The Role of Macrophages in the Development of Human Renal Allograft Fibrosis in the First Year after Transplantation. Homo sapiens

The aim of this study was to investigate the role of infiltrating macrophages in renal allograft fibrosis. Forty-six protocol renal allograft biopsies obtained one-year after transplantation were stained with Sirius Red to quantify fibrosis and double stained with CD68 and CD206 to identify the proportion of alternately activated (M2) macrophages. 23 protocol biopsies obtained 12 months post transplant were analyzed for gene expression by microarray, which was correlated with macrophage infiltration and the severity of fibrosis. Phenotypic analysis showed 92% of infiltrating macrophages exhibited an M2 phenotype with CD68+CD206+ dual staining. Gene microarrays demonstrated a distinct alloimmune response despite the lack of rejection and inflammatory infiltrate with upregulation of interferon-γ-response genes. This suggests that following initiation of Th1 driven macrophage proliferation or infiltration, M2 macrophages contribute to tubular injury and progression of fibrosis. Overall design: 23 protocol renal biopsies were obtained from patients at 12 month post transplant. The study population was divided into two groups according to the number of infiltrating macrophages (CD68 positive cells) (Group I: Recipients with a low number of infiltrating macrophages, CD68 positive cells 1) and those without. To correlate gene expression with kidney fibrosis, or intensity of CD68 infiltrate, Spearman correlations analysis of the gene expression data with 12 month IFTA was performed and the correlation co-efficiency and its p value calculated. Gene Ontology enrichment and IPA pathway and network analysis (Ingenuity System Inc.) were performed on the associated genes. All p-values were two-sided, and p < 0.05 was considered significant.本研究旨在探讨浸润性巨噬细胞在肾移植纤维化中的作用。本研究纳入46份移植术后1年的方案性肾移植活检标本，采用天狼星红（Sirius Red）染色量化纤维化程度，并通过CD68与CD206双染色鉴定交替活化（M2型）巨噬细胞的占比。其中23份移植术后12个月的方案性活检标本通过微阵列（microarray）进行基因表达检测，并将检测结果与巨噬细胞浸润程度及纤维化严重程度进行关联分析。表型分析结果显示，92%的浸润性巨噬细胞呈CD68+CD206+双阳性表型，即M2型巨噬细胞。基因微阵列分析表明，尽管未观察到排斥反应及炎性浸润，但仍存在显著的同种免疫应答，且干扰素-γ（interferon-γ）应答基因表达上调。上述结果提示，在Th1驱动的巨噬细胞增殖或浸润启动后，M2型巨噬细胞可介导肾小管损伤并促进纤维化进展。总体实验设计：从23例肾移植术后12个月的患者中获取方案性肾活检标本。根据浸润性巨噬细胞（CD68阳性细胞）数量将研究对象分为两组：第I组为浸润性巨噬细胞数量较低的受者，其余为浸润性巨噬细胞数量较高的受者。为探究基因表达与肾纤维化程度或CD68阳性细胞浸润强度的关联，本研究对基因表达数据与术后12个月的间质纤维化与肾小管萎缩（Interstitial Fibrosis and Tubular Atrophy, IFTA）评分进行斯皮尔曼（Spearman）相关分析，并计算相关系数及其p值。对筛选出的关联基因进行基因本体（Gene Ontology, GO）富集分析、IPA通路与网络分析（Ingenuity系统公司）。所有p值均采用双侧检验，p<0.05被认为具有统计学显著性。