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.

Local and Systemic Oxidative Stress in Balkan Endemic Nephropathy Is Not Associated with Xanthine Oxidase Activity.

Balkan endemic nephropathy (BEN) represents a chronic tubulointerstitial nephropathy which is followed by the progression of kidney fibrosis to end-stage kidney failure. The critical involvement of poisons in food (aristolochic acid (AA), ochratoxin, and heavy metals) and selenium deficiency are among nutritive factors which contribute to the pathogenesis of BEN, due to reactive oxygen species (ROS) liberation and/or decreased antioxidative defence system. The aim of the study is to distinguish a possible systemic and local origin of ROS through the measurement of xanthine oxidase (XO) activity in urine and plasma, along with the determination of the oxidative changes in lipids and proteins. The study included 50 patients with BEN and 38 control healthy subjects. We noted increased levels of both thiobarbituric acid-reactive substances (TBARS) and advanced oxidation protein products (AOPPs) in the plasma of patients with BEN, compared to the control group (p < 0.001). The urinary levels of AOPPs were higher in patients with BEN in comparison to the control (p < 0.001). The specific activity of XO was significantly lower in plasma and urine in BEN samples, compared to controls (p < 0.005). Based on these results, we hypothesize that XO might not be considered a direct systemic or local contributor to ROS production in BEN, most probably because of the diminished kidney functional tissue mass and/or AA-induced changes in purine nucleotide conformation. The increased AOPP and TBARS level in both plasma and urine in BEN may predict ROS systemic liberation with toxic local effects.

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.

Critical review of renal tubule karyomegaly in non-clinical safety evaluation studies and its significance for human risk assessment.

Scientific databases were searched for terms applicable to karyomegaly in renal tubules of laboratory animals used in preclinical safety evaluation studies, and in humans. Renal tubule karyomegaly was more frequently reported in the rat in response to chemical exposure compared to other laboratory animal species. Renal tubule karyomegaly also occurred in the mouse in response to chemical insult, but much less commonly than in the rat. This nuclear lesion was recorded infrequently for hamster, dog, guinea pig, rabbit, pig, and non-human primate. Most instances of renal karyomegaly reported in humans represented cases of the genetic syndrome, karyomegalic interstitial nephritis, known to be caused by a mutation in the FAN1 gene. Human reports of karyomegaly in the kidney associated with chemical exposure are rare, and linked mainly to chemotherapeutic or antiviral therapies. The rat appears to be highly predisposed to developing karyomegaly as a renal response on exposure to diverse chemical agents, but karyomegaly in the rat is not consistently associated with renal tubule tumor development. Because of this inconsistency, renal tubule karyomegaly is an inaccurate predictor of renal tubule neoplasia, and there is no evidence that karyomegalic cells are involved in tumor development as a form of preneoplasia. A chemically induced karyomegalic response in the rat does not necessarily predict a similar alteration in human kidneys. Because modest nuclear enlargement of kidney tubule cells can occur as physiological or functional responses, it is recommended that the threshold for diagnosing renal tubule karyomegaly in animal studies should be accepted as at least four times normal nuclear size or larger.Abbreviations: BEN: Balkan Endemic Nephropathy; DMN: dimethylnitrosamine; GLP: Good Laboratory Practice; KIN: karyomegalic interstitial nephritis; LAL: lysinoalanine; MeCCNU: 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea; NTP: National Toxicology Program; OSOM: outer stripe of outer medulla; OTA: ochratoxin A; RTT: renal tubule tumor.

The Phytoalexin Resveratrol Ameliorates Ochratoxin A Toxicity in Human Embryonic Kidney (HEK293) Cells.

