Molecular profiles and urinary biomarkers of upper tract urothelial carcinomas associated with aristolochic acid exposure.

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.

CHRONIC KIDNEY DISEASE IN RURAL POPULATION.

The aims of the study were to provide data on chronic kidney disease (CKD) prevalence in rural population and to analyze the association with cardiovascular risk factors and aging. A random sample of 2193 farmers (1333 female (F) and 860 male (M), mean age 50.61±17.12) were enrolled. Questionnaire and clinical examination were conducted. Participants provided a spot urine and fasting blood sample. Estimated glomerular filtration rate (eGFR) was estimated using Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation. Subjects were classified according to the KDIGO guidelines. The overall prevalence of CKD (eGFR <60 mL/min/1.73 m2) was 8.83% (F vs. M 9.9% vs. 6.3%; p<0.001). Albuminuria (albumin-to-creatinine ratio >30 mg/g) was found in 8.45% (F vs. M p>0.05). Sharp increase in CKD prevalence was found to begin after the sixth decade (29.44% in subjects older than 65 years; F vs. M 30.9% vs. 26.8%; p<0.01). The strongest predictor factors for CKD were age >65 years (OR 22.12), hypertension (OR 6.53), albuminuria (OR 5.71), fasting blood glucose >7 mmol/L (OR 5.49), diabetes (OR 3.07), abdominal obesity (OR 2.05) and non-smoking (OR 0.41). In multivariate analysis, age (OR 1.13), female gender (OR 0.60) and diabetes (OR 1.75) were the independent predictor factors for CKD. In conclusion, CKD prevalence is high in rural population, being higher in women than in men. In both genders, eGFR significantly decreased with aging. Aging is a significant independent predictor of CKD.

Renal cell carcinomas of chronic kidney disease patients harbor the mutational signature of carcinogenic aristolochic acid.

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 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.

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.

Factitious proteinuria - the most dominant feature in a young female patient with Munchausen syndrome.

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.

Obesity and systolic blood pressure in young adult men born small for gestational age.

Individuals born small for gestational age (SGA) are supposed to be at higher risk to develop cardiovascular disorders, and recent report showed that concurrent obesity influences blood pressure (BP) in SGA children. Our aim was to investigate the impact of obesity and birth weight on blood pressure values in young adult men born SGA and controls born after normal pregnancy, Normotensive, non-treated adult men were enrolled (N = 185; mean age 21.29 +/- 0.9 years). Birth parameters were obtained from medical records and 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) non-obese SGA (N = 67), (3) obese with normal BW (N = 40), (4) obese SGA (N = 28). BP was measured using Omron M6 and Spacelab 90207 device following the ESH/ESC guidelines. Systolic BP, 24-hour BP variability and pulse pressure were significantly higher in SGA subjects than in those with normal BW (p < 0.05). The highest 24-hour and daytime systolic BP values as well as 24-hour pulse pressure were found in the subgroup of obese SGA subjects (p < 0.001). Significant differences for the above parameters were observed between obese SGA group and non-obese SGA group (p < 0.05). Obese SGA subjects had higher 24-hour and daytime systolic BP values compared to obese normal BW group. No difference was found in BP between non-obese SGA and non-obese group with normal BW (p > 0.05). In addition to BW and shorter pregnancy duration, obesity concurrently and significantly determines systolic BP in young normotensive men and point to a need for more aggressive implementation of healthy lifestyle as early as possible.

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.

Prevalence, treatment and control of hypertension in a Croatian endemic nephropathy area.

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.

Two novel variants in the lecithin:cholesterol acyltransferase gene resulted in classic LCAT deficiency.

