Editorial Nephron 1992;62:1-5
Department of Environmental Health Science, Case Western Reserve University, Cleveland, Ohio, USA
Balkan Endemic Nephropathy: More Questions than Answers
The earliest description of Balkan endemic nephropathy (BEN) appears in the local medical literature of Vratza, Bulgaria, in 1956. Public health physicians noted that pa tients from that region were being admitted to the hospital with symptoms of chronic uremia at a remarkably high frequency. This prompted the central authorities to or ganize systematic epidemiologic and morphologic studies of the involved population. It was suggested, in 1956, that the disease was caused by a chronic metal intoxication or a virus. Some investigators, on reviewing the morphologic characteristics, felt that it was chronic pyelonephritis. Others suggested that some agents o f ‘mould’ type, were the causative agents. In 1960, a meeting devoted to BEN was held in Sofia [1], At that time, chronic infectious etiology was ruled out on the grounds that; the geographic distribution of cases remained isolated for a decade; overt evidence of the disease was not seen in children; hypertension and leukocyturia were absent and, most significantly, the mor phology was not at all similar to any infectious renal dis ease. The first international symposium on BEN was held in Sofia, in September of 1963. At that time, it was pointed out by the late Prof. Puchlev [2] that epidemiologic investiga tions in Bulgaria. Rumania and Yugoslavia had been in progress for almost a decade. In setting the stage for that meeting, he stated the problems which investigators faced commencing with that of the etiology as being most impor tant. He noted that ‘this would not be forthcoming’ at that meeting. He listed three questions which he felt should be answerable. Was the disease described in the three coun tries the same disease? Was the ‘exceptionally great fre quency of upper urethelial tract tumors’ described in Bulga ria seen in the other two countries? Was BEN nothing more than the chronic interstitial nephritis characteristic of pyelonephritis?
At the end of that meeting, there was general agreement that the disease was the same in the three involved coun tries, based on the epidemiology and clinical picture. The question of the tumors was left open as the coincidental occurence of both diseases in the same patient had not been described in Yugoslavia or Rumania. The question of the similarity of the postmortem histomorphology remained controversial. A final quote from Puchlev’s [2] 1963 opening address seems an appropriate starting point for this editorial: ‘I would like to remind you that our task is not exhausted by giving a careful hearing to the reports and by taking active part in the discussion. We must end this symposium by drafting a programme, which should, above all, define our future assignments in our joint work, through which we may hope to solve, as soon as possible, the basic problem of the cause of endemic nephropathy.'
Over the past three decades, many international meet ings have taken place and many independent groups in each of the three countries have continued to investigate various etiologies with little success. Regularly scheduled symposia have been held in Yugoslavia and Bulgaria with ‘open’ invitation to participate given to all investigators in each of the countries and to their international collabora tors. Groups of investigators attended these meetings, pre sented their material and went back to their respective institutions without attempting to reconcile the marked differences in the clinical course, epidemiology and mor phology reported by the different groups. Monographs have been published in which BEN has been presented as a primary glomerulonephritis with the typical picture of an epidemic of postinfectious glomerulonephritis occurring in children [3]. The role of silicates in the induction of ex perimental renal disease and clinical BEN has been advo-
Philip W. Hall. 3rd Department of Environmental Health Science Case Western Reserve University. 10900 Euclid Avenue Cleveland. OH 44106 4940 (USA)
© l992S.K argerAO .Basd 0028-2766/92/ 0621-0001$2.75/0
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Philip W. Hall, 3rd
2
Hall
Of particular interest were the series of reports which examined 50 patients from a region of Bosnia where BEN is endemic. The clinical and epidemiologic studies were per formed by the Department of Nephrology at the University of Tuzla in Bosnia [12]. Urine samples from these patients were studied by Serbian investigators at the Institute of General and Physical Chemistry, where specific expertise in the protein analysis of small polypeptides and proteins exists [13]. The biopsy material was studied in Slovenia, using light, immunofluorescent and electron microscopy by Ferluga et. al [14] at the Institute of Pathology, Ljubjana University. The clinical criteria used to define these cases were; (a) residence in an endemic focus; (b) family history of renal disease; (c) presence of azotemia or hypercreatininemia in the absence of significant proteinuria, hypertension or he maturia, and (d) no signs or symptoms of any recognizable primary or secondary renal disease. This diagnosis was derived by the process of exclusion. All participants agreed that there was no single clinical or laboratory finding pa thognomonic for BEN. The meticulous detailed analysis of the biopsy material was performed without knowledge of the clinical or labora tory data. The results yielded information that may poten tially explain some of the discrepancies that appeared in previous reports of the pathology of BEN. It was concluded ‘that the histopathology is predomi nantly tubulointerstitial sclerosis without infiltrates. Com bined with the histology, the characteristic tubular protein uria, the geographic origin and familiar occurrence qualifies BEN as a unique disease" [14]. The location was determined by the criteria of Radovanovic[15]. It was based on the incidence of ESRD in villages as determined by public health statistics. The clinical course is characterized by the insidious onset of uremia without an associated high incidence of hypertension and without hematuria or a ne phrotic phase. The disease has its onset usually in the 5th decade of life with no characteristic first symptoms, only those of uremia [12]. If one studies the light and electron biopsy material from different stages of the disease, as was done by Ferluga et al. [14], without reference to the clinical or laboratory findings, diabetes mellitus, the aging kidney and focal sclerosing glomerulonephritis (FSGS) would all be seriously con sidered. From personal observation, diabetic nephropathy is a very unusual cause of ESRD in patients receiving dialysis in centers serving the endemic population. The age of onset of clinical BEN is too young to be consistent with the pathology. Typically focal global glomerulosclerosis accompanies chronic glomerulonephritis and pyelone
