Clinical markers in adult offspring of families with and without Balkan Endemic Nephropathy
2006; Elsevier BV; Volume: 69; Issue: 4 Linguagem: Inglês
10.1038/sj.ki.5000120
ISSN1523-1755
AutoresPlamen Dimitrov, Svetla Tsolova, R. Georgieva, D. Bozhilova, Valeri Simeonov, Angel Bonev, Wilfried Karmaus,
Tópico(s)Amyloidosis: Diagnosis, Treatment, Outcomes
ResumoBalkan Endemic Nephropathy (BEN) is a kidney disease that progresses slowly. Only a few studies have investigated renal clinical markers in offspring of BEN families before the onset of the disease. This project aimed to determine whether kidney function and structure are altered in BEN offspring compared with non-BEN offspring. The study population consisted of 102 adult BEN offspring and a control group of 99 non-BEN offspring. We collected urine and blood samples, and conducted face-to-face interviews, physical examinations and ultrasound measurements of the kidney. Total protein, albumin, β2-microglobulin and creatinine in urine, creatinine and urea in serum, and creatinine clearance (CCR) were determined. Two risk factors were assessed: first, the overall status of being an offspring from a BEN family, and second, the specific status of a mother and/or father with BEN. The data were analyzed using linear regression. After adjusting for confounders, we found that kidney length and minimal cortex width in BEN offspring were significantly decreased. Urine concentrations of total protein, albumin, and β2-microglobulin were higher in BEN offspring. Regarding parental history, the associations were statistically significant only for the offspring of mothers who had BEN, with the exception of minimal cortex width, which showed no parental difference. For CCR, we did not identify a statistically significant effect for BEN offspring status nor for parental history. In conclusion, adult offspring of BEN families can be characterized by shorter kidney length and an increased excretion of albumin, total protein, and β2-microglobulin, in particular, when the mother had BEN. Balkan Endemic Nephropathy (BEN) is a kidney disease that progresses slowly. Only a few studies have investigated renal clinical markers in offspring of BEN families before the onset of the disease. This project aimed to determine whether kidney function and structure are altered in BEN offspring compared with non-BEN offspring. The study population consisted of 102 adult BEN offspring and a control group of 99 non-BEN offspring. We collected urine and blood samples, and conducted face-to-face interviews, physical examinations and ultrasound measurements of the kidney. Total protein, albumin, β2-microglobulin and creatinine in urine, creatinine and urea in serum, and creatinine clearance (CCR) were determined. Two risk factors were assessed: first, the overall status of being an offspring from a BEN family, and second, the specific status of a mother and/or father with BEN. The data were analyzed using linear regression. After adjusting for confounders, we found that kidney length and minimal cortex width in BEN offspring were significantly decreased. Urine concentrations of total protein, albumin, and β2-microglobulin were higher in BEN offspring. Regarding parental history, the associations were statistically significant only for the offspring of mothers who had BEN, with the exception of minimal cortex width, which showed no parental difference. For CCR, we did not identify a statistically significant effect for BEN offspring status nor for parental history. In conclusion, adult offspring of BEN families can be characterized by shorter kidney length and an increased excretion of albumin, total protein, and β2-microglobulin, in particular, when the mother had BEN. Balkan Endemic Nephropathy (BEN) was first characterized in the Vratza District, Bulgaria, in 1956.1.Tanchev Y. Evstatiev Z. Dorossiev D. et al.Studies on the nephritides in the District of Vratza.Savremenna Med. 1956; 7: 14-29Google Scholar Later, similar nephropathies were described in Yugoslavia in 19572.Danilovic V. Djurisic M. Mokranjac M. et al.Porodicna oboljenia bubrega u selu Sopic izvazvana hronicnom intoksikacijom olovom.Srpski Arh Tcelok Lek. 1957; 85: 1115-1125PubMed Google Scholar, 3.Danilovic V. Djurisic M. Mokranjac M. et al.Chronic nephritis caused by poisoning with lead via the digestive tract (flour).Presse Med. 1957; 65: 2039-2040PubMed Google Scholar and in Romania in 1961.4.Fortza N. Negoescu M. Nefrita cronica azotemia endo-epidemica.Stud Cercet Med. 1961; 1: 217-221Google Scholar As a result, in 1964, the disease was recognized as a new nosological entity and was named Balkan Endemic Nephropathy. BEN is a tubulointerstitial kidney disease that develops with no inflammation or hypertension, and progresses slowly over many years. The final disease stage is characterized by renal failure and shrinkage of both kidneys to the size of walnuts.5.Vukelic M. Sostaric B. Belicza M. Pathomorphology of Balkan endemic nephropathy.Food Chem Toxicol. 