Unusual Urothelial Tumors and Refractory Uremia Due to Balkan Endemic Nephropathy: A Case Report.

Balkan endemic nephropathy (BEN) is a rare progressive chronic renal disease found in residents living along the Balkan peninsula. We present a 92-year-old female who complained initially of cardio-respiratory symptoms and was found to have an acute hypoxemic respiratory failure with hypervolemia. The patient underwent computed tomography imaging and was found to have bilateral pleural effusions and moderate left-sided renal atrophy with left-sided hydronephrosis. The patient underwent diuresis for fluid overload and was treated with broad-spectrum antibiotics for hospital-acquired pneumonia. Further urological work-up revealed masses in the posterior bladder wall and left ureteropelvic junction. A biopsy of the posterior bladder wall mass confirmed high-grade papillary urothelial carcinoma. A review of the epidemiological history revealed the patient lived in Kosovo/former Yugoslavia for several decades following birth. A review of old records revealed the patient had chronic kidney disease (CKD) that was not fully explained by other causes, such as hypertension or diabetes. Given the epidemiological history, accelerated CKD, and unusual locations of urothelial carcinoma, the patient was diagnosed with BEN. Despite medical management and hemodialysis, the patient's renal function and mental status continued to deteriorate, and the decision was made to proceed with palliative care measures.

Effects of Heavy Metal Co-Exposure on the formation of DNA Adducts from Aristolochic Acid I: Implications for Balkan Endemic Nephropathy Development.

Accumulated evidence has shown that Balkan endemic nephropathy (BEN) is a multifactorial environmental disease, with exposure to aristolochic acids (AA), and the associated DNA adduct formation, as a key causative factor of BEN development. Here, we show that coexposure to arsenic, cadmium, and iron increases the DNA adduct formation of AA in cultured kidney cells, while exhibiting both an exposure concentration and duration dependence. In contrast, coexposure to calcium and copper showed a decreasing DNA adduct formation. Because DNA damage is responsible for both the nephrotoxicity and carcinogenicity of AA, these results shed greater light on the endemic nature of BEN.

Assessment of Retinal Microangiopathy in Patients with Balkan Endemic Nephropathy Using Optical Coherence Tomography Angiography-A Pilot Study.

Background and Objectives: It is well known that alterations in microvascular structure and function contribute to the development of ocular, renal, and cardiovascular diseases. Accordingly, the presence of fundus vascular changes in patients suffering from chronic kidney disease (CKD) and Balkan endemic nephropathy (BEN) may provide information of prognostic value regarding the progression of renal disease. This study aimed to examine the associations between clinical characteristics and retinal optical coherence tomography angiography (OCTA) parameters in patients with BEN and compare them with those in CKD. Materials and Methods: This pilot study, conducted from March 2021 to April 2022, included 63 patients who were divided into two groups: the first group consisted of 29 patients suffering from BEN, and the second was a control group of 34 patients with CKD. Demographic, laboratory, clinical, and medication data were noted for all the patients included in this study. Each eye underwent OCT angiography, and the results were interpreted in accordance with the practical guide for the interpretation of OCTA findings. Results: Statistically significantly higher levels of total serum protein and triglycerides were recorded in the BEN group than in the CKD group, while the level of HDL cholesterol was lower. Based on the performed urinalysis, statistically significantly higher values of total protein and creatinine were detected in patients with CKD compared to the BEN group. It was demonstrated that the OCTA vascular plexus density of certain parts of the retina was in significant association with systolic and diastolic blood pressure, creatinine clearance, urinary creatinine, total cholesterol, diabetes mellitus type 2, age, body mass index, total serum and urinary protein, sCRP, and diuretic and antihypertensive treatment. Conclusions: In comparison with CKD, BEN leads to more significant disturbances in retinal vasculature density.

SGLT2 inhibitor dapagliflozin protects the kidney in a murine model of Balkan nephropathy.