BEN:More Questions than Answers
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cated by Markovic [4], No other investigators have con firmed his experimental observations. The concentration of silicates found in the water sources of the villages where BEN is endemic are common in many areas of the world. The histomorphologic studies have failed to show any increase in silica in kidney tissue. While no direct evidence has supported Markovic’s [4] hypothesis, the intriguing statistical correlation between occupational exposure to silica and end-stage renal disease (ESRD) has been re ported by Steenland et al. [6]. These data were derived from the United States Renal Data System [7] which also contains a record of the type of kidney disease of each patient. Among the diagnoses recorded, there is a category listed as cause unknown. If this proved to be the listed ‘cause' of a significant number of silica-exposed patients, could this be the USA equivalent of BEN? Research into the role of viruses and mycotoxins in the etiology of BEN has followed several lines. Early reports of viral-like particles seen by electron microscopy in a few cases have not been confirmed by others [8], Stoian et al. [9] found antibody levels to BK virus in 95.2% of BEN patients and in 74.7% of apparently healthy individuals from an endemic area. The SV40 titers were absent in BEN patients and 27.7% of the healthy group. This contrasted with their findings in the control population of healthy individuals who averaged 40% SV40 virus titers. Using the BHK.-21 cell line, Voiculescu et al. [8] demonstrated the presence of cytoplasmic inclusions resulting from studies of the urine of BEN patients. Follow-up studies are needed. Mycotoxins have been incriminated as casual agents of BEN. An excellent review of this research can be found elsewhere [9]. In June 1991, at the meeting held under the auspices of the IARC, several investigators presented a research that focused on the roll of mycotoxins in BEN and urinary tract tumors [10], While high levels of ochratoxin A have been found in the blood and urine of patients with BEN or the urinary tract tumors, similar levels have been found in individuals from other countries where no such endemic nephropathy exists [II]. In 1989 and 1990, new attemps were made to foster international cooperative re search on BEN. Under the sponsorship of the IUPAB special commission on Radiation and Environmental Bio physics and the Yugoslav Biophysical Society, Bulgarian and Yugoslavian investigators reported on the current re search programs. Attempts to involve Rumanian investiga tors were unsuccessful. The proceedings of these two meet ings have been published [5]. For the first time, collabora tion of multiple centers of excellence put together the clinical, epidemiologic and histomorphology of clearly defined groups of patients with BEN.
The association of papillary transitional cell carcinoma of the upper urinary tract and BEN continues to stimulate research. Cukuranovic et al. [20] showed that the frequency rate for urinary tract tumors in an endemic population was 29.7 mean annual incidence calculated per 100,000 inhabi tants. This compares to a rate of 0.52/100,000 in a control population. The follow-up studies of the relationship of the tumors and BEN were presented at the Belgrade meetings by Petronic et al. [21]. There is no doubt about the very unique association of these two diseases. Comparing fre quency rates of occurrence of the tumors in 1988 with those of the late 1960s indicated that the high risk of developing the tumor was still prevalent. Here again, the evidence indicated that the causative factor(s) of BEN and urinary tract tumors persist(s). Studies involving the endemic popu lation in Bulgaria have shown that a significant number of individuals who were otherwise healthy had an elevation in urinary and/or serum p2M [22, 23]. Populations of BEN patients, urinary tract tumor patients and otherwise healthy indivuduals, all living in endemic foci, had abnormal urine and/or serum p2M when compared to controls. Because of the similarity of p2M to IgG light chains and the evidence that [TM production may be increased in both BEN and tumor patients, it has been speculated that p2M may cause proximal tubular damage [22], Morozov et al. [24] have demonstrated that (TM strongly binds polyvalent cations, i.e., Cu, Ca, Zn, Be and La. Such binding releases fi2M from the HLA complexes. This results in an increased load of free P2M available for filtration. These observations, coupled with those of Batuman [25] showing that light chains bind to the high-capacity, low-affinity sites on the brush border membrane, indicate a mechanism for p2M toxicity. Multiple nephrotoxic chemical elements, i.e., lead, cad mium and mercury have been ruled out as primary cau sative agents [26]. Organic solvents, however, have not been the object of extensive research. Federet al. [27] have shown that there is a correlation between the presence of lowranked coal deposits and the geographic distribution of BEN. They postulate that the presence of such coal and organic-rich shales in the vicinity of endemic villages yields organic compounds to the water shed. Studies of such coal indicate that polycyclic aromatic hydrocarbons and aro matic amines are readily leeched into aqueous environ ments. The expertise of another scientific discipline has been brought to bear on this fascinating medical mystery! It is generally accepted that BEN is primarily a disease resulting from some environmental exposure. The geo graphically restricted distribution of the diseased popula tions, the acquisition of the disease by individuals moving into affected areas and the emigrant-twin cases are strong
