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Early diagnosis of endemic nephropathy
Clinica Chimica Acta 297 (2000) 85–91 www.elsevier.com / locate / clinchim
Early diagnosis of endemic nephropathy ˇ ˇ´ * Dubravka Cvoriscec Clinical Institute of Laboratory Diagnosis, Zagreb University School of Medicine, ˇ ´ Clinical Hospital Center, Kispaticeva 12, 10000 Zagreb, Croatia Received 20 September 1999; received in revised form 1 December 1999; accepted 10 February 2000
Abstract Urinary a 1 -microglobulin as a tubular marker and albumin as a glomerular marker were ˇ Croatia, previously measured in 373 subjects living in a typical endemic village of Kaniza, classified as diseased, suspect, ‘at risk’ and others according to the criteria used for the diagnosis of endemic nephropathy. Based on the excretion pattern of a 1 -microglobulin and albumin and its extent, significant tubular with significant glomerular proteinuria was found in seven diseased subjects. Significant tubular proteinuria with slight glomerular proteinuria was found in one diseased subject, while another diseased subject had slight tubular with significant glomerular proteinuria. Significant tubular with significant glomerular proteinuria was found in only one suspect subject. Also, significant tubular proteinuria was established in one other subject. 2000 Elsevier Science B.V. All rights reserved. Keywords: Endemic nephropathy; Tubulointerstitial lesion; Excretion pattern of a 1 -microglobulin and albumin
1. Introduction Endemic nephropathy is a chronic tubulointerstitial renal disease. Its etiology and pathogenesis are still unknown. Also, its association with papillary transitional cell tumors of the renal pelvis and ureters is unclear [1–3]. One of the early signs of endemic nephropathy is proteinuria, which is mild, sometimes *Tel. / fax: 1 385-1-212-079. 0009-8981 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0009-8981( 00 )00236-9
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ˇ ˇ´ / Clinica Chimica Acta 297 (2000) 85 – 91 D. Cvoriscec
intermittent, and characterized by increased excretion of low molecular weight proteins such as b 2 -microglobulin, retinol-binding protein, lysozyme, immunoglobulin light chains and a 1 -microglobulin, and normal or slightly increased excretion of albumin. During the past three decades it has been detected by sulphosalicylic acid test or test strip and low molecular weight test (low molecular weight test is a semiquantitative radial immunodiffusion test for the detection of b 2 -microglobulin and other low molecular weight proteins in native urine) or b 2 -microglobulin test [4–7]. A few years ago, a 1 -microglobulin was recommended as the most reliable marker of tubular dysfunction and is now included together with albumin and total proteins, immunoglobulin G and a 2 -macroglobulin in a new diagnostic strategy for detection and differentiation of proteinuria in various renal diseases [8–10]. The aim of this study was to evaluate the value of urinary a 1 -microglobulin as a tubular marker and albumin as a glomerular marker in the detection of proteinuria in subjects living in an area of endemic nephropathy.
