Role of proteolytic suPAR fragment in insulin dependent diabetes and kidney disease

慢性肾脏疾病（CKD）的全球流行正以惊人的速度发展。据估计，美国有3700万人患有慢性肾病（占成年人口的15%，超过七分之一的成年人），在过去20年里，这一数字翻了一番多。肾脏相关疾病正在迅速摆脱目前的治疗方案和资源。CKD的三个常见病因是糖尿病、高血压和肾小球肾炎。我们和其他人认为可溶性尿激酶激活受体（suPAR）是CKD发病和进展的重要危险因素之一，无论其病因如何。除了作为生物标志物的作用外，我们和其他人已经提出suPAR是一种致病/瘢痕因子，通过激活足细胞上的v3整合素而成为足细胞损伤的基础。足细胞是维持肾滤过器特异性所必需的终末分化细胞。我们的初步数据表明，血浆中存在的suPAR蛋白水解片段D2D3片段通过激活足细胞上的v3整合素诱导肾小球损伤。此外，出乎意料的是，我们的初步研究表明，D2D3片段也会损伤胰腺的-细胞。总之，这些见解提出了一个假设，即循环D2D3片段同时导致肾脏和胰腺两个器官损伤。我们在3个具体目标中检验了这一假设。在Specific Aim 1中，我们通过检查D2D3- tg小鼠的器官功能，确定了D2D3诱导肾脏和胰腺损伤的生理机制，D2D3小鼠在正常饮食和高脂肪饮食中受到肾毒性血清或阿霉素的刺激。此外，我们将D2D3-Tg小鼠与非肥胖糖尿病（NOD）小鼠杂交，以研究胰腺损伤的协同作用。抗supar抗体和靶向调节GTPases （dynamin, Cdc42, Rac1）的小分子，其作用将在Aims 2和3中阐明，将作为潜在的治疗方法进行测试。由于D2D3-Tg小鼠表现出双器官损伤，针对不同细胞类型的不同途径的联合治疗也将被研究。在特异性目标2中，我们研究了D2D3片段对肌动蛋白细胞骨架、调节gtpase、线粒体功能和终分化和初级足细胞中基因表达的影响，无论存在或不存在脂毒刺激或高葡萄糖水平。我们关注D2D3对肌动蛋白细胞骨架和线粒体功能之间反馈回路的影响，以及足细胞生理学变化对其的修改。在Specific Aim 3中，我们研究了D2D3对-细胞、分离的小鼠胰岛和组织切片中葡萄糖摄取、细胞内Ca2+动力学、细胞骨架动力学和基因表达的影响。该研究揭示了双器官损伤的分子机制和生理相关性，是开发针对CKD和胰岛素依赖型糖尿病的新型机制特异性治疗方法的关键一步。

The global epidemic of chronic kidney disease (CKD) is progressing at an alarming rate. CKD affects an estimated 37 million people in the U.S. (15% of the adult population; more than 1 in 7 adults), and this number has more than doubled in the last two decades. Kidney-related diseases are rapidly eluding present treatment options and resources. The three common causes of CKD are diabetes mellitus, hypertension, and glomerulonephritis. We and others have implicated soluble urokinase activating receptor (suPAR) as one of the significant risk factors for both, the onset as well as the progression of CKD, regardless of its etiologies. In addition to its role as a biomarker, we and others have suggested that suPAR is a pathogenic/scarring factor that underlies podocyte injury by activating v3 integrin on podocytes. Podocytes are terminally differentiated cells essential for maintaining the specificity of the kidney filter. Our preliminary data suggest that the presence of the proteolytic fragment of suPAR, D2D3 fragment, in the plasma induces glomerular injury by activating v3 integrin on podocytes. In addition, and highly unexpectedly, our preliminary studies suggest that the D2D3 fragment also injures -cells of the pancreas. Together, these insights suggest a hypothesis that circulating D2D3 fragment simultaneously causes injury to two organs, the kidney and pancreas. We test this hypothesis in 3 Specific Aims. In Specific Aim 1, we define the physiological mechanisms by which D2D3 induces kidney and pancreas injuries by examining organ functions in D2D3-Tg mice on regular- and high-fat- diet challenged by nephrotoxic serum or Adriamycin. In addition, we will cross D2D3-Tg mice with Non-Obese Diabetic (NOD) mice to examine synergy upon pancreas injury. Anti-suPAR antibodies and small molecules that target regulatory GTPases (dynamin, Cdc42, Rac1), whose roles will be elucidated in Aims 2 and 3, will be tested as potential therapeutics. As D2D3-Tg mice exhibit dual organ injury, combination treatments targeting diverse pathways in distinct cell types will also be investigated. In Specific Aim 2, we examine the effects of the D2D3 fragment on the actin cytoskeleton, regulatory GTPases, mitochondrial function, and gene expression in terminally differentiated and primary podocytes in the presence and absence of lipotoxic stimuli or high glucose levels. We focus on the effects of D2D3 on a feedback loop between the actin cytoskeleton and mitochondrial function, and its modifications by changes in podocyte physiology. In Specific Aim 3, we examine the effects of D2D3 on glucose uptake, intracellular Ca2+ dynamics, cytoskeleton dynamics, and gene expression in -cells, isolated mouse islets, and tissue slices. The proposed study, delineating molecular mechanisms and the physiological relevance of dual organ injury, is a critical step in developing novel mechanism-specific therapeutics for CKD and insulin-dependent diabetes mellitus.