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phritis as well as arteriolar nephrosclerosis. None of these diseases have the clinical picture of BEN. It is apparent that, at the current state of the art, attempts to characterize the morphologic progression of the disease have not helped to discover the etiology. This, in part, may be due to the fact that much less attention has been given to the tubulointersti tial tissue changes compared to those of the glomerulus. If the tubulointerstitial component of the nephron is the site of primary involvement in BEN, we must await a better under standing of the significance of alterations shown in these tissues. The unusual nature of proteinuria in BEN has attracted researchers for more than 20 years. Tubular proteinuria has been a consistent finding in BEN patients reported from Bulgaria, Rumania and all three involved Republics of Yugoslavia as reviewed elsewhere [16]. Epidemiologic studies, comparing protein excretion patterns of popula tions at risk to develop BEN to control populations have shown that otherwise healthy individuals in the at-risk group had increased concentrations of (T-microglobulin (P2M) in their urine [17, 18], Hrabar et al. [19] showed that otherwise healthy individuals living in areas where BEN is endemic, had a 9.9 times greater chance of developing clinical BEN than those with normal urinary p2M, in a 15-year follow-up study. Fourteen-year follow-up studies showed that the frequency, sex and age distribution of tubular proteinuria in the population at risk was the same in 1988 as it was in 1974 [17]. This finding was interpreted to indicate that the causative agent was still present in the environment. There was a group of clinically healthy indi viduals with increased urine p2M concentration as the only detectable laboratory abnormality. There were also individ uals who showed persistent tubular proteinuria 14 years later without developing any overt evidence of renal dis ease. In addition, there were individuals who, having been persistently positive for tubular proteinuria 14 years earlier, had no abnormal finding. There was a group which was persistently negative during the initial study that had become positive 14 years later. These fascinating prelimi nary findings suggested to the investigators that the expo sure of the different groups had changed. New studies are planned to explore what changes have occurred over the 14 years. The above studies suggested to Jevremovic et al. [17] that BEN is not a ‘universally progressive and fatal' disease which has been the accepted premise. On the contrary, BEN may actually be reversible in that the tubular protein uria may disappear with time leaving no detectable abnor mality. This conclusion is based on the assumption that tubular proteinuria is a marker of BEN.
points of evidence in support of this. There are two lines of evidence suggesting a hereditary component which add additional intrigue to this Balkan puzzle. Pavlovic et al. [28] studied two groups of individuals who were living in an ‘endemic village’. Both groups were healthy, one group had a strong family history of BEN, the other was without any history of kidney disease. The group with a positive family history had a low serum lecithin: cholesterol acyltransferase (LCAT) level averaging 39 nm ol/m l/h compared to 65.9 nm ol/m l/h in the group without a positive family history (p < 0.0001). Familial LCAT deficiency is associated with renal disease [29]. The lipid profiles described by Pavlovic et al. [28] in the indi viduals with LCAT deficiency are quite different than those seen in patients with the familial disease. The chro mosome and LCAT studies await confirmation. What role these lipid abnormalities have in the progression of BEN remains to be discovered. Toncheva et al. [30] have demonstrated a higher fre quency of spontaneous aberrations and chromosome le sions in medium TC 199, and radiation-induced breakages then were found in a control group. The investigators speculate that the ‘results support the idea that 3q25 may
play a specific role and be a marker for BEN’. They also demonstrated that 3 of the additional 5 bands with in creased frequencies of lesions in BEN patients contained oncogenes: lq36-c src, 3p25-raf-l and 6q23-myb. These findings suggested to them that the frequent association of BEN and cancer can be explained by the chromosomal hypothesis of oncogenesis. At the close of the international workshop in 1989, it was agreed to initiate combined international research. This was to include, for example, LCAT studies to be performed on populations in villages where BEN is en demic in Croatia and Bosnia, with the cooperation of Pavlovic’s laboratory in Nis, in the Republic of Serbia. Chromosome studies were to be performed on suitable populations from the area near Belgrade in conjuntion with Toncheva’s group in Sofia, Bulgaria. This was part of the plan that the late Prof. Puclev proposed 30 years ago. In late 1990, it looked as if it would finally come about. Unfortunately, due to the current political situation, this is on hold now. As this editorial was being written, a peace ful solution was in the offing. Hopefully, resumption of research with significant international cooperation will be directed at this fascinating medical mystery.
1 Puclec A (ed): Endemic Nephritis in Bulgaria. Sofia, State Publishing Co. Medicine & Physical Culture, 1960. 2 Puchlev A: Opening address at he International Symposium on Endemic Nephropathy. Sofia. Bulgarian Academy of Science Press. 1965, pp 10-12. 3 Susa S: Endemska Nefropatija 1979, Savremena Administracija. Beograd 1979; ARC Mycotoxins, Nephropathy and Urinary Tract Tumors, Lyon, June 1991. 4 Markovic B. Arambasic MD; Experimental chronic interstitial nephritis compared with endemic human nephropathy. J Pathol 1971; 103:35-40. 5 IUPAB Special Commission on Radiation and Environmental Biophysics, the Yugoslav Biophysical Society: Balkan endemic ne phropathy. Kidney Int 1991:40(suppl 34):I—104. 6 Steenland NK, Thun JM, Eerguson CW, Port FK: Occupational and other exposures associ ated with end-stage renal disease: A case-con trol study. Am J Public Health 1990:80: 153-157.