Background and aims: We report the first two cases of familial lecithin:cholesterol acyltransferase (LCAT) deficiency in Croatia with classical clinical and biochemical features. Patients and methods: A 30-year-old man with nephrotic syndrome, corneal opacities, hepatosplenomegaly, anemia, low high-density lipoprotein (HDL)-cholesterol levels and arterial hypertension (blood pressure >200/100 mmHg) was admitted to our department. At admission, he had an elevated creatinine serum level (233 µmol/L), proteinuria of 12 g in 24-h urine (g/24 h), 3-7 erythrocytes in urine sediment and notable anemia (hemoglobin level 90 g/l). His HDL-cholesterol was significantly low (0.42 mmol/L). Besides chronic kidney disease (CKD), other secondary causes of hypertension were ruled out. The patient was previously diagnosed with membranous nephropathy and treated unsuccessfully with immunosuppressive agents (steroids, cyclosporine, cyclophosphamide). Re-evaluation of histopathological findings of kidney biopsy revealed massive deposition of lipid material in the glomerular basal membrane and in the mesangial region. His 4-year younger brother was also evaluated due to corneal opacities and new-onset arterial hypertension. Nephrotic range proteinuria with preserved global renal function was determined. He also had very low HDL-cholesterol levels. Results: Kidney biopsies from both patients were consistent with LCAT deficiency. The disease was confirmed by measurement of LCAT enzyme activity, plasma cholesterol esterification rate, and genetic testing. Two novel missense variants in the LCAT gene (c.496G > A and c.1138T  >  C) were found. Conclusions: To our knowledge, the presented cases are the first reported cases of genetic LCAT deficiency in Croatia. Given the clinical presentation, the complete lack of LCAT activity and cholesterol esterification rate, diagnosis of familial LCAT deficiency was made.

Blood pressure dipping and salivary cortisol as markers of fatigue and sleep deprivation in staff anesthesiologists.

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, 5pm, 8pm, 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 impact of a single 24 h working day on cognitive and psychomotor performance in staff anaesthesiologists.

BACKGROUND AND OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Arterial Stiffness in Balkan Endemic Nephropathy, an Environmental Form of Aristolochic Acid Nephropathy.

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.

Reliability of CKD-EPI predictive equation in estimating chronic kidney disease prevalence in the Croatian endemic nephropathy area.

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.

Chronic dietary exposure to aristolochic acid and kidney function in native farmers from a Croatian endemic area and Bosnian immigrants.

BACKGROUND AND OBJECTIVES: 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. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: 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<60 ml/min per 1.73 m(2) were considered to be abnormal. RESULTS: CKD and proximal tubule damage prevalence was significantly lower in Bosnian immigrants than inhabitants of endemic nephropathy villages (6.9% versus 16.6%; P<0.001; 1.3% versus 7.3%; P=0.003, respectively); 20 years ago, Bosnian immigrants observed fewer Aristolochia clematitis in cultivated fields (41.9% versus 67.8%) and fewer seeds among wheat seeds (6.1% versus 35.6%) and ate more purchased than homemade bread compared with Croatian farmers from endemic nephropathy villages (38.5% versus 14.8%, P<0.001). Both Croatian farmers and Bosnian immigrants observe significantly fewer Aristolochia plants growing in their fields compared with 15-30 years ago. Prior aristolochic acid exposure was associated with proximal tubule damage (odds ratio, 1.64; 95% confidence interval, 1.04 to 2.58; P=0.02), whereas present exposure was not (odds ratio, 1.31; 95% confidence interval, 0.75 to 2.30; P=0.33). Furthermore, immigrant status was an independent negative predictor of proximal tubule damage (odds ratio, 0.40; 95% confidence interval, 0.19 to 0.86; P=0.02). CONCLUSIONS: Bosnian immigrants and autochthonous Croats residing in endemic areas are exposed significantly less to ingestion of aristolochic acid than in the past. The prevalence of endemic nephropathy and its associated urothelial cancers is predicted to decrease over time.

Low-Coverage Exome Sequencing Screen in Formalin-Fixed Paraffin-Embedded Tumors Reveals Evidence of Exposure to Carcinogenic Aristolochic Acid.

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.

Endemic (Balkan) nephropathy is aristolochic acid nephropathy.

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.

Adiponectin is not associated with blood pressure in normotensives and untreated hypertensives with normal kidney function.

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 (ß=-0.040; P=0.43) or diastolic BP (ß=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.