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Aristolochic acid (AA) is a potent dietary cytotoxin and carcinogen, and an established etiological agent underlying severe human nephropathies and associated upper urinary tract urothelial cancers, collectively designated aristolochic acid nephropathy (AAN). Its genome-wide mutational signature, marked by predominant A:T > T:A transversions occurring in the 5′-CpApG-3′ trinucleotide context and enriched on the nontranscribed gene strand, has been identified in human upper urinary tract urothelial carcinomas from East Asian patients and in experimental systems. Here we report a whole-exome sequencing screen performed on DNA from formalin-fixed, paraffin-embedded renal cell carcinomas (RCC) arising in chronic renal disease patients from a Balkan endemic nephropathy (EN) region. In the EN regions, the disease results from the consumption of bread made from wheat contaminated by seeds of Aristolochia clematitis, an AA-containing plant. In five of eight (62.5%) tested RCC tumor specimens, we observed the characteristic global mutational signature consistent with the mutagenic effects of AA. This signature was absent in the control RCC samples obtained from patients from a nonendemic, metropolitan region. By identifying a new tumor type associated with the AA-driven genome-wide mutagenic process in the context of renal disease, our results suggest new epidemiological and public health implications for the RCC incidence worldwide, particularly for the high-risk regions with unregulated use of AA-containing traditional herbal medicines.

Endemic nephropathy is a syndrome that comprises two entities: chronic interstitial nephropathy and urothelial cell cancers predominantly of the upper urinary tract. The etiological agent for the disease is aristolochic acid, a compound found in the plants of Aristolochia spp. The development of urothelial cancers is characterized by the formation of aristolactam DNA adducts leading to mutations, predominantly A: T-T: A transversions. In order to comprehensively understand the gene regulation programs in upper urothelial cancers we performed integrated miRNA and mRNA expression profiling of paired tumours and unaffected urothelium samples. The obtained data will help us to understand the carcinogenesis caused by aristolochic acid and might be the source for the design of a diagnostic biomarker.

Endemic (Balkan) nephropathy (EN) is a chronic tubulointerstitial nephropathy frequently associated with upper urothelial cancer exclusively affecting farming villagers [1–3]. Based on our results, EN is considered to be an environmental form of aristolochic acid nephropathy (AAN) [1–3]. AAN was first reported in 1993 in Belgium and subsequently more AAN cases were reported worldwide as AA has been an integral part of traditional herbal medicines [4]. The extent of this problem was recently documented in Taiwan where precise data on prescriptions of herbal products containing AA is available [5]. Aristolochia spp. has been used for more than 2,000 years in the practice of traditional medicine and European physicians were familiar with the use of this plant as well. After its intrinsic toxicity became known, importing Aristolochia herbs was banned in many countries, including Croatia. Nevertheless, products containing AA remain a part of traditional medicine and are sold in many countries that do not have strict control protocols. Recently we reported that AA DNA adducts were present in 95 % of patients with EN who underwent surgery for upper urothelial cancers [2] and affirmed the idea that bread contaminated with AA might be the cause of EN [6, 7]. However, the causative relationship between AA and EN again raised the question whether bread intake is the only route of ingestion or whether AA was ingested also in EN as a part of folkloric medicine. Gluhovschi et al. [8] reported that although therapeutic remedies based on AA products are used in the EN affected area in Romania, no relationship between these remedies and the development of EN or of tumors was observed. However, they used HPLC for detection of AA in plasma, which is less sensitive than the mass spectrometry we recently used in tissue samples [1, 2]. In addition, when re-analyzing their data, it does appear that AA was used more frequently in the endemic area. In our opinion, this leaves the question whether herbal tea may play a role in EN still unanswered. In our preliminary study we failed to find any evidence in the group of 1,041 Croatian farmers that herbal tea or traditional medicine use is related to EN [9]. The observed differences between

Objective:Data on prevalence of metabolic syndrome (MS) in rural regions are still inconclusive and several reports showed it to be higher in urban than in rural areas. The aim of this study was to determine the prevalence of MS in a rural, continental Croatian part comparing 3 most widely used defi

Munchausen syndrome is a factitious disorder with predominantly physical signs and symptoms, resulting from the patient’s high motivation for assuming a sick role, without any external incentives or boundaries. We report the case of a young female patient with factitious proteinuria in the nephrotic range and a fairly eventful medical history. After performing many expensive and unnecessary investigations and procedures, the real origin of the proteinuria was determined ; it was found to be caused by the patient carefully adding calibrated egg albumin to her urine samples. This discovery roused suspicions about multiple, non-corroborated conditions from her history (e.g., multiple miscarriages, breast cancer, and thyroid disorders).The diversity of diseases presented by a single Munchausen patient tends to be bizarre, and thus is a challenge for health care providers to diagnose the condition. Teamwork is therefore of the utmost necessity to diagnose Munchausen syndrome.

