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.

Balkan Endemic Nephropathy and the Causative Role of Aristolochic Acid.

Balkan endemic nephropathy is a chronic tubulointerstitial disease with insidious onset, slowly progressing to end-stage renal disease and frequently associated with urothelial carcinoma of the upper urinary tract (UTUC). It was described in South-East Europe at the Balkan peninsula in rural areas around tributaries of the Danube River. After decades of intensive investigation, the causative factor was identified as the environmental phytotoxin aristolochic acid (AA) contained in Aristolochia clematitis, a common plant growing in wheat fields that was ingested through home-baked bread. AA initially was involved in the outbreak of cases of rapidly progressive renal fibrosis reported in Belgium after intake of root extracts of Aristolochia fangchi imported from China. A high prevalence of UTUC was found in these patients. The common molecular link between Balkan and Belgian nephropathy cases was the detection of aristolactam-DNA adducts in renal tissue and UTUC. These adducts are not only biomarkers of prior exposure to AA, but they also trigger urothelial malignancy by inducing specific mutations (A:T to T:A transversion) in critical genes of carcinogenesis, including the tumor-suppressor TP53. Such mutational signatures are found in other cases worldwide, particularly in Taiwan, highlighting the general public health issue of AA exposure by traditional phytotherapies.

How common is Balkan endemic nephropathy among immigrants in endemic regions?

PURPOSE: In the early 1970s, a number of authors described the development of Balkan endemic nephropathy (BEN) in immigrants in endemic regions. The aim of this study was to examine whether immigrants in endemic regions are suffering from BEN today. METHODS: The study involved 193 residents of two endemic regions divided into three groups: two groups of native residents-(1) members of BEN families, (2) members of non-BEN families, and (3) immigrants, who had moved from non-affected settlements to the endemic regions of Kolubara (38 years ago) or Semberia (20 years ago). All persons were subjected to an interview, objective examination, kidney ultrasound, and laboratory analysis to detect the presence of BEN consensus diagnostic criteria. RESULTS: The number of immigrants with BEN biomarkers outside cutoff values was significantly lower than for BEN family members. Five BEN family members met diagnostic criteria for BEN and four for suspected BEN. Although five non-BEN family members had different combinations of BEN biomarkers, all of them had diseases other than BEN in which these biomarkers also occurred. None of the immigrants met the criteria for BEN. Nevertheless, one descendant of an immigrant, a 78-year-old male, whose mother was from a non-BEN family in the Kolubara district, exhibited all the criteria for BEN: alpha1-microglobulinuria, chronic renal failure, and anemia. CONCLUSION: While 30 years ago, BEN was reported equally among immigrants and natives, currently it is diagnosed in some BEN family members in the eighth decade of life, but extremely rarely in immigrants also in old age.

An Integrated View of Aristolochic Acid Nephropathy: Update of the Literature.

The term "aristolochic acid nephropathy" (AAN) is used to include any form of toxic interstitial nephropathy that is caused either by ingestion of plants containing aristolochic acids (AA) as part of traditional phytotherapies (formerly known as "Chinese herbs nephropathy"), or by the environmental contaminants in food (Balkan endemic nephropathy). It is frequently associated with urothelial malignancies. Although products containing AA have been banned in most of countries, AAN cases remain regularly reported all over the world. Moreover, AAN incidence is probably highly underestimated given the presence of AA in traditional herbal remedies worldwide and the weak awareness of the disease. During these two past decades, animal models for AAN have been developed to investigate underlying molecular and cellular mechanisms involved in AAN pathogenesis. Indeed, a more-in-depth understanding of these processes is essential to develop therapeutic strategies aimed to reduce the global and underestimated burden of this disease. In this regard, our purpose was to build a broad overview of what is currently known about AAN. To achieve this goal, we aimed to summarize the latest data available about underlying pathophysiological mechanisms leading to AAN development with a particular emphasis on the imbalance between vasoactive factors as well as a focus on the vascular events often not considered in AAN.

[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.

Herbs and hazards: risk of aristolochic acid nephropathy in Iran.

Herbs are usually considered as inherently harmless products. Nonetheless, various renal injuries have been reported in association with several herbs. The best-known herb-induced chronic kidney disease is aristolochic acid nephropathy. Aristolochic acid is found in Chinese slim herbs. Balkan endemic nephropathy is nowadays considered as an aristolochic acid nephropathy. Plants of Aristolochiaceae (also known as birthwort, dutchman's pipe, and somersworth) is named zaravand or chopoghak in Persian and it grows in different mountainous and rural areas of Iran. The fruit and the steam of the Aristolochiacae are named zaravand gerd (nokhod alvand) and zaravand dearaz, respectively, and have different usage in Iranian teadirional such as treatment of headache, back pain, and anxiety. Some patients with end-stage renal disease and bilateral small kidneys have a history of exposure to some herbal remedies. We need to consider the possibility of environmental toxins and even Aristolochia nephrotoxicity as a potential danger in Iran.

