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.

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.

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.

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 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.

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.

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 (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.

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.

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.