Balkan Endemic Nephropathy Patients Research Articles

Positive Feedback Mechanism in Aristolochic Acid I Exposure-Induced Anemia and DNA Adduct Formation: Implications for Balkan Endemic Nephropathy.

Balkan endemic nephropathy (BEN) is a chronic kidney disease that predominantly affects inhabitants of rural farming communities along the Danube River tributaries in the Balkans. Long-standing research has identified dietary exposure to aristolochic acids (AAs) as the principal toxicological cause. This study investigates the pathophysiological role of anemia in BEN, noting its earlier and more severe manifestation in BEN patients compared to those with other chronic kidney diseases. Utilizing a mouse model, our research demonstrates that prolonged exposure to aristolochic acid I (AA-I) (the most prevalent AA variant) leads to significant red blood cell depletion through DNA damage, such as DNA adduct formation in bone marrow, prior to observable kidney function decline. Furthermore, in vitro experiments with kidney cells exposed to lowered oxygen and pH conditions mimicking an anemia environment show enhanced DNA adduct formation, suggesting increased AA-I mutagenicity and carcinogenicity. These findings indicate for the first time a positive feedback mechanism of AA-induced anemia, DNA damage, and kidney impairment in BEN progression. These results not only advance our understanding of the underlying mechanisms of BEN but also highlight anemia as a potential target for early BEN diagnosis and therapy.

GPX3 rs8177412 Polymorphism Modifies Risk of Upper Urothelial Tumors in Patients with Balkan Endemic Nephropathy.

Current data suggest that aristolochic acid (AA) exposure is a putative cause of Balkan endemic nephropathy (BEN), a chronic kidney disease strongly associated with upper tract urothelial carcinoma. The cellular metabolism of AA is associated with the production of reactive oxygen species, resulting in oxidative distress. Purpose: Therefore, the aim of this study was to analyze individual, combined and cumulative effect of antioxidant gene polymorphisms (Nrf2 rs6721961, KEAP1 rs1048290, GSTP1AB rs1695, GSTP1CD rs1138272, GPX3 rs8177412 and MDR1 rs1045642), as well as GSTP1ABCD haplotypes with the risk for BEN development and associated urothelial cell carcinoma in 209 BEN patients and 140 controls from endemic areas. Experimental method: Genotyping was performed using polymerase chain reaction (PCR) and PCR with confronting two-pair primers (PCR-CTTP) methods. Results: We found that female patients carrying both variant GPX3 rs8177412 and MDR1 rs1045642 genotypes in combination exhibited significant risk towards BEN (OR 1 = 3.34, 95% CI = 1.16-9.60, p = 0.025; OR 2 = 3.79, 95% CI = 1.27-11.24, p = 0.016). Moreover, significant association was determined between GPX3rs8174412 polymorphism and risk for urothelial carcinoma. Carriers of variant GPX3*TC + CC genotype were at eight-fold increased risk of BEN-associated urothelial tumors development. There was no individual or combined impact on BEN development and BEN-associated tumors among all examined polymorphisms. The haplotype consisting of variant alleles for both polymorphisms G and T was associated with 1.6-fold increased risk although statistically insignificant (OR = 1.64; 95% CI = 0.75-3.58; p = 0.21). Conclusions: Regarding GPX3 rs8177412 polymorphism, the gene variant that confers lower expression is associated with significant increase in upper urothelial carcinoma risk. Therefore, BEN patients carrying variant GPX3 genotype should be more frequently monitored for possible upper tract urothelial carcinoma development.

LONGER TELOMERE LENGTH IN BALKAN ENDEMIC NEPHROPATHY PATIENTS UNDERGOING CHRONIC HEMODIALYSIS IS ASSOCIATED WITH LOWER CARDIOVASCULAR MORTALITY

