Division of Nephrology, Maisonneuve-Rosemont Hospital, 5415 Boulevard de l'Assomption, Montreal, QC, H1T 2M4, Canada.
Research Center, Maisonneuve-Rosemont Hospital, Montreal, QC, Canada.
Inflamm Res. 2024 Mar;73(3):447-457. doi: 10.1007/s00011-023-01848-3. Epub 2024 Jan 31.
Immunoglobulin A nephropathy (IgAN) is a kidney disease characterized by the accumulation of IgA deposits in the glomeruli of the kidney, leading to inflammation and damage to the kidney. The inflammatory markers involved in IgAN remain to be defined. Gene expression analysis platforms, such as the NanoString nCounter system, are promising screening and diagnostic tools, especially in oncology. Still, their role as a diagnostic and prognostic tool in IgAN remains scarce. In this study, we aimed to validate the use of NanoString technology to identify potential inflammatory biomarkers involved in the progression of IgAN.
A total of 30 patients with biopsy-proven IgAN and 7 cases of antineutrophil cytoplasmic antibody (ANCA)-associated pauci-immune glomerulonephritis were included for gene expression measurement. For the immunofluorescence validation experiments, a total of 6 IgAN patients and 3 controls were included.
Total RNA was extracted from formalin-fixed paraffin-embedded kidney biopsy specimens, and a customized 48-plex human gene CodeSet was used to study 29 genes implicated in different biological pathways. Comparisons in gene expression were made between IgAN and ANCA-associated pauci-immune glomerulonephritis patients to delineate an expression profile specific to IgAN. Gene expression was compared between patients with low and moderate risk of progression. Genes for which RNA expression was associated with disease progression were analyzed for protein expression by immunofluorescence and compared with controls.
IgAN patients had a distinct gene expression profile with decreased expression in genes IL-6, INFG, and C1QB compared to ANCA patients. C3 and TNFRSF1B were identified as potential biomarkers for IgAN progression in patients early in their disease course. Protein expression for those 2 candidate genes was upregulated in IgAN patients compared to controls. Expression of genes implicated in fibrosis (PTEN, CASPASE 3, TGM2, TGFB1, IL2, and TNFRSF1B) was more pronounced in IgAN patients with severe fibrosis compared to those with none.
Our findings validate our NanoString mRNA profiling by examining protein expression levels of two candidate genes, C3 and TNFRSF1B, in IgAN patients and controls. We also identified several upregulated mRNA transcripts implicated in the development of fibrosis that may be considered fibrotic markers within IgAN patients.
免疫球蛋白 A 肾病（IgAN）是一种以肾脏肾小球中 IgA 沉积为特征的肾脏疾病，导致肾脏炎症和损伤。IgAN 涉及的炎症标志物仍有待确定。基因表达分析平台，如 NanoString nCounter 系统，是有前途的筛选和诊断工具，特别是在肿瘤学领域。然而，它们在 IgAN 中的诊断和预后工具的作用仍然很少。在这项研究中，我们旨在验证使用 NanoString 技术来识别与 IgAN 进展相关的潜在炎症生物标志物。
总共纳入 30 例经活检证实的 IgAN 患者和 7 例抗中性粒细胞胞质抗体（ANCA）相关性少免疫性肾小球肾炎患者进行基因表达测量。为了进行免疫荧光验证实验，总共纳入了 6 例 IgAN 患者和 3 例对照者。
从福尔马林固定石蜡包埋的肾活检标本中提取总 RNA，并使用定制的 48 plex 人类基因 CodeSet 研究 29 个涉及不同生物学途径的基因。比较 IgAN 患者和 ANCA 相关性少免疫性肾小球肾炎患者之间的基因表达，以描绘出 IgAN 特有的表达谱。比较低危和中危进展风险患者之间的基因表达。对与疾病进展相关的 RNA 表达的基因进行免疫荧光分析，并与对照者进行比较。
与 ANCA 患者相比，IgAN 患者的基因表达谱具有明显的特征，即 IL-6、INFG 和 C1QB 基因的表达下调。C3 和 TNFRSF1B 被鉴定为 IgAN 患者疾病早期进展的潜在生物标志物。与对照组相比，2 个候选基因的蛋白表达在 IgAN 患者中上调。与无严重纤维化的 IgAN 患者相比，纤维化相关基因（PTEN、CASPASE 3、TGM2、TGFB1、IL2 和 TNFRSF1B）的表达在有严重纤维化的 IgAN 患者中更为明显。
通过检查 IgAN 患者和对照者中两个候选基因 C3 和 TNFRSF1B 的蛋白表达水平，我们验证了我们的 NanoString mRNA 分析结果。我们还鉴定了几个上调的 mRNA 转录本，这些转录本与纤维化的发展有关，可能被认为是 IgAN 患者中的纤维化标志物。
