typical EN changes. AA-DNA adducts and specific p53 mutational spectra (A:T→ T:A transversion) were found in tissues of cases 1 and 2. CONCLUSION: Diverse clinical courses seem to be related not to differences in exposure but to differences in metabolic activation or detoxification of AA and/or DNA repair resulting from different genetic polymorphisms.

Aristolactam-DNA adducts are a biomarker of environmental exposure to aristolochic acid.

Endemic (Balkan) nephropathy is a chronic tubulointerstitial disease frequently accompanied by urothelial cell carcinomas of the upper urinary tract. This disorder has recently been linked to exposure to aristolochic acid, a powerful nephrotoxin and human carcinogen. Following metabolic activation, aristolochic acid reacts with genomic DNA to form aristolactam-DNA adducts that generate a unique TP53 mutational spectrum in the urothelium. The aristolactam-DNA adducts are concentrated in the renal cortex, thus serving as biomarkers of internal exposure to aristolochic acid. Here, we present molecular epidemiologic evidence relating carcinomas of the upper urinary tract to dietary exposure to aristolochic acid. DNA was extracted from the renal cortex and urothelial tumor tissue of 67 patients that underwent nephroureterectomy for carcinomas of the upper urinary tract and resided in regions of known endemic nephropathy. Ten patients from nonendemic regions with carcinomas of the upper urinary tract served as controls. Aristolactam-DNA adducts were quantified by (32)P-postlabeling, the adduct was confirmed by mass spectrometry, and TP53 mutations in tumor tissues were identified by chip sequencing. Adducts were present in 70% of the endemic cohort and in 94% of patients with specific A:T to T:A mutations in TP53. In contrast, neither aristolactam-DNA adducts nor specific mutations were detected in tissues of patients residing in nonendemic regions. Thus, in genetically susceptible individuals, dietary exposure to aristolochic acid is causally related to endemic nephropathy and carcinomas of the upper urinary tract.

Could disappearance of endemic (Balkan) nephropathy be expected in forthcoming decades?

BACKGROUND/AIMS: An epidemiological survey of endemic nephropathy (EN) was performed in endemic Croatian areas and the current prevalence was compared to that reported for the same villages several decades ago. METHODS: A total of 2,487 adult farmers from 6 endemic villages and 3 non-endemic villages were enrolled. An extensive epidemiological questionnaire, clinical examination and laboratory analyses of blood and urine were performed. According to the modified WHO criteria, participants were classified into diseased, suspected of having EN, and those at risk of developing EN. RESULTS: The overall prevalence of EN in the Croatian areas was 1.0%, ranging between 0.3 and 2.3% in different villages. Those suspected of having EN amounted to 3.9%. In the endemic villages a decreasing trend in the prevalence of EN was observed comparable to the results obtained in previous surveys. It is interesting to note that no EN patients were recorded in the endemic village of Dubocac. CONCLUSION: The prevalence of EN in the endemic Croatian areas appears to be decreasing. For the first time, we failed to detect any EN patients in a village that was previously considered endemic, which might indicate that EN is diminishing.

Fifty years of Balkan endemic nephropathy in Romania: some aspects of the endemic focus in the Mehedinti county.

Balkan endemic nephropathy (BEN) is a chronic tubulointerstitial nephritis seen primarily in countries in the Balkan Peninsula. The disease, which was first described in Romania 50 years ago, often manifests as a form of chronic nephritis that is also associated with upper urothelial cancers (UUC). This review summarizes the observations and studies performed in Romania regarding this disease during the last 50 years with particular emphasis on Mehedinti county. The paper analyzes current data on the epidemiology of the disease in this area, specifically in relation to the observations made in dialysis centers in the same area. It also discusses the diagnostic criteria of patients with BEN stemming from collaborations between specialists working in other countries affected by the disease. Moreover, the paper analyzes the main etiological factors suspected to play a role in BEN: aristolochic acid (the disease has many similarities to aristolochic nephropathy caused by Chinese herbs), mycotoxins, toxic substances from pliocene lignite, genetic factors, and viruses. Studies performed by Romanian authors are presented briefly in comparison to studies performed by other authors. Finally, given that BEN is an important health problem in the region, the relationship between BEN and UUC is further analyzed.