Ochratoxin A (OTA) is a nephrotoxic mycotoxin produced by Aspergillus and Penicillium fungi. It contaminates human and animal food products, and chronic exposure is associated with renal fibrosis in humans (Balkan endemic nephropathy). Resveratrol, a phytoalexin, possesses anti-cancer and antioxidant properties. We investigated the mechanism of cellular oxidative stress induced by OTA, and the effect of resveratrol in human embryonic kidney (HEK293) cells over 24 and 48 h. Cells were exposed to OTA [IC50 = 1.5 µM (24 h) and 9.4 µM (48 h) determined using MTT assay] and 25 µM resveratrol. Glutathione was quantified by luminometry and gene expression of Nrf2 and OGG1 was determined by qPCR. Protein expression of Nrf2, LonP1, SIRT3, and pSIRT1 was assessed by Western blot, DNA damage (comet assay), and intracellular reactive oxygen species (flow cytometry). At 24 h, resveratrol increased mRNA expression of the DNA repair enzyme, OGG1 (P < 0.05), whereas OTA and OTA+resveratrol significantly decreased OGG1 expression (P < 0.05). OGG1 expression increased during 48-h exposure to resveratrol and OTA+resveratrol (P < 0.05). Comet tail lengths doubled in 48-h OTA-treated cells, whereas at both time periods, OTA+resveratrol yielded shorter comet tails (P < 0.0001). During 24- and 48-h exposure, OTA, resveratrol, and OTA+resveratrol significantly decreased mRNA expression of Nrf2 (P < 0.05). Luminometry analysis of GSH revealed an increase by OTA+resveratrol for 24 and 48 h (P < 0.05 and P < 0.001, respectively). Western blot analysis showed decreased Nrf2 protein expression during 24-h exposure, but increased Nrf2 expression during 48 h. LonP1 protein expression increased during 24-h exposure to OTA (P < 0.05) and OTA+resveratrol (P < 0.0011) and during 48-h exposure to resveratrol (P < 0.0005).

Ochratoxin A and human health risk: a review of the evidence.

Ochratoxin A (OTA) is a mycotoxin produced by several fungal species including Aspergillus ochraceus, A. carbonarius, A. niger, and Penicillium verrucosum. OTA causes nephrotoxicity and renal tumors in a variety of animal species; however, human health effects are less well-characterized. Various studies have linked OTA exposure with the human diseases Balkan endemic nephropathy (BEN) and chronic interstitial nephropathy (CIN), as well as other renal diseases. This study reviews the epidemiological literature on OTA exposure and adverse health effects in different populations worldwide, and assesses the potential human health risks of OTA exposure. Epidemiological studies identified in a systematic review were used to calculate unadjusted odds ratios for OTA associated with various health endpoints. With one exception, there appears to be no statistically significant evidence for human health risks associated with OTA exposure. One Egyptian study showed a significantly higher risk of nephritic syndrome in those with very high urinary OTA levels compared with relatively unexposed individuals; however, other potential risk factors were not controlled for in the study. Larger cohort or case-control studies are needed in the future to better establish potential OTA-related human health effects, and further duplicate-diet studies are needed to validate biomarkers of OTA exposure in humans.

NGS nominated CELA1, HSPG2, and KCNK5 as candidate genes for predisposition to Balkan endemic nephropathy.

Balkan endemic nephropathy (BEN) is a familial chronic tubulointerstitial disease with insidious onset and slow progression leading to terminal renal failure. The results of molecular biological investigations propose that BEN is a multifactorial disease with genetic predisposition to environmental risk agents. Exome sequencing of 22 000 genes with Illumina Nextera Exome Enrichment Kit was performed on 22 DNA samples (11 Bulgarian patients and 11 Serbian patients). Software analysis was performed via NextGene, Provean, and PolyPhen. The frequency of all annotated genetic variants with deleterious/damaging effect was compared with those of European populations. Then we focused on nonannotated variants (with no data available about them and not found in healthy Bulgarian controls). There is no statistically significant difference between annotated variants in BEN patients and European populations. From nonannotated variants with more than 40% frequency in both patients' groups, we nominated 3 genes with possible deleterious/damaging variants--CELA1, HSPG2, and KCNK5. Mutant genes (CELA1, HSPG2, and KCNK5) in BEN patients 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.

Incidence and predictive factors of Balkan endemic nephropathy: a longitudinal study.

Balkan endemic nephropathy (BEN) is a chronic kidney disease that progresses slowly. There are no known clinical markers to identify an early disease development. We evaluated the relationship between parental history of BEN and clinical markers as predictors of new occurrences of BEN. A 5-year prospective study in the offsprings of BEN and control patients was conducted in Vratza, Bulgaria, between 2003 and 2009 using markers in years one and three to predict new cases of BEN in the year five. We defined incident cases of BEN based on parental history, reduced kidney size and reduced kidney function, distinguishing probable and definite BEN, both combined as total incidence. The data were analyzed by Cox regression models using age as time scale and controlling for gender. We estimated hazard ratios and their 95% confidence intervals. The incidence of BEN was 17.4%. Paternal history was strongly associated with all three incidence groups (hazards ratio: 27-68, P <0.05). A reduction of kidney size of 1 mm resulted in a 5% increased hazard. However, taking parental history of BEN into account, these associations lost their significance. No kidney function measures were associated with new onset of BEN. A parental history of BEN is more important than clinical markers predicting the incidence of BEN. Without this information, kidney length forecasts probable BEN and the total incidence, while none of any clinical markers was related to definite BEN.

