Bojan Jelaković

Aristolochic acid and the etiology of endemic (Balkan) nephropathy

Endemic (Balkan) nephropathy (EN), a devastating renal disease affecting men and women living in rural areas of Bosnia, Bulgaria, Croatia, Romania, and Serbia, is characterized by its insidious onset, invariable progression to chronic renal failure and a strong association with transitional cell (urothelial) carcinoma of the upper urinary tract. Significant epidemiologic features of EN include its focal occurrence in certain villages and a familial, but not inherited, pattern of disease. Our experiments test the hypothesis that chronic dietary poisoning by aristolochic acid is responsible for EN and its associated urothelial cancer. Using 32P-postlabeling/PAGE and authentic standards, we identified dA-aristolactam (AL) and dG-AL DNA adducts in the renal cortex of patients with EN but not in patients with other chronic renal diseases. In addition, urothelial cancer tissue was obtained from residents of endemic villages with upper urinary tract malignancies. The AmpliChip p53 microarray was then used to sequence exons 2–11 of the p53 gene where we identified 19 base substitutions. Mutations at A:T pairs accounted for 89% of all p53 mutations, with 78% of these being A:T → T:A transversions. Our experimental results, namely, that (i) DNA adducts derived from aristolochic acid (AA) are present in renal tissues of patients with documented EN, (ii) these adducts can be detected in transitional cell cancers, and (iii) A:T → T:A transversions dominate the p53 mutational spectrum in the upper urinary tract malignancies found in this population lead to the conclusion that dietary exposure to AA is a significant risk factor for EN and its attendant transitional cell cancer.

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.

TP53 Mutational signature for aristolochic acid: an environmental carcinogen

This study was designed to establish the TP53 mutational spectrum of aristolochic acid (AA), examined in the context of endemic (Balkan) nephropathy, an environmental disease associated with transitional cell (urothelial) carcinomas of the upper urinary tract (UUC). Tumor tissue was obtained from residents of regions in Bosnia, Croatia and Serbia where endemic nephropathy has been prevalent for over 50 years. Fifty‐nine TP53 mutations were detected in 42 of the 97 tumors analyzed. Mutational spectra were dominated by A:T to T:A transversions with the mutated adenines located almost exclusively on the nontranscribed strand. This marked strand bias is attributed to selective processing of aristolactam‐dA adducts by transcription‐coupled nucleotide excision repair. Hotspots for A:T to T:A mutations include codons 131 and 179 and the 5′‐AG acceptor splice site of intron 6. The unique TP53 mutational signature for AA identified in this study can be used to explore the hypothesis that botanical products containing this human carcinogen and nephrotoxin are responsible, in part, for the high prevalence of UUC and chronic renal disease in countries where Aristolochia herbal remedies traditionally have been used for medicinal purposes.

p53 mutations as fingerprints for aristolochic acid: an environmental carcinogen in endemic (Balkan) nephropathy

The activation of protooncogenes and inactivation of tumor suppressor genes are considered to be the main molecular events in the multistep process of carcinogenesis. Mutations of the TP53 tumor suppressor gene have been found in nearly all tumor types and are estimated to contribute to more than 50% of all cancers. Most mutations lead to the synthesis of highly stable, inactive proteins that accumulate in the nucleus of cancer cells. Among the 393 codons of the human p53 gene, 222 are targets of 698 different types of mutations. Alterations of codons 175, 248, 273 and 282 correspond to 19% of all mutations and are considered general hot spot mutations. Dietary exposure to aristolochic acid (AA), an established nephrotoxin and human carcinogen found in all Aristolochia species was shown to be the causative agent of aristolochic acid nephropathy (previously called Chinese herbs nephropathy). This syndrome is characterized by proximal tubular damage, renal interstitial fibrosis, slow progression to the end stage renal disease and a high prevalence of upper urinary tract urothelial carcinoma (otherwise a highly unusual location). AA preferentially binds to purines in DNA and is associated with a high frequency of A-->T transversions in the p53 gene. Rats treated with AA develop A:T-->T:A mutations in codon 61. The pathological and clinical features of endemic (Balkan) nephropathy closely resemble those associated with aristolochic acid nephropathy except for the slower progression to end stage renal disease and longer cumulative period before the appearance of urothelial cancer. Recently, we reported the presence of AA-DNA adducts in renal cortex and A-->T p53 mutations in tumor tissue of patients from Croatia and Bosnia with endemic nephropathy. These data support the hypothesis that dietary exposure to AA is a major risk factor for endemic (Balkan) nephropathy.

Diagnostic criteria for Balkan endemic nephropathy: proposal by an international panel.

Balkan endemic nephropathy (BEN) is a familial chronic tubulointerstitial disease with insidious onset and slow progression to terminal renal failure. Diagnostic criteria for BEN have been described more than 40 years ago. Research groups on BEN use one of at least three described lists of criteria. Comparison of studies using such criteria is difficult, and a recent meeting of investigators (Zagreb, October 2006) has suggested that unified criteria have to be elaborated. In this paper, an International Panel of BEN Investigators agreed on criteria appropriate to epidemiologic studies and clinical investigations of BEN. A screening procedure of BEN in endemic settlements is proposed.

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.