Objective: Balkan endemic nephropathy (BEN) is characterized with later onset, milder forms of hypertension and lower pulse wave velocity (PWV) than other end-stage kidney disease (ESKD). We hypothesized that telomere length (TL) would be longer in BEN patients and associated with lower PWV and less cardiovascular (CV) mortality. Design and method: 124 patients undergoing hemodialysis (HD) (68 BEN, 56 non-BEN; 48.4% men; 65±14 years) were enrolled and followed-up for 72 months. Blood pressure and PWV were determined before mid-week dialysis. TL was measured in leukocytes by Southern blot. Results: Age and sex-adjusted TL was significantly longer in the BEN group (7.19 kb vs 6.79 kb; p<0.001). TL was associated in the BEN group with PWV, and in the non-BEN group with age. BEN patients presented significantly lower CV mortality than non-BEN patients (p<0.001). In the BEN group shorter TL was the only determinant of shorter survival (HR 0.11 [95% CI 0.03, 0.35]). To analyze the diagnostic value of TL for CV mortality we used ROC analysis that revealed TL < 6.21 kb to be useful for prediction of CV mortality in patients undergoing chronic HD. Conclusions: Shorter TL is associated with CV mortality in patients undergoing chronic HD. BEN patients had longer TL and longer survival than other ESKD patients. In BEN patients, TL was negatively associated with PWV. This study confirmed our hypothesis that BEN is associated with slower vascular aging and that longer TL may partially explain this phenomenon.

Urine Metabolomic Signature of People Diagnosed with Balkan Endemic Nephropathy and Other Types of Chronic Kidney Disease Compared with Healthy Subjects in Romania

Metabolomic analysis methods were employed to determine biomarkers for various chronic kidney diseases (CKDs). Modern analytical methods were developed and applied successfully to find a specific metabolomic profile in urine samples from CKD and Balkan endemic nephropathy (BEN) patients. The aim was to explore a specific metabolomic profile defined by feasible/easy-to-identify molecular markers. Urine samples were collected from patients with CKDs and BEN, and from healthy subjects from endemic and nonendemic areas in Romania. Metabolomic analysis of urine samples, extracted by the liquid-liquid extraction (LLE) method, was performed by gas chromatography-mass spectrometry (GC-MS). The statistical exploration of the results was performed through a principal component analysis (PCA) evaluation. Urine samples were statistically analyzed using a classification based on six types of metabolites. Most urinary metabolites are distributed in the center of a loading plot, meaning that these compounds do not represent significant markers for BEN. One of the most frequent and higher-concentration urinary metabolites in BEN patients was p-Cresol, a phenolic compound that implies a severe injury of the renal filtration function. The presence of p-Cresol was associated with protein-bound uremic toxins, which have specific functional groups such as indole and phenyl. In prospective studies for future investigation, prevention, and disease treatment, we suggest a larger sample size, sample extraction using other methods, and analysis using other chromatography techniques coupled with mass spectrometry, which can generate a more significant data set for statistical analysis.

BALKAN ENDEMIC NEPHROPATHY, PULSE WAVE VELOCITY AND INCREASED TELOMERE LENGTH-ASSOCIATED CARDIOVASCULAR MORTALITY IN PATIENTS UNDERGOING CHRONIC HEMODIALYSIS

Objective: Balkan endemic nephropathy (BEN) is characterized with later onset, milder forms of hypertension and lower pulse wave velocity (PWV)). We hypothesized that telomere length (TL) would be longer in BEN patients and associated with lower PWV and less cardiovascular (CV) mortality. Design and method: On total 124 hemodialysed (HD) patients (68 BEN, 56 non-BEN; 48.4% men; 65 ± 14 years) were enrolled and followed-up for 25 months. Blood pressure and PWV were determined before mid-week dialysis. TL was determined by telomere restriction fragment analysis. Results: Age-sex adjusted TL was significantly longer in the BEN group (7.19 vs 6.79; p < 0.001) despite being significantly older. In the BEN group TL was associated with PWV (beta = -0.373, p < 0.05), and in the non-BEN group with age (beta = -0.680, p < 0.01). In the full-adjusted model of multivariate linear regression analyses significant predictors of TL were systolic BP/PWV, and age in the BEN and the non-BEN group, respectively. BEN patients died significantly less frequently from CV events than non-BEN patients (p < 0.001) and lived significantly longer (log-rank p = 0.04). Patients who died had significantly shorter TL than survivors (p < 0.001). In the Cox Regression analysis older age, diagnosis of non-BEN and shorter TL were determinants of shorter survival in the entire cohort. In the BEN group shorter TL was the only determinant of shorter survival (HR 0.11 [95% CI 0.03, 0.35]). To analyze the diagnostic value of TL for CV mortality we used ROC analysis that revealed TL < 6.21 Kb to be useful for prediction of CV mortality in patients undergoing chronic HD. Conclusions: Shorter TL is associated with CV mortality in patients undergoing chronic HD. BEN patients had longer TL and longer survival than other ESKD patients. PWV was significant predictor of TL in BEN patients. This study confirmed our hypothesis that BEN is associated with slower vascular aging.