1992; 30: 193-200Crossref PubMed Scopus (54) Google Scholar The disease shows familial clustering and develops only in certain areas in the Balkan countries – Bulgaria, Romania, Serbia and Montenegro, Croatia, and Bosnia and Herzegovina. The spatial distribution of BEN shows a mosaic-like pattern, with some villages afflicted for decades, whereas others situated in the same vicinity have remained free of BEN.6.Tanchev Y. Dorossiev D. The first clinical description of Balkan Endemic Nephropathy (1956) and its validity 35 years later.in: Castenagro M. Plestina R. Dirheimer G. Chernozemsky I. Bartasch H. Mycotoxins, Endemic Nephropathy and Urinary Tract Tumours. International Agency for Research on Cancer, Lyon1991: 21-28Google Scholar, 7.Radovanovic Z. Epidemiological characteristics of Balkan Endemic Nephropathy in Eastern regions in Yugoslavia.in: Castenagro M. Plestina R. Dirheimer G. Chernozemsky I. Bartasch H. Mycotoxins, Endemic Nephropathy and Urinary Tract Tumours. International Agency for Research on Cancer, Lyon1991: 11-20Google Scholar, 8.Ganev V. Petropoulos E. Balkan Endemic Nephropathy and its putative environmental and genetic causes: a review.in: Lekkas T. Pythagorion Samos Sixth International Conference of Environmental Science and Technology. University of Aegen, Greece1999: 11-21Google Scholar The causes of BEN are still unclear. Usually, BEN patients are diagnosed in the late stages of the disease. There is a shortage of studies describing how the disease develops in its earlier stages. A recent study reported a lower creatinine clearance (CCR) in family members of patients with BEN, but no other differences in the frequency of renal function disorders were found when comparing them with members of non-BEN families.9.Arsenovic A. Bukvic D. Trbojevic S. et al.Detection of renal dysfunctions in family members of patients with Balkan Endemic Nephropathy.Am J Nephrol. 2005; 25: 50-54Crossref PubMed Scopus (21) Google Scholar One prior investigation from Nis, Serbia, reported that protein and albumin excretion was increased in children who had a family member with BEN.10.Stefanovic V. Mitic-Zlatkovic M. Cukuranovic R. Vlahovic P. Increased urinary protein excretion in children from families with Balkan Endemic Nephropathy.Nephron Clin Pract. 2003; 95: 116-120Crossref Scopus (17) Google Scholar, 11.Stefanovic V. Cukuranovic R. Mitic-Zlatkovic M. Hall P.W. Increased urinary albumin excretion in children from families with Balkan Nephropathy.Pediatr Nephrol. 2002; 17: 913-916Crossref PubMed Scopus (15) Google Scholar This Serbian project also showed that offspring of BEN families excreted significantly less creatinine and that CCR was decreased.12.Mitic-Zlatkovic M. Cukuranovic R. Lecic N. Stefanovic V. Urinary creatinine excretion in children from families with Balkan endemic Nephropathy: evidence for genetic predisposition to the disease.Pathol Biol. 2000; 48: 554-557PubMed Google Scholar Another study from Lazarevac, Serbia, reported that BEN patients in the early phase of the disease have significantly higher CCR and smaller kidneys in comparison to healthy controls or to patients with renal disease other than BEN.13.Djukanovic L. Bukvic D. Maric I. Creatininne clearance and kidney size in Balkan endemic nephropathy patients.Clin Nephrol. 2004; 61: 384-386Crossref PubMed Scopus (15) Google Scholar It is known that the size of both kidneys decreases as the disease progresses.14.Stefanovic V. Polenakovic M. Balkan nephropathy. Kidney disease beyond the Balkans? [editorial].Am J Nephrol. 1991; 11: 1-11Crossref PubMed Scopus (55) Google Scholar, 15.Radovanovic Z. Sindic M. Polenakovic M. et al.Endemic Nephropathy. Zavod za udzbenike i nastavna sredstva, Belgrade2000: 447Google Scholar However, it is unknown when the process of shrinkage starts and how it progresses over the years. Other BEN studies suggest that β2-microglobulin may be the first tubular protein excreted in increased amounts.16.Hall P. Vasilevic M. Beta2-microglobulin excretion as an index of renal tubular disorders with special reference to endemic Balkan Nephropathy.J Lab Clin Med. 1973; 81: 897-904PubMed Google Scholar, 17.Karlsson F. Lenkei R. Urinary excretion of albumin and beta 2-microglobulin in a population from an area where Balkan Nephropathy is endemic.Scand J Clin Lab Invest. 1977; 37: 169-173PubMed Google Scholar, 18.Hrabar A. Aleraj B. Ceovic S. et al.Beta 2-microglobulin studies in endemic Balkan Nephropathy.Kidney Int Suppl. 1991; 34: S38-S40PubMed Google Scholar, 19.Stefanovic V. Mitic-Zlatkovic M. Cukuranovic R. et al.Beta 2-microglobulin in patients with Balkan Nephropathy and in healthy members of their families.Kidney Int Suppl. 