Balkan Endemic Nephropathy is Still Present in the Kolubara Region, Serbia

Abstract

Background. Almost 50 years ago Balkan Endemic Nephropathy (BEN) was first described in Serbia in the village of Šopić where the first field examination was carried out in 1971. Our aim was to find out whether BEN is still present in this region. Methods. Prevalence data on BEN from a field examination run in 1971 were compared with the results of a cross-sectional study conducted in the same village in 1992. In addition, every new case of the disease diagnosed between 1971 and 1992 was recorded retrospectively. The prospective study included 50 members of five BEN families randomly selected from 28 BEN families registered in the village Šopić in 1992. The objective survey and examination of global and tubular kidney function was carried out in all examined persons once yearly in 1998, 1999, and 2000. Results. The overall prevalence of BEN was 6.4% in 1971 and 8.9% in 1992. In the period of 21 years, 161 new BEN patients were detected in 28 families in which the disease had already been recorded. No new family affected by BEN and none of the new patients in 47 families registered previously as nonaffected were discovered. In the prospective study of five BEN families, three new BEN cases were discovered among 50 members, and two patients fulfilled criteria for BEN-suspected ones. Conclusion. Balkan Endemic Nephropathy (BEN) is still present in the village of Šopić, but the clinical course of the disease became more protracted over time. New cases of BEN appeared only in the affected families.

Keywords:

• Balkan Endemic Nephropathy
• cross-sectional study
• epidemiology
• follow-up study
• Serbia

Introduction

Balkan Endemic Nephropathy (BEN) is a familial, chronic, tubulointerstitial disease of unknown etiology affecting the rural population in recognized endemic areas of some regions in Southeastern Europe (Serbia, Croatia, Bosnia and Herzegovina, Rumania, and Bulgaria). Forty-eight years ago BEN was first described on the territory of the Serbian municipality of Lazarevac (Kolubara River) in the village of Šopić where the first field examination was carried out in 1971.[1&2] The incidence and prevalence of BEN were stable over many years,[2&3] but recently some authors have suggested that the incidence of BEN has been decreasing,[4&5] while the others described the stable incidence of the disease.[6&7]

With the aim of examining whether BEN is disappearing or if the disease continues to be found in the Kolubara region, two studies were undertaken: the first examined the prevalence of BEN in the village of Šopić in 1992 and compared it with the prevalence determined in 1971; the second prospective study examined frequency of renal dysfunctions in the members of five BEN families selected from the previous study.

Methods

The first systematic examination of the population in order to determine the prevalence of BEN in the village of Šopić was conducted in 1971. Twenty-one years later, in 1992, the population of the same village was investigated again. Both studies involved the whole population of the village and the same method was used (the same epidemiological questionnaire was used, proteinuria checked with 20% sulfosalicylic acid in both studies, etc.).

Both cross-sectional studies consisted of one phase conducted in the field and two phases conducted at the Institute of Endemic Nephropathy, Lazarevac, and the Institute of Urology and Nephrology, Clinical Center Serbia, Belgrade.

The first phase of the study covered the entire population of the endemic village of Šopić including the epidemiological questionnaire and qualitative determination of proteinuria in the morning urine sample (test with 20% sulfosalicylic acid). All subjects with a positive test with sulfosalicylic acid for proteinuria, those with a personal and familial history of renal disease, and whole families in which two or more members were found to have proteinuria were selected for the subsequent phase of the study (phase II).

In the second phase conducted in 1992, all selected persons were subjected to further examination, including blood pressure measurements, complete urine analysis, including measurement of urine osmolality and quantitative measurement of proteinuria; complete blood count; serum urea; and creatinine measurement. Peripheral blood cell count and serum and urine levels of urea were measured using commercially available kits (Beckman, Germany). Serum creatinine was determined on a Beckman Creatinine Analyzer II (Brea, California) with the modified Jaffe rate method. Anemia was defined as hemoglobin below 13.0 g/dL for males and postmenopausal women and below 12 g/dL for premenopausal women.[8] Azotemia is defined as abnormal levels of urea (> 7.2 mmol/L) and creatinine (> 124 µmol/L). The 24 hour endogenous creatinine clearance was calculated and values above 90 mL/min/1.73 m² were considered normal.[9] Urine protein was measured by the biuret method in 24 hour urine collection and values over 0.2 g/day were considered abnormal. Urine osmolality and specific gravity were measured in a morning specimen after 8 hours with no fluid intake. The urine osmolality, determined cryoscopically, was considered to be normal when it reached the age-related values proposed by Tryding and coworkers as normal.[10] Urine specific gravity (Assistant Urinprober hydrometer model 242; Sondheim/Rhon, Germany) below 1005 was considered abnormal.