Predictive Markers for Malignant Urothelial Transformation in Balkan Endemic Nephropathy: A Case-Control Study.

Simple SummaryBalkan endemic nephropathy (BEN) is chronic kidney disease caused by intoxication with Aristolochia plant. Apart from subtle decline of renal function that eventually results in kidney failure, the patients are at increased risk for urothelial carcinoma (UC) development. Based on the observed UC markers, the aim of this study was to examine urinary and plasma levels of some these markers in BEN patients without carcinoma, in order to potentially identify those with predictive value. Our study revealed either plasma or urinary survivin levels as a potential predictors of future malignant transformation of urothelium. Balkan endemic nephropathy (BEN) is a chronic tubulointerstitial disease frequently accompanied by urothelial carcinoma (UC). In light of the increased UC incidence and the markers observed in BEN patients with developed UC, the aim of the current case–control study is to assess survivin, p53 protein, growth factors and receptors (VEGF, VEGFR1, IGF I, IGF-1R and IGFBP5), tumor marker (TF)/CD142, circulating soluble Fas receptor and neopterin, as potentially predictive markers for UC in patients with BEN (52 patients), compared to healthy, age-matched subjects (40). A threefold increase was registered in both circulating and urinary survivin level in BEN patients. Especially noticeable was the ratio of U survivin/U Cr level five times the ratio of BEN patients associated with standard renal markers in multivariate regression models. The concentrations of VEGF, VEGFR1, (TF)/CD142, (sFas) were not significantly different in BEN patients, while urinary/plasma level demonstrated a significant decrease for VEGF. The levels of IGF I, IGFBP5 and IGF-1R were significantly reduced in the urine of BEN patients. Plasma concentration of neopterin was significantly higher, while urinary neopterin value was significantly lower in BEN patients compared to healthy controls, which reflected a significantly lower urine/plasma ratio and low local predictive value. As BEN is a slow-progressing chronic kidney disease, early detection of survivin may be proposed as potential predictor for malignant alteration and screening tool in BEN patients without the diagnosis of UC.

The role of HLA in Balkan endemic nephropathy

Balkan endemic nephropathy (BEN), a progressive chronic tubulointerstitial disease, occurs in the endemic focus of Croatia in a population of about 10,000 inhabitants. One of its most peculiar characteristics is a strong association with upper tract urothelial carcinoma (UTUC). Despite a high number of studies, currently there are insufficient data about the association of BEN and HLA genes. The aim of this study was to investigate the polymorphism of HLA-A, -B, and -DRB1 alleles and haplotypes among BEN patients and to determine whether an association between HLA and BEN exists. In this study, we investigated HLA-A, -B, and -DRB1 alleles and haplotypes in a population of patients with BEN (N=111) and matched healthy controls (N=190). All individuals were tested by PCR-SSO and PCR-SSP methods to assess the possible contribution of HLA alleles and haplotypes to the development of/protection from BEN. Our results showed a positive association between the presence of HLA-B*35:02 and DRB1*04:02 alleles and BEN (P=0.0179 and P=0.0151, respectively) in contrast to the protective effect of HLA-A*01:01, B*27:05 and B*57:01 alleles (P=0.0111, P=0.0330 and P=0.0318, respectively). Moreover, when BEN patients' HLA haplotypes were compared to controls, two haplotypes were associated with BEN susceptibility among Croatians (HLA-A*02:01~B*08:01~DRB1*03:01 and HLA-A*02:01~B*27:02~DRB1*16:01, P=0.0064 and P=0.0023, respectively), while haplotypes HLA-A*02:01~B*27:05~DRB1*01:01 and HLA-A*02:01~B*38:01~DRB1*13:01 each showed a possible protective effect (P=0.0495). Our results point toward genetic susceptibility to BEN and observed differences in both susceptible/protective HLA profiles indicate the necessity of further studies in order to elucidate the pathogenesis of this disease.

Association of SOD2 (rs4880) and GPX1 (rs1050450) Gene Polymorphisms with Risk of Balkan Endemic Nephropathy and its Related Tumors.