Urothelial carcinoma associated with Balkan endemic nephropathy. A worldwide disease.

Balkan endemic nephropathy (BEN), a familial chronic tubulo-interstitial disease with a slow progression to terminal renal failure, affects people living in the alluvial plains along the tributaries of the Danube River. One of its most peculiar characteristics is a strong association with upper urothelial cancer. An increased incidence of upper urinary tract (UUT) transitional cell cancer (TCC) was discovered among the inhabitants of endemic settlements and in families affected by BEN. In areas where BEN is endemic, the incidence of upper tract TCC is significantly higher, even 100 times, than in non-endemic regions. A high incidence of urothelial cancer in end-stage BEN patients strongly suggests preventive nephro-ureterectomy in all end-stage patients with BEN treated with either transplantation or dialysis. Better understanding of the molecular mechanisms involved in carcinogenesis and tumor progression, has provided a large number of molecular markers of TCC, with a potential diagnostic and prognostic value. Markers that distinguish among TCC, normal urothelium, and benign urothelial conditions are potentially diagnostic, prognostic, and therapeutic targets. The geographic correlation and presence of AA-DNA adducts in both BEN and associated urothelial cancer, support the speculation that these diseases share a common etiology. Dietary exposure to AA is a significant risk factor for BEN and its attendant transitional cell cancer. These are cases of well-known AA induced urothelial carcinoma, and could be detected worldwide. The presence of more than one risk factors is possible and it is important to test etiological hypotheses in different endemic foci, preferably as a multicentric research.

Beta2-microglobulin and alpha1-microglobulin as markers of Balkan endemic nephropathy, a worldwide disease.

BACKGROUND: Urine beta2-microglobulin (beta2-MG) was mainly used as a tubular marker of Balkan endemic nephropathy (BEN) but recently alpha1-microglobulin (alpha1-MG) was proposed for the diagnosis of BEN. In this study, the potential of urine beta2-MG, alpha1-MG, albumin, and total protein in the differentiation of BEN from healthy persons and patients with glomerulonephritis (GN) and nephrosclerosis (NS) was examined. METHODS: This study involved 47 patients with BEN, 36 with GN, 11 with NS, 30 healthy subjects from BEN families, and 46 healthy subjects from non-BEN families. RESULTS: In BEN patients area under the curve (AUC) for urine beta2-MG (0.828) and alpha1-MG (0.782) was higher than for urine albumin (0.740), but in GN patients AUC for urine protein (0.854) and albumin (0.872) was significantly higher than for the two low molecular weight proteins. AUC for all four urinary markers in NS patients was significantly lower than in BEN patients, ranging between 500 and 595. Median urine beta2-MG excretion in BEN patients was 17.5 times higher than in GN patients and 18.3 times higher than in controls; median alpha1-MG excretion was higher only 3.0 and 2.25 times, respectively. In the differentiation of BEN from healthy controls, beta2-MG had higher sensitivity and specificity at the cutoff levels (p < 0.001) than alpha1-MG (p < 0.05). In the differentiation of BEN from GN, beta2-MG was the best marker. CONCLUSION: All four urinary markers can be used for the differential diagnosis of BEN, beta2-MG being the best. Like in aristolochic acid nephropathy, beta2-MG seems to be an early marker of tubular damage in BEN.

Fifty years of research in Balkan endemic nephropathy: where are we now?