Morphology of Balkan endemic nephropathy: current state.

Balkan endemic nephropathy (BEN) is interesting renal disease, because of its unique clinical, epidemiological and morphological characteristics: intensive interstitial fibrosis and tubular atrophy without any inflammation. In the present paper we evaluate the incidence of BEN from the morphological point of view for the last decade. Therefore we analyzed material obtained from autopsies, kidney biopsies and nephrectomy due to upper urothelial cancer (UUC) from the patients which were divided into two groups: those with permanent residence in BEN areas and those from nonendemic areas. At the Institute of Pathology, University of Belgrade for the last 15 years we had only 1 autopsy due to BEN out of 6,825. More than 30 years ago there were over 50 autopsy cases of BEN at the same institute. For the last decade we had only 2 kidney biopsies suspected for BEN out of 2,182, but morphologically not confirmed as BEN. However, previously we had over 40 kidney biopsies diagnosed as early or late stage of BEN. At the Clinical Center of Serbia 180 nephrectomies were performed due to UUC. The incidence of UUC for the last five years in BEN regions has significantly decreased, whereas at the same time in non-BEN regions it has remained on the same level. There was no morphological difference of the renal tissue adjacent to tumor between patients from BEN and non-BEN regions. According to our study based on routine pathological work, we could clearly conclude that BEN today is more clinical and epidemiological than a morphological entity.

Higher apoptotic cell rate in Balkan endemic nephropathy--stereologic analysis.

The aim of this study was to correlate apoptotic cell rate from different nephron segments between control group and groups of patients with Balkan endemic nephropathy (BEN). Kidney specimens of20 patients with clinically and epidemiologically confirmed BEN were compared with biopsy material of 10 patients (group I, non BEN) without glomerular or tubulointerstitial disease. Out of 20 patients with BEN, 10 suffered and died from BEN (group II, BEN) and 10 patients (group III, BEN/CV) suffered from BEN but died from cardiovascular disease. Patient age ranged from 40 to 50 years. The apoptotic cell rate was measured in proximal and distal tubules and in collecting ducts using the 40X objective with a calibrated eyepiece multipurpose M 42 test system according to Weibel. Comparison of all three nephron segments yielded statistically significant differences in volume density of apoptotic cells in proximal tubules and in collecting ducts among all three patient groups (non BEN vs. BEN, non BEN vs. BEN/CV and BEN vs. BEN/CV, P<0.001 all). Statistically significant difference in apoptotic cell rate was also found in distal tubules between non BEN and BEN groups and non BEN and BEN/CV groups, but not between BEN and BEN/CV groups. Our results showed a statistically significant increase of apoptotic cells in all three nephron segments in patients with BEN (BEN and BEN/CV) compared to control group. The highest number of apoptotic cells was found in distal tubules in the groups of patients with BEN and BEN with coexisting cardiovascular disease, suggesting that these cells might be most frequently and most severely injured in patients with BEN.

Presence of ochratoxin A in Tunisian blood nephropathy patients. Exposure level to OTA.

Ochratoxin A (OTA) produced by Aspergillus and Penicillium genera contaminates cereals and different food compounds. OTA presents a wide range of toxic effects, especially nephrotoxicity. It is also considered to be the main causal agent of Balkan Endemic Nephropathy (BEN) which is similar to the Chronic Interstitial Nephropathy with unknown aetiology seen in Tunisia. In this study, we attempted to confirm the relationship between OTA blood levels and the development of renal pathology. Hence, serum OTA levels were measured in several groups of patients having different renal diseases: a group presenting Chronic Interstitial Nephropathy (CIN) with unknown aetiology, a group presenting Chronic Interstitial Nephropathy (CIN) with known aetiology, a group presenting Chronic Glomerular Nephropathy (CGN), and a group presenting Chronic Vascular Nephropathy (CVN). Each group was compared to a healthy control group. In the healthy group, 49% of individuals showed OTA concentrations ranging from 1.7 to 8.5 ng/ml, with a mean value of 3.3±1.5 ng/ml. However, among nephropathic patients, the group with CIN of unknown aetiology showed the highest incidence (76%), ranging from 1.8 to 65 ng/ml with a mean value of 18±7 ng/ml. Even in the healthy group, the calculated Daily Intake (DI) ranged from 5.0 to 24.9 ng/kgb.w./day when compared to the recommended DI by the scientific committee on foods of 5 ng/kgb.w./day, indicating a high degree of exposure to OTA in the Tunisian population. Our study confirms the involvement of this nephrotoxic mycotoxin, present at high blood levels in the Tunisian population, in the outcome of this particular human nephropathy (CIN with unknown aetiology) which is similar to BEN.

