Magistroni Riccardo, Ligabue Giulia, Lupo Valentina, Furci Luciana, Leonelli Marco, Manganelli Luca, Masellis Mario, Gatti Valentina, Cavazzini Fabrizio, Tizzanini Walter, Albertazzi Alberto
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.
Nephrol Dial Transplant. 2009 May;24(5):1672-81. doi: 10.1093/ndt/gfp020. Epub 2009 Feb 11.
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.
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.
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.
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.
肾病综合征是一种临床上与不良预后相关的病症。在本研究中，我们比较了不同的尿液样本制备技术，以制定一个可靠的分析方案，来确定与肾病性蛋白尿相比，尿液异常情况下的差异尿蛋白质组。
我们招募了5名正常对照受试者、16名尿液异常患者和16名肾病综合征患者。正常尿液中的蛋白质采用三种不同方案（丙酮、超滤和三氯乙酸沉淀）进行处理，去除白蛋白和免疫球蛋白，然后通过二维聚丙烯酰胺凝胶电泳（2D-PAGE）凝胶和质谱分析。
二维凝胶电泳后通过肉眼观察凝胶比较三种提取方法，丙酮沉淀法和三氯乙酸法产生的蛋白质提取质量最佳，而丙酮沉淀法效率最高。此外，我们测试了三种用于去除白蛋白和免疫球蛋白的商业试剂盒。我们应用优化后的丙酮提取方案，将肾病患者（NP）的尿液样本与尿液异常患者（UAP）的尿液样本进行比较。我们观察到一种针对白蛋白的蛋白水解活性。这种观察在NP的尿液样本中比在UAP的尿液样本中更普遍。在两组中，观察到的蛋白水解活性存在一些个体间差异。在NP的尿液中还观察到α1抗胰蛋白酶浓度升高。我们在相同样本中通过一维和二维蛋白质印迹分析白蛋白片段化，跳过白蛋白和免疫球蛋白去除步骤，以避免白蛋白片段去除可能产生的混淆。分析证实肾病组的蛋白水解活性更强。
针对白蛋白的蛋白水解活性和α1抗胰蛋白酶的抗蛋白水解活性可能相关，并可能在肾病过程中起重要作用。如果在更大样本中得到重复验证，这种方法可能有助于更好地理解肾病综合征的潜在病理生理过程。