Background and Objectives: Experimental data show that superoxide dismutase 2 (SOD2) is involved in ochratoxin (OTA)-induced nephrotoxicity, whereas clinical data indicate the role of SOD2 rs4880 or glutathione peroxidase 1 (GPX1) rs1050450 polymorphisms in end-stage renal disease and urothelial carcinoma risk, known to be the major complications of Balkan endemic nephropathy (BEN). Therefore, we hypothesized that SOD2 and GPX1 gene polymorphisms would influence the risk of BEN and its associated tumors. Materials and Methods: The study was conducted in 207 BEN patients and 86 controls from endemic areas. Results: Individuals with both copies of variant SOD2 allele, known for lower mitochondrial antioxidant protection, are at a significantly higher BEN risk (OR = 2.6, p = 0.021). No association was observed between GPX1 gene polymorphism and BEN risk. Combining SOD2 and GPX1 genotypes did not alter the risk of BEN development. Regarding the risk of urothelial tumors in BEN patients, none of the polymorphisms studied was significantly associated with the risk of these tumors. Conclusions: Polymorphism in SOD2 rs4880 gene affects the risk of BEN development. Hence, SOD2 genotyping could, together with a panel of other enzymes, be used as a biomarker of susceptibility in BEN areas.

Differential methylation pattern of xenobiotic metabolizing genes and susceptibility to Balkan endemic nephropathy, in a cohort of Romanian patients.

A severe, chronic and irreversible kidney disease affecting discrete rural populations in the Balkan Peninsula countries, Balkan endemic nephropathy (BEN) has been a scientific puzzle for more than half a century. Many environmental and other factors have been suggested as the primary cause and recent significant findings have linked BEN to aristolochic acids, phytotoxins derived from the plant Aristolochia clematitis, found in high density in the endemic areas. However, given that the incidence of BEN is less than 10% in affected villages, and it tends to have a family aggregation, as yet unidentified genetic factors may also play a role. To further explore this possibility, a pilot study was initiated to investigate the DNA methylation of CYP1A1, CYP1A2, NAT1, NQO1 and GSTT1 in blood samples from a group of Romanian BEN patients, compared to healthy controls and non-BEN chronic kidney disease (CKD) subjects. Our study revealed a more pronounced hypomethylation pattern in BEN and non-BEN CKD groups, compared to the healthy control group at specific CpGs across all five genes interrogated. Average methylation across the five regions investigated indicated significant differences only at GSTT1, in both BEN patients (p = 0.028) and non-BEN disease subjects (p = 0.015), relative to healthy individuals. Since GSTT1 active genotype appears to be a common feature of Serbian and Romanian BEN patients, GSTT1 epigenetic variation and increased gene activity could act as a predisposing (co)factor in BEN populations from the affected countries. BEN and non-BEN CKD groups show similar methylation patterns with exception of GSTT1 CpG8 (p = 0.046).

Increased Glomerular Filtration Rate in Early Stage of Balkan Endemic Nephropathy.

Background: A previous study indicated that Balkan endemic nephropathy (BEN) patients in the early stage of the disease had significantly higher creatinine clearance (Ccr) than healthy persons. The aim of the study was to assess whether tubular creatinine secretion affects Ccr in early stages of BEN and to check the applicability of serum creatinine-based glomerular filtration rate (GFR) equations in these patients. Methods: The study involved 21 BEN patients with estimated GFR (eGFR) above 60 mL/min/1.73 m2, excluding any conditions that could affect GFR or tubular creatinine secretion, and 15 healthy controls. In all participants Ccr with and without cimetidine and iohexol clearance (mGFR) were measured and eGFR calculated using Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) and Modification of Diet in Renal Disease Study (MDRD) equations. Glomerular hyperfiltration cutoff (GFR-HF) was calculated. Results: There was no significant difference between the groups in Ccr before and after cimetidine or for eGFR, but mGFR was significantly higher in BEN patients than in controls (122.02 ± 28.03 mL/min/1.73 m2 vs. 101.15 ± 27.32 mL/min/1.73 m2; p = 0.032). Cimetidine administration reduced Ccr by 10% in both groups. The ratio of Ccr to mGFR was significantly above one in seven BEN patients and five controls and their mGFR values were similar. Seven other patients and eight controls had this ratio equal to one, while values below one were recorded for seven more patients and two controls. mGFR of all these 14 patients was significantly higher than that of healthy controls (129.88 ± 27.52 mL/min/1.73 m2 vs. 107.43 ± 19.51 mL/min/1.73 m2; p = 0.009). Mean GFR-HF was significantly higher than mGFR in controls, but these two values were similar in BEN patients. eGFR underestimated mGFR in both BEN patients and controls. Conclusion: The ratio of Ccr to mGFR and mGFR to GFR-HF indicated that elevated mGFR in early stages of BEN could be explained by increased glomerular filtration, but tubular creatinine secretion augmented Ccr in a smaller proportion of patients, who did not differ from healthy subjects.