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7 US Renal Data System: USRDS 1989 Annual Data Report. Natl Inst Health, Natl Inst Diabetes Dig Kidney Dis, Bethesda, MD. Au gust 1989. 8 Ferluga D, Hvala A. Vizjak A. Tmacevic S. Halilbasic A: Renal function, protein excre tion and pathology of Balkan endemic ne phropathy. IV. Light- and electron-micro scopic study. Kidney Int 199! :40(suppl 34): 57-67. 9 Gastegnarao MK, Chemozemsky IN, Hietanen E. Bartsch H: Are mycotoxins risk fac tors for endemic nephropathy and associated urothelial cancers? Arch Geschwulstforsch 1990:60:295-303. 10 International Agency for Research on Cancer (IARC): Mycotoxins. Nephropathy and Uri nary Tract Tumours (abstracts). Lyon. 1991. 11 Hald B: Ochratoxin A in human blood in european countries in mycotoxins: in Interna tional Agency for Research on Cancer (IARC): Nephropathy and urinary tract tu mours (abstracts). Lyon, June 1991.
12 Tmacevic S, Halilbasic A, Ferluga D, Plavljanic D, Vizjak A. Durakovic H. Habul V. Mesic E, Imamovic G, Hranisavljevic J. Pasic M, Paunovic G: Renal function, protein excre tion and pathology of Balkan endemic ne phropathy. I. Renal funciton: Kidney Int 1991 ;40(suppl 34):49-51. 13 Raicevic S, Tmacevic S. Hranisavljevic J, Vucelic D: Renal function, protein excretion and pathology of Balkan endemic nephropa thy. 11. Protein excretion. Kidney Int 1991: 40(suppl 34): 52-56. 14 Ferluga D. Hvala A. Vizjak A, Tnaccvic S, Halilbasic A: Renal function, protein excre tion and pathology of Balkan endemic nephropathy. III. Light- and electron-micro scopic study. Kidney Int I99l;40(suppl 34): 57-67. 15 Radovanovic Z: Topographical distribution of Balkan endemic nephropathy in Serbia (Yugoslavia). Trop Gcogr Med 1979:31:185189. 16 Hall P. Dammin G: Balkan nephropathy. Ne phron 1978:22:281-300.
Hall
BEN:More Questions than Answers
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References
21 Petronic VJ, Bukurov NS, Djokic MR, Milenkovic DZ, Vuksanovic AM, Avramovic AD, Nale DP: Balkan endemic nephropathy (ben) and papillary transitional cell tumors of the re nal pelvis and ureters (UTT): A follow-up study. Kidney Int 1991 ;40(suppl 34):77— 79. 22 Saltier TA, Dimitrov TS. Hall PW: Relation between endemic (Balkan) nephropathy and urinary tract tumours. Lancet 1977; i :278—280. 23 Hall PW, Doichinov D, Dinev I, Dimitrov TS, Radovanovic Z, Dammin G: Balkan nephropa thy: Relationship with transitional cell cancer: in Zurukzoglu W, Papadimitrious M, Pyrpasopoulos M, Sion M, Zamboulis C (eds): Proc 8th Int Congr Nephrol. Athens, 1981, pp 1210-1216. 24 Morozov VN, Morozova TVa, Bray P, Hranisavljevic J, Vucelic D: Survey of small molecule and ion binding to (L-microglobulin. Kidney Int 1991:40(suppl 34): 85-89. 25 Batuman V: Possible pathogenetic role of lowmolecular-weight proteins in Balkan nephropa thy. Kidney int 1991 ;40(suppl 34):89-92. 26 Wedeen RP: Environmental renal disease: Lead, cadmium and Balkan endemic nephropa thy. Kidney Int 1991:40(suppl 34):4-8.
27 Feder GL, Radovanovic Z, Finkelman RB: Re lationship between weathered coal deposits and the etiology of Balkan endemic nephropathy. Kidney Int 1991:40(suppl 34):9—11. 28 Pavlovic NM, Varghese Z, Persaud JW, Stefa novic V, Strahinjic S, Savic V, Moorehead JF: Partial lecithin: Cholesterol acyltransferase (LCAT) deficiency in Balkan endemic nephro pathy (BEN). Kidney Int I991;40(suppl 34): 102-104. 29 Norum KR, Gjone E: Familial plasma lecithin: Cholesterol acyltrasferase deficiency: Bio chemical study of a new inborn error of meta bolism. Scand J Clin Lab Invest 1967:20:231243. 30 Toncheva DI. Gcrgov TD, Tzoneva MT, Bouchakliev ZP: Spontaneous and induced chro mosome aberrations in Balkan endemic ne phropathy. Kidney Int 1991:40(suppl 34):97101.
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17 Jevremovic I, Jankovic S, Radovanovic Z, Danilovic V, Velimirovic D, Naumovic T, Vacca C. Stamenkovic M, Bukvic D, Vaclava Stojanovic, Trbojcvic S, Hall PW: Beta-2microglobulinuria ((LM) in a population ex posed to Balkan endemic nephropathy. Inferen ces from repeated cross-sectional studies. Kid ney Ini 1991 ;40(suppl 34):35-37. 18 Hrabar A. Aleraj B. Ceovic S, Cvoriscec, Vacca C, Hall PW: (b-Microglobulin studies in en demic Balkan nephropathy. Kidney Int 1991: 40(suppl 34):38-40. 19 Hrabar A. Aleraj B. Ceovic S, Cvoriscec D, Vacca C, Hall PW: A fifteen-year cohort-based evaluation of |L-microglobuIin as an early sign of Balkan endemic nephropathy. Kidney Int 1991:40(suppl 34):4I— 43. 20 Cukuranovic R, Ignjatovic M, Stefanovic V: Urinary tract tumors and Balkan nephropathy in the south Morava river basin: Kidney Int 1991:40{suppl 34):80-84.