2. Materials and methods a 1 -Microglobulin and albumin were measured in random urine samples of ˇ The subjects were 373 subjects living in the typical endemic village of Kaniza. classified into four groups according to the criteria for the diagnosis of endemic nephropathy [11]: diseased, suspect, subjects ‘at risk’ and other subjects. Subjects were considered diseased if they had proteinuria, i.e. sulphosalicylic acid test . 0.2 g / l or test strip positive ( . 0.3 g / l) and / or low molecular weight test positive ( . 1 mg / l) or b 2 -microglobulin test positive ( . 0.3 mg / l), anemia (hemoglobin , 120 g / l in males and , 113 g / l in females) and plasma creatinine . 132.6 mmol / l, or proteinuria, anemia and a positive family history of endemic nephropathy, or all four together, provided that no other renal disease was present. Subjects were considered suspect if they had proteinuria and anemia without clinical and laboratory signs of other renal disease, or proteinuria and a positive family history of endemic nephropathy. Subjects with a positive family history of endemic nephropathy but without clinical and laboratory signs of renal disease were considered to be ‘at risk’. All other ˇ with a negative family history of endemic nephropathy subjects living in Kaniza and without laboratory signs of renal disease were considered as others. Urinary a 1 -microglobulin and albumin concentrations were measured by latex-enhanced nephelometry on a Behring Nephelometer II (Behring Diagnostics GmbH, Marburg, Germany). Protein Standard Urine Concentrate was used for construction of a reference curve for a 1 -microglobulin. To check the plausibility of single protein measurements, urinary total proteins were measured by the pyrogallol red-molybdate method on an Olympus AU 600 analyzer
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(Mishima Olympus Co., Japan). Urinary creatinine concentration was measured by the Jaffe method on the same analyzer. The concentrations of urinary a 1 -microglobulin and albumin were expressed per gram of urinary creatinine. The excretion values of 15 mg / g creatinine for a 1 -microglobulin and 30 mg / g creatinine for albumin were used as the upper normal limits [12]. The relation of the a 1 -microglobulin excretion value to the albumin excretion value was presented on logarithmic coordinates, with albumin on the x-axis and a 1 microglobulin on the y-axis [8]. The upper normal limits were represented as dashed lines. Such a diagram of a single a 1 -microglobulin and albumin excretion pattern allows visual interpretation of proteinuria and its connection with different kinds of nephropathies based on knowledge of the physiology of protein filtration and reabsorption, and its specific handling by the kidney in tubulointerstitial and glomerular nephropathies. The extent of tubular and glomerular proteinuria was described as slight when a 1 -microglobulin and albumin were below 50 and 100 mg / g creatinine, and as significant when a 1 -microglobulin and albumin were above 50 and 100 mg / g creatinine, respectively [9].
3. Results Table 1 presents the age and gender distribution, and the frequency of pathological findings in 373 subjects from the area of endemic nephropathy grouped into diseased subjects, suspect subjects, subjects ‘at risk’ and other subjects. An excretion value of a 1 -microglobulin . 15 mg / g creatinine was found in 90.0% of diseased subjects, in 41.6% of suspect subjects, in 7.1% of subjects ‘at risk’ and in 8.9% of other subjects. An albumin excretion value . 30 mg / g creatinine was found in 100.0% of diseased subjects, in 33.3% of suspect subjects, in 4.7% of subjects ‘at risk’ and in 7.5% of other subjects. The distribution of a 1 -microglobulin and albumin excretion patterns in diseased subjects, suspect subjects, subjects ‘at risk’ and other subjects is presented in Fig. 1. Table 1 Age and gender distribution, and frequency of pathological findings in 373 subjects from an area of endemic nephropathy Group
N (m / f)
Age (years)
a 1 -Microglobulin . 15 mg / g creatinine
Albumin . 30 mg / g creatinine
Diseased Suspect ‘At risk’ Others
10 (4 / 6) 24 (11 / 13) 127 (57 / 70) 212 (104 / 108)
46–75 27–77 4–74 4–74
90.0% 41.6% 7.1% 8.9%
100.0% 33.3% 4.7% 7.5%
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Fig. 1. Urinary excretion of a 1 -microglobulin in relation to albumin in 373 subjects from the area of endemic nephropathy.
