Evaluation of Renal Damage by Urinary Beta-Trace Protein in Patients with Chronic Kidney Disease

Transcription

1 Clin. Lab. 2011;57:XXX-XXX Copyright ORIGINAL ARTICLE Evaluation of Renal Damage by Urinary Beta-Trace Protein in Patients with Chronic Kidney Disease MARIJANA DAJAK 1, SVETLANA IGNJATOVIĆ 2, BILJANA STOJIMIROVIĆ 3, SNEŽANA GAJIĆ 4, NADA MAJKIĆ-SINGH 2 1 Center of Medical Biochemistry, Clinical Center of Serbia, Belgrade, Serbia 2 Center of Medical Biochemistry, Clinical Center of Serbia and School of Pharmacy, Belgrade, Serbia 3 Center of Urology and Nephrology, Clinical Center of Serbia and School of Medicine, Belgrade, Serbia 4 Institute of Urology and Nephrology Department, Dr Dragiša Mišović Clinic, Belgrade, Serbia SUMMARY Background: Beta-trace protein (BTP) was found to be increased in the serum and urine of patients with renal diseases. The aim of this study was to compare the urinary levels of beta-trace protein with levels of other urinary proteins: albumin, β 2 -microglobulin (B2M), α 1 -microglobulin (A1M), and cystatin C and to determine its clinical usefulness for detection of renal dysfunction in chronic kidney disease (CKD). Methods: These markers were measured in 24-hour urine samples from 134 patients with CKD. Results: BTP correlated significantly with A1M (r = 0.871), cystatin C (r = 0.759), total protein (r = 0.684), B2M (r = 0.497), and albumin (r = 0.448) in 24-hour urine samples (P < 0.05). Urinary BTP concentrations in patients with albuminuria below 30 were significantly lower than in patients with albuminuria above 30 (P < ). ROC analysis showed high diagnostic accuracy of BTP for detection of > 30 albuminuria (AUC 0.908). Urinary BTP was also in significant correlation with the estimated glomerular filtration rate (r = 0.580). Conclusions: The results of our study suggest that BTP may be a useful and reliable urinary marker of renal dysfunction and may have a place in addition to urinary α 1 -microglobulin and albumin as an alternative marker for tubular damage and the magnitude of renal impairment in patients with chronic kidney disease. (Clin. Lab. 2011;57:XXX-XXX) KEY WORDS Chronic kidney disease, urinary beta-trace protein, urinary proteins INTRODUCTION Early diagnosis and treatment of chronic kidney disease (CKD) may delay need for renal replacement therapy and improve outcome since preventive and renoprotective interventions are available. Current recommendations for evaluating the presence of renal disease are measurement of urinary albumin in order to assess kidney damage and estimation of the glomerular filtration rate (GFR) using an equation based on the level of serum creatinine (1). Increased excretion of proteins in the urine is the hallmark of almost any type of kidney disease. Urinary protein analysis is of particular value in disease prevention, screening, and monitoring of renal disease (2). Beta-trace protein (BTP) is a brain-specific protein, primarily isolated as prostaglandin D2 synthase (EC ) from cerebrospinal fluid, but it is also found in low concentrations in serum, urine, amniotic fluid, and seminal plasma. It has a molecular weight of kda depending on the degree of glycosylation. ΒTP catalyses the conversion of prostaglandin H 2 to prostaglandin D 2 (PGD 2 ). It is a secretory and transporter protein of the lipocalin family that binds retinoids, thyroid hormones, and bile pigments. BTP is almost completely excreted via the kidneys and it is stable at the PH of urine (3). Beta-trace protein has emerged as a promising novel marker of renal impairment (4-6). Manuscript accepted July 3, 2010 Clin. Lab. 1+2/2011 1

