Role of p66Shc in Regulation of Microvascular Reactivity of Renal Blood Vessels

p66Shc 在肾血管微血管反应性调节中的作用

• 批准号: 10198033
• 负责人: ANDREY SOROKIN
• 金额: $ 38.41万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-22 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10198033
• 关键词: Adaptor Signaling Protein; Address; Aging; Blood Vessels; Cells; Chronic Kidney Failure; Dahl Hypertensive Rats; Data; Development; Diabetes Mellitus; Diabetic Nephropathy; Disease; Disease Marker; Electrophysiology (science); Evaluation; General Population; Genes; Guanine; Guanine Nucleotide Exchange Factors; Healthcare; Homeostasis; Human; Hypertension; Image; Impairment; Injury; Injury to Kidney; Kidney; Kidney Diseases; Knock-out; Knowledge; Lead; Location; Longevity; Mediating; Medical History; Microvascular Dysfunction; Mitochondria; Molecular; Oxidative Stress; Pathogenesis; Patients; Peptides; Phosphorylation; Play; Proteins; Rattus; Regulation; Renal Blood Flow; Renal function; Resistance; Role; SHC1 gene; SHetA2; Sampling; Serine; Signal Pathway; Signal Transduction; Site-Directed Mutagenesis; Smooth Muscle Myocytes; Streptozocin; Structure; Testing; Up-Regulation; Vascular Diseases; Vasoconstrictor Agents; Vasodilation; age related; arteriole; base; combat; diabetic; diabetic rat; dietary salt; experimental study; kidney cell; kidney cortex; kidney vascular structure; mutant; novel; novel therapeutic intervention; overexpression; p66(ShcA) protein; pressure; prevent; protein protein interaction; renal damage; response; restoration; salt intake; salt sensitive; two-photon; type I diabetic; vasoconstriction

Renal microvascular injury occurs in a majority of patients with diabetes and hypertension-induced nephropathy. Thus, the uncovering of the molecular mechanisms of changes in microvascular reactivity of renal blood vessels is necessary for developing new therapeutic strategies to combat these diseases, which contribute significantly to escalation of health care. Based on our preliminary data we hypothesize that overexpression of adaptor protein p66Shc is implicated in the loss of microvascular reactivity during the progression of both hypertension-induced nephropathy and diabetic nephropathy. We will use genetically modified Dahl salt sensitive (SS) rats, generated by targeted modification of Shc1 gene, and primary renal vascular smooth muscle cells (SMC) derived from these rats. p66Shc-dependent regulation of microvascular reactivity will be studied in rat afferent arterioles and human renal microvessels from deceased donors with medical history of either diabetes or hypertension-induced nephropathy. Specific Aim 1 will test the hypothesis that loss of renal microvascular reactivity in hypertension induced nephropathy is caused by p66Shc- dependent inhibition of Ca2+ influx mediated by TRPC channels and will analyze molecular mechanisms of TRPC regulation by p66Shc. We will test the role of p66Shc interaction with guanine exchange factor beta-Pix in regulation of TRPC channels activity and channel subcellular distribution. Using 2-photon imaging we will study the role of p66Shc in regulation of spontaneous intracellular Ca2+ oscillations in SMC embedded in the vascular wall of human renal resistance vessels. We will also test compound SHetA2, known to interfere with p66Shc action, for its ability to prevent p66Shc-induced decline of renal function in hypertension nephropathy. The restoration of renal vascular function will be evaluated by studying microvascular responses to purinergic activation and perfused pressure in the control SS rats and SS rats treated with compound SHetA2. We will also define whether SHetA2 has beneficial effect on vascular function in samples from diseased donors with and without hypertension. Specific Aim 2 will test the hypothesis that p66Shc regulates arteriolar KATP channel activity and causes hyperfiltration in diabetic nephropathy. Effect of upregulation of KATP activity, in concert with impaired Ca2+ influx responses to modulators of myogenic tone, is likely to promote vasodilation of renal afferent arterioles, causing hyperfiltration. We will study whether p66Shc regulates KATP channels in human renal microvessels. We will also employ type 1 diabetic rat model of STZ-induced diabetic nephropathy, which display markers of the disease similar to those observed in human patients. p66Shc-dependent KATP channel activity will be tested by electrophysiological recording in renal SMC. We will test whether p66Shc stimulates KATP channel activity via inducing protein-protein interactions with adaptor protein 14-3-3. The proposed experiments will provide direct evidence for the role of p66Shc signaling cascade in the regulation of renal microvascular reactivity and progression of renal injury, associated with hypertension and diabetes.

肾脏微血管损伤发生在大多数糖尿病患者和高血压诱导的患者中 肾病。因此，发现了微血管反应性变化的分子机制 肾血管对于制定新的治疗策略是必要的，以对抗这些疾病，这些疾病 为医疗保健的升级做出了重大贡献。根据我们的初步数据，我们假设 适配器蛋白p66SHC的过表达与在此期间的微血管反应性丧失有关 高血压诱导的肾病和糖尿病性肾病的进展。我们将在基因上使用 通过针对SHC1基因的靶向修饰和初级肾脏产生的改性DAHL盐敏感（SS）大鼠 源自这些大鼠的血管平滑肌细胞（SMC）。 p66SHC依赖于微血管的调节 反应性将在大鼠传入小动脉和已故供体的人类肾微血管中进行研究 糖尿病或高血压引起的肾病的病史。具体目标1将检验假设 高血压诱导的肾病中肾脏微血管反应性的丧失是由p66shc-引起的 依赖于TRPC通道介导的Ca2+涌入的抑制作用，并将分析分子机制 P66SHC的TRPC调节。我们将测试p66SHC与鸟嘌呤交换因子beta-pix的作用 在调节TRPC通道活性和通道亚细胞分布中。使用2光子成像，我们将 研究p66SHC在嵌入在SMC中的自发细胞内Ca2+振荡中的作用 人肾耐药容器的血管壁。我们还将测试化合物Sheta2，已知会干扰 P66SHC的作用，其能够预防P66SHC诱导的高血压肾病中肾功能下降。 肾血管功能的恢复将通过研究微血管对嘌呤能的反应来评估 用化合物Sheta2处理的对照SS大鼠和SS大鼠的激活和灌注压力。我们将 还定义了sheta2是否对患病供体的样品的血管功能有益作用 并且没有高血压。具体目标2将检验p66SHC调节小动脉KATP通道的假设 活性并引起糖尿病性肾病的过滤。 KATP活动上调的影响 CA2+涌入对肌吻合调节剂的反应受损，可能会促进肾脏的血管舒张 传入小动脉，导致过滤。我们将研究P66SHC是否调节人类的KATP渠道 肾脏微连续剧。我们还将采用STZ诱导的糖尿病性肾病的1型糖尿病大鼠模型，该模型 该疾病的显示标记类似于人类患者中观察到的标记。 P66SHC依赖性KATP通道 活性将通过肾脏SMC中的电生理记录来测试。我们将测试P66SHC是否刺激 KATP通道活性通过诱导蛋白质 - 蛋白质相互作用与衔接蛋白14-3-3。提议 实验将为P66SHC信号级联反应在肾脏调节中的作用提供直接证据 与高血压和糖尿病有关的肾损伤的微血管反应性和进展。