Purinergic control of calcium flux in podocytes

足细胞钙流的嘌呤能控制

• 批准号: 10292941
• 负责人: ALEXANDER STARUSCHENKO
• 金额: --
• 依托单位: JAMES A. HALEY 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 2023-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10292941
• 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.

由于其在 调节肾小球渗透性并维持肾小球结构。据信足细胞损伤是 在糖尿病性肾病（DN）中，病原体和预后很重要。决定的主要因素之一 肾小球形态和渗透率的病理变化与足细胞内的升高有关 钙（[Ca2+] i）。瞬态受体潜力典型（TRPC）渠道是重要的参与者 肾脏和心血管疾病的发病机理。 ATP是关键的信号分子在 足细胞功能。但是，我们对肾小球中嘌呤能信号传导的了解及其对TRPC的调节 DN环境中的足细胞中的通道和[Ca2+] i是基本的，因此是电流的重点 提议。 该提议的核心假设是，在糖尿病中，[Ca2+] I稳态的重大变化 发生足细胞，由以下介导：1）细胞外ATP的浓度增加； 2）重塑 从代谢性P2Y到离子P2X受体的嘌呤能信号传导； 3）ROS的产生过多；和4） TRPC通道的多动症；这些事件总共导致肾小球损伤，蛋白尿和 因此，ESRD。我们进一步假设，足细胞中的[Ca2+] I流入增加导致病理学 增加肾小球对白蛋白的渗透性。基于初步数据和发布的发现，主要是 该项目的目的是定义介导ATP和ROS对TRPC影响的特定机制 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靶标 DN发病机理中的渗透性。为了探索这个想法，我们开发了允许的新方法 用酶促生物传感器离体和体内评估ATP和H2O2释放；测量内源性TRPC 通道活性在完整肾小球的足细胞中；量化新鲜分离的钙通量 肾小球；研究肾小球白蛋白渗透性离体。 T2DN和链霉菌素处理的DAHL盐敏感 大鼠将用于在1型和2型糖尿病的模型中测试我们的假设。在这里，我们将测试 以下特定目的：1）确定糖尿病中的基础和ANG II诱导的细胞外ATP 动物，并定义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）确定响应肾小球渗透性改变的结果 到细胞外ATP和TRPC依赖性钙的流入； 4）定义特定P2受体的贡献 通过测试它们抑制对糖尿病肾病发展的影响。这项研究 自然界的基本基础将开始填补知识的巨大空白，并影响两者的健康和福利 美国军事人员和所有受益人。希望通过描述的研究更好 了解钙稳态对糖尿病肾病进展的影响。这 该应用程序中提出的研究是新颖的，因为它是第一个直接定义TRPC渠道和 DN中的嘌呤能信号传导。这项研究将导致重大发现，以提高我们对 这种疾病。它可以确定TRPC通道或特定P2受体作为治疗控制的新目标 DN并转向根除这种疾病。因此，该应用与医疗保健有直接相关性 美国退伍军人人口及其家人的需求。