In subjects with an established diagnosis of endemic nephropathy, tubular proteinuria with glomerular proteinuria was found in 9 / 10 subjects. In eight subjects the excretion value of a 1 -microglobulin was . 100 mg / g creatinine, and in one subject it was , 50 mg / g creatinine. In one subject the glomerular proteinuria was slight, and in others it was significant, but the albumin excretion value never exceeded 500 mg / g creatinine. An albumin excretion value of only 31 mg / g creatinine was found in one subject previously diagnosed as a diseased subject. In suspect subjects, the a 1 -microglobulin excretion values , 15 mg / g creatinine and albumin excretion values , 30 mg / g creatinine, i.e. physiological proteinuria, were found in 11 / 24 subjects. Slight tubular proteinuria was found in 5 / 24 suspect subjects. In 5 / 24 suspect subjects, tubular proteinuria with glomerular proteinuria was found. An excretion value of a 1 -microglobulin . 50 mg / g creatinine was found in only one of them, while an albumin excretion value of . 100 mg / g creatinine was found in four suspect subjects. Glomerular
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proteinuria was found in another 3 / 24 suspect subjects and was significant in one of them. Among subjects ‘at risk’, excretion values of a 1 -microglobulin and albumin below 15 and 30 mg / g creatinine, i.e. physiological proteinuria, were found in 114 / 127 subjects. Slight tubular proteinuria was established in 7 / 127 subjects ‘at risk’, and slight tubular proteinuria with slight glomerular proteinuria in 2 / 127 subjects ‘at risk’. Glomerular proteinuria was found in 4 / 127 subjects ‘at risk’. It was significant in one of them. In other subjects, physiological proteinuria, i.e. excretion values of a 1 microglobulin and albumin below 15 and 30 mg / g creatinine, was found in 181 / 212 subjects. Significant tubular proteinuria was found in 1 / 212 of other subjects, and slight tubular proteinuria in 14 / 212 of other subjects. In 4 / 212 of other subjects, slight tubular proteinuria with slight glomerular proteinuria was found. Glomerular proteinuria was found in 12 / 212 of other subjects, in six of them it was significant and in the other six it was slight.
4. Discussion The diagnostics of endemic nephropathy and detection of subjects suffering from endemic nephropathy in population screening are rather difficult. In the phases of azotemia or uremia it is difficult to differentiate endemic nephropathy from other chronic tubulointerstitial nephropathies, and a specific test for the diagnosis of endemic nephropathy is not available. Numerous investigations of endemic nephropathy including measurements of single proteins in urine, and determinations of the type of proteinuria by zone electrophoresis on acetate cellulose and immunoelectrophoresis on agarose as well as SDS-electrophoresis on polyacrylamide have shown that excretion of low molecular weight proteins such as b 2 -microglobulin, retinol-binding protein, lysozyme, immunoglobulin light chains and a 1 -microglobulin is increased in the early phase of the disease [5–7,13–17]. In the later phase, when glomeruli are also affected, mixed tubular and glomerular proteinuria is present. This finding has resulted in the opinion that early diagnosis could be considered if a tubular type of proteinuria is found in subjects living in the endemic area. Conventional tests such as the sulphosalicylic acid test and test strip are of limited value in detection of proteinuria in endemic nephropathy, especially in the early phase of the disease. These tests may be negative in subjects with endemic nephropathy and, on the contrary, they may be positive in subjects without endemic nephropathy. The LMW test was found positive in more than 70% of cases with a clinical diagnosis of endemic nephropathy [5]. b 2 Microglobulin, which is a sensitive marker of tubular dysfunction and was used for the diagnosis of endemic nephropathy, was found positive in 75% of subjects
90
ˇ ˇ´ / Clinica Chimica Acta 297 (2000) 85 – 91 D. Cvoriscec