Department of Environmental Health Science, Case Western Reserve University, Cleveland, Ohio, USA
Balkan Endemic Nephropathy: More Questions than Answers
The earliest description of Balkan endemic nephropathy (BEN) appears in the local medical literature of Vratza, Bulgaria, in 1956. Public health physicians noted that pa tients from that region were being admitted to the hospital with symptoms of chronic uremia at a remarkably high frequency. This prompted the central authorities to or ganize systematic epidemiologic and morphologic studies of the involved population. It was suggested, in 1956, that the disease was caused by a chronic metal intoxication or a virus. Some investigators, on reviewing the morphologic characteristics, felt that it was chronic pyelonephritis. Others suggested that some agents o f ‘mould’ type, were the causative agents. In 1960, a meeting devoted to BEN was held in Sofia [1], At that time, chronic infectious etiology was ruled out on the grounds that; the geographic distribution of cases remained isolated for a decade; overt evidence of the disease was not seen in children; hypertension and leukocyturia were absent and, most significantly, the mor phology was not at all similar to any infectious renal dis ease. The first international symposium on BEN was held in Sofia, in September of 1963. At that time, it was pointed out by the late Prof. Puchlev [2] that epidemiologic investiga tions in Bulgaria. Rumania and Yugoslavia had been in progress for almost a decade. In setting the stage for that meeting, he stated the problems which investigators faced commencing with that of the etiology as being most impor tant. He noted that ‘this would not be forthcoming’ at that meeting. He listed three questions which he felt should be answerable. Was the disease described in the three coun tries the same disease? Was the ‘exceptionally great fre quency of upper urethelial tract tumors’ described in Bulga ria seen in the other two countries? Was BEN nothing more than the chronic interstitial nephritis characteristic of pyelonephritis?
At the end of that meeting, there was general agreement that the disease was the same in the three involved coun tries, based on the epidemiology and clinical picture. The question of the tumors was left open as the coincidental occurence of both diseases in the same patient had not been described in Yugoslavia or Rumania. The question of the similarity of the postmortem histomorphology remained controversial. A final quote from Puchlev’s [2] 1963 opening address seems an appropriate starting point for this editorial: ‘I would like to remind you that our task is not exhausted by giving a careful hearing to the reports and by taking active part in the discussion. We must end this symposium by drafting a programme, which should, above all, define our future assignments in our joint work, through which we may hope to solve, as soon as possible, the basic problem of the cause of endemic nephropathy.'
Over the past three decades, many international meet ings have taken place and many independent groups in each of the three countries have continued to investigate various etiologies with little success. Regularly scheduled symposia have been held in Yugoslavia and Bulgaria with ‘open’ invitation to participate given to all investigators in each of the countries and to their international collabora tors. Groups of investigators attended these meetings, pre sented their material and went back to their respective institutions without attempting to reconcile the marked differences in the clinical course, epidemiology and mor phology reported by the different groups. Monographs have been published in which BEN has been presented as a primary glomerulonephritis with the typical picture of an epidemic of postinfectious glomerulonephritis occurring in children [3]. The role of silicates in the induction of ex perimental renal disease and clinical BEN has been advo-
Philip W. Hall. 3rd Department of Environmental Health Science Case Western Reserve University. 10900 Euclid Avenue Cleveland. OH 44106 4940 (USA)
© l992S.K argerAO .Basd 0028-2766/92/ 0621-0001$2.75/0
Downloaded by: King's College London 137.73.144.138 - 5/16/2018 11:58:56 AM
Philip W. Hall, 3rd
2
Hall
Of particular interest were the series of reports which examined 50 patients from a region of Bosnia where BEN is endemic. The clinical and epidemiologic studies were per formed by the Department of Nephrology at the University of Tuzla in Bosnia [12]. Urine samples from these patients were studied by Serbian investigators at the Institute of General and Physical Chemistry, where specific expertise in the protein analysis of small polypeptides and proteins exists [13]. The biopsy material was studied in Slovenia, using light, immunofluorescent and electron microscopy by Ferluga et. al [14] at the Institute of Pathology, Ljubjana University. The clinical criteria used to define these cases were; (a) residence in an endemic focus; (b) family history of renal disease; (c) presence of azotemia or hypercreatininemia in the absence of significant proteinuria, hypertension or he maturia, and (d) no signs or symptoms of any recognizable primary or secondary renal disease. This diagnosis was derived by the process of exclusion. All participants agreed that there was no single clinical or laboratory finding pa thognomonic for BEN. The meticulous detailed analysis of the biopsy material was performed without knowledge of the clinical or labora tory data. The results yielded information that may poten tially explain some of the discrepancies that appeared in previous reports of the pathology of BEN. It was concluded ‘that the histopathology is predomi nantly tubulointerstitial sclerosis without infiltrates. Com bined with the histology, the characteristic tubular protein uria, the geographic origin and familiar occurrence qualifies BEN as a unique disease" [14]. The location was determined by the criteria of Radovanovic[15]. It was based on the incidence of ESRD in villages as determined by public health statistics. The clinical course is characterized by the insidious onset of uremia without an associated high incidence of hypertension and without hematuria or a ne phrotic phase. The disease has its onset usually in the 5th decade of life with no characteristic first symptoms, only those of uremia [12]. If one studies the light and electron biopsy material from different stages of the disease, as was done by Ferluga et al. [14], without reference to the clinical or laboratory findings, diabetes mellitus, the aging kidney and focal sclerosing glomerulonephritis (FSGS) would all be seriously con sidered. From personal observation, diabetic nephropathy is a very unusual cause of ESRD in patients receiving dialysis in centers serving the endemic population. The age of onset of clinical BEN is too young to be consistent with the pathology. Typically focal global glomerulosclerosis accompanies chronic glomerulonephritis and pyelone
