Peritoneal dialysis effluent (PDE) is an attractive biochemical window into the peritoneum. Albumin, serotransferrin, α1-antitrypsin, α1-microglobulin and immunoglobulins are the predominant proteins in PDE, representing more than 60% of total protein amount. The abundance of these proteins may limit the ability to identify potential biomarkers of peritoneal membrane function or damage. In this study, combinatorial peptide ligand library (CPLL) was used to enrich low abundance proteins and simultaneously reduce the highly abundant proteins. The CPLL elutions of PDE (CPLL-PDE) obtained from paediatric patients at different time duration of automated peritoneal dialysis (APD) were compared by two-dimensional electrophoresis.
Patients and Methods
We studied 19 paediatric patients whose characteristic are reported in Table 1. At the moment of the study, patients had been on APD with glucose-based solutions for a median of 13 months (range 1-38 months). Patients had not suffered from any infection episode for at least 1 month before the study. Dialysate samples were taken at the end of each dwell, when patient’s abdomen was accurately drained and dialysate volume was measured. Sample protein concentration was determined using bicinchoninic acid assay.
Materials and Methods
Combinatorial peptide ligand library. Each dialyzed PDE sample was loaded onto a column of 75 μL peptide library beads equilibrated in 25 mM phosphate buffer, pH 7.4. By using a peristaltic pump the samples were left in constant re-circulation overnight at room temperature. The column was then washed with 25 mM phosphate buffer, pH 7.4. Absorbed proteins were eluted with a solution of 2% SDS, 2.5% DTE and 100 mM Tris/HCl pH 7.4 and boiled for 10 min.
Two-Dimensional Electrophoresis. 2D was performed using homemade diluting gel matrix strips in a non linear pH 3-10 interval. The re-swelling of strips was carried out overnight at RT in a focusing solution, i.e. 7 M urea, 2 M thiourea, 2% (w/v) CHAPS, 15 mM DTE and a 0.6% (v/v) carrier ampholyte cocktail. Samples were cleaned and precipitated and finally dissolved in the focusing solution, i.e. 7 M urea, 2 M thiourea, 4% CHAPS and 65 mM DTE. The second dimension was run on T % 8-16 gels. Image was digitized by means of GS-800 (Bio-Rad). Each experiment was repeated in triplicate.
Protein identification by LC-ESI MS-MS/MS. All mass spectrometric measurements were performed using a LTQ linear ion trap mass spectrometer (Thermo Electron, San Jose, USA) coupled to a HPLC.
Image and statistical analysis. All digitalized images were analysed with PDQuest software (Bio-Rad). Spot optical density value were analysed by nonparametric U Mann-Whitney test for unpaired samples. Differences were considered statistically significant at p values < 0.05.
Results and Discussion
Image gel analysis revealed about 1000 spots in the PDE (Figure 1) and more than 1700 spots in the CPLL-PDE (Figure 2a-b). Therefore, combined proteomic approach highlighted a mean of 700 new proteins in each gel. Twenty-nine spots in CPLL-PDE proteome profile were expressed with a significantly different optical density in relation to the duration of APD treatment (Table 2). Among the 29 identified spots, 3 were particularly attractive from the clinical point of view. These spots correspond to gelsolin (spot 7) and intelectin-1 (spots 20 and 21). Changes of optical density of these spots in relation to the duration of APD treatment were showed in Figure 3.
Gelsolin is a calcium regulated multifunctional actin binding protein. Its cytoplasmic isoform is a regulator of actin organization, while the secreted isoform was implicated in a number of processes, such as the extracellular actin scavenging system and the presentation of lysophasphatidic acid and other inflammatory mediators to their receptors, in addition to its function as a substrate for extracellular matrix-modulating enzymes. Blood gelsolin levels decrease markedly in a variety of clinical conditions such as acute respiratory distress syndrome, sepsis, prolonged hyperoxia and liver injury.
Intelectin-1 is a homo-trimer disulfide linked and N-glycosylated protein. It is also named Endothelian lectin HL-1, Galactofuranose binding lectin, Intestinal lactoferrin receptor and Omentin. Intelectin-1 has been shown to: enhance insulin-stimulated glucose uptake in adipocytes; increase the phosphorylation of protein kinase B in the absence and presence of insulin; play a role in the defense system against microorganisms; specifically recognize carbohydrate chains of pathogens and bacterial components containing galactofuranosyl residues, in a calcium-dependent manner; be involved in iron metabolism. Cell intelectin-1 levels increase in malignant pleura mesotheliona, in the airway epithelium of asthmatic subjects, and in intestinal epithelial cell during chronic inflammation.
Both gelsolin and intelectin-1 are involved in chronic inflammation. Therefore, the changes that we observed in the PDE concentrations of these two proteins in relation to the duration of APD treatment may suggest their potential utility as a prognostic marker of chronic inflammation and/or fibrosis of the peritoneal membrane. To this regard, further investigations will be carried out in our laboratory to better characterize these proteins in plasma, PDE and mesothelial cells of a larger number of uremic patients undergoing peritoneal dialysis.