with an established diagnosis of endemic nephropathy when 0.3 mg / l was used as the upper normal limit [18]. Preliminary results obtained by measurement of a 1 -microglobulin as the most reliable marker of tubular dysfunction in subjects living in the endemic nephropathy area showed values of a 1 -microglobulin . 20 mg / l, used as the upper normal limit, in 85.7% diseased, 50.0% suspect, 10.2% at risk, and in 8.1% of other subjects. The a 1 -microglobulin determination was performed by radial immunodiffusion [12]. In this study, excretion patterns of a 1 -microglobulin and albumin were investigated in subjects living in an area of endemic nephropathy. Tubular proteinuria with glomerular proteinuria was found in 9 / 10 subjects with an established diagnosis of endemic nephropathy. The extent of excretion of a 1 -microglobulin and albumin was in the ranges characteristic for tubulointerstitial diseases, which is consistent with results of a previous study of proteinuria in endemic nephropathy. In one subject diagnosed as an endemic nephropathy case, tubular proteinuria was not found. It seems that the diagnosis of endemic nephropathy was incorrect in this subject, and should be revised. Among suspect subjects, tubular proteinuria alone or with glomerular proteinuria was found in 10 / 24 subjects, and in only one suspect subject was tubular proteinuria significant. Slight tubular proteinuria with or without slight glomerular proteinuria was also found in 9 / 127 subjects ‘at risk’, and in 18 / 212 other subjects. Among other subjects, one case of significant tubular proteinuria was found. Such slight tubular proteinuria with or without slight glomerular proteinuria found in suspect, at risk and other subjects could be interpreted as a latent, early subclinical phase of endemic nephropathy providing that other possible causes of elevated a 1 -microglobulin and albumin excretion are excluded. Therefore, the retesting of a 1 -microglobulin and albumin under standardized conditions together with measurement of other protein markers for differentiation of proteinuria should be performed in the second step of laboratory examination. Based on the results of this study, simultaneous determination of a 1 -microglobulin as a tubular marker and albumin as a glomerular marker appears to be a sensitive screening procedure for the detection of proteinuria in subjects living in an endemic nephropathy area and allows more reliable detection of suspect cases of endemic nephropathy. The definitive clinical diagnosis of a single patient has to be established after an extensive clinical and laboratory examination of suspect subjects.
References ˇ ˇ ´ Z, Zupanic ´ V. Endemic nephropathy in Yugoslavia. In: Mostofi FK, [1] Radonic´ M, Radosevic Smith DE, editors, The kidney, Baltimore: Williams and Wilkins, 1966, pp. 503–22.
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ˇ ´ Z. Clinical features of Balkan endemic nephropathy. Food Chem [2] Radonic´ M, Radosevic Toxicol 1992;30:89–92. ˇ ˇ ˇ´ D, Ceovic ´ S, Borso ˇ G, Stavljenic´ Rukavina A. Endemic nephropathy in Croatia. [3] Cvoriscec Clin Chem Lab Med 1998;36:271–7. ˇ ˇ ´ Z, Radonic´ M, Keler-Bacoka ˇ ˇ ´ N, Ceovic ´ S et al. Report on [4] Hrabar A, Radosevic M, Mihelcic investigation of endemic nephropathy. Phase I. Public Law 1971–1979;83:480. [5] Radonic´ M. b 2 -Microglobulin in Balkan endemic nephropathy. Pathol Biol 1978;26:317–20. ˇ ˇ ´ S, Cvoriscec ˇ´ D, Vacca C, Hall PW. A fifteen year cohort based [6] Hrabar A, Aleraj B, Ceovic evaluation of b 2 -microglobulin as an early sign of Balkan endemic nephropathy. Kidney Int 1991;40(Suppl 34):41–3. ˇ ˇ ´ S, Cvoriscec ˇ´ D, Vacca C, Hall PW. b 2 -Microglobulin studies in [7] Hrabar A, Aleraj B, Ceovic endemic Balkan nephropathy. Kidney Int 1991;40(Suppl 34):38–40. ¨ [8] Hofmann W, Rossmuller B, Guder WG, Edel HH. A new strategy for characterizing proteinuria and haematuria from a single pattern of defined proteins in urine. Eur J Clin Chem Clin Biochem 1992;30:707–12. [9] Ivandic M, Hofmann W, Guder WG. Development and evaluation of a urine protein expert system. Clin Chem 1996;42:1214–22. [10] Guder WG, Ivandic´ M, Hofmann W. Physiopathology of proteinuria and laboratory diagnostic strategy based on single protein analysis. Clin Chem Lab Med 1998;36:935–9. [11] World Health Organization. Memorandum. The endemic nephropathy of South-Eastern Europe. Bull WHO 1965;32:441–8. ˇ ˇ´ D. Urinary a 1 -microglobulin levels