2 M. DAJAK et al. Proteinuria is one of the most commonly used markers of kidney damage. Urinary albumin excretion is a sensitive diagnostic and prognostic marker of renal disease. Albumin has a relatively low molecular weight (66.3 kda) and its high excretion suggests increased glomerular permeability, proximal tubular damage, or a combination of these (7). Microalbuminuria is the term which refers to the urinary excretion of Microalbuminuria is now widely acknowledged as an independent predictor and risk marker for cardiovascular disease, diabetes, stroke, and kidney failure (8). Alpha 1 -microglobulin (A1M), β 2 -microglobulin (B2M), and cystatin C are low molecular weight proteins with molecular weights of 27 kda, 11.8 kda, and 12.8 kda, respectively. They are almost completely filtered at the glomerulus and then reabsorbed and metabolized in the proximal tubule. These proteins are some of the best characterized tubular proteins to detect proximal tubular injury (9-11). Beta 2 -microglobulin is present on the surface of most nucleated cells, and cystatin C is produced at a constant rate by nucleated cells. Because of its small size and high isoelectric point (pi = 9.2), cystatin C is more freely filtered at the glomerulus than other low molecular weight proteins (12,13). The diagnostic utility of B2M is hampered by its poor stability at acid ph and increased production in other diseases such as autoimmune diseases, infections, and tumors (13). Alpha 1 -microglobulin, also called protein HC, is produced by the liver. It is stable in urine, and its levels are very stable in pathological conditions such as tumors, infections, and rheumatoid arthritis. Urinary A1M is a well established early marker of tubular damage and its excretion has been successfully used in the diagnosis of various urological conditions involving kidney damage (11). The aim of this study was to investigate the clinical usefulness of the urinary levels of beta-trace protein for detection of renal dysfunction in patients with chronic kidney disease and to compare them with the levels of other urinary proteins. MATERIALS AND METHODS Subject samples The study included 134 patients (72 females and 62 males), aged years, with chronic kidney disease. The causes of kidney disease were: focal segmental glomerulosclerosis (n = 17), other glomerulosclerosis (n = 6), membranous glomerulonephritis (n = 24), other glomerulonephritis (n = 11), systemic lupus erythematosus (n = 17), hypertension (n = 19), diabetic nephropathy (n = 9), nephrolithiasis (n = 5), polycystic kidney disease (n = 7), and other (n = 19). The patients had a wide range of renal function that encompassed all five stages of the CKD according to the American National Kidney Foundation classification system (1). Twenty-four-hour urine samples were collected from each patient according to the routine regimen (13). The urine samples were collected without adding preservatives and were stored at 4 C during collection period. According to literature data (13), all measured urinary proteins, including BTP, are stabile in such collection conditions, except B2M which is unstable in urine with a ph < 6.0. The ph values of all urine samples were checked and were above ph 7 and therefore suitable for measurement of B2M. BTP was also measured in 24- hour urine samples of 50 healthy volunteers (25 females and 25 males) aged years. The study was conducted in accordance with the Helsinki Declaration. All study participants gave written informed consent. Urinary concentrations of creatinine, total protein, albumin, and Β2M were determined on the day of inclusion. Urine samples for Β2M were measured after the addition of stabilization buffer. The stabilization buffer is a solution containing detergent and was added according to manufacturer's instructions. Cystatin C, α 1 -microglobulin, and BTP were measured later in urine samples stored at -70 C. Results for urinary proteins were expressed in mg per day and for urinary creatinine in mmol per day. The glomerular filtration rate is widely accepted as the best overall index of kidney function. We also measured the serum creatinine concentrations in these patients and estimated GFR (egfr) with the creatinine-based MDRD study formula (14). Blood sera were obtained at the end of the urine collections. The values of egfr were used for classification into the CKD stages. Methods BTP was measured by a latex particle-enhanced immunonephelometric assay on a Behring Nephelometer II analyzer (Dade Behring, Marburg, Germany). Interassay imprecision (n = 20) was calculated for one control material (N/A Protein Control LC, Dade Behring) and three urine samples with concentrations of mg / L was between 2.15 % and 3.91 %. A Behring Nephelometer II analyzer was also used to measure albumin, A1M, cystatin C, and B2M in urine samples by immunonephelometry. The detection limits, specified by the manufacturer, for these proteins in urine were as follows: cystatin C, 0.05 mg / L; B2M, 0.21 mg / L; albumin, 10.4 mg / L and A1M, 5.64 mg / L. Urinary and serum creatinine was determined by a kinetic alkaline picrate method and urinary total protein by a turbidimetric method using benzethonium chloride, both on an ARCHITECT ci8200 analyzer (Abbott Diagnostics, Wiesbaden, Germany). The detection limit for total protein in urine was 0.07 g / L. Statistical analysis Adherence to Gaussian distributions was assessed using the Kolmogorov-Smirnov test, taking P < 0.05 as a significant result. Whereas the variables in groups showed mainly nongaussian-distribution, median and range were used for descriptive statistics. The Mann-Whitney U-test and Spearman s test were used for comparison and correlation analysis. A P value less than 0.05 was 2 Clin. Lab. 1+2/2011

