Injury Induced Paracrine Effects on the Podocyte's Transcriptome

Although injury in glomerular disease might only damage a subset of podocytes in any given glomerulus, the response of the healthy neighboring podocytes to the injured podocytes oftentimes determines the course of the disease. To investigate this relationship, we designed a dual chamber open microfluidic co-culture device to specifically examine paracrine signaling to healthy podocytes from podocytes targeted injured by either Adriamycin, Puromycin Aminonucleoside or a cytopathic anti-podocyte antibody. Global transcriptomic analysis measured by RNA-sequencing revealed shared and unique pathways between the three forms of targeted injury, with temporal differences in the transcriptomic responses to each form of injury. Paracrine-induced injury to neighboring podocytes was similar to the targeted primary injured podocytes and was specific for each podocyte injury model. Ligand-receptor analysis of ligands secreted by the insult targeted podocytes and receptors expressed by the responsive, paracrine injured counterparts identified 23 candidate mediator pairs. These findings critically define a new concept for future studies to understand the pathways involved in secondary cellular injury fields in animal models and ultimately human studies. Overall design: Primary, human podocytes were seeded into a two-chamber microfluidic device. The podocytes in the outer chamber were injured using either (i) a cytopathic anti-podocyte antibody (IgG), (ii) puromycin aminonucleoside (PAN), or (iii) Adriamycin/doxorubicin (ADR). The podocytes in the inner chamber remained naïve (uninjured). After 24-hours post-injury, the two chambers were connected by flooding with shared media. Autocrine and paracrine effects of the injury models were assessed with mRNA sequencing at 24- and 96-hours post-injury.

尽管肾小球疾病所致的损伤可能仅累及单个肾小球内的部分足细胞（podocytes），但健康邻近足细胞对受损足细胞的应答往往决定了疾病的病程。为探究这一关联，我们设计了双腔开放式微流控共培养装置，以专门研究经阿霉素（Adriamycin）、嘌呤霉素氨基核苷（Puromycin Aminonucleoside）或细胞致病性抗足细胞抗体靶向损伤的足细胞，向健康足细胞传递的旁分泌信号。通过RNA测序（RNA-sequencing）开展的全局转录组分析显示，三种靶向损伤模型间存在共通与独特的通路，且不同损伤模型的转录组应答存在时间差异。旁分泌诱导的邻近足细胞损伤与靶向原发性受损足细胞的损伤特征相似，且针对每种足细胞损伤模型具有特异性。对损伤靶向足细胞分泌的配体，以及应答性旁分泌受损足细胞所表达的受体进行配体-受体分析，共鉴定出23个候选介导因子对。本研究发现首次明确了全新研究理念，可为未来解析动物模型乃至最终人类研究中继发性细胞损伤微环境相关通路提供指导。总体实验设计：将原代人足细胞接种于双腔微流控装置中。对外腔足细胞采用以下三种方式进行损伤处理：(i) 细胞致病性抗足细胞抗体（IgG）、(ii) 嘌呤霉素氨基核苷（PAN）、(iii) 阿霉素/多柔比星（ADR）。内腔足细胞则保持未损伤状态。损伤后24小时，通过灌注共享培养基连接两个腔室。分别于损伤后24小时和96小时，通过mRNA测序评估各损伤模型的自分泌与旁分泌效应。