Diagnostic Criteria for Balkan Endemic Nephropathy: Proposal by an International Panel

Abstract

Balkan endemic nephropathy (BEN) is a familial chronic tubulointerstitial disease with insidious onset and slow progression to terminal renal failure. Diagnostic criteria for BEN have been described more than 40 years ago. Research groups on BEN use one of at least three described lists of criteria. Comparison of studies using such criteria is difficult, and a recent meeting of investigators (Zagreb, October 2006) has suggested that unified criteria have to be elaborated. In this paper, an International Panel of BEN Investigators agreed on criteria appropriate to epidemiologic studies and clinical investigations of BEN. A screening procedure of BEN in endemic settlements is proposed.

Keywords:

• Balkan endemic nephropathy
• tubulointerstitial disease
• urothelial cancer
• diagnostic criteria
• screening

INTRODUCTION

Balkan endemic nephropathy (BEN) is a familial chronic tubulointerstitial disease with insidious onset and slow progression to terminal renal failure. It affects people living in the alluvial plains along the tributaries of the Danube River in Bosnia, Bulgaria, Croatia, Romania, and Serbia. BEN was first described in 1956 in Bulgaria, then in Serbia in 1957, and in Croatia in 1959.[1–3] Over the past fifty years, much progress has been made in the understanding of the disease.[4–6]

Precise diagnostic criteria of BEN are not well established and vary in their interpretation and application between studies. At least three sets of criteria have been applied by various research groups: the 1965 WHO criteria, Danilovic's criteria (1973), and those described by Stefanovic in 1983.[6–9] Because nephrology diagnostics has improved and the comparison of studies using different criteria is difficult, various leaders of BEN centers agreed to review current diagnostic criteria at a recent Meeting of Investigators (Zagreb, October 2006) in an effort to promote collaborative work in the years to come. The objective of the present paper is to bring an overview of BEN's diagnostic criteria and screening procedures as agreed upon by this international panel of investigators. After reviewing the important characteristics of BEN, diagnostic procedures are described in details, with an emphasis on difficult or ambiguous cases. A screening procedure of BEN in endemic settlements is proposed.

EPIDEMIOLOGICAL CHARACTERISTICS OF BEN

BEN affects people living in the alluvial plains along the tributaries of the Danube River in Bosnia, Bulgaria, Croatia, Romania, and Serbia. BEN has a familial character, initially described in 1957.[5] In a single household, several members of one or several generations may be affected. Within the same village, affected and spared households live in close proximity. The incidence of BEN is thus very heterogeneous both within and between endemic villages. BEN occurs in some but not all villages in the endemic area. Affected villages are sometimes separated from disease-free villages by only a few kilometers. So far, the disease has been identified in 142 villages in Bosnia, Croatia, and Serbia, with a local prevalence ranging between 0.5 and 4%, in 40 settlements in Bulgaria with a local mean morbidity rate of 3%, as well as in 40 villages and small towns clustered in a limited south-west territory of Romania (districts of Mehedinti and Caras-Severin) where the prevalence of manifest disease was over 2%.[5] A high prevalence of tumors of the renal pelvis and ureter was described in patients with BEN and in affected families.[5]

The epidemiological characteristics of BEN are summarized in Table 1.

Table 1 Epidemiological, clinical and functional characteristics of BEN

Epidemiological characteristics
• Residence in an endemic settlement
• Family history of renal disease and of renal deaths
• Urothelial tumors
• Occupational history of farming
Clinical characteristics
• Slowly progressive renal insufficiency
• Anemia: normochromic or slightly hypochromic
• Edema absent
• Hypertension: rare in early, common in advanced renal failure
• Urothelial tumors: common
• Abnormalities on urinalysis
• Reduced kidney size, normal size in early stages
Functional changes
• Impaired concentrating capacity
• Decreased glomerular filtration rate
• Impaired urinary acidification
• Glycosuria, aminoaciduria
• Increased uric acid excretion
• Renal salt wasting
• Proteinuria of the tubular type

Modified from Stefanovic (1983).

Residence in an Endemic Settlement

BEN was first described first by Danilovic et al.[2] in some villages around the valley of the Kolubara river. Soon after, several endemic regions in former Yugoslavia, Bulgaria, and Romania, were identified. BEN was also found along the Sava and its tributaries, in flood areas of Podrinje and Macva, in some areas of Bosnia (near Bijeljina), and in Croatia (around Slavonski Brod). Later reports described BEN in the valleys of Velika Morava, Juzna Morava, Binacka Morava, and Zapadna Morava. BEN occurs only in village inhabitants. However, not all villages are affected in endemic areas: those affected are sometimes at a distance of only a few kilometers from unaffected ones.[5]

