Department of Internal Medicine, The University of Texas Health Science Center at Houston, Houston, Texas, United States of America.
PLoS One. 2013;8(1):e53342. doi: 10.1371/journal.pone.0053342. Epub 2013 Jan 10.
Dot1l encodes histone H3 K79 methyltransferase Dot1a. Mice with Dot1l deficiency in renal Aqp2-expressing cells (Dot1l(AC)) develop polyuria by unknown mechanisms. Here, we report that Aqp5 links Dot1l deletion to polyuria through Aqp2. cDNA array analysis revealed and real-time RT-qPCR validated Aqp5 as the most upregulated gene in Dot1l(AC) vs. control mice. Aqp5 protein is barely detectable in controls, but robustly expressed in the Dot1l(AC) kidneys, where it colocalizes with Aqp2. The upregulation of Aqp5 is coupled with reduced association of Dot1a and H3 dimethyl K79 with specific subregions in Aqp5 5' flanking region in Dot1l(AC) vs. control mice. In vitro studies in IMCD3, MLE-15 and 293Tcells using multiple approaches including real-time RT-qPCR, luciferase reporter assay, cell surface biotinylation assay, colocalization, and co-immunoprecipitation uncovered that Dot1a represses Aqp5. Human AQP5 interacts with AQP2 and impairs its cell surface localization. The AQP5/AQP2 complex partially resides in the ER/Golgi. Consistently, AQP5 is expressed in none of 15 normal controls, but in all of 17 kidney biopsies from patients with diabetic nephropathy. In the patients with diabetic nephropathy, AQP5 colocalizes with AQP2 in the perinuclear region and AQP5 expression is associated with impaired cellular H3 dimethyl K79. Taken together, these data for the first time identify Aqp5 as a Dot1a potential transcriptional target, and an Aqp2 binding partner and regulator, and suggest that the upregulated Aqp5 may contribute to polyuria, possibly by impairing Aqp2 membrane localization, in Dot1l(AC) mice and in patients with diabetic nephropathy.
Dot1l 编码组蛋白 H3 K79 甲基转移酶 Dot1a。在肾 Aqp2 表达细胞中缺乏 Dot1l 的小鼠（Dot1l(AC)）通过未知机制发生多尿。在这里，我们报告 Aqp5 通过 Aqp2 将 Dot1l 的缺失与多尿联系起来。cDNA 芯片分析显示并通过实时 RT-qPCR 验证 Aqp5 是 Dot1l(AC)与对照小鼠相比最上调的基因。在对照中几乎检测不到 Aqp5 蛋白，但在 Dot1l(AC)肾脏中强烈表达，在那里它与 Aqp2 共定位。Aqp5 的上调与 Dot1a 和 H3 二甲基 K79 与 Dot1l(AC)与对照小鼠相比在 Aqp5 5'侧翼区的特定亚区的结合减少有关。在 IMCD3、MLE-15 和 293T 细胞中的体外研究使用包括实时 RT-qPCR、荧光素酶报告基因测定、细胞表面生物素化测定、共定位和共免疫沉淀在内的多种方法表明 Dot1a 抑制 Aqp5。人 AQP5 与 AQP2 相互作用并损害其细胞表面定位。AQP5/AQP2 复合物部分位于 ER/Golgi 中。一致地，AQP5 在 15 个正常对照中的任何一个中都不表达，但在所有 17 个糖尿病肾病患者的肾活检中都表达。在糖尿病肾病患者中，AQP5 在核周区域与 AQP2 共定位，并且 AQP5 表达与细胞内 H3 二甲基 K79 的损害相关。总之，这些数据首次将 Aqp5 鉴定为 Dot1a 的潜在转录靶标，以及 Aqp2 的结合伴侣和调节剂，并表明上调的 Aqp5 可能通过损害 Dot1l(AC)小鼠和糖尿病肾病患者的 Aqp2 膜定位导致多尿。
