Renal diabetic complications mediated by the PAR1 signaling in podocytes

项目摘要 足细胞作为肾脏疾病的靶点已成为研究和临床工作的关键焦点 由于其在调节肾小球渗透性和维持肾小球结构中的重要作用，因此它被认为是一种有效的干预措施。 足细胞损伤在糖尿病肾病（DKD）中具有重要的病理和免疫学意义。之一 决定肾小球形态学和通透性病理变化的主要因素是 足细胞中的基础细胞内钙（[Ca 2 +]i）水平，这可能是由于各种信号传导的激活而发生的 瀑布蛋白酶激活受体（PARs）由于其潜在的生物学活性而成为感兴趣的蛋白质。 调节足细胞[Ca 2 +]i水平，特别是在病理条件下，如DKD。临床研究 证明PAR活化蛋白酶的循环浓度与DKD相关。 此外，最近的前瞻性OPTIMUS-5研究揭示了FDA批准的 PAR 1拮抗剂Vorapaxar治疗2型糖尿病然而，尽管有关键证据表明， DKD足细胞中的PAR信号通路，这一领域仍然研究不足。我们的初步数据显示 大鼠和人类足细胞中PAR-GPCR-TRPC 6信号通路的功能性存在增加 在糖尿病的情况下。与这些发现相一致，我们发现丝氨酸蛋白酶促进 PAR 1-TRPC 6级联反应在来自新鲜分离的大鼠肾小球的足细胞中，其触发[Ca 2 +]i的快速升高。 此外，我们的初步研究表明，这些信号通路在大鼠模型中高度上调， 2型糖尿病肾病（T2 DN大鼠）的临床观察结果，与人类患者的临床观察结果相似。中央 该建议的假设是，在2型糖尿病DKD的发展过程中，当尿凝血酶和 尿激酶浓度迅速增加，PAR 1的过度刺激促进了尿激酶中[Ca 2 +]i水平的过度升高。 足细胞通过激活TRPC 6通道，最终导致细胞凋亡，发展成 蛋白尿和肾小球损伤。因此，PAR 1活性的抑制将减轻足细胞损伤，并可能 在DKD中具有治疗益处。几种创新的方法和独特的大鼠模型将用于测试 以下具体目的：目的1将检验PAR 1表达及其活性在 DKD在2型糖尿病中的进展，并且该途径有助于Ca 2+稳态的改变 Aim 2将为PAR-1的激活提供机制性的见解 足细胞介导的信号传导以及相关的肾小球结构和功能变化。此外该 将研究足细胞中PAR信号与T2 DN大鼠DKD发展中性别差异的相关性。 探讨了

Project Summary The podocyte has become a crucial focus of research and clinical efforts as a target for kidney disease interventions due to its vital role in regulating glomerular permeability and maintaining glomerular structure. Podocyte injury is pathogenetically and prognostically important in diabetic kidney disease (DKD). One of the main factors determining pathological changes in glomerular morphology and permeability is the elevation of basal intracellular calcium ([Ca2+]i) levels in podocytes, which can occur due to activation of various signaling cascades. Protease-activated receptors (PARs) are emerging as proteins of interest for their potential to modulate podocyte [Ca2+]i levels, especially under pathological conditions, such as DKD. Clinical studies have demonstrated that circulating concentrations of PAR-activating proteases are associated with DKD. Furthermore, the recent prospective OPTIMUS-5 study revealed several beneficial effects of the FDA-approved PAR1 antagonist Vorapaxar in type 2 diabetes mellitus. However, despite crucial evidence for the importance of PAR signaling pathways in podocytes in DKD, this area is still understudied. Our preliminary data demonstrate the functional presence of a PAR-GPCR-TRPC6 signaling pathway in rat and human podocytes that is increased under diabetic conditions. Consistent with these findings, we found that serine proteases promote activation of PAR1-TRPC6 cascade in podocytes from freshly isolated rat glomeruli, which triggers a rapid elevation of [Ca2+]i. Furthermore, our pilot studies have revealed that these signaling pathways are highly upregulated in a rat model of type 2 Diabetic Nephropathy (T2DN rats), similar to clinical observations in human patients. The central hypothesis of this proposal is that during the development of DKD in type 2 diabetes, when urinary thrombin and urokinase concentrations increase rapidly, overstimulation of PAR1 promotes excessive [Ca2+]i levels in podocytes through activation of TRPC6 channels, ultimately leading to cell apoptosis, development of albuminuria and glomerular damage. Thus, inhibition of PAR1 activity will mitigate podocyte damage and may be of therapeutic benefit in DKD. Several innovative approaches and unique rat models will be utilized to test the following Specific Aims: Aim 1 will test the hypothesis that PAR1 expression and its activity increase during the progression of DKD in type 2 diabetes and that this pathway contributes to the alterations in Ca2+ homeostasis in podocytes and glomerular damage; and Aim 2 will provide mechanistic insight into the activation of PAR-1 mediated signaling in podocytes and associated glomerular structure and function changes. In addition, the correlation of PAR signaling in podocytes and sex difference in the development of DKD in T2DN rats will be explored.