Nephrology Department, Sechenov First Moscow State Medical University, Trubezkaya, 8, 119048 Moscow, Russia.
Department of Internal Medicine, Lomonosov Moscow State University, GSP-1, Leninskie Gory, 119991 Moscow, Russia.
Int J Mol Sci. 2022 Oct 20;23(20):12607. doi: 10.3390/ijms232012607.
Primary focal segmental glomerulosclerosis (FSGS), along with minimal change disease (MCD), are diseases with primary podocyte damage that are clinically manifested by the nephrotic syndrome. The pathogenesis of these podocytopathies is still unknown, and therefore, the search for biomarkers of these diseases is ongoing. Our aim was to determine of the proteomic profile of urine from patients with FSGS and MCD. Patients with a confirmed diagnosis of FSGS (n = 30) and MCD (n = 9) were recruited for the study. For a comprehensive assessment of the severity of FSGS a special index was introduced, which was calculated as follows: the first score was assigned depending on the level of eGFR, the second score-depending on the proteinuria level, the third score-resistance to steroid therapy. Patients with the sum of these scores of less than 3 were included in group 1, with 3 or more-in group 2. The urinary proteome was analyzed using liquid chromatography/mass spectrometry. The proteome profiles of patients with severe progressive FSGS from group 2, mild FSGS from group 1 and MCD were compared. Results of the label free analysis were validated using targeted LC-MS based on multiple reaction monitoring (MRM) with stable isotope labelled peptide standards (SIS) available for 47 of the 76 proteins identified as differentiating between at least one pair of groups. Quantitative MRM SIS validation measurements for these 47 proteins revealed 22 proteins with significant differences between at least one of the two group pairs and 14 proteins were validated for both comparisons. In addition, all of the 22 proteins validated by MRM SIS analysis showed the same direction of change as at the discovery stage with label-free LC-MS analysis, i.e., up or down regulation in MCD and FSGS1 against FSGS2. Patients from the FSGS group 2 showed a significantly different profile from both FSGS group 1 and MCD. Among the 47 significantly differentiating proteins, the most significant were apolipoprotein A-IV, hemopexin, vitronectin, gelsolin, components of the complement system (C4b, factors B and I), retinol- and vitamin D-binding proteins. Patients with mild form of FSGS and MCD showed lower levels of Cystatin C, gelsolin and complement factor I.
原发性局灶节段性肾小球硬化症（FSGS）和微小病变病（MCD）是一类以足细胞损伤为主要特征的疾病，临床上表现为肾病综合征。这些足细胞病的发病机制尚不清楚，因此，对这些疾病的生物标志物的研究仍在进行中。我们的目的是确定 FSGS 和 MCD 患者尿液的蛋白质组特征。我们招募了确诊为 FSGS（n=30）和 MCD（n=9）的患者进行研究。为了全面评估 FSGS 的严重程度，我们引入了一个特殊的指数，该指数的计算方法如下：第一个分数根据 eGFR 水平分配，第二个分数根据蛋白尿水平分配，第三个分数根据对类固醇治疗的反应性分配。将这些分数总和小于 3 的患者纳入第 1 组，总和为 3 或更高的患者纳入第 2 组。使用液相色谱/质谱法分析尿液蛋白质组。比较了第 2 组中严重进展性 FSGS 患者、第 1 组中轻度 FSGS 患者和 MCD 患者的蛋白质组图谱。使用基于稳定同位素标记肽标准（SIS）的靶向 LC-MS 对无标记分析的结果进行了验证，对于在至少一对组中具有区分能力的 76 种蛋白质中的 47 种，可用 SIS 进行标记。对这 47 种蛋白质进行定量 MRM SIS 验证测量时，发现有 22 种蛋白质在至少一对组之间存在显著差异，并且有 14 种蛋白质在两种比较中都得到了验证。此外，在第 2 组 FSGS 患者中，通过 MRM SIS 分析验证的所有 22 种蛋白质与无标记 LC-MS 分析在发现阶段的变化方向相同，即在 MCD 和 FSGS1 中上调或下调，而在 FSGS2 中下调。第 2 组 FSGS 患者的蛋白质组谱与第 1 组 FSGS 和 MCD 患者的蛋白质组谱明显不同。在 47 种具有显著差异的蛋白质中，最显著的是载脂蛋白 A-IV、血红素结合蛋白、纤连蛋白、转铁蛋白、补体系统成分（C4b、因子 B 和 I）、视黄醇和维生素 D 结合蛋白。轻度 FSGS 和 MCD 患者的胱抑素 C、凝胶蛋白和补体因子 I 水平较低。
