Department of Electron Microscopy/Molecular Pathology, The Cyprus Institute of Neurology and Genetics, Iroon Avenue 6, Agios Dometios, 2371, P.O. Box 23462 / 1683, Nicosia, Cyprus.
Cyprus School of Molecular Medicine, Iroon Avenue 6, Agios Dometios, 2371, P.O. Box 23462 / 1683, Nicosia, Cyprus.
Arthritis Res Ther. 2020 Jun 18;22(1):147. doi: 10.1186/s13075-020-02236-6.
Approximately 50% of systemic lupus erythematosus (SLE) patients develop nephritis, which is among the most severe and frequent complications of the disease and a leading cause of morbidity and mortality. Despite intensive research, there are still no reliable lupus nephritis (LN) markers in clinical use that can assess renal damage and activity with a high sensitivity and specificity. To this end, the aim of this study was to identify new clinically relevant tissue-specific protein biomarkers and possible underlying molecular mechanisms associated with renal involvement in SLE, using mass spectrometry (MS)-based proteomics.
Kidneys were harvested from female triple congenic B6.NZMsle1/sle2/sle3 lupus mice model, and the respective sex- and age-matched C57BL/6 control mice at 12, 24 and 36 weeks of age, representing pre-symptomatic, established and end-stage LN, respectively. Proteins were extracted from kidneys, purified, reduced, alkylated and digested by trypsin. Purified peptides were separated by liquid chromatography and analysed by high-resolution MS. Data were processed by the Progenesis QIp software, and functional annotation analysis was performed using DAVID bioinformatics resources. Immunofluorescence and multiple reaction monitoring (MRM) MS methods were used to confirm prospective biomarkers in SLE mouse strains as well as human serum samples.
Proteomic profiling of kidney tissues from SLE and control mice resulted in the identification of more than 3800 unique proteins. Pathway analysis revealed a number of dysregulated molecular pathways that may be mechanistically involved in renal pathology, including phagosome and proximal tubule bicarbonate reclamation pathways. Proteomic analysis supported by human transcriptomic data and pathway analysis revealed Coronin-1A, Ubiquitin-like protein ISG15, and Rho GDP-dissociation inhibitor 2, as potential LN biomarkers. These results were further validated in other SLE mouse strains using MRM-MS. Most importantly, experiments in humans showed that measurement of Coronin-1A in human sera using MRM-MS can segregate LN patients from SLE patients without nephritis with a high sensitivity (100%) and specificity (100%).
These preliminary findings suggest that serum Coronin-1A may serve as a promising non-invasive biomarker for LN and, upon validation in larger cohorts, may be employed in the future as a screening test for renal disease in SLE patients.
约 50%的系统性红斑狼疮（SLE）患者会发展为肾炎，这是该疾病最严重和最常见的并发症之一，也是发病率和死亡率的主要原因。尽管进行了深入研究，但目前仍没有在临床上可用于评估肾脏损伤和活动的可靠狼疮肾炎（LN）标志物，这些标志物具有较高的灵敏度和特异性。为此，本研究旨在使用基于质谱（MS）的蛋白质组学方法，鉴定与 SLE 肾脏受累相关的新的临床相关组织特异性蛋白生物标志物和可能的潜在分子机制。
从小鼠模型中获取雌性三重基因敲入 B6.NZMsle1/sle2/sle3 狼疮鼠的肾脏，并获取相应的性成熟和年龄匹配的 C57BL/6 对照鼠在 12、24 和 36 周龄时的肾脏，分别代表无症状前、建立后和终末期 LN。从肾脏中提取蛋白质，进行还原、烷基化和胰蛋白酶消化。用液相色谱分离纯化肽段，并进行高分辨率 MS 分析。使用 Progenesis QIp 软件处理数据，并使用 DAVID 生物信息学资源进行功能注释分析。使用免疫荧光和多重反应监测（MRM）MS 方法在 SLE 小鼠品系和人类血清样本中确认候选生物标志物。
对 SLE 和对照小鼠肾脏组织的蛋白质组学分析鉴定出 3800 多种独特蛋白质。途径分析显示，许多失调的分子途径可能与肾脏病理学有关，包括吞噬体和近端肾小管碳酸氢盐回收途径。基于人类转录组数据和途径分析的蛋白质组学分析表明，微丝相关蛋白 1A、泛素样蛋白 ISG15 和 Rho GDP 解离抑制剂 2 可能是 LN 的潜在生物标志物。使用 MRM-MS 在其他 SLE 小鼠品系中进一步验证了这些结果。最重要的是，在人类中的实验表明，使用 MRM-MS 测量人血清中的微丝相关蛋白 1A 可以将 LN 患者与无肾炎的 SLE 患者区分开来，灵敏度（100%）和特异性（100%）均很高。
这些初步发现表明，血清微丝相关蛋白 1A 可能是 LN 的一种有前途的非侵入性生物标志物，在更大的队列中验证后，它可能在未来被用作 SLE 患者肾脏疾病的筛查试验。