Despite broad investigations into the possible role of genetic factors, environmental agents and immune mechanisms, the etiology of Balkan endemic nephropathy (BEN) is only partially understood. An increased incidence of upper urothelial cancer in patients with BEN and in populations from endemic settlements has been demonstrated. Genetic studies have landed support for genetic predisposition to BEN. The similarity of the morphological and clinical pattern of BEN and Chinese herbs nephropathy has raised the possibility of a common etiologic agent, aristolochic acid (AA), described in 1969 by Ivic and confirmed by a recent study of AA-DNA adducts. Ochratoxin A (OTA) is studied extensively as the etiologic agent of BEN. Weathering of low-rank coals nearby the endemic villages produces water-soluble polycyclic aromatic hydrocarbons and aromatic amines, similar to metabolic products of acetaminophen, which has a causal relationship with analgesic nephropathy. AA is confirmed as the etiologic agent of BEN; however, it may not be the sole risk factor. More research is needed on the patterns of BEN over time and between different endemic places. Therefore, it is important to test etiological hypotheses in different endemic foci, preferably as a multicentric research. An international approach to solving the etiology of BEN is needed in the coming years. The geographic correlation and presence of AA-DNA adducts in both BEN and associated urothelial cancer support the hypothesis that these diseases share a common etiology.

Molecular markers in upper urothelial carcinoma associated to Balkan endemic nephropathy. Aristolochic acid as the major risk factor of the worldwide disease.

The role of aristolochic acid in the etiology of Balkan endemic nephropathy (BEN) and associated upper urothelial carcinoma (UUC) was recently confirmed. The aim of this study was to determine the marker(s) specific for BEN-associated UUC. A total of 82 patients with UUC (38 from the BEN region and 44 control tumors) were included in the study. The Ki-67 index in BEN tumors correlated with the grade and multifocality (p < 0.05), but in regression analysis, only the grade of BEN tumor. The p53 index was significantly higher in BEN than in control tumors (p < 0.05), as well as the alteration of p53 (p < 0.05). BEN low-stage tumors, tumors without limphovascular invasion (LVI), and tumors of the renal pelvis had a higher p53 index than the control tumors (p < 0.05, 0.01, 0.05, respectively). The Ki-67 index was higher in control tumors with high-stage and solid growth than in BEN UUC (p < 0.050, 0.005). The Ki-67 correlated with the grade, growth, stage, LVI, and multifocality of UUC on the best way, but not with the group. In regression analysis, only multifocality of UUC had predictive influence on Ki-67 activity (p < 0.001). P53 correlated with the grade, growth, and group (p < 0.05). This investigation identifies the p53 pathway as the specific cell cycle marker involved in BEN-associated UUC.

Visual assessment of endemic nephropathy markers relationship.

The aim of this paper was to assess relationship between possible endemic nephropathy (EN) markers visually by the CoPlot methodology, and to illustrate this promising data analysis approach. From 912 screened persons in 3 Croatian endemic villages, 25 persons were diagnosed as confirmed EN patients, 371 as non-EN, and the remainder were classified as suspected of having EN, or at risk. Data on 25 confirmed EN patients were matched with appropriate non-EN examinees. All records with missing data were excluded, resulting in 35 subjects with complete data on the 13 key EN variables for CoPlot mapping. CoPlot solution met the accepted goodness of fit measure thresholds. Result showed relationship between EN markers, identifying some nearly duplicated variables, and possible outliers needing some subsequent analysis.

Balkan Endemic Nephropathy and the Causative Role of Aristolochic Acid.

Balkan endemic nephropathy is a chronic tubulointerstitial disease with insidious onset, slowly progressing to end-stage renal disease and frequently associated with urothelial carcinoma of the upper urinary tract (UTUC). It was described in South-East Europe at the Balkan peninsula in rural areas around tributaries of the Danube River. After decades of intensive investigation, the causative factor was identified as the environmental phytotoxin aristolochic acid (AA) contained in Aristolochia clematitis, a common plant growing in wheat fields that was ingested through home-baked bread. AA initially was involved in the outbreak of cases of rapidly progressive renal fibrosis reported in Belgium after intake of root extracts of Aristolochia fangchi imported from China. A high prevalence of UTUC was found in these patients. The common molecular link between Balkan and Belgian nephropathy cases was the detection of aristolactam-DNA adducts in renal tissue and UTUC. These adducts are not only biomarkers of prior exposure to AA, but they also trigger urothelial malignancy by inducing specific mutations (A:T to T:A transversion) in critical genes of carcinogenesis, including the tumor-suppressor TP53. Such mutational signatures are found in other cases worldwide, particularly in Taiwan, highlighting the general public health issue of AA exposure by traditional phytotherapies.

