Balkan Endemic Nephropathy and the Causative Role of Aristolochic Acid †,‡ § Bojan Jelakovic, MD, PhD * Zivka Dika, MD, PhD * Volker M. Arlt, PhD Marie Stiborova, PhD Nikola M. Pavlovic, MD, PhD jj Jovan Nikolic, MD ¶ Jean-Marie Colet, PhD # Jean-Louis Vanherweghem, MD, PhD ** and Joelle€ L. Nortier, MD, PhD**,†† Summary: 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 aristolac- tam-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 carcino- genesis, including the tumor-suppressor TP53. Such mutational signatures are found in other cases worldwide, particu- larly in Taiwan, highlighting the general public health issue of AA exposure by traditional phytotherapies. Semin Nephrol 39:284−296 Ó 2019 Elsevier Inc. All rights reserved. Keywords: Balkan endemic nephropathy, aristolochic acid nephropathy, upper tract urothelial carcinoma, aristolo- chic acid, Aristolochia species *School of Medicine, University of Zagreb, Department of Nephrology, n 1993, the occurrence of a rapidly progressive form Hypertension, Dialysis and Transplantation, University Hospital of renal interstitial fibrosis associated with a weight Centre Zagreb, Zagreb, Croatia loss diet that included the ingestion of pulverized y I Department of Analytical, Environmental and Forensic Sciences, Medi- plant extracts used in traditional Chinese medicine led to cal Research Council-Public Health England Centre for Environment the description of a new toxic nephropathy.1 The identifi- and Health, King’s College London, London, United Kingdom zNational Institute for Health Research Unit, Health Impact of Envi- cation of aristolochic acid (AA) in these powders ronmental Hazards, King’s College London, in partnership with brought to attention the severe toxicity of certain species Public Health England and Imperial College London, London, of Aristolochia.2 Since then, secondary nephropathies United Kingdom resulting from the toxicity of plants containing AA have x Department of Biochemistry, Faculty of Science, Charles University, been described worldwide.3 The similarity between the Prague 2, Czech Republic jjSerbian Medical Society, Branch Nis,̌ Nis,̌ Serbia histologic aspects of this particular nephropathy and the {Clinical Centre Serbia, Belgrade, Serbia so-called Balkan endemic nephropathy (BEN) proved #Department of Human Biology and Toxicology, Faculty of Medicine instrumental in reviving an old hypothesis of the etiology and Pharmacy, University of Mons, Mons, Belgium of BEN.1,4 In 1969, Ivic5 suggested that the latter, occur- **Nephrology Department, Erasme Hospital, Universite Libre de ring in certain villages throughout the Danube Valley, Bruxelles, Brussels, Belgium yyLaboratory of Experimental Nephrology, Faculty of Medicine, might be caused by the chronic ingestion of the seeds of Universite Libre de Bruxelles, Brussels, Belgium Aristolochia clematitis, a common plant growing in the Financial support: Work at Charles University was supported by the wheat fields of these endemic regions. This hypothesis Grant Agency of the Czech Republic (GACR 17−12816S); work at now has been confirmed by the discovery of specific King’s College London was supported by the Wellcome Trust DNA adducts that are formed by the metabolites of AA (101126/Z/13/Z and 101126/B/13/Z), and in part by the National Institute for Health Research, Health Protection Research Unit, (aristolactams) in the renal tissue and the urothelial Health Impact of Environmental Hazards at King’s College Lon- tumors of those patients suffering from BEN, as well as don, in partnership with Public Health England and Imperial Col- in the initial cohort of Belgian patients.6-9 lege London. The views expressed are those of the authors and not Today, the term aristolochic acid nephropathy (AAN) necessarily those of the National Health Service, the National Insti- is used to include any form of toxic interstitial nephropa- tute for Health Research, the Department of Health and Social Care, or Public Health England. thy that is caused either by the ingestion of plants con- Conflict of interest statement: none. taining AA as part of traditional phytotherapies Correspondence to be addressed to Joelle€ Nortier, MD, PhD, (formerly known as Chinese herb nephropathy), or by Nephrology