Family History of Kidney Disease

The familial character of this disease was described by Danilovic et al.[2] Later reports confirmed this observation. BEN can sometimes affect several members of a household in one generation or in several generations.[5] In Bulgaria, Mihailov established that BEN was confined to certain families and appeared to have a genetic background.[10] However, studies of BEN in villages around Slavonski Brod, Croatia, pointed to environmental factors as key determinants of individual susceptibility to BEN.[11] This statement was confirmed by the observation that in endemic villages, both autochthonous Croat population and immigrant Ukrainians were affected by BEN. On the other hand, Ukrainians who immigrated to nonendemic villages did not develop BEN.[11]

Urothelial Tumors

Bulgarian authors have first described a high prevalence of tumors of the renal pelvis and ureter in patients with BEN.[12–14] This has been confirmed in inhabitants of other settlements where the disease is endemic and in affected families.[15–17] The most extensive studies of UUC in Serbia were done by Petkovic et al.[15] Recent data from Croatia revealed that the prevalence of UUC were several times higher in endemic area than in other parts.[17]

Occupational History

BEN was found among farmers of the affected villages. Farming is a common occupation among villagers, and we were able to prove that farming, at least at some time in life, was characteristic of patients with BEN. Thus, the putative etiologic agent could relate to farming procedures or to dietary habits peculiar to a given rural area.

CLINICAL CHARACTERISTICS OF BEN

BEN is a chronic tubulointerstitial disease with an insidious onset and gradual slow development of renal failure. The clinical picture is one of progressive renal failure unaccompanied by salt retention or early hypertension (see Table 1). The disease is relatively asymptomatic and can remain latent for years or even decades, the patient becoming symptomatic only in advanced renal failure, when anemia is usually marked. Normo- or hypochromic normocytic anemia is a usual finding. Kidney size assessed by ultrasonography or radiography varies from normal to the size of a small contracted kidney.

Renal Insufficiency

BEN is a chronic tubulointerstitial disease inexorably leading to chronic renal failure. Polyuria and nocturia are features of a decreased concentrating capacity of kidneys due to the marked tubulointerstitial damage and progressive renal impairment. End-stage renal disease develops after the fifties, necessitating kidney-supporting therapy. In some patients, upper urothelial cancers can develop even prior to any renal impairment.

Reduced Kidney Size

In the early stages of BEN, kidneys have a normal size, whereas BEN patients with renal failure have small or shrinking kidneys. Symmetric reduction in kidney size is the most common finding, both kidneys appearing small with a smooth outline.[5],[6]

Anemia

Anemia is a constant feature of BEN. An anemia disproportionate to the degree of renal failure has been described by some authors. It can be demonstrated in the early stage of BEN, progresses with functional nephron loss, and is very pronounced in uremia.[5],[6] It is of the normochromic or slightly hypochromic type. During the initial stages of renal insufficiency due to BEN, significantly lower red cell concentrations have been observed in comparison with control subjects from the endemic region.[18] Importantly, BEN patients undergoing dialysis require higher doses of erythropoietin for anemia treatment than other patients with end-stage kidney disease (ESKD).[19],[20]

Absent Edema

No peripheral edema of the renal origin has been observed during the whole course of the disease.

Abnormalities in Urine Analysis

Relatively scant and intermittent proteinuria has been described. In the course of the disease, proteinuria increases, being around 1 g/L or less in advanced renal failure.[5],[6] Low molecular proteinuria is a hallmark of BEN and is usually accompanied by enzymuria and other markers of tubular damage.

Scarce sediment of urine is also characteristic. A few red and white blood cells per high power field could be found. Macroscopic hematuria usually reflects the presence of a urinary tract tumor.[5],[6]

Blood cells or granular casts might be very occasionally observed.

Uric acid, urate, and some other crystal formation is frequent, especially during summer months. However, a paucity of abnormalities in urine is a characteristic of BEN.

Hypertension

High blood pressure has occasionally been observed in BEN patients in the early stages of the disease. If hypertension is established, it is usually mild or moderate. In patients with advanced renal failure, blood pressure is frequently increased as a consequence of salt and water excess.[5] However, it has recently been reported that the prevalence of hypertension in endemic villages was high, although it is the same as in nonendemic rural parts.[21],[22] Hypertension was found in 43% of BEN and 35% of non-BEN family members.[21] In the endemic villages, the prevalence of hypertension was the same as in control (51.7% vs 49.2%).[22] It was suggested that the higher prevalence of hypertension is likely due, in addition to older age, to the fact that that this group contains the greatest number of obese individuals.

Urinary Tract Infection

In a study comprising 236 patients (133 females and 103 males) with BEN on follow-up from 6 months to 10 years, urinary tract infection was observed in 13.5% of patients in the early stages of the disease and 16.7% of patients in asymptomatic renal failure.[23] A urinary tract infection in BEN patients could further compromise renal function and should be searched for in every patient with rapidly progressing renal failure. Further studies are needed to establish the role of urinary tract infection in the progression of kidney damage in BEN.

