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Supplementary Figure legends

Supplementary Figure S1. Identification of AA signature by ultra-low coverage sequencing reads.

Supplementary Table S1: Clinicopathological and epidemiological patient data and urothelial tumor sample description. Supplementary Table S2: Complete list of annotated SBS identified in all LC-WES analyzed urothelial tumors (HiSeq2500 data only). Supplementary Table S3: GO and KEGG Classification of genes mutated by non-synonymous A:T>T:A mutations. Supplementary Table S4: List of cancer driver genes found recurrently mutated in the EN and Taiwan UTUC sample sets. Supplementary Table S5: GO and KEGG classification of the recurrently mutated cancer driver genes (see Figure 3B).

Supplementary Methods

Recurrent upper tract urothelial carcinomas (UTUCs) arise in the context of nephropathy linked to exposure to the herbal carcinogen aristolochic acid (AA). Here we delineated the molecular programs underlying UTUC tumorigenesis in patients from endemic aristolochic acid nephropathy (AAN) regions in Southern Europe. We applied an integrative multiomics analysis of UTUCs, corresponding unaffected tissues and of patient urines. Quantitative microRNA (miRNA) and messenger ribonucleic acid (mRNA) expression profiling, immunohistochemical analysis by tissue microarrays and exome and transcriptome sequencing were performed in UTUC and nontumor tissues. Urinary miRNAs of cases undergoing surgery were profiled before and after tumor resection. Ribonucleic acid (RNA) and protein levels were analyzed using appropriate statistical tests and trend assessment. Dedicated bioinformatic tools were used for analysis of pathways, mutational signatures and result visualization. The results delineate UTUC-specific miRNA:mRNA networks comprising 89 miRNAs associated with 1, 862 target mRNAs, involving deregulation of cell cycle, deoxyribonucleic acid (DNA) damage response, DNA repair, bladder cancer, oncogenes, tumor suppressors, chromatin structure regulators and developmental signaling pathways. Key UTUC- specific transcripts were confirmed at the protein level. Exome and transcriptome sequencing of UTUCs revealed AA-specific mutational signature SBS22, with 68% to 76% AA-specific, deleterious mutations propagated at the transcript level, a possible basis for neoantigen formation and immunotherapy targeting. We next identified a signature of UTUC- specific miRNAs consistently more abundant in the patients' urine prior to tumor resection, thereby defining biomarkers of tumor presence. The complex gene regulation programs of AAN-associated UTUC tumors involve regulatory miRNAs prospectively applicable to noninvasive urine-based screening of AAN patients for cancer presence and recurrence.

Objective. To assess the forearm skin microvascular reactivity and anthropometric parameters in randomly selected population and seek the differences between a group of people who had elevated blood pressure at the time of measurement and those who were normotensive. Design and method. Total of 148 randomly selected individuals (50 men/98 women) participated in this study. The exclusion criteria were less than 1l of urine collected at 24h, and creatinine values below the reference values. Forthy eight individuals included in this study had arterial hypertension (HT) and 44 had normal arterial blood pressure values (NT). Post-occlusive reactive hyperemia (PORH) in skin microcirculation following 1 minute of vascular occlusion period was assessed using laser Doppler flowmetry. Anthropometric parameters, blood pressure, heart rate were measured, and 24-hour urine was collected to sodium excretion and creatinine measurement. P< 0, 05 was considered statistically significant. Results. Sistolic (SBP), diastolic (DBP) and mean (MAP) arterial blood pressure were significantly higher, while PORH was significantly lower in HT group compared to NT individuals. HT had higher body mass index (BMI) and waist circumference, while there were no differences in heart rate between HT and NT group. In HT individuals, PORH was negatively associated with waist circumference (r=-0, 438), and positively associated with heart rate (r=0, 316). Sodium excretion was positively associated with DBP (r=0, 361). In NT group, PORH was negatively associated with SBP (r=-0, 362) and MAP (r=-0, 37). SBP and MAP were positively associated with BMI (r=0, 484, r=0, 312) and waist circumference (r=0, 421, r=0, 319). Sodium excretion was positively associated with BMI (r=0, 546) and waist circumference (r=0, 523). Analyzes of all individuals together showed that PORH was negatively associated with BMI (r=-0, 253), SBP (r=-0, 274) and MAP (r=-0, 24), while BMI was positively associated with SBP (r=0, 359), DBP (r=0, 219) and MAP (r=0, 312). Conclusions. High blood pressure attenuated microvascular function. Arterial blood pressure is related to obesity (BMI, waist circumference).

doi: 10.1097/01.hjh.0000571716.53390.4b

Objective:Our aim was to determine trends in salt and potassium intake in general rural population during the 7.5 years of follow-up and to analyze differences in results obtained using various equations.Design and method:Out of 1582 subjects (random rural sample) enrolled in ENAH study conducted in