BEN:More Questions than Answers
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cated by Markovic [4], No other investigators have con firmed his experimental observations. The concentration of silicates found in the water sources of the villages where BEN is endemic are common in many areas of the world. The histomorphologic studies have failed to show any increase in silica in kidney tissue. While no direct evidence has supported Markovic’s [4] hypothesis, the intriguing statistical correlation between occupational exposure to silica and end-stage renal disease (ESRD) has been re ported by Steenland et al. [6]. These data were derived from the United States Renal Data System [7] which also contains a record of the type of kidney disease of each patient. Among the diagnoses recorded, there is a category listed as cause unknown. If this proved to be the listed ‘cause' of a significant number of silica-exposed patients, could this be the USA equivalent of BEN? Research into the role of viruses and mycotoxins in the etiology of BEN has followed several lines. Early reports of viral-like particles seen by electron microscopy in a few cases have not been confirmed by others [8], Stoian et al. [9] found antibody levels to BK virus in 95.2% of BEN patients and in 74.7% of apparently healthy individuals from an endemic area. The SV40 titers were absent in BEN patients and 27.7% of the healthy group. This contrasted with their findings in the control population of healthy individuals who averaged 40% SV40 virus titers. Using the BHK.-21 cell line, Voiculescu et al. [8] demonstrated the presence of cytoplasmic inclusions resulting from studies of the urine of BEN patients. Follow-up studies are needed. Mycotoxins have been incriminated as casual agents of BEN. An excellent review of this research can be found elsewhere [9]. In June 1991, at the meeting held under the auspices of the IARC, several investigators presented a research that focused on the roll of mycotoxins in BEN and urinary tract tumors [10], While high levels of ochratoxin A have been found in the blood and urine of patients with BEN or the urinary tract tumors, similar levels have been found in individuals from other countries where no such endemic nephropathy exists [II]. In 1989 and 1990, new attemps were made to foster international cooperative re search on BEN. Under the sponsorship of the IUPAB special commission on Radiation and Environmental Bio physics and the Yugoslav Biophysical Society, Bulgarian and Yugoslavian investigators reported on the current re search programs. Attempts to involve Rumanian investiga tors were unsuccessful. The proceedings of these two meet ings have been published [5]. For the first time, collabora tion of multiple centers of excellence put together the clinical, epidemiologic and histomorphology of clearly defined groups of patients with BEN.
The association of papillary transitional cell carcinoma of the upper urinary tract and BEN continues to stimulate research. Cukuranovic et al. [20] showed that the frequency rate for urinary tract tumors in an endemic population was 29.7 mean annual incidence calculated per 100,000 inhabi tants. This compares to a rate of 0.52/100,000 in a control population. The follow-up studies of the relationship of the tumors and BEN were presented at the Belgrade meetings by Petronic et al. [21]. There is no doubt about the very unique association of these two diseases. Comparing fre quency rates of occurrence of the tumors in 1988 with those of the late 1960s indicated that the high risk of developing the tumor was still prevalent. Here again, the evidence indicated that the causative factor(s) of BEN and urinary tract tumors persist(s). Studies involving the endemic popu lation in Bulgaria have shown that a significant number of individuals who were otherwise healthy had an elevation in urinary and/or serum p2M [22, 23]. Populations of BEN patients, urinary tract tumor patients and otherwise healthy indivuduals, all living in endemic foci, had abnormal urine and/or serum p2M when compared to controls. Because of the similarity of p2M to IgG light chains and the evidence that [TM production may be increased in both BEN and tumor patients, it has been speculated that p2M may cause proximal tubular damage [22], Morozov et al. [24] have demonstrated that (TM strongly binds polyvalent cations, i.e., Cu, Ca, Zn, Be and La. Such binding releases fi2M from the HLA complexes. This results in an increased load of free P2M available for filtration. These observations, coupled with those of Batuman [25] showing that light chains bind to the high-capacity, low-affinity sites on the brush border membrane, indicate a mechanism for p2M toxicity. Multiple nephrotoxic chemical elements, i.e., lead, cad mium and mercury have been ruled out as primary cau sative agents [26]. Organic solvents, however, have not been the object of extensive research. Federet al. [27] have shown that there is a correlation between the presence of lowranked coal deposits and the geographic distribution of BEN. They postulate that the presence of such coal and organic-rich shales in the vicinity of endemic villages yields organic compounds to the water shed. Studies of such coal indicate that polycyclic aromatic hydrocarbons and aro matic amines are readily leeched into aqueous environ ments. The expertise of another scientific discipline has been brought to bear on this fascinating medical mystery! It is generally accepted that BEN is primarily a disease resulting from some environmental exposure. The geo graphically restricted distribution of the diseased popula tions, the acquisition of the disease by individuals moving into affected areas and the emigrant-twin cases are strong