1991; 34: S21-S26PubMed Google Scholar, 20.Jevremovic I. Jankovic S. Radovanovic Z. et al.Beta2-microglobulinemia in a population exposed to Balkan Endemic Nephropathy: inferences from repeated cross-sectional studies.Kidney Int Suppl. 1991; 34: S35-S37PubMed Google Scholarβ2-microglobulin is filtered readily through the glomerular capillary wall and almost completely resorbed by cells in the proximal tubules, where it is metabolized.21.Berggard I. Bearn A. Isolation and properties of a low molecular weight beta2-microglobulin occuring in human biological fluids.J Biol Chem. 1968; 243: 4095-4103Abstract Full Text PDF PubMed Google Scholar, 22.Karlsson F. Groth T. Sege K. et al.Turnover of human beta2-microglobulin, the constant chain of HLA antigens.Eur J Clin Invest. 1980; 10: 293-300Crossref PubMed Scopus (113) Google Scholar, 23.Peterson P. Ervin P. Berggard I. Differentiation of glomerular tubular and normal proteinuria: determination of urinary excretion of beta2-microglobulin, albumin and total protein.J Clin Invest. 1969; 48: 1189-1198Crossref PubMed Scopus (451) Google Scholarβ2-microglobulin is a major constituent of tubular proteinuria: elevated urinary concentration of β2-microglobulin is a sensitive index of proximal renal tubular function.24.Schadurin G. Statius van Eps L. Beta2-microglobulin: its significance in the evaluation of renal function.Kidney Int. 1987; 32: 635-641Abstract Full Text PDF PubMed Scopus (218) Google Scholar The aim of this study was to determine whether clinical markers of BEN are altered in adult offspring of BEN patients compared with non-BEN offspring. We hypothesized that, controlling for confounders, in as yet unaffected adult offspring of BEN families:Kidney length and minimal cortex width are reduced.CCR is lower.Excretion of total protein, albumin and β2-microglobulin is higher. Of 122 eligible BEN offspring, 102 participated (83.6%), and of 120 control subjects, 99 took part (82.5%). All cases and controls resided in Vratza, Bistretz, or Beli Izvor, but not all were born in these settlements (Table 1). Ages ranged from 30 to 73 years. The mean age of BEN offspring was 49 years, and for the control group, 47 years. For gender and the three age groups, the proportion of BEN offspring and controls did not differ statistically. BEN offspring included more current smokers (39.2 vs 34.4%) and ex-smokers (22.6 vs 12.1%), and more BEN offspring had a history of hypertension (22.6 vs 5.1%). Kidney stones and pyelonephritis were more often reported by BEN offspring – 10.8 vs 4.0 and 10.8 vs 0%, respectively. Of the 11 participants who had a history of pyelonephritis, eight are women and three are men. We adjusted for pyelonephritis in linear regression analyses (Table 3). Pyelonephritis was of no importance for kidney length and cortex width, or CCR (Cockcroft and Gault). However, a history of pyelonephritis was weakly associated with total protein excretion in urine (P=0.059) and with albumin excretion (P=0.029), and there was no association with β2-microglobulin.Table 1Characteristics of the sampleAdult offspring of BEN patients, n=102Adult offspring of control patients, n=99n%n%Gender Men5150.04747.5 Women5150.05252.5Age (years) 30–391716.72323.2 40–545553.94848.5 55 plus3029.42828.3Place of birthBEN village9695.07272.7Smoking status Current smoker4039.23434.4 Ex-smoker2322.61212.1 Non-smoker3938.25353.5Other urinary tract diseases Kidney cancer22.000 Kidney stones1110.844.0 Pyelonephritis1110.800 Hydronephrosis1100 Cystitis43.955.1Diabetes mellitus87.866.1Hypertension2322.655.1Parental history of BEN Mother3938.200 Father2625.500 Both3736.300Parental history of other kidney diseases Mother109.822.0 Father109.800BEN, Balkan Endemic Nephropathy. Open table in a new tab BEN, Balkan Endemic Nephropathy. Diabetes mellitus was equally frequent among both groups. A parental history of other kidney diseases (other than BEN) was more frequent in BEN offspring. Table 2 describes the non-adjusted mean and median, and the 5 and 95% confidence limits of the clinical markers in BEN and non-BEN offspring. Table 3 provides the adjusted (geometric) mean values of the clinical markers that were found to be significantly different in offspring of BEN and non-BEN parents. Table 3 focuses on the differences between no parental history, maternal history, paternal history or both parents with a history of BEN.Table 2Distributions of clinical markers in BEN and non-BEN offspringAdult offspring of BEN patientsAdult offspring of control patientsVariableNMean or medianaFor kidney length and cortex width, we used the mean, for other markers the median, since these were not normally distributed.5% value95% valueNMean or medianaFor kidney length and cortex width, we used the mean, for other markers the