Objective:Our aim was to analyze association of sodium and potassium intake on clinical course i.e. changes of blood pressure (BP) category in general population during the 7.5 years of follow up.Design and method:Out of 1582 subjects (random rural sample) enrolled in ENAH study conducted in 2008–20

Objective:Our aim was to analyze association of sodium and potassium intake with new onset chronic kidney disease (CKD) in general population during the 7.5 years of follow up.Design and method:Out of 1582 subjects (random rural sample) enrolled in ENAH study conducted in 2008–2010, 739 were invited

Balkan endemic nephropathy (BEN), an environmental form of aristolochic acid nephropathy is characterized with later onset and milder forms of hypertension (HT). Thus, we hypothesized that arterial stiffness progresses slower in BEN patients resulting in lower CV mortality. A total of 186 hemodialysed (HD) patients (90 BEN, 96 non-BEN; 67.3 + 13.0 years) were enrolled and followed-up for 25 months. Brachial blood pressure (BP) and pulse wave velocity (PWV) were determined before mid-week dialysis. BEN patients were older (72.1 ± 37.1 vs. 62.8 ± 15.1; p < 0.001), had shorter duration of HT prior commencement of HD than non-BEN patients (36 vs. 84 months; p < 0.001). There were no differences in BP, but BEN patients were treated with less antihypertensive drugs (p < 0.01). BEN patients had lower PWV values at baseline and at the end of follow-up period despite being chronologically older (p < 0.001). Baseline PWV > 10 m/s was associated with higher risk for CV mortality (aHR 1.8 [1.4, 2.4]). In multivariate analyses BEN was predictor of lower PWV. During the follow-up period significantly less CV deaths were observed in BEN vs. on-BEN patients (12 vs. 31; p = 0.001). CV mortality adjusted for other risk factors was significantly lower in BEN group (aHR 0.2 [0.1, 0.5]). Overall BEN patients had longer mean survival time on HD (22.3 vs. 18.2 months; p < 0.001). Observed slower vascular aging (i.e., lower PWV) in BEN patients compared to other ESRD patients is related to the later onset of HT and milder stages of HT during predialytic clinical course and better control of BP and phosphate during HD.

Objective: Our aim was to analyze sodium and potassium intake in obese normotensive and hypertensive subjects from rural area. Design and method: Out of 1582 subjects (random rural sample) enrolled in ENAH study conducted in 2008–2010, 739 were invited for the second visit in 2015 and 377 subjects with reliable both urine samples (urine creatinine in normal ranges) were included in further analyses s (80m 297w ; average age 52 ± 13.5). Blood pressure was measured three times (Omron M6) and mean values were calculated. Fasting blood and first morning (spot) urine samples were collected. Sodium and potassium were determined using Tanaka, Kawasaki and Intersalt equations. Potassium intake was further adjusted for intestinal intake (77%) and renal excretion (92%). Optimal, normal, high normal BP, untreated and treated hypertension (HT) were diagnosed in 20.9%, 15.3%, 12.9%, 19.1% and 31.5%, respectively. There were 21.2%, 35.3% and 42.8% subjects in the BMI 1 (30) group, respectively. Results: Significant differences in HT prevalence were observed among the BMI groups (46.9%, 63.6%, 80 %, respectively ; p = 0.001). Sodium excretion were significantly lower in BMI1 vs BMI3 groups (BMI < 0.0001). More than 10 g/day of salt intake was most seldom present in BMI1 and more frequently in BMI3 group (41.9%, 54.5%, 63.1%, respectively ; p < 0.05). Compared to normotensive in hypertensive subjects salt intake >10 g/day was significantly more frequently presented in overweight (X2 = 12.68 ; p = 0.0003) but not in obese subjects. Potassium intake was significantly higher in BMI3 than in BMI1 group (p < 0.001). No differences in Na/K ratio were observed among BMI groups. Conclusions: Higher salt intake contributes to high prevalence of hypertension and according to our results is more closely associated with hypertension in overweight than in obese subjects. No difference in Na/K ratio among BMI groups reflects high sodium and poor potassium intake in all subjects.