Objective:Chronic kidney disease(CKD) is established CV risk factor, and already early renal impairment(RI) increases risk for hypertension(HT) and loss of renal function. It was reported that blood pressure (BP) and metabolic derangements are associated with glomerular hyperfiltration(GHF), and GHF

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.

Background: Hypertension is not considered to be a characteristic of endemic nephropathy (EN). Recent observations suggested that it might be more prevalent than it was reported before. Aim: The aim of our study was to analyze prevalence, treatment and control of hypertension in a Croatian endemic area. Methods: In the present cross-sectional study, 1,602 farmers were enrolled, 1,246 from EN and 356 from control villages. Epidemiological and medical histories were taken and clinical and laboratory examinations performed for kidney function. Blood pressure was measured following the ESH/ESC guidelines. Results: The prevalence of hypertension in EN villages was higher than in control (50.8 vs. 46.5%, p = 0.005). There was no difference in overall treatment, control of all and treated hypertensives between the villages. In all villages, women were treated more than men (EN 41.6 vs. 28.4%, p < 0.001; control 46.4 vs. 27.3%, p < 0.001), but better control of treated was achieved in men (EN 24.7 vs. 17.4%, p = 0.002; control 29.6 vs. 15.5%, p = 0.002). Women had lower income and level of education than men (p < 0.01). Conclusion: Hypertension is highly prevalent in endemic villages. In all villages, men had better blood pressure control despite being treated less. This finding could be explained by poorer education and income in women.

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.

Anesthesiologists often work extended duty shifts that result in acute and chronic sleep loss and circadian disruption. Stress caused by sleep deprivation, together with excessive workload could contribute to acute increases in blood pressure (BP) and sympathetic nervous system activity. Non-dipping pattern of BP is considered an additional risk factor for cardiovascular events and target organ damage. We hypothesized that there would be significant changes of cardiovascular parameters when comparing work on call during the 24-hour in-hospital shift (24-HD) versus ordinary working day (8-HD) combined with changes of dipping pattern and altered diurnal cortisol secretion, measured by salivary cortisol (SC). Following local Medical Ethics Committee approval, 12 out of 36 staff anesthesiologists (8 male, 4 female), 33–61 years old, participated in this study. Ambulatory BP monitor was used for noninvasive 24-hour ambulatory BP and heart rate (HR) monitoring. Each participant was monitored continuously during the 8-HD, as well as during the 24-HD. Saliva for analysis of cortisol levels was collected six times a day (at 8 am, 11 am, 2 pm, 5 pm, 8 pm, and 11 pm) both during 8-HD and on 24-HD. There was a significant decrease in number of diastolic dippers on call vs. diastolic dippers on ordinary working day (4/12 vs. 10/12, p=0.036), and non significant decrease of systolic dippers (3/12 vs. 7/12, p=0.214). There were no significant differences in SC values between 8-HD and 24-HD at all observed time points. However, the SC values measured during the night were markedly elevated on both days compared with reference values and the shapes of SC curves were altered. The lack of diastolic BP dipping could be more sensitive indicator of stress among staff anesthesiologists than systolic BP dipping. The shape of SC diurnal curve in terms of elevated night values could be another indicator of their chronic fatigue.

The profession of anaesthesiologist is demanding and potentially hazardous. Extended work shifts combined with intensive work load may adversely affect physicians' performance. The aim of this study was to explore the impact of a single in- hospital 24 h shift on the cognitive and psychomotor performance of anaesthesiologists in a surgical emergency department. Following ethical and institutional approval, 11 staff anaesthesiologists [six men, five women, age 48 (35-50), years of experience 17 (7-20), median (range)] successfully completed the study protocol. Four computer-generated psychological tests (CRD, Complex Reactionmeter Drenovac, Croatia) consisting of light signal position discrimination (CRD 311), simple visual orientation (CRD 21), simple arithmetic operations (CRD 11), and complex psychomotor coordination (CRD 411) were used to measure objective parameters of cognitive and psychomotor performance at four time points (D1 = 8:00 a.m., D2 = 3:00 p.m., D3 = 11:00 p.m. ; and D4 = 7:00- 8:00 a.m. next day) during the 24 h working day. The control testing on an ordinary working day was performed at two time points (C1 = 8:00 a.m., C2 = 3:00 p.m.). Three parameters were recorded: total test solving time (TTST), total variability, and total number of errors for all four tests. TTST was significantly impaired during the 24 h shift in all tests, and TTST was prolonged in CRD 21 test at different time points from 1.6 +/- 1.4 to 5.5 +/- 1.6 s compared with the control (F = 6.39, P = 0.001). The reaction times were prolonged from 1.3 +/- 1.8 to 5.4 +/- 1.2 s (F = 3.49, P = 0.009) in CRD 311, from 3.8 +/- 9.0 to 34.3 +/- 5.8 s (F = 5.05, P = 0.002) in CRD 11 TTST, and from 0.8 +/- 3.0 to 16.3 +/- 8.6 s (F = 2.67, P = 0.034) in CRD 411. Total variability was significantly altered during the 24 h shift only in CRD 411 (F = 2.63, P = 0.036). There was no difference in the total number of errors between the 24 h shift and the ordinary working day. Anaesthesiologists' 24 h working day in the emergency department altered cognitive and psychomotor function in comparison with ordinary working days. Speed, reliability and mental endurance (measured by TTST) were significantly impaired in all four tests. Stability and reaction time (measured by total variability) were only slightly impaired. Paradoxically, attention and alertness (measured by total number of errors) were not adversely affected. In conclusion, anaesthesiologists' psychomotor performance was impaired during the single 24 h shift.