in surveys of [12] Itoh Y, Cooper EH, Stavljenic´ A, Cvoriscec Balkan endemic nephropathy. Biomed Pharmacother 1986;40:341–4. ˇ ´ Z, Traeger J, Radonic´ M, Manuel Y, Revillard JP. Etude electrophoretique ´ ´ [13] Radosevic de la ´ ´ ´ proteinurie de 31 sujets croates vivant en pays de nephropatie endemique. J Urol Nephrol Paris 1968;74:703–10. ˇ ˇ ´ Z, Keler-Bacoka ˇ ˇ ´ N, Ceovic ´ S. Electrophoretic and [14] Radonic´ M, Radosevic M, Mihelcic immunoelectrophoretic analysis of urine in patients and healthy subjects coming from areas of endemic nephropathy. Lijecˇ Vjesn 1975;97:547–55. ˇ ˇ ˇ´ D, Radonic´ M, Ceovic ´ S, Aleraj B. Characteristics of proteinuria in endemic [15] Cvoriscec nephropathy. J Clin Chem Clin Biochem 1983;21:569–71. ˇ ˇ´ D, Stavljenic´ A, Radonic´ M. Characteristics of proteinuria in Balkan endemic [16] Cvoriscec nephropathy. Acta Pharm Jugosl 1985;35:127–32. ˇ ˇ´ D, Stavljenic´ A, Radonic´ M. Relationship between tubular and Tamm–Horsfall [17] Cvoriscec proteinuria in Balkan endemic nephropathy. Nephron 1986;42:152–5. ˇ ˇ´ D, Stavljenic´ Rukavina A. Laboratory diagnosis of endemic nephropathy. In: [18] Cvoriscec ˇ ˇ ˇ´ D, Ceovic ´ S, Stavljenic´ Rukavina A, editors, Endemic nephropathy in Croatia, Cvoriscec Zagreb: Academia Croatica Scientiarum Medicarum, 1996, pp. 77–86.
Early diagnosis of endemic nephropathy ˇ ˇ´ * Dubravka Cvoriscec Clinical Institute of Laboratory Diagnosis, Zagreb University School of Medicine, ˇ ´ Clinical Hospital Center, Kispaticeva 12, 10000 Zagreb, Croatia Received 20 September 1999; received in revised form 1 December 1999; accepted 10 February 2000
Abstract Urinary a 1 -microglobulin as a tubular marker and albumin as a glomerular marker were ˇ Croatia, previously measured in 373 subjects living in a typical endemic village of Kaniza, classified as diseased, suspect, ‘at risk’ and others according to the criteria used for the diagnosis of endemic nephropathy. Based on the excretion pattern of a 1 -microglobulin and albumin and its extent, significant tubular with significant glomerular proteinuria was found in seven diseased subjects. Significant tubular proteinuria with slight glomerular proteinuria was found in one diseased subject, while another diseased subject had slight tubular with significant glomerular proteinuria. Significant tubular with significant glomerular proteinuria was found in only one suspect subject. Also, significant tubular proteinuria was established in one other subject. 2000 Elsevier Science B.V. All rights reserved. Keywords: Endemic nephropathy; Tubulointerstitial lesion; Excretion pattern of a 1 -microglobulin and albumin
1. Introduction Endemic nephropathy is a chronic tubulointerstitial renal disease. Its etiology and pathogenesis are still unknown. Also, its association with papillary transitional cell tumors of the renal pelvis and ureters is unclear [1–3]. One of the early signs of endemic nephropathy is proteinuria, which is mild, sometimes *Tel. / fax: 1 385-1-212-079. 0009-8981 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0009-8981( 00 )00236-9
86
ˇ ˇ´ / Clinica Chimica Acta 297 (2000) 85 – 91 D. Cvoriscec
intermittent, and characterized by increased excretion of low molecular weight proteins such as b 2 -microglobulin, retinol-binding protein, lysozyme, immunoglobulin light chains and a 1 -microglobulin, and normal or slightly increased excretion of albumin. During the past three decades it has been detected by sulphosalicylic acid test or test strip and low molecular weight test (low molecular weight test is a semiquantitative radial immunodiffusion test for the detection of b 2 -microglobulin and other low molecular weight proteins in native urine) or b 2 -microglobulin test [4–7]. A few years ago, a 1 -microglobulin was recommended as the most reliable marker of tubular dysfunction and is now included together with albumin and total proteins, immunoglobulin G and a 2 -macroglobulin in a new diagnostic strategy for detection and differentiation of proteinuria in various renal diseases [8–10]. The aim of this study was to evaluate the value of urinary a 1 -microglobulin as a tubular marker and albumin as a glomerular marker in the detection of proteinuria in subjects living in an area of endemic nephropathy.