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.

Patients with Balkan endemic nephropathy from Mehedinti County, Romania: An epidemiological approach

Introduction Balkan endemic nephropathy (BEN) is a tubulointerstitial nephropathy, which evolves to symmetric kidney atrophy, renal failure and frequent upper urinary tract malignancies. Patients’ survival is conditioned by dialysis or renal transplantation. The causative agent is considered to be aristolochic acid, but the mode of natural exposure in the endemic Danube region (Romania, Bulgaria, Serbia, Croatia, Bosnia) was not exactly established. In Romania no confirmatory studies have been done so far, but the endemic area is considered to be the rural localities from Mehedinți and Caraș Severin counties. The purpose of this study was to describe the epidemiological characteristics of Mehedinți BEN cases in order to create the premises for future studies about disease's determinants identification, the ways for achieving the exposure in the natural environment and suggesting paths to identify possible diagnostic criteria in an early stage of the disease. Method A cross-sectional study was conducted between July and September 2009, including the patients diagnosed with BEN and assisted in three dialysis centres from Drobeta Turnu-Severin. Data were collected using an original questionnaire, which included demographic data, symptomatology and investigations. Statistical analysis with descriptive indicators was performed in Epi info and Excel. Results A total of 74 patients were included in the study sample being in dialysis treatment in all three Drobeta Turnu-Severin dialysis centres. Patients, more women than men (gender ratio male to female of 0.81), were aged between 38 and 83 years, with an average age of 66.2 ± 9.5 years (± standard deviation: SD). They lived different time period in 38 villages in Mehedinti County, except for only one patient. Only in endemic area lived 57.7% of BEN patients and 22.6% of them have had interposed periods of staying in outside area. Others, 19.7% of them, after having originally lived in the endemic area, they permanently moved to non-endemic areas where they developed the disease. The mean age at diagnosis was 58.2 ± 11.5 years (range: 23–79 years), in men 59.4 ± 11 years (range: 23–71), and in women 57.5 ± 11.8 years (range: 33–79), without significant differences (P = 0.532) between the two genders. If in those living continuously in the endemic area BEN was diagnosed at an average age of 57.8 ± 11.6 years (range: 23–75 years), in the others who permanently left the endemic area the disease appeared after an average of 40.4 years (range: 10–69 years) after leaving the area, respectively to an average age of 59 ± 11.7 years (range: 33–79 years), differences being insignificant (P > 0.05) as time interval. Symptomatology reported by patients before diagnosis was installed slowly, with the most frequent being asthenia (93.1%), nycturia (81.9%), inappetence (77.8%), nausea (59.7%), polydipsia (54.2%), vomiting (52.8%), dysuria (45.8%). Of the laboratory parameters, beside those revealing renal dysfunction, consistently low values for hemoglobin (64.9%), erythrocytes (81.1%) and hematocrit (86.5%) were reported in patients. Concomitant malignancies had 11 patients (14.9%), mainly as urinary tract tumours. One patient had lung cancer, one colorectal and one ovarian cancer. Conclusions BEN affects only adult persons who live or have lived in the endemic area, especially in the 60th and 70th decade of life, regardless of their gender. Installation of the disease was similar regardless of whether or not the person left the endemic area. Patients presented as main associated pathology anemia and malignancies of urinary tract and other localizations.

Biomarkers of oxidative damage and antioxidant enzyme activities in pre-dialysis Balkan endemic nephropathy patients.

To obtain more insight into molecular mechanisms underlying oxidative stress in Balkan endemic nephropathy (BEN), biomarkers of oxidative stress and antioxidant enzyme activities were studied in 38 pre-dialysis BEN patients, 21 healthy BEN family members and 36 healthy subjects from non-endemic areas. Protein thiol groups (P-SH), antioxidant enzyme activities [superoxide dismutase (SOD) and glutathione peroxidase (GPX)], were determined in plasma spectrophotometrically, while malondialdehyde adducts (MDA) by enzyme immunoassay. BEN patients had significantly lower plasma GPX activity in comparison with values for both control groups (p=0.016), gradually decreasing with kidney function impairment estimated by glomerular filtration rate (r=0.53, p=0.002). GPX activity was inversely correlated with serum urea (r=-0.627, p<0.001), creatinine (r=-0.53, p<0.05), urinary excretion of protein and α1-microglobulin (r=-0.44, p=0.012; r=-0.50, p<0.007). Significant upregulation of SOD activity was observed in healthy BEN family members (p<0.05). While the concentration of MDA adducts was similar in all three groups, BEN patients and healthy BEN family members exhibited increased protein damage, based on fewer P-SH groups in comparison with subjects from non-BEN areas (p=0.085; p=0.014, respectively). Based on our results on increased oxidative protein damage in both pre-dialysis BEN patients and healthy BEN family members, it can be speculated that individuals from BEN areas, in general, are chronically exposed to some prooxidant environmental compounds. Moreover, decrease in plasma GPX activity, as a consequence of impaired kidney function, could further affect oxidative status in BEN patients.

