Increased Blood Pressure in Adult Offspring of Families With Balkan Endemic Nephropathy: A Prospective Study

Plamen S. Dimitrov; Valeri A. Simeonov; Svetlana D. Tsolova; Angel G. Bonev; Rossitza B. Georgieva; Wilfried J. Karmaus


BMC Nephrology 

In This Article



In the period of October, 2003, to April, 2004, we recruited adult offspring (102 study subjects) whose father and/or mother were included in the Vratza Hospital registry of BEN patients in 2001 and who resided in one of three communities (Vratza, Bistretz, or Beli Izvor, Bulgaria). The diagnosis of BEN in the parent generation is based on three groups of criteria: epidemiological, clinical/laboratory, and pathological/anatomical.[20] Regarding epidemiology, the region is known to have a higher prevalence of BEN, thus, the only individual criterion is onset after the age of 20. Clinical and laboratory criteria are a follows: obscure onset, chronic course, no sense of edema, and normochromic type of anemia in more advanced phases. Pathologically, an almost symmetrical macroscopic shrinkage of the two kidneys was required in the parent BEN population. A control group of equal size comprised of adult offspring (99 study subjects) of non-BEN hospitalized patients was enrolled in the study during the same time period. Diagnoses in control parents included diabetes mellitus, cardiovascular disorders, and liver problems. Only three of the 99 parents had kidney disorders (one paternal kidney cancer not related to BEN and two maternal pyelonephritis cases). Subjects of both groups were matched according to gender and ten-year age groups. All participants provided written or verbal consent (witnessed) through a procedure approved by the Institutional Review Board (human-subject research committee) of the National Center of Public Health Protection, Sofia, Bulgaria. The population was enrolled and examined in 2003/04 and investigated again in 2004/05, which provided us with two repeated measurements one year apart.


We conducted face-to-face interviews with all participants either in the hospital, or by visiting them in their home villages. The standardized questionnaire asked for the place of living, type of water supply, diet, smoking and drinking habits, medical symptoms, family history of BEN, family history of other kidney diseases and kidney tumors, and occupational history.

Physical Examination

The physical examination was performed by an experienced physician with board certification in internal diseases and nephrology. It was aimed at assessing the general health status of the study subjects and at revealing symptoms of BEN and/or other internal diseases. Blood pressure was measured according to standards set by the World Health Organization.[21] Participants were in the seated position; three measurements were taken on the right arm at 5 minute intervals. We recorded systolic and diastolic blood pressure and calculated pulse pressure as the difference between these two values.

Determination of the Kidney Sizes

Ultrasound investigations of both kidneys took 20-30 minutes. The patient was investigated lying on both the left and right sides. After finding a suitable image, measurements were taken. The longest dimension of the kidney was determined. The thickness of the kidney parenchyma (in the thinnest or minimal part), parenchyma structure, and the relationship of parenchyma and pyelon were measured. Information gathered also included the location, size, and morphology (cysts, stones, and tumors) of the kidneys. The images were saved electronically for future reference. The ultrasonographer (AGB) worked in the Department of Image Diagnostics, Vratza District Hospital, and was blinded to the clinical status of the participant (BEN or control offspring).

Blood Collection and Analysis of Lead

Blood samples were drawn in K2EDTA vacutainers. Lead in blood was determined by using atomic absorption spectrometry. A Model 4110 ZL atomic absorption spectrometer (Bodenseewerk Perkin-Elmer, Ueberlingen, Germany) with a transverse heated graphite atomizer and longitudinal Zeeman-effect background correction equipped with an AS-72 autosampler, electrodeless discharge lamps System II, software WinLab (Version 1.2) and both "Standard"(Part No B 300-0643) and "End-capped" (Part No B 300-0644) THGA tubes with integrated platforms was used for direct electrothermal AAS measurements. To measure lead, blood was diluted 10-fold with 0.25% v/v Triton X-100; 10 μL of the sample and 25 μg of (NH4)2HPO4 as a chemical modifier were injected in the THGA, pre-treated with 250 μg of zirconium and 20 μg iridium. Matrix-matched calibrations were applied.[22] Certified reference materials (Seronorm Trace Elements Whole Blood, Level 1, Cat. No 201505, and Level 2, Cat. No 201605, Sero AS, Norway) were used for internal laboratory control.

Statistical Analyses

Outcome variables were diastolic, systolic blood pressure, and pulse pressure. For descriptive purposes, we defined moderate hypertension as a systolic blood pressure ≥150 mm Hg and a diastolic blood pressure ≥100 mm Hg. However to investigate average increases, the explanatory models used the three continuous outcome variables.

To answer the question whether minimal kidney cortex width is related to higher blood pressure, we controlled for potentially confounding factors such as gender, age, history of smoking, diabetes mellitus, parental history of hypertension or of Balkan Endemic Nephropathy, body surface area, and blood lead level. For smoking, we categorized a subject as being a current smoker, ex-smoker, or non-smoker. A parental history of high blood pressure fell into one of three groups: mother, father, or both affected. We asked for presence of diabetes mellitus in the first investigation (2003/04) and for a new occurrence in the second investigation. The body surface area (BSA) was calculated as ([height * weight]/3600)2. This variable is used in the model to control for the effect of weight and height on kidney size and on blood pressure. In each investigation (2003/04 and 2004/05) the average kidney cortex width and kidney length of both kidneys were used as predictors.

To test the hypothesis that offspring of BEN parents have increased blood pressure, we grouped parental disease status into three groups: mother, father, or both parents affected. None of the comparison offspring fell into any of these groups (reference). The effect of cortex width and parental BEN status was tested in the same model.

In order to estimate the association of minimal kidney width and parental history of BEN with blood pressure based on repeated measurements, we applied linear mixed models. Measurements of the two investigations are not independent and mixed models allow adjusting for within-participant effects.[23] The mixed model assumes that the random effects and the error vector are normally distributed, which was the case for the blood pressure variables. SAS PROC MIXED was used to perform the regression analysis.[24] We used Akaike information criteria and the likelihood ratio test to examine the significance of serial correlation (in repeated statement) as well as to model random effects, along with a suitable variance-covariance matrix structure.

To compare the agreement between measurements of continuous variables in 2003/04 and 2004/05 we used the intra-class correlation coefficient (ICC). This is the between-subject minus the within-subject variance divided by the sum of the two variances. The ICC quantifies the proportion of total outcome variance that is due to inter-individual variation.


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