Center for Biological Systems Analysis, Albert-Ludwigs-University of Freiburg, Freiburg, Germany.
Spemann Graduate School of Biology and Medicine, Albert-Ludwigs-University of Freiburg, Freiburg, Germany.
Metabolomics. 2019 Apr 30;15(5):71. doi: 10.1007/s11306-019-1535-0.
Nephronophthisis (NPH) is an inherited form of cystic kidney disease with various extrarenal manifestations accounting for the largest amount of endstage renal disease in childhood. Patient mutations of Anks6 have also been found to cause NPH like phenotypes in animal models. However, little is known about functionality of Anks6.
OBJECTIVES/METHODS: We investigated the impact of Anks6 depletion on cellular metabolism of inner medullary collecting duct cells by GC-MS profiling and targeted LC-MS/MS analysis using two different shRNA cell lines for tetracycline-inducible Anks6 downregulation, namely mIMCD3 krab shANKS6 i5 and mIMCD3 krab shANKS6 i1.
In combination, we could successfully identify 158 metabolites of which 20 compounds showed similar alterations in both knockdown systems. Especially, large neutral amino acids, such as phenylalanine, where found to be significantly downregulated indicating disturbances in amino acid metabolism. Arginine, lysine and spermidine, which play an important role in cell survival and proliferation, were found to be downregulated. Accordingly, cell growth was diminished in tet treated mIMCD3 krab shANKS6 i5 knockdown cells. Deoxynucleosides were significantly downregulated in both knockdown systems. Hence, PARP1 levels were increased in tet treated mIMCD3 krab shANKS6 i5 cells, but not in tet treated mIMCD3 krab shANKS6 i1 cells. However, yH2AX was found to be increased in the latter.
In combination, we hypothesise that Anks6 affects DNA damage responses and proliferation and plays a crucial role in physiological amino acid and purine/pyrimidine metabolism.
肾单位肾痨（NPH）是一种遗传性囊性肾病，具有多种肾外表现，是儿童终末期肾病的主要原因。ANKs6 的患者突变也被发现会导致动物模型出现类似于 NPH 的表型。然而，ANKs6 的功能知之甚少。
目的/方法：我们通过 GC-MS 谱分析和使用两种不同的四环素诱导 ANKS6 下调的 shRNA 细胞系（即 mIMCD3 krab shANKS6 i5 和 mIMCD3 krab shANKS6 i1）进行靶向 LC-MS/MS 分析，研究了 ANKS6 耗竭对内髓集合管细胞细胞代谢的影响。
我们联合使用这两种细胞系成功鉴定出了 158 种代谢物，其中 20 种化合物在两种敲低系统中表现出相似的变化。特别是，大中性氨基酸，如苯丙氨酸，明显下调，表明氨基酸代谢紊乱。精氨酸、赖氨酸和亚精胺，它们在细胞存活和增殖中起着重要作用，被发现下调。因此，在 tet 处理的 mIMCD3 krab shANKS6 i5 敲低细胞中，细胞生长受到抑制。脱氧核苷在两种敲低系统中均显著下调。因此，在 tet 处理的 mIMCD3 krab shANKS6 i5 细胞中，PARP1 水平增加，但在 tet 处理的 mIMCD3 krab shANKS6 i1 细胞中没有增加。然而，在后一种细胞中，yH2AX 被发现增加。
综上所述，我们假设 ANKS6 影响 DNA 损伤反应和增殖，并在生理氨基酸和嘌呤/嘧啶代谢中发挥关键作用。
