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.

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.

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.

Glomerular filtration rate in examined population of Bosnian Posavina - region of Balkan Endemic Nephropathy.

Balkan endemic nephropathy (BEN) is chronic tubulointersticial nephritis of unknown aetiology characterized by an insidious onset and gradual progression to end stage renal disease (ESRD). Endemic regions of Bosnia and Herzegovina are Posavina and Semberija, sited at basin of Sava River. In BEN, just like in other chronic renal diseases (CKD), glomerular filtration rate (GFR), is assumed a marker of overall renal function. The aim of this study was to compare GFR in examinees of endemic and non-endemic region for BEN, and between examinees with and without risk factors for BEN within endemic region. Study included 603 inhabitants of Bosnian Posavina, out of whom 386 (65%) from endemic (Domaljevac) and 217 (36%) from non-endemic (Svilaj) village, and it was performed in two phases. The first phase encompassed obtaining anamnestic data (demographic, personal and family history), measurement of arterial blood pressure, and urine dipstick testing (specific gravity, pH, proteins, leukocytes, glucose, ketones, and microalbuminuria). In the second phase, besides repeated urine dipstick test, laboratory blood testing and abdominal ultrasound, with special attention to urinary tract, was also performed. We have compared GFR between examinees of endemic and non-endemic regions for BEN, and between examinees with and without family burden for BEN within endemic region, using MDRD formula for calculating GFR, with cut-off value (5th percentile) based on result of studies performed in European Caucasians in screening for CKD and for establishing stages of CKD in BEN. Medical was used for statistical testing. Out of total number of examined inhabitants (603), 145 examinees were included in the second phase. After exclusion of 17 diabetic patients, 94 (73%) examinees from endemic and 34 (27%) examinees from non-endemic region remained. In the endemic region there were 46 (49%) examinees with and 48 (51%) without family burden for BEN. Overall GFR in examined groups was within physiologic range. There was not statistically significant difference in calculated GFR between examinees of endemic and non-endemic regions for BEN (Mann-Whitney test p=0.104; Fisher's test p=1), neither between examinees with and without family burden for BEN within endemic region (Mann-Whitney test p=0,7393; Fisher's test p=0,263). Overall GFR in examined groups was within physiologic range. There wasn't statistically significant difference in calculated GFR between examinees of endemic and non-endemic regions for BEN, neither between examinees with and without family burden for BEN within endemic region. GFR, no matter how accurately calculated and estimated, does not represent significant biomarker for diagnosis, especially early diagnosis, of BEN, until maybe its overt advanced form.

[Balkan endemic nephropathy in Bosnia and Herzegovina--renal registry report]. / Balkanska endemska nefropatija u Bosni i Hercegovini--izvjestaj renalnog registra.

INTRODUCTION: Balkan Endemic Nephropathy (BEN) is still dominant cause of the end stage renal disease (ESRD) in North-Eastern Bosnia. The aim of this paper was to analyze the patients with BEN diagnosis on chronic dialysis treatment in Bosnia and Herzegovina. METHODS: In this study we used data from individual questionnaires which we collected for Renal Registry. Individual questionnaires include: sex, age, place of birth and address, primary renal disease, data of the first dialysis treatment, type of dialysis, kidney transplantation, co-morbid diseases, erythropoietin therapy and outcome. For patients with BEN diagnosis we gathered additional data: history of urothelial tumor and family history of similar kidney diseases and renal replacement therapy. We compared these data with data about others dialysis patients in Bosnia and Herzegovina. STATISTICAL ANALYSIS: descriptive statistical analysis. RESULTS: Prevalence of the chronic dialysis patients in Bosnia and Herzegovina in 2003 was 474 pmp, 70 pmp for patients with BEN and 54 pmp for patients with diabetic nephropathy. In North-Eastern Bosnia prevalence of chronic dialysis patients was 844 and of patients with BEN 520 pmp. Incidence of the new chronic dialysis patients in Bosnia and Herzegovina in 2003 was 113 pmp, 11 pmp for BEN, and 19 pmp for diabetic nephropathy. Mortality of the chronic dialysis patients in Bosnia and Herzegovina in 2003 was 11.24 %, and mortality of the BEN patients 10.75 %. CONCLUSION: From the total number of the chronic dialysis patients in Bosnia and Herzegovina 14.7 % are BEN patients and 11.3 % are patients with diabetes. BEN is still big medical and social problem in Bosnia and Herzegovina, especially in the North-Eastern Bosnia. There are certain indicators that the incidence of the BEN patients is in decrease such as decreased difference between the prevalence of the patients with BEN and diabetic nephropathy; as well as increase of average age of patients with BEN.