Objective:Arterial stiffness (AS) is an independent risk factor of cardiovascular mortality in general and haemodialysis (HD) population. Endemic (Balkan) nephropathy (EN) is a chronic tubulointerstitial salt wasting nephropathy characterized with later onset of arterial hypertension (AH) which coul

The role of adiponectin in hypertension is still a matter of debate. Obtained conflicting results could be mostly explained with diversity of subjects included in different studies. Our aim was to analyze association of adiponectin with blood pressure (BP) in a group of normotensive and untreated hypertensive subjects. Participants (N = 257) were selected from a random sample of 2487 subjects enrolled in an observational cross-sectional study. Subjects with diabetes and chronic kidney diseases were excluded. BP was measured using Omron M6 device following ESH/ESC guidelines. Adiponectin concentration was determined by ELISA. There were no differences in adiponectin values (mg/L) between hypertensives and normotensives (median 9.75; iqr: 7.44–17.88 vs 11.35; iqr: 7.43–12.63; P = 0.17). On univariate linear regression adiponectin was not associated with systolic or diastolic BP (P > 0.05). Furthermore, multivariate analysis did not show significant contribution of log-transformed adiponectin either to systolic (β = −0.040; P = 0.43) or diastolic BP (β = 0.066; P = 0.33). In our group of normotensives and untreated hypertensives with normal kidney function adiponectin was not associated with BP even after adjustment for other risk factors. Our results and conclusions should not be extrapolated to subjects with other characteristics.

Dietary intake of Aristolochia plants containing aristolochic acid (AA, IARC Group 1 carcinogen), leads to renal disease and to recurrent urinary tract transitional cell carcinomas (TCC). Mutation screens identified frequent A>T transversions, alterations underrepresented in other cancer types, as the signature of AA exposure. AA-associated TCC is a preventable cancer yet millions are estimated at risk due to unregulated medicinal use of Aristolochia. Here we present an ultra-low coverage modification of whole-exome sequencing (ULC-WES) as a novel molecular epidemiology method that reliably detects the AA mutation signature in small amounts of DNA from FFPE tumor tissues and can identify new exposed populations. DNA was isolated from paraffin sections of 18 TCC tumors from endemic nephropathy (EN) patients from regions in Croatia and Bosnia linked to exposure to AA. Four non-EN upper tract TCCs were used as controls. 250 ng of DNA was used for sequencing libraries and exome enrichment (Nimblegen, Roche). Libraries were sequenced on Illumina HiSeq2500 at 10-12x coverage. After data processing, GATK was used to call single nucleotide variants (SNV). To assess platform independence, 7 samples were run on SOLID 5500XL (Life), using proprietary chemistry and pipeline for data processing and variant calling. Normal genetic variants were filtered out after SNV annotation. ULC-WES at 10x identified the A>T predominance in 17 of 18 EN tumors (94%), and at T pattern in 14 (78%) samples. In contrast, the A>T presence was T mutations in the genome (R^2=0.83 for mutation count correlated with the chromosome length) but also identified recurrent non-synonymous A>T mutations in tumor suppressors and oncogenes including TP53, ARID1A, BRCA1/2, AXIN1, GATA3 and NRAS, DNMT1, FLT3, CSF1R and PIK3CA. We next detected recurrent A>T mutations in a number of histone and DNA modifiers suggesting epigenetic deregulation of genome activity in the AA-associated TCCs. In 2 patients, pairs of distinct TCCs from the same surgery had markedly overlapping mutation patterns (624 SNVs shared between same-side pelvic and ureteric tumors in one case, and 191 shared between renal pelvic and bladder tumors in the other). Thus, ULC-WES provided evidence supporting the concept of tumor cell seeding along the urinary tract as a mechanism of TCC spread and recurrence. We conclude that ULC-WES is a sensitive and specific method to identify AA exposure in tumor tissue and is directly applicable to identification of new populations at risk. The method can ultimately facilitate evidence-based preventive measures in the affected regions. Acknowledgments: Genetic Services Platform, IARC; Genome Technology Center, NYU Langone Medical Center; NIH P01 ES04068, NIH Fogarty International Center 1R03TW007042, Croatian Ministry of Science. Citation Format: Xavier Castells, Sandra Karanovic, Magali Olivier, Maude Ardin, Florence Le Calvez-Kelm, Catherine Voegele, James McKay, Damir Dittrich, Karla Medak Tomic, Kathleen Dickman, Arthur P. Grollman, Bojan Jelakovic, Jiri Zavadil. Ultra-low coverage exome sequencing of FFPE tumor specimens identifies exposure to carcinogenic aristolochic acid. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 305. doi:10.1158/1538-7445.AM2014-305