Contribution to the definition of diagnostic criteria for Balkan endemic nephropathy.

BACKGROUND: Diagnostic criteria for Balkan endemic nephropathy (BEN) have not been precisely established. In the present study the predictive value of variables previously proposed as diagnostic criteria for BEN was examined. METHODS: The study involved 182 patients: 98 patients with BEN, 57 patients with other kidney diseases (20 with glomerulonephritis, 17 with tubulointerstitial diseases and 20 with hypertensive nephrosclerosis) and 27 healthy subjects. The BEN group comprised patients who fulfilled criteria for BEN and suspected BEN, together with patients with proteinuria and at least two tubular abnormalities or one tubular abnormality and a history of urothelial tumour. Demographic, clinical, laboratory and ultrasound variables of examined groups were combined in univariate/multivariate logistic regression analysis. RESULTS: Out of 28 analysed variables only urine alpha1-microglobulin (MG) and kidney length were selected as significant predictors in differentiating BEN from other kidney diseases and healthy controls. Using ROC curves the cutoff values of these variables and proteinuria and kidney volume, variables collinear with them, were found. Moderate sensitivity and specificity characterized all these cutoff values except for proteinuria, which provided high sensitivity and specificity in combination of BEN and healthy persons. The predictive value of different combinations of selected variables was not significantly different from the predictive value of each variable individually. CONCLUSIONS: Proteinuria, urine alpha1-MG, kidney length and volume were selected as significant predictors of BEN. Variables related to kidney failure as well as several tubular disorders (urine specific gravity, FENa and TRP) had an insignificant predictive value and could not be used for differential diagnosis of BEN.

Balkan endemic nephropathy: a still unsolved puzzle.

Balkan endemic nephropathy (BEN) is a chronic tubulointerstitial renal disease, occurring in certain regions in 5 countries of the Balkan peninsula. Its etiology is largely unknown, though several hypotheses have been formulated and are discussed in this review. In several cases, etiological hypotheses (e.g., viral, ochratoxin or trace element involvement) are verified only in local endemic areas and can not be confirmed when tested elsewhere. Only certain families in the endemic areas are affected. An exposure of at least 20 years to the unknown factors in the endemic areas seems to be mandatory for the development of the disease, but a genetic predisposition to this disease also seems to be mandatory. Prominent clinical features are severely shrunken kidneys, a more severe anemia relative to the level of renal function, and a slow progression to end-stage renal failure. An international approach to solving the etiological and pathogenetic enigma of BEN is needed in the coming years. It is also time to reevaluate other chronic, slowly progressive tubulointerstitial nephropathies diagnosed elsewhere in the world and to search for possible etiological similarities with BEN.

Balkan endemic nephropathy and associated urothelial cancer.

Balkan endemic nephropathy (BEN) is a chronic tubulointerstitial disease described only in some rural parts of southeastern Europe. One of its most peculiar characteristics is a strong association with upper urothelial cancer (UUC). BEN-related UUC has the same histological features as other forms of UUC in general, but is more frequently bilateral, less frequently affects the bladder and has a sex ratio close to 1. BEN and BEN-associated UUC share the same etiology. Over time, incidence of these conditions has been declining. Since BEN was first described, around half a century ago, socioeconomic changes (in housing, farming, living standards, etc.) have been profound and have obscured the factors responsible for the observed reduction in incidence. Whatever the causes of BEN, the disease might not be restricted only to southeastern Europe. Rather, the intensity of exposure to risk factors for BEN and, consequently, clustering of cases has more likely determined our knowledge of topographical distribution of an etiological entity that is much more widespread, or that might even be ubiquitous in its sporadic form.