Molecular Mechanisms of Peritoneal Fibrosis- Proteinkinase C alpha and Peritoneal Dialysis

在我们以前的实验中，我们能够证明PKCα是MPMCs中表达的唯一经典PKC亚型。在体外模拟PD条件下和体内长期暴露于PDF后，MPMCs中PKCα蛋白表达和活化上调。然而，在WT小鼠中，慢性PDF给药导致PM的严重形态、结构和功能变化，用Go 6976功能性阻断PKCα活性完全保护PM的结构和功能。PKCα缺乏显示出相当的益处，表明PKCα在PM紊乱中起关键作用。基于上一个资助期的这些发现，我们提出了一种新的治疗方法来预防PD流体诱导的炎症、新血管生成和纤维化。1.使用针对PKC亚型α的反义策略，在透析期间定期与PD液一起使用，我们希望抑制葡萄糖诱导的炎症、新血管生成和纤维化。特异性反义方法 防止其他PKC抑制剂（如Gö）的副作用，这些抑制剂也可能影响来自各种蛋白激酶家族的多种激酶，并且通常具有其他药理作用，导致体外和体内不同的作用，与PKC抑制无关。2.我们将分析我们的策略对腹膜不同细胞类型的治疗效果。我们有证据表明，从我们以前的研究，间皮细胞是一个主要的治疗目标。 然而，我们以前也看到PKC-α在内皮细胞、巨噬细胞和成纤维细胞中起重要作用。 因此，我们计划使用单核基因表达分析来确定我们的治疗策略。在体内使用，特异性高， 这些研究的结果将使我们能够确定损伤腹膜中的分子和细胞机制，这些机制通过我们的新治疗策略来解决，并使用特定的纳米颗粒开发靶向治疗方法。为了改善我们的反义方法的药代动力学和药效学特征，我们将使用纳米颗粒。这种方法将使我们能够开发复杂的靶向策略，并降低我们使用的反义化合物的浓度。

In our previous experiments we were able to demonstrate in the experiments supported by that PKCα is the only classical PKC isoform expressed in MPMCs. PKCα protein expression and activation is upregulated in MPMCs in vitro under PD-simulating conditions and in vivo after prolonged exposure to PDF. Whereas in WT mice chronic PDF administration leads to severe morphologic, structural, and functional changes of the PM, the functional blockade of PKCα activity with Go6976 completely protected PM structure and function. PKCα deficiency showed a comparable benefit, suggesting a crucial role of PKCα in PM disturbances. Based on these findings in the last funding period we propose a novel therapeutic approach to prevent PD-fluid induced inflammation, neoangiogenesis and fibrosis. 1. Using an antisense strategy against PKC isoform alpha regularly applied with the PD fluid during dialysis we want to inhibit the glucose-induced inflammation, neoangiogenesis and fibrosis. The specific antisense approach prevents the side effects of other PKC inhibitors such as Gö, which may also affect multiple kinases from a variety of protein kinase families and usually have other pharmacological actions leading to different effects in vitro and in vivo independently from PKC inhibition. 2. We will analyze the therapeutic effect of our strategy on the different cell types of the peritoneum. We have evidence from our previous research that mesothelial cells are a main therapeutic target. However, we have also seen previously that PKC-a in endothelial cells, macrophages and fibroblasts plays an important role. We therefore plan to use single nucleus gene expression analysis to define our therapeutic strategy. be used in vivo, have a high specificity and can. The findings from these studies will allow us to identify the molecular and cellular mechanisms in the injured peritoneum which are addressed by our novel therapeutic strategy and to develop a targeted therapeutic approach using specific nanoparticles 3. To improve the pharmacokinetic and pharmacodynamic profile of of our antisense approach we will use nanoparticles. This approach will allow us to develop sophisticated targeting strategies and reduces the concentration of our antisense compound used.