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.

Genetic, Genomic and Epigenomic Studies of Balkan Endemic Nephropathy (Ben).

BEN is a primary, chronic tubulointerstitial nephritis characterized with chronic anemia, absence of edema, xantoderma, normal blood pressure and normal findings on the fundus oculi. The disease is distributed in restricted areas in Bulgaria, Romania, Croatia, Bosnia, Former Yugoslavia. Despite numerous studies on genetic and environmental factors and their possible involvement in BEN, its etiopathogenesis still remains elusive. Our recent study aim to elucidate the possible epigenetic component in BEN development. Whole genome DNA array methylation analysis was applied to compare the methylation profiles of male and female BEN patients from endemic regions in Bulgaria and Serbia and healthy controls. All three most prominent candidate genes with aberrations in the epigenetic profile discovered with this study are involved in the inflammatory/immune processes and oncogenesis. These data are in concordance with the reported pathological alterations in BEN. This research supports the role of epigenetic changes in BEN pathology. Exome sequencing of 22.000 genes with Illumina Nextera Exome Enrichment Kit revealed three mutant genes (CELA1, HSPG2, and KCNK5) in BEN patients which encode proteins involved in basement membrane/extracellular matrix and vascular tone, tightly connected to process of angiogenesis. We suggest that an abnormal process of angiogenesis plays a key role in the molecular pathogenesis of BEN.

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.

Proteomic studies in endemic nephropathy.

Endemic nephropathy (EN) is a chronic tubulointerstitial nephropathy with an early insidious and slow development into terminal renal failure. Proteomics is the systematic study of a proteome, which is the total protein content of a cell, organism or body fluids. Application of proteomic technologies in nephrology has enabled more detailed analyses of protein functions and examined their importance in various physiological and pathological states. Biomarkers with high specificity and sensitivity to early diagnosis are needed for a better understanding of the mechanisms of EN development and its consequences. Urine beta2-microglobulin (B2M) was mainly used as a tubular marker of EN but recently alpha1-microglobulin (AMBP) was proposed for the diagnosis of EN. We studied the urine proteins of 360 patients with EN, diabetic nephropathy (DN) and acute kidney injury (AKI) and the healthy population using proteomic tools. Protein maps from the urine of patients with EN showed significant differences in comparison to the healthy subjects and patients with DN and AKI. Our study highlights six proteins in urine that were differentially excreted in the urine of EN patients compared with the other groups and have potential to be markers for EN prediction. In one of our studies, using routine biomarkers, we investigated the potential of urine B2M, AMBP, albumin and total protein as diagnostic markers for EN, in comparison to glomerulonephritis, nephrosclerosis and a healthy state. Modern proteomic technologies are still robust investigation tools, but can access a vast amount of information from one set of experiments in comparison to a classic diagnostic approach.

Evaluation of diagnostic criteria for endemic nephropathy.

Diagnosis of endemic nephropathy (EN) is based on the combination of several clinical and laboratory criteria. Despite extensive research no specific diagnostic biomarker for EN has yet been identified. The aim of the study was to evaluate the diagnostic significance of the variables previously proposed as diagnostic criteria, but also new ones. After an extended questionnaire, the clinical and laboratory examination population in EN villages was classified according to the modified WHO criteria. The urinary active form of TGF-ß was measured with a bioassay using a cell line which expresses luciferase activity. In the study we used ROC analysis to examine the predictive value of the tested variables. In the study there was no difference in haemoglobin level between the study subgroups. Leucine aminopeptidase (LAP) in urine and active urinary TGF-ß levels were increased in the EN diseased group when compared to other subgroups, but they did not fulfil the statistical criteria needed for differentiating a diseased form from other study subgroups. Both kidney length and parenchima thickness, alfa1-microglobulinuria, and kidney function assessed by MDRD formula were the variables that differentiated the study subgroups well. Based on our results the cut-off value of alfa1-microglobulin for screening should be 23.5 mg/g creatinine instead of 15 mg/g creatinine in the present criteria, and for making a diagnosis of EN 31,5 mg/g creatinine. Persons with a positive family history for EN had a 5.8 times greater risk of developing EN when compared to a negative one. Taken together, the above-mentioned variables should be implemented in new uniform diagnostic criteria for EN.

