| Abstract|| |
In patients with the nephrotic syndrome, it is often desirable to assess the disease process, not only by proteinuria but also by indices of glomerular inflammatory process. We investigated the importance of β-microglobulin (βM) as a means of assessing renal function in patients with the nephrotic syndrome with normal or abnormal values of creatinine clearance. There were 46 patients (mean age, 42.2 + 10.4 years; male/female (M/F) ratio = 31/15) and 35 healthy controls (mean age 39 + 4.5 years, M/F ratio 25/10). We subdivided the study patients into group A (n = 18, mean age 39.6 + 10.6 years, M/F ratio 8/10) and group B patients (n = 28, mean age 45.6 + 8.9 years, M/F ratio 23/5) who had normal and abnormal values of creatinine clearance respectively. An enzyme-linked-immunosorbent assay (ELISA) was used to quantitate plasma β2M in the study patients and controls. The median 132M levels of the study patients and controls were 44.0 and 1.7 mg/l respectively (p < 0.0001). Beta-2-M levels correlated significantly with serum creatinine (r = 0.56, p < 0.0001), and creatinine clearance (r = -0.6, p < 0.0001). In group A patients, the median β2M level was significantly higher than normal (4.1 vs. 1.7 mg/1, p < 0.01). Plasma β2M levels did not correlate well with any other parameter measured in group A patients. When groups A and B were compared, the median plasma β2M level in group B was significantly higher than group A (20.3 vs. 4.1 mg/1, p < 0.0001). The urinary β2M (expressed per mg urine creatinine) was also higher in group B than group A patients (6.8 vs. 0.7 p < 0.05). We conclude that elevation of β2M-microglobulin in patients with the nephrotic syndrome who have normal creatinine clearance suggests early abnormal renal function in these patients. It may be used to assess the rate of normalisation of renal function or progression to chronic renal failure.
Keywords: Plasma beta-2M-microglobulin, Nephrotic syndrome, Renal function.
|How to cite this article:|
Adeyemi EO, Obineche EN, Abdulle AS, Osman AR. Evaluation of Plasma Beta-2-microglobulin in Patients with the Nephrotic Syndrome. Saudi J Kidney Dis Transpl 2001;12:151-6
|How to cite this URL:|
Adeyemi EO, Obineche EN, Abdulle AS, Osman AR. Evaluation of Plasma Beta-2-microglobulin in Patients with the Nephrotic Syndrome. Saudi J Kidney Dis Transpl [serial online] 2001 [cited 2019 Nov 22];12:151-6. Available from: http://www.sjkdt.org/text.asp?2001/12/2/151/33804
| Introduction|| |
The nephrotic syndrome (NS) is characterised, among other features, by heavy proteinuria, usually associated with peripheral edema. As the underlying causes may vary widely, from multisystemic diseases such as diabetes mellitus to neoplasms such as multiple myeloma, it is often desirable to assess the disease process, not only by proteinuria or the clinical signs on physical examination, but also by indices of glomerular inflammatory process. β2-microglobulin (β2M) is a globular low molecular weight protein (11.8 kilo Daltons) which is found on all nucleated cells and shed into the blood stream, especially when the lymphocytes are activated during an inflammation or infection. , It is a sub-unit of class 1 human leukocyte antigen (HLA) molecule, which in conjunction with class II molecules, encoded by the HLA-D region, directs self and non-self discrimination at the beginning of an immune response.  This association of β2M with the HLA system and, in particular, the antigen presenting cells (APC), is important for cellular immune response in chronic renal disease. β2M, by virtue of its small molecular size is filtered by the glomerular apparatus and part of it is absorbed by the renal tubules. The fate of the absorbed portion is not known. β2M has also been viewed as an indicator of T cell activation  and its biologic importance in patients with NS and normal creatinine clearance has not been studied extensively. Therefore, the main aim of this study is to test the hypothesis, that plasma β2M is higher in NS with normal creatinine clearance than in healthy controls.