Chapter 7 Aristolochic Acid Nephropathy: An Environmental and Iatrogenic Disease

Publisher Summary This chapter reviews the molecular and clinical toxicology of aristolochic acid (AA), nephrotoxic chemical carcinogen. Recently, AA, a principal component of all Aristolochia sp., is shown to be the toxin responsible for the clinical syndromes known as Chinese herb nephropathy (CHN) and endemic (Balkan) nephropathy (EN). The epidemiology and pathophysiology of CHN and EN are reviewed extensively and the association of these diseases with human cancer is the subject of several comprehensive reports. Both disorders are associated with a high incidence of urothelial (transitional cell) cancer and appear to constitute a single disease entity, designated aristolochic acid nephropathy (AAN). The dramatic revelation that AA is a powerful nephrotoxin and carcinogen for humans drew attention to the worldwide distribution and extensive use of Aristolochia sp. as herbal remedies. The subsequent reports described almost 200 patients outside of Belgium in whom chronic renal failure followed ingestion of Aristolochia herbs. In addition based on the traditional use of Aristolochia in herbal remedies, the chapter posits that AAN represents a long-overlooked iatrogenic disease and an international public health problem of considerable magnitude.

Management of the hypertensive patient with elevated heart rate: Statement of the Second Consensus Conference endorsed by the European Society of Hypertension.

In June 2015, a panel of experts gathered in a consensus conference to plan updating recommendations on the management of the hypertensive patient with elevated heart rate (HR), previously released in 2006. The issues examined during that meeting and further discussed by the participants during the following months involved the assessment of HR, the relevance of HR as a cardiovascular risk factor, the definition of tachycardia and the treatment of the hypertensive patient with high HR. For the measurement of resting HR the panel experts recommended that scientific investigations focusing on HR should report information on length of resting period before measurement, information about temperature and environment, method of measurement, duration of measurement, number of readings, time interval between measurements, body position and type of observer. According to the panellists there is convincing evidence that HR is an important risk factor for cardiovascular disease and they suggest to routinely include HR measurement in the assessment of the hypertensive patient. Regarding the definition of tachycardia, the panellists acknowledged that in the absence of convincing data any threshold used to define tachycardia is arbitrary. Similarly, as there are no outcome studies of HR lowering in tachycardia hypertension, the panellists could not make practical therapeutic suggestions for the management of such patients. However, the experts remarked that absence of evidence does not mean evidence against the importance of tachycardia as a risk factor for cardiovascular disease and that long-term exposure to a potentially important risk factor may impair the patients prognosis. The main aims of the present document are to alert researchers and physicians about the importance of measuring HR in hypertensive patients, and to stimulate research to clarify unresolved issues.

Blood pressure and low-density lipoprotein-cholesterol lowering for prevention of strokes and cognitive decline: a review of available trial evidence.

Background and objectives: It is well established by a large number of randomized controlled trials that lowering blood pressure (BP) and low-density lipoprotein cholesterol (LDL-C) by drugs are powerful means to reduce stroke incidence, but the optimal BP and LDL-C levels to be achieved are largely uncertain. Concerning BP targets, two hypotheses are being confronted: first, the lower the BP, the better the treatment outcome, and second, the hypothesis that too low BP values are accompanied by a lower benefit and even higher risk. It is also unknown whether BP lowering and LDL-C lowering have additive beneficial effects for the primary and secondary prevention of stroke, and whether these treatments can prevent cognitive decline after stroke. Results: A review of existing data from randomized controlled trials confirms that solid evidence on optimal BP and LDL-C targets is missing, possible interactions between BP and LDL-C lowering treatments have never been directly investigated, and evidence in favour of a beneficial effect of BP or LDL-C lowering on cognitive decline is, at best, very weak. Conclusion: A new, large randomized controlled trial is needed to determine the optimal level of BP and LDL-C for the prevention of recurrent stroke and cognitive decline.

Continuation of the ESH-CHL-SHOT trial after publication of the SPRINT: rationale for further study on blood pressure targets of antihypertensive treatment after stroke.

Alberto Zanchetti, Lisheng Liu, Giuseppe Mancia, Gianfranco Parati, Guido Grassi, Marco Stramba-Badiale, Vincenzo Silani, Grzegorz Bilo, Giovanni Corrao, Antonella Zambon, Lorenza Scotti, Xinhua Zhang, Ting Rui Guan, Yuqing Zhang, Xuezhong Zhang, Eivind Berge, Josep Redon, Krzysztof Narkiewicz, Anna Dominiczak, Peter Nilsson, Margus Viigimaa, Stéphane Laurent, Enrico Agabiti-Rosei, Zhaosu Wu, Dingliang Zhu, José Luis Rodicio, Luis Miguel Ruilope, Nieves Martell-Claros, Fernando Pinto Roland E. Schmieder, Michel Burnier, Maciej Banach, Renata Cifkova, Csaba Farsang, Alexandra Konradi, Irina Lazareva, Yuriy Sirenko, Maria Dorobantu, Arman Postadzhiyan, Rok Accetto, Bojan Jelakovic, Dragan Lovic, Athanasios J. Manolis, Philippos Stylianou, Dror Dicker, Gangzhi Wei, Chengbin Xu, Hengge Xie, Antonio Coca, John O’Brien, Gary Ford, on behalf of the ESH-CHL-SHOT trial investigators