Endemic nephropathy and upper urothelial carcinoma--new insights in molecular biology.

Upper Tract Urothelial Carcinoma (UTUC) is an uncommon disease which occurs more frequently in some regions of Balkan countries than in other areas in the world. Investigation of UTUC in the South Morava River basin and its tributaries where BEN is endemic revealed increased frequency not only of tumour of the renal pelvis and ureter but also of urinary bladder tumours. A comparative morphological and immunohistochemical study of UTUC in the BEN region and control rural and city populations free of BEN, identify growth pattern as the best morphological characteristic which differentiated BEN and control tumours, i.e. solid growth for BEN tumours and papillary for control tumours. Overexpression of tumour suppressor p53 as well as decreased expression of E-CD was detected in BEN tumours. Other cells cycle related molecular markers--Cyclin D1, p16, and HER-2 showed no difference in expression between groups, as well as the proliferative marker Ki-67. Investigation of apoptosis-related markers identifies Bax as a specific marker of BEN-associated UTUC. Decrease of the pro-apoptotic protein Bax together with alteration of Survivin may be indicative of specific disturbances of an intrinsic apoptotic pathway in UTUC arising in endemic areas.

Update on endemic nephropathies.

PURPOSE OF REVIEW: A large number of patients worldwide suffer from chronic kidney disease (CKD) of unknown cause. Endemic nephropathies possibly contribute to this. The purpose of this review is to give a brief review of endemic nephropathies and to summarize what is known about their cause. RECENT FINDINGS: The cause of Balkan endemic nephropathy was eventually resolved, after 50 years of research. The cause was exposure to aristolochic acid from food. A new type of endemic nephropathy has recently been identified in Central America; Mesoamerican nephropathy. This kidney disease mainly affects agricultural workers in hot climates. Renal biopsy studies suggest that repeated dehydration and kidney ischemia is involved in the pathogenesis. SUMMARY: Endemic nephropathies may comprise an important cause of CKD. Epidemiological studies are needed to describe the occurrence and distribution of the diseases. However, biopsy studies, in combination with careful clinical evaluation of the patients, are necessary to find out the cause of endemic nephropathies and thereby help in their prevention.

Consensus statement on screening, diagnosis, classification and treatment of endemic (Balkan) nephropathy.

Currently used diagnostic criteria in different endemic (Balkan) nephropathy (EN) centers involve different combinations of parameters, various cut-off values and many of them are not in agreement with proposed international guidelines. Leaders of EN centers began to address these problems at scientific meetings, and this paper is the outgrowth of those discussions. The main aim is to provide recommendations for clinical work on current knowledge and expertise. This document is developed for use by general physicians, nephrologists, urologist, public health experts and epidemiologist, and it is hoped that it will be adopted by responsible institutions in countries harboring EN. National medical providers should cover costs of screening and diagnostic procedures and treatment of EN patients with or without upper urothelial cancers.

The epidemiology, diagnosis, and management of aristolochic acid nephropathy: a narrative review.

It has been 20 years since the first description of a rapidly progressive renal disease that is associated with the consumption of Chinese herbs containing aristolochic acid (AA) and is now termed aristolochic acid nephropathy (AAN). Recent data have shown that AA is also the primary causative agent in Balkan endemic nephropathy and associated urothelial cancer. Aristolochic acid nephropathy is associated with a high long-term risk for renal failure and urothelial cancer, and the potential worldwide population exposure is enormous. This evidence-based review of the diagnostic approach to and management of AAN draws on the authors' experience with the largest and longest-studied combined cohort of patients with this condition. It is hoped that a better understanding of the importance of this underrecognized and severe condition will improve epidemiologic, preventive, and therapeutic strategies to reduce the global burden of this disease.

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.

Angiogenesis in upper tract urothelial carcinoma associated with Balkan endemic nephropathy.

Upper tract urothelial carcinoma (UTUC) associated with Balkan endemic nephropathy (BEN) is characterized by a number of aberrations in cell-cycle regulation and apoptosis. The aim of this study was to detect angiogenesis-related marker(s) specific for BEN UTUC, and to examine the influence of HIF 1α upon angiogenesis and apoptosis in UTUC. Present investigation included 110 patients with UTUC, 50 from BEN region and 60 control tumors. Altered expression of VEGFR1 was more often present in control UTUC than in BEN tumors (p<0.005). It was associated with high grade, low and high stage, solid growth, and metaplastic change of control UTUC. Microvessel density assessed by CD31 (MVD CD31) was significantly higher in UTUC with lymphovascular invasion (p<0.05), and in BEN tumors with papillary growth (p<0.05). Discriminant analysis indicated that BEN and control tumors do not differ significantly in expression of angiogenesis related markers. The most important discriminant variable that determined control UTUC was expression of VEGFR1 (p=0.002). HIF 1α in UTUC significantly correlated with the low stage, papillary growth and expression of Bcl-2, Caspase-3 index, and MVD CD34 (p<0.001; 0.0005; 0.01; 0.005; 0.01, respectively). HIF-1α may be helpful marker in evaluation of UTUC, especially when combined with angiogenesis and apoptosis.