Placental growth factor and placental protein 13 in patients with Balkan endemic nephropathy, a worldwide disease

Background: Balkan endemic nephropathy (BEN) is a chronic tubulointerstitial kidney disease occurring in people living in along the tributaries of the Danube River. The aim of the study was to determine serum level and urinary excretion of placental growth factor (PlGF) and placental protein 13 (PP13) in patients with BEN. Methods: Thirty patients with BEN from the South Morava River region of Serbia and 18 controls were studied. Age of patients was 74 yr (53–87) and 73 yr (66–83) in controls. Results: In patients with BEN, serum creatinine was significantly higher than in controls (129.7 vs. 83.2 µmol/L, respectively), but GFR was lower in patients than in controls (40.7 vs. 54.6 mL/min). Serum PlGF was significantly higher in BEN patients than in controls (9.90 vs. 6.80 pg/mL), urinary excretion being significantly lower in patients (0.20 vs. 0.90 pg/mmol creat.). Serum PP13 was significantly lower in BEN patients (208.2 vs. 291.0 pg/mL). Urinary excretion of PP13 was also significantly lower in BEN patients than in controls (32.5 vs. 182.5 pg/mmol creat). In multivariate regression analysis BEN, sex and age were significant determinants of the observed changes in PlGF and PP13. Conclusion: Important changes of PlGF and PP13 in patients with BEN were demonstrated, where kidney disease, female sex, and the age have been significant determinants.

Balkan nephropathy.

Balkan endemic nephropathy (BN), frequently associated to upper urothelial cancer, is a familial chronic tubulointerstitial disease with insidious onset and slow progression to end-stage renal disease. After 60 years of research, its cause remains the major unanswered question. Etiology assumes polygenic susceptibility to the disease in interaction with multiple environmental factors. Chronic intoxication with Aristolochia is the major environmental risk factor for this disease. The mycotoxin hypothesis considers that BN is produced by ochratoxin A. The Pliocene lignite hypothesis assumes that the disease is caused by long-term exposure to organic toxins leached from coal nearby the endemic villages. Exome sequencing of 22,000 genes revealed that mutant genes (CELA1, HSPG2, and KCNK5) in BN patients encode proteins involved in basement membrane/extracellular matrix and vascular tone, which are tightly connected to the process of angiogenesis. SEC61G, IL17RA, and HDAC11 proved to be differently methylated throughout all patient-control pairs. The acetylation of histone lysine residues was detected and found increased at specific sites of H3 and total H4 histones isolated from urothelial cells of patients with BN. The results of molecular biological research will allow the discovery of genetic markers of BN and associated urothelial cancer, permitting early detection of BN-predisposing mutations and identification of susceptible individuals who might be at risk of exposure to environmental agents. The research of gene-gene and gene-environment interactions could lead to further studies to determine the precise risk for BN.

Familial versus environmental factors in Balkan endemic nephropathy in Mehedinti county, Romania, by means of albuminuria and tubular biomarkers: preliminary study

Introduction and aims: Balkan endemic nephropathy (BEN), a regional tubulointerstitial kidney disease encountered in South-Eastern Europe, with still undefined etiology and inexorable evolution towards end stage renal disease, raises the question of the relative contribution of family and environmental factors in its etiology. In order to evaluate the intervention of these factors, markers of tubular injury have been assessed, this lesion being considered an early renal involvement in BEN. Methods: The paper studies relatives of BEN patients currently included in dialysis programmes (for involvement of the family factor) and their neighbors (for involvement of environmental factors) and analyzes them with regard to tubular injury by means of tubular biomarkers (N-acetyl-beta-d-glucosaminidase—NAG and alpha-1-microglobulin), and albuminuria. At the same time, glomerular filtration rate (GFR) (CKD-EPI) was measured. It is considered that, in order to acquire the disease, one should have lived for 20 years in the BEN area. The relatives have been classified according to this criterion. Results: More evident tubular injury was found in the neighbors of BEN patients living for more than 20 years in the endemic area, which argues in favor of environmental factors. Higher levels of urinary alpha-1-microglobulin and albumin in relatives of BEN patients who had been living for more than 20 years in the area than in relatives with a residence under 20 years, plead for the same hypothesis. GFR was lower in persons who had been living for more than 20 years in the BEN area (neighbors and relatives). Conclusions: Environmental factors could be more important in BEN than family factors.