| Materials and Methods|| |
Forty-six patients (mean age, 42.2 + 10.4; male/female (M/F) ratio = 31/15), who were admitted to the department of medicine of the UAE University hospital, for further assessment of their peripheral edema and excessive proteinuria were studied. Diagnosis of NS was based on a detailed history, careful physical examination, exclusion of intermittent or postural proteinuria, ultrasound scan of the kidneys, measurement of 24hour urinary protein excretion in addition to creatinine clearance. Serum urea, creatinine and electrolytes were measured. Urinary excretion of Bence Jones' protein was excluded where appropriate. Detection of Hepatitis B and C viral (HBV, HCV) infections was provided by the hospital laboratory services.
Ethylene-diamine-tetra-acetic acid (EDTA) blood samples (5 mls each) and urine specimens from 24-hour urine samples were obtained from the patients and an enzymelinked-immunosorbent assay (ELISA) was performed to measure plasma β2M (Immunotech International, France) levels. The samples and standard plasma were incubated for ninety minutes in microtiter well plates, coated with monoclonal anti-β2M antibody, in the presence of β2Malkaline phosphatase conjugate. Excess β2M-alkaline phosphatase conjugate was removed by washing the wells with phosphate buffered saline containing 0.1% Tween 20. The colour was developed with a freshly prepared substrate solution comprising para-nitrophenyl phosphate (PNPP) in diethanolamine buffer and the absorbance values were determined at 410 nm. The colour intensity was indirectly proportional to the β2M concentrations. The lower detection limit was 0.1 µg/ml. The urinary β2M excretion was expressed per g of urinary creatinine. The intra- and inter-assay coefficients of variation were less than 8 and 10% respectively. When β2M was added to a plasma sample with a known β2M concentration, recovery of the added β2M was greater than 95%. Multiple freezing and thawing did not affect the concentration of β2M in serum or plasma. β2M levels were also measured in the blood of healthy controls (n = 35; mean age 39 + 4.5 years, male: female (M/F) ratio 25/10).
| Data Analysis|| |
The data were entered into the spreadsheets of Microsoft Excel for Windows version 5.0 (Microsoft, USA). Statistical analysis was performed using the Microsoft Excel-based Analyse-It software, U.K. Ltd. (www.analyse-it.com). The results were validated with SPSS (Statistical Package for Social Sciences). As β2M levels did not conform to the Gaussian distribution, comparisons between patients and controls and various data sets were performed by the Mann-Whitney U test and interdependence between the variables was with the Spearman correlation. Repeated measurements in the second group of patients were assessed with the Wilcoxon test. A twotailed p < 0.05 was considered significant.
| Results|| |
The median β2M level in normal controls was 1.7 (0.7-3.4 mg/1) [Figure - 1]. In all patients the median β2M level was significantly higher than normal (44 versus 1.7 mg/1, p < 0.0001). It also correlated significantly with serum creatinine (r = 0.56, p < 0.0001, n = 46) [Figure - 2], and creatinine clearance (r = -0.6, p < 0.0001, n = 46) [Figure - 3]. When the patients were divided into those with normal (group A: n = 18, mean age 3 9.6 + 10.6 years, M/F ratio 8/10) and abnormal (group B: n = 28, mean age 45.6 + 8.9 years, M/F ratio 23/5) values of creatinine clearance, the median β2M level in group A patients was significantly higher than normal (4.1 vs 1.7 mg/1, p<0.01). The plasma β2M levels in group A did not correlate well with any of the measured parameters. When groups A and B were compared, both the median plasma β2M level (20.3 vs. 4.1 mg/1, p < 0.0001), and the urinary β2M concentrations (6.8 vs. 0.7 p < 0.05, expressed in per gram of excreted creatinine, [Table - 1]) were significantly higher in group B than in group A patients.
| Discussion|| |
The cause of idiopathic nephrotic syndrome is unknown. The syndrome is characterised by proteinuria, usually in excess of three grams in 24 hours and fusion of the epithelial cells with loss of sialoglycoprotein.  Many studies have shown that lack of adequate response to prednisolone, defined as inability to reduce the rate of urinary protein excretion, often heralds, or at least initiates disease progression to endstage renal failure. , Therefore, it is of paramount importance to have biochemical markers, apart from excreted urinary protein, which may be used to monitor progression of this disease.
