Protease dependent signalling at the glomerular filtration barrier

糖尿病肾病(DNP)的流行范围正在扩大，给受影响的个人、卫生保健提供者和保险公司带来了巨大的负担。已有的治疗方法，如血糖和血压控制延迟，但不能避免DNP。近年来，我们确立了凝血酶在调节肾小球功能中的重要作用。特别是，我们证明了丝氨酸蛋白酶激活的蛋白C(APC)对DNP具有保护作用。这一发现已得到其他人的证实，临床研究也支持APC的这种作用。然而，凝血酶依赖的信号是如何在肾小球滤过屏障局部调节的仍是个谜。值得注意的是，不仅凝血因子的受体(例如，蛋白酶激活的受体，PARs)，而且它们的调节因子，如血栓调节蛋白(TM)和组织因子(Tf)，甚至蛋白本身(例如，蛋白C，PC)，都由肾小球细胞(如足细胞和内皮细胞)局部表达。然而，目前尚不清楚局部表达的凝血因子和调节因子是否以及如何通过凝血酶作用于信号转导。此外，目前尚不清楚内皮细胞和足细胞是否以及如何通过凝血酶相互作用。在初步工作中，我们发现TM的足细胞特异性表达对DNP具有保护作用-至少部分地不依赖于PC的激活。此外，我们检测到APC和与足细胞信号相关的α-β3-整合素之间的相互作用。同样，Tf对DNP的肾小球功能也有调节作用。TM和TF协调调节肾小球功能的潜在机制将在当前的提案中进行评估。我们希望确定TM是否在PAR-1和整合素依赖的信号之间提供了一个功能性的信号开关，以及TM是否通过与TF或通过其细胞质结构域与细胞内蛋白进行功能性相互作用来调制信号。具体地说，我们希望1)表征局部表达的凝血调节因子的表达和活性，2)评估TM是否在PAR-1和PAR-3-avb3信号之间提供功能开关，3)确定TMS胞浆结构域在肾小球细胞功能和DNP中的作用，4)表征TM依赖的补体调节对TF活性的功能相关性，5)描述TF调节肾小球细胞功能的机制，6)表征TM和TF介导的受体形成，包括与整合素的相互作用，7)分析异构酶开关与肾小球细胞内的蛋白酶依赖性信号传导的相关性。这些全面的研究将为肾小球疾病中凝血酶依赖的信号转导提供新的见解。我们希望这些发现将为翻译工作提供新的动力，并将对解决其他组织中凝血酶信号的研究产生影响。

Diabetic nephropathy (dNP) is gaining pandemic dimensions, putting an enormous burden on affected individuals, health care providers, and insurances. Established therapies, such as blood glucose and blood pressure control delay, but do not avert dNP. In recent years we established an important function of coagulation proteases in regulating glomerular function. In particular, we demonstrated that the serine protease activated protein C (aPC) protects against dNP. This finding has been confirmed by others and clinical studies support such a role of aPC. However, how coagulation protease dependent signalling is regulated locally at the glomerular filtration barrier remains largely enigmatic. Of note, not only receptors for coagulation factors (e.g. protease activated receptors, PARs), but also their regulators, such as thrombomodulin (TM) and tissue factor (TF), and even the proteases themselves (e.g. protein C ,PC), are locally expressed by glomerular cells such as podocytes and endothelial cells. Yet, it remains unknown whether and how locally expressed coagulation factors and regulators contribute to signalling via coagulation proteases. Furthermore, it is not known whether and how endothelial cells and podocytes interact via coagulation proteases. In preliminary work we identified that podocyte specific expression of TM protects against dNP - at least in part independent of PC-activation. Furthermore, we detected an interaction between aPC and alphavbeta3-integrin, which is relevant for podocyte signalling. Likewise, TF modulates glomerular function in dNP. Potential mechanism through which TM and TF may coordinately modulate glomerular function will be evaluated within the current proposal. We wish to determine whether TM provides a functional signaling switch between PAR-1 and integrin dependent signalling and whether TM modulates signalling either by functionally interacting with TF or with intracellular proteins via its cytoplasmic domain. Specifically, we wish to 1) characterize the expression and activity of locally expressed coagulation regulators, 2) evaluate whether TM provides a functional switch between PAR-1 and PAR-3 - avb3 signalling, 3) identify the role of TMs cytoplasmic domain for glomerular cell function and dNP, 4) characterize the functional relevance of TM-dependent complement regulation for TF-activity, 5) delineate the mechanism through which TF modulates glomerular cell function, 6) characterize TM- and TF-mediated receptorsome formation, including the interaction with integrins, 7) analyze the relevance of isomerase switches for protease dependent signalling in podocytes. These comprehensive studies will provide novel insight into coagulation protease dependent signalling in glomerular disease. We expect that these findings will provide new impetus for translational efforts and will have implications for studies addressing coagulation protease signalling in other tissues.