Aristolactam-DNA adducts are a biomarker of environmental exposure to aristolochic acid.

Endemic (Balkan) nephropathy is a chronic tubulointerstitial disease frequently accompanied by urothelial cell carcinomas of the upper urinary tract. This disorder has recently been linked to exposure to aristolochic acid, a powerful nephrotoxin and human carcinogen. Following metabolic activation, aristolochic acid reacts with genomic DNA to form aristolactam-DNA adducts that generate a unique TP53 mutational spectrum in the urothelium. The aristolactam-DNA adducts are concentrated in the renal cortex, thus serving as biomarkers of internal exposure to aristolochic acid. Here, we present molecular epidemiologic evidence relating carcinomas of the upper urinary tract to dietary exposure to aristolochic acid. DNA was extracted from the renal cortex and urothelial tumor tissue of 67 patients that underwent nephroureterectomy for carcinomas of the upper urinary tract and resided in regions of known endemic nephropathy. Ten patients from nonendemic regions with carcinomas of the upper urinary tract served as controls. Aristolactam-DNA adducts were quantified by (32)P-postlabeling, the adduct was confirmed by mass spectrometry, and TP53 mutations in tumor tissues were identified by chip sequencing. Adducts were present in 70% of the endemic cohort and in 94% of patients with specific A:T to T:A mutations in TP53. In contrast, neither aristolactam-DNA adducts nor specific mutations were detected in tissues of patients residing in nonendemic regions. Thus, in genetically susceptible individuals, dietary exposure to aristolochic acid is causally related to endemic nephropathy and carcinomas of the upper urinary tract.

Could disappearance of endemic (Balkan) nephropathy be expected in forthcoming decades?

BACKGROUND/AIMS: An epidemiological survey of endemic nephropathy (EN) was performed in endemic Croatian areas and the current prevalence was compared to that reported for the same villages several decades ago. METHODS: A total of 2,487 adult farmers from 6 endemic villages and 3 non-endemic villages were enrolled. An extensive epidemiological questionnaire, clinical examination and laboratory analyses of blood and urine were performed. According to the modified WHO criteria, participants were classified into diseased, suspected of having EN, and those at risk of developing EN. RESULTS: The overall prevalence of EN in the Croatian areas was 1.0%, ranging between 0.3 and 2.3% in different villages. Those suspected of having EN amounted to 3.9%. In the endemic villages a decreasing trend in the prevalence of EN was observed comparable to the results obtained in previous surveys. It is interesting to note that no EN patients were recorded in the endemic village of Dubocac. CONCLUSION: The prevalence of EN in the endemic Croatian areas appears to be decreasing. For the first time, we failed to detect any EN patients in a village that was previously considered endemic, which might indicate that EN is diminishing.

Identification and validation of six proteins as marker for endemic nephropathy.

Endemic nephropathy (EN) is defined as a slow progressive renal tubulointestitial disease that mainly occurs in the restricted areas of the Balkan Peninsula. The complexity of the pathogenesis of EN makes its earlier diagnosis very difficult. Urine samples from healthy volunteers from EN regions, EN patients with proteinuria less than 150 mg/L and EN patients with proteinuria more than 150 mg/L, patients with acute kidney injury, patients with diabetic nephropathy and healthy volunteers from Germany were collected. The urinary proteome analyses were performed using 2-D DIGE and mass spectrometry. The validation of biomarkers was investigated by two approaches (Western blot (WB) and dot blot) in successively increasing size - and partially overlapping - sample sets. Comparative and statistical analyses of the proteomics data from the different patient groups allowed the identification of six proteins (alpha-1-microglobulin, alpha-2-glycoprotein-1, beta-2-microglobulin, mannose-binding-lectin-2, protection-of-telomeres-protein-1, and superoxide-dismutase [Cu-Zn]), which were able to discriminate EN with low and high proteinuria from the other groups with high significance (p<0.05). The reliability of the identified proteins as EN marker was underlined with high statistical significance using WB analyses (sensitivity 66.7-98% and specificity 70-100%), whereas the dot blot analyses revealed a decrease in the sensitivity and specificity of these biomarkers.