Proximal tubular uptake of aristolochic acid (AA) forms aristolactam (AL)-DNA adducts, which cause a p53/p21-mediated DNA damage response and acute tubular injury. Recurrent AA exposure causes kidney function loss and fibrosis in humans (Balkan endemic nephropathy) and mice and is a model of (acute kidney injury) AKI to chronic kidney disease (CKD) transition. Inhibitors of the proximal tubule sodium-glucose transporter SGLT2 can protect against CKD progression, but their effect on AA-induced kidney injury remains unknown. C57BL/6J mice (15-wk-old) were administered vehicle or AA every 3 days for 3 wk (10 and 3 mg/kg ip in females and males, respectively). Dapagliflozin (dapa, 0.01 g/kg diet) or vehicle was initiated 7 days prior to AA injections. All dapa effects were sex independent, including a robust glycosuria. Dapa lowered urinary kidney-injury molecule 1 (KIM-1) and albumin (both normalized to creatinine) after the last AA injection and kidney mRNA expression of early DNA damage response markers (p53 and p21) 3 wk later at the study end. Dapa also attenuated AA-induced increases in plasma creatinine as well as AA-induced up-regulation of renal pro-senescence, pro-inflammatory and pro-fibrotic genes, and kidney collagen staining. When assessed 1 day after a single AA injection, dapa pretreatment attenuated AL-DNA adduct formation by 10 and 20% in kidney and liver, respectively, associated with reduced p21 expression. Initiating dapa application after the last AA injection also improved kidney outcome but in a less robust manner. In conclusion, the first evidence is presented that pretreatment with an SGLT2 inhibitor can attenuate the AA-induced DNA damage response and subsequent nephropathy.NEW & NOTEWORTHY Recurrent exposure to aristolochic acid (AA) causes kidney function loss and fibrosis in mice and in humans, e.g., in the form of the endemic Balkan nephropathy. Inhibitors of the proximal tubule sodium-glucose transporter SGLT2 can protect against CKD progression, but their effect on AA-induced kidney injury remains unknown. Here we provide the first evidence in a murine model that pretreatment with an SGLT2 inhibitor can attenuate the AA-induced DNA damage response and subsequent nephropathy.

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.

Simultaneous toxicokinetic studies of aristolochic acid I and II and aristolactam I and II using a newly-developed microdialysis liquid chromatography-tandem mass spectrometry.

Aristolochic acids (AAs) are naturally occurring genotoxic carcinogens linked to Balkan endemic nephropathy and aristolochic acid nephropathy. Aristolochic acid I and II (AA-I and AA-II) are the most abundant AAs, and AA-I has been reported to be more genotoxic and nephrotoxic than AA-II. This study aimed to explore metabolic differences underlying the differential toxicity. We developed a novel microdialysis sampling coupled with solid-phase extraction liquid chromatography-tandem mass spectrometry (MD-SPE-LC-MS/MS) to simultaneously study the toxicokinetics (TK) of AA-I and AA-II and their corresponding aristolactams (AL-I and AL-II) in the blood of Sprague Dawley rats co-treated with AA-1 and AA-II. Near real-time monitoring of these analytes in the blood of treated rats revealed that AA-I was absorbed, distributed, and eliminated more rapidly than AA-II. Moreover, the metabolism efficiency of AA-I to AL-I was higher compared to AA-II to AL-II. Only 0.58% of AA-I and 0.084% of AA-II was reduced to AL-I and AL-II, respectively. The findings are consistent with previous studies and support the contention that differences in the in vivo metabolism of AA-I and AA-II may be critical factors for their differential toxicities.

Differential Cytokine Responses and the Clinical Severity of Adult and Pediatric Nephropathia Epidemica.

Nephropathia epidemica (NE), caused by the hantavirus infection, is endemic in Tatarstan Russia. The majority of patients are adults, with infection rarely diagnosed in children. This limited number of pediatric NE cases means there is an inadequate understanding of disease pathogenesis in this age category. Here, we have analyzed clinical and laboratory data in adults and children with NE to establish whether and how the disease severity differs between the two age groups. Serum cytokines were analyzed in samples collected from 11 children and 129 adult NE patients during an outbreak in 2019. A kidney toxicity panel was also used to analyze urine samples from these patients. Additionally, serum and urine samples were analyzed from 11 control children and 26 control adults. Analysis of clinical and laboratory data revealed that NE was milder in children than in adults. A variation in serum cytokine activation could explain the differences in clinical presentation. Cytokines associated with activation of Th1 lymphocytes were prominent in adults, while they were obscured in sera from pediatric NE patients. In addition, a prolonged activation of kidney injury markers was found in adults with NE, whilst only a short-lasting activation of these markers was observed in children with NE. These findings support previous observations of age differences in NE severity, which should be considered when diagnosing the disease in children.

Effects of Diet on Aristolochic Acid-DNA Adduct Formation: Implications for Balkan Endemic Nephropathy Etiology.