2. Materials and methods a 1 -Microglobulin and albumin were measured in random urine samples of ˇ The subjects were 373 subjects living in the typical endemic village of Kaniza. classified into four groups according to the criteria for the diagnosis of endemic nephropathy [11]: diseased, suspect, subjects ‘at risk’ and other subjects. Subjects were considered diseased if they had proteinuria, i.e. sulphosalicylic acid test . 0.2 g / l or test strip positive ( . 0.3 g / l) and / or low molecular weight test positive ( . 1 mg / l) or b 2 -microglobulin test positive ( . 0.3 mg / l), anemia (hemoglobin , 120 g / l in males and , 113 g / l in females) and plasma creatinine . 132.6 mmol / l, or proteinuria, anemia and a positive family history of endemic nephropathy, or all four together, provided that no other renal disease was present. Subjects were considered suspect if they had proteinuria and anemia without clinical and laboratory signs of other renal disease, or proteinuria and a positive family history of endemic nephropathy. Subjects with a positive family history of endemic nephropathy but without clinical and laboratory signs of renal disease were considered to be ‘at risk’. All other ˇ with a negative family history of endemic nephropathy subjects living in Kaniza and without laboratory signs of renal disease were considered as others. Urinary a 1 -microglobulin and albumin concentrations were measured by latex-enhanced nephelometry on a Behring Nephelometer II (Behring Diagnostics GmbH, Marburg, Germany). Protein Standard Urine Concentrate was used for construction of a reference curve for a 1 -microglobulin. To check the plausibility of single protein measurements, urinary total proteins were measured by the pyrogallol red-molybdate method on an Olympus AU 600 analyzer
ˇ ˇ´ / Clinica Chimica Acta 297 (2000) 85 – 91 D. Cvoriscec
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(Mishima Olympus Co., Japan). Urinary creatinine concentration was measured by the Jaffe method on the same analyzer. The concentrations of urinary a 1 -microglobulin and albumin were expressed per gram of urinary creatinine. The excretion values of 15 mg / g creatinine for a 1 -microglobulin and 30 mg / g creatinine for albumin were used as the upper normal limits [12]. The relation of the a 1 -microglobulin excretion value to the albumin excretion value was presented on logarithmic coordinates, with albumin on the x-axis and a 1 microglobulin on the y-axis [8]. The upper normal limits were represented as dashed lines. Such a diagram of a single a 1 -microglobulin and albumin excretion pattern allows visual interpretation of proteinuria and its connection with different kinds of nephropathies based on knowledge of the physiology of protein filtration and reabsorption, and its specific handling by the kidney in tubulointerstitial and glomerular nephropathies. The extent of tubular and glomerular proteinuria was described as slight when a 1 -microglobulin and albumin were below 50 and 100 mg / g creatinine, and as significant when a 1 -microglobulin and albumin were above 50 and 100 mg / g creatinine, respectively [9].