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phritis as well as arteriolar nephrosclerosis. None of these diseases have the clinical picture of BEN. It is apparent that, at the current state of the art, attempts to characterize the morphologic progression of the disease have not helped to discover the etiology. This, in part, may be due to the fact that much less attention has been given to the tubulointersti tial tissue changes compared to those of the glomerulus. If the tubulointerstitial component of the nephron is the site of primary involvement in BEN, we must await a better under standing of the significance of alterations shown in these tissues. The unusual nature of proteinuria in BEN has attracted researchers for more than 20 years. Tubular proteinuria has been a consistent finding in BEN patients reported from Bulgaria, Rumania and all three involved Republics of Yugoslavia as reviewed elsewhere [16]. Epidemiologic studies, comparing protein excretion patterns of popula tions at risk to develop BEN to control populations have shown that otherwise healthy individuals in the at-risk group had increased concentrations of (T-microglobulin (P2M) in their urine [17, 18], Hrabar et al. [19] showed that otherwise healthy individuals living in areas where BEN is endemic, had a 9.9 times greater chance of developing clinical BEN than those with normal urinary p2M, in a 15-year follow-up study. Fourteen-year follow-up studies showed that the frequency, sex and age distribution of tubular proteinuria in the population at risk was the same in 1988 as it was in 1974 [17]. This finding was interpreted to indicate that the causative agent was still present in the environment. There was a group of clinically healthy indi viduals with increased urine p2M concentration as the only detectable laboratory abnormality. There were also individ uals who showed persistent tubular proteinuria 14 years later without developing any overt evidence of renal dis ease. In addition, there were individuals who, having been persistently positive for tubular proteinuria 14 years earlier, had no abnormal finding. There was a group which was persistently negative during the initial study that had become positive 14 years later. These fascinating prelimi nary findings suggested to the investigators that the expo sure of the different groups had changed. New studies are planned to explore what changes have occurred over the 14 years. The above studies suggested to Jevremovic et al. [17] that BEN is not a ‘universally progressive and fatal' disease which has been the accepted premise. On the contrary, BEN may actually be reversible in that the tubular protein uria may disappear with time leaving no detectable abnor mality. This conclusion is based on the assumption that tubular proteinuria is a marker of BEN.
points of evidence in support of this. There are two lines of evidence suggesting a hereditary component which add additional intrigue to this Balkan puzzle. Pavlovic et al. [28] studied two groups of individuals who were living in an ‘endemic village’. Both groups were healthy, one group had a strong family history of BEN, the other was without any history of kidney disease. The group with a positive family history had a low serum lecithin: cholesterol acyltransferase (LCAT) level averaging 39 nm ol/m l/h compared to 65.9 nm ol/m l/h in the group without a positive family history (p < 0.0001). Familial LCAT deficiency is associated with renal disease [29]. The lipid profiles described by Pavlovic et al. [28] in the indi viduals with LCAT deficiency are quite different than those seen in patients with the familial disease. The chro mosome and LCAT studies await confirmation. What role these lipid abnormalities have in the progression of BEN remains to be discovered. Toncheva et al. [30] have demonstrated a higher fre quency of spontaneous aberrations and chromosome le sions in medium TC 199, and radiation-induced breakages then were found in a control group. The investigators speculate that the ‘results support the idea that 3q25 may
play a specific role and be a marker for BEN’. They also demonstrated that 3 of the additional 5 bands with in creased frequencies of lesions in BEN patients contained oncogenes: lq36-c src, 3p25-raf-l and 6q23-myb. These findings suggested to them that the frequent association of BEN and cancer can be explained by the chromosomal hypothesis of oncogenesis. At the close of the international workshop in 1989, it was agreed to initiate combined international research. This was to include, for example, LCAT studies to be performed on populations in villages where BEN is en demic in Croatia and Bosnia, with the cooperation of Pavlovic’s laboratory in Nis, in the Republic of Serbia. Chromosome studies were to be performed on suitable populations from the area near Belgrade in conjuntion with Toncheva’s group in Sofia, Bulgaria. This was part of the plan that the late Prof. Puclev proposed 30 years ago. In late 1990, it looked as if it would finally come about. Unfortunately, due to the current political situation, this is on hold now. As this editorial was being written, a peace ful solution was in the offing. Hopefully, resumption of research with significant international cooperation will be directed at this fascinating medical mystery.
1 Puclec A (ed): Endemic Nephritis in Bulgaria. Sofia, State Publishing Co. Medicine & Physical Culture, 1960. 2 Puchlev A: Opening address at he International Symposium on Endemic Nephropathy. Sofia. Bulgarian Academy of Science Press. 1965, pp 10-12. 3 Susa S: Endemska Nefropatija 1979, Savremena Administracija. Beograd 1979; ARC Mycotoxins, Nephropathy and Urinary Tract Tumors, Lyon, June 1991. 4 Markovic B. Arambasic MD; Experimental chronic interstitial nephritis compared with endemic human nephropathy. J Pathol 1971; 103:35-40. 5 IUPAB Special Commission on Radiation and Environmental Biophysics, the Yugoslav Biophysical Society: Balkan endemic ne phropathy. Kidney Int 1991:40(suppl 34):I—104. 6 Steenland NK, Thun JM, Eerguson CW, Port FK: Occupational and other exposures associ ated with end-stage renal disease: A case-con trol study. Am J Public Health 1990:80: 153-157.
