Purinergic control of calcium flux in podocytes

足细胞由于在肾脏疾病中的关键作用，已成为肾脏疾病干预的关键焦点。 调节肾小球通透性，维持肾小球结构。足细胞损伤被认为是 在糖尿病肾病中具有重要的病理意义和预后意义。决定的主要因素之一 肾小球形态和通透性的病理改变与细胞内足细胞的升高有关 [Ca~(2+)]i)。瞬时受体电位规范(TRPC)通道在 肾脏和心血管疾病的发病机制。三磷酸腺苷是一种重要的信号分子，在细胞周期中起关键作用 足细胞功能。然而，我们对肾小球中的嘌呤能信号及其对TRPC的调控的了解 在糖尿病肾病模型中，足细胞内的通道和[Ca~(2+)]_i是基本的，因此是目前研究的焦点 求婚。 这一建议的中心假设是，糖尿病患者体内[Ca~(2+)]i动态平衡发生显著变化 足细胞的发生是由：1)细胞外ATP浓度增加；2)细胞重构。 从亲代谢性P2Y到亲离子性P2X受体的嘌呤能信号；3)过量产生ROS；以及4) TRPC通道过度活跃；总之，这些事件会导致肾小球损伤、蛋白尿和， 因此，ESRD。我们进一步假设足细胞内[Ca~(2+)]i内流增加会导致病理 肾小球对白蛋白的通透性增加。根据初步数据和公布的调查结果，主要 本项目的目的是明确ATP和ROS对TRPC影响的具体机制 新分离肾小球中的通道并鉴定控制肾小球白蛋白的药理靶点 通透性在糖尿病肾病发病机制中的作用为了探索这一想法，我们开发了新的方法，允许 用酶生物传感器评估体内外ATP和H_2O_2的释放；测量内源性TRPC 完整肾小球足细胞膜片钳通道活动；新分离的钙离子流量的定量 肾小球；体外研究肾小球白蛋白通透性。T2DN和链脲佐菌素治疗Dahl盐敏感 大鼠将被用来在1型和2型糖尿病模型中检验我们的假设。在这里我们将测试 具体目的如下：1)测定糖尿病患者基础和血管紧张素Ⅱ诱导的细胞外ATP浓度 确定ATP对TRPC通道功能的影响；2)确定ROS在脑内ROS的作用 ATP驱动的信号通路；3)确定肾小球通透性改变的反应后果 细胞外ATP和依赖TRPC的钙内流；4)确定特定的P2受体的作用 通过检测其抑制作用对糖尿病肾病发生发展的影响。这项研究在 自然界的基本面将开始填补知识的巨大缺口，并影响 美国军事人员和所有受益者。希望通过所描述的研究，更好地 了解钙稳态在糖尿病肾病进展中的影响将会实现。这个 本申请中提出的研究是新颖的，因为它是第一个直接定义TRPC通道和 糖尿病肾病中的嘌呤能信号转导。这项研究将带来重大发现，将促进我们对 这种病。它可能确定TRPC通道或特异性P2受体作为治疗控制的新靶点。 并朝着根除这种疾病的方向迈进。因此，这一应用程序与医疗保健具有直接的相关性 美国退伍军人及其家庭成员的需求。

The podocyte has become a crucial focus as a target for interventions in kidney disease due to its key role in regulating glomerular permeability and maintaining glomerular structure. Podocyte injury is believed to be pathogenetically and prognostically important in diabetic nephropathy (DN). One of the main factors determining pathological changes of glomerular morphology and permeability are linked to elevation of podocyte intracellular calcium ([Ca2+]i). Transient receptor potential canonical (TRPC) channels are important players in the pathogenesis of renal and cardiovascular diseases. ATP is a critical signaling molecule playing key role in podocyte function. However, our knowledge about purinergic signaling in glomeruli and their regulation of TRPC channels and [Ca2+]i in podocytes in the setting of DN is rudimentary and therefore is the focus of the current proposal. The central hypothesis of this proposal is that in diabetes significant changes in [Ca2+]i homeostasis in podocytes occur, which are mediated by: 1) increased concentration of extracellular ATP; 2) remodeling of purinergic signaling from metabotropic P2Y to ionotropic P2X receptors; 3) excessive production of ROS; and 4) hyperactivity of TRPC channels; altogether these events lead to glomeruli damage, proteinuria and, consequently, ESRD. We further hypothesize that increased [Ca2+]i influx in podocytes results in a pathological increase in glomeruli permeability to albumin. Based on the preliminary data and published findings, the main objective of this project is to define the specific mechanisms mediating the effect of ATP and ROS on TRPC channels in freshly isolated glomeruli and to identify the pharmacological targets that control glomerular albumin permeability in the pathogenesis of DN. To explore this idea, we have developed novel approaches that allow assessing ATP and H2O2 release with enzymatic biosensors ex vivo and in vivo; measuring endogenous TRPC channels activity with patch clamp in podocytes of intact glomeruli; quantifying calcium flux in freshly isolated glomeruli; studying glomerular albumin permeability ex vivo. T2DN and streptozotocin treated Dahl salt-sensitive rats will be used to test our hypotheses in models of both type 1 and type 2 diabetes. Here we will test the following Specific Aims: 1) To determine basal and Ang II-induced concentrations of extracellular ATP in diabetic animals, and to define the effects of ATP on TRPC channels function; 2) To identify the contribution of ROS in ATP driven signaling pathways; 3) To determine a consequence of altered glomerular permeability in response to extracellular ATP and TRPC-dependent calcium influx; 4) To define the contributions of specific P2 receptors by testing the effects of their inhibition on the development of diabetic nephropathy. This research while fundamental in nature will begin to fill a large gap in knowledge and impact the health and welfare of both the U.S military personnel and all beneficiaries. It is the hope, that through the described studies a better understanding of the impact of calcium homeostasis in progression of diabetic nephropathy will be realized. The research proposed in this application is novel for it is the first to directly define the role of TRPC channels and purinergic signaling in DN. This research will result in significant findings that will advance our understanding of this disease. It may determine TRPC channels or specific P2 receptors as new targets for therapeutic control of DN and move towards eradication of this disease. Thus, this application has direct relevance to the health care needs of the U.S. Veteran population and their family members.