Understanding the molecular basis of the peritoneal solute transport to improve peritoneal dialysis efficacy and outcome

腹膜透析（PD）是越来越多的终末期慢性肾病（CKD 5）患者的一种挽救生命、具有成本效益的治疗方法。腹膜透析利用腹膜作为半透膜的优势，从患者体内清除毒素和水分。然而，PD的有限净化能力和PD液体的非生理成分会影响技术和患者生存。定义腹膜溶质和水运输的分子结构在很大程度上是未知的。我们已经建立了一个大型的腹膜和网膜组织的生物库，提供有关健康腹膜的年龄相关的解剖信息，对PD诱导的腹膜转化过程和相关的全身血管病变。本项目旨在全面了解腹膜溶质和水转运穿过内皮和间皮细胞屏障的分子机制，其基本上定义了PD膜功能及其对局部和全身血管病变的影响。基于先前的研究，我们确定了潜在的分子候选物，即连接蛋白，其是邻近上皮和内皮细胞屏障的关键组分，调节密封和细胞旁转运功能。在非靶向人离体方法中，我们将对来自非CKD患者、来自CKD 5和PD患者以及来自最近接受肾移植和标准化免疫抑制的PD患者的腹膜和网膜小动脉进行全外显子组和蛋白质组分析。将在独立患者队列中验证关键发现，并与腹膜炎症、血管生成和纤维化的已知病理机制、血管病变和PD转运功能相关。在Transwell/Ussing室系统中，人间皮细胞和内皮细胞单层将经历钠、水和4- 70 kDa分子的跨上皮阻力和分子量依赖性转运测量以及连接丰度和定位研究。将研究不同的PD液体类型、相应的液体组分、反应性代谢产物和充分描述的尿毒症毒素以及已确定和计算机识别的连接调节剂和药物。CRISPR/Cas9基因敲除研究将确定关键连接组件的功能影响。超分辨率显微镜将证明特定连接组分的共定位和连接-细胞骨架相互作用，冷冻断裂电子显微镜将可视化连接链网络的连续性和延伸以及处理诱导的链断裂。然后将离体和体外结果转化为PD的尿毒症小鼠模型。将测试连接功能的调节剂，以促进新型PD液体原型和治疗概念的开发，从而改善PD解毒功效和可持续性，从而改善PD患者结局。六个国家和国际合作伙伴将为这项研究作出贡献。

Peritoneal dialysis (PD) is a life-saving, cost-effective therapy for an increasing number of patients with end stage chronic kidney disease (CKD5). PD takes advantage of the peritoneum as a semipermeable membrane to remove toxins and water from the patient. The limited purification capacity of PD and the nonphysiological composition of PD fluids, however, compromise technique and patient survival. The molecular structures defining peritoneal solute and water transport are largely unknown.We have established a large biobank of peritoneal and omental tissues providing information on the age related anatomy of the healthy peritoneum, on the PD induced peritoneal transformation process and on associated systemic vasculopathy. The present project aims at a comprehensive understanding of molecular mechanisms of peritoneal solute and water transport across the endothelial and mesothelial cell barrier, which essentially define PD membrane function and their impact on local and systemic vasculopathy. Based on previous studies we identified potential molecular candidates, i.e. junction proteins, which are key components of adjacent epithelial and endothelial cell barriers, regulating sealing and paracellular transport functions. In an untargeted human ex vivo approach we will perform whole exome and proteome analyses of the peritoneum and of omental arterioles from non-CKD patients, from patients with CKD5 and on PD, and from PD patients, who recently underwent renal transplantation and standardized immunosuppression. Key findings will be validated in independent patient cohorts, and related to known pathomechanisms of peritoneal inflammation, angiogenesis and fibrosis, to vasculopathy and PD transport function. In Transwell/Ussing chamber systems human mesothelial cell and endothelial cell monolayers will undergo transepithelial resistance and molecular weight dependent transport measurements of sodium, water and 4-70kDa molecules together with junction abundance and localization studies. Different PD fluid types, respective fluid components, reactive metabolites and well-described uremic toxins as well as established and in silico identified junction modulators and medications will be investigated. CRISPR/Cas9 gene knockout studies will define the functional impact of key junction components. Super resolution microscopy will demonstrate the co-localization of specific junction components and the junction-cytoskeleton-interaction, freeze fracture electron microscopy will visualize the junction strand network continuity and extension and treatment-induced strand breaks. Ex vivo and in vitro findings will then be translated into a uremic mouse model of PD. Modulators of junction function will be tested in order to promote the development of novel PD fluid prototypes and therapeutic concepts improving PD detoxification efficacy and sustainability and thus PD patient outcome. Six national and international cooperating partners will contribute to the study.