Abstract：
Dent disease is a rare renal tubular disease with X-linked recessive inheritance characterized by low molecular weight proteinuria (LMWP), hypercalciuria, and nephrocalcinosis. Mutations disrupting the 2Cl-/1H+ exchange activity of chloride voltage-gated channel 5 (CLCN5) have been causally linked to the most common form, Dent disease 1 (DD1), although the pathophysiological mechanisms remain unclear. Here, we conducted the whole exome capture sequencing and bioinformatics analysis within our DD1 cohort to identify two novel causal mutations in CLCN5 (c.749 G > A, p. G250D, c.829 A > C, p. T277P). Molecular dynamics simulations of ClC-5 homology model suggested that these mutations potentially may induce structural changes, destabilizing ClC-5. Overexpression of variants in vitro revealed aberrant subcellular localization in the endoplasmic reticulum (ER), significant accumulation of insoluble aggregates, and disrupted ion transport function in voltage clamp recordings. Moreover, human kidney-2 (HK-2) cells overexpressing either G250D or T277P displayed higher cell-substrate adhesion, migration capability but reduced endocytic function, as well as substantially altered transcriptomic profiles with G250D resulting in stronger deleterious effects. These cumulative findings supported pathogenic role of these ClC-5 mutations in DD1 and suggested a cellular mechanism for disrupted renal function in Dent disease patients, as well as a potential target for diagnostic biomarker or therapeutic strategy development.
摘要：
Dent病是一种罕见的以低分子量蛋白尿（LMWP）为特征的X连锁隐性遗传性肾小管疾病，高钙尿症,和肾钙质沉着症。破坏氯电压门控通道5（CLCN5）的2Cl-/1H+交换活性的突变与最常见的形式有因果关系，凹陷病1（DD1），尽管病理生理机制尚不清楚。这里,我们在DD1队列中进行了全外显子组捕获测序和生物信息学分析，以鉴定CLCN5中的两个新的因果突变（c.749G>A，p.G250D,c.829A>C，p.T277P)。ClC-5同源模型的分子动力学模拟表明，这些突变可能会引起结构变化，使ClC-5不稳定。变异体在体外的过表达揭示了内质网（ER）中的异常亚细胞定位，不溶性聚集体的显著积累，电压钳记录中的离子传输功能中断。此外，过表达G250D或T277P的人肾-2（HK-2）细胞表现出更高的细胞-底物粘附力，迁移能力，但内吞功能降低，以及G250D显著改变的转录组概况，导致更强的有害作用。这些累积发现支持这些ClC-5突变在DD1中的致病作用，并提示了Dent病患者肾功能破坏的细胞机制。以及诊断生物标志物或治疗策略开发的潜在目标。