median, since these were not normally distributed.5% value95% valueLength of the kidney (mm)102116.5102.6126.599119.4109.5131.7Minimal cortex width (mm)10215.311.518.09915.812.019.0Creatinine in serum (μmol/l)10268.355.092.99971.049.996.0Creatinine clearance (Cockcroft and Gault) (ml/min per 1.73m2)100111.270.5156.599103.972.0168.7Total urine protein (mg/g creatinine)102181.129.61839.399101.114.91120.2Urine albumin (mg/g creatinine)10223.96.05390.39916.51.8343.0β2-microglobulin (μg/g creatinine)10199.044.61350.259988.9940.63346.59BEN, Balkan Endemic Nephropathy.a For kidney length and cortex width, we used the mean, for other markers the median, since these were not normally distributed. Open table in a new tab Table 3Adjusted means and their 5–95% values of the kidney and urinary markersaAdjusted for sex, place of birth (BEN and non-BEN village), current place of residence (Vratza, Bistretz, Beli Izvor), age, history of smoking (current, ex- and non-smokers), diabetes, hypertension, and pyelonephritis.Kidney length (mm)Minimal width of the kidney cortex (mm)Creatinine clearance (Cockcroft and Gault) (ml/min per 1.73 m2)Total urine protein (mg/g creatinine)Urine albumin (mg/g creatinine)β2-microglobulin (μg/g creatinine)nMean5–95%Mean5–95%Geom. mean5–95%Geom. mean5–95%Geom. mean5–95%Geom. mean5–95%Parental BEN None99119.2116.0–122.315.814.8–16.7111.199.1–123.2177.1102.6–305.725.113.7–45.886.962.3–121.1 Father26119.1115.5–122.615.114.0–16.1111.697.9–125.2279.5150.7–518.525.012.6–49.4102.870.6–149.7 Mother39113.8*P≤0.05.110.7–116.815.214.4–16.110796.1–119.4354.7*P≤0.05.209.3–601.244.9*P≤0.05.25.1–80.5124.1*P≤0.05.90.0–171.0 Both37116.6113.4–119.715.214.3–16.1119.6107.5–131.6193.8112.7–333.335.619.5–64.8100.472.2–139.7BEN, Balkan Endemic Nephropathy.Geom. mean=geometric mean.* P≤0.05.a Adjusted for sex, place of birth (BEN and non-BEN village), current place of residence (Vratza, Bistretz, Beli Izvor), age, history of smoking (current, ex- and non-smokers), diabetes, hypertension, and pyelonephritis. Open table in a new tab BEN, Balkan Endemic Nephropathy. BEN, Balkan Endemic Nephropathy. Geom. mean=geometric mean. The mean length of the kidneys in offspring of BEN patients was 116.5 mm, whereas in the control offspring it was 119.4 mm (Table 2). The minimal cortex width was 15.3 mm in BEN offspring, whereas in the control offspring, it was 15.8 mm. After adjustment for confounders, the kidney length showed a substantial difference between the offspring of BEN patients and control offspring: 115.9 vs 118.8 mm, respectively (P=0.007, data not shown). With regard to parental history of BEN, having a mother with BEN was related to a significant reduction in kidney length (Table 3). Regarding minimal cortex width, after adjusting for confounders (sex, place of birth, current place of residence, age, history of smoking, diabetes, hypertension, and pyelonephritis), BEN offspring had a significantly smaller width compared to the controls: 15.2 vs 15.8 mm (P=0.049, data not shown). In the case of minimal cortex width, there was no difference owing to the parental history of BEN. For the CCR according to Cockcroft and Gault, we did not detect any statistically significant difference for the two risk factors under consideration: being an offspring from a BEN family or having a mother and/or father with BEN. Total urine protein excretion in BEN offspring was higher than in the control offspring with median values of 181.1 and 101.1 mg/g creatinine, respectively (Table 2). Table 3 shows that total urine protein excretion is statistically significantly higher among offspring whose mothers had BEN than offspring with no affected parent. The same tendency was found for urine albumin and β2-microglobulin excretion (Tables 2 and 3). The fact that both parents had BEN (indicator variable) had no further effect after maternal BEN was taken into consideration. We additionally investigated whether the various markers are correlated and thus measure comparable characteristics. Most of the markers are only weakly correlated; however, their correlation is significant (Table 4). CCR (Cockcroft and Gault) showed a significantly positive rank correlation with kidney length and cortex width.Table 4Spearman's rank correlation of the clinical markersVariableCCR (Cockcroft and Gault) (ml/min per 1.73 m2)β2-microglobulin (μg/g creatinine)Total urine protein (mg/g creatinine)Urine albumin (mg/g creatinine)Width of the kidney cortex (mm)Kidney length (mm)Creatinine in serum (μmol/l) Correlation coefficient-0.23-0.05-0.25-0.24-0.0020.15 Probability0.0010.470.00030.00060.980.036 n199201201201201201CCR (Cockcroft and Gault) (ml/min per 1.73 m2) Correlation coefficient-0.11-0.040.070.290.21 Probability0.130.550.34<0.00010.003 n199199199199199β2-microglobulin Correlation coefficient0.170.03-0.220.005 