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.

由于足细胞在肾脏疾病中的关键作用，足细胞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（DN）中具有重要的病理学和药理学意义。其中一个主要因素决定了 肾小球形态和通透性的病理变化与足细胞内 钙离子（[Ca 2 +]i）。瞬时受体电位典型（TRPC）通道是在细胞内的重要参与者。 肾脏和心血管疾病的发病机制。ATP是一种重要的信号分子， 足细胞功能然而，我们对肾小球中嘌呤能信号传导及其对TRPC的调节的了解， 在DN的背景下，足细胞中的[Ca 2 +] i和[Ca 2 +]i是基本的，因此是目前研究的重点。 提议 这一建议的中心假设是，在糖尿病中，[Ca 2 +]i稳态的显著变化， 足细胞发生，这是由以下介导的：1）细胞外ATP浓度增加; 2） 从代谢型P2 Y到离子型P2 X受体的嘌呤能信号传导; 3）ROS的过度产生;以及4） TRPC通道的过度活跃;这些事件共同导致肾小球损伤、蛋白尿， 因此，ESRD。我们进一步假设足细胞中[Ca 2 +]i内流的增加导致病理性的 肾小球对白蛋白通透性增加。根据初步数据和已公布的调查结果， 本研究的目的是明确ATP和ROS介导TRPC作用的具体机制 通道，并确定控制肾小球白蛋白的药理学靶点 糖尿病肾病的发病机制。为了探索这个想法，我们开发了新的方法， 用离体和体内酶生物传感器评估ATP和H2 O2释放;测量内源性TRPC 完整肾小球足细胞中膜片钳通道活性;定量新鲜分离的 肾小球;离体研究肾小球白蛋白渗透性。T2 DN和链脲佐菌素治疗Dahl盐敏感性 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 以下具体目的：1）测定糖尿病患者基础和Ang II诱导的细胞外ATP浓度， 2）确定ROS在TRPC通道功能中的作用，并确定ATP对TRPC通道功能的影响。 ATP驱动的信号通路; 3）确定肾小球通透性改变的结果， 细胞外ATP和TRPC依赖的钙内流; 4）确定特异性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。这项研究虽然 将开始填补知识上的巨大空白，并影响人类的健康和福利。 美国军事人员和所有受益人。这是希望，通过所描述的研究， 了解钙稳态在糖尿病肾病进展中的作用。的 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信道的作用， 嘌呤能信号在DN中的作用。这项研究将产生重大的发现，将促进我们对 这种疾病。它可能决定TRPC通道或特异性P2受体作为治疗控制的新靶点。 DN并努力根除这种疾病。因此，该应用与医疗保健直接相关， 美国退伍军人及其家庭成员的需求。