Department of Development and Regeneration, Organ System Cluster, Group of Biomedical Sciences, KU Leuven, Belgium.
Department of Pediatrics, University Hospitals Leuven, Belgium. (Herestraat 49. 3000, Leuven, Belgium).
Curr Pharm Des. 2017;23(38):5911-5918. doi: 10.2174/1381612823666171009143146.
Finding the right drug-dosage for neonates is still a challenge. Until now, neonatal doses are extrapolated from adults and children doses. However, there are differences between neonatal and adult kidney physiology that should be considered, especially when it comes to drug metabolism and/or transport. Studying renal drug disposition in neonates is limited by the lack of reliable human cell models.
To illustrate the feasibility of developing an in vitro model for neonatal proximal tubule epithelial cells (nPTECs) to study renal drug disposition at this age.
nPTECs were isolated from urine samples of neonates of different gestational ages and were conditionally immortalized using a temperature sensitive SV40T antigen and human telomerase hTERT. Cell clones were characterized on gene expression level for PTEC markers such as P-glycoprotein (ABCB1), aquaporin1 (AQP1), and organic cation transport protein 2 (SLC22A2), and for kidney progenitor cell and podocyte markers. In addition, protein expression and functional assessment were performed for P-gp and OCT2.
We established 101 clonal cell lines of conditionally immortalized nPTECs derived from neonatal urines. Characterization of primary cells lines showed expression of genes from different cell types such as progenitors, PTECs and podocytes, however the developed conditionally immortalized nPTECs only expressed proximal tubule markers. Quantitative PCR analysis confirmed the expression of proximal tubule markers in nPTECs similar to the adult control PTECs. P-gp was expressed in nPTECs derived from the different gestational ages with a similar functionality compared with adult derived PTECs. In contrast, OCT2 functionality was significantly lower in nPTEC cell lines compared with adult PTECs.
We demonstrate the feasibility of culturing proximal tubule epithelial cells with high efficiency from urine of neonates. These cells expressed PTEC-specific genes and functional drug transporters. The cell model presented is a valuable tool to study proximal tubule physiology and pharmacology in newborns. In addition, we demonstrate the physiological differences between the neonatal and adult kidney, which emphasizes the importance of studying drug disposition in neonatal models instead of extrapolating from adult data.
为新生儿找到合适的药物剂量仍然是一个挑战。到目前为止，新生儿的剂量是从成人和儿童的剂量推断出来的。然而，新生儿和成人肾脏生理学之间存在差异，这些差异应该被考虑在内，尤其是在药物代谢和/或转运方面。研究新生儿的肾脏药物处置受到缺乏可靠的人类细胞模型的限制。
说明开发用于研究该年龄段肾脏药物处置的体外新生儿近端肾小管上皮细胞（nPTEC）模型的可行性。
从不同胎龄新生儿的尿液样本中分离 nPTEC，并使用温度敏感的 SV40T 抗原和人端粒酶 hTERT 条件性永生化。通过基因表达水平对细胞克隆进行鉴定，鉴定出 PTEC 标志物，如 P 糖蛋白（ABCB1）、水通道蛋白 1（AQP1）和有机阳离子转运蛋白 2（SLC22A2），以及肾脏祖细胞和足细胞标志物。此外，还对 P-糖蛋白和 OCT2 进行了蛋白表达和功能评估。
我们从新生儿尿液中建立了 101 个条件性永生化 nPTEC 克隆细胞系。原代细胞系的鉴定表明，不同细胞类型的基因表达，如祖细胞、PTEC 和足细胞，但发育的条件性永生化 nPTEC 仅表达近端小管标志物。定量 PCR 分析证实，nPTEC 中表达的近端小管标志物与成人对照 PTEC 相似。从不同胎龄的 nPTEC 中表达 P-糖蛋白，其功能与成人来源的 PTEC 相似。相比之下，nPTEC 细胞系中 OCT2 的功能明显低于成人 PTEC。
我们证明了从新生儿尿液中高效培养具有高效率的近端肾小管上皮细胞的可行性。这些细胞表达了 PTEC 特异性基因和功能药物转运体。所提出的细胞模型是研究新生儿近端小管生理学和药理学的有价值的工具。此外，我们证明了新生儿和成人肾脏之间的生理差异，这强调了在新生儿模型中研究药物处置而不是从成人数据推断的重要性。
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