Probability0.0180.690.0010.95 n201201201201Total urine protein Correlation coefficient0.43-0.12-0.18 Probability<0.00010.0950.01 n201201201Urine albumin Correlation coefficient-0.08-0.28 Probability0.24<0.0001 n201201Kidney cortex width Correlation coefficient0.28 Probability 300 mg/g creatinine) was detected in 8.8% of BEN and 6.1% of the control offspring. The β2-microglobulin reference value of our laboratory goes up to 300 μg/l.28.Sims H. Miller L. Beta2-microglobulin in laboratory diagnosis.Lab Med. 1984; 15: 803-805Google Scholar In the literature, the upper clinical limit for β2-microglobulin is 0.2 mg/g creatinine.25.Clinical Laboratory Diagnostics Use and Assessment of Clinical Laboratory Results, Lothar Thomas. 1st English edn. TH-Books Verlagsgeselschaft, 1998: 1727Google Scholar In our sample, 5.9% of the BEN offspring and 1% of the control offspring had values above the laboratory reference value (300 μg/l). For serum creatinine, the laboratory reference interval was 62–124 μmol/l for men and 44–106 μmol/l for women. In the literature, clinical references are 74–110 μmol/l for men and 58–96 μmol/l for women.25.Clinical Laboratory Diagnostics Use and Assessment of Clinical Laboratory Results, Lothar Thomas. 1st English edn. TH-Books Verlagsgeselschaft, 1998: 1727Google Scholar A serum creatinine concentration of >110 μmol/l in men and >96 μmol/l in women was recorded in 1% of the BEN offspring and in 3% of the control offspring. The results from our study show that adult offspring of BEN families can be characterized by smaller kidney length as well as higher excretion of albumin, total protein and β2-microglobulin. The CCR values did not show a statistically significant difference between BEN offspring and the control group. Increased β2-microglobulin urine concentration is a nonspecific indicator of altered tubular function. Tubular damage is generally considered the primary pathological stage in BEN development. The increased β2-microglobulin excretion in clinically healthy members of families with BEN, found in this study, indicates that tubular damage may have an early adverse effect on kidney function in population groups at risk for BEN. Increased β2-microglobulin excretion along with increased albumin excretion have also been reported in other investigations of populations in BEN regions.17.Karlsson F. Lenkei R. Urinary excretion of albumin and beta 2-microglobulin in a population from an area where Balkan Nephropathy is endemic.Scand J Clin Lab Invest. 1977; 37: 169-173PubMed Google Scholar, 19.Stefanovic V. Mitic-Zlatkovic M. Cukuranovic R. et al.Beta 2-microglobulin in patients with Balkan Nephropathy and in healthy members of their families.Kidney Int Suppl. 1991; 34: S21-S26PubMed Google Scholar Increased protein excretion, including modest albuminuria, is a marker of tubulointerstitial lesions. The increased excretion of proteins points to possible early tubular lesions in the population at higher risk of BEN development. However, the early detection of increased albuminuria in clinically healthy persons can also be a sign for glomerular lesions. Thus, alteration of these parameters, compared to the control group, may be a sign that in addition to tubulointerstitial dysfunction, early glomerular dysfunction may have occurred in the offspring of BEN patients. We cannot rule out this possibility, and it would be consistent with a prior report of early glomerular lesions in BEN patients.29.Dimitrov T. Balkan Endemic Nephropathy. Bulgarian Academy of Sciences, Sofia1984: 190Google Scholar Regarding CCR (Cockcroft and Gault), we did not detect any significant differences when comparing offspring of BEN and non-BEN parents. Mitic-Zlatkovic et al.12.Mitic-Zlatkovic M. Cukuranovic R. Lecic N. Stefanovic V. Urinary creatinine excretion in children from families with Balkan endemic Nephropathy: evidence for genetic predisposition to the disease.Pathol Biol. 2000; 48: 554-557PubMed Google Scholar reported a reduced creatinine excretion, but Djukanovic et al.13.Djukanovic L. Bukvic D. Maric I. Creatininne clearance and kidney size in Balkan endemic nephropathy patients.Clin Nephrol. 2004; 61: 384-386Crossref PubMed Scopus (15) Google Scholar reported a higher CCR in BEN patients before the disease is manifested. Thus, there is not enough evidence to conclude whether CCR is altered before the clinical manifestation of BEN. The ultrasound examinations in our study revealed reduced kidney length and cortex width in the BEN offspring group compared to the controls. One of the most important diagnostic criteria of BEN is the decrease in size in both kidneys.15.Radovanovic Z. Sindic M. Polenakovic M. et al.Endemic Nephropathy. Zavod za udzbenike i nastavna sredstva, Belgrade2000: 447Google Scholar However, it is not clear when the process of symmetrical shrinkage begins. There are two different opinions about the time when kidney size starts to decrease: (1) along with the development of renal failure30.Radonic M. Radosevic Z. Clinical features of Balkan Endemic Nephropathy.Food Chem Toxicol. 