The diagnosis of BEN was made on the basis of criteria proposed by Danilovic and coauthors,[2] described in details elsewhere.[6] According to these criteria BEN-suspected persons were considered to be farmers who lived in the endemic village for over 20 years, had a positive family history of BEN, had proteinuria above 0.2 g/day, and had at least one of the three following criteria: lowered urine specific gravity, lowered urine osmolality, and anemia. They also had a normal creatinine clearance. Diagnosis of BEN was established in the presence of at least five out of six previously mentioned criteria and azotemia. Both, BEN and BEN-suspected persons were subjected to more detailed examination that was conducted in order to exclude all other renal diseases (phase III). This examinations, according to the need, involved cardiologic and ophthalmologic surveys, laboratory analysis of serum and urine including determination of the type of proteinuria (glomerular and tubular), urine bacteriological analysis, immunological tests, ultrasonography and x-ray methods, and kidney biopsy in patients with suspected glomerulonephritis.

In addition, every new case of the disease diagnosed between 1971 and 1992 was recorded retrospectively using family history of all families included in the cross-sectional study and medical documentation of the Institute of Endemic Nephropathy.

The second prospective study included all family members (28 males and 22 females, 44 ± 15 years) of five BEN families. The families were randomly selected from 28 BEN families registered in the previous cross-sectional study. We assigned each family a number, starting with 1, 2, 3 and so on to 28. Then numbers were selected at random, until the desired sample size (5 families) was attained. The clinical and laboratory examinations used in the second phase of the previous study were carried out in all 50 family members in the beginning of 1998, 1999, and 2000. In addition, fractional sodium excretion (FENa; normal values < 1%) and percentage of tubular phosphate reabsorption (TRP; normal values > 84%) were determined. Serum and urine levels of sodium and phosphate were measured using commercially available kits (Beckman, Germany).

Results

The population of the village of Šopić comprised by the repeated cross-sectional studies is presented in Table 1. The response rates in all three phases were higher in the study conducted in 1992 as compared to the study conducted in 1971. Nonparticipants were people of different ages, and they were the members of both BEN and non-BEN families.

Table 1. Population of the village of Šopić comprised by two cross sectional studies

Phase of the study	Year of investigation
	1971			1992
	Summoned	Responded		Summoned	Responded
First	1927	1520	78.9%	1548	1326	85.7%
Second	343	264	77.0%	607	503	82.9%
Third	144	123	85.2%	140	131	93,6%

The overall prevalence of BEN (Table 2) was higher in the cross-sectional study conducted in 1992. Patients who manifested BEN were more prevalent in the study conducted in 1971, while the suspected ones were more prevalent in 1992. The average age of BEN-suspected patients in 1971 was 40.68 ± 2.85 years, and for BEN-manifested patients it was 54.13 ± 6.66 years. In the study conducted in 1992, the average age of BEN-suspected patients was 54.02 ± 12.85; and it was 63.36 ± 13.22 years for BEN-manifested patients.

Table 2. Prevalence of Balkan endemic nephropathy in the village of Šopić determined in two cross-sectional studies

Population	Year of investigation
	1971		1992
	No	Prevalence	No	Prevalence
Examined	1520		1326
BEN suspected	30	2.0%	84	6.3%
BEN manifested	67	4.4%	35	2.6%
All BEN patients	97	6.4%	119	8.9%

In 1971 97 BEN patients were recorded in 34 families. In the period of 21 years, 161 new patients were detected in 28 families in which the disease previously had been recorded. New cases of BEN were not found in six of the BEN-affected families and in none of the 47 families in which the disease had not been present in 1971.

The results obtained by clinical and laboratory examinations of 50 BEN family members, examined in the second prospective study, are presented in Figure 1. In three checkups carried out in the three-year period no abnormality was revealed in about one third of the persons. The number of these persons diminished in the follow-up period. At the same time the number of persons with proteinuria and/or tubular disorders (low urine specific gravity, low urine osmolality, increased FENa, decreased TRP) increased. In this group two persons fulfilled criteria for BEN-suspected patients. In eight BEN family members' proteinuria associated with tubular disorders and hypertension was found at the outset of the study and all of them fulfilled criteria for BEN. In five of them BEN was diagnosed before the study, but three new patients were detected in the present study. In one patient with BEN diagnosed before the study proteinuria was not found in the third checkup. Remaining patients had isolated proteinuria or tubular disorders with or without hypertension.

Figure 1 Summary of the results obtained by prospective study of 50 members of five BEN families from the village of Šopić. Tubular disorders considered as the following disorders: low urine specific gravity, low urine osmolality, increased FENa, decreased TRP.