3 EVALUATION OF RENAL DAMAGE BY URINARY BETA-TRACE PROTEIN Table 1. Median and range values of urinary markers in patients with chronic kidney disease. Urinary marker n Median Range Creatinine, mmol / day Total protein, g / day Albumin, Cystatin C, α 1 -Microglobulin, β 2 -Microglobulin, Beta-trace protein, Table 2. Median and range values of specific urinary proteins in different chronic kidney diseases. Chronic kidney disease (CKD) Specific urinary protein Albumin Cystatin C A1M B2M BTP Glomerulosclerosis Glomerulonephritis Systemic lupus erythematosus Hypertension Diabetic nephropathy Nephrolithiasis Polycystic kidney disease Other CKD (n = 20) (n = 31) (n = 14) (n = 11) (n = 7) (n = 22) (n = 7) (n = 26) (n = 10) (n = 11) The concentrations were under detection limit (n = 4) (n = 5) (n = 8) (n = 14) (n = 25) (n = 10) (n = 10) (n = 4) (n = 9) (n = 23) (n = 35) (n = 17) (n = 19) (n = 9) (n = 5) (n = 7) (n = 19) considered statistically significant. A 95 % confidence interval (CI) was also shown in reported data. The diagnostic value of urinary BTP for identifying albuminuria under 30 in patients with chronic kidney diseases was evaluated using receiver operating characteristic (ROC) curve analysis. The data are expressed as area under the curve (AUC; 95 % confidence interval, 95 % CI; standard error, SE). Statistical analysis was performed using MedCalc for Windows, version (MedCalc Software, Mariakerke, Belgium). Clin. Lab. 1+2/2011 3

4 M. DAJAK et al. Table 3. Median and range values of specific urinary proteins in different stages of chronic kidney diseases. CKD stages (egfr, ml / min per 1.73 m 2 ) Specific urinary protein Albumin Cystatin C A1M B2M BTP Stage 1 ( 90) Stage 2 (60-89) Stage 3 (30-59) Stage 4 (15-29) Stage 5 (< 15) (n = 35) (n = 27) (n = 17) (n = 18) (n = 10) (n = 21) (n = 24) (n = 14) (n = 7) (n = 21) (n = 24) (n = 14) (n = 21) (n = 50) (n = 36) RESULTS The median concentration of BTP in 24-hour urine samples of 50 healthy volunteers was 2.03 and ranged (median, mg / mmol creatinine; Range, mg / mmol creatinine). There was no difference (P = ) between ΒTP excretions for males (median, 2.04 ; range, ) and females (median, 2.02 ; range, ). When the concentrations of BTP in urine samples were expressed in mg per mmol of creatinine, males showed slightly higher concentrations (median, 0.188; range, ) than females (median, 0.143; range, ). This between-gender difference was not statistically significant (P = ). The median (range) urine concentration of BTP in patients with CKD was 3.93 ( ). Concentrations of BTP in patients were significantly higher than in healthy individuals (P < ). Median and range values of proteins in the investigated patient population are presented in Table 1. The values of specific urinary proteins in different chronic kidney diseases and in different stages of CKD are summarized in Table 2 and Table 3. Mainly, concentrations of urinary proteins were significantly higher in diabetic nephropathy than in other disease groups. BTP significantly increased from CKD stage 2 to stage 5 and cystatin C from CKD stage 3 to stage 5. Albumin and B2M only showed significant higher urinary concentrations in later stages than in early ones. The highest correlation was observed between BTP and A1M urinary concentrations (r = 0.871, 95 % CI ). Likewise, beta-trace protein correlated significantly (Spearman test, P < ) with cystatin C and total protein (TPU) in 24-hour samples (cystatin C, r = 0.759, 95 % CI ; TPU, r = 0.684, 95 % CI ). Slightly weaker correlation was obtained with B2M (r = 0.497, 95 % CI , P < ) and albumin (r = 0.448, 95 % CI , P < ) concentrations. The correlation between urinary BTP and urinary creatinine was very poor, but significant (r = 0.175, P = 0.044) (Figure 1). The concentrations of urinary BTP were also in negative significant correlation with calculated egfr (r = , 95 % CI from to , P < ). Additionally, patients were divided into 3 subgroups according to urinary albumin concentrations: normal, microalbuminuria, and albuminuria, where urinary albumin concentrations were below 30, from 30 to 300, and above 300, respectively. The urinary concentrations of A1M, cystatin C, and B2M were below the method detection limit in the group of patients with albumin below 30. The median excretions of BTP were: 1.03 (n = 33, range: ), 4.29 (n = 25, range: ) and 6.67 (n = 76, range: ), for normal, microalbuminuria, and albuminuria groups, respectively (Figure 2). The median (range) concentrations of BTP in urine samples expressed in mg per mmol of creatinine were also calculated and they were: ( ), ( ) and ( ), for the normal, microalbuminuria, and albuminuria group, respectively. Urinary BTP concentrations in the group with albuminuria below 30 mg / day were significantly lower than in the other two groups (P < ). Patients with microalbuminuria 4 Clin. Lab. 1+2/2011

