Binding capacity and pathophysiological effects of IgA1 from patients with IgA nephropathy on human glomerular mesangial cells
Binding capacity and pathophysiological effects of IgA1 from patients with IgA nephropathy on human glomerular mesangial cells
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DOI：
10.1111/j.1365-2249.2004.02408.x
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发表时间：
2004-04
影响因子：
4.6
通讯作者：
Y. Wang;M. Zhao;Y.‐K. Zhang;X. Li;H-y Wang
中科院分区：
文献类型：
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作者：
Y. Wang;M. Zhao;Y.‐K. Zhang;X. Li;H-y Wang
IgA 在肾小球系膜中的沉积以及与系膜细胞的相互作用很可能是 IgA 肾病 (IgAN) 的最终共同途径。 IgAN 患者 IgA1 铰链区 O 糖基化的改变可能导致 IgA1 通过新型 IgA1 受体发生系膜沉积和系膜细胞 (MC) 激活，并且可能是 IgAN 发病机制中的关键事件。本研究的目的是调查 IgAN 患者和健康对照的 IgA1 对人类系膜细胞 (HMC) 的结合能力和生物效应。使用 Jacalin 亲和层析分离血清 IgA1，加热至聚集形式 (aIgA1) 并用 125 I 标记。通过放射性配体结合测定评估 aIgA1 与培养的原代 HMC 的体外结合能力，并通过竞争性抑制测定测定结合的特异性。通过共聚焦分析研究细胞内钙释放，通过蛋白质印迹分析测定细胞外信号调节激酶（ERK）的磷酸化。通过流式细胞术证明细胞周期的变化，并通过直接细胞计数评估HMC增殖。分别通过 RT-PCR 和间接竞争 ELISA 检测 TGF-β mRNA 的表达和上清液纤连蛋白的产生。来自 IgAN 患者和正常对照的 aIgA1 以剂量依赖性、可饱和的方式与 HMC 结合，并且在每 0·5 ml aIgA1 大约 500 pmol 时饱和。然而，来自 IgAN 患者的 aIgA1 以更高的速度与 HMC 结合，Scatchard 分析显示，来自健康对照的 aIgA1 的 Kd 为 (8·89 ± 2·1) × 10−8m，而健康对照的 aIgA1 为 (4·3 ± 1·2) × 10−7m (P = 0·026)。这种结合是特异性的，因为它仅被未标记的 Mono-IgA1 抑制。 (mIgA1) 而不是血清白蛋白或 IgG。 IgAN 患者的 aIgA1 可以诱导细胞内钙的释放、ERK 磷酸化、DNA 合成、HMC 增殖、TGF-βmRNA 表达和 HMC 中纤连蛋白的分泌，其时间依赖性与健康对照的 aIgA1 类似，但效果更强，持续时间更长（分别 P < 0·05）。我们得出的结论是，与健康对照的 aIgA1 相比，IgAN 患者的 aIgA1 与 HMC 具有更高的结合能力和更强的生物学效应。这表明 IgA1 和 HMC 之间的直接相互作用以及随后的病理生理反应可能在 IgAN 的发病机制中发挥重要作用。
IgA deposition in glomerular mesangium and the interaction with mesangial cells may well be the final common pathway to IgA nephropathy (IgAN). Altered hinge‐region O‐glycosylation of IgA1 from patients with IgAN may predispose to mesangial deposition and activation of the mesangial cell (MC) by IgA1, via a novel IgA1 receptor, and may be a key event in the pathogensis of IgAN. The aim of this study was to investigate the binding capacity and biological effects of IgA1, from both patients with IgAN and healthy controls, on human mesangial cells (HMC). Serum IgA1 was isolated with jacalin affinity chromatography, heated to aggregated form (aIgA1) and labelled with 125I. Binding capacity of aIgA1 in vitro to cultured primary HMC was evaluated by a radioligand binding assay and the specificity of binding was determined by a competitive inhibition assay. Intracellular calcium release was studied by confocal analysis and phosphorylation of extracellular signal‐regulated kinase (ERK) was determined by Western blot analysis. Change of cell cycles was demonstrated by flow cytometry and HMC proliferation was evaluated by direct cell count. Expression of TGF‐β mRNA and production of supernatant fibronectin were tested by RT‐PCR and indirect competitive ELISA, respectively. aIgA1 from both the patients with IgAN and normal controls bound to HMC in a dose‐dependent, saturable manner, and was saturated at approximately 500 pmoles per 0·5 ml of aIgA1. aIgA1 from patients with IgAN, however, bound to HMC at a higher speed and Scatchard analysis revealed a Kd of (8·89 ± 2·1) × 10−8mversus (4·3 ± 1·2) × 10−7m for aIgA1 from healthy controls (P = 0·026).The binding was specific because it was only inhibited by unlabelled Mono‐IgA1 (mIgA1) and not by serum albumin or IgG. aIgA1 from patients with IgAN could induce release of intracellular calcium, phosphorylation of ERK, DNA synthesis, proliferation of HMC, expression of TGF‐βmRNA and secretion of fibronectin in HMC in a similar time‐dependent manner as aIgA1 from healthy controls, but the effects were much stronger and the durations were much longer (P < 0·05, respectively). We conclude that aIgA1 from patients with IgAN has a higher binding capacity to HMC and stronger biological effects than aIgA1 from healthy controls. This suggests that direct interaction between IgA1 and HMC and subsequential pathophysiological responses may play an important role in the pathogenesis for IgAN.