Reduced kidney size in adult offspring of Balkan endemic nephropathy patients and controls: a prospective study.

INTRODUCTION: Reduced kidney size has been proposed as a criterion for clinical diagnosis of Balkan endemic nephropathy (BEN). Some studies suggest that smaller kidneys are found in advanced stages of BEN, whereas others reported them in earlier stages. To investigate the clinical course of kidney sizes in the offspring of BEN and non-BEN parents, we followed up a cohort of adult offspring over 5 years. We hypothesized that parental history affects kidney dimensions. METHODS: Four repeated ultrasound measurements of kidney length and cortex width were conducted in 121 offspring of BEN and 98 offspring of non-BEN parents. Repeated measurements were analyzed using mixed models adjusting for gender and time-dependent information on other kidney diseases, diabetes, age, height and year of follow-up. RESULTS: A reduction of kidney length was associated with maternal BEN (-4 mm, P = 0.001). We detected a parallel decline in kidney length in the various offspring groups. However, kidney cortex width was significantly smaller when both parents or the mother had BEN and offspring age > or =60 years (-1.88 mm, P = 0.0003; -1.03 mm, P = 0.05). In the 5th year of follow-up, 37 participants developed BEN (14 confirmed, 23 suspected). Kidney cortex width at baseline was smaller in offspring who developed BEN (P = 0.0001). CONCLUSIONS: The development of kidney dimensions depends on the parental BEN status and offspring age. In BEN offspring, ultrasound measurements of the kidney cortex width seem to have a prognostic value.

Characteristics of upper urothelial carcinoma in an area of Balkan endemic nephropathy in south Serbia. A fifty-year retrospective study.

AIMS AND BACKGROUND: Upper urinary tract transitional cell carcinoma, a relatively rare tumor, is up to 100 times more frequent in regions with Balkan endemic nephropathy. Characteristics of transitional cell carcinoma in the endemic South Morava Region in Serbia in the previous 50 years were evaluated. PATIENTS: We analyzed 477 cases with pathologically confirmed transitional cell carcinoma who underwent surgery from 1957 to 2006: 91 from endemic, 106 from adjacent and 280 from control settlements. Cases in the study came from 10 endemic villages, 46 adjacent villages, 51 control villages and the city of Nis. RESULTS: The increase in number of transitional cell carcinoma from 1957 was followed by a peak between 1967 and 1978 (yearly incidence 21.9 per 100,000) and a slow decrease thereafter to 7.4 (1997-2006). In the control settlements, the increase was steady. Reduced kidney function at surgery was found in 58% of patients from endemic and in 20% from control settlements. Age at surgery has significantly increased from 52.3 and 51.5 (1957-1966) to 70.9 and 66.1 (1997-2006) for endemic and control settlements, respectively. The female sex was predominant in endemic and adjacent settlements and the male sex in control settlements. Transitional cell carcinoma from endemic settlements was of a lower grade in the period from 1957-1986, but in the period from 1987-2006 they were predominantly high grade. Low tumor stage (pTa-pT1) predominated in transitional cell carcinoma from the endemic and adjacent but not the control settlements in the period from 1957 to 1986. However, in the last 20 years, upper urinary tract transitional cell carcinoma stage increased, the highest in the period from 1997 to 2006 in all settlements studied. Conservative surgery was advocated for transitional cell carcinoma in Balkan endemic nephropathy areas up to 1996. Transitional cell carcinoma are now more malignant and more advanced than before, and a less aggressive approach is used only for absolute indications. CONCLUSIONS: An increased number of transitional cell carcinoma in endemic settlements was observed, markedly decreasing in the last decade. An increasing age and a shorter survival were recorded in patients both from Balkan endemic nephropathy and control settlements. Sporadic cases upper urinary tract transitional cell carcinoma in settlements adjacent to endemic settlements were demonstrated.

A pilot study of nuclear instability in archived renal and upper urinary tract tumours with putative ochratoxin aetiology.