Is increased susceptibility to Balkan endemic nephropathy in carriers of common GSTA1 (*A/*B) polymorphism linked with the catalytic role of GSTA1 in ochratoxin a biotransformation? Serbian case control study and in silico analysis.

Although recent data suggest aristolochic acid as a putative cause of Balkan endemic nephropathy (BEN), evidence also exists in favor of ochratoxin A (OTA) exposure as risk factor for the disease. The potential role of xenobiotic metabolizing enzymes, such as the glutathione transferases (GSTs), in OTA biotransformation is based on OTA glutathione adducts (OTHQ-SG and OTB-SG) in blood and urine of BEN patients. We aimed to analyze the association between common GSTA1, GSTM1, GSTT1, and GSTP1 polymorphisms and BEN susceptibility, and thereafter performed an in silico simulation of particular GST enzymes potentially involved in OTA transformations. GSTA1, GSTM1, GSTT1 and GSTP1 genotypes were determined in 207 BEN patients and 138 non-BEN healthy individuals from endemic regions by polymerase chain reaction (PCR). Molecular modeling in silico was performed for GSTA1 protein. Among the GST polymorphisms tested, only GSTA1 was significantly associated with a higher risk of BEN. Namely, carriers of the GSTA1*B gene variant, associated with lower transcriptional activation, were at a 1.6-fold higher BEN risk than those carrying the homozygous GSTA1*A/*A genotype (OR = 1.6; p = 0.037). In in silico modeling, we found four structures, two OTB-SG and two OTHQ-SG, bound in a GSTA1 monomer. We found that GSTA1 polymorphism was associated with increased risk of BEN, and suggested, according to the in silico simulation, that GSTA1-1 might be involved in catalyzing the formation of OTHQ-SG and OTB-SG conjugates.

Global and specific histone acetylation pattern in patients with Balkan endemic nephropathy, a worldwide disease

Background: Balkan endemic nephropathy (BEN) is a chronic tubulointerstitial nephropathy present in the Danube river regions in several Balkan countries. There appears to be a polygenic susceptibility to the disease in interaction with multiple environmental factors (aristolochic acid, ochratoxin A). In a previous study SEC61G, IL17RA, HDAC11 proved to be differently methylated throughout all patient-control pairs of BEN patients from Serbia and Bulgaria. Emerging connections between DNA methylation and histone acetylation prompted the present study on histone acetylation in patients with BEN. Methods: The study involved 39 patients with BEN, and 39 controls collected from non-endemic regions in Serbia. The EpiSeeker Histone H3 and H4 Total Acetylation Detection colorimetric Kits and specific acetylated at lysine 18 H3K18 and H3K36 acetylated at lysine 36 detection kits were used. Results: It was documented that total H4 histone acetylation level was increased significantly, while total H3 histone acetylation did not differ significantly. Specific histone structure and functional properties may be affected by the observed derangement of H3 histone acetylation pattern, since H3K36 site was significantly more acetylated, while H3K18 tended to be less acetylated than in control subjects. Multiple regression analysis revealed a statistically significant relationship between H4, H3T and H3K36 in BEN patients. Conclusion: This preliminary study suggests that the acetylation of histone lysine residues was detectable and found increased at specific sites of H3 and total H4 histones isolated from urothelial cells of patients with BEN. Having in mind a possible mechanism and biological role of epigenetic chromatin modification in urothelial tumor development they obtained results may open opportunity for selective therapeutic interventions in patients with BEN.

Exceptionally long-term persistence of DNA adducts formed by carcinogenic aristolochic acid I in renal tissue from patients with aristolochic acid nephropathy.

