Genomewide transcriptional analysis of growth hormone-treated human podocytes. Genomewide transcriptional analysis of growth hormone-treated human podocytes

The growth hormone plays a significant role in normal renal function and overactive growth hormone signaling has been implicated in proteinuria in diabetes. Earlier studies from our group have shown that the glomerular podocytes, which play an essential role in renal filtration, express the growth hormone receptor, suggesting the direct action of growth hormone on these cells. Nevertheless, the precise mechanism and the downstream pathways that are induced by the excess growth hormone in these podocytes leading to diabetic nephropathy are not clearly established. To compressively understand the growth hormone’s effect on podocytes at transcript level we performed RNA-Sequencing. Conditionally immortalized human podocytes were employed in this study. Overall design: Human podocytes in duplicates were treated with (500 ng/ml) or without growth hormone for 30 min and total RNA was isolated. Both by running on an agarose gel and using the bioanalyzer confirmed the quality of RNA. High-quality RNA libraries were constructed using mRNA templates, random hexamers, oligo-dT primers, and Illumina second strand synthesis buffer. RNA sequencing was performed on Illumina 2500 Hi-Seq devise by pair-end reads at Nucleome Informatics Facility, Hyderabad, India.

生长激素（growth hormone）在正常肾功能中发挥关键作用，而过度激活的生长激素信号通路已被证实与糖尿病性蛋白尿相关。我们团队此前的研究显示，在肾脏滤过过程中承担核心功能的肾小球足细胞（glomerular podocytes）表达生长激素受体，这提示生长激素可直接作用于此类细胞。然而，过量生长激素在这些足细胞中诱导的、最终引发糖尿病肾病的确切机制及下游通路仍未明确。为全面解析生长激素在转录层面对足细胞的影响，我们开展了RNA测序（RNA-Sequencing）实验。本研究采用条件永生化的人肾小球足细胞作为实验材料。实验整体设计：将人肾小球足细胞设置重复组别，分别以500 ng/ml浓度的生长激素处理与不做处理，处理时长为30分钟，随后提取总RNA。通过琼脂糖凝胶电泳及生物分析仪两种方法验证了RNA的质量。以mRNA为模板，使用随机六聚体、寡聚dT引物与Illumina第二链合成缓冲液构建高质量RNA文库。测序实验在印度海得拉巴的Nucleome信息学设施中完成，采用Illumina HiSeq 2500仪器进行双端读长测序。