DNA ploidy measurement has been applied uniquely to wax-embedded tissue of primary renal cell and metastatic tumours of a key experimental researcher on porcine ochratoxicosis, a control, and four transitional cell carcinomas from cases of Balkan endemic nephropathy. Primary renal tumour was diploid, and hyperdiploid metastasis was within the lower ploidy range for typical renal cell carcinoma. Three Balkan primary tumours showed extensive aneuploidy indicating marked nuclear instability, similar to model rat renal carcinoma caused by ochratoxin A. In contrast, much less nuclear instability in the putative occupational ochratoxicosis case fitted poorly with the ochratoxin A model.

Molecular markers in upper urothelial carcinoma associated to Balkan endemic nephropathy. Aristolochic acid as the major risk factor of the worldwide disease.

The role of aristolochic acid in the etiology of Balkan endemic nephropathy (BEN) and associated upper urothelial carcinoma (UUC) was recently confirmed. The aim of this study was to determine the marker(s) specific for BEN-associated UUC. A total of 82 patients with UUC (38 from the BEN region and 44 control tumors) were included in the study. The Ki-67 index in BEN tumors correlated with the grade and multifocality (p < 0.05), but in regression analysis, only the grade of BEN tumor. The p53 index was significantly higher in BEN than in control tumors (p < 0.05), as well as the alteration of p53 (p < 0.05). BEN low-stage tumors, tumors without limphovascular invasion (LVI), and tumors of the renal pelvis had a higher p53 index than the control tumors (p < 0.05, 0.01, 0.05, respectively). The Ki-67 index was higher in control tumors with high-stage and solid growth than in BEN UUC (p < 0.050, 0.005). The Ki-67 correlated with the grade, growth, stage, LVI, and multifocality of UUC on the best way, but not with the group. In regression analysis, only multifocality of UUC had predictive influence on Ki-67 activity (p < 0.001). P53 correlated with the grade, growth, and group (p < 0.05). This investigation identifies the p53 pathway as the specific cell cycle marker involved in BEN-associated UUC.

Upper urothelial carcinoma in Balkan endemic nephropathy and non-endemic regions: a comparative study of pathological features.

Upper urothelial carcinoma (UUC), a rare neoplasm, occurs more frequently in some regions of Balkan countries than in other areas in the world. The aim of this study is to compare phenotypic morphological characteristics of UUC in Balkan endemic nephropathy (BEN) region and control rural and city populations free of BEN, and to determine the characteristic(s) that could discriminate tumors in endemic and non-endemic regions. The authors analyzed biopsies from 88 patients with UUC, 40 patients who live in Balkan endemic (BEN) settlements and 48 control subjects. The histological sections were used to assess morphological variables: histologic grade, pathologic stage (pT), growth pattern, pattern of invasion, lympho-vascular invasion (LVI), presence of necrosis and metaplastic changes (squamous or glandular) within the tumor. Statistically significant differences between the groups were found concerning tumor grade, pattern of invasion, growth pattern and metaplastic changes. High-grade tumors and trabecular/infiltrative patterns of invasion were more frequent in the group of BEN tumors (chi(2)=4.583, p<0.05; chi(2)=8.064, p<0.05). Moreover, solid growth and metaplastic changes are significant in BEN tumor, chi(2)=9.696, p<0.01; chi(2)=9.35, p<0.01, respectively. Discriminant analysis of morphological variables had indicated that BEN and control tumors are significantly different (Wilks' lambda=0.833, chi(2)=15.044 and p<0.05). The best characteristic that differentiated them was growth pattern; i.e., solid growth for BEN tumors and papillary for control tumors.

Balkan endemic nephropathy: a still unsolved puzzle.

Balkan endemic nephropathy (BEN) is a chronic tubulointerstitial renal disease, occurring in certain regions in 5 countries of the Balkan peninsula. Its etiology is largely unknown, though several hypotheses have been formulated and are discussed in this review. In several cases, etiological hypotheses (e.g., viral, ochratoxin or trace element involvement) are verified only in local endemic areas and can not be confirmed when tested elsewhere. Only certain families in the endemic areas are affected. An exposure of at least 20 years to the unknown factors in the endemic areas seems to be mandatory for the development of the disease, but a genetic predisposition to this disease also seems to be mandatory. Prominent clinical features are severely shrunken kidneys, a more severe anemia relative to the level of renal function, and a slow progression to end-stage renal failure. An international approach to solving the etiological and pathogenetic enigma of BEN is needed in the coming years. It is also time to reevaluate other chronic, slowly progressive tubulointerstitial nephropathies diagnosed elsewhere in the world and to search for possible etiological similarities with BEN.