Prolonged exposure to aristolochic acids (AAs) through AA-containing herbal medicine or AA-contaminated food is associated with the development of aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN), both public health risks to which the World Health Organization is calling for global action to remove exposure sources. The AA exposure-induced DNA damage is believed to be related to both the nephrotoxicity and carcinogenicity of AA observed in patients suffering from BEN. While the chemical toxicology of AA is well-studied, we investigated in this study the understated effect of different nutrients, food additives, or health supplements on DNA adduct formation by aristolochic acid I (AA-I). By culturing human embryonic kidney cells in an AAI-containing medium enriched with different nutrients, results showed that cells cultured in fatty acid-, acetic acid-, and amino acid-enriched media produced ALI-dA adducts at significantly higher frequencies than that cultured in the normal medium. ALI-dA adduct formation was most sensitive to amino acids, indicating that amino acid- or protein-rich diets might lead to a higher risk of mutation and even cancer. On the other hand, cells cultured in media supplemented with sodium bicarbonate, GSH, and NAC reduced ALI-dA adduct formation rates, which sheds light on their potential use as risk-mitigating strategies for people at risk of AA exposure. It is anticipated that the results of this study will help to better understand the effect of dietary habits on cancer and BEN development.

Broad-specificity indirect competitive enzyme-linked immunosorbent assay for aristolochic acids: Computer-aided hapten design and molecular mechanism of antibody recognition.

Long-term dietary exposure of aristolochic acids (AAs)-contaminated food proved to be one of the main culprits of Endemic Nephropathy, renal failure; and urothelial cancer. The antibodies utilized in immunoassays for AAs suffer from low affinity and failure of recognition to the family of AAs. This study, we prepared a broad-specificity monoclonal antibody (mAb) 5H5 with highly and uniform affinity for AAs by help of computational chemistry fully exposing the AAs common structures of methoxy and hydroxyl groups. The mAb 5H5 exhibited half inhibitory concentrations of AAA, AAB, AAC, AAD were 0.03, 0.06, 0.05, 0.03 ng/mL. To explain the broad-specificity profile of mAb 5H5, molecular docking was performed. Results shown that multiple conformations of AAs can be flexibly oriented in the spacious cavity of single-chain variable fragment antibody (scFv) 5H5 and the specific hydron bonds were formed by ASN62 and GLY64 of scFV 5H5 to the nitro group of AAs which gave an explanation of the high cross-reactivity of mAb 5H5. The ELISA based on the broad-specificity mAb 5H5with detection limits of 0.04-0.11 µg/kg and 0.02-0.06 µg/kg for four AAs in flour and soil samples, respectively. The study provided a promising method for the family of AAs in environmental and food samples.

Optimised Fermentation Production of Radiolabelled Ochratoxin A by Aspergillus ochraceus with Maximum 14C in the Pentaketide Moiety for Exploring Its Rat Renal Toxicology.

In the context of the mysterious Balkan endemic nephropathy of the 1900s, and the discovery in the 1960s of the potent mycotoxin ochratoxin A, experimental research projects sought to explore any inter-relationship. Experimental lifetime administration of the toxin to male rats had revealed renal DNA adducts with the toxin, correlated with renal tumours, confirmation of which required molecular evidence. Consequently, production of 14C-ochratoxin A of a high specific radioactivity was required, practical biosynthetic detail of which had not previously been published. A fermentation study of Aspergillus ochraceous was carried out during 2002 for a European project, to select for the production of high-quality 14C-ochratoxin A, necessarily exploring for the maximum diversion of 14C-sodium acetate into the pentaketide portion of mycotoxin. Experimentation necessarily had to optimise the competitive context of fungal growth dynamics and addition of the biosynthetic precursor in the early days of shaken-flask fermentation before adding the radiolabelled precursor. From optimal fermentation, 50 mg of the 14C ochratoxin A was supplied within a European project for DNA adduct experimentation, but that proved negative as subsequently published. Experimental description of the radiolabelled ochratoxin A production was later made in a doctoral thesis, but is first publicised here. Further review of the literature reveals an explanation for the published failure to confirm rat DNA/ochratoxin A adduct formation, for which further experimentation is now recommended.

Detection and Removal of Aristolochic Acid in Natural Plants, Pharmaceuticals, and Environmental and Biological Samples: A Review.