Animal experiments have shown clearly, that supernatants of cultured lymphocytes of patients with NS, contained a factor which, when injected into the skin of a guinea pig, caused increased vascular permeability.  When these products of cultured lymphocytes of patients with NS were injected into the rat renal artery, proteinuria, fusion of the epithelial cells and reduction of the basal membrane charges, were observed. ,,, Recently Bustos et al  demonstrated increased production of tumour necrosis factor-α and expression of its gene in the peripheral blood mononuclear (PBMN) cells of nephrotic patients. This suggests that PBMN cellular activation plays a pathogenetic role in the nephrotic syndrome. As β2M is found on all nucleated cells and shed into the blood stream, especially when the lymphocytes are activated in inflammation or in infection,  it is attractive to evaluate its role in patients with nephrotic syndrome.
We approached the hypothesis, that β2M is elevated in patients with nephrotic syndrome with normal creatinine clearance, by determining plasma β2M levels in patients with the nephrotic syndrome with normal and abnormal creatinine clearance and in normal controls.
The results are interesting, in that β2M separated clearly between normal controls and nephrotic syndrome patients with normal creatinine clearance on the one hand and between nephrotic syndrome patients with normal and abnormal values of creatinine clearance on the other. The statistically significantly higher β2M levels measured in nephrotic patients with normal creatinine clearance, when compared with control subjects, can not be explained by the creatinine levels, as these and the values of creatinine clearance were within normal limits. The higher β2M levels may be ascribed to PBMN cell activation in nephrotic syndrome and damage to the renal tubules may be due to release of the pro-inflammatory cytokines, such as interleukin-12, and other cytokines; , and probably the reactive oxygen metabolites.  We did not study in vitro β2M production by the PBMN cells. This would have been a useful addition to this study
Urinary β2M excretion was significantly ower in group A than in group B patients. The glomerular filtration and tubular reabsorption mechanism of β2M in man may help to explain this finding. The relatively small molecular weight of β2M permits filtration through the glomerular membrane in health. It is also filtered through the synthetic dialysis membranes in patients on hemodiafiltration due to chronic renal failure.  It is then reabsorbed and broken down by the proximal tubular cells.  Appearance of β2M in the urine depends on plasma β2M elevation exceeding its renal reabsorptive threshold of 5 mg/1 18 and/or from proximal tubular damage, which can be assessed by measuring the concentration of N-acetyl-beta-D-glucosaminidase (NAG) in the urine.  Urinary excretion of NAG, as a marker of proximal renal tubule function was not studied, because the main objective was to evaluate the role of β2M in the nephrotic syndrome.
The renal threshold of 5 mg/1 for β2M was higher than the median β2M value in group A but lower than the median plasma β2M value of 20.3 mg/1 measured in group B patients. There is increased excretion and renal tubular catabolism of β2M in patients whose plasma β2M levels exceed the threshold of 5 mg/1, but we have found no evidence in the literature to suggest that β2M per se is toxic to the renal tubule. The present data do not support the hypothesis either.
In conclusion, our study may support the hypothesis that β2M is elevated in nephritic patients with normal creatinine clearance and may be a useful tool for monitoring progression of renal disease to chronic renal failure.
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Edward O Adeyemi
Department of Medicine, U.A.E. University, P.0. Box 17666, AI Ain
United Arab Emirates
[Figure - 1], [Figure - 2], [Figure - 3]
[Table - 1]