Objective: Subjects with low birth weight and those born small for gestational age (SGA) have higher risk for hypertension, kidney disease and cardiovascular events as adults. Arterial stiffness (AS) is a marker of vascular ageing. Our aim was to analyse whether birth weight (BW) determines AS in young men. Design and Method: Normotensive non-treated, healthy adult men (n = 159 ; age 21.27 ± 0.9 years) were enrolled. Birth parameters were obtained from medical records. SGA was defined as BW under 10th percentile for gestational age and obesity as body mass index (BMI) > 25 kg/m2. Subjects were divided into the four groups: non-obese with normal BW (N = 55), obese with normal BW (N = 43), non-obese SGA (N = 40), obese SGA (N = 21). Blood pressure (BP) was measured with oscillometric device following ESH guidelines while aortic BP (SBPAO) and AS markers (pulse wave velocity (PWV), brachial and aortic augmentation index (AIxBR, AIxAO)) were determined using Arteriograph. Results: Overall, SGA subjects had higher brachial systolic BP (SBP) (p = 0.021) and HR (p = 0.003) values than those born with normal BW. However, there were no differences in PWV, AIxBR, AIxAO and SBPAO between subjects with SGA and normal BW(p > 0.05). When subjects were further divided according to the BMI, we found that SBP was the highest in obese SGA subjects (p = 0.035). Obese SGA had higher SBPAO and PP values than non-obese SGA (p = 0.017 ; p = 0.036, respectively) and non-obese subjects with normal BW (p = 0.027 ; p = 0.001, respectively). There were no significant correlations between BMI and AS markers in all subgroups. Conclusion: In young men, birth weight influences brachial SBP and HR, but according to our results it does not increase arterial stiffness determined with this technique. It might be concluded that higher values of SBP and HR observed in this group could be rather markers of hyperkinetic circulation than consequences of early vascular ageing. Obesity additionally affects BP values increasing even central SBP. SGA subjects should be informed about importance of life-style changes and monitored very closely.

Objective: Recent reports have shown that concurrent obesity influences blood pressure (BP) phenotype in children born small for gestational age (SGA). Our aim was to investigate the impact of obesity and birth weight on blood pressure (BP) values in young adult men. Subjects and Methods: Young, healthy, normotensive non-treated adult men (N = 185 ; mean age 21.29 + 0.9) years were enrolled. Birth parameters were obtained from medical records. SGA was defined as birth weight (BW) under 10th percentile for gestational age and obesity as BMI > 25 kg/m2. According to the presence or absence of obesity and BW the subjects were divided into four groups: (1) non- obese with normal BW (N = 50), (2) obese with normal BW (N = 40), (3) non-obese SGA (N = 67), (4) obese SGA (N = 28). BP was measured using oscillometric manometer (Omron M6) and Spacelab 90207 device following the ESH/ESC guidelines, and the average 24-hour, daytime and nighttime systolic and diastolic BP values, blood pressure load (BPL), dipper vs. non-dipper status, BP variability (estimated as standard deviation), heart rate and pulse pressure (PP) values were analysed. Results: Systolic BP, 24-hour BP variability and pulse pressure were significantly higher in subjects with SGA than in those born with normal BW (p 0.05). Conclusion: In addition to birth weight and shorter pregnancy duration obesity concurrently and significantly determines systolic BP in young normotensive men and may contribute to the early vascular ageing. Our findings are in agreement with the results obtained in children and point to a need for aggressive implementation of healthy lifestyle as early as possible in the population subset particularly prone to develop hypertension.