College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 151-742, South Korea.
Cell Biol Toxicol. 2015 Apr;31(2):121-30. doi: 10.1007/s10565-015-9299-4. Epub 2015 Mar 26.
Measurable indicators of renal injury are required for the assessment of kidney function after toxicant challenge. In our previous study, pleckstrin homology-like domain, family A, member 3 (Phlda3) was a most greatly up-regulated molecule downstream from p53, culminating with kidney tubular injury. This study investigated the positive feedforward effect of Phlda3 on p53 in an effort to explain the largest increase of Phlda3 in injured tubules and the potential of its urine excretion. qRT-PCR assays confirmed a rapid and substantial increase in Phlda3 messenger RNA (mRNA) in the kidney cortex of mice treated with a single dose of cisplatin. Cisplatin overexpression of Phlda3 was verified by gene set analyses of three different microarray databases. In the immunohistochemistry, Phlda3 staining intensities were augmented in the tubules as kidney injury worsened. Moreover, the urinary content of Phlda3 was increased after cisplatin treatment, as were those of other kidney injury markers (Kim-1 and Timp-1). By contrast, cisplatin failed to increase Phlda3 mRNA in the liver despite hepatocyte necrosis and ensuing increases in serum transaminase activities. In NRK52E tubular cells, siRNA knockdown of Phlda3 enhanced the ability of cisplatin to increase p-Mdm2 presumably via Akt, enforcing the interaction between Mdm2 and p53. Consistently, a deficiency in Phlda3 abrogated p53 increase by cisplatin, indicating that Phlda3 promotes p53 accumulation. Phlda3 overexpression had the opposite effect. In addition, treatment with cyclosporine A or CdCl2, other nephrotoxicants, increased Phlda3 mRNA and protein levels in NRK52E cells, as did cisplatin treatment. Overall, Phlda3 may cause p53 accumulation through a feedforward pathway, facilitating tubular injury and its urine excretion.
需要可衡量的肾损伤指标来评估毒物暴露后肾脏功能。在我们之前的研究中，pleckstrin homology-like domain, family A, member 3（Phlda3）是 p53 下游上调最显著的分子，最终导致肾小管损伤。本研究试图解释为什么在损伤的肾小管中 Phlda3 上调幅度最大，以及其尿液排泄的潜在机制，研究了 Phlda3 对 p53 的正反馈作用。qRT-PCR 检测证实，顺铂单次处理后小鼠肾皮质中 Phlda3 信使 RNA（mRNA）迅速且大量增加。基因集分析证实了三种不同微阵列数据库中 Phlda3 的顺铂过表达。免疫组化结果显示，随着肾损伤的加重，Phlda3 染色强度在肾小管中增强。此外，顺铂处理后，Phlda3 的尿含量增加，其他肾损伤标志物（Kim-1 和 Timp-1）也是如此。相比之下，尽管顺铂导致肝细胞坏死和血清转氨酶活性升高，但未能增加肝中 Phlda3 mRNA 的表达。在 NRK52E 肾小管细胞中，Phlda3 的 siRNA 敲低增强了顺铂增加 p-Mdm2 的能力，可能通过 Akt 增强了 Mdm2 和 p53 之间的相互作用。一致地，Phlda3 缺陷消除了顺铂对 p53 的增加，表明 Phlda3 促进了 p53 的积累。Phlda3 的过表达则产生相反的效果。此外，环孢素 A 或 CdCl2 等其他肾毒性药物处理以及顺铂处理均增加了 NRK52E 细胞中 Phlda3 mRNA 和蛋白水平。总之，Phlda3 可能通过正反馈途径导致 p53 积累，从而促进肾小管损伤及其尿液排泄。
