Data_Sheet_4_JMJD1A/NR4A1 Signaling Regulates the Procession of Renal Tubular Epithelial Interstitial Fibrosis Induced by AGEs in HK-2.ZIP

Diabetic kidney disease (DKD) is one of the most serious complications of diabetic patients. Advanced glycation end products (AGEs) induce epithelial-mesenchymal transformation (EMT) of renal tubular epithelial cells (HK-2), resulting in renal tubulointerstitial fibrosis. However, the underlying epigenetic mechanisms remain to be further investigated. In this work, we investigated the functional role of JMJD1A involved in DKD progression. The molecular mechanism study was performed in AGEs-induced HK-2 cells by gene expression analysis, RNA sequencing (RNA-seq), and JMJD1A lentiviral knockdown and overexpression particle transfection. The results showed that AGEs could upregulate JMJD1A, and the expressions of related fibrotic factor were also increased. At the same time, in the DKD animal model induced by unilateral nephrectomy plus streptozotocin (STZ), IHC immunohistochemical staining showed that compared with the control group, the expressions of JMJD1A, FN, and COL1 in the model group were all increased, masson staining results also show that the model group has typical fibrotic changes. This is consistent with the results of our in vitro experiments. In order to determine the downstream pathway, we screened out JMJD1A downstream transcription factors by RNA-seq. Further analysis showed that JMJD1A overexpression could accelerate the progression of AGEs-induced renal fibrosis by reducing the expression of NR4A1 in HK-2 cells. Meanwhile, NR4A1 inhibitor can promote the expression of fibrosis-related factors such as VIM, a-SMA in HK-2 cells, and aggravate the process of fibrosis. Taken together, JMJD1A/NR4A1 signaling can regulate the procession of renal tubular epithelial interstitial fibrosis induced by AGEs in HK-2.

糖尿病肾病（Diabetic kidney disease, DKD）是糖尿病患者最严重的并发症之一。晚期糖基化终末产物（Advanced glycation end products, AGEs）可诱导肾小管上皮细胞（HK-2）发生上皮-间质转化（Epithelial-mesenchymal transformation, EMT），进而引发肾间质纤维化，但其潜在的表观遗传调控机制仍有待进一步探究。本研究探讨了JMJD1A在DKD进展中的功能作用。通过基因表达分析、RNA测序（RNA-seq）以及JMJD1A慢病毒敲低与过表达颗粒转染，在AGEs诱导的HK-2细胞中开展分子机制研究。实验结果显示，AGEs可上调JMJD1A的表达，相关纤维化因子的表达水平亦同步升高。此外，在单侧肾切除联合链脲佐菌素（Streptozotocin, STZ）诱导的DKD动物模型中，免疫组化（Immunohistochemistry, IHC）染色结果表明，与对照组相比，模型组中JMJD1A、纤连蛋白（Fibronectin, FN）、Ⅰ型胶原（Collagen type 1, COL1）的表达均显著上调；马松（Masson）染色结果亦显示，模型组出现典型的纤维化改变，这与本研究的体外实验结果一致。为明确下游调控通路，本研究通过RNA-seq筛选得到JMJD1A的下游转录因子。进一步分析表明，在HK-2细胞中，JMJD1A过表达可通过降低NR4A1的表达，加速AGEs诱导的肾纤维化进程。同时，NR4A1抑制剂可上调HK-2细胞中波形蛋白（Vimentin, VIM）、α-平滑肌肌动蛋白（α-smooth muscle actin, α-SMA）等纤维化相关因子的表达，加重纤维化过程。综上，JMJD1A/NR4A1信号轴可调控HK-2细胞中AGEs诱导的肾小管上皮间质纤维化进程。