Molecular forms of adiponectin in uraemic plasma

Sir,

Adiponectin is a recently found anti-atherogenic plasma protein secreted by adipocytes. Plasma adiponectin level is reduced in patients with coronary artery disease [1], type 2 diabetes mellitus [1] and obesity [2]. In contrast to these high-risk groups, plasma adiponectin has been reported to be elevated in haemodialysis patients [3]. Since uraemic plasma is known to contain not only intact forms but also fragments of some peptide hormones such as parathyroid hormone, it is an important question whether adiponectin in uraemic plasma is intact or not. To answer the question, we analysed its molecular forms.

Plasma samples were taken from two patients (patients A and B) on maintenance haemodialysis and a healthy volunteer; both 42-year-old men without diabetes, obesity or coronary artery disease. Fresh plasma was fractioned by gel filtration using a 10 x 300 mm column of Superose 6 HR (Amarsham Biosciences, Tokyo) and 31 mM Tris–HCl buffer (pH 7.2). An aliquot of each 0.5 ml fraction was assayed for adiponectin by ELISA [2]. Subsequently, another aliquot of the fractioned plasma was subjected to SDS–polyacrylamide gel electrophoresis (PAGE) in reducing condition. Western blotting was done using anti-adiponectin monoclonal antibody ANOC 4908 (a generous gift from Dr Funahashi, Osaka University Graduate School of Medicine) as the first antibody and peroxidase-labelled rabbit anti-mouse IgG polyclonal antibody as the second antibody. Plasma adiponecitin levels of the haemodialysis patients and the healthy subject were 25.1, 9.1 and 5.1 µg/ml, respectively. Reference range of adiponectin was 5.5±2.0 µg/ml (mean±SD, n = 51) for healthy men in our laboratory.

Upon gel filtration chromatography of plasma from the healthy subject, immunoreactive adiponectin migrated as macromolecules larger than IgG (150 kDa) showing three peaks. A similar gel filtration pattern was found for the uraemic plasma samples. No adiponectin immunoreactivity was detected in fractions corresponding to monomeric adiponectin or smaller fragments.

Western blotting of the fractioned healthy plasma (Figure 1) showed one major band at 30 kDa and another faint band at 80 kDa in reducing condition. In contrast, the 80 kDa band predominated in non-reducing condition. The extra band noticed at 28 kDa was non-specific staining for IgG light chain due to the known cross-reactivity of the second antibody (labelled polyclonal rabbit anti-IgG antibody) with human IgG light chain [2]. No other bands corresponding to adiponectin fragments were detected. These results agreed well with a previous report [2], and fit the proposed structure of adiponectin in the circulation. Adiponectin monomers form homo-trimers by disulfide bond, which further assemble as larger complex forms. The immunoblotting patterns of uraemic plasma samples were not different from that of the normal plasma, showing no adiponectin fragments.



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Fig. 1. Western blotting of plasma adiponectin. Healthy (top) and uraemic (bottom) plasma samples were fractioned by gel chromatography, and an aliquot of each fraction was subjected to SDS–PAGE in reducing condition. Adiponectin was visualized by western blotting using anti-adiponectin antibody. Pre-stained SDS–PAGE molecular standards included lysozyme (21.4 kDa), soybean trypsin inhibitor (28.8 kDa), carbonic anhydrase (35.5 kDa), ovalbumin (50.3 kDa), bovine serum albumin (93 kDa) and phosphoylase B (113 kDa). The gel filtration fraction numbers and molecular size markers for gel filtration (IgM, 900 kDa; IgG, 150 kDa; albumin, 67 kDa) are indicated below each lane. The major bands of 30 kDa correspond to adiponectin monomer.

 
The data suggest that uraemic plasma contains increased levels of intact adiponectin. This explains the significant correlation between plasma adiponectin and lipid levels, and the predictive value of adiponectin for a lower risk of cardiovascular events reported for haemodialysis patients [3]. However, it is still unknown why adiponectin levels are increased in uraemic plasma. Since an increased body mass index is paradoxically associated with a reduced risk of cardiovascular death in haemodialysis patients [4], fat cell functions may be altered in uraemia to be more anti-atherogenic rather than atherogenic. Further studies are needed to clarify the mechanisms of the altered levels of apparently intact adiponectin in renal failure.

Conflict of interest statement. None declared.

Tetsuo Shoji, Eiji Kimoto, Kayo Shinohara, Sawako Hatsuda and Yoshiki Nishizawa

Department of Metabolism, Endocrinology and Molecular Medicine Osaka City University Graduate School of Medicine 1-4-3, Asahi-machi Abeno-ku Osaka 545-8585 Japan Email: t-shoji{at}med.osaka-cu.ac.jp

References

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  2. Arita Y, Kihara S, Ouchi N et al. Paradoxical decrease of an adipose-specific protein, adiponectin, in obesity. Biochem Biophys Res Commun 1999; 257: 79–83[CrossRef][ISI][Medline]
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