FUNCTIONAL CHANGES IN BEN

Although clinical observations have indicated which renal functions could be affected in BEN, there are only a few reports of detailed renal functional investigations. A short summary of the gathered information from previous studies on renal blood flow, glomerular filtration rate, and specific tubule functions in BEN patients can be found henceforth. Beside the changes in renal blood flow and glomerular filtration rate, BEN is characterized by significant changes in the renal tubular handling of a variety of solutes as well as in the regulatory control of water excretion. It seems that the major site of injury in BEN lies in the nephron, predominantly affecting the proximal tubule. Glycosuria and aminoaciduria have been reported. A tubular proteinuria was found to occur in BEN before routine tests for proteinuria become positive.[24]

Impaired Concentrating Capacity

Impairment of the concentrating capacity of the kidney was found late in the course of BEN. A comparison of the concentrating capacity estimated by measuring the maximal urinary specific gravity and creatinine clearance in BEN patients has shown that the concentration capability of the kidney and the glomerular filtration rate decrease slowly and progressively over the years.[5]

Renal Insufficiency

Renal insufficiency usually develops in the adult after having lived in an endemic region for more than 15 years, and slowly progresses for several years up to the terminal stage of the disease. A rapid deterioration of renal function should arouse suspicion of a urinary tract or other type of infection, water and electrolyte depletion, drug nephrotoxicity, or other precipitating factors.

Renal Salt Wasting

Renal salt wasting has been observed in many patients, as BEN is primarily a tubulointerstitial disease. Dietary restriction of salt in BEN patients could therefore lead to hypovolemia and a further decrease in renal function.

Increased Uric Acid Excretion

Uric acid clearance and uric acid/creatinine clearance ratios were found to be significantly increased in individuals with early tubular proteinuria of BEN.[24] The increased uric acid clearance could be the result of decreased proximal tubular reabsorption or altered distal tubular secretion.

Impaired Urinary Acidification

An acidifying defect has also been described in patients with BEN who appeared to fail to make ammonia normally, suggesting an additional distal tubular defect.[24]

Glycosuria and Aminoaciduria

Glycosuria and aminoaciduria have been reported, both linked to proximal tubular lesions.[24],[25]

Tubular Proteinuria

Tubular proteinuria has been reported by many authors. Three low-molecular weight proteins have been observed to be increased in BEN patients, namely ß₂-microglobulin, lysozyme, and ribonuclease.[26] The determination of ß₂-microglobulin in urine is reported to be a useful tool in the screening procedure of BEN. Alpha-1-microglobulin, which is much more stable than β2-microglobulin in urine, has also recently been described as a useful screening tool in BEN.[27]

MORPHOLOGIC CHANGES IN BEN

The characteristic morphologic changes in BEN have recently been reviewed by Stefanovic and Cosyns.[6] Data from renal biopsies performed in BEN patients for several decades indicate that BEN is a chronic tubulointerstitial disease.[6],[28–33] Light microscopical analyses reveal that BEN is characterized by diffuse cortical interstitial fibrosis, which is hypocellular in the majority of the cases, and by tubular atrophy, with both features decreasing from the outer to the inner cortex. At early stages, the lesions are focal and associated with interstitial edema as well as proximal tubule epithelial cell degeneration. Morphologic features considered as accessory or secondary include glomerular ischemia or obsolescence of the collapsing type. Vascular lesions include arteriolar hyalinosis, sclerosis of interlobular arteries, and thickened peritubular capillary basement membranes. Immunofluorescence and electron microscopy findings are non-specific. Glomerular immune deposits are either absent or limited to likely non-significant deposits of IgM and/or C3.

BIOCHEMICAL INVESTIGATIONS

Blood and urine analyses should be performed in every patient suspected of having BEN.

Urine

A fresh morning sample of urine should be sent to the laboratory for the determination of the specific gravity, pH, protein, and red blood cells. Reagent strips can be used, and if positive, a quantitative determination of the amount of total proteinuria has to be performed. Urinary sediments should be examined, preferably by phase-contrast microscopy. Daily protein excretion studies require a 24-hour urine collection and protein determination by biuret or some other, more sensitive method.

In several specialized laboratories, the type of proteinuria can be determined by using electrophoretic separation of urinary proteins. Agar-gel, cellulose acetate, or polyacrylamide gel electrophoresis methods are used. Urinary levels of ß₂-microglobulin can be determined by the radioimmunological method as an indicator of tubular proteinuria.[34] However, as alpha-1-microglobulin is more stable in acid urine, its role in the diagnostic procedure of BEN requires further investigation. Urinary albumin excretion, as a consequence of tubular damage, was also found to be increased in BEN patients.[34] Microalbuminuria is not pathognomonic for glomerular injury, and indeed there is some evidence that it could also be seen as a result of early tubular injury. Thus, it might be a useful tool in screening for early cases of BEN similar to alpha1-microglobulinuria, beta₂-microglobulinuria, and retinol binding proteinuria, among others. Urinary N-acetyl-β-D-glucosaminidase (NAGA) excretion was found higher in BEN patients than in healthy controls.[35]

Blood Cell Count

Blood cell count and hemoglobin should never be omitted.

