ApoL1 Overexpression Drives Variant-Independent Cytotoxicity
ApoL1 Overexpression Drives Variant-Independent Cytotoxicity
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DOI：
10.1681/asn.2016121322
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发表时间：
2018-03-01
影响因子：
13.6
通讯作者：
Sedor, John R.
中科院分区：
文献类型：
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作者：
O'Toole, John F.;Schilling, William;Sedor, John R.
APOL1基因的编码变异与非洲祖先人群中的肾脏疾病有关；然而，其潜在的生物机制仍不确定。变异体依赖的自噬和细胞毒性细胞死亡被认为是介导肾损伤的致病途径。为了检验这种可能性，我们在HEK293细胞中使用四环素调控系统有条件地表达了APOL1-G0(参考)、-G1和-G2(变体)。用生化方法监测自噬，用多种方法测定细胞死亡。用原子吸收光谱法测定细胞内Na+、K+含量，用全细胞膜片钳技术测定APOL1依赖电流。参考和变异的APOL1均不能诱导自噬。在高表达水平，APOL1-G0、-G1和-G2插入质膜并形成pH敏感的阳离子通道，导致细胞Na+和K+梯度的崩溃，p38丝裂原活化蛋白激酶的磷酸化，以及细胞死亡，没有变异依赖的差异。在全细胞膜片钳实验中，APOL1-G0和-G2表现出相似的通道特性。在低表达水平，无论是参考的还是变异的APOL1都不定位在质膜上，Na+和K+的梯度保持不变，细胞仍然存活。我们的结果表明，APOL1介导的孔道形成对APOL1的胰酶分解活性至关重要，并在过表达系统中驱动APOL1介导的细胞毒作用。在生理表达水平上没有细胞毒性表明细胞内K+丢失依赖于变异，细胞毒性不会导致肾脏疾病的进展。
Coding variants in the APOL1 gene are associated with kidney diseases in African ancestral populations; yet, the underlying biologic mechanisms remain uncertain. Variant-dependent autophagic and cytotoxic cell death have been proposed as pathogenic pathways mediating kidney injury. To examine this possibility, we conditionally expressed APOL1-G0 (reference), -G1, and -G2 (variants) using a tetracycline-regulated system in HEK293 cells. Autophagy was monitored biochemically and cell death was measured using multiple assays. We measured intracellular Na+ and K+ content with atomic absorption spectroscopy and APOL1-dependent currents with whole-cell patch clamping. Neither reference nor variant APOL1s induced autophagy. At high expression levels, APOL1-G0, -G1, and -G2 inserted into the plasma membrane and formed pH-sensitive cation channels, causing collapse of cellular Na+ and K+ gradients, phosphorylation of p38 mitogen-activated protein kinase, and cell death, without variant-dependent differences. APOL1-G0 and -G2 exhibited similar channel properties in whole-cell patch clamp experiments. At low expression levels, neither reference nor variant APOL1s localized on the plasma membrane, Na+ and K+ gradients were maintained, and cells remained viable. Our results indicate that APOL1-mediated pore formation is critical for the trypanolytic activity of APOL1 and drives APOL1-mediated cytotoxicity in overexpression systems. The absence of cytotoxicity at physiologic expression levels suggests variant-dependent intracellular K+ loss and cytotoxicity does not drive kidney disease progression.