Aristolochic acids (AAs) are a toxic substance present in certain natural plants. Direct human exposure to these plants containing AAs leads to a severe and irreversible condition known as aristolochic acid nephropathy (AAN). Additionally, AAs accumulation in the food chain through environmental mediators can trigger Balkan endemic nephropathy (BEN), an environmental variant of AAN. This paper presents a concise overview of the oncogenic pathways associated with AAs and explores the various routes of environmental exposure to AAs. The detection and removal of AAs in natural plants, drugs, and environmental and biological samples were classified and summarized, and the advantages and disadvantages of the various methods were analyzed. It is hoped that this review can provide effective insights into the detection and removal of AAs in the future.

Synergistic Interaction of Polycyclic Aromatic Hydrocarbons, Phthalate Esters, or Phenol on DNA Adduct Formation by Aristolochic Acid I: Insights into the Etiology of Balkan Endemic Nephropathy.

Balkan endemic nephropathy (BEN) is a multifactorial environmental disease, with chronic exposure to aristolochic acids (AAs) through AA-contaminated food being one of the major etiological mechanisms. However, the bulk of previous research has only focused on investigating the possible roles of individual pollutants in disease development and the etiological mechanism of BEN remains controversial. In this study, we investigated the exposure concentration and duration dependence of coexposure to phthalate esters and lignite coal-derived phenol and polycyclic aromatic hydrocarbons (PAHs) on the metabolism and DNA adduct formation of aristolochic acid I (AAI). Results showed that both the metabolic activation and DNA adduct formation of AAI in cultured human kidney cells were affected by their coexposure to the above-mentioned environmental pollutants. Furthermore, our results suggest that chemicals leached from lignite coal likely played a role by triggering AA-activating enzymes to produce more of the promutagenic DNA adducts, thus further elevating the nephrotoxicity and carcinogenicity of AAs and increasing the risk of BEN. It is believed that the results of this study provide a better understanding of the etiological mechanism of BEN and offer insights into methods and policies to lower the risk of this devastating disease.

Aristolochic acid I as an emerging biogenic contaminant involved in chronic kidney diseases: A comprehensive review on exposure pathways, environmental health issues and future challenges.

Described in the 1950s, Balkan Endemic Nephropathy (BEN) has been recognized as a chronic kidney disease (CKD) with clinical peculiarities and multiple etiological factors. Environmental contaminants - aromatic compounds, mycotoxins and phytotoxins like aristolochic acids (AAs) - polluting food and drinking water sources, were incriminated in BEN, due to their nephrotoxic and carcinogenic properties. The implication of AAs in BEN etiology is currently a highly debated topic due to the fact that they are found within the Aristolochiaceae plants family, used around the globe as traditional medicine and they were also incriminated in Aristolochic Acid Nephropathy (AAN). Exposure pathways have been investigated, but it is unclear to what extent AAs are acting alone or in synergy with other cofactors (environmental, genetics) in triggering kidney damage. Experimental studies strengthen the hypothesis that AAI, the most studied compound in the AAs class, is a significant environmental contaminant and a most important causative factor of BEN. The aim of this review is to compile information about the natural exposure pathways to AAI, via traditional medicinal plants, soil, crop plants, water, food, air. Data that either supports or contradicts the AAI theory concerning BEN etiology was consolidated and available solutions to reduce human exposure were discussed. Because AAI is a phytotoxin with physicochemical properties that allow its transportation in environmental matrices from different types of areas (endemic, nonendemic), and induce CKDs (BEN, AAN) and urinary cancers through bioaccumulation, this review aims to shed a new light on this compound as a biogenic emerging pollutant.

Mesoamerican Endemic Nephropathy (MeN): A Disease Reported in Adults That May Start Since Childhood?

Mesoamerican endemic nephropathy (MeN) is a type of chronic kidney disease (CKD) of uncertain etiology that occurs along the Pacific coast of the southern part of Mexico and Central America. During the past 20 years MeN has become a leading cause of death in the region, clamming close to 50,000 lives, with 40% of these deaths occurring in young people. The cause remains unknown, but most researchers believe in a multifactorial etiology that includes social determinants of poverty. Existing evidence suggests that subclinical kidney injury begins early in life and leads to a higher than expected prevalence of CKD among children in Central America. Access to health services in the region, specifically kidney replacement therapy, remains limited. We proposed a strategy to address the perceived needs and urge coordinated efforts of governments, academic organizations, and international bodies to develop a comprehensive plan of action to mitigate this situation among the vulnerable and economically disadvantaged population.

Analysis of Polycyclic Aromatic Hydrocarbons and Phthalate Esters in Soil and Food Grains from the Balkan Peninsula: Implication on DNA Adduct Formation by Aristolochic Acid I and Balkan Endemic Nephropathy.