Blood Chemistry

Serum creatinine and blood urea are the classic indicators of kidney function. Creatinine is freely filtered by the glomerulus, but is also secreted by the proximal tubule. Hence, the amount of creatinine excreted in the urine is the composite of both the filtered and secreted creatinine. Factors other than the level of GFR can also influence creatinine secretion. Creatinine secretion is inhibited by some common medications, such as cimetidine and trimethoprim. In patients with normal creatinine values, renal function tests should be applied. In patients with renal failure, creatinine could be used as a rough estimate of renal function. Serum electrolytes (Na, K, Cl, HCO₃, Ca, and P) are determined particularly in patients with renal failure, as EBN patients can present water and electrolytes in later stages, as well as acid-base disorders, along with renal osteodystrophy.

Measurement of Renal Function

The normal level of GFR varies according to age, gender, and body size. The normal mean (standard deviation) GFR in young adults is approximately 120–130 (20–25) mL/min/1.73 m². Normal values in women are assumed to be 8% lower at all ages. After approximately age 20–30 years, the normal mean value for GFR declines with age in both men and women, with a mean decrease of approximately 1 mL/min/1.73 m² per year. Thus, by age 70, the normal mean value is approximately 70 mL/min/1.73 m². Data from NHANES III, 1988–1994, suggest that almost 75% of individuals 70 years old may have GFR <90 mL/min/1.73 m², and almost 25% may have GFR <60 mL/min/1.73 m². The fraction of elderly individuals with decreased GFR who truly have chronic kidney disease has not been systematically studied. Moreover, the health outcomes of decreased GFR in the elderly, with or without chronic kidney disease, are also not known. This is important because the clinical presentation of BEN is moving to the older age group.[5]

The direct measurement of creatinine clearance is not easy. Urinary clearance measurements require timed urine collections, which are difficult to obtain and often involve errors in collection. Furthermore, day-to-day variations in creatinine excretion exist, making the estimation of GFR, even from a valid 24-hour urine collection, imprecise. The most frequently used equation for estimating GFR in adults is the Cockcroft-Gault equation, which was developed for estimating creatinine clearance but has been tested widely in its prediction of GFR. Among adults, the MDRD Study equation may perform better than the Cockroft-Gault equation, and incorporation of this formula as a measure of GFR should be considered in patients with GFR up to 60 mL/min.[36]

IMMUNOLOGICAL INVESTIGATIONS

As the precise etiology of BEN is to date still unknown, several authors have studied the likely role of some immunological events.

Immunofluorescent microscopy studies of the kidney have shown scarce immunoglobulin and complement deposits in some tubules and glomeruli.[31]

No significant activation of either the classic or alternate pathways of the complement system occurs in the early stages of BREN.[37] Circulating autoantibodies against glomerular and tubular basement membranes have not been detected.[38]

Cell-mediated immunity does not play any major role in BEN.[5]

DIAGNOSTIC IMAGING

Renal Ultrasonography

Diagnostic ultrasound is another important method of examining the urinary tract of BEN patients, especially due to a high prevalence of UUC. Ultrasonography can replace radiology for the estimation of kidney size and can be helpful in the early diagnosis of some morphological abnormalities and renal pelvis/ureter dilatation. This is an easy and safe method. The major limitations of ultrasonography are its relative inability to provide precise information about the renal parenchyma and the ureter, as well as the fact that it largely depends on the skills of the person performing examination.

Plain Film

Plain abdominal radiography will usually reveal the size and the outline of the kidneys. In addition, it may reveal abdominal and renal calcifications and provide an assessment of the bladder size.

Intravenous Urography

Intravenous urography (IVU) is an examination of choice for BEN patients, following ultrasound examination. IVU permits an accurate morphological description of the kidney, renal pelvis, ureter, and urinary bladder. Because renal excretion is dependent on function, IVU gives an estimate of the overall kidney function.

Kidney size varies from normal to severely reduced, depending on the stage of the disease. The renal outline is usually smooth. Normal pelvicaliceal and ureteral morphology are the usual findings in BEN patients, but IVU could be useful for the diagnosis of renal pelvis or ureteral cancer. In patients with advanced renal failure, satisfactory opacification of the collecting system cannot be obtained by standard procedures. A late urogram with tomography should then be performed.

Any superimposed chronic pyelonephritis, urinary tract calculi, or tumor may then be demonstrated.

Computed Tomography

Computed tomography (CT), although providing detailed anatomical information, has only been used in BEN patients occasionally. CT introduces new elements: it gives a clear picture of the outline of the hilum and of the peri-renal space. Sequential sections enable the visualization of a dilated ureter. Contrast-enhanced CT scans may show minimal kidney function loss that may not have been visible by excretory urography. Its usefulness in revealing the nature of space-occupying lesions of the kidney is very helpful in the diagnosis of urinary tract tumors commonly encountered in BEN patients.

Retrograde Ureteropyelography

Retrograde ureteropyelography is required in suspected urothelial cancer of renal pelvis and ureter.

Renal Arteriography

Renal arteriography has been used primarily for research purposes but has a limited and decreasing role in the management of BEN. Its main application remains in cases of suspected renal tumor and in pretransplant evaluation of end-stage BEN patients.