Aristolochic acid (AA) causes aristolochic acid nephropathy (AAN), first described in women in Belgium accidently prescribed Aristolochia fangchi in a slimming treatment, and also Balkan endemic nephropathy (BEN), through probable dietary contamination with Aristolochia clematitis seeds. Both nephropathies have a high risk of urothelial cancer, with AA being the causative agent. In tissues of AAN and BEN patients, a distinct DNA adduct, 7-(deoxyadenosin-N6-yl)-aristolactam I (dA-AAI), has been detected. DNA adducts can be removed through DNA repair, they can result in mutations through erroneous DNA replication or they can cause cell death. The dA-AAI adduct induces AT to TA transversions in the tumor-suppressor TP53 gene in experimental systems, matching TP53 mutations observed in urothelial tumors from AAN cancer cases. Using thin-layer chromatography 32P-postlabeling and mass spectrometric analysis we report the detection of dA-AAI in renal DNA from 11 Belgian AAN patients over 20 years after exposure to AA had ceased. Our results showed that dA-AAI is an established biomarker of AA exposure, and that this biomarker can be demonstrated to be persistent decades after a distinct AA exposure. Further, the persistence of dA-AAI adducts appears to be a critical determinant for the AA mutational fingerprint frequently found in oncogenes and tumor suppressor genes recently identified by whole genome sequencing of AA-associated urothelial tumors. The potential for exposure to AA worldwide is high; the unprecedented long-term persistence of dA-AAI provides a useful long-term biomarker of exposure and attests to the role of AA in human urothelial malignancy.

Chronic Kidney Disease and Upper Tract Urothelial Carcinomas

Upper tract urothelial carcinomas (UTUC) have high prevalence rates in patients with chronic kidney disease (CKD), including dialysis patients and kidney transplant recipients. Analgesic nephropathy, Balkan endemic nephropathy, and aristolochic acid (AA) nephropathy share the common association with the development of CKD and UTUC. Genome studies allow identification of patients with genetic predisposition to Balkan endemic nephropathy and AA nephropathy. Furthermore, the identification of aristolactam deoxyribonucleic acid (DNA) adducts now provides robust evidence for AA exposure in UTUC patients, even in the absence of recallable exposure history. Diagnosis and treatment of UTUC in CKD patients are challenging. Clinical diagnosis of UTUCs in CKD patients with the available urological and imaging methods is much more difficult than in patients with normal renal function due to atrophic kidneys and poor excretory functions in these patients. High prevalence rates of contralateral upper tract and urinary bladder involvements of UC in CKD, either at presentation or recurrence, raise concerns of the necessity and techniques as well as perioperative risk of complete urinary tract exenteration. The standard treatment of radical nephroureterectomy with bladder cuff excision for UTUC patients may result in deterioration of renal function, rendering adjuvant chemotherapy unsuitable. This special issue collectively addresses these important topics regarding UTUC in CKD patients. The authors in one paper nominate three mutant genes (CELA1, HSPG2, and KCNK5) in Balkan endemic nephropathy patients, which are associated with angiogenesis. This finding suggests that angiogenesis might play an important role in the development of Balkan endemic nephropathy. Another paper shows an elevated risk of urothelial carcinomas (UC) in ESRD patients with the analysis of a national wide cohort in Taiwan. Female patients have 9–18 folds of increased risk of UC as compared to 4–14 folds in male ESRD patients, which may reflect female predominance in AA consumption. One paper discusses the challenges of UTUC diagnosis in CKD patients. It reviews the detection rate of UTUCs in dialysis patients and kidney transplant recipients with the use of a variety of imaging and urological methods in the literature. Combination use of urological and imaging methods has been suggested for diagnosing UTUCs in symptomatic dialysis patients and kidney transplant recipients. Another paper develops a predictive model for postoperative renal insufficiency in UTUC patients undergoing radical nephroureterectomy, which help in selecting eligible patients for cisplatin based adjuvant chemotherapy. Older age, lower estimated glomerular filtration rate before surgery, smaller tumor size, renal pelvis tumor, and absence of hydronephrosis or multifocal tumors are predictors for ineligibility for adjuvant chemotherapy. One paper describes modified incision complete urinary tract exenteration for UC in dialysis patients, providing short operative time, early oral feeding, and no major perioperative complications of this modified surgical procedure. Another paper reviews epidemiological evidence of AA exposure associated with occurrence of nephropathy and UC, including AA exposure scenarios in Belgium, Taiwan, and the Danube River as well as occupational exposure in Chinese herbalists. The identification of aristolactam DNA adducts in these patients further corroborates biological plausibility of AA exposure contributing to the occurrence of UTUC. Li-Jen Wang Joelle L. Nortier Bin Tean Teh Cheng-Keng Chuang Shen-Yang Lee