Balkan endemic nephropathy (BEN) is a chronic tubulointerstitial nephropathy affecting residents of rural farming areas in many Balkan countries. Although it is generally believed that BEN is an environmental disease caused by multiple geochemical factors with much attention on aristolochic acids (AAs), its etiology remains controversial. In this study, we tested the hypothesis that environmental contamination and subsequent food contamination by polycyclic aromatic hydrocarbons (PAHs) and phthalate esters are AA toxicity factors and important to BEN development. We identified significantly higher concentrations of phenanthrene, anthracene, diethyl phthalate (DEP), dibutyl phthalate (DBP), and benzyl butyl phthalate (BBP) in both maize and wheat grain samples collected from endemic villages than from nonendemic villages. Other PAHs and phthalate esters were also detected at higher concentrations in the soil samples from endemic villages. Subsequent genotoxicity testing of cultured human kidney cells showed an alarming phenomenon that phenanthrene, DEP, BBP, and DBP can interact synergistically with AAs to form elevated levels of AA-DNA adducts, which are associated with both the nephrotoxicity and carcinogenicity of AAs, further increasing their disease risks. This study provides direct evidence that prolonged coexposure to these environmental contaminants via dietary intake may lead to greater toxicity and accelerated development of BEN.

Prognostic value of Balkan endemic nephropathy and gender on upper tract urothelial carcinoma outcomes after radical nephroureterectomy: A cohort study.

BACKGROUND: To identify the prognostic impact of residence in a BEN-endemic area and gender on upper tract urothelial carcinoma (UTUC) outcomes in Serbian patients treated with radical nephroureterectomy (RNU). METHODS: The study included 334 consecutive patients with UTUC. Patients with permanent residence in Balkan endemic nephropathy (BEN) or non-endemic areas from their birth to the end of follow-up were included in the analysis. Cox regression analyses were used to address recurrence-free (RFS) and cancer-specific survival (CSS) estimates. RESULTS: Female patients were more likely to have preoperative pyuria (P = 0.01), tumor multifocality was significantly associated with the female gender (P = 0.003). Gender was not associated with pathologic stage and grade, lymph node metastasis, lymphovascular invasion, adjuvant chemotherapy, bladder cancer history, tumor size, distribution of tumor location, preoperative anemia and demographic characteristics. A total of 107 cases recurred, with a median time to bladder recurrence of 24.5 months. History of bladder tumor (HR, 1.98; P = 0.005), tumor multifocality (HR, 3.80; P < 0.001) and residence in a BEN-endemic area (HR, 1.81; P = 0.01) were independently associated with bladder cancer recurrence. The 5-year bladder cancer RFS for the patients from areas of BEN was 77.8 % and for the patients from non-BEN areas was 64.7 %. The 5-year CSS for the men was 66.2% when compared to 66.6% for the women (P = 0.55). CONCLUSIONS: Residence in a BEN-endemic area represents an independent predictor of bladder cancer recurrence in patients who underwent RNU. Gender cannot be used to predict outcomes in a single-centre series of consecutive patients who were treated with RNU for UTUC.

Aristolochic acid I: an investigation into the role of food crops contamination, as a potential natural exposure pathway.

Aristolochic acid I (AAI) is a potent nephrotoxic and carcinogenic compound produced by plants of the Aristolochiaceae family and thoroughly investigated as a main culprit in the etiology of Balkan endemic nephropathy (BEN). So far, the AAI exposure was demonstrated to occur through the consumption of Aristolochia clematitis plants as traditional remedies, and through the contamination of the surrounding environment in endemic areas: soil, food and water contamination. Our study investigated for the first time the level of AAI contamination in 141 soil and vegetable samples from two cultivated gardens in non-endemic areas, A. clematitis being present in only one of the gardens. We developed and validated a simple and sensitive ultra-high-performance liquid chromatography-ion trap mass spectrometry method for qualitative and quantitative AAI analysis. The results confirmed the presence of AAI at nanogram levels in soil and vegetable samples collected from the non-endemic garden, where A. clematitis grows. These findings provide additional evidence that the presence of A. clematitis can cause food crops and soil contamination and unveil the pathway through which AAI could move from A. clematitis to other plant species via a common matrix: the soil. Another issue regarding the presence of AAI, in a non-endemic BEN area from Romania, could underlie a more widespread environmental exposure to AAI and explain certain BEN-like cases in areas where BEN has not been initially described.

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.

Mutations in Collagen Genes in the Context of an Isolated Population.

