Fibrocyte Contribution to Systemic Fibrosis in Chronic Kidney Disease

描述（由申请人提供）： 暴露于钆基磁共振成像（MRI）造影剂是发生肾源性系统性纤维化（NSF）的主要风险因素，NSF是一种在急性或慢性肾损害患者中首次发现的严重衰弱性疾病。迄今为止，MRI造影剂在这些患者中被认为是安全的，因此被不加区别地使用。基于钆的造影剂仍然是MR成像的主流，并且仍然缺乏替代品。申请人的初步数据显示，MRI造影剂在体外对人成纤维细胞产生直接增殖作用，增加细胞外基质合成。暴露于MRI造影剂的肾功能不全大鼠表现出显著的皮肤异常，包括骨髓源性细胞的募集。MRI对比剂处理的小鼠类似地表现出皮肤纤维化和真皮细胞过多。此外，在受影响的大鼠和小鼠皮肤以及MRI造影剂治疗动物的其他器官（如肾脏和肝脏）中检测到钆。本提案的总体目标是确定在肾功能不全的情况下暴露于基于钆的MRI造影剂后触发异常纤维化模式的候选机制。中心假设是，在肾功能不全的情况下，MRI造影剂暴露触发靶器官（如皮肤）的促纤维化状态。随后产生的特异性趋化因子激活并募集骨髓来源的和循环的间充质前体细胞或纤维细胞到受影响的区域。这些细胞通过合成和减少细胞外基质的产生并诱导驻留细胞的增殖来复合纤维化过程。实验将解决以下具体目标：1a确定钆基造影剂诱导NSF的机制。申请人已经证明，骨髓来源的细胞占皮肤病变中真皮细胞结构的约44%。皮肤中的钆沉积是否促进促纤维化细胞因子的产生 这导致纤维细胞募集，或者钆刺激在其他部位，如骨髓，编程成纤维细胞前体骨特定部位是未知的。申请方将通过对骨髓供体（转基因，“标记”）大鼠进行MRI造影剂暴露“预处理”，然后将该骨髓移植到致死性辐照动物体内来对此进行测试。1b：证明钆基造影剂诱导循环纤维细胞增加。将在大鼠中定量MRI造影剂对特定时间点循环纤维细胞数量的影响。图2a：确定负责将骨髓来源的细胞募集到特定组织的趋化因子和受体。假设是单核细胞趋化蛋白-（MCP-）1/趋化因子受体2（CCR 2）轴调节MRI造影剂诱导的系统性纤维化中纤维细胞的募集。导致循环细胞募集至NSF病变的化学介质尚未充分探索。将在具有遗传缺陷的动物中检查对NSF的抗性，将探索CCR 2。该蛋白的作用将通过骨髓移植实验进行验证，其中将CCR 2缺陷型骨髓移植到正常（野生型）小鼠中，反之亦然。2b：检验钆的生物分布在小鼠和大鼠的正常肾功能和肾功能不全之间存在差异的假设。尽管先前的出版物明确指出肝脏是MRI造影剂治疗的大鼠和小鼠中钆的主要储存库，但皮肤总是涉及NSF，并且其程度远远大于申请人迄今为止发现的实验动物中的任何肝脏病理学。如果钆的沉积是引发疾病的病灶，那么可能钆的生物分布在肾功能不全和肾功能不全的状态之间是不同的。将在肾功能不全（肾次全切除术）动物和假手术对照动物中比较NSF可能影响的器官（皮肤、肺、心脏、隔膜、肝、脾、骨、肌肉）中的钆蓄积。这些发现将有助于更好地了解系统性纤维化是如何发生的，以及为什么某些器官是靶向的。

DESCRIPTION (provided by applicant): Exposure to gadolinium-based magnetic resonance imaging (MRI) contrast is a major risk factor in the acquisition of nephrogenic systemic fibrosis (NSF), a severely debilitating disease first recognized in patients with acute or chronic renal impairment. MRI contrast agents heretofore were considered safe in these patients and thus indiscriminately used. Gadolinium-based contrast agents remain a mainstay for MR imaging and alternatives are still lacking. The applicant's preliminary data show that MRI contrast elicits a direct proliferative effect on human fibroblasts with an increase in extracellular matrix synthesis in vitro. Rats with renal insufficiecy exposed to MRI contrast demonstrate significant skin abnormalities, including recruitment of bone marrow-derived cells. MRI contrast-treated mice similarly demonstrate skin fibrosis with dermal hypercellularity. Furthermore, gadolinium has been detected in affected rat and mouse skin as well as other organs (such as the kidneys and livers) from MRI contrast-treated animals. The overall objective of this proposal is to identify candidate mechanisms that trigger the aberrant pattern of fibrosis after exposure to gadolinium-based MRI contrast in the setting of renal insufficiency. The central hypothesis is that in the setting of renal insufficiency, MRI contrast exposure triggers a pro-fibrotic state in target organs, such as the skin. Subsequent generation of specific chemokines activate and recruit bone marrow-derived and circulating mesenchymal precursor cells, or fibrocytes, to the affected areas. These cells compound the fibrotic process by synthesizing and reducing the generation of extracellular matrix and inducing the proliferation of resident cells. Experiments will address the following Specific Objectives: 1a Determine the mechanism by which gadolinium-based contrast induces NSF. The applicant has proven that bone marrow-derived cells constitute around 44% of the dermal cellularity in skin lesions. Whether gadolinium deposition in the skin elicits the generation of pro-fibrotic cytokines that lead to fibrocyte recruitment or if gadolinium stimulation in other sites, such as the bone marrow, programs fibroblast precursors to hone to specific sites is unknown. The applicant will test this by "priming" bone marrow donor (transgenic, "tagged") rats with exposure to MRI contrast and then transplanting this bone marrow into lethally-irradiated animals. 1b: Demonstrate that gadolinium-based contrast induces an increase in circulating fibrocytes. The effect of MRI contrast on the number of circulating fibrocytes at specific time points will be quantified in rats. 2a: Determine the chemokines and receptors responsible for recruitment of bone marrow-derived cells to specific tissues. The hypothesis is that the monocyte chemoattractant protein- (MCP-) 1/chemokine receptor 2 (CCR2) axis modulates the recruitment of fibrocytes in MRI contrast-induced systemic fibrosis. The chemical mediators that lead to the recruitment of circulating cells to NSF lesions is not well-explored. Resistance to NSF will be examined in animals with a genetic deficiency CCR2 will be explored. The role of this protein will be validated by using bone marrow transplant experiments where CCR2-deficient bone marrow is transplanted to normal (wild-type) mice and vice-versa. 2b: Test the hypothesis that biodistribution of gadolinium differs between normal renal function and renal insufficiency in mice and rats. Even though prior publications definitively indicate that the liveris a major reservoir for gadolinium in MRI-contrast treated rats and mice, the skin is invariably involved in NSF and to a far greater extent than any liver pathology in experimental animals found to date by the applicant. If the deposition of gadolinium is the nidus for triggering the disease, perhaps biodistribution of gadolinium differs between the states of renal sufficiency and insufficiency. Gadolinium accumulation in organs potentially affected in NSF (skin, lungs, heart, diaphragm, liver, spleen, bone, muscle) will be compared in animals with renal insufficiency (subtotal nephrectomies) and sham-treated controls. The findings will lead to a better understanding of how systemic fibrosis occurs and why certain organs are targeted.