Radionuclide Investigation

Radionuclide examination has come into practice as a simple and non-invasive method giving valuable information primarily on separate kidney function.[39],[40] Though morphology on scintigraphic examination is inferior to the IVU, it is complementary and most valuable in patients sensitive to iodinated contrast agents.

Renal Biopsy

When available, renal biopsy is of high diagnostic value. Indeed, observation of the typical hypocellular interstitial fibrosis decreasing in severity from the outer to the inner cortex is highly suggestive of BEN. However, at the present state of knowledge, therapy of BEN does not benefit much from biopsy studies. It is therefore important to stress that renal biopsy is no longer recommended as a routine diagnostic procedure, especially not for patients with advanced renal failure. For diagnostic purposes, biopsy could be considered in the following situations:

1. in family members previously not affected by BEN but living in an endemic settlement;

2. in patients with nephropathy of unknown etiology living in settlements adjacent to the BEN areas;

3. in immigrants to the BEN regions who lived more than 15 years in endemic villages; and

4. in emigrants from them.[5]

DIFFICULT DIAGNOSIS

Diagnosis of BEN in the Early Stage

The early stages of BEN are not easily detectable clinically, as the disease is asymptomatic until a significant decline in renal function occurs, and even then symptoms are usually non-specific. The sensitivity of routinely used markers of BEN such as serum urea/creatinine is poor, so by the time such tests show an abnormality, significant renal function has been lost, making study of the initiation of early renal damage difficult. The natural history of BEN is complex, possibly with multiple risk factors operating both at the stage of initiation of renal damage and in its progression to ESRD. In BEN, genetic susceptibility is due to multiple genes of small effects, gene-gene interactions, and gene-environment interactions of a complex nature that render it difficult to assess with current study designs. There are furthermore many risk factors such as smoking, alcohol consumption, obesity, physical inactivity, and genetic factors that may be partly responsible for the familial aggregation of BEN.

Early diagnosis of BEN requires a positive family history, residence in the endemic settlements for more than 15 years, tubular type of proteinuria, microalbuminuria, enzymuria (increased urinary NAGA excretion), and, if feasible, renal biopsy. Although of high diagnostic value, renal biopsy is not performed due to the fact that positive histology does not result in any specific therapy. As UUC might evolve prior to manifest tubulointerstitial damage, urine cytology should also be included in the diagnostic procedure. Furthermore, to the suggestions of the experts who met in Zagreb in October 2006, detection of DNA adducts originating from environmental nephrotoxins/carcinogens in urothelial cells are currently under investigation.

Diagnosis of BEN in Advanced Renal Failure

Very often, patients from endemic settlements and who have a strong family history of kidney disease are observed for the first time in advanced renal failure. Polyuria, nocturia, cramps, and pruritus are the most common symptoms. The lack of other data (i.e., acute renal disease, kidney stone, major urinary tract anomalies) in a farmer is suggestive of BEN. All other causes of chronic kidney damage, such as ischemic nephropathy, analgesic nephropathy, diabetic nephropathy, and chronic primary glomerulonephritis, should be ruled out. There is strong evidence against the hypothesis that BEN is a form of chronic glomerulonephritis. However, we should remain aware that some BEN patients might also have diabetes, hypertension, or any other contributing pathology. Bilaterally contracted kidneys with a smooth outline point to BEN but may be produced by a chronic, previously unrecognized, glomerulonephritis. In 7 out of 72 cases suspected of having BEN at an early stage, renal biopsy demonstrated IgA glomerulonephritis.[28],[30] Of note, mesangial IgA deposition is found in 9% of an apparently healthy population.[41] On the other hand, mesangial IgA has been found in a patient, fulfilling in addition all the diagnostic criteria of advanced aristolochic acid nephropathy (AAN).[42] This could also occur in advanced BEN cases. Abnormality of the lower urinary tract should be excluded, but even when present, diagnosis is not made easier, as it could be superimposed on BEN. Findings of one unilateral scarred kidney while the other kidney is contracted with a smooth outline could also point to BEN and superimposed pyelonephritis. Radiologically, very small kidneys, as in bilateral renal hypoplasia, have been found characteristic of end-stage BEN.

Immunological investigations could be misleading in advanced renal failure. Kidney biopsy showing a typical hypocellular interstitial fibrosis decreasing from the outer to the inner cortex (if the biopsy can be oriented) would be highly suggestive. However, it might be associated with adverse effects inherent to this invasive diagnostic method, and because it will not help its therapeutic management, it is not a recommended diagnostic procedure for BEN.

Hypertension is absent from the early stages of disease but frequent in advanced renal failure.

Normal-size kidneys in a patient with advanced renal failure are not compatible with BEN.

Diagnosis in patients with a strong family history of BEN in advanced renal failure is made after exclusion of known, easily recognized kidney diseases, such as polycystic kidney disease and obstructive nephropathy. Establishing a diagnosis of BEN can be difficult and is susceptible to several errors. For example, it would be wrong to jump to a diagnosis of BEN in a patient coming from an endemic area but who does not have a positive family history of BEN and is showing signs of advanced renal failure at the first examination.

