Koeppert Sina, Ghallab Ahmed, Peglow Sarah, Winkler Camilla Franziska, Graeber Steffen, Büscher Andrea, Hengstler Jan Georg, Jahnen-Dechent Willi
Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University, Aachen, Germany.
Leibniz Research Centre for Working Environment and Human Factors, Dortmund, Germany.
Front Cell Dev Biol. 2021 Apr 29;9:633925. doi: 10.3389/fcell.2021.633925. eCollection 2021.
The liver-derived plasma protein fetuin A is a systemic inhibitor of ectopic calcification. Fetuin-A stabilizes calcium phosphate mineral initially as ion clusters to form calciprotein monomers (CPM), and then as larger multimeric consolidations containing amorphous calcium phosphate (primary CPP, CPP 1) or more crystalline phases (secondary CPP, CPP 2). CPM and CPP mediate excess mineral stabilization, transport and clearance from circulation.
We injected i.v. synthetic fluorescent CPM and studied their clearance by live two-photon microscopy. We analyzed organ sections by fluorescence microscopy to assess CPM distribution. We studied cellular clearance and cytotoxicity by flow cytometry and live/dead staining, respectively, in cultured macrophages, liver sinusoidal endothelial cells (LSEC), and human proximal tubule epithelial HK-2 cells. Inflammasome activation was scored in macrophages. Fetuin A monomer and CPM charge were analyzed by ion exchange chromatography.
Live mice cleared CPP in the liver as published previously. In contrast, CPM were filtered by kidney glomeruli into the Bowman space and the proximal tubules, suggesting tubular excretion of CPM-bound calcium phosphate and reabsorption of fetuin A. Fetuin-A monomer clearance was negligible in liver and low in kidney. Anion exchange chromatography revealed that fetuin A monomer was negatively charged, whereas CPM appeared neutral, suggesting electrochemical selectivity of CPM versus fetuin A. CPM were non-toxic in any of the investigated cell types, whereas CPP 1 were cytotoxic. Unlike CPP, CPM also did not activate the inflammasome.
Fetuin-A prevents calcium phosphate precipitation by forming CPM, which transform into CPP. Unlike CPP, CPM do not trigger inflammation. CPM are readily cleared in the kidneys, suggesting CPM as a physiological transporter of excess calcium and phosphate. Upon prolonged circulation, e.g., in chronic kidney disease, CPM will coalesce and form CPP, which cannot be cleared by the kidney, but will be endocytosed by liver sinusoidal endothelial cells and macrophages. Large amounts of CPP trigger inflammation. Chronic CPM and CPP clearance deficiency thus cause calcification by CPP deposition in blood vessels and soft tissues, as well as inflammation.
肝脏来源的血浆蛋白胎球蛋白A是异位钙化的全身抑制剂。胎球蛋白A最初将磷酸钙矿物质稳定为离子簇，形成钙蛋白单体（CPM），然后形成包含无定形磷酸钙（初级CPP，CPP 1）或更多结晶相（次级CPP，CPP 2）的更大的多聚体聚集体。CPM和CPP介导多余矿物质的稳定、运输和从循环中的清除。
我们静脉注射合成荧光CPM，并通过实时双光子显微镜研究其清除情况。我们通过荧光显微镜分析器官切片以评估CPM分布。我们分别通过流式细胞术和活/死染色研究培养的巨噬细胞、肝窦内皮细胞（LSEC）和人近端肾小管上皮HK-2细胞中的细胞清除和细胞毒性。对巨噬细胞中的炎性小体激活进行评分。通过离子交换色谱分析胎球蛋白A单体和CPM电荷。
活体小鼠如先前报道的那样在肝脏中清除CPP。相比之下，CPM被肾小球滤过进入鲍曼腔和近端小管，提示CPM结合的磷酸钙经肾小管排泄以及胎球蛋白A的重吸收。胎球蛋白A单体在肝脏中的清除可忽略不计，在肾脏中清除率较低。阴离子交换色谱显示胎球蛋白A单体带负电荷，而CPM呈中性，提示CPM相对于胎球蛋白A具有电化学选择性。CPM在任何研究的细胞类型中均无毒性，而CPP 1具有细胞毒性。与CPP不同，CPM也不激活炎性小体。
胎球蛋白A通过形成CPM来防止磷酸钙沉淀，CPM会转变为CPP。与CPP不同，CPM不会引发炎症。CPM在肾脏中易于清除，提示CPM是过量钙和磷的生理转运体。在长时间循环时，例如在慢性肾脏病中，CPM会聚集并形成CPP，CPP不能被肾脏清除，但会被肝窦内皮细胞和巨噬细胞内吞。大量的CPP会引发炎症。因此，慢性CPM和CPP清除缺陷会通过CPP在血管和软组织中的沉积以及炎症导致钙化。
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