Comparison of kidney size between patients with Balkan endemic nephropathy and other kidney diseases.

BACKGROUND: The aim of this study was to assess the relationship between kidney dimensions and creatinine clearance (Ccr) in patients with Balkan endemic nephropathy (BEN), nephrosclerosis (NSc), glomerulonephritis (GN), diabetic nephropathy (DN) and in healthy persons. The main objective was to find out at which stage of BEN the kidneys start to shrink. METHODS: The study involved 84 patients with BEN, 39 with NSc, 56 with GN, 55 with DN, and 52 healthy subjects, allocated to group 1 (n = 28) sex- and age-matched with BEN/NSc patients, or group 2 (n = 24) sex- and age-matched with GN/DN patients. Based on Ccr, patients were classified according to the NKF/DOQI guidelines. RESULTS: The kidney dimensions of BEN patients in all stages of the disease were significantly shorter than those of healthy controls and patients with GN and DN. In stages 3-5, BEN patients had significantly smaller kidneys than patients with NSc. Patients with NSc had smaller kidney dimensions than controls and GN/DN patients but all of these differences were not significant. CONCLUSION: BEN patients had significantly smaller kidneys than sex- and age-matched healthy persons and patients with GN and DN in all stages of the disease and patients with NSc in stages 3-5 of the disease.

Balkan nephropathy: evolution of our knowledge.

Balkan endemic nephropathy (BEN), originally described in the late 1950s as a chronic tubulointerstitial kidney disease, is identified by its unique epidemiological features. The most remarkable characteristic of BEN is the focal topographical nature that characterizes its occurrence at the global, national, and even household level. BEN affects only certain endemic rural foci along tributaries of the Danube River in the Balkan countries of Bosnia, Bulgaria, Croatia, Romania, and Serbia. The spatial distribution has remained astonishingly unchanged with time because the disease affects the same endemic clusters as 50 years ago. The natural course of the disease is characterized by universal development of end-stage renal disease and the frequent development of upper urinary tract tumors, posing a substantial disease burden to the afflicted areas. The greatest challenge in the study of BEN has been the elucidation of its cause. The unique features of the disease, in particular its endemic nature and the long incubation period required for the disease to develop, have led to the proposal that BEN represents a unique environmental disease. The quest for the responsible environmental factor has been long and diverse, and although no definitive answer has been provided to date, converging lines of evidence support the theory that long-term consumption of food contaminated with aristolochic acid underlies the pathogenesis of BEN. The present review describes the evolution of our knowledge of BEN in relation to the development of the main theories for its pathogenesis.

Contribution to the definition of diagnostic criteria for Balkan endemic nephropathy.

BACKGROUND: Diagnostic criteria for Balkan endemic nephropathy (BEN) have not been precisely established. In the present study the predictive value of variables previously proposed as diagnostic criteria for BEN was examined. METHODS: The study involved 182 patients: 98 patients with BEN, 57 patients with other kidney diseases (20 with glomerulonephritis, 17 with tubulointerstitial diseases and 20 with hypertensive nephrosclerosis) and 27 healthy subjects. The BEN group comprised patients who fulfilled criteria for BEN and suspected BEN, together with patients with proteinuria and at least two tubular abnormalities or one tubular abnormality and a history of urothelial tumour. Demographic, clinical, laboratory and ultrasound variables of examined groups were combined in univariate/multivariate logistic regression analysis. RESULTS: Out of 28 analysed variables only urine alpha1-microglobulin (MG) and kidney length were selected as significant predictors in differentiating BEN from other kidney diseases and healthy controls. Using ROC curves the cutoff values of these variables and proteinuria and kidney volume, variables collinear with them, were found. Moderate sensitivity and specificity characterized all these cutoff values except for proteinuria, which provided high sensitivity and specificity in combination of BEN and healthy persons. The predictive value of different combinations of selected variables was not significantly different from the predictive value of each variable individually. CONCLUSIONS: Proteinuria, urine alpha1-MG, kidney length and volume were selected as significant predictors of BEN. Variables related to kidney failure as well as several tubular disorders (urine specific gravity, FENa and TRP) had an insignificant predictive value and could not be used for differential diagnosis of BEN.