Diagnosis of BEN in Children

EBN is a slow-developing, long-lasting kidney disease. Diagnosis is usually made when patients are in their forties or fifties, and terminal renal failure usually develops when they are over fifty years of age. No case of BEN has ever been documented in children. However, an increased urinary excretion of albumin and total protein has been demonstrated in children coming from families with BEN.[43],[44] Moreover, congenital renal abnormalities have been found more commonly in children from nephropathic families.[45]

BEN in Subjects Living in Settlements Adjacent to Nephropathic Areas

BEN is found in some villages but not in others that are yet located only a few kilometers away. This pattern of distribution has been recognized by many authors. Sporadic cases of BEN in such villages were described. Another problem has arisen in cases of tubulointerstitial disease resembling BEN in settlements more or less distant from the endemic area. Many cases of BEN have been found in such villages and usually involved a woman from the endemic village and endemic family who got married and settled there. Members of the nephropathic families or those suspected of having a kidney disease encounter definite problems in getting a partner in their native villages, so they often marry someone who has no full knowledge of the disease, mainly from the villages where BEN is not a problem.

Diagnosis of BEN in such emigrants requires a positive family history, residence in the endemic settlements for more than 15 years, tubular type of proteinuria, microalbuminuria, enzymuria (increased urinary NAGA excretion) and, if feasible, a renal biopsy.

Diagnosis of BEN in Emigrants

There have been several reports of BEN in people leaving their native (endemic) villages.[5],[46] BEN appeared several years after they had settled into the new environment. Because the development of renal disease can take several years or even decades, such cases are quite understandable. Some of these people left the country and settled abroad in Australia, Switzerland, or even Sweden, where a diagnosis of chronic interstitial nephritis of unknown etiology was made.

Diagnostic procedures should be perfected and include an extensive family history, residence in the endemic settlement for more than 15 years, tubular type of proteinuria, microalbuminuria, enzymuria (increased urinary NAGA excretion), and morphology compatible with BEN. The family history of kidney disease/UUC and previous residence in the endemic settlement are of the utmost importance.

Diagnosis of BEN in Immigrants

BEN was described in immigrants from the Ukraine who settled some 70 years ago in villages near Slavonski Brod.[11] Such evidence is very important for the etiology of BEN, which points to environmental rather than genetic factors. BEN has also been described in immigrants to several other endemic settlements.[4]

Because we need ample demonstration of kidney disease in immigrants, any likely BEN case should have a complete work-up including family history, exposure to the endemic environment, tubular type of proteinuria, microalbuminuria, enzymuria (increased urinary NAGA excretion), and morphology compatible with BEN. Such cases should be examined in a few units specialized for BEN, before advanced renal failure develops. For cases with advanced renal failure, we would not suggest undocumented claims of BEN.

BEN in City Population

The authors have not observed any case of BEN in people born and raised in cities.[5] However, sporadic cases of BEN in subjects born in towns are described. Mihailov reported on autochthonous cases of BEN from the town of Vratza, but their kinship with endemic families in adjacent endemic villages was established by the genealogical method.[10] Because this question has important etiological implications, the precise diagnosis is mandatory.

It must be borne in mind that after the Second World War, cities had far fewer inhabitants, and a massive migration of farmers and their families occurred in the last forty years. Furthermore, it should be remembered that the suburban population of almost all towns was in part engaged in farming. Many cases of BEN are registered in Vrapcane, a suburban settlement of the city of Paracin, Serbia.

BEN outside of the Balkans

BEN might not be confined exclusively to the Balkans. Like lecithin cholesterol acyltransferase (LCAT) deficiency, originally described in Scandinavia but occurring all over the world, BEN, clustered in the Balkans, could probably also spread elsewhere in Europe and the world. This is an intriguing idea, but it should be further elaborated upon. According to the current literature, there does not appear to be any other such limited geographic areas with such a high prevalence of chronic kidney disease and UUC. However, outside of the endemic regions, in Europe and overseas, BEN should be suspected in any case of chronic interstitial nephritis of unknown etiology. An increased incidence of both BEN and urothelial tumors not associated with analgesic abuse should draw attention to the possibility of BEN.[47],[48] Only once the etiological factor(s) have been identified will it be possible to make an etiological diagnosis with the appropriate laboratory tests (Zagreb Symposium, 2006).

DIFFERENTIAL DIAGNOSIS OF BEN

Chronic interstitial fibrosis is associated with upper urothelial malignancy in BEN, in analgesic nephropathy (AN), and AAN. All three diseases share a usually normal blood pressure in the early phase, early and severe anemia, low molecular weight tubular proteinuria, glycosuria, and the absence of recurrence after renal transplantation.[37]

BEN is characterized by a familial and environmental clustering, affects equally both genders, and runs a very slow, decade-long course.[5],[6],[48] Clinical features of BEN, AN, and AAN are presented in Table 2.

