Purinergic control of calcium flux in podocytes

足细胞钙流的嘌呤能控制

• 批准号: 10047722
• 负责人: ALEXANDER STARUSCHENKO
• 金额: --
• 依托单位: CLEMENT J. ZABLOCKI VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-10-01 至 2022-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10047722
• 关键词: Address; Adenosine; Adult; Albumins; Albuminuria; American; Angiotensin II; Animals; Apoptosis; Biochemistry; Biosensor; Calcium; Calcium Channel; Cardiovascular Diseases; Cardiovascular system; Caring; Centers for Disease Control and Prevention (U.S.); Chronic; Dahl Hypertensive Rats; Data; Development; Diabetes Mellitus; Diabetic Nephropathy; Disease; Electrophysiology (science); End stage renal failure; Epidemic; Epithelial Cells; Event; Family member; Foot Process; Gene Expression; Growth; Health; Health Benefit; Healthcare; Homeostasis; Hydrogen Peroxide; Hyperactivity; In Vitro; Injury; Insulin-Dependent Diabetes Mellitus; Intervention; Kidney; Kidney Diseases; Knowledge; Lead; Link; Measurement; Measures; Mediating; Medical; Microscopy; Military Personnel; Modeling; Morbidity - disease rate; Morphology; Nature; Non-Insulin-Dependent Diabetes Mellitus; Oxidative Stress; P2X-receptor; Paracrine Communication; Patch-Clamp Techniques; Pathogenesis; Pathologic; Patients; Permeability; Pharmacology; Play; Population; Prediabetes syndrome; Production; Proteinuria; Publishing; Rattus; Reactive Oxygen Species; Regulation; Research; Risk; Role; Scheme; Signal Pathway; Signal Transduction; Signaling Molecule; Stimulus; Streptozocin; Structure; System; Testing; Tissues; Type 2 diabetic; Veterans; base; beneficiary; cell injury; clinically significant; defined contribution; diabetic; experimental study; extracellular; genetic strain; in vivo; military veteran; mortality; new therapeutic target; novel; novel strategies; paracrine; patch clamp; podocyte; prognostic; receptor; release of sequestered calcium ion into cytoplasm; response; salt sensitive; tripolyphosphate; welfare

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.

足细胞因其在肾脏疾病干预中的关键作用而成为肾脏疾病干预的一个重要焦点。 调节肾小球通透性，维持肾小球结构。足细胞损伤被认为是 对糖尿病肾病（DN）的发病机制和预后具有重要意义。决定的主要因素之一 肾小球形态和通透性的病理变化与足细胞胞内升高有关 钙 ([Ca2+]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。 ATP 是一种重要的信号分子，在 足细胞功能。然而，我们对肾小球嘌呤能信号传导及其对 TRPC 调节的了解 DN 背景下足细胞中的通道和 [Ca2+]i 还处于初级阶段，因此是当前研究的焦点 提议。 该提案的中心假设是，在糖尿病中，[Ca2+]i 稳态发生显着变化。 足细胞的发生是由以下因素介导的：1) 细胞外 ATP 浓度增加； 2）改造 从代谢型 P2Y 到离子型 P2X 受体的嘌呤能信号传导； 3）ROS产生过多；和 4) TRPC 通道过度活跃；总的来说，这些事件会导致肾小球损伤、蛋白尿， 因此，终末期肾病（ESRD）。我们进一步假设足细胞中 [Ca2+]i 流入的增加导致病理性 肾小球对白蛋白的通透性增加。根据初步数据和已发表的调查结果，主要 该项目的目标是确定介导 ATP 和 ROS 对 TRPC 影响的具体机制 新鲜分离的肾小球中的通道并确定控制肾小球白蛋白的药理学靶点 DN 发病机制中的渗透性。为了探索这个想法，我们开发了新的方法，允许 使用酶生物传感器体外和体内评估 ATP 和 H2O2 释放；测量内源性TRPC 使用膜片钳在完整肾小球的足细胞中通道活动；量化新鲜分离的钙通量 肾小球;离体研究肾小球白蛋白通透性。 T2DN 和链脲佐菌素处理 Dahl 盐敏感 大鼠将用于在 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 具体目标如下： 1) 确定糖尿病患者细胞外 ATP 的基础浓度和 Ang II 诱导浓度 动物，并确定 ATP 对 TRPC 通道功能的影响； 2）确定ROS的贡献 ATP驱动的信号通路； 3) 确定肾小球通透性改变的反应结果 细胞外 ATP 和 TRPC 依赖性钙流入； 4) 定义特定P2受体的贡献 通过测试它们对糖尿病肾病发展的抑制作用。这项研究同时 本质上是基本的，将开始填补知识方面的巨大空白，并影响人类的健康和福祉 美国军人和所有受益人。希望通过所描述的研究能够更好地 了解钙稳态对糖尿病肾病进展的影响将得到实现。这 该申请中提出的研究是新颖的，因为它是第一个直接定义 TRPC 通道的作用和 DN 中的嘌呤能信号传导。这项研究将产生重大发现，促进我们对 这种病。它可能确定 TRPC 通道或特定 P2 受体作为治疗控制的新靶点 DN 并朝着根除这种疾病的方向迈进。因此，该应用程序与医疗保健直接相关 美国退伍军人及其家人的需求。