Department, Erasme Hospital, Route de Lennik 808, the environmental contaminants in food (BEN).10 1070 Brussels, Belgium. E-mail: [email protected] Although the initial Belgian cohort included more 0270-9295/ - see front matter than 100 patients, it is estimated that exposure to AA © 2019 Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.semnephrol.2019.02.007 affects 100,000 people in the Balkans (where the total 284 Seminars in Nephrology, Vol 39, No 3, May 2019, pp 284−296 Balkan endemic nephropathy and aristolochic acid 285 number of patients with kidney disease amounts to were recognized in the middle of the 20th century. Dis- approximately 25,000), 8 million people in Taiwan, and ease was observed only in harvesting farmers and more than 100 million in mainland China.3,11 Given the affected only certain villages. Family, or, more precisely, fact that the nephrotoxic effects of AA are irreversible household aggregation, was reported in all BEN coun- and that its carcinogenic effects may be very slow in tries (Bosnia and Herzegovina, Bulgaria, Croatia, Roma- manifesting itself after the patient’s initial exposure, nia, and Serbia) (Fig. 1A). An inherited pattern of the AAN and associated cancers are likely to become a disease was ruled out by the fact that BEN often affected major public health issue in the years to come.12 several members of the same household who were not The Aristolochia species is a genus of herbaceous, necessarily blood-related. At the World Health Organi- perennial plants that include more than 500 species. zation meeting that was held in 1964 in Dubrovnik, Cro- They are widespread in the warm regions of the Mediter- atia, the disease was named endemic nephropathy. The ranean, Africa, and Asia. In France, A clematitis or birth- average prevalence of diseased subjects in the past years wort, Aristolochia rotunda, and Aristolochia pistolochia has ranged between 2% and 5%. However, the preva- grow mainly in limestone soil and can be found on road- lence of farmers suspected to have BEN is much higher sides, in coppices, vineyards, and other agricultural and was reported to be 10% to 15%. There were no gen- areas. Furthermore, A clematitis is a parasitic plant that der differences, although a slight insignificant female grows alongside wheat in the local wheat fields in the predominance was found (1:1.2). Importantly, it was warm and humid regions of the Danube Valley.13 never reported in children, indicating that a long period The accidental ingestion of these species of Aristolo- of exposure to the environmental agent is needed. In past chia not only explains the existence of BEN, but also decades, the age when BEN patients started to receive clarifies incidents of severe livestock poisoning, occur- dialysis was shifted to older ages, raising the question of ring among horses in the Balkans and goats in Africa, whether the etiologic agent is still present or active. This respectively.5,14 In Spain, moreover, the regular con- is in concordance with the results obtained in Croatian sumption of an infusion with A pistolochia led to a case field surveys conducted between 2005 and 2015, when of chronic interstitial nephritis.15 neither new BEN nor new UTUC patients were detected In the past, Aristolochia were widely used in Western in some previously established BEN villages.21 Similar medicine.13 In fact, their first use to stimulate the expul- trends were observed in Serbia.22,23 sion of the placenta during childbirth was responsible for An early hallmark of BEN is proximal tubule damage, coining the name “Aristos lokos” or “excellent deliv- which is manifested as low-molecular-weight (tubular) ery.”16 Because the plants also were recommended for proteinuria and enzymuria that are in line with the patho- treating snake bites, Aristolochia were included in the logic findings: good preservation of glomeruli and a gradi- preparation of theriac. As a result, they were prescribed ent of tubular atrophy with severe interstitial fibrosis that for treating gout (Dutchman’s pipe). decreases in severity from outer-to-inner cortex. Two tar- In addition to the dramatic history of AA environmen- get tissues were identified in BEN patients: proximal renal tal exposure in the Balkans that