Introduction: Chronic kidney disease (CKD) is a significant public health problem and it is not possible to precisely predict its progression to terminal renal failure. According to current guidelines, CKD stages are classified based on the estimated glomerular filtration rate (eGFR) and albuminuria. Aims of this study were to determine the reliability of predictive equation in estimation of CKD prevalence in Croatian areas with endemic nephropathy (EN), compare the results with non-endemic areas, and to determine if the prevalence of CKD stages 3-5 was increased in subjects with EN. Materials and methods: A total of 1573 inhabitants of the Croatian Posavina rural area from 6 endemic and 3 non-endemic villages were enrolled. Participants were classified according to the modified criteria of the World Health Organization for EN. Estimated GFR was calculated using Chronic Kidney Disease Epidemiology Collaboration equation (CKD-EPI). Results: The results showed a very high CKD prevalence in the Croatian rural area (19%). CKD prevalence was significantly higher in EN then in non EN villages with the lowest eGFR value in diseased subgroup. Conclusions: eGFR correlated significantly with the diagnosis of EN. Kidney function assessment using CKD-EPI predictive equation proved to be a good marker in differentiating the study subgroups, remained as one of the diagnostic criteria for EN.

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.

Objective:Our aim was to analyze characteristics of subjects with new onset hypertension (NOHT) and impact of sodium and potassium intake on development of NOHT.Design and method:Out of 1582 subjects (random rural sample) enrolled in ENAH study conducted in 2008–2010, 739 were invited for the second

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 Dubočac. 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.

Improvements in agricultural practices in Croatia have reduced exposure to consumption of aristolochic acid- contaminated flour and development of endemic (Balkan) nephropathy. Therefore, it was hypothesized that Bosnian immigrants who settled in an endemic area in Croatia 15-30 years ago would be at lower risk of developing endemic nephropathy because of reduced exposure to aristolochic acid. To test this hypothesis, past and present exposure to aristolochic acid, proximal tubule damage as a hallmark of endemic nephropathy, and prevalence of CKD in Bosnian immigrants were analyzed. In this cross- sectional observational study from 2005 to 2010, 2161 farmers were divided into groups: indigenous inhabitants from endemic nephropathy and nonendemic nephropathy villages and Bosnian immigrants ; α-1 microglobulin-to- creatinine ratio >31.5 mg/g and eGFR

Background: Dietary exposure to cytotoxic and carcinogenic aristolochic acid (AA) causes severe nephropathy typically associated with urologic cancers. Monitoring of AA exposure uses biomarkers such as aristolactam-DNA adducts, detected by mass spectrometry in the kidney cortex, or the somatic A>T transversion pattern characteristic of exposure to AA, as revealed by previous DNA-sequencing studies using fresh-frozen tumors. Methods: Here, we report a low-coverage whole-exome sequencing method (LC-WES) optimized for multisample detection of the AA mutational signature, and demonstrate its utility in 17 formalin-fixed paraffin-embedded urothelial tumors obtained from 15 patients with endemic nephropathy, an environmental form of AA nephropathy. Results: LC-WES identified the AA signature, alongside signatures of age and APOBEC enzyme activity, in 15 samples sequenced at the average per-base coverage of approximately 10×. Analysis at 3 to 9× coverage revealed the signature in 91% of the positive samples. The exome-wide distribution of the predominant A>T transversions exhibited a stochastic pattern, whereas 83 cancer driver genes were enriched for recurrent nonsynonymous A>T mutations. In two patients, pairs of tumors from different parts of the urinary tract, including the bladder, harbored overlapping mutation patterns, suggesting tumor dissemination via cell seeding. Conclusions: LC-WES analysis of archived tumor tissues is a reliable method applicable to investigations of both the exposure to AA and its biologic effects in human carcinomas. Impact: By detecting cancers associated with AA exposure in high-risk populations, LC-WES can support future molecular epidemiology studies and provide evidence-base for relevant preventive measures. Cancer Epidemiol Biomarkers Prev; 24(12); 1873–81. ©2015 AACR.

Endemska nefropatija (EN) kronična je tubulointersticijska nefropatija aristolohične kiseline (AAN) koja se javlja u stanovnika određenih sela u dolinama velikih pritoka Dunava na jugoistoku Europe uključujući i Hrvatsku. Oboljeli od EN-a imaju znatno veću učestalost karcinoma prijelaznog epitela mokraćovoda nego opća populacija. Danas se smatra da je A:T transverzija gena p53 »potpisna« mutacija za aristolohičnu kiselinu koja je uzrok endemske nefropatije. Postojeći kriteriji za EN su zastarjeli, neujednačeni (tri vrste kriterija) te nisu u skladu s novim preporukama za klasifikaciju bubrežnih bolesti. Stoga su na temelju sadašnjih znanja i stručnosti grupe znanstvenika i stručnjaka iz svih zemalja s EN-om, kao i svijeta u kojem je AAN opisan stvoreni novi dijagnostički kriteriji i nova klasifikacija stanovništva endemskih sela na simpoziju o EN-u. EN je velik javnozdravstveni problem pa bi nam sadašnja znanja o ovoj bolesti, kao i novi dijagnostički kriteriji trebali pomoći u njezinu ranom otkrivanju i liječenju, a možda u nekoj bližoj budućnosti i njezinoj eradikaciji., 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.

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 (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