Table 2 Clinical features and kidney morphology in BEN, analgesic nephropathy, and aristolochic acid nephropathy

	Balkan endemic nephropathy	Aristolochic acid nephropathy	Analgesic nephropathy
Familial character	Important feature	No	No
Associated urothelial cancer	Yes	Yes	Yes
Rate of progression	Very slow	Rapidly progressive	Slow
	(>20 years)	(6 months–2 years)	(>5 years)
Kidney imaging
Kidney size	Shrunken kidneys	Shrunken kidneys	Shrunken kidneys
Outline	Smooth surface	Smooth surface	Irregular contours
Calcifications	No	No	Medullary and papillary
Urinary deposit	Scarce deposit	Aseptic leukocyturia	Leukocyturia, microhematuria
Gross morphology
Kidney atrophy	Symmetric	Symmetric	Asymmetric
Outline	Smooth	Smooth	Irregular
Kidney histology
Tubular atrophy	3, Mainly outer cortex	3, Mainly outer cortex	3, Multifocal
Interstitial fibrosis	2, Mainly outer cortex	3, Mainly outer cortex	2, Multifocal
Interstitial cellular infiltration	1	1	2
Papillary necrosis	Absent	Absent	Hallmark
Etiology	Unknown	AA	Analgesics
Corticosteroid treatment	Not described	Slowed decrease of GFR	Not described

* Graded from 1 (present) to 3 (extensive).

Analgesic Nephropathy

AN is characterized by a history of analgesic abuse, medullary and papillary calcification or necrosis, chronic interstitial nephritis, urothelial tumors, and atheromatous renal artery stenosis. AN is part of a wider clinical syndrome, including AN, gastric ulcer, anemia, psychiatric disturbances, cardiovascular and gonadal disturbances, and premature aging.[48]

Aristolochic Acid Nephropathy

AAN has been reported throughout the world, mainly in females, and runs a more rapid, often subacute course.[49–51] Patients usually become symptomatic only when renal failure is advanced. Clinical examination is usually normal. The urine sediment is virtually normal except for mild aseptic leukocyturia. Normoglycemic glucosuria is often present. Proteinuria is usually mild (<2 g/day) but characteristic of the tubular type. On x-ray, kidneys have a symmetric, reduced size with a smooth outline. AAN is characterized by hypocellular (in the vast majority of the patients) interstitial fibrosis, decreasing from the outer to the inner cortex, and by the development of UUC in around 50% of the patients, which is amazingly similar to BEN. AAN is therefore proposed as a clue to BEN.[32]

Ochratoxin Nephropathy

Produced by several species of Aspergillus and Penicillium, ochratoxin A (OTA) is nephrotoxic in animals, but no definitive case of nephrotoxicity in humans has been reported. Pigs fed OTA develop proteinuria and normoglycemic glucosuria associated with polyuria, a loss of concentrating capacity, as well as interstitial fibrosis and possible kidney/liver tumors.[52] OTA has been incriminated in BEN for several reasons. For example, samples of staple foods more frequently contain high levels of OTA in affected families or in endemic areas than in unaffected families or non-endemic areas. The number of blood samples with high levels of the toxin is higher in patients with nephropathy and/or urinary tract tumors than in unaffected people from endemic and control subjects from nonendemic areas.[53] However, a European Research Group, convened to review the evidence linking OTA and BEN, recently concluded that “there is no convincing evidence from human epidemiology to confirm the association between OTA exposure and the prevalence of BEN or upper urinary tract tumors.”[54]

Rare Forms of Chronic Interstitial Nephritis

Beside the chronic interstitial nephropathy induced by the abuse of analgesics, a number of toxic compounds or drugs have been associated not only with acute renal failure but also with chronic interstitial renal disease.[50]

Heavy Metals

• Chronic lead intoxication

• Chronic lithium exposure

• Chronic cadmium intoxication

• Chronic arsenic intoxication

Drugs

A number of drugs (listed below) introduced in the last 30 years may induce severe interstitial nephritis, leading occasionally to end-stage renal failure. Prevention requires minimal dosage and constant monitoring of renal function and, whenever feasible, prevailing serum drug levels.

• Cyclosporin A

• Ifosfamide

• Phytotherapy-associated interstitial nephropathy (PAIN)

• Pamidronate

• Analgesics

• Lithium

• Arsenic

• Nitrosoureas

• Tacrolimus (FK506)

• cis-Diamminedichloride platinum (CDDP)

DIAGNOSTIC PROCEDURE

Diagnosis of BEN is made in inhabitants from endemic settlements using the following:

1. Epidemiologic criteria

2. Demonstration of

   • GFR decrease

   • proteinuria generally bellow 1 g/24 h

   • microalbuminuria

   • little urinary sediment

   • tubular markers (renal glucosuria, increased urinary excretion of β2 microglobulin or alpha-1- microglobulin, and NAGA)

   • A typical renal histology showing hypocellular cortical interstitial fibrosis decreasing from the outer to the inner cortex (if renal biopsy feasible).