1992; 30: 189-192Crossref PubMed Scopus (46) Google Scholar and (2) in the early stages with normal glomerular filtration rate.31.Trnacevic S. Halilbasic A. Ferluga D. et al.Renal function, protein excretion and pathology of Balkan Endemic Nephropathy. I. Renal function.Kidney Int Suppl. 1991; 34: S49-S51PubMed Google Scholar Our findings suggest that kidney length is shorter, and cortex width is smaller, before the development of renal failure. Thus, we support the assumption that the process of shrinkage may begin early in life, as reported by Djukanovic et al.13.Djukanovic L. Bukvic D. Maric I. Creatininne clearance and kidney size in Balkan endemic nephropathy patients.Clin Nephrol. 2004; 61: 384-386Crossref PubMed Scopus (15) Google Scholar The reported biochemical and morphological changes suggest that preclinical renal dysfunction, both tubular and possibly glomerular, seems to be characteristic of BEN offspring. Surprisingly, the differences in total kidney lengths, as well as protein, albumin and β2-microglobulin excretion, are statistically significant in relation to a maternal history of BEN, not a paternal one. The status of both parents having BEN did not contribute significantly once maternal history is taken into consideration. We did not find that birth in a BEN village was related to clinical markers in the offspring sample of BEN and non-BEN patients. This, in our opinion, indicates that the village environment during pregnancy may not be of importance. However, for all clinical markers, we found that the maternal history of BEN is more important than the paternal. To the best of our knowledge, BEN-related genetic polymorphisms are not based on sex chromosomes. The dominant maternal effect may, however, be initiated during the fetal period, a time window when the fetus is exposed to the environment, metabolism, kidney function and immune responses of the mother. A challenge of this finding is, however, that women in the childbearing age are not as yet diagnosed with BEN, as the disease typically manifests after the age of 50. As in every study, this investigation has strengths and limitations. We do not believe that the sample is affected by a selection bias because 83.6% of the eligible BEN offspring and 82.5% of the control subjects participated. A strength of this study is that the hospital registry in Vratza, established in 1963, provided documentation of BEN patients who had repeated clinical assessments. This registry facilitated the recruitment of well-defined groups of BEN offspring. However, offspring of BEN patients and offspring controls included a wide age range. From our results, we can conclude that BEN offspring have altered kidney function and length, but we cannot determine the age of earliest detection. It would be advantageous to investigate a birth cohort of BEN offspring of similar age. However, cohorts of more homogeneous age groups can best be achieved by transnational collaboration among the Balkan countries. Another strength of our study is that we were able to simultaneously determine markers of renal function and morphology. This will facilitate the future assessment of the clinical development of the disease. In conclusion, our data indicate that kidney function and size in the offspring of BEN patients are already reduced before manifestation of the disease. Future studies are warranted to determine the development of clinical markers and kidney morphology over time. Given a linear development, this information will facilitate backward extrapolation to determine the age of onset. The strong associations of maternal BEN with kidney function and size in their adult offspring is intriguing and has not been reported before. This finding indicates that future studies are needed to determine whether maternal conditions during pregnancy, at an age before the mother is diagnosed with BEN, may alter the development of the kidneys and their function in the newborn. This, in turn, may provide a new explanation of the family disposition found for BEN. Maternal influences during pregnancy may either directly alter the offspring's kidney (direct delayed effect) or may initiate gene expression that results in altered function (epigenetic effect). In the period of October 2003 to April 2004, we recruited adult offspring (102 study subjects) whose father or mother were included in the Vratza hospital registry of BEN patients in 2001 and resided in three communities (Vratza, Bistretz and Beli Izvor, Bulgaria). An equal-sized control group of adult offspring of non-BEN parents (99 study