5 EVALUATION OF RENAL DAMAGE BY URINARY BETA-TRACE PROTEIN Figure 1. The correlations between beta-trace protein (BTP) and α 1 -microglobulin (A1M), cystatin C, total protein (TPU), β 2 - microglobulin (B2M), albumin, and creatinine in 24-hour urine samples from patients with chronic kidney disease. Figure 2. The urinary concentrations of beta trace protein (BTP) in groups of patients with chronic kidney disease according to urinary albumin concentrations: normal (below 30 ), microalbuminuria (30 to 300 ) and albuminuria (above 300 ). Data are presented as box-plots showing the 10th, 25th, 50th, 75th, and 90th percentiles. Clin. Lab. 1+2/2011 5

6 M. DAJAK et al. Figure 3. ROC analysis of beta-trace protein (BTP) for the detection of > 30 mg/day albuminuria in patients with chronic kidney disease. had slightly lower urinary BTP concentrations than patients with albuminuria above 300, but the difference was not statistically significant (P = ). In order to determine the diagnostic accuracy of BTP for the detection of > 30 albuminuria in patients with chronic kidney diseases, the ROC plot was calculated and AUC was (95 % CI, ; SE, ) (Figure 3). The sensitivity of 78.2 % and the specificity of 87.9 % were obtained for the optimum cutoff value of BTP (2.62 ). When the concentrations of BTP in urine samples were expressed in mg per mmol of creatinine, the calculated parameters of ROC analysis were: AUC, 0.868; the sensitivity of 82.2 %, and the specificity of 75.8 % for the cutoff value of mg / mmol creatinine. DISCUSSION Beta-trace protein is a multi-functional protein: it acts as a PGD 2 -producing enzyme within the cells and functions as a lipophilic ligand-binding protein after secretion into the extracellular spaces. It is a very stable enzyme and is highly resistant to heat treatment and protease digestion (3). Remarkably increased serum and urinary levels of beta-trace protein have been reported in patients with various renal diseases (4-6). In this study, BTP concentrations in 24-hour urine samples in patients with CKD were markedly higher than in healthy individuals. Healthy males had higher BTP concentrations (median, 0.188; range mg / mmol creatinine) than females (median, 0.143; range mg / mmol creatinine), but not statistically significant. The upper reference limit of 0.45 mg / mmol creatinine in adults has been reported using the same assay (15). Slightly higher reference values for BTP in urine for males were also found in other studies (6,15). The presence of BTP in seminal plasma may be the reason for the higher urinary BTP levels in males (16). Mainly, urinary concentrations of BTP, as well as concentrations of albumin, A1M, B2M, and cystatin C, did not differ in different CKD groups except for diabetic nephropathy (Table 2). All disease groups encompassed all five stages of the CKD. Therefore, high variability of results was obtained in these groups. Impaired tubular function is often accompanied by decreased glomerular filtration rate. The excretion rate of tubular markers increases slowly with progression of the glomerular diseases. High excretion of BTP is caused by increased glomerular permeability and/or, as for other tubular markers, by the exhaustion of the tubular reabsorptive function or primary tubular disease (6,13,17). The precise mechanism of BTP actions in renal pathophysiology has not yet been clarified. In 24-hour urine samples from patients with CKD, BTP was significantly correlated with all the measured urinary renal markers. Of the various proteins freely filtered, but reabsorbed in the proximal tubule, α 1 -microglobulin may be the best marker of tubular damage 6 Clin. Lab. 1+2/2011

