Study of urinary proteomes in patients with nephrotic syndrome.
Heavy proteinuria may be caused by either increased glomerulal basement membrane permeability or membrane or podocyte structural damage, and also by impairment of secretion-reabsorption tubular processes. The precise composition of modified or degraded urine proteins in proteinuria is not known. However, a possible toxic effect of proteins on tubular cells and disease progression is assumed. In this study, 15 patients with nephrotic proteinuria and other diagnoses (systemic lupus erythematodes with renal involvement (lupus nephritis) and AAV) were analysed by the 2D electrophoresis method. We have studied sample stability during storage, the albumin separation effect on sample analyses using ammonium sulphate, and the effect of proteases on the protein spectrum. In the first step, the proteins were divided by the isoelectric focusing method using polyacrylamide strips (pH 3-10 linear). The second step involved two-dimensional SDS electrophoresis performed in 12% polyacrylamide gel, which separated proteins according to their molecular weight. The proteins were visualized by the silver method. The gels were evaluated by Phoretix 2D expression software 2005. We found out that samples are stable for more than 6 months provided that they are frozen to -80 degrees C. The separation of albumin caused higher lucidity of the urinary proteomes. Without adding protease inhibitors we could detect proteolysis with increased quantity of proteins manifested in the area of about 10 kDa and decreased quantity of proteins detectable in the area with molecular weights about 50 kDa.
Vojtová
L
L
Institute of Clinical Biochemistry and Laboratory Diagnostics, 1 st Faculty of Medicine, Charles University, and General Teaching Hospital, Prague, Czech Republic. lucie.vojtova@vfn.cz
Zima
T
T
Tesar
V
V
Kazderová
M
M
D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D009404:Nephrotic Syndrome / Q000652:urine*; D010447:Peptide Hydrolases / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D020543:Proteome / Q000032:analysis*; D012709:Serum Albumin / Q000302:isolation & purification; D012984:Software; D013816:Thermodynamics
D011480:Protease Inhibitors; D020543:Proteome; D012709:Serum Albumin; D010447:Peptide Hydrolases