Proteomic analysis of urine from proteinuric patients shows a proteolitic activity directed against albumin.
BACKGROUND
Nephrotic syndrome is a condition that is clinically associated with poor outcome. In this study, we compared different techniques of urine sample preparation in order to develop a robust analytical protocol to define the differential urinary proteome of urinary abnormalities compared to nephrotic proteinuria.


METHODS
We recruited 5 normal control subjects, 16 patients with urinary abnormalities and 16 patients with nephrotic syndrome. Proteins from normal urine were processed using three different protocols [acetone, ultrafiltration and trichloroacetic acid (TCA) precipitation], depletion of albumin and IgGs and then analysed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) gels and mass spectrometry.


RESULTS
Comparing the three extraction methods by visual inspection of gels after 2D gel electrophoresis, the acetone precipitation and TCA methods yielded the best quality of protein extraction, while the acetone precipitation method was the most efficient. Furthermore, we tested three commercial kits for albumin and IgG depletion. We applied the optimized acetone extraction protocol to compare urinary samples from nephrotic patients (NP) to urinary samples obtained from patients presenting with urinary abnormalities (UAP). We observed a proteolytic activity directed against albumin. This observation was more prevalent in urinary samples from NP than from UAP. Within both groups, there was some inter-individual variability in the observed proteolytic activity. An increased concentration of alpha1 antitrypsin was also observed in urine of NP. We analysed albumin fragmentation by 1D and 2D western blots in the same samples skipping the albumin and IgG depletion steps to avoid the possible confound of albumin fragment removal. The analysis confirmed a stronger proteolytic activity in the nephrotic group.


CONCLUSIONS
The proteolytic activity against albumin and the anti-proteolytic activity of alpha1 antitrypsin are likely linked and could play an important role in the nephrotic process. If replicated in larger samples, this methodology may lead to a better understanding of the underlying pathophysiological process of nephrotic syndrome.
Magistroni
Riccardo
R
Division of Nephrology Dialisys and Transplantation, Dipartimento Integrato Medicine e Specialità Mediche, Universià di Modena e Reggio Emilia, via del Pozzo 71 41100 Modena, Italy. riccardo.magistroni@unimore.it
Ligabue
Giulia
G
Lupo
Valentina
V
Furci
Luciana
L
Leonelli
Marco
M
Manganelli
Luca
L
Masellis
Mario
M
Gatti
Valentina
V
Cavazzini
Fabrizio
F
Tizzanini
Walter
W
Albertazzi
Alberto
A
D000328:Adult; D000368:Aged; D000418:Albumins / Q000378:metabolism*; D016022:Case-Control Studies; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D006801:Humans; D007074:Immunoglobulin G / Q000652:urine; D008297:Male; D013058:Mass Spectrometry; D008875:Middle Aged; D009404:Nephrotic Syndrome / Q000503:physiopathology / Q000652:urine*; D015588:Observer Variation; D010447:Peptide Hydrolases / Q000652:urine*; D011507:Proteinuria / Q000503:physiopathology / Q000652:urine*; D040901:Proteomics; D000515:alpha 1-Antitrypsin / Q000652:urine
D000418:Albumins; D007074:Immunoglobulin G; D000515:alpha 1-Antitrypsin; D010447:Peptide Hydrolases