Nephrin研究的机遇和挑战（上）

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未明原因的pFSGS

近来研究显示，抗-Nephrin自身抗体（Anti-nephrin Ab）在微小病变（MCD）和局灶性节段性肾小球硬化（FSGS）的发生中扮演重要的作用，本文概述了抗-Nephrin抗体作为MCD和原发性FSGS（pFSGS）的血液循环渗透因子的研究进展，并对将来的研究方向提出了展望。

循环因子理论与FSGS

当1972年Hoyer等[6]第一次报道了肾移植术后复发FSGS的患者开始，研究人员就推测血液中某种循环渗透因子可能引起了FSGS的产生。随之流行病学研究显示，移植后FSGS具有较高的复发率，有时在移植后几小时内就会发生，这结果进一步促使研究人员相信，致病因子可能不是“肾脏内的局部现象”，而是一种血液循环渗透因子，它通过改变肾小球屏障来损伤足细胞[7-8]。直到2012年Gallon等[9]在一个FSGS复发的患者案例研究中发现，当移植肾被切除并再次移植给另一个没有FSGS病史的受者时，肾脏的功能得到了恢复，且FSGS特有的组织病理学损害也发生了逆转，这基本确定了是血液中存在的某些循环渗透因子导致了FSGS的发生。

随后几年，大量的研究资源投入到寻找在MCD和pFSGS蛋白尿形成中起关键作用的循环渗透因子[10-11]。尽管投入了非常大的资源和研究努力，这些未知的循环通透性因子的鉴定一直是艰苦漫长的，研究者并没有找到可靠一致的生物标志物。

一些被认为增加血清蛋白血管透过性的候选生物标志物分子（表1），包括可溶性尿激酶型纤溶酶原激活物受体(suPAR)[12]、心肌营养素样细胞因子-1 (CLCF-1)[13]、可溶性CD40配体[14]等等，已经发现与MCD和pFSGS的进展和复发相关，但是验证其临床效用的研究表明，这些标记物并不在MCD和FSGS的发展中起到决定性作用，它们也可以在健康人和非肾病综合征患者血清中检测到[15-17]。

表1. FSGS和MCD相关循环渗透因子及其局限性

Nephrin蛋白介绍

1966年，在一组患有先天性肾病综合征（NPHS1)[39]的芬兰家系中，研究发现这是一种罕见的遗传基因突变造成的肾脏疾病，但导致这种疾病的具体基因未知。直到1998年NPHS1基因所在的关键染色体区域被鉴定和测序， NPHS1基因的表达产物被命名为Nephrin，其基因座定位于染色体19q12-q13.1[40-41]。虽然Nephrin蛋白的精确结构和功能在此时仍然未知，但据推测，其结构域结构类似于属于免疫球蛋白家族的一大类细胞粘附受体，它可以作为粘附受体和信号蛋白[40]发挥作用。在后续研究中[42-43]，证实Nephrin是形成足细胞裂孔隔膜相关复合物（slit diaphragm, SD）的组成部分，其与肾小球内皮细胞和基底膜（GBM）一起形成肾小球滤过屏障。（图1）

图1. Nephrin抗体诱导纵膈改变、足细胞损伤和蛋白质渗漏入尿液中

Nephrin是一种跨膜蛋白，其N-末端为胞外片段，C-末端为胞内结构域，使用对人Nephrin的N-末端特异的抗体检测证实了其位于肾的纵膈[44]。整个蛋白由八个免疫球蛋白样胞外结构域、一个纤连蛋白III型样结构域、一个跨膜结构域和一个短的胞内结构域组成，它通过与肌动蛋白细胞骨架[40,45]的相互作用而维持足细胞的组织形态结构和功能。作为免疫球蛋白超家族的细胞表面受体蛋白，Nephrin还参与细胞间粘附和信号传导功能。当Src激酶家族的Fyn磷酸化Nephrin的胞内结构域的六个酪氨酸残基(Tyr1114、Tyr1136、Tyr1176、Tyr1183、Tyr1193、Tyr1217)中的一个或多个时，由Nephrin的胞内尾部介导的下游信号传导被激活[46]。具体表现为磷酸化的胞内结构域会与几种足细胞胞质蛋白相互作用，包括podocin、CD2AP、NEPH1和磷脂酰肌醇3-激酶(PI3K)，以将下游信号传递到肌动蛋白细胞骨架，从而调节足细胞的结构完整性和肾小球滤过狭缝的功能[47-48]。磷酸化Nephrin促进Nck衔接蛋白、ZO-1、连环蛋白、桶蛋白、足细胞素、CD2-AP和PI3K等的募集并与之相互作用，Nck衔接蛋白发出肌动蛋白重塑的信号，并参与多种细胞内信号通路的调节，从而影响对足细胞稳定至关重要的肌动蛋白的聚合动力学[48-49]（图2）。

图2. 相邻足突之间的缝隙层示意图。胞内结构域在磷酸化时通过级联反应与胞质蛋白相互作用，诱导信号传递，调节肌动蛋白聚合动力学并调整足细胞结构。抗-Nephrin抗体和Nephrin之间的相互作用通过导致Nephrin远离狭缝层，重新定位而失去狭缝屏障功能。

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