Discussion

The data on the prevalence of BEN are quite unreliable and vary between different foci, depending upon the diagnostic criteria used. Review of various reports reveals the range of 0.3 to 10%,[11-14] rarely over 10%. Stojimirović and coauthors studied the villages of Šopić, Petka, and Vreoci and reported 7.0% of affected inhabitants.[15] The systematic screening of the whole population of the municipality of Lazarevac in three most severe foci (Petka, Šopić, and Vreoci) resulted in identification of 8.3% BEN patients in different stages of the disease.[2] Velimirović and collaborators performed the systematic screening of the most severe foci in the municipality of Lajkovac and recorded 2.7%–7.0% of inhabitants affected by BEN.[3] C˘eović and collaborators reported a prevalence of 0.4% to 8.3% in endemic settlements in Croatia, showing a mild falling trend in recent years; but they concluded that in none of the endemic foci had the disease been eliminated.[13] No reliable data on the increase of BEN have been revealed, but no evidence of significant fall of the number of newly discovered cases in the focal areas was found either. Nevertheless, the increase of BEN patients who are placed on the chronic dialysis program has been recorded in the endemic foci, where 80% of dialyzed patients belong to this group.[16] Opposite to our finding, Čukuranović and coauthors established that the mean annual rate of BEN patients on maintenance dialysis in the dialysis centers in South Serbia (Niš and Leskovac) showed a decreasing trend in the period 1988–1997.[4] In Bulgaria, Dimitrov and coauthors attributed the decrease from 1985–1987 to some decline in the interest in record keeping and active screening.[5] These controversial results directed us to undertake the cross-sectional study in village of Šopić in 1992, 21 years after the first cross-sectional study in the same village. Similar cross-sectional studies in the other endemic villages involved in the first study are in progress and they could give a better survey of BEN prevalence in the region.

According to our results the overall prevalence of BEN and the prevalence of BEN-suspected persons were higher in the study conducted in 1992 than in the 1971 study. Therefore, the prevalence of BEN manifested was higher in the first study. Our results also proved that the average age of BEN patients moved to an older age over time. In the light of these results it is logical to assume that the cause or causes of the BEN are still present in the endemic areas, and that the clinical course of the disease has become more protracted over time. According to Dimitrov and coauthors, the age of onset of the disease in Bulgaria did not change, but the age of case-identification moved to an older age, probably because cases were not identified at an early stage, but they were identified only after they sought medical treatment.[5]

The screening of the population of the village of Šopic in 1971 revealed 97 BEN patients originating from 34 families. In the period between two cross-sectional studies we failed to reveal any new family affected by BEN, but we did discover new cases in 28 of 34 already identified BEN families. These results suggested that all BEN-affected families necessitate regular check-ups and, therefore, prospective study of five BEN families was carried out. The study was started five years after the cross-sectional study in the village of Šopic and at the outset of the study three persons, who had not been detected previously, fulfilled criteria for diagnosis of BEN. All of them had azotemia that confirmed the previously mentioned fact that diagnosis of BEN is most commonly established in the advanced phase.[2], [7] In addition, more than one-half of examined BEN family members had proteinuria and/or tubular disorders with or without hypertension. These patients did not fulfill criteria for even BEN-suspected ones. These data suggest that routine laboratory methods proposed as diagnostic criteria are insufficient for diagnosis of BEN in the early phase of the disease. Nevertheless, both BEN-suspected persons and persons with renal disorders that did not fulfill criteria for suspicion of BEN should be regularly controlled and treated with well-known measures for slowing down chronic kidney disease progression. As has been already mentioned, in the 5-year period after the cross-sectional study in village of Šopic, three new BEN cases appeared. In 1993 these persons had proteinuria and/or tubular disorders but not enough criteria for even suspected BEN. However, in the following five years they developed manifested BEN. Familial clustering of cases is one of the most conspicuous features of the disease.[17] Many authors have suggested the possible role of genetic factors in the etiology of BEN.[18-20] Extensive epidemiological and genetic studies disclose characteristics of multifactorial (polygenic) inheritance of BEN. Toncheva and coauthors cited that the evidences of “familial tendency,” the development of BEN in individuals from at-risk families who were born in nonendemic areas and the expressions of 3q25 cytogenetic marker suggest that the genetic factor plays an important role as causative factors in BEN development.[19] The same authors did not exclude the possible impact of environmental triggers on individuals genetically predisposed to BEN.[17]

In summary, the repeated cross-sectional studies in the village of Šopić (Kolubara River) demonstrated that BEN maintained that the prevalence of BEN changed insignificantly during the 21 years between studies. The clinical course of the disease became more protracted over that time. New cases of BEN appeared in the affected families during three year follow-up.