Shao Xiaohang, Xu Huaming, Kim Hyojin, Ljaz Sadaf, Beier Fabian, Jankowski Vera, Lellig Michaela, Vankann Lucia, Werner Jan Niklas, Chen Lu, Ziegler Susanne, Kuppe Christoph, Zenke Martin, Schneider Rebekka K, Hayat Sikander, Saritas Turgay, Kramann Rafael
Department of Nephrology and Clinical Immunology, RWTH Aachen University, Medical Faculty, Aachen, Germany.
Department of Cell Biology, Institute of Biomedical Engineering, RWTH Aachen University Medical School, Aachen, Germany.
Cell Death Discov. 2024 Aug 14;10(1):364. doi: 10.1038/s41420-024-02131-y.
Emerging evidence highlights cellular senescence's pivotal role in chronic kidney disease (CKD). Proximal tubule epithelial cells (PTECs) and fibroblasts are major players in CKD and serve as cellular sources of senescence. The generation of a conditionally immortalized human kidney cell model would allow to better understand the specific mechanisms and factors associated with cellular senescence in a controlled setting, devoid of potential confounding factors such as age and comorbidities. In addition, the availability of human kidney cell lines for preclinical research is sparse and most cell lines do not reflect their in vivo counterparts due to their altered behavior as immortalized cancer-like cells. In this study, PTECs and fibroblasts from human kidneys were isolated and transduced with doxycycline-inducible simian virus 40 large T antigen (SV40LT) vector. By comparing their gene expression with single-cell RNA sequencing data from human kidneys, the newly produced human kidney cell lines demonstrated significant resemblances to their in vivo counterparts. As predicted, PTECs showed functional activity and fibroblasts responded to injury with fibrosis. Withdrawal of the immortalizing factor doxycycline led to p21 cell-cycle arrest and the key hallmarks of senescence. The obtained senescence gene set largely overlapped between both cell lines and with the previously published SenMayo set of senescence-associated genes. Furthermore, crosstalk experiments showed that senescent PTECs can cause a profibrotic response in fibroblasts by paracrine actions. In 76 human kidney sections, the number of p21 cells correlated with the degree of fibrosis, age and reduced glomerular filtration, validating the role of senescence in CKD. In conclusion, we provide a novel cellular ex vivo model to study kidney senescence which can serve as a platform for large scale compounds testing.
新出现的证据突显了细胞衰老在慢性肾脏病（CKD）中的关键作用。近端肾小管上皮细胞（PTECs）和成纤维细胞是CKD的主要参与者，也是衰老的细胞来源。构建一个条件永生化的人肾细胞模型将有助于在可控环境中更好地理解与细胞衰老相关的具体机制和因素，避免年龄和合并症等潜在混杂因素的影响。此外，用于临床前研究的人肾细胞系非常稀少，而且大多数细胞系由于其作为永生化癌样细胞的行为改变，无法反映其体内对应细胞的情况。在本研究中，从人肾中分离出PTECs和成纤维细胞，并用强力霉素诱导的猿猴病毒40大T抗原（SV40LT）载体进行转导。通过将它们的基因表达与来自人肾的单细胞RNA测序数据进行比较，新产生的人肾细胞系与其体内对应细胞表现出显著的相似性。正如预期的那样，PTECs显示出功能活性，而成纤维细胞对损伤产生纤维化反应。去除永生化因子强力霉素会导致p21细胞周期停滞和衰老的关键特征。在两个细胞系之间以及与先前发表的SenMayo衰老相关基因集之间，所获得的衰老基因集在很大程度上重叠。此外，相互作用实验表明，衰老的PTECs可通过旁分泌作用在成纤维细胞中引起促纤维化反应。在76个人肾切片中，p21阳性细胞的数量与纤维化程度、年龄和肾小球滤过率降低相关，证实了衰老在CKD中的作用。总之，我们提供了一种用于研究肾衰老的新型体外细胞模型，可作为大规模化合物测试的平台。
