Proteomic analysis of urine from proteinuric patients shows a proteolitic activity directed against albumin

Riccardo Magistroni, Giulia Ligabue, Valentina Lupo, Luciana Furci, Marco Leonelli, Luca Manganelli, Mario Masellis, Valentina Gatti, Fabrizio Cavazzini, Walter Tizzanini, Alberto Albertazzi

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)1672-1681
Number of pages10
JournalNephrology Dialysis Transplantation
Volume24
Issue number5
DOIs
Publication statusPublished - May 2009

Fingerprint

Proteomics
Albumins
Urine
Acetone
Electrophoresis, Gel, Two-Dimensional
Nephrotic Syndrome
Trichloroacetic Acid
Immunoglobulin G
Gels
Ultrafiltration
Proteome
Proteinuria
Mass Spectrometry
Proteins
Western Blotting

Keywords

  • Albumin
  • Kidney
  • Nephrotic syndrome
  • Proteomic
  • Urine

ASJC Scopus subject areas

  • Nephrology
  • Transplantation

Cite this

Magistroni, R., Ligabue, G., Lupo, V., Furci, L., Leonelli, M., Manganelli, L., ... Albertazzi, A. (2009). Proteomic analysis of urine from proteinuric patients shows a proteolitic activity directed against albumin. Nephrology Dialysis Transplantation, 24(5), 1672-1681. https://doi.org/10.1093/ndt/gfp020

Proteomic analysis of urine from proteinuric patients shows a proteolitic activity directed against albumin. / Magistroni, Riccardo; Ligabue, Giulia; Lupo, Valentina; Furci, Luciana; Leonelli, Marco; Manganelli, Luca; Masellis, Mario; Gatti, Valentina; Cavazzini, Fabrizio; Tizzanini, Walter; Albertazzi, Alberto.

In: Nephrology Dialysis Transplantation, Vol. 24, No. 5, 05.2009, p. 1672-1681.

Research output: Contribution to journalArticle

Magistroni, R, Ligabue, G, Lupo, V, Furci, L, Leonelli, M, Manganelli, L, Masellis, M, Gatti, V, Cavazzini, F, Tizzanini, W & Albertazzi, A 2009, 'Proteomic analysis of urine from proteinuric patients shows a proteolitic activity directed against albumin', Nephrology Dialysis Transplantation, vol. 24, no. 5, pp. 1672-1681. https://doi.org/10.1093/ndt/gfp020
Magistroni, Riccardo ; Ligabue, Giulia ; Lupo, Valentina ; Furci, Luciana ; Leonelli, Marco ; Manganelli, Luca ; Masellis, Mario ; Gatti, Valentina ; Cavazzini, Fabrizio ; Tizzanini, Walter ; Albertazzi, Alberto. / Proteomic analysis of urine from proteinuric patients shows a proteolitic activity directed against albumin. In: Nephrology Dialysis Transplantation. 2009 ; Vol. 24, No. 5. pp. 1672-1681.
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AU - Leonelli, Marco

AU - Manganelli, Luca

AU - Masellis, Mario

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AB - 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.

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