Renal diabetic complications mediated by the PAR1 signaling in podocytes

项目摘要 足细胞已成为研究和临床工作的关键焦点，成为肾脏疾病的靶点。 干预由于其在调节肾小球通透性和维持肾小球结构方面的重要作用。 足细胞损伤在糖尿病肾病(DKD)中具有重要的病理意义和预后意义。其中一个 决定肾小球形态和通透性病理改变的主要因素是 足细胞的基础细胞内钙([Ca~(2+)]i)水平，这可能是由于激活各种信号而发生的 瀑布。蛋白水解酶激活受体(Pars)因其潜在的作用而成为人们关注的蛋白质。 调节足细胞[Ca~(2+)]i水平，特别是在病理条件下，如DKD。临床研究表明 证明循环中PAR激活蛋白水解酶的浓度与DKD有关。 此外，最近的前瞻性Optimus-5研究揭示了FDA批准的几种有益效果 PAR1拮抗剂伏拉帕沙治疗2型糖尿病。然而，尽管有关键证据表明 在DKD的足细胞中，PAR信号通路仍未得到充分的研究。我们的初步数据显示 大鼠和人足细胞中存在功能增强的PAR-GPCRTRPC6信号通路 在糖尿病的情况下。与这些发现一致的是，我们发现丝氨酸蛋白酶促进了 PAR1-TRPC6在新鲜分离的大鼠肾小球的足细胞中级联，从而触发[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(T2DN大鼠)的临床观察与临床观察相似。中环 这一建议的假设是，在2型糖尿病DKD的发展过程中，当尿凝血酶和 尿激酶浓度快速升高，PAR1过度刺激促进[Ca~(2+)]i升高 足细胞通过激活TRPC6通道，最终导致细胞凋亡，发展为 蛋白尿和肾小球损害。因此，抑制PAR1活性将减轻足细胞损伤，并可能 对DKD有治疗作用。将利用几种创新的方法和独特的老鼠模型来测试 以下具体目标：目标1将检验PAR1表达及其活性在 DKD在2型糖尿病中的进展及其在钙稳态改变中的作用 在足细胞和肾小球损伤中；目标2将提供对PAR-1激活的机械性洞察 足细胞介导的信号传递及相关的肾小球结构和功能变化。此外， 糖尿病肾病大鼠足细胞PAR信号与性别差异在DKD发生发展中的相关性 探索过了。

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.