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7 US Renal Data System: USRDS 1989 Annual Data Report. Natl Inst Health, Natl Inst Diabetes Dig Kidney Dis, Bethesda, MD. Au gust 1989. 8 Ferluga D, Hvala A. Vizjak A. Tmacevic S. Halilbasic A: Renal function, protein excre tion and pathology of Balkan endemic ne phropathy. IV. Light- and electron-micro scopic study. Kidney Int 199! :40(suppl 34): 57-67. 9 Gastegnarao MK, Chemozemsky IN, Hietanen E. Bartsch H: Are mycotoxins risk fac tors for endemic nephropathy and associated urothelial cancers? Arch Geschwulstforsch 1990:60:295-303. 10 International Agency for Research on Cancer (IARC): Mycotoxins. Nephropathy and Uri nary Tract Tumours (abstracts). Lyon. 1991. 11 Hald B: Ochratoxin A in human blood in european countries in mycotoxins: in Interna tional Agency for Research on Cancer (IARC): Nephropathy and urinary tract tu mours (abstracts). Lyon, June 1991.
12 Tmacevic S, Halilbasic A, Ferluga D, Plavljanic D, Vizjak A. Durakovic H. Habul V. Mesic E, Imamovic G, Hranisavljevic J. Pasic M, Paunovic G: Renal function, protein excre tion and pathology of Balkan endemic ne phropathy. I. Renal funciton: Kidney Int 1991 ;40(suppl 34):49-51. 13 Raicevic S, Tmacevic S. Hranisavljevic J, Vucelic D: Renal function, protein excretion and pathology of Balkan endemic nephropa thy. 11. Protein excretion. Kidney Int 1991: 40(suppl 34): 52-56. 14 Ferluga D. Hvala A. Vizjak A, Tnaccvic S, Halilbasic A: Renal function, protein excre tion and pathology of Balkan endemic nephropathy. III. Light- and electron-micro scopic study. Kidney Int I99l;40(suppl 34): 57-67. 15 Radovanovic Z: Topographical distribution of Balkan endemic nephropathy in Serbia (Yugoslavia). Trop Gcogr Med 1979:31:185189. 16 Hall P. Dammin G: Balkan nephropathy. Ne phron 1978:22:281-300.
Hall
BEN:More Questions than Answers
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References
21 Petronic VJ, Bukurov NS, Djokic MR, Milenkovic DZ, Vuksanovic AM, Avramovic AD, Nale DP: Balkan endemic nephropathy (ben) and papillary transitional cell tumors of the re nal pelvis and ureters (UTT): A follow-up study. Kidney Int 1991 ;40(suppl 34):77— 79. 22 Saltier TA, Dimitrov TS. Hall PW: Relation between endemic (Balkan) nephropathy and urinary tract tumours. Lancet 1977; i :278—280. 23 Hall PW, Doichinov D, Dinev I, Dimitrov TS, Radovanovic Z, Dammin G: Balkan nephropa thy: Relationship with transitional cell cancer: in Zurukzoglu W, Papadimitrious M, Pyrpasopoulos M, Sion M, Zamboulis C (eds): Proc 8th Int Congr Nephrol. Athens, 1981, pp 1210-1216. 24 Morozov VN, Morozova TVa, Bray P, Hranisavljevic J, Vucelic D: Survey of small molecule and ion binding to (L-microglobulin. Kidney Int 1991:40(suppl 34): 85-89. 25 Batuman V: Possible pathogenetic role of lowmolecular-weight proteins in Balkan nephropa thy. Kidney int 1991 ;40(suppl 34):89-92. 26 Wedeen RP: Environmental renal disease: Lead, cadmium and Balkan endemic nephropa thy. Kidney Int 1991:40(suppl 34):4-8.
27 Feder GL, Radovanovic Z, Finkelman RB: Re lationship between weathered coal deposits and the etiology of Balkan endemic nephropathy. Kidney Int 1991:40(suppl 34):9—11. 28 Pavlovic NM, Varghese Z, Persaud JW, Stefa novic V, Strahinjic S, Savic V, Moorehead JF: Partial lecithin: Cholesterol acyltransferase (LCAT) deficiency in Balkan endemic nephro pathy (BEN). Kidney Int I991;40(suppl 34): 102-104. 29 Norum KR, Gjone E: Familial plasma lecithin: Cholesterol acyltrasferase deficiency: Bio chemical study of a new inborn error of meta bolism. Scand J Clin Lab Invest 1967:20:231243. 30 Toncheva DI. Gcrgov TD, Tzoneva MT, Bouchakliev ZP: Spontaneous and induced chro mosome aberrations in Balkan endemic ne phropathy. Kidney Int 1991:40(suppl 34):97101.
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17 Jevremovic I, Jankovic S, Radovanovic Z, Danilovic V, Velimirovic D, Naumovic T, Vacca C. Stamenkovic M, Bukvic D, Vaclava Stojanovic, Trbojcvic S, Hall PW: Beta-2microglobulinuria ((LM) in a population ex posed to Balkan endemic nephropathy. Inferen ces from repeated cross-sectional studies. Kid ney Ini 1991 ;40(suppl 34):35-37. 18 Hrabar A. Aleraj B. Ceovic S, Cvoriscec, Vacca C, Hall PW: (b-Microglobulin studies in en demic Balkan nephropathy. Kidney Int 1991: 40(suppl 34):38-40. 19 Hrabar A. Aleraj B. Ceovic S, Cvoriscec D, Vacca C, Hall PW: A fifteen-year cohort-based evaluation of |L-microglobuIin as an early sign of Balkan endemic nephropathy. Kidney Int 1991:40(suppl 34):4I— 43. 20 Cukuranovic R, Ignjatovic M, Stefanovic V: Urinary tract tumors and Balkan nephropathy in the south Morava river basin: Kidney Int 1991:40{suppl 34):80-84.