subjects) was enrolled in the study. Study subjects of both groups were matched according to gender and 10-year age groups. All participants provided written consent through a procedure approved by the Institutional Review Board (human–subject research committee) of the National Center of Public Health Protection, Sofia, Bulgaria. We conducted face-to-face interviews of all participants either in the hospital or by visiting them in their home villages. The standardized questionnaire asked for their place of residence, type of water supply, diet, smoking, and drinking habits, medical symptoms, family history of BEN, other kidney diseases, and kidney tumors, and occupational history. The physical examination was performed by a physician with board certification in internal medicine and nephrology. It was aimed at assessing the general health of the study subjects and to reveal symptoms of BEN and/or other internal diseases, which could influence the results of laboratory tests. The examination was to determine the status of the pulmonary, cardiovascular, and urinary systems; gastrointestinal tract and liver; spleen and lymph nodes; thyroid gland; and skin and visible mucous membranes. Blood pressure was measured according to standards set by the World Health Organization.32.WHO World Health Organization – International Society of Hypertension guidelines for the management of hypertension. 1999http://new.euromise.org/mgt/who1999/who1999.htmlGoogle Scholar Ultrasound investigations of both kidneys took 20–30 min. The patient was investigated lying on both the left and right sides. After finding a suitable image, the measurements were taken. The longest dimension of the kidney was determined. Thickness of the kidney parenchyma (in the thinnest or minimal part), the parenchyma structure and the relationship of parenchyma and pyelon were measured. Information also included the location, size, and morphology (cysts, stones and tumors) of the kidneys. The images were saved electronically for future reference. Figure 1 shows the time line of the urine and blood collections. Urine samples of the second urination in the morning were collected at the laboratory (Figure 1), at the beginning of the 4-h sampling period. In this urine sample, relative density, glucose, protein, bilirubin, urobilinogen, ketones and blood were measured by strip tests and urine sediment was examined by microscope. After providing the second urine sample of the morning, the study subjects had to drink 0.5 l of mineral water. From that moment on, they collected all their urine in polyethylene containers, which were then refrigerated. At the end of the 4-h collection period, the urine volume was measured and recorded, and a sample was frozen at -20°C. Total protein and albumin were measured in the 4-h urine samples. In addition, study subjects were asked to collect their urine at the end of the first hour. This sample and any other urine collected during the first hour were used for the determination of β2-microglobulin. The pH of the urine was measured by pH-indicator paper strips (Merck, NJ, USA). If the pH was lower than 7, a portion of the urine was alkalized by adding droplets of 1 N NaOH until a pH equal to or higher than 7 and lower than 8 was reached. A sample from the alkalized urine was frozen at -20°C for the determination of β2-microglobulin. The rest of the urine was returned to the 4-h urine container. The volume of the urine sample for β2-microglobulin testing was taken into account when the 4-h urine volume was recorded. Urine creatinine was measured twice: in the 1- and 4-h urine samples. All clinical markers measured in urine were calculated as amount per gram creatinine. Venous blood for the determination of serum creatinine was taken in the middle of the 4-h urine collection period. Blood was centrifuged, serum was separated and samples were frozen at -20°C. We determined total protein, albumin, β2-microglobulin and creatinine in urine, creatinine and urea in serum, as well as CCR. Total protein and albumin were analyzed in the 4-h urine samples. The preparation of albumin was carried out using latex immunoturbidimetric assays (Cat. No. 31924; BioSystems S.A., Spain). The following samples were prepped with commercially available kits: serum and urine creatinine (Jaffe photometric method, Cat. No. 0060); total urine protein (pyrogallol red photometric method, Cat. No. 0074) and serum urea (urease/glutamate dehydrogenase method, Cat. No. 4077). All kits were provided by Giesse Diagnostics, Italy. Total protein, albumin, urine creatinine, serum creatinine and serum urea were quantified with the photometric colorimetric optical system (Roche Cobas-Mira Plus CC chemistry analyzer; F Hoffmann-La Roche Ltd, Switzerland). The urine collected during the first hour of the 4-h period was