7 EVALUATION OF RENAL DAMAGE BY URINARY BETA-TRACE PROTEIN because it is stable, easily measurable, not significantly affected by diabetic and other pathological urine matrices, and its behavior is consistent and well-documented in various types of renal tubular disease (9,11). In addition, quantification of A1M together with albumin, immunoglobulin G, and α 2 -macroglobulin has been shown to be useful in the differentiation of the causes of proteinuria (17). In this study, the highest correlation was obtained between urinary BTP and urinary A1M (r = 0.871). Cystatin C is currently under investigation as a replacement for serum creatinine in estimating GFR. It has been suggested that combined measurement of cystatin C in serum and urine is useful to detect the mild reducetion of GFR (18). B2M was shown to be useful as a renal marker in specific situations (19,20). Among the low molecular weight proteins, only B2M, which causes dialysis-related amyloidosis, has known toxicity (21). In contrast to A1M, these proteins may have lower diagnostic applicability because of their instability in urine at physiological ph (B2M) or very low levels in urine of patients with normal renal function (cystatin C) (22). After A1M, the most significant correlation was obtained between urinary BTP and urinary cystatin C (r = 0.759), followed by correlation with urinary total protein and B2M (correlation coefficients were and 0.497). In contrast, urinary BTP concentrations showed very poor correlation with urinary creatinine concentrations. These results are in accordance with the recent findings where similar significant correlations between urinary BTP and urinary A1M and total protein were obtained but no correlation with urinary creatinine (15). BTP seems to be useful as a diagnostic marker for the early detection of renal tubular damage, because it correlates highly with A1M and other tubular proteins, has a constant production rate, and its concentrations do not vary in situations with an acute-phase reaction. Unlike B2M, it is stable over a wide ph range and, unlike A1M, does not bind other proteins (15,23). According to American National Kidney Foundation guidelines (1) measured or estimated GFR is used for the diagnosis and classification of CKD. In this study, urinary BTP correlated significantly with the values of egfr (r = ) and significantly increased from CKD stage 2 to stage 5 (Table 3). Therefore, urinary BTP concentrations were also relative to the severity of renal dysfunction in patients with CKD. Albuminuria is an early and sensitive marker of kidney damage in many types of CKD. It is an established marker of glomerular permeability. According to the National Kidney Disease Education Program, besides the estimation of GFR, measuring and reporting the albumin excretion is crucial for the detection and treatment of CKD (1,24). In this study, the correlation between BTP and albumin concentrations in 24-hour urine samples was significant but not very high (r = 0.448). Furthermore, we found significantly lower concentrations of BTP in patients with albuminuria below 30 mg / day than in patients with albuminuria above 30 mg / day. Figure 2 showed overlap in BTP excretion among the three groups according the degree of albuminuria. This may be explained by better correlation of BTP with tubular markers than with albumin and BTP's increased excretion in a manner which is more similar to tubular than glomerular markers. The data from ROC analysis showed that urinary BTP has a high diagnostic value (AUC = 0.908) for detection of > 30 albuminuria in patients with chronic kidney diseases. The limitations of this analysis were the small number of patients, especially with albuminuria below 30 and the heterogeneity of the patient population. BTP is similar to albumin in the chemical properties including anionic charges at ph 7.4, but it has a much smaller molecular weight than albumin. Thus, it passes more easily through glomerular capillary walls of the kidney than albumin. Recently published data show that urinary BTP excretions were superior to the other urinary markers except for albuminuria in assessing the predictability of renal diseases. The authors also described how the urinary BTP excretions reflect the currently increased permeability of injured glomerular capillary walls and better predict the future status of renal injury in patients with type-2 diabetes and albuminuria with less of 30 mg / g creatinine (6). They obtained the AUC ROC of for BTP for predicting > 30 mg / g Cr albuminuria in patients. In our study AUC was for detecting albuminuria in patients with CKD (BTP values were expressed in mg per mmol of creatinine). Therefore, the results of our study support other reported data in stating that BTP may be a useful and reliable urinary marker of renal dysfunction and may have a place beside urinary α 1 -microglobulin and albumin as an alternative marker for tubular damage and the magnitude of renal impairment in patients with chronic kidney disease. Acknowledgements: This study was conducted as a part of the project No , financially supported by the Ministry of Science and Technological Development of the Republic of Serbia. Declaration of Interest: No conflict of interest. Clin. Lab. 1+2/2011 7

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