Tubular Epithelial Cell Senescence Associated Secretory Phenotype: Impact on Regeneration in Acute and Chronic Kidney Disease

急性肾损伤(AKI)是临床常见而严重的问题，具有较高的发病率。AKI的发病率正在稳步上升，这在很大程度上是由于老龄化人口的增加。这一点特别令人担忧，因为随着年龄的增长，AKI后的恢复率明显变差。老年人更有可能发展为慢性肾功能衰竭，并在急性肾损伤后依赖透析。已经确定了几种不同的与年龄相关的机制，这些机制可能是这个问题的基础。在这些机制中，体细胞衰老(SCS)已成为最强大的途径之一。SCS是一种不可逆的细胞周期停滞状态，在这种状态下，细胞仍然活着，但功能和代谢活动发生了变化。这包括一种特定的分泌蛋白表达模式，称为衰老相关分泌表型(SASP)。最近的研究表明，SASP导致细胞微环境的显著旁分泌变化，导致正常组织内环境的严重破坏。在肾脏方面，到目前为止还没有关于SASP作用的研究。在初步研究中，我们发现，在诱导SCS时，肾小管细胞的分泌体发生了显著的变化。肾成纤维细胞与衰老的肾小管细胞共培养可迅速激活成纤维基质，产生肌成纤维细胞。基于这些发现，我们假设肾小管上皮细胞，尤其是肾小管上皮细胞-SASP(TEC-SASP)在不良适应性肾修复中起重要作用。这项拟议的项目的目标是结合体外和体内策略来研究这一假说。作为概念研究的证明，我们将研究允许AKI后选择性消除衰老细胞的转基因小鼠。我们的假设是，SCS的清除将改善促衰老和促纤维化的机制，并将促进肾脏修复。围绕衰老肾小管细胞与白细胞的相互作用，我们将研究TEC-SASP对AKI损伤肾脏巨噬细胞极化的影响。此外，我们还将研究在衰老肾小管细胞微阵列研究中差异调节的新候选因子。特别是，我们将研究纤溶酶原激活因子-2(PAI-2)和Klotho的作用，使用体外遗传过度表达或敲除策略，用于共培养系统和具有靶向基因缺失的小鼠进行体内分析。最终，我们将把我们的数据与当地肾移植计划的患者肾脏活检相关联。我们建议的长期目标不仅是更好地从生物学上了解SCS和SASP，而且还将识别新的分子靶点，这些靶点可能对改善急慢性肾脏疾病的肾脏预后具有潜在的治疗作用。

Acute kidney injury (AKI) is a common and serious clinical problem which is associated with a high morbidity. The incidence of AKI is steadily rising, which is largely due to an increase in the aging population. This is of particular concern because the recovery rate after AKI is significantly worse with advancing age. Older people are more likely to develop chronic renal failure and to become dialysis dependent after AKI. Several distinct age-dependent mechanisms have been identified that might underlie this problem. Among these mechanisms, Somatic Cellular Senescence (SCS) has emerged as one of the most powerful pathways. SCS is a state of irreversible cell cycle arrest in which the cell remains viable but develops altered functional and metabolic activity. This includes a specific expression pattern of secreted proteins which is referred to as the Senescence-Associated-Secretory-Phenotype (SASP). Recent studies have shown that the SASP is responsible for significant paracrine changes in the cellular microenvironment leading to a profound disruption of normal tissue homeostasis. In the kidney, no research on the role of the SASP has been conducted so far. In preliminary studies, we found that renal tubular cells develop a significantly altered secretome when SCS is induced. Co-culturing of kidney fibroblasts with senescent tubular cells caused a rapid activation into pro-fibrotic matrix producing myofibroblasts. Based on these findings we hypothesize that tubular SCS and in particular the Tubular Epithelial Cell - SASP (TEC-SASP) are important contributors to maladaptive renal repair. It is the goal of the proposed project to investigate this hypothesis using a combination of in vitro and in vivo strategies. As a proof of concept study we will investigate transgenic mice which allow for a selective elimination of senescent cells after AKI. Our hypothesis is that the clearance of SCS will ameliorate pro-aging and pro-fibrotic mechanisms and will improve kidney repair. Focussing on the cross-talk of senescent tubular cells with infiltrating leukocytes we will study the effects of TEC-SASP on macrophage polarization in AKI-damaged kidneys. Moreover, we will study novel candidate factors that were differentially regulated in a microarray study of senescent tubular cells. In particular, we will study the role of plasminogen-activating-factor-2 (PAI-2) and of Klotho using in vitro genetic overexpression or knock-down strategies for co-culture systems and mice with targeted gene deletion for in vivo analysis. Ultimately, we will correlate our data to patient renal biopsies from the local kidney transplant program. The long-term objective of our proposal will not only be a better biological understanding of SCS and SASP but also the identification of new molecular targets that could be of potential therapeutic use for improving renal outcome in acute and chronic kidney disease.