3. Exclusion of other known kidney disease (e.g., chronic pyelonephritis—obstructive and atrophic, adult dominant polycystic kidney disease, glomerulonephritis)

Diagnosis is made in patients from the early stage to advanced renal failure, including staging, according to KDOQI.[55] Defining stages of chronic kidney disease requires the “categorization” of continuous measures of kidney function, and the cutoff levels between stages are inherently arbitrary. Nonetheless, staging of chronic kidney disease will facilitate application of clinical practice guidelines, clinical performance measures, and quality improvement efforts to the evaluation and management of chronic kidney disease.

Normal Protein Excretion

Normal mean value for urine albumin excretion in adults is approximately 10 mg/day. Albumin excretion is increased by physiological variables, such as upright posture, exercise, pregnancy, and fever. Normal mean value for urine total protein is approximately 50 mg/day.

SCREENING OF BEN

Despite the availability of improved medical therapy to slow the progression of nephropathy, true epidemics of ESRD can be observed in some regions. BEN is still the major problem in several endemic regions in Bosnia, Croatia, and Serbia. Many patients are not diagnosed until the late stages of disease, as early kidney disease may be asymptomatic. Ideally, all adults ought to be routinely screened for evidence of early BEN and associated risk factors.[56]

Evaluation of Patients at Increased Risk

Clinical evaluation of patients at increased risk of chronic kidney disease includes assessment of markers of kidney damage, estimated GFR, and blood pressure. Abnormal urinary excretion of albumin and total protein is a highly sensitive indicator of glomerular disease. The results of urine sediment examination and of imaging studies of the kidney, however, can also suggest other types of chronic kidney diseases, including vascular, tubulointerstitial, and cystic diseases of the kidney. In addition, proteins other than albumin in the urine may indicate tubulointerstitial injury. At present, there are no clinically proven markers that are specific for tubulointerstitial or vascular diseases of the kidney. Due to high incidence of UUC, urine cytology must be performed.

Markers of Kidney Disease

Markers of kidney damage in addition to proteinuria include abnormalities in the urine and abnormalities on imaging studies. New markers are needed to detect kidney damage prior to a reduction in GFR in other types of chronic kidney diseases, including BEN.

Screening Recommendations

Members of a working party for the management of CKD have identified several recommendations for the screening of patients at risk of CKD.[41] These could be used for the screening of patients at risk of BEN, in addition of tubular markers.

How Do You Screen for Chronic Kidney Disease, Including BEN?

• Take a short history, including family history, and measure blood pressure. Use a dipstick (untimed spot urine sample) for proteinuria, albuminuria, WBC, and RBC.

  • If positive for proteinuria, measure total protein to creatinine ratio in an untimed spot urine sample.

  • If positive for albuminuria: measure albumin to creatinine ratio in an untimed spot urine sample.

  • In the screening of BEN: measure β2-microglobulin to creatinine ratio in an untimed spot urine sample.

  • If positive for WBC or RBC: perform a sediment analysis in an untimed spot urine sample.

• Estimate creatinine clearance from serum creatinine using Cockcroft-Gault equation or MDRD Study equation. Renal ultrasonography is usually needed.

How Often Should Screening Be Performed?

• If screening is negative: every 1–3 years, depending on risk factors.

• If abnormality is evidenced at screening: perform diagnostic and therapeutic work-up.

CONCLUSION AND PERSPECTIVES

According to reports from several endemic regions, prevalence of BEN is still high, and the number of patients who need to start dialysis treatment in dialysis centers near endemic focuses has not decreased. Equally, the prevalence of UUC is still several folds higher in endemic areas than in any other region in Europe or worldwide.

A lack of criteria agreed upon for the diagnosis of the disease may contribute to our inability to discover the etiologic agent. Conversely, the definition of these criteria is hampered by our ignorance concerning the etiology of the disease. Accordingly, the International Panel of BEN Investigators agreed to establish criteria based on the most recent evidence and new diagnostic possibilities.

During the symposium on BEN in Zagreb (October 2006), a large body of evidence pointed to an environmental disease with few possible agents, among which aristolochic acid seems to be the most likely culprit. Further establishment of the responsibility of this alkaloid, and possibly of ochratoxin A (for its possible fibrogenicity), in the development of BEN might provide the diagnostic criteria that we need. We might anticipate that the diagnostic criteria of BEN could be all summed up within the near future in:

• proven exposure (epidemiology, intake, exposure, DNA adducts, other biologic markers),

• typical renal interstitial disease (focused on key clinical and biologic features), or

• typical renal histologic picture (focused on key features).

Hopefully, the 50^th anniversary of the first description of endemic nephropathy will be marked by the establishment of diagnostic criteria that will improve clinical work and further scientific investigations of BEN.

ACKNOWLEDGMENTS

The authors wish to thank Dr. Claire de Burbure for proofreading the manuscript. This work was supported by a grant from the Ministry of Science of Serbia