STEM CELL THERAPY FOR ACUTE KIDNEY INJURY

描述(申请人提供)：急性肾损伤(AKI)具有很高的发病率和死亡率。目前对AKI可用的唯一治疗方法是支持性措施。该项目的总体目标是开发基于干细胞的疗法来治疗AKI。我们之前已经证明，新鲜分离的小鼠造血干细胞(HSC)可以被移植并整合到再生的肾小管中。然而，并入率低，没有观察到功能上的好处。为了提高HSC的治疗潜力，我们开发了一种新的方法，在移植前，造血干和造血祖细胞在移植前用细胞因子、肾源性因子和HDAC抑制剂曲古抑素A诱导肾分化。诱导细胞具有肾脏发育基因的从头表达和造血细胞分化基因的下调。注射诱导细胞或诱导细胞条件培养液可改善AKI小鼠的肾功能，提示通过内分泌/旁分泌效应来保护肾脏。此外，阻断诱导细胞上的钙敏感受体CAR会增加细胞的肾脏定位。这一结果表明，调节CAR可能有助于增加细胞在肾内的输送，以达到更好的治疗效果。为了推广这些有希望的发现，我们建议用额外的肾脏标记物来表征诱导细胞，以确认肾脏细胞命运的选择。我们将通过体外实验和移植研究来确定诱导细胞是否能分化为有功能的肾上皮细胞。将分析细胞转化所涉及的分子途径，以指导发现细胞转化的最佳条件(目标1)。我们将研究诱导细胞是否通过减少内源性肾小管上皮细胞和/或内皮细胞的死亡和促进增殖而发挥内分泌/旁分泌作用。由于存活的肾小管细胞是肾脏修复的主要来源，诱导细胞释放的肾营养因子可能是治疗AKI的重要因素(目标2)。此外，我们将用抗体阻断CAR，或使用CAR缺陷小鼠来测试CAR的调节是否会增加细胞在肾内的迁移。这一策略可通过增加直接细胞替代的肾小管整合和向受损肾小管提供更持久的肾营养因子输送来提高诱导细胞的治疗效果(目标3)。总之，我们采取了新的方法来开发细胞治疗药物，通过对造血干细胞和造血祖细胞的序贯治疗来治疗AKI，这些细胞可以很容易地获得用于供者指导的治疗。

DESCRIPTION (provided by applicant): Acute kidney injury (AKI) carries high morbidity and mortality. The only treatment that is currently available for AKI consists of supportive measures. The overall goal of this project is to develop stem cell- based therapy to treat AKI. We have previously shown that freshly isolated mouse hematopoietic stem cells (HSC) can be transplanted and incorporated into regenerating renal tubules. However, the incorporation rate is low and no functional benefit is observed. To enhance the therapeutic potential of HSC, we have developed a novel approach in which hematopoietic stem and progenitor cells are treated sequentially with cytokines, nephrogenic factors, and an HDAC inhibitor trichostatin A to induce renal differentiation prior to transplantation. Induced cells have de novo expression of renal developmental genes and down-regulation of hematopoietic differentiation genes. Injection of induced cells or induced cell-conditioned medium improves renal function in mice with AKI, suggesting renal protection by endocrine/paracrine effects. Furthermore, blocking the calcium sensing receptor, CaR, on induced cells increases renal localization of the cells. This result suggests that modulation of the CaR may be useful to increase intrarenal delivery of the cells to achieve greater therapeutic effects. To extend these promising findings, we propose to characterize induced cells with additional renal markers to confirm the selection of renal cell fate. We will determine whether induced cells can differentiate into functional renal epithelial cells by in vitro assays and transplantation studies. The molecular pathways involved in cell conversion will be analyzed to guide the discovery of optimal conditions for cell conversion (Aim 1). We will examine whether induced cells exert endocrine/paracrine effects by decreasing death and increasing proliferation of endogenous tubular epithelial cells and/or endothelial cells. Since surviving tubular cells are the main source for renal repair, renotrophic factors released by induced cells can be important agents to treat AKI (Aim 2). Furthermore, we will block the CaR with an antibody or use CaR-deficient mice to test whether modulation of the CaR could increase intrarenal transmigration of the cells. This strategy may increase the therapeutic utility of induced cells by increasing tubular integration for direct cell replacement and providing more sustained delivery of renotrophic factors to the injured tubules (Aim 3). In conclusion, we take novel approaches to develop cell therapeutic agents to treat AKI by sequential treatment of hematopoietic stem and progenitor cells that can be obtained easily for donor-directed therapy.