Genetic studies of population isolates have great potential to provide a unique insight into genetic differentiation and phenotypic expressions. Galicnik village is a population isolate located in the northwest region of the Republic of North Macedonia, established around the 10th century. Alport syndrome-linked nephropathy with a complex inheritance pattern has been described historically among individuals in the village. In order to determine the genetic basis of the nephropathies and to characterize the genetic structure of the population, 23 samples were genotyped using a custom-made next generation sequencing panel and 111 samples using population genetic markers. We compared the newly obtained population data with fifteen European population data sets. NGS analysis revealed four different mutations in three different collagen genes in twelve individuals within the Galicnik population. The genetic isolation and small effective population size of Galicnik village have resulted in a high level of genomic homogeneity, with domination of R1a-M458 and R1b-U106* haplogroups. The study explains complex autosomal in cis digenic and X-linked inheritance patterns of nephropathy in the isolated population of Galicnik and describes the first case of Alport syndrome family with three different collagen gene mutations.

Prof. Dimitar Hrisoho, Md, Phd, Founder of Nephrology in the R. N. Macedonia.

Prof. Dr. Dimitar T. Hrisoho was born on June 11, 1924 in Bitola, R. Macedonia. He died in Struga on September 22, 1986, and was buried in Skopje. He completed primary and secondary education in Bitola. He graduated from the Medical Faculty in Belgrade in 1951 as one of the best students of his generation (average grade of 9.75). In 1953 he was employed at the Internal Clinic of the Medical Faculty in Skopje, where in 1955 he passed the specialist exam in internal medicine. He successfully defended his habilitation "Polyarthritis chronica evolutiva" and his doctoral dissertation "Clinical features of Vitina nephropathy". The doctoral dissertation indicates that Vitina nephropathy is a new site of the Balkan Endemic Nephropathy entity and that more genetic testing of patients were needed. Based on numerous clinical and scientific researches published in over 200 papers, he was elected a Full Professor of internal medicine at the Medical Faculty of the Ss. Cyril and Methodius University in Skopje in 1971. In 1970, he formed the nephrology section of the Macedonian Medical Association (MMA), which grew into the nephrology Association of MMA. Through the Association, the education of the medical staff from the field of nephrology was performed. He also set up a bio-cybernetics association. He achieved his vision and desire to transfer and apply the achievements of modern nephrology in the diagnosis and treatment of kidney patients in Macedonia at the Clinic of Nephrology of the Medical Faculty in Skopje, which was the first specialized institution established for examination and treatment of kidney patients in the former Yugoslavia and the Balkans. The Clinic educated nephrological staff and examined and treated kidney patients with new methods and drugs that positively affected the development of nephrology as a subspecialty of the internal medicine. D. Hrisoho was actively involved in the introduction of new methods for examination of kidney patients, as well as in the treatment of patients with acute and chronic renal insufficiency with dialysis since 1965. He also participated in the first two kidney transplantations from living donors performed in 1977. He wrote a chapter on "kidney examination", printed in the book of Prof. A. J. Ignjatovski "Fundamentals of Internal Propedeutics" Part III, published by "Prosvetno delo", 1963, in Skopje. This is the first text to investigate a patient with kidney disease published in a textbook in R. Macedonia. In 1984 he published the textbook "Clinical Nephrology" printed by the University of the Ss. Cyril and Methodius University in Skopje. Prof. D. Hrisoho organized the First Scientific Meeting of Yugoslav Nephrologists with international participation, from 26 to 28 September 1977, in Struga, R. Macedonia. The meeting was attended by prominent nephrologists from the former Yugoslavia, the Balkans, Europe and the United States, among them: J.S. Cameron from UK, J.L. Funck-Brentano from France, M. Burg and P. Ivanovich from the USA, R. Kluthe from Germany and A. Puchlev from Bulgaria. The scientific meeting was the largest nephrology event until then organized in the former Yugoslavia. The meeting provided an exchange of experiences with world-renowned nephrologists. D. Hrisoho presented the paper Artificial intelligence in nephrology. The author tried to apply bio-cybernetics in nephrology. Prof. D. Hrisoho was Vice Dean of the Medical Faculty in Skopje in the period 1963-1965 and Vice Rector of the University of the Ss. Cyril and Methodius University in Skopje in the period 1974-1975. Prof. Hrisoho was also active in socio-political organizations. For his medical, educational and scientific activities he received several awards and recognitions in the country and abroad. Thus, the work of Prof. D. Hrisoho was permanently embedded in the nephrology of R. Macedonia.