analyzed for β2-microglobulin using a chemiluminescent immunoassay (Cat. No. LKBM1; DPC, Los Angeles, CA, USA) with the Immulite analyzer (DPC). Internal laboratory controls were performed with reference materials analyzed parallel to each series of samples. For the control of β2-microglobulin, three-level control samples were provided by DPC, USA (Cat. No. BMCM). Control serum (two levels), produced by F Hoffmann-La Roche Ltd, Switzerland, was used for quality control of creatinine and urea measurements (Precinorm U, Cat. No. 171735; Precipath U, Cat. No. 171760). Control urine (normal level) was tested in parallel with the analyses of total protein, albumin and creatinine in urine (Cat. No. AU2352; Randox, Crumlin, UK). CCR was determined from serum creatinine values by using Cockcroft and Gault formula. Specifically, it was calculated in males as CCR Cockcroft and Gault ((140-age) × (body weight in kg))/(72 × serum creatinine) expressed in (ml/min per 1.73 m2).33.Cockcroft D. Gault M. Prediction of creatinine clearance from serum creatinine.Nephron. 1976; 16: 31-41Crossref PubMed Scopus (13103) Google Scholar Further, for women, a correction factor of 0.85 was used. Whenever clinical markers were normally distributed, we calculated means and their 5 and 95% confidence limits. When distributions were not normal, we used the median or conducted log transformations and used geometric means and their confidence limits. To answer the primary question, whether offspring of BEN parents had altered kidney morphology and/or function, we grouped the parental disease status: first, the overall status of being an offspring from a BEN family, and second, the specific status of mother, father, or both affected. In all comparisons, the reference group was comprised only of non-BEN offspring. In addition, parents in either group could have had other kidney disorders. As it has been reported10.Stefanovic V. Mitic-Zlatkovic M. Cukuranovic R. Vlahovic P. Increased urinary protein excretion in children from families with Balkan Endemic Nephropathy.Nephron Clin Pract. 2003; 95: 116-120Crossref Scopus (17) Google Scholar, 11.Stefanovic V. Cukuranovic R. Mitic-Zlatkovic M. Hall P.W. Increased urinary albumin excretion in children from families with Balkan Nephropathy.Pediatr Nephrol. 2002; 17: 913-916Crossref PubMed Scopus (15) Google Scholar, 12.Mitic-Zlatkovic M. Cukuranovic R. Lecic N. Stefanovic V. Urinary creatinine excretion in children from families with Balkan endemic Nephropathy: evidence for genetic predisposition to the disease.Pathol Biol. 2000; 48: 554-557PubMed Google Scholar that offspring from villages that were affected by occurrences of BEN had altered clinical markers, we statistically controlled for place of birth. Whenever an offspring was born in Vratza, Bistretz, or Beli Izvor, place of birth was labeled 'BEN village'; all others were 'non-BEN village'. Other confounders used in all explanatory models include age, gender, history of smoking, diabetes, and hypertension. For descriptive purposes, age was grouped into three categories, 30–39, 40–54, and 55 years and older. In the statistical analyses, we used age as a continuous variable. For smoking, we distinguished current smoker, ex-smoker, and non-smoker. Other non-BEN urinary tract disorders were classified as kidney cancer, kidney stones, pyelonephritis, hydronephrosis and cystitis. A participant was categorized as having a history of hypertension if (a) increased values for blood pressure were detected for the first time in the last 12 months, (b) higher blood pressures were measured at several occasions, or (c) a physician diagnosed hypertension. We used linear regression analyses (procedure generalized linear model (PROC GLM) of the Statistical Analysis System, SAS),34.SAS-Institute SAS/STAT Software. SAS Institute Inc., Cary, NC2000Google Scholar adjusting for confounders, to determine the effect of parental status (BEN offspring and status of the parents). To compare the effect of a parental history of BEN, we estimated the adjusted geometric means of the clinical markers in groups with no BEN parent, BEN mother, BEN father, or both parents with BEN. In addition, to compare to what extent the various markers are associated, we estimated Spearman's rank correlations. All statistical analyses were performed using SAS 8.0.34.SAS-Institute SAS/STAT Software. SAS Institute Inc., Cary, NC2000Google Scholar The research was supported by NIH Research Grant No. R01 TW006192 funded by the Fogarty International Center and the National Institute of Environmental Health Sciences, National Institutes of Health, USA, awarded to the National Center of Public Health Protection (formerly National Center of Hygiene, Medical Ecology, and Nutrition), Sofia, Bulgaria.
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