3. Results Table 1 presents the age and gender distribution, and the frequency of pathological findings in 373 subjects from the area of endemic nephropathy grouped into diseased subjects, suspect subjects, subjects ‘at risk’ and other subjects. An excretion value of a 1 -microglobulin . 15 mg / g creatinine was found in 90.0% of diseased subjects, in 41.6% of suspect subjects, in 7.1% of subjects ‘at risk’ and in 8.9% of other subjects. An albumin excretion value . 30 mg / g creatinine was found in 100.0% of diseased subjects, in 33.3% of suspect subjects, in 4.7% of subjects ‘at risk’ and in 7.5% of other subjects. The distribution of a 1 -microglobulin and albumin excretion patterns in diseased subjects, suspect subjects, subjects ‘at risk’ and other subjects is presented in Fig. 1. Table 1 Age and gender distribution, and frequency of pathological findings in 373 subjects from an area of endemic nephropathy Group
N (m / f)
Age (years)
a 1 -Microglobulin . 15 mg / g creatinine
Albumin . 30 mg / g creatinine
Diseased Suspect ‘At risk’ Others
10 (4 / 6) 24 (11 / 13) 127 (57 / 70) 212 (104 / 108)
46–75 27–77 4–74 4–74
90.0% 41.6% 7.1% 8.9%
100.0% 33.3% 4.7% 7.5%
88
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Fig. 1. Urinary excretion of a 1 -microglobulin in relation to albumin in 373 subjects from the area of endemic nephropathy.
In subjects with an established diagnosis of endemic nephropathy, tubular proteinuria with glomerular proteinuria was found in 9 / 10 subjects. In eight subjects the excretion value of a 1 -microglobulin was . 100 mg / g creatinine, and in one subject it was , 50 mg / g creatinine. In one subject the glomerular proteinuria was slight, and in others it was significant, but the albumin excretion value never exceeded 500 mg / g creatinine. An albumin excretion value of only 31 mg / g creatinine was found in one subject previously diagnosed as a diseased subject. In suspect subjects, the a 1 -microglobulin excretion values , 15 mg / g creatinine and albumin excretion values , 30 mg / g creatinine, i.e. physiological proteinuria, were found in 11 / 24 subjects. Slight tubular proteinuria was found in 5 / 24 suspect subjects. In 5 / 24 suspect subjects, tubular proteinuria with glomerular proteinuria was found. An excretion value of a 1 -microglobulin . 50 mg / g creatinine was found in only one of them, while an albumin excretion value of . 100 mg / g creatinine was found in four suspect subjects. Glomerular
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proteinuria was found in another 3 / 24 suspect subjects and was significant in one of them. Among subjects ‘at risk’, excretion values of a 1 -microglobulin and albumin below 15 and 30 mg / g creatinine, i.e. physiological proteinuria, were found in 114 / 127 subjects. Slight tubular proteinuria was established in 7 / 127 subjects ‘at risk’, and slight tubular proteinuria with slight glomerular proteinuria in 2 / 127 subjects ‘at risk’. Glomerular proteinuria was found in 4 / 127 subjects ‘at risk’. It was significant in one of them. In other subjects, physiological proteinuria, i.e. excretion values of a 1 microglobulin and albumin below 15 and 30 mg / g creatinine, was found in 181 / 212 subjects. Significant tubular proteinuria was found in 1 / 212 of other subjects, and slight tubular proteinuria in 14 / 212 of other subjects. In 4 / 212 of other subjects, slight tubular proteinuria with slight glomerular proteinuria was found. Glomerular proteinuria was found in 12 / 212 of other subjects, in six of them it was significant and in the other six it was slight.
4. Discussion The diagnostics of endemic nephropathy and detection of subjects suffering from endemic nephropathy in population screening are rather difficult. In the phases of azotemia or uremia it is difficult to differentiate endemic nephropathy from other chronic tubulointerstitial nephropathies, and a specific test for the diagnosis of endemic nephropathy is not available. Numerous investigations of endemic nephropathy including measurements of single proteins in urine, and determinations of the type of proteinuria by zone electrophoresis on acetate cellulose and immunoelectrophoresis on agarose as well as SDS-electrophoresis on polyacrylamide have shown that excretion of low molecular weight proteins such as b 2 -microglobulin, retinol-binding protein, lysozyme, immunoglobulin light chains and a 1 -microglobulin is increased in the early phase of the disease [5–7,13–17]. In the later phase, when glomeruli are also affected, mixed tubular and glomerular proteinuria is present. This finding has resulted in the opinion that early diagnosis could be considered if a tubular type of proteinuria is found in subjects living in the endemic area. Conventional tests such as the sulphosalicylic acid test and test strip are of limited value in detection of proteinuria in endemic nephropathy, especially in the early phase of the disease. These tests may be negative in subjects with endemic nephropathy and, on the contrary, they may be positive in subjects without endemic nephropathy. The LMW test was found positive in more than 70% of cases with a clinical diagnosis of endemic nephropathy [5]. b 2 Microglobulin, which is a sensitive marker of tubular dysfunction and was used for the diagnosis of endemic nephropathy, was found positive in 75% of subjects
90
ˇ ˇ´ / Clinica Chimica Acta 297 (2000) 85 – 91 D. Cvoriscec
with an established diagnosis of endemic nephropathy when 0.3 mg / l was used as the upper normal limit [18]. Preliminary results obtained by measurement of a 1 -microglobulin as the most reliable marker of tubular dysfunction in subjects living in the endemic nephropathy area showed values of a 1 -microglobulin . 20 mg / l, used as the upper normal limit, in 85.7% diseased, 50.0% suspect, 10.2% at risk, and in 8.1% of other subjects. The a 1 -microglobulin determination was performed by radial immunodiffusion [12]. In this study, excretion patterns of a 1 -microglobulin and albumin were investigated in subjects living in an area of endemic nephropathy. Tubular proteinuria with glomerular proteinuria was found in 9 / 10 subjects with an established diagnosis of endemic nephropathy. The extent of excretion of a 1 -microglobulin and albumin was in the ranges characteristic for tubulointerstitial diseases, which is consistent with results of a previous study of proteinuria in endemic nephropathy. In one subject diagnosed as an endemic nephropathy case, tubular proteinuria was not found. It seems that the diagnosis of endemic nephropathy was incorrect in this subject, and should be revised. Among suspect subjects, tubular proteinuria alone or with glomerular proteinuria was found in 10 / 24 subjects, and in only one suspect subject was tubular proteinuria significant. Slight tubular proteinuria with or without slight glomerular proteinuria was also found in 9 / 127 subjects ‘at risk’, and in 18 / 212 other subjects. Among other subjects, one case of significant tubular proteinuria was found. Such slight tubular proteinuria with or without slight glomerular proteinuria found in suspect, at risk and other subjects could be interpreted as a latent, early subclinical phase of endemic nephropathy providing that other possible causes of elevated a 1 -microglobulin and albumin excretion are excluded. Therefore, the retesting of a 1 -microglobulin and albumin under standardized conditions together with measurement of other protein markers for differentiation of proteinuria should be performed in the second step of laboratory examination. Based on the results of this study, simultaneous determination of a 1 -microglobulin as a tubular marker and albumin as a glomerular marker appears to be a sensitive screening procedure for the detection of proteinuria in subjects living in an endemic nephropathy area and allows more reliable detection of suspect cases of endemic nephropathy. The definitive clinical diagnosis of a single patient has to be established after an extensive clinical and laboratory examination of suspect subjects.
References ˇ ˇ ´ Z, Zupanic ´ V. Endemic nephropathy in Yugoslavia. In: Mostofi FK, [1] Radonic´ M, Radosevic Smith DE, editors, The kidney, Baltimore: Williams and Wilkins, 1966, pp. 503–22.
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