Protease dependent signalling at the glomerular filtration barrier

糖尿病肾病（dNP）正在获得大流行的规模，给受影响的个人，医疗保健提供者和保险带来巨大的负担。既定的治疗，如血糖和血压控制延迟，但不能避免dNP。近年来，我们确定了凝血蛋白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蛋白C（aPC）保护dNP。这一发现已被其他人证实，临床研究支持aPC的这种作用。然而，凝血蛋白酶依赖性信号传导如何在肾小球滤过屏障局部调节仍然是个谜。值得注意的是，肾小球细胞如足细胞和内皮细胞不仅局部表达凝血因子的受体（例如蛋白酶激活受体，PAR），而且还表达其调节剂，如血栓调节蛋白（TM）和组织因子（TF），甚至蛋白酶本身（例如蛋白C，PC）。然而，它仍然是未知的，是否以及如何局部表达的凝血因子和调节剂有助于通过凝血蛋白酶的信号。此外，还不知道内皮细胞和足细胞是否以及如何通过凝血蛋白酶相互作用。在初步工作中，我们确定足细胞特异性表达TM保护免受dNP-至少部分独立于PC激活。此外，我们检测到aPC和α v β 3-整联蛋白之间的相互作用，这与足细胞信号传导有关。同样，TF调节dNP中的肾小球功能。TM和TF可能协调调节肾小球功能的潜在机制将在当前提案中进行评估。我们希望确定TM是否提供PAR-1和整合素依赖性信号之间的功能性信号转换，以及TM是否通过与TF或胞内蛋白质通过其胞质结构域功能性相互作用来调节信号。具体而言，我们希望1）表征局部表达的凝血调节剂的表达和活性，2）评估TM是否提供PAR-1和PAR-3-avb3信号传导之间的功能性开关，3）鉴定TM胞质结构域对肾小球细胞功能和dNP的作用，4）表征TM依赖性补体调节对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介导的受体体形成，包括与整合素的相互作用，7）分析足细胞中蛋白酶依赖性信号传导的异构酶开关的相关性。这些全面的研究将为肾小球疾病中凝血蛋白酶依赖性信号传导提供新的见解。我们希望这些发现将为翻译工作提供新的动力